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Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use (1989)

Chapter: 2 HEREDITARY IMMUNODEFICIENCIES

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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 36
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 37
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 38
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 39
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 40
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 41
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 42
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 43
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 44
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 45
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 46
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 47
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 48
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 49
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 50
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 51
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 52
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 53
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 54
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 55
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 56
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 57
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 58
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 59
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 60
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 61
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 62
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 63
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 64
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 65
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 66
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 67
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 68
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 69
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 70
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 71
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 72
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 73
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 74
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 75
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 76
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 77
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 78
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 79
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 80
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 81
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 82
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 83
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 84
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 85
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 86
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 87
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 88
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 89
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 90
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 91
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 92
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 93
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 94
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 95
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 96
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 97
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 98
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 99
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 100
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 101
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 102
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 103
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
×
Page 104
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 105
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 106
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 107
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 108
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 109
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 110
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 111
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 112
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 113
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 114
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 115
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 116
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 117
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 118
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 119
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 120
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 121
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Page 125
Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Suggested Citation:"2 HEREDITARY IMMUNODEFICIENCIES." National Research Council. 1989. Immunodeficient Rodents: A Guide to Their Immunobiology, Husbandry, and Use. Washington, DC: The National Academies Press. doi: 10.17226/1051.
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Hereditary Immunodeficiencies This chapter describes the genetics, pathophysiology, husbandry, and re- production of 64 inbred, hybrid, and mutant strains of rodents with hereditary immunodeficiencies. It is not an exhaustive review, but rather, it summarizes current knowledge and provides key references as a starting point for the reader. Most of the animals described do not need extraordinary methods of isolation and maintenance, but do require procedures that prevent the intro- duction of rodent pathogens. The procedures necessary for the care of those animals susceptible to ubiquitous organisms are detailed in Chapter 4. Strains to be discussed are listed in Table 2-1. MICE WITH SINGLE MUTATIONS Beg:, Begs (Resistance and Susceptibility to Mycobacterium bovis); Ityr, Ids (Resistance and Susceptibility to Salmonella typhimurium); [star, [shS (Resistance and Susceptibility to L`eishmania Donovan Genetics Resistance and susceptibility alleles of Beg, Itcy, and Lsh define relative resistance to in vivo infection with Mycobacterium bovis (Gros et al., 1981) or M. Iepraemurium (Skamene et al., 1984; Brown and Glynn, 1987), Sal- monella t~yphimurium (Plant and Glynn, 1979), and Leishmania donovani (Bradley et al., 1979), respectively. Phenotyping of several inbred, recom 36

HEREDITARY IMMUNODEFICIENCIES 37 TABLE 2-1 List of Immunodeficient Rodents Dysfunctions Other than Care Immunodeficiency Special Special Page Auto- Non- Breeding Husbandry Mutation or Strain No. immunity immune Techniques Procedures Mice with single mutations Bcgr; Bcgs (resistance or susceptibility to Mycobacterium bovis) 36 - - - - bg (beige) 40 - + db (diabetes) 43 + + + + df (Ames dwarf) 46 - + Dh (dominant hemimelia) 47 - + + dw (dwarf) 49 - + + + gl (gray-lethal) 51 - + + gld (generalized lympho proliferative disease) 52 + Hc° (hemolytic complement) 54 hr (hairless); hrrh (rhino) 55 - + + Ityr; ItyS (resistance or susceptibility to Salmonella typhimurium) 36 Ih (lethargic) 58 - + + Ipr (lymphoproliferation) 59 + - + Lps ~ ( lip op olyac charide response, defective) 62 Lshr; Lshs (resistance or susceptibility to Leishmania donovani) 36 me (motheaten); meV (viable motheaten) 64 + + mi (microphthalmia) 67 - + + + nu (nude); nuS'r (streaker) 69 + + + + ob (obese) 72 + + + oc (osteosclerotic) 73 - + + + op (osteopetrosis) 75 - + + + scid (severe combined immunodeficiency) 77 - - - + Tol-1 (tolerance to bovine gamma-globulin) 80 Continued

38 IMMUNODEFICIENT RODENTS TABLE 2-1 (continue~ Dysfunctions Other than Care Immunodeficiency Mutation or Strain Page Auto- Non No. immunity immune Special Breeding Techniques Special Husbandry Procedures Mice with single mutations (continued) vit (vitilago) 80 W (dominant spotting); wv (viable dominant spotting) wst (wasted) xid (X-linked immune deficiency) Yaa (Y-linked autoimmune accelerator) S-region-linked genes controlling murine C4 Mice with multiple mutations gld xid Ipr nu Ipr xid Ipr Yaa nu bg nu xid nu bg xid nu Dh Yaa bg Yaa xid Inbred mice BSVR, BSVS BXSB/Mp (females) DBA/2Ha MRL/Mp NOD NON NZB NZB x NZW F~ (BWFi) NZB x SWRF~or SWR x NZB F~, both abbreviated SNF~ PN SAM-P SJL/J SL/Ni / 108 112 82 84 - + 85 87 89 90 90 91 91 91 92 92 93 93 94 94 96 98 98 100 + 103 104 + + + + + + + _ + _ - + + + 1 + + + + + + _ _ + + + + + + + + _ _ + - + + - _ + +

HEREDITARY IMMUNODEFICIENCIES 39 TABLE 2-1 (continued) Dysfunctions Other than Immunodeficiency Page Auto- Non Mutation or Strain No. immunity immune Care Special Breeding Techniques Special Husbandry Procedures Outbred mice SWAN Rat mutants ia (incisor absent) op (osteopetrosis) rnu (Rowett nude); rnuN (n nu, New Zealand nude) It (toothless) C4 deficiency Inbred rats BB/Wor LOU/C Guinea pig mutants C2 deficiency C3 deficiency C4 deficiency Hamster mutants nu C6 deficiency 122 123 124 125 128 129 130 134 134 135 136 138 139 + + + _ + + + + NOTE: A discussion of each positive ( + ) listing follows in the narrative describing each specific mutant or strain. binant inbred, and congenic strains indicates that the resistance and susceptibility alleles for all of these unrelated, obligate, intracellular pathogens are identical or closely linked on chromosome 1 (Plant et al., 19821. The BALB/c, C57BL/6, C57BL/lOScSn, and DBA/1 strains carry the susceptibility al- lelefs); the A/J, C3H/HeJ, C57L, and DBA/2 strains have the resistance allelefs). BALB/c (Potter et al., 1983) and C57BL/lOScSn (Blackwell, 1985) congenic strains carrying the resistance allelefs) have been developed using DBA/2 and C57L, respectively, as donor strains. Although these loci do not affect the immune response per se, they are unique in affecting macrophage function. There are other loci that affect resistance to Mycobacterium bovis, M. Iepraemurium, Salmonella typhi- murium, and Leishmania donovani; however, these have not yet been mapped (Curtis et al., 1982; Curtis and Turk, 19841.

40 IMMUNODEFICIENT RODENTS Pathophysiology Short-term culture of splenic and hepatic cells indicates that the phenotype is expressed in macrophages, which vary in their ability to control the rate of intracellular replication of pathogens (Lissner et al., 1983~. Denis et al. (1988) evaluated the efficiency of mouse splenic macrophages from strains congeneic for the Bcg locus and found that macrophages from Bcgr mice were superior to those from Bcgs mice in presenting bacteria-derived soluble and particulate antigens. In addition to imparting resistance to infection to Salmonella typhimurium and Leishmania donovani, the Bcg locus plays a less significant role in the protection of the closely related agents Salmonella typhi and Leishmania major. In the case of L. major, Davies et al. (1988) found that the parasite has developed a sophisticated method for escaping the effects of the Bcg gene. It induces increased monocyte infiltration into the site of infection, thereby providing safe targets in which the parasite can survive and multiply. Husbandry Special husbandry procedures are not required. Reproduction These animals reproduce normally. bg (Beige) Genetics Beige (formerly also called slate) is a recessive mutation located on chro- mosome 13 that arose independently several times. The symbol bg was assigned to a mutation, probably radiation induced, found at Oak Ridge National Laboratory, Oak Ridge, Tenn. (Kelly, 19571. A spontaneous mu- tation called slate (sit) was discovered at Brown University in 1955 (Chase, 1959) and was first described in 1963 (Pierro and Chase, 19631. In 1965 sit was recognized to be allelic with bg, and Chase (1965) recommended that the symbol sit be dropped if the mutant proved to be identical to bg. Another mutant allele, Egg, occurred spontaneously in the C57BL/6J strain at the Jackson Laboratory, Bar Harbor, Maine (Lane, 1962~. Beige-2J (bg2~) arose spontaneously in strain C3H/HeJ at the Jackson Laboratory in 1972.

HEREDITARY IMMUNODEFICIENCIES 41 A C57BL/6J-bg/bg mouse (rear) and a C57BL/6J control (front). Mice homozygous for the beige mutation show a dilution in pigmentation. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine. Pa thop hys to logy Homozygous beige mice have a light coat color and reduced ear and tail pigmentation. Eye color is light at birth, changing to a color that varies from ruby to almost black in adults. The phenotypic manifestations in bgibg mice closely resemble the Chediak-Higashi syndrome in humans, the Aleutian trait in mink, and a syndrome of partially albino Hereford cows (Lutzner et al., 1967). Beige homozygotes have been reported to have cytotoxic T-cell and mac- rophage defects (Mahoney et al., 1980; Saxena et al., 1982; Halle-Pannenko and Bruley-Rosset, 1985~. They have abnormally large lysosomal granules in a wide variety of cells, including leukocytes of bone marrow and peripheral blood, thyroid follicular cells, type II pneumocytes, mast cells, pyramidal cells and pericytes of the cerebral cortex, Purkinje cells of the cerebellum, spinal cord neurons, islet and acinar cells of the pancreas, liver parenchymal cells, and proximal tubule cells of the kidneys (Oliver and Essner, 1973; Chi et al., 1978; Prueitt et al., 19781. The defective granules are thought to contribute to lowered chemotaxis and a general motility defect (Gallin et al., 19741. Peritoneal exudate leukocytes of C57BL/6J-bg/bg mice contain very low or undetectable levels of neutral protease (Vassalli et al., 1978), and cir- culating polymorphonuclear leukocytes (PMNs) in bg homozygotes have

42 IMMUNODEFICIENT RODENTS impaired chemotactic and bactericidal activities. Kaplan et al. (1978) showed that the addition of small numbers of normal platelets or serotonin to whole blood of beige homozygotes increased the bactericidal activity of the PMNs. They theorized that normal platelets or serotonin enhanced the formation of cellular cGMP, thereby promoting polymerization of tubulin to microtubules and reversing abnormal bactericidal activity. A selective impairment of NK-cell function in bgibg mice prevents the initiation of antibody-dependent or antibody-independent cytolysis of tumor cells. The exact nature of the defect is not known, but it is believed to lie in the cell's lyric mechanism (Roder and Duwe, 19791. In vitro, these NK cells can be activated by treatment with interferon (Brunda et al., 19801. Defects have also been reported in cytotoxic T cells and macrophages (Ma- honey et al., 1980; Saxena et al., 1982; Halle-Pannenko and Bruley-Rosset, 19851. Beige homozygotes show a greater susceptibility to infection with pyogenic bacteria than do normal controls. This appears to be due to a defect in lysosomal function. The impairment is not of humoral origin because serum immunoglobulin levels are comparable in homozygotes, heterozygotes, and normal controls (Elin et al., 19744. Lane and Murphy (1972) demonstrated that the bg gene confers a greater susceptibility to spontaneous pneumonitis. In their study with SB/Le mice, which are homozygous for bg, sa (satin), and Aw (white-bellied agouti), and backcross offspring from an outcross to C57BL/6J-AW-~, beige homozygotes (sa bgisa bg and + bgisa bg) had a significantly higher incidence of spontaneous pneumonitis than did nonbeige mice (sa +Isa bg and + +Isa by). C57BL/6J-bg'/bg~ and C3H/HeJ- bg2'lbg2' mice do not develop spontaneous pneumonitis under similar ex- perimental conditions (J. B. Roths, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). C57BL/6 mice carrying the bg mutation or the pigment mutations pallid (pa), pearl (pe), light ear (le), pale ears (ep), maroon (ru-2mr), or ruby eye (ru) have hypopigmentation, prolonged bleeding time, normal platelet num- bers accompanied by reduced platelet granules, and decreased levels of plate- let serotonin. These seven mutations map to separate chromosomal sites; however, bone marrow transplantion from normal C57BL/6 mice to irradiated mutants corrects the defects in platelet serotonin and bleeding time. This suggests that in each case there is a cellular basis to the deficiency (Novak et al., 1985; McGarry et al., 19861. Experimentally, beige mice have played an important role in studies of hematopoietic differentiation. The giant lysosomal granules of beige mice provide an exquisite cytoplasmic marker for mast cells, PMNs, and osteo- clasts in bone marrow chimeras (Murphy et al., 1973; Ash et al., 1980; Kitamura et al., 19811. C3H/HeJ-bg27/bg9~ mice that survive to 17 months of age show a pro

HEREDITARY IMMUNODEFICIENCIES 43 gressive neurological disorder accompanied by a nearly complete loss of cerebellar Purkinje cells (Murphy and Roths, 1978b). This lesion is less severe in similarly aged C57BL/6J-bgi/bg~ mice. The pigment mutations ep, pa, and rp (reduced pigmentation) affect ly- sosomal functions and lead to suppressed NK cell activity (Orn et al., 1982~. An additional 29 pigment mutations have been described in mice, all of which are known to affect lysosomal biogenesis. However, little is known about the function of the immune system in these mice (Brands et al., 198 1~. Husbandry Beige mice are more susceptible than immunocompetent mice to challenge by a wide variety of infectious agents, but they do not suffer from infections caused by indigenous microorganisms that are not pathogenic to immuno- competent mice. These animals survive well in a pathogen-free environment. Barrier isolation, as described in Chapter 4, is not required for the mainte- nance of these animals. Reproduction Beige mice of both sexes will breed. db (Diabetes) Genetics The mutation diabetes (db) on chromosome 4 is a spontaneous autosomal recessive obesity gene discovered at the Jackson Laboratory, Bar Harbor, Maine, in the C57BL/KsJ inbred strain (Hummer et al., 19661. The gene has been transferred to the C57BL/6J and a number of other inbred strains. Several other alleles are present at the db locus: dab arose in an inbred brown (b) whirler (wi) stock (Lane, 1968), db3~ arose in strain 129/J (Letter et al., 1980), and dbPaS arose in strain DW/J (Aubert et al., 19851. Another allele, dba4, arose in a stock selected for large size and was called adipose (ad) (Falconer and Isaacson, 19591. In 1972 the gene was found to be allelic with db and was redesignated dboa' (Hummer et al., 19731. Pathophysiology Regardless of inbred strain background, dbidb mice always exhibit a marked obesity associated with hyperphagia, hyperinsulinemia, and severe insulin resistance. However, ultimate development in dbidb mice of a permanent diabetic condition (typified by chronic hyperglycemia, reduced serum insulin

44 IMMUNODEFICIENT RODENTS A 129/J-db3J/db3J mouse (left) and its normal littermate (right). Mice homozygous for the diabetes mutation develop non-insulin-dependent diabetes and become obese. Photograph cour- tesy of the Jackson Laboratory, Bar Harbor, Maine. levels, beta-cell necrosis, and pancreatic islet atrophy) depends entirely on the inbred strain background and gender (Coleman, 1 978; Leiter et al., 1 98 1 ). The mutation was named diabetes because the C57BL/KsJ strain of origin proved susceptible to its diabetogenic action. In this strain, dbldb mice of both sexes develop an early-onset diabetes resembling in some respects the human non-insulin-dependent (type II) diabetes mellitus. On the contrary, the same mutation on the diabetes-resistant C57BL/6J inbred background did not produce beta-cell necrosis or permanent hyperglycemia; instead, the dia- betogenic action of the mutation was well compensated by unrestricted hy- perplasia of pancreatic beta cells and sustained hyperinsulinemia (Coleman, 19789. Although type II diabetes in humans is not generally associated with an autoimmune etiology, the reports of both humoral and cell-mediated autoim- munity against pancreatic beta cells in C57BL/KsJ-db/db mice (Debray-Sachs et al., 1983), as well as the finding of immune complex deposition in the kidneys (Meade et al., 1981), suggested that this model might be an amalgam of features of type I (autoimmune) as well as type II diabetes in humans. An association between the H-2 haplotype and diabetes susceptibility and resistance was initially suggested by a comparison of db gene expression on a variety of inbred strain backgrounds (Letter et al., 1981~. However, seg- regation analyses have shown that H-2 does not control susceptibility; instead, male gender-linked factors, and possibly endogenous retroviral genes, appear to be the major modifiers of diabetes severity (Letter, 1985; Leiter et al., 1987a). The question of the pathogenic role of the humoral and cell-mediated anti-beta-cell reactivities in dbldb mice has also been resolved by combining -

HEREDITARY IMMUNODEFICIENCIES 45 this mutation with immunodeficiency genes such as severe combined im- munodeficiency (scid) to compromise T- and B-lymphocyte function (Letter et al., 1987b). This study showed that immunodeficient dbidb mice still developed diabetes, indicating that autoimmunity is probably a reflection of islet cell destruction rather than its cause. The C57BL/KsJ-db/db mouse has proven to be exceptionally useful as a model for analyzing the effects of chronic non-insulin-dependent diabetes on the immune system. There is early thymic involution and T lymphopenia from 8 weeks onward (Boillot et al., 19861; the disturbed metabolic milieu leads to depressed cell-mediated immunity and lymphokine production (Fer- nandes et al., 1978; Mandel and Mahmoud, 1978; Kazura et al., 1979; Pasko et al., 19811. Many of the T-cell functions that are impaired in vivo (for example, generation of alloreactive cells) are normal when assessed in vitro (Fernandes et al., 1978), which demonstrates the suppressive effect of the imbalanced metabolic environment in vivo. In contrast to the suppression of T-lymphocyte functions, certain B-lymphocyte functions are increased, in- cluding autoantibody production against islet cell cytoplasmic antigens, thymic hormones, and insulin (Dardenne et al., 1984; Serreze et al., 1988b; Yoon et al., 1988~. Transfer of dbldb marrow cells into lethally irradiated +/+ recipients rescued recipient mice from radiation death but did not transfer the diabetes syndrome (Letter et al., 1987b). Husbandry Special procedures are not required to maintain these animals; however, their life span can be prolonged by dietary restriction, and the severity of the syndrome can be significantly diminished by feeding carbohydrate-free, protein-enriched defined diets (Letter et al., 19831. Reprodluction Diabetic mice of both sexes on all backgrounds will not mate, and females are hypogonadal. Therefore, breeding is accomplished with heterozygotes. To aid in the identification of heterozygous breeders on the C57BL/KsJ and C57BL/6J inbred backgrounds, the db gene has been placed in repulsion with the coat color mutation misty (m). Black, lean mice obtained from a cross of db + / + m heterozygotes are used as breeders, whereas lean mice with grey coats ~ + m/ + m genotype) are discarded. Black mice that become obese at weaning are the presumptive mutants (db + Idb + genotype). When it is desirable to identify presumptive dbidb mice as early as 3 days post- partum, breeding stocks are utlized in which db and m are maintained in coupling (db ml + + J. The mutant pups can be recognized by the absence of pigment in the paws and on the tip of the tail.

46 IMMUNODEFICIENT RODENTS df (Ames Dwarf) Genetics Ames dwarf (did appeared first in 1961 as an autosomal recessive mutation in a line of extreme nonagouti (ae/ae) mice derived from a cross between Goodale's giant and a pink-eyed stock (Schaible and Gowen, 19611. Although phenotypically similar to the mutation dwarf (dw) (see page 49), df is not allelic with dw. The df mutation, which maps to chromosome 11, is main- tained on an outbred and on the NFR/N inbred backgrounds. Pa thophys lo logy Homozygous Titmice resemble dwldw mice. Growth retardation is observed after 1 week of age, and by 2 months of age they are only one-half the weight of controls. The anterior pituitary lacks cells that produce either prolactin (Barkley et al., 1982) or growth hormone (Duquesnoy and Pedersen, 19811. Ames dwarf mice have a deficit of the T-cell component of the immune system, which becomes apparent by 3 weeks of age. Morphological abnor- malities include depletion of lymphocytes in the thymus-dependent regions of lymph nodes and periarteriolar sheaths of the spleen follicles and a grad- ually progressive lymphopenia. This abnormality culminates in the devel- opment of progressive atrophy of lymphoid tissue, signs of wasting and infection, and early death. Other features of the T-cell deficit are depressed ability of spleen cells to induce graft-versus-host reaction, impaired ability to reject allogeneic skin grafts, and impaired phytohemagglutinin (PHA)- induced blastogenesis. Both dwarf mutations have a similar deficit of T-cell function, although the deficit is more severe in df than in dw mice. There are subtle differences found in the kinetics of the immune response to sheep red blood cells (SRBCs). Although dw mice always develop more feeble plaque-forming cell (PFC) responses compared with control mice, they show an increase in PFC re- sponses with increasing doses of SRBCs. In contrast, df mice do not show a dose-related dependency of immune response to SRBCs. The reason for this difference between the two dwarf mutations is not well understood, and numerous theoretical explanations have been postulated (Duquesnoy and Pedersen, 19811. Husbandry Mice carrying the df mutation should be maintained as described for those carrying dw (see page 491.

HEREDITARY IMMUNODEFICIENCIES 47 Reproduction Females and nearly all male df homozygotes are sterile (Bartke, 19649; therefore, the stock must be maintained by breeding heterozygotes. Dh (Dominant Hemimelia) Genetics The autosomal dominant mutant gene Dh arose spontaneously in a crossbred stock at the Institute of Animal Genetics, Edinburgh, Scotland (Carter, 19541. Dh is located on chromosome 1, approximately 3 centimorgans (cM) prox- imal to Pep-3. Dhl+ mice are maintained on the BALB/cJBoy inbred and C57BL/6J x C3HeB/FeJLe-a/a Fit hybrid backgrounds. Pathophysiology Both hetero- and homozygous Dh mice are congenitally asplenic and have multiple gastrointestinal, urogenital, and skeletal anomalies (polydactyly and oligodactyly) (Searle, 19591. Homozygotes, in which these anomalies are very severe, die shortly after birth. The abnormalities have been traced to a defect in the splanchnic mesoderm (Green, 19671. Heterozygotes have enlarged lymph nodes and elevated numbers of cir- culating lymphocytes, granulocytes, and thrombocytes (Lozzio, 19721. They show decreased serum levels of IgM and IgG2 immunoglobulins but normal levels of IgG1. There is no evidence that Dh mice spontaneously produce autoantibodies (Fletcher et al., 19771. Both primary and secondary immune responses against SRBCs are reduced compared with those in eusplenic controls. Cell-mediated immunity, as measured by allograft rejection, is not impaired, and the response of lymph node cells to the mitogens concanavalin A (ConA), PHA, and lipopolysaccharide (LPS) is normal. Lymph nodes of Dhl + mice have a significantly reduced number of B cells, with a com- pensatory increase in the number of T cells. Hereditary asplenia is also associated with a significant reduction in the rate of T-cell maturation (helper T cells specifically) (Fletcher et al., 19771. The immunocompetence of Dh mice has been thoroughly reviewed by Welles and Battisto (19811. Husbandl7y Special husbandry procedures are not required for maintaining heterozy- gotes. Special husbandry procedures will not prolong the life of Dh homo- zygotes.

48 ~7 ~ ... ............................... ... ..... .. ..................... ...... .......... .. .. .. . . ... . . , . . YE . ,: a. . A B6C3/Fe-~/~ Ad/ + mouse. Mice china the gene dominant hemimelia commonly have polydac[yly, as shown on the animal's hat Hot. which has six logs. Photograph courtesy of the Jackson Laboralo~, Bar Harbor. Maine. The ~A mutation is maintained by breeding hcterozygous Add/ +) ~- males with homozygous normal ~ + / + ~ males or + / + Smalls with Ad/ + males.

HEREDITARY IMMUNODEFICIENCIES 49 dw (Dwarf) Genetics The autosomal recessive mutation dwarf (dw) was discovered by Snell (1929) in a stock of mice obtained from an English mouse fancier. The mutation, which maps to chromosome 16, is now propagated in the segregating DW/J inbred strain. It has also been transferred to C3H:/HeJ, C3H/HeN, C57BL/6J, and NFR/N backgrounds. C57BL/6J-dw/dw mice are preserved as frozen em- bryos. A point mutation producing dwarfed offspring on the C3H/HeJ back- ground has been found to be a new allele, dim, at the dwarf locus (Etcher and Beamer, 19801. Pathophysiology Homozygous dw mice are one-fourth to one-third the size of normal mice. The anterior hypophysis, as in df mice, is deficient in acidophils and thy- rotropic hormone-producing cells (Elftman and Wegelius, 19599. The im- mune system deficit was first described by Baroni and coworkers (Baron), 1967; Baroni et al., 1967, 1969) and further characterized by Duquesnoy and coworkers (Duquesnoy and Good, 1970; Duquesnoy and Pedersen, 198 1; Duquesnoy et al., 1970), who showed the deficiency to be in the T-cell system. The thymus of dw homozygotes is characterized by atrophy and a marked A DW/J-dw/dw mouse (left) and its normal littermate (right). Dwarf homozygotes are much smaller than control mice. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Ma~ne.

50 IMMUNODEFICIENT RODENTS loss of lymphocytes in both the medulla and the cortex. A specific deficiency of lymphocytes in the cortex alone is observed in 25 percent of dwarf mice. Periodic acid-Schiff (PAS)-positive cells are prominent in the cortex. Pe- ripheral lymph nodes are small, and the spleen has only one-tenth the nor- mal number of lymphoid cells. Peripheral lymphoid tissues show hypocellu- larity, especially in the T-cell-dependent areas (paracortical areas of the lymph nodes and perifollicular sheaths in the spleen), but structurally they are similar to the lymphoid follicles of control mice. Dwarf mice have a marked reduction in peripheral blood lymphocytes but normal numbers of PMNs. Duquesnoy et al. (1970) noted that the immunologic deficiency becomes particularly evident after weaning at 21 days of age and showed that prolonged nursing delays its development. These observations raised the question of whether a factor present in the milk contributes to maintenance of the lym- phoid system. Dwarf mice have nodal levels of serum IgG1, IgG2, IgM, and IgA. Hemagglutinating antibody titers to SRBCs (T cell dependent) and agglutin- ating antibody responses to Brucella antigen (T cell independent) are con- siderably lower in dwldw mice than in controls. Spleen cells of homozygotes show a depressed capacity to initiate graft-versus-host reaction and a dimin- ished response to PHA stimulation. Pelletier et al. (1976) have found that dwarf mice have very low levels of serum thymic hormone activity (thymulin or facteur thymique serique) and minimal kidney disease. Littermate controls have normal levels of thymulin, and by 14 weeks they develop severe kidney lesions characterized by deposits of IgG1, IgG2, IgA, IgM, and C3 (a component of complement), which are found mainly in the glomerular mesangium. The serum of the heterozygous littermates contains antinuclear antibodies (ANAs) and anti-DNA autoanti- bodies, while dwarf homozygotes do not develop these autoantibodies. It has been postulated that dwl + mice have a precocious decrease of suppressor cells (Pelletier et al., 19761. Schneider (1976) found normal delayed-type hypersensitivity and blasto- genic responses in T cells of dw homozygotes, and Dumont et al. (1979) reported that dwldw mice do not have major abnormalities in either T- or B- cell function. These findings, although contrary to most work done with these mice, demonstrate that the immunologic deficiencies described previously might not automatically accompany the dwarfism. In their recent review, Shultz and Sidman (1987) suggest that differences in genetic background and husbandry conditions might explain the wide differences seen in the immune competences of these mice. Recent studies report a mean life span in dwarf mice of 18 months (Schneider, 1976) to 26 months (Etcher and Beamer, 1980), compared with earlier studies reporting a life span of only 2-4 months (Baron), 1967; Duquesnoy et al., 19701.

HEREDITARY IMMUNODEFICIENCIES 51 Husbandry Investigators are encouraged to maintain homozygous dw mice in a path- ogen-free environment because of the reported variability in levels of immune competence. Other special husbandry procedures that should be used because of the small size of these animals are to place food pellets in the bottom of the cages, as well as in feeders, and to use longer than normal sipper tubes for water. Reproduction Homozygous dwarf mice of both sexes are infertile. The mutation is prop- agated by matings of tested heterozygotes. g! (Gray-Lethal) Genetics Grey-lethal (al) is a recessive lethal mutation that arose spontaneously in a stock segregating for ce (extreme dilution) (Gruneberg, 19351. It has been mapped to chromosome 10 (Lane, 19711. It is maintained on the inbred GL strain, whose genetic background has not been typed. Pathophysiology Grey-lethal is one of four osteopetrotic mutations in mice (see also mi, page 67; oc, page 73; and Op. page 751. Animals have a grey coat, unless the mutation is carried on a homozygous nonagouti (a/a) background. The teeth of homozygotes fail to erupt, the animals develop severe osteopetrosis, and death occurs by 20-40 days of age (Gruneberg, 1936, 1938; Marks, 19841. Gruneberg (1938) reported a marked reduction in cortical thymocytes; however, since the teeth fail to erupt, it is likely that this thymic defect was caused by starvation. Wiktor-Jedrzejczak et al. (1983) observed normal thymic cellularity at 3 weeks of age and thymic involution, apparently associated with ill health, by 5 weeks of age. The gilgl mouse is anemic and has a reduced white cell count, although the granulocyte count is slightly elevated. Spleen cellularity is reduced; how- ever, the number of splenic stem cells is increased (Wiktor-Jedrzejczak et al., 19811. Osteoclasts are small and reduced in number, and there appear to be two subpopulations, one with a normal acid phosphatase content and one with a reduced content (Marks and Walker, 19761. The rate of bone deposition is higher, the number of parafollicular (calcitonin-secreting) cells

52 IMMUNODEFICIENT RODENTS of the thyroid is greater (by eightfold,, and the level of serum calcium is lower in gilgl mice than in normal littermates. Parathyroid hormone admin- istration does not cure the disease and only slightly raises serum calcium (Marks and Walker, 19691. The gilgl mouse does not form normal bone marrow cavities in long bones. Responsiveness of splenic lymphocytes to T- and B-cell mitogens and of thymocytes to ConA is equal to or better than that of normal littermates, at least early in life (Wiktor-Jedrzejczak et al., 19811. The response of the gilgl mouse to transplanted hematopoietic stem cells has not been completely characterized; however, cure can be effected by transplantation of normal spleen or bone marrow cells from histocom- patible donors (Walker, 1975a,c). The defect can be transferred with spleen cells from gilgl donors (Walker, 1975b). The gilgl mouse might provide a model for studying the association between the immune system, especially T lymphocytes, and early events leading to osteopetrosis (Wiktor-Jedrzejczak etal., 19811. Husbandry Special husbandry procedures are not required for maintaining heterozy- gotes. Special husbandry procedures will not prolong the life of homozygotes. Reproduction The gl mutation is maintained by brother x sister mating of heterozygotes in a balanced stock. Litter sizes are normal, and there is no evidence for in utero deaths. alar (Generalized Lymphoproliferative Disease) Genetics Generalized lymphoproliferative disease (gid) is an autosomal recessive mutation that occurred spontaneously in the C3H/HeJ strain (Murphy et al., 19821. The gene determines the development of early-onset lymphoid hy- perplasia with autoimmunity. Although phenotypically similar to C3H/HeJ- lpr/lpr, early tests indicated that aid and Ipr were not allelic. The aid mutation has been mapped to chromosome 1 between Pep-3 and Lp. It has been transferred to inbred strains C57BL/6J and SJL/J by multiple (more than eight) cross-intercross cycles of matings.

HEREDITARY IMMUNODEFICIENCIES 53 ~ ~ i. ~ :.:~:: ::: :: . .. ~ ~ ~ ~ i. i ...... .. ....... Ad, ~ ,~ ~ ~ ~ ... , ~.~.~. ~.~,. ~ ~i. ,~ .. ~ .. . . . .,,,.., ,. ~ ~ i,. .~.. . ~ .~, ~.~,~.~., Ace. ,. i. Ad, . i. ~ ~ , ...... ~ ~ . ~,~ , ..~ ~ , , ~,~.,....~...~.~.~.~.~ ~ : . ~.~,~. ~ ~ ... ~.,~,,...~..,~,~.......... ~,.~.~,.~.~....~ ..~. ~. ~.~.~. ~ ~ ~ ... I... At. . ... ~ ~ ~ ~ ~ . ~ ~ ~ . ... A C3H/HeJ-gld/gld mouse. Mice affected with generalized lymphoproliferative disease develop enlarged lymph nodes, as shown by this animal's enlarged prescapular lymph node. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine. Pathophysiology The mean life span of C3H/HeJ-gld/gld mice is 53 weeks for females and 57 weeks for males. They appear healthy, except for enlarged lymph nodes, and are active until several weeks prior to death. Significant lymph node enlargement is apparent as early as 12 weeks of age, and by 20 weeks, lymph nodes are 60-fold heavier than those in cois- ogeneic C3H/HeJ controls. A major regression of lymph node mass occurs at about 30 weeks of age (reduction from 60- to 25-fold heavier), followed by a second proliferative phase in which the lymph node mass becomes 96- fold heavier than normal by 47 weeks of age. Histological analysis reveals a loss of nodular architecture with a predom- inant proliferation of lymphocytes, including an admixture of plasma cells and histiocytes. Fibrosis and multinucleated giant cells are frequently ob- served in gld mice that reach more than 40 weeks of age (Roths et al., 19841. Fluorescence-activated cell sorting (FACS) analysis of enlarged lymph nodes from C3H/HeJ-gld/gld mice reveals an aberrant population of Thy-1+, Ly-1 +, L3T4-, and Lyt-2- T cells that also bear the B-cell marker Ly-5 but not surface immunoglobulin (sIg) or Ia (Davidson et al., 19851. DNA probe analysis for the immunoglobulin heavy chain and the T-cell antigen receptor beta-chain rearrangements demonstrates that abnormal cells found in lymph nodes of gld homozygotes are of T-cell origin (Davidson et al.,

54 IMMUNODEFICIENT RODENTS 19861. Ishida et al. (1987) postulate, based on investigations comparing normal and gld mutant mice, that the aberrant cell population in gld mice is a normal regulatory T cell that is proliferating abnormally. Lymphocytes of gld mutant mice are deficient in their ability to produce IL-2 and have reduced proliferative responses to alloantigens in mixed lymphocyte reactions (MLRs) (Davidson et al., 19851. However, clones of T cells with a normal surface phenotype that are established from the lymph nodes of gld homozygotes are fully capable of recognizing class I molecules on target cells and func- tioning as cytotoxic lymphocytes (Yui et al., 19871. Splenomegaly, in which the spleens become eight times heavier than those of controls by 47 weeks of age, is primarily caused by the expansion of white pulp. Neonatal thy- mectomy prevents the lymphadenopathy in gldlgld mice, thus implicating a major role for thymic-derived lymphocytes in the pathogenesis of this disease. Only 14 percent of autopsied C3H/HeJ-gld/gld homozygotes have signif- icant lupuslike renal disease. Immune complexes are found in the kidneys (primarily the mesangium) of all adult C3H/HeJ-gld/gld mice. Histological analysis of kidneys of the majority of gldlgld mice, however, reveals only a minor focal renal pathology. Serologically, C3H/HeJ-gld/gld mice develop antinuclear antibodies, in- cluding anti-double-stranded DNA (anti-dsDNA), thymus-binding autoanti- body, and hyperimmunoglobulinemia with major increases in several isotypes. Recent evidence suggests that expression of the xid gene can decrease the B-cell manifestations of autoimmunity without affecting the abnormal T-cell expansion (Seldin et al., 19871. Interstitial pneumonitis is found in virtually all gld mice and is a likely cause of morbidity (Roths et al., 19841. Husbandry Special husbandry procedures are not required. Reprodfuction Productivity of gld homozygotes is similar to that of their normal ~ + / + counterparts. Hc° (Hemolytic Complement Absent) Genetics Hemolytic complement (Hc) is located on chromosome 2. Two alleles are known: Hc~ determines the presence of the fifth component of complement (C5) in serum; Hc° determines its absence (Erickson et al., 19641. Hetero- zygotes have half the amount of complement as do homozygous Hc~ mice.

HEREDITARY IMMUNODEFICIENCIES 55 Pathophysiology Lack of complement activity was discovered by Rosenberg and Tachibana (1962) in two strains of mice, DB A/2 and B10.D2/oSnJ. In the early liter- ature, C5 was called beta-globulin, MuB1 (Cinader and Dubiski, 1964), or ,B1F glycoprotein (Nilsson and Muller-Eberhard, 19651. In humans, total C5 deficiency corresponds to a genetic locus linked to the major histocompati- bility complex (Rosenfeld et al., 19771. Studies by Patel and Minta (1979) demonstrated the incorporation of l4C- labeled amino acids into a C5-like protein by C5-deficient cells in vitro. The protein elaborated by C5-deficient macrophages was the 200-kDa pro-C5 molecule deficient in carbohydrate and not secreted (Ooi and Colten, 1979~. The absence of circulating C5 prohibits the formation of the membrane attack complex (CSb-9) during complement activation. Strains of mice that bear the Hc° allele have low serum hemolytic com- plement activity only because of a deficiency of C5. There are no circulating complement inhibitors (Nilsson and Muller-Eberhard, 19671. When strains of mice carrying the homozygous Hc° allele (complement deficient) are compared with strains homozygous for the Hcl allele, they appear to be more susceptible to a variety of infectious agents inoculated experimentally (Caren and Rosenberg, 1966; Glynn and Medhurst, 1967; Morelli and Rosenberg, 1971; Easmon and Glynn, 1976; Hicks et al., 19781. Congenic strains dif- fering only by the Hc allele have been used to demonstrate delayed pulmonary clearance of Staphylococcus aureus in C5-deficient mice (Cerquetti et al., 19831. There is little evidence, however, that C5-deficient mice are more susceptible to spontaneous infectious diseases. Husbandry Special husbandry procedures are not required based on the presence of Hc° alone. However, specific-pathogen-free conditions are recommended for certain strains bearing Hc° (e.g., DBA/2) that appear to be extremely sus- ceptible to viral pathogens. Reproduct~on Most Hc° strains reproduce normally. hr (Hairless); hrrh (RhinoJ Genet~cs The original recessive mutation to hr. located on chromosome 14, was discovered in a wild mouse captured in a London aviary in 1924 (Brooke,

56 IMMUNODEFICIENT RODENTS HRS/J-hrlhr (top) and RHJ/Le-hrrh/hrrh (bottom) mice. Hairless homozygotes are born with pelage but lose their hair after 10 days. Rhino homozygotes are hairless and have excessive skin folding. Photographs courtesy of the Jackson Laboratory, Bar Harbor, Maine. 19261. It has recently been reported that the mutation results from the in- tegration of a provirus into the normal allele at the hairless locus (Stoye et al., 1988J. Studies of abnormalities of the immune system in hrlhr mice have focused on the HRS/J strain, which originated from a cross between an outbred stock carrying the hr mutation and the BALB/cGn strain (Heiniger et al., 19749. Homozygosity for a second allele at the hr locus, called rhino (hrrh), also causes immunologic dysfunction.

HEREDITARY IMMUNODEFICIENCIES 57 Pathophysiology Unlike nude mice, which are hairless throughout life, homozygotes for mutant alleles at the hr locus develop a normal pelage up to 10 days of age, at which time they lose their hair. Although a few thin hairs grow in at monthly intervals, the mice are essentially hairless by 5-6 weeks of age. Alopecia in these mice appears to be related to the malpositioning of the internal root sheath. The most striking feature of the skin following hair loss is the formation of cutaneous cysts from hair follicles and isolated sebaceous glands (Mann, 19711. Investigations of the immune system of hairless mice were stimulated by the finding that they develop a high incidence of thymic lymphomas. HRS/J-hr/hr mice show a 45 percent incidence of thymic lymphoma by 10 months of age; HRS/J-hr/+ mice show a 1 percent incidence (Meter et al., 19691. Although hairless mice are euthymic, by 6 months of age there is marked thymic cortical atrophy (Heiniger et al., 19741. Defective immune function in hairless mice is associated with T-cell abnormalities. Splenic T cells from this mutant have a depressed proliferative response to I-region alloantigens (Morrissey et al. ,1980) . In addition, spleens show an inversion in the normal proportions of Ly-1+ and Ly-123+ T cells (Reske-Kunz et al., 19791. Reduced hu- moral immune responses to thymic-dependent antigens have also been reported in these mice (Heiniger et al., 19749. An association between defective immune function and lymphomagenesis in hairless mice is sug- gested by the reduced immune responsiveness of this mutant to syngeneic lymphoma cells and to purified murine leukemia viruses (MuLVs) (John- son and Meter, 1981). HRSlJ-hrlhr mice and their heterozygous littermates have been found to be unusually susceptible to infection with Listeria monocytogenes (Archinal and Wilder, 1988a). During studies on oxidative metabolic activities associated with the respiratory burst in mutant and normal mice, the liberation of superoxide anion and the chemiluminescence response of macrophages were both signifi- cantly diminished in the caseinate-elicited cells from hrlhr and hrl + mice (Ar- chinal and Wilder, 1988b). These results are compatible with the hypothesis that the defect in resistance to L. monocytogenes in mutant mice is caused by an intrinsic deficiency in the mobilization and the listericidal activity of the macrophage. Homozygosity for a second deleterious allele at the hr locus, called rhino (hrrh), also results in the loss of hair and reduced responsiveness to thymic- dependent antigens. Rhino mice are similar in appearance to hairless mice, except that their skin becomes thickened and markedly wrinkled (Mann, 19711. As with hairless mice, decreased humoral immune responses to thymic- dependent antigens in rhino mice appear to be due to reduced helper T-cell function (Takaoki and Kawaji, 19801.

58 IMMUNODEFICIENT RODENTS Husbandry Special husbandry procedures are not required to maintain hairless and rhino mice, which, with the exception of occasional skin abscesses, show no increased susceptibility to infection. Reproduction Hairless and rhino mice are fertile, but females do not nurse their young well. The most effective breeding system uses homozygous mutant males and heterozygous females. [h (Lethargic) Genetics The recessive mutation lethargic (Ih), first described by Dickie (1964), occurred spontaneously at the Jackson Laboratory, Bar Harbor, Maine, in 1962 in strain BALB/cGn. The Ih locus is approximately 24 cM from the centromere on chromosome 2. Pathophysiology Homozygous Ih mice demonstrate an instability of gait by 15 days of age, and the majority (68-80 percent) die by 45 days. The pleiotropic effects of Ih include an absence of subcutaneous fat and abnormalities of the lymphoid system. At 4 weeks of age Ih mice are less than 50 percent of the weight of their normal littermates. The neuropathologic phenotype has been reviewed by Sidman et al. (19651. Lymphoid organs of Ihllh mice, including thymus, spleen, lymph nodes, and Peyer's patches, are smaller than those of normal littermates. Lethargic mice have reduced erythrocyte packed-cell volumes (36 percent) and severe leukocytopenia. Homozygous Ih mice have defects in cell-mediated immune functions. For example, the ability to reject skin allografts is defective in 30-day-old homozygotes but is normal in 40-day-old homozygotes. This transitional defect in cell-mediated immunity also is found in the ability to induce graft-versus-host reactions. Serum IgG1 and IgG2a levels are higher in mutant mice than they are in controls. Extensive studies by Dung and colleagues on the anatomic, necrologic, hematologic, and immunologic as- pects of the lethargic mutation have been reviewed (Dung, 19811.

HEREDITARY IMMUNODEFICIENCIES 59 Husbandry Compared with controls, Ihllh mice have a reduced capacity to resist hypothermia when exposed to extremely cold (4°C) ambient temperature. Even under conventional mouse room conditions, young (less than 45-day- old) Ihllh mice are hypothermic (Dung, 19811. Reproduction Homozygous survivors of both sexes can breed, but their reproductivity is low (Green, 19811. At the Jackson Laboratory, Bar Harbor, Maine, this mutation is maintained by continued backcrosses to C57BL/6J x C3H/HeSnJ F1 hybrid mice (see Chapter 51. Ipr (Lymphoproliferation) Genetics The mutant gene lymphoproliferation (Ipr) is a single autosomal recessive gene that arose spontaneously in the twelfth generation of inbreeding of the developing MRL/Mp strain (Murphy and Roths, 19771. The origin of this strain and several of its sublines has been reviewed (Roths, 19871. Despite numerous attempts to demonstrate linkage, the location of the Ipr gene is unknown. The gene has been transferred by multiple cross-intercross matings to inbred strains AKR J. BALB/cBy, C3H/HeJ, C57BL/6J, C57BL/lOSn, and SJL/J. An MRL/MpJ-lpr/lpr mouse. Mice homozygous for the mutation lymphoproliferation develop enlarged lymph nodes, as shown by this animal's enlarged prescapular node. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine.

60 IMMUNODEFICIENT RODENTS Pathophysiology The mean life span of MRL/Mp-lpr/lpr females and males is 17 and 22 weeks, respectively. Homozygotes die with massive generalized enlargement of the lymph nodes. Subcutaneous lymph nodes can be palpated as early as 10 weeks of age (Murphy and Roths, 1978a). Enlarged lymph nodes of homozygotes are characterized predominantly by lymphocytic proliferation with an admixture of histiocytes, plasma cells, and immunoblasts. There is also blurring of the nodal architecture. Multiple attempts to transplant the lymphoid mass to congeneic normal recipients have been unsuccessful. The Ipr-induced lymphoproliferation is presumed to be hyperplastic, not neoplastic. Lymphoproliferation in 4-month-old MRL/Mp- lprllpr mice is composed of approximately 90 percent T cells. Although the frequency of B cells is reduced to 7 percent from the normal ~ +/+) level of 28 percent, there is an absolute increase in the numbers of B cells at this age. Definitive FACS analyses employing monoclonal antibodies indicate that hyperplastic lymph node cells stain for Thy- 1 (dull), Ly- 1 (dull), H- 11, and Ly-5. Such cells are negative for L3T4, Ly-2, sIg, ThB, and Ia-associated antigens. Thus, Iprllpr mice have abnormal populations of T cells that express the Ly-5 marker usually confined to B cells (Morse et al., 19821. Treatment of MRL-lpr/lpr mice from birth to 11 weeks of age with the monoclonal antibody Mel-14 results in a 10- to 20-fold reduction in lymph- adenopathy and induces marked splenomegaly. Since Mel-14 recognizes the lymphocyte surface receptor gp90, which is responsible for lymph node homing, it is speculated that lymphadenopathy occurs in MRL-lpr/lpr mice because of increased homing of gp90+ T cells to the lymph nodes (Mountz et al., 19881. The spleen weight of MRL-lprllpr mice is sevenfold larger than that of controls. While lymphoid tissue in the area of the thymus appears enlarged in moribund mice, most of the mass is due to enlarged lymph nodes rather than to an enlarged thymus. MRL/Mp-lpr/lpr mice develop immune complex glomerulonephritis. Gen- eralized arterial disease, characterized by polyarteritis and degenerative ar- teriolar lesions without cellular inflammation, is also found. The lungs of Iprllpr mice show extensive perivascular and peribronchial lymphocytic in- filtration with only occasional atelectasis and exudate. Severe bronchopneu- monia can occur, but interstitial pneumonitis is not characteristic (Murphy and Roths, 1978a). Joints of MRL/Mp-lprllpr mice show arthritic changes resembling those of rheumatoid arthritis (Andrews et al., 19781. Coronary artery disease and myocardial infarction can be a contributing cause of death (Hang et al., 19821. Near the end of their life span, MRL/Mp-lpr/lpr mice develop erythematous skin lesions, necrosis of the ears, swollen feet, and, frequently, generalized edema (Murphy and Roths, 1978a).

HEREDITARY IMMUNODEFICIENCIES 61 Serologically, Iprllpr mice have a fivefold increase in gamma-region pro- teins. Twofold increases in IgA, IgM, and IgG2b and sixfold increases in IgG1 and IgG2a have been identified by radial immunodiffusion. S era of Iprllpr mice are uniformly positive for antinuclear autoantibodies by 12 weeks of age. The presence of anti-single-stranded DNA (anti-ssDNA), anti-dsDNA, and anti-Smith (anti-Sin) autoantibodies is characteristic. Autoantibodies di- rected against thymocyte or erythrocyte surface antigens are detected infre- quently (Murphy and Roths, 1978a). Retroviral gp70 immune complexes and IgM and IgG rheumatoid factors are found in significant concentrations in Iprllpr mice (Andrews et al., 19781. Muraoka and Miller (1988) found that MRL-lpr/lpr and C57BL/6-lprllpr mice, but not MRL/Mp or C57BL/6 mice, develop lymph node and bone marrow-derived cytotoxic lymphocytes directed against self (H-2k) deter- minants in a slCr release assay with mitogen-induced lymphoblasts. They concluded that at least some of the self-reactive cells are generated as part of the Ipr defect. Both the longevity and pathologic sequelae associated with the Ipr gene depend on the background of the host. Life spans of female Ipr homozygotes on the AKR/J, C3H/HeJ, and C57BL/6J inbred strains are between 42 and 52 weeks. SJLlJ-lprllpr homozygotes have life spans similar to those of MRLlMp-lprllpr mice ~ 17-22 weeks) (Roths, 19871. The degree of lymphoproliferation varies greatly among strains. At 26 weeks of age the mean weight of the lymph nodes is 20-fold greater in AKR/J- lprllpr mice than in congeneic normal AKR/J mice. This compares with a 116-fold increase over control lymph node weight in C3HlHeJ-lprllpr mice (Morse et al., 19851. The rank order of increasing lymphoid hyperplasia for five congenic strains is AKRlJ-lprllpr < C57BL16J-lprllpr < SJLlJ-lprllpr MRLlMp-lprllpr < C3HlHeJ-lprllpr. Genetic background also affects spontaneous production of anti-dsDNA autoantibodies. At 6 months of age the percentage binding of radiolabeled dsDNA in Iprllpr mice has been found to be 5 percent on C57BL/6J, 20 percent on AKR/J, 26 percent on C3H/HeJ, and 49 percent on MRL/Mp (Izui et al., 19841. Renal pathology is extensive in MRLlMp-lprllpr mice at 4-7 months of age, compared with negligible kidney disease in C57BL/6J- lprllpr and C3HlHeJ-lprllpr mice as old as 14-16 months of age. AKR/J- lprllpr mice have mild renal disease. Levels of urinary protein parallel the degree of renal histopathology (Kelley and Roths, 19851. Husbandry Special husbandry procedures are not required for maintaining MRL/Mp- lprllpr, C57BL16J-lprllpr, or C3HlHeJ-lprllpr mice. The severity of disease and

62 IMMUNODEFICIENT RODENTS early morbidity make it extremely difficult to maintain a colony of SJL/J- lprllpr mice. Reproduction MRL/Mp- +/+ and MRL/Mp-lpr/lpr are independently inbred with pe- riodic crosses (every 5-10 inbreeding generations) to the MRL/Mp-+/+ reference strain. MRL/Mp mice are large and docile; males rarely fight. MRL/Mp-lpr/lpr breeders produce an average of seven pups per litter, with 96 percent of the offspring surviving to weaning. Because of the early mor- tality of both males and females, however, maintaining a colony of MRL- lprllpr breeders requires careful management. An average of only two litters per breeding female should be expected. Approximately one-tenth of the breeding colony should be retired and replaced by newly weaned breeders each week. Trio matings are successful but require breeding boxes adequate to house the increased population of preweanling mice. [pad (Lipopolysaccharide Response, Defective) Genetics The Lps locus in mice controls a number of dominant or codominant traits that are elicited in response to the complex glycolipid component (endotoxin) of the outer membrane of gram-negative bacteria (Mergenhagen and Pluznik, 19841. By common usage, the term lipopolysaccharide (LPS) or lipid A is considered to be synonymous with this endotoxic component. The Lps gene is located on chromosome 4 (Watson et al., 1978a). Mice homozygous for the Lpsn locus have normal in vivo and in vitro responses to endotoxin, while mice carrying the Lps~ allele have defective responses. The first documented allele for defective LPS responses occurred in the C3H/HeJ strain between 1960 and 1968, presumably by mutation (Glode and Rosenstreich, 1976; Watson and Riblet, 19741. Subsequently, additional- perhaps identical defective mutations at the Lps locus have been detected in C57BL/lOScCr and C57BL/lOScN strains (Green, 1981; Morrison and Ryan, 1979; Vogel etal.,1981~. Pa thop hys io logy C3H/HeJ mice, which have the allele Lps4, show a 20- to 38-fold increase in resistance to endotoxin lethality over the highly susceptible A/HeJ strain (Sultzer, 19681. At the same time they show a greater susceptibility to Sal- monella typhimur~um administered systemically than do strains bearing the

HEREDITARY IMMUNODEFICIENCIES 63 Lpsn allele (O'Brien et al., 1985). In addition, the initial clearance of both S. typhimurium and Escherichia cold from mucosal surfaces is significantly impaired in Lps~ mice (Eden et al., 19881. The increased susceptibility to S. typhimurium and E. cold regulated by the Lps~ locus is independent of the expression of the Itys locus in mice (see page 36J. Spleen cells from the Apse strain C3H/HeJ cannot support LPS-induced herpes simplex virus (HSV) replication, and the C3H/HeJ strain is resistant to lethal challenge with HSV (Kirchner et al., 19781. The dichotomy seen between resistance to endotoxin and HSV and susceptibility to S. typhimurium in C3H/HeJ is reflected in the numerous abnormal responses to LPS conferred by Lps4. To date, these abnormalities have been studied best in the immunologic and reticuloen- dothelial systems. Watson and Riblet (1975) found that B lymphocytes from C3H/HeJ mice were unable to proliferate in the presence of LPS. This defect was shown to reside in a single codominantly expressed autosomal gene (Coutinho et al., 1975~. In addition to these defective mitogenic and polyclonal PFC responses, Skidmore et al. (1976) demonstrated that C3H/HeJ mice were deficient in the LPS-induced adjuvant response to antibody production. These investigators also demonstrated that the nodal abrogation of tolerance to human IgG associated with LPS was not observed in the Apse strains. The LPS-induced phenotypic differentiation of B cells (immunoglobulin, Ia expression) and T cells (Thy-1 expression) was shown to be lacking in Lps~ strains (Koenig et al., 1977; Watson, 19779. Taken collectively, these ob- servations suggest a significant role for the Lpsn gene product in the response of B cells (and some T cells) to LPS. Glode et al. (1977) found that macrophages collected from C3H/HeJ mice were intrinsically unresponsive to the cytotoxic effects of endotoxin. Mac- rophages from C3H/HeJ mice have no LPS-induced inhibition of phagocy- tosis and produce very low levels of IL-1 and prostaglandin E2 on exposure to LPS (Vogel et al., 19791. Chedid et al. (1976) reported that macrophages collected from Lps~ strains of mice were not activated by endotoxin to kill tumor cells. Backcross linkage analysis conducted between Apse and Lpsn strains demonstrated complete concordance in the expression of macrophage tumoricidal capacity and B-cell proliferative responses (Ruco et al., 19781. A wide variety of additional abno~al biological responses are conferred by Lps4. Augmentation of neutrophil migration in response to LPS administered intraperitoneally has been described (Sultzer, 1968; Moeller et al., 19781. Lps~ strains fail to make type I interferon (~/~) in vivo (Apse et al., 1977), and Lps~ fibroblasts have decreased glucose utilization in response to LPS in vitro (Ryan and McAdam, 19771. Using backcross linkage analysis in- volving the C3H/HeJ and C57BL/6J (LpsnlLpsn) strains, Watson et al. (1978b) observed that Lps~ mice failed to produce serum amyloid A (SAA) and

64 IMMUNODEFICIENT RODENTS granulocyte-macrophage colony-stimulating factor in response to LPS. They also discovered that Apse mice develop hypothermic responses following LPS injection. The low responsiveness of C3H/HeJ mice to lipid A is the result of an inability to activate target cells rather than an inability of cells to respond to endogenous mediators (Mergenhagen and Pluznik, 19841. Selective mito- genic responses have been elicited in Apse mice by using detergent-dissociated fractions of LPS (Vukajlovich and Morrison, 19841. Vogel et al. (1984) MA +~+ 1;~:~ A : : ~ ~ . . aemonslrateG Inal llplO A detective in 3-deoxy-D-mannooctulosonic acid as well as ester-linked lauryl and myristoyl residues is capable of inducing many of the biological effects of intact lipid A in both Lps'2- and Lps~-containing macrophages. They speculate that the defect in C3H/HeJ mouse cells as- sociated with the Lps~ gene may be related to the processing of lipid A to a suitable stimulatory form. Although the nature of LPS cell binding and activation is not completely known, it appears that in Lps~ macrophages, endotoxin fails to produce protein phosphorylation and, thus, complete signal transduction (Prpic et al., 19871. Husbandry Special husbandry procedures are not required. Reproduction These animals reproduce normally. me (Motheaten); met (Viable Motheaten) Genetics The recessive mutation motheaten (me) occurred spontaneously in 1965 in the C57BL/6J strain at the Jackson Laboratory, Bar Harbor, Maine (Dickie, 19671. The me locus has been mapped to chromosome 6, 21.9 cM distal to MiWh. A second spontaneous mutation at the me locus occurred in C57BL/6J mice at the Jackson Laboratory in 1980 (Shultz et al., 19841. This mutant allele was designated viable motheaten (met) because of its increased lon- gevity. In addition to the strain of origin, motheaten is available on strains C3H/HeN, C3HeB/FeJLe, C57BL/6N, NFS/N, NZB/N, and PIN. Viable motheaten has been partially or fully backcrossed onto several inbred strains? including AKR/J, C3H/HeJ, DBA/2J, MRL/Mp, and SJL/J.

HEREDITARY IMMUNODEFICIENCIES 65 ~ ~ ... ~.~ ~ .. of. ~..~ A C3HeB/FeJLe-a/a melme mouse. Motheaten homozygotes have patchy absence of hair, are small, and develop terminal pneumonia (hunched position). Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine. Pathophysiology C57BL/6J-me/me mice are recognized by 3-4 days of age by neutrophilic aggregates in the skin. Later, subepidermal accumulations of neutrophils displace hair follicles and result in the patchy absence of pigment. Motheaten mice are considerably smaller than normal littermates from 4 days of age onward. C57BL/6J-me/me mice have the shortest life span of any mouse strain with a single mutation affecting the immune system. Only one-fifth of these homozygotes survive to weaning, and none survive longer than 8 weeks. The mean longevity of C57BL/6J-me/me and C57BL/6-met/meV mice is 3.1 and 8.7 weeks, respectively (Green and Shultz, 1975; Shultz et al., 19841. However, the average life span of other inbred strains carrying the me locus can be as long as 6-8 weeks (C. T. Hansen, Division of Research Services, National Institutes of Health, unpublished data). The immediate cause of death appears to be pneumonia. Large numbers of macrophages, which often contain fine crystalline material associated with extravasation of erythrocytes, are found in the alveoli. Until approximately 3 weeks of age, the thymuses of motheaten mice appear to be histologically normal, although they are smaller in size than those of controls. Thereafter, the thymuses show marked cortical invo- lution and are absent in the oldest survivors. Splenomegaly, characterized by an increase in erythropoiesis and myelopoiesis and a loss of white pulp,

66 IMMUNODEFICIENT RODENTS is seen in both me and met homozygotes. Lymph nodes might be slightly enlarged, but follicles are absent. Large numbers of atypical plasma cells with Russell bodies are found in meV/mev mice. Peyer's patches are reduced in size and have no follicles. Bone marrow shows reduced erythropoiesis and increased myelopoiesis. The peripheral blood is characterized by an increased number of neutrophils and a decreased number of lymphocytes. In the oldest me and met homozygotes, renal disease is evident. By 20 weeks of age, meVlmev mice have marked glomerulonephritis with an associated increase in blood urea nitrogen. Male met'lmeV mice are sterile because of depletion of Leydig's cells, lowered testosterone levels, and impaired spermatogenesis. Motheaten mice have severe deficiencies in both humoral and cellular immunity. Homozygous me mice have increased levels of all major classes of immunoglobulins, and IgM levels in particular are up to 25 times higher in 6-week-old melme mice than they are in controls. Viable motheaten mice have increased numbers of Ly-1 + B cells (Sidman et al., 1986b), impaired proliferative responses to B-cell mitogens (Sidman et al., 1978), and poly- clonal B-cell activation (Sidman et al., 19781. Although numbers of T lym- phocytes are normal, melme mice have impaired proliferative responses to T-cell mitogens and lack the capacity to develop cytotoxic killer cells against allogeneic cells. Greiner et al. (1986a) have postulated that a subset of TdT+ bone marrow cells are almost totally depleted in me homozygotes, and pro- thymocytes appear to be developmentally arrested at the pre-TdT + -cell stage. It has been postulated that the maturation of prothymocytes in metimeV mice is arrested because of a defect in the radiosensitive compartment of the bone marrow microenvironment (Komschlies et al., 1987J. There is an early onset of autoimmunity characterized by the presence of autoantibodies against thymocytes and dsDNA (Shultz and Zurier, 19781. Immune complexes (gran- ular deposition of IgM and IgG) have been identified in renal glomeruli by immunofluorescence microscopy (Shultz and Green, 19761. Sherr et al. (1987) postulated that the increased population of Ly-1 + B cells has a direct role in the autoimmune disease of meVlmev mice by secreting autofidiotype)- reactive antibodies and elaborating helper lymphokines that drive autoreactive subsets to secretion. Painter et al. (1988) evaluated the antibodies produced by several hundred hybridomas derived from C57BL/6JSmn-meV/mev mice. Of 33 immunoglob- ulin-secreting hybridomas, 17 exhibited reactivity to autoantigens. While some of the monoclonal antibodies (mAbs) recognized a single autoantigen, others recognized multiple antigenic specificities. Northern blotting analysis for V-gene families in the meV-derived autoantibodies was interpreted as showing random VH family selection but biased use of VK gene families. They concluded that the immunoregulatory defect in meV mutants operated at a more generalized level than at the VH or VK loci.

HEREDITARY IMMUNODEFICIENCIES 67 Using Whitlock-Witte cultures for lymphocytes and Dexter cultures for mye- loid cells, Hayashi et al. (1988) found that neither lymphocytes nor granulocytes were formed from mutant meVlmev bone marrow cells alone or together with normal cells. There was no evidence for soluble mediators of suppression. Thus, the meVlmev mouse might be a good model for investigating cell-associated molecules that normally limit progenitor cell replication. Husbandry Special husbandry procedures are not required for maintaining me and met mice. Life span is not increased by maintaining these animals under germfree conditions (Lutzner and Hansen, 19761. Reproduction Propagation of me and met mice is difficult, but can be done by breeding heterozygotes (mel +) or by transplanting ovaries of C57BL/6J-me/me fe- males to histocompatible hosts with subsequent mating to C57BL/6J males to produce known heterozygotes (see Chapter 5~. mi (Microphthalmia) Genetics Microphthalmia (my is a semidominant gene that arose in the descendants of an irradiated male (Hertwig, 19421. Numerous alleles occur at this locus, A mouse carrying the mu- tation microphthaImia (ml) and its normal littermate. Mutants of microphthaImic stock (right) can be identi- fied at birth by the absence of retinal pigment, which is shown with an arrow on the normal littermate (left).

68 IMMUNODEFICIENT RODENTS which has been mapped to chromosome 6, and some combinations show interallelic complementation (Green, 19811. The gene is maintained on C57BL/6 and CBA/H-T6 backgrounds. Pa thophys to logy Heterozygous mi mice often have white spotting on the head, tail, and belly and exhibit reduced iris pigment. Homozygotes are devoid of eye and coat pigments, have small eyes, and lack incisors. Microphthalmic mice (like gilgl, ocloc, and oplop mice) exhibit osteopetrosis. Most homozygotes die at about the weaning age or a little older. Osteoclasts in milmi mice are small and numerous, have fewer nuclei, lack cytoplasmic vacuoles, and have rudimentary ruffled borders (Marks and Walker, 1981~. Like gilgl mice, milmi mice appear to have two populations of osteoclasts, one with normal and one with reduced acid phosphatase (Marks and Walker, 19761. Homozygotes have wide expanses of osteoid, unminer- alized bone matrix. Osteoblasts are numerous and active (Marks and Walker, 19699. Like the other osteopetrotic mutants, milmi mice show hyperplasia of the calcitonin-secreting cells of the thyroid, hypocalcemia, and hypo- phosphatemia (Marks and Walker, 19691. Serum levels of 1,25 dihydroxy- vitamin D are elevated, and levels of the 24,25 metabolite of vitamin D are reduced (Zerwekh et al., 1987J. The milmi mouse is anemic and has a leukocyte count in the low normal range. Spleen stem cell populations are markedly increased, and spleno- megaly is present. Marrow hemopoietic stem cell populations are normal (Wiktor-Jedrzejczak et al., 19811. Studies using cell isolates from whole spleens have demonstrated defects in monocyte (Chambers and Loutit, 1979; Minkin, 1981), macrophage (Chambers and Loutit, 1979; Minkin, 1981), and lymphocyte (Olsen et al., 1978; Minkin et al., 1982) function; however, Schneider and Marks (1983), using ficoll-hypaque separated splenic lymphoid cells, did not observe re- duced lymphocyte function. They suggest that the reduced immune respon- siveness might be due to the dilution of immunocompetent cells by stem cells and immature hematopoietic cells. This suggestion is supported by their finding of increased erythropoietic tissue relative to lymphoid tissue in the spleens of milmi mice. A chemotactic defect in peritoneal macrophages has also been demon- strated (Minkin and Pokress, 19801. In vivo, milmi mice respond to the administration of thioglycollate with only half the influx of macrophages observed in mil + or +/+ controls. In vitro, milmi macrophages respond poorly to chemotactic stimuli. The hematopoietic and lymphoid defects caused by the mi mutation can be transmitted to normal mice by transplantation of affected spleen or bone

HEREDITARY IMMUNODEFICIENCIES 69 marrow cells (Walker, 1975b). Mutant mice can be cured by transplanting bone marrow or spleen cells from normal litterrnates (Walker, 1975a,c). Husbandry Marks (1987) has reviewed the husbandry for this mutant. Most homo- zygotes die between 30 and 60 days of age, but with special care they can survive to adulthood. Soft diets are essential. Reproduction Only heterozygotes make effective breeders. nu (Nude); nuStr (Streaker) Genetics Nude (nu) is an autosomal recessive mutation located on chromosome 11. The first recorded mutation at this locus occurred in a closed, but not inbred, stock of mice at the Ruchill Hospital Virus Laboratory, Glascow, Scotland (Flanagan, 19661. A second spontaneous mutation to nude occurred in the AKR/J strain at the Jackson Laboratory, Bar Harbor, Maine. This mutation was given the new name streaker and the gene symbol nuStr, which indicates both the independent occurrence of the mutation and its allelism with nu A BALB/cByJ-nu/nu mouse. Mice homozygous for the mutation nude are athymic and hairless and vibrissae are lacking or crinkled by the time the animals are 24 hours old. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine.

70 IMMUNODEFICIENT RODENTS (Etcher, 19761. The nude gene has been transferred to many inbred strains. The streaker gene has also been transferred to a number of inbred strains. Pathophysiology The two major defects of nude homozygotes are failure of hair growth and dysgenesis of the thymic epithelium. The underlying basis for the pleio- tropic effects of the nude mutation is unknown. Abnormal hair growth in nude mice has been described in detail (Eaton, 19761. Thymic dysgenesis is traceable to a developmental failure of the thymic anlage, which arises from the third pharyngeal pouch. The thymic rudiment remains small and cystic throughout life, and there are severely reduced numbers of mature functional T cells (Wortis et al., 19711. As a result, nude homozygotes do not reject allografts and often do not reject xenografts. The finding that human neo- plasms can grow in nude mice has resulted in the wide use of this model for studying the mechanisms of transplantable human tumor growth and metas- tasis. There is no intrinsic defect of T-cell precursors in nude mice; the T-cell defect can be corrected by transplanting mature T cells, thymocytes, or normal thymic epithelium (Wortis et al., 19711. Cytotoxic T-cell activity can be induced in nude mice by the administration of IL-2 (Hunig and Bevan, 1980), and older mice, especially if they have a microbial infection, often have some functional mature T cells. It has been suggested that the failure of stromal thymic elements to interact with lymphocytic precursors to form T cells is due to an abnormal distribution or expression of Ia antigens by epithelial components (Jenkinson et al., 19811. This is consistent with the recent finding that Ia+ cells are absent from the thymic rudiment of nude mice (Van Vliet et al., 19851. Nude mice respond poorly to thymus-dependent antigens because of a defect in helper T-cell activity. When responses can be detected to such antigens, antibody is largely limited to IgM. Responses to thymus-indepen- dent antigens in nude mice are normal. Levels of serum IgG1, IgG2a, IgG2b, and IgA are reduced, while IgM levels tend to be slightly elevated and IgG3 is present in normal or slightly reduced amounts. A B-cell defect has been reported, but it has not been demonstrated unequivocally (Mona et al., 1982; Wortis et al., 19821. Increased NK cell activity has also been described (Minato et al., 1980; E. A. Clark et al., 19811. Spontaneous autoimmunity has been reported in nude mice by Monier et al. (1974), who found that approximately one-third of nude homozygotes have circulating ANAs as early as 6-8 weeks of age. ANAs were present in the eluates from kidneys con- taining immune complexes. Limited investigations have shown that differences in phenotypic expres- sion between nu and nuStr homozygotes do not appear to be any greater than

HEREDITARY IMMUNODEFICIENCIES 71 the reported variation among different stocks or strains of nude mice. Like nude mice, streaker homozygotes show severely reduced numbers of T cells, lack measurable T-cell activity, and show a reduction in all major classes of serum immunoglobulins (Shultz et al., 1 978, 1 982a). Streaker mice also have elevated NK cell activity (E. A. Clark et al., 19811. AKRlJ-nustrlnustr mice do not show the high rate of spontaneous thymic lymphoma and chronic infection with endogenous MuLV normally present in AKR/J mice (Bedigian et al., 19791. This mutant, therefore, is a manipulatable experimental system with which to investigate the relationships among thymus function, MuLV expression, and lymphomagenesis. Although AKR-nustrlnustr mice do not develop T-cell lymphomas, this mutant has high numbers of preleukemic cells in the bone marrow (Shultz et al., 1983) and develops spontaneous reticulum cell sarcomas (Shultz et al., 1982a). The mean life span of male and female streaker mice under pathogen-free conditions is 34 weeks, compared with 44 weeks for littermate controls. Moribund streaker mice show symptoms of intercurrent infections and wast- ing disease (Shultz et al., 19781. Husbandry Inbred mice homozygous for nu are highly susceptible to infection by a broad spectrum of bacterial and viral pathogens, and they should be main- tained in a germfree, defined-flora, or pathogen-free environment (see Chap- ter 41. Under these conditions their life span approaches that of normal littermates. Outbred nude mice are hardier than inbred nude mice and can be maintained under less stringent conditions if isolated from conventionally housed mice. However, it should be recognized that any nude mouse housed in a conventional environment probably has a microbial infection and that this infection might influence experimental data considerably. Streaker mice should be maintained under conditions appropriate for nude mice. Reproduction Neither nude nor streaker females are efficient breeders. The most effective breeding scheme uses homozygous mutant males and heterozygous females. The homozygous pups can be identified within 24 hours postpartum by their lack of vibrissae or poorly developed, crinkled vibrissae. It might be advan- tageous to cull some normal litte~ates to optimize survival of the mutant mice; however, reducing litter size below four to five pups can lead to diminished lactation. To maintain a breeding colony in conventional animal rooms, it might be desirable to graft a normal thymus under the kidney capsule of the nude males (Hetherington and Hegan, 19751.

72 IMMUNODEFICIENT RODENTS ob (Obese) Genetics The mutation obese (ob) on chromosome 6 is a spontaneous autosomal recessive mutation discovered in 1949 at the Jackson Laboratory, Bar Harbor, Maine (Ingalls et al., 19501. The mutation is maintained on the C57BL/6J and C57BL/KsJ inbred backgrounds. Pathophysiology The ob mutation produces a marked obesity state associated with hyper- phagia and hyperinsulinemia. The obesity syndrome is characterized by adi- pocyte hyperplasia and insulin resistance. This insulin resistance, associated with decreased numbers of high-affinity insulin receptors on liver cells, adipocytes, and lymphocytes, becomes more severe as the mice age (Kahn et al., 19731. Because manipulations, such as food restriction, that reduce hyperinsulinemia also improve insulin binding to its receptor, the reduced numbers of plasma membrane insulin receptors may be a secondary reflection of hyperinsulinemia (Soil et al., 19751. Like the unlinked obesity gene diabetes (db) on chromosome 4, the path- ophysiological effects of the ob mutation are strikingly dependent on the inbred strain background. C57BL16J-oblob mice of both sexes develop an obesity syndrome that is well compensated by unrestricted hyperplasia of pancreatic beta cells and sustained hyperinsulinemia; males develop a mild, transient hyperglycemia between 2 and 4 months of age that is apparently corrected by the increasing pancreatic output of insulin into the serum (Hum- mel et al., 19721. In contrast, a severe hyperglycemia becomes established in C57BL/KsJ-ob/ob mice of both sexes shortly after weaning. This is as- sociated with necrosis of pancreatic beta cells, islet atrophy, and relative insulinopenia (normal levels of serum insulin in the presence of severe insulin resistance). Unlike the transiently hyperglycemic C57BL/6J-ob/ob mice, which have a life span of approximately 2 years, the severely diabetic C57BL/KsJ- oblob mice usually die between 6 and 8 months of age. Many of the perturbations in immune function in C57BL16J-oblob mice appear to be secondary consequences of the metabolic imbalances associated with the pleiotropic mutation, because discrepancies observed in vivo be- tween control and mutant lymphoid cell functions usually do not persist when these cells are removed from the abnormal metabolic milieu and studied in vitro. Those anomalies observed in vivo include reductions in lymphoid organ weights and in lymphocyte and monocyte numbers (Chandra and Au, 19801; depressed T-cell-mediated immunity in vivo was reflected by a reduced ability to reject allografts or to mount delayed-type hypersensitivity reactions (Sheena

HEREDITARY IMMUNODEFICIENCIES 73 and Meade, 1978; Meade et al., 1979; Chandra and Au, 19801. NK cell activity and antibody-dependent cell-mediated cytotoxicity were elevated in C57BL16J-oblob mice (Chandra and Au, 19801. C57BL16J-oblob mice differ from C57BL/KsJ-db/db mice by not exhib- iting a markedly elevated PFC response after immunization with SRBCs when data are expressed on a per-cell basis. This difference very likely reflects differences in the inbred background, which include differences at the major histocompatibility complex (MHC). Both C57BL16J-oblob and C57BL/KsJ- dbldb mice exhibit immune complex depositions (containing antibodies against insulin) in renal glomeruli (Meade et al., 19811. Circulating autoantibodies against islet cell cytoplasmic antigens are more prevalent in C57BL/KsJ- dbldb than in C57BL16J-oblob mice (Yoon et al., 19881. Islet cell surface antibodies are present only at a low frequency and after initiation of the hyperglycemic phase (Flats et al., 19851. There is no evidence that these autoantibodies are of primary pathogenic significance. However, MHC con- trol of autoantibody expression is apparently involved because these auto- antibodies are not found in a C57BL/KsJ.B6-H-2b dbldb congenic strain (Yoon et al., 19881. C57BL16J-oblob mice show an accelerated deterioration in immune func- tion with age compared with mice with a normal genotype (Harrison et al., 19841. Presumably, compromised immune function accounts for the in- creased susceptibility of these mutants to diabetogenic encephalomyocarditis virus (D ' Andrea et al., 19811. One contrary report claims increased im- munocompetence in C57BL16J-oblob mice, as evidenced by enhanced re- sistance to the B16 melanoma (Black et al., 19831. Husbandry Special procedures are not required to maintain these animals, although life span might be prolonged by dietary restriction. Reproduction Obese mice of both sexes are infertile, and breeding is done with heter- ozygotes. The heterozygotes are generally produced by ovarian transplan- tation (see Chapter 51. Oc (Osteosclerotic) Genetics Osteosclerotic (oc) is an autosomal recessive mutation located on chro- mosome 19 (Marks et al., 19851. It arose spontaneously in the C57BL/6J- bf stock at the Jackson Laboratory, Bar Harbor, Maine (Dickie, 19671.

74 IMMUNODEFICIENT RODENTS A B6C3/Fe-a/a ocloc mouse. Mice homozygous for the mutation osteosclerotic are small and have clubbed feet and a kinked tail. Photograph courtesy of the Jackson Laboratory, Bar Harbor, Maine. Pa thop hys lo logy Homozygous oc mice can be recognized at about 10 days of age. Mice are small, facial growth is stunted producing a foreshortened snout, incisors fail to erupt, feet are often clubbed, and the tail is kinked. Vestibular ab- normalities cause homozygotes to circle. Homozygotes die within 30-40 days of birth. Long bones exhibit a failure of secondary bone resorption and an absence of marrow cavities (Marks et al., 19851. Bone marrow cells are absent and leukocyte and lymphocyte counts are decreased. Erythrocyte counts, as well as the numbers of spleen and thymus cells, are within the normal range. The spleens of oc homozygotes are enlarged, but splenic hematopoietic stem cells are approximately normal in number, as are monoblast populations (Wiktor- Jedrzejczak et al., 19811. Osteoclasts have poorly developed ruffled borders and lack cytoplasmic vacuolization (C. R. Marks et al., 19841. They are unable to resorb calcified cartilage (Seifert and Marks, 19851. Homozygotes exhibit rickets and have wide expanses of osteoid, unmineralized bone matrix. There is hyperplasia of the calcitonin-secreting cells of the thyroid. Serum calcium is lower than normal and does not respond to exogenous parathyroid hormone (Marks and Walker, 19691. Like the other osteopetrotic mutants, ocloc mice are hypo- phosphatemic (Marks and Walker, 1969), have elevated serum levels of 1,25 dihydroxyvitamin D (Zerwekh et al., 1987), and have depressed levels of the 24,25 metabolite of vitamin D (Zerwekh et al., 19871.

HEREDITARY IMMUNODEFICIENCIES 75 Osteosclerotic mice are not cured by bone marrow or spleen cell transplants (Seifert and Marks, 19871. Transferability of this disease has not been studied, and little is known about immunologic dysfunction. Husbandry Marks (1987) has reviewed the husbandry for this mutant. Soft diets and disease-free conditions prolong the survival of homozygotes. Reproduction This mutation is maintained at the Jackson Laboratory in a manner identical to that of the op mutant (see pages 76-771. op (Osteopetrosis) Genetics Osteopetrosis (op.) is an autosomal recessive mutation that arose sponta- neously in a C57BL/6J-dw stock maintained at the Jackson Laboratory, Bar Harbor, Maine (Lane, 19731. It has been mapped to chromosome 3 (Lane, 1979~. Pa thop hys to logy Approximately 60 percent of osteopetrotic mice that survive weaning live longer than 12 months (Marks and Lane, 19761. Homozygotes lack incisors, i.. ~ ~ .~ ~ ~ .~ 1 ~'~ : ~ ~ ~ .~.~.~ : _ .., ~.~.~.~.~.~.~ ~ ~ ~. _. ,~.~.~.~. , ~ ~ .. ~ , ~ _ ~ ~ . ~ ~ ~ . ~ ~ _ ~ ~. ~ ~ A... . A B6C3/Fe-a/a op/op mouse. Mice homozygous for the mutation osteopetrosis are underweight and have a dome-shaped skull.

76 IMMUNODEFICIENT RODENTS are small, have stunted facial growth, and have a domed skull. Occasionally, hydrocephalus is found. Like the other osteopetrotic mouse mutants, total leukocyte counts are decreased. Marrow cells are reduced to one-tenth normal. Spleen cellularity is normal, but stem cell populations are elevated (Wiktor-Jedrzejczak et al., 1981). Peritoneal macrophages and peripheral blood monocytes are markedly reduced in number (Wiktor-Jedrzejczak et al., 19821. Bone in op homozygotes contains large lipoid droplets of unknown origin. Osteoclasts are small and markedly reduced in number, but they have elab- orate ruffled borders and extensive cytoplasmic vacuolization (Marks, 19829. They exhibit an abnormal acid phosphatase distribution within the cell and have lipoid inclusions that might be related to the larger extracellular lipoid masses (Marks and Lane, 19764. Bone matrix formation and parafollicular (calcitonin-secreting) cells of the thyroid are increased, serum phosphate levels are decreased, and serum calcium levels are normal (Marks and Walker, 19691. Olsen et al. (1978) have reported that spleen cells from op homozygotes have defective in vitro responses to PHA, LPS, and poly(I-C); however, it has been suggested more recently (Schneider and Marks, 1983) that the reduced immune responsiveness might be due to the dilution of immuno- competent cells by stem cells and immature hematopoietic cells (see also mi, page 671. The disease induced by the op mutation undergoes spontaneous partial resolution in adulthood; however, these mice cannot be cured by transplan- tation of normal bone marrow or spleen cells (Marks et al., 19841. Con- versely, the transfer of spleen cells from op homozygotes to lethally irradiated, normal littermates does not transfer the defect (S. C. Marks, Jr., University of Massachusetts Medical School, unpublished data). Normal mouse fibro- blast culture medium has been reported to stimulate spleen cells from oplop mice to differentiate into monocytes and macrophages, which suggests that the op defect might be an abnormality of the microenvironment (Wiktor- Jedrzejczak et al., 19821. Husband~ry Marks (1987) has reviewed the husbandry for this mutant. Homozygous op mice survive weaning if they are provided soft food; however, viability is reduced, despite the partial resolution of the defect in adulthood. Specific- pathogen-free (SPF) conditions are recommended. Reprodfuction Homozygotes do not breed; breeding can be accomplished by using het- erozygotes. This mutation is propagated at the Jackson Laboratory by a

HEREDITARY IMMUNODEFICIENCIE5 77 system of ovarian transplantation using compatible sets of hybrids (see Chapter 51. scia' (Severe Combined Immunodeficiency) Genetics Severe combined immunodeficiency (scion is an autosomal recessive mu- tation that occurred spontaneously in the C.B-Igh-lb (N17F34) (usually ab- breviated CB-17) congenic strain (Bosma et al., 1983J. In this congenic strain the inbred BALB/c strain carries the immunoglobulin heavy-chain allele (Igh-lb) of the C57BL/Ka strain. The scid locus has recently been mapped to the centromeric end of chromosome 16 (Bosma et al., 19891. Pa thophys lo logy Homozygous scid mice have little or no immunoglobulin in their serum. The lymph nodes and thymus are abnormally small, as is the spleen of most animals. The thymus consists of a rudimentary medulla without a cortex. Spleen and lymph node follicles are virtually devoid of lymphocytes. All of these lymphoid organs consist primarily of vascularized supportive tissue with variable numbers of fibroblasts, histiocytes, and macrophages. Bone marrow, although lacking lymphocytes and plasma cells, appears otherwise to be morphologically normal (Bosma et al., 1983; Custer et al., 19851. Mice with the scid mutation lack dendritic Thy- 1 + epidermal cells (Nixon- Fulton et al., 19871. They cannot reject allogeneic grafts or produce an A C.B-Igh-lblIgh-lb scidiscid mouse. Mice homozygous for the mutation severe combined immunodeficiency have no abnormal external characteristics when they are maintained under germfree conditions. Photograph courtesy of the Jackson Laboratory. Bar Harbor, Maine.

78 IMMUNODEFICIENT RODENTS tibodies to common laboratory antigens, and their spleen cells do not pro- liferate in response to T- or B-cell-specific mitogens. Fluorochrome-conju- gated antibody reagents that specifically stain B and pre-B cells fail to detect such cells in the spleen or bone marrow (Bosma et al., 1983; Dorshkind et al., 19841. The presence of early B cells is, nonetheless, evident from the ability of Abelson murine leukemia virus (A-MuLV) to transform scid bone marrow cells (Fulop et al., 19881. The resulting A-MuLV transformants show a pre-B-cell phenotype, that is, a rearrangement of both Igh alleles with the Igl alleles remaining in the reline configuration (Schuler et al. 19861. There is also evidence for the presence of early T cells, as about 15 percent of scid mice spontaneously develop thymic lymphomas (Custer et al., 1985) that contain rearranged Tony and Tcri3 alleles and express T-cell-specific cell surface antigens (L3T4, Ly-2) (Custer et al., 1985; Schuler et al., 19861. However, the observed Igh and Tcr rearrangements in the thymic lymphomas and A-MuLV transformants, respectively, are abnormal, consisting of aber- rant deletions of variable-region-coding elements (V, D, or J). The deletions appear to result from the attempted rearrangement of these elements, sug- gesting that the VDJ recombinase mechanism is defective (Schuler et al., 1986~. This idea is further supported by several recent findings. First, un- rearranged Igh, Igl, and Tcr genes become transcriptionally active in lym- phopoietic tissues, but fail to produce functional transcripts (Schuler et al., 19881. Second, rearranged Igh alleles in A-MuLV transformants lack D elements, J elements, or both at the recombination sites (Hendrickson et al., 1988; Kim et al., 1988; Malynn et al., 19889. Similar observations have been made in B-cell lines of long-term scid bone marrow cultures (Okazaki et al., 19881. Finally, transformed pre-B and pre-T cells contain an active but abnormal VDJ recombinase activity that is unable to catalyze the for- mation of functional joints between V(D)J elements (Lieber et al., 19881. As an inbred mutant strain, all scid mice share the same single genetic disease, and yet not all mice lack functional lymphocytes. A variable per- centage of young adult mice (2-20 percent) appear "leaky" in that they develop low numbers of functional B and T cells (Bosma et al., 1988; Carroll and Bosma, in press). This condition is not inherited and may reflect a very low rate of somatic reversions at either scid allele, such that the VDJ re- combinase activity is normalized in some developing lymphocytes in scid homozygotes. Alternatively, the leaky phenomenon may reflect a low but finite chance of a good rearrangement at two critical antigen receptor loci (e.g., Igh and Igl) in cells with a highly defective VDJ recombinase system. The scid mutation does not appear to affect the differentiation of myeloid cells (e.g., erythrocytes, granulocytes, and macrophages). The capacity of scid splenic cells to generate mixed myeloid colonies in vitro is equivalent to that of normal C.B-Igh-lb mice (Dorshkind et al., 19841. Moreover,

HEREDITARY IMMUNODEFICIENCIE5 79 macrophage activation and antigen-presenting function are unimpaired (Czi- trom et al., 1985; Bancroft et al., 19861. Interestingly, NK cell activity is also unaffected by the scid mutation (Dorshkind et al., 1985; Hackett et al., 19861. These NK cells are capable of mediating the rejection of allogeneic bone marrow grafts transplanted into irradiated scid hosts (Murphy et al., 19871. Homozygotes can be "cured" of their lymphocyte deficiency by engraft- ment with histocompatible bone marrow or fetal liver cells of normal mice (Bosma et al., 19831. For example, injection of neonatal scid mice with -0.5 x 106 fetal liver cells results in full lymphoid reconstitution at 8-12 weeks of age (R. P. Custer, G. C. Bosma, and M. J. Bosma, Institute for Cancer Research, Fox Chase, Pa., unpublished data). Spleen and lymph node follicles become repopulated with lymphocytes, and Peyer's patches and solitary follicles of the intestinal tract return to normal. The thymus shows a prominent cortex and corticomedullary delineation, and there is a normal morphologic gradient of developing lymphocytes that proceeds from capsule to medulla. Injection of adult scid mice with 3 x 106 to 5 x 106 normal bone marrow cells also results in lymphocyte repopulation, although the reconstitution is often incomplete (Custer et al., 19851. This problem can be circumvented by sublethal irradiation of adult mice prior to cell transfer (Fulop and Phillips, 19861. Recently, the scid mouse has been used as a recipient for transplanted human tumors (Ready et al., 1987), human fetal lymphoid tissues (McCune et al., 1988), and peripheral blood lymphocytes (Mosier et al., 19881. Thus, the scid mouse represents a potential new model for engraftment of xenogeneic cells and tissues. The scid mouse is also useful for examining the relationship between immunity and disease. Specifically, it serves as a model for studying human severe combined immune deficiency and its associated infections. As in humans, scid mice develop a severe interstitial pneumonitis resulting from infection by Pneumocystis carinii. This microorganism is easily demonstrated with Gomori's methenamine silver stain (J. B. Roths and C. L. Sidman, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). In addition, the high incidence of spontaneous thymic lymphomas makes the scid mouse a potential model for understanding the basis of increased lymphoid malig- nancies in certain immune deficiency states. Husband~ry Homozygous scid mice readily succumb to microbial infections because of their lack of an immune system and must be maintained in a pathogen- free environment (see Chapter 41. Those animals maintained in a single-user, SPF, barrier-protected room can survive to 9-12 months of age.

80 IMMUNODEFICIENT RODENTS Reproduction Homozygous scid mice can be bred without difficulty, although the average litter size (four to six) is smaller than that of the congeneic C.B-Igh-lb strain (six to nine). Tot-] (Tolerance to Gamma-GIobulin) Genetics The Tol-1 locus controls the induction of tolerance to bovine gamma- globulin (BOG) or human gamma-globulin (HGG). Induction of tolerance to BGG is easy in the DBA/2 strain and difficult in the BALB/c strain (Des and Leskowitz, 19701. F1 hybrid and backcross analyses suggest that the difference is due to alleles at a single autosomal locus (Lukic et al., 1975~. Pathophysiology Cell transfer studies indicate that the difference in induction of tolerance to gamma-globulin is a function of macrophages (Des and Leskowitz, 19741. In vitro studies show that macrophages of the BALB/c mouse can efficiently remove a minor component of BGG, which renders the BGG capable of easily inducing tolerance (Cowing et al., 19771. Preliminary studies with HOG suggest that IgG3 is the critical component (Cowing et al., 19791. Husbandry Special husbandry procedures are not required. Reproduction These animals reproduce no ally. Fit (Vitiligo) Genetics / Vitiligo (vit) is an autosomal recessive mutation that was first identified by E. S. Russell at the Jackson Laboratory, Bar Harbor, Maine, as a spon- taneous mutation that occurred in the C57BL/6J strain. It is now maintained as C57BL/6JLer-vit/vit (Palkowski et al., 1987~.

HEREDITARY IMMUNODEFICIENCIES 81 Pathophysiology Mice homozygous for the vit locus have congenital white spotting of the back and abdomen and a progressive depigmentation with each spontaneous hair molt. By 2 years of age the mutant mouse is white. The epidermis, including that of the tail and ear, loses all pigment with age (M. Bell et al., 19841. Light and electron microscopic changes include degeneration of fol- licular melanocytes without inflammation. There is also destruction of me- lanocytes with the choroid and pigment epithelium of the eyes (Palkowski et al., 1987~. C57BL/6JLer-vit/vit mice have fewer Ia-positive Langerhans' cells in the interfollicular epidermis of the back and ear than do C57BL/6J control mice (Rheins et al., 19861. However, the number of Langerhans' cells present in the follicullar epidermis is normal and unchanged during depigmentation (Palkowski et al., 19871. The density of epidermal Thy-1+ cells increases during depigmentation (Amornsiripanitch et al., 19881. While young pig- mented C57BL/6JLer-vit/vit mice were capable of developing moderate con- tact sensitivity (allergic contact dermatitis) to 2,4-dinitro-1-fluorobenzene (DNFB) and picryl chloride, older depigmented animals were not (Rheins et al., 19861. Transplantion of normal C57BL/6 mouse skin onto a depig- mented C57BL/6JLer-vit/vit mouse restores the ability to induce contact sen- sitivity (Palkowski et al., 1 987), and transplantation of vitlvit skin onto normal C57BL/6 mice fails to allow the induction of contact sensitivity, suggesting that the contact sensitivity defect in depigmented mice is cutaneous, probably epidermal, and not systemic in origin (Amornsiripanitch et al., 19881. Since the contact sensitivity reaction is a form of cell-mediated immunity, the delayed-type hypersensitivity reaction was evaluated in vitlvit mice. Both C57BL/6JLer-vit/vit and normal C57BL/6J mice were sensitized with SRBCs given intravenously and challenged with intradermal SRBC injec- tions. Young vitlvit mice had delayed hypersensitivity reactions comparable to those of age-matched controls; however, 24-week-old depigmented vitlvit mice demonstrated enhanced reponsiveness (Amornsiripanitch et al., 19881. The C57BL/6JLer-vit/vit mouse was also comparable to C57BL/6J in the production of humoral immunity to T-cell-dependent and -independent an- tigens. Skin "raft rejection also appears to be normal in vitlvit mice (Amorn- siripanitch et al., 19881. When pigmented skin from a C57BL/6J mouse is transplanted onto a depigmenting vitlvit mouse, it does not depigment, suggesting that the process of melanocyte degeneration is not systemic (Lerner et al., 19861. Husband~ry Information has not been published.

82 IMMUNODEFICIENT RODENTS Reproduction . Information has not been published. W (Dominant Spotting); we (Viable Dominant Spotting) Genetics The W locus is found on chromosome 5. Associated with this locus are many mutant alleles, most of which, including W and We, are semidominant in expres- sion. The W mutation occurred many years ago and was preserved by mouse fanciers (Dunn, 19371. The we mutation was discovered in the C57BL strain in 1937 (Little and Cloudman, 19371. Dominant spotting is maintained as a heterozygote (W/ + ~ on the C57BL/6J and WB/ReJ backgrounds. Viable dom- inant spotting is maintained on the C57BL/6J, MWT/Le, NIXON, NFS/N, and WB/ReN backgrounds. The compound mutant (W/W~) is maintained on the WB/ReJ x C57BL/6J Fit (abbreviated WBB6F~) hybrid background. Pathophysiology W homozygotes or W/W~ compound mutant mice are white with black eyes, are sterile, and have macrocytic anemia. Although W homozygotes have severe anemia from day 12 of gestation and die within the first week A mouse carrying the mutation viable dominant spotting (W'). Affected mice have white spotting and a slightly dilute coat color. Photograph courtesy of the Jackson Laboratory, Bar Harbor. Maine.

HEREDITARY IMMUNODEFICIENCIES 83 of birth (Russell, 1970), we homozygotes can survive to maturity. Hetero- zygotes (W/ + or WV/ + ~ have white spotting and a slightly diluted coat color, have normal erythrocyte numbers (W/+) or a slight macrocytic anemia (WV/+), and are fully viable and fertile (Russell, 19491. Macrocytic anemia and pigment defects are due to intrinsic defects in progenitor cells of erythrocytes and melanocytes. Some evidence suggests that the defect operates at a stage before the proliferation of melanoblasts in the skin (Mayer, 19701. The defect is also present in W/+ heterozygotes (Gordon, 1977~. The genetic basis for anemia probably resides in erythro- poietic cells at an erythropoietin-sensitive stage (Bannerman et al., 1973) and appears to involve a Thy-1 + regulatory cell that is necessary for stem cell differentiation (Sharkis et al., 19781. Lymphopoietic cells also appear to be abnormal. A lymphocytic stem cell population absent in We homozy- gotes can be demonstrated in lymph nodes of homozygotes transplanted with normal bone marrow (Harrison and Astle, 19761. The W/WV compound mutant has a severe reduction in the number of mast cells (Kitamura et al., 19781. Aplastic anemia in W homozygotes is corrected by transplantation of bone marrow, spleen, or fetal liver stem cells from syngeneic (W/ + or + / + ~ donors. Irradiation is not required prior to transplantation. Erythrocyte, as well as gran- uloctye, populations become the donor type following transplantation (Murphy et al., 19731. Recently a new mutation, my, has occurred at this locus. Homozygotes have normal viability and fertility and no anemia. These mice, however, still lack mast cells (Stevens and Loutit, 19821. W/WV mice have a delayed expulsion of primary nematode (Nippostron- gylus brasiliensis) infestation but are refractory to secondary infestation (Crowle and Reed, 19811. When reconstituted with bone marrow or spleen cells from normal littermates, they develop normal connective tissue mast cells. They also acquire mucosal mast cells in response to parasitic infection, but the mucosal mast cells do not accelerate parasite rejection (Crowle, 19831. Like- wise, W/WV mice have a delayed expulsion of Trichinella spiralis (Ha et al., 19831; however, in this instance bone marrow transplantation, which recon- stitutes the mast cell population, does accelerate parasite rejection. Husbandry Special husbandry procedures are not required for maintaining W/Wt com- pound mutants or We homozygotes. Special husbandry procedures will not prolong the life of W homozygotes. Reprodluction The mutation dominant spotting is maintained by breeding heterozygotes. W/Wt mice are produced by mating WB/ReJ-W/ + females with C57BL/6J

84 IMMUNODEFICIENT RODENTS Wt/+ males. These mice are easily distinguished from We/+, W/+, and + / + littermates by the distinctive white coat color (Crowle and Reed, 1981). Wails and W/ + heterozygotes can be distinguished from the wild type + / + ~ by the presence of a white spot on the abdomen. wst (Wasted) Genetics Wasted (grist) is an autosomal recessive, lethal mutation that arose spon- taneously in 1972 in the HRS/J strain at the Jackson Laboratory, Bar Harbor, Maine (Lane, 19811. It has been mapped to chromosome 2 (Sweet, 19841. The wst mutation was crossed to a segregating F1 hybrid background (C3HeB/FeLe-a/a x C57BL/6J Fit to increase the viability of progeny (Shultz et al., 1982b). The gene is being transferred to strain C57BL/6J (N1 1 as of December 1988) (L. D. Shultz, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). Pathophysiology Homozygous wst mice are recognized by 20 days of age by neurological abnormalities, including tremors and ataxia. Paralysis precedes death at 30 days of age or less. Wasted mice gain weight only until 20 days of age. By 28 days, wst homozygotes are one-half as large as +/-controls. Homozygous wst mice show pathologic changes similar to those of patients with ataxia telangiectasia. Central nervous system abnormalities include de- generation of cerebellar Purkinje cells and focal demyelination in the cere- bellar cortex and ventral columns of the spinal cord. There is a fourfold greater incidence of chromosome damage in bone marrow cells of wst ho- mozygotes than in those of heterozygous littermates. Both thymus-dependent and thymus-independent areas of the lymph nodes are markedly hypoplastic. The follicles of the lymph nodes and the spleen are poorly developed, Peyer's patches are reduced in size, and the thymic cortex has reduced cellularity. In addition, wstlwst mice have a threefold decrease in numbers of circulating leukocytes. Analysis of lymphocyte sub- sets by immunofluorescence microscopy indicates that the percentages of B and T cells are within the normal range. Homozygous wst mice have significantly impaired delayed-type hyper- sensitivity to SRBCs when tested 4 days following priming (Shultz et al., 1982b). The response of thymic and splenic lymphocytes to ConA or LPS is markedly reduced (Goldowitz et al., 19851. Wasted mice have normal levels of serum IgA, and the spleen has normal numbers of sIgA-bearing B cells and plasma cells. However, IgA plasma cells are totally absent from

HEREDITARY IMMUNODEFICIENCIE5 85 the intestine, and IgA-specific B-cell precursors are absent from the Peyer's patches. Kaiserlian et al. (1985) suggest that the wst mutation might he useful as a model for the regulation of IgA production. Nordeen et al. (1984) have expressed reservations about wasted mice being considered as a model for human ataxia telangiectasia. The gamma irradia- tion- or bleomycin-resistant component of DNA replication characteristic of fibroblasts in humans with ataxia telangiectasia (Cramer and Painter, 1981) has not been observed in fibroblast cell lines of wasted mice (Nordeen et al., 1984~. Recently, Abbott et al. (1986) described abnormally low levels of aden- osine deaminase (ADA) in wasted mice and proposed that wst represents a mutation in the structural gene for this enzyme. However, investigations by Geiger and Nagy (1986) indicate that these mice have measurable ADA levels in blood and lymphoid tissues and increased ADA activity in the spleen and cerebellum. Thus, the status of the wasted mouse as an animal model for human ADA deficiency is still in question. O ~ Husbandry Special husbandry procedures are not required. Reproduction Wasted mice can be perpetuated by mating heterozygotes. At the Jackson Laboratory the mutation is maintained by ovarian transplantation (see Chap- ter 51. xia, (X-Linket1 Immune Deficiency) Genetics X-linked immune deficiency (xid) is a sex-linked recessive mutation that originated on the CBA/MN substrain (Amsbaugh et al., 1972; Huber et al., 19771. This mutation is now available on several genetic backgrounds. Pathophysiology The mutation xid causes functional defects of B lymphocytes. Homozygous females and hemizygous males (hereafter called.xid mice) fail to respond to thymus-independent antigens such as haptenated-Ficoll, dextran, pneumo- coccal polysaccharide, and dsDNA (Scher et al., 1973; Cohen et al., 19761. Furthermore, they are nonresponsive to certain thymus-dependent antigens (Press, 19811. Affected mice have low levels of the immunoglobulin isotypes

86 IMMUNODEFICIENT RODENTS 11 and y3 and extremely low levels of A. The B-cell defect is believed to be intrinsic, because the functional lesions are overcome when normal bone marrow is transferred to an xid host. Conversely, Did marrow transplanted to an irradiated, normal, syngeneic host retains its abnormal phenotype. Affected mice lack a subpopulation of B cells that expresses small amounts of surface IgM and large amounts of surface IgD. This subpopulation also expresses a unique surface component (Lyb-3) that, when bound by antibody, induces an enhanced response to antigen, which suggests that it functions as a lymphokine receptor (Huber, 19821. The same subset of B cells expresses the alloantigen Lyb-5 (Smith et al., 19861. In normal mice this B-cell subset responds to thymus-independent antigens, is polyclonally stimulated by anti- immunoglobulin, forms colonies in vitro (Kincade, 1977), binds to macro- phage surfaces, and is exquisitely radiation sensitive (Lee and Woodland, 19851. It is a matter of controversy whether these B cells form a unique differentiation pathway or represent a late maturation stage of all normal B cells (see nu xid, page 921. It has also been argued that those B cells that are present in xid mice are not normal (Sprent and Bruce, 19841. The Ly- 1 + B-cell population is also missing in these mice (Hayakawa et al., 19831. Mice of the H-2b haplotype carrying the xid mutation fail to express the surface antigen IaW39 but do have IaW39-positive cytoplasmic protein. This suggests that the failure to express IaW39 as a surface molecule is due to a deficiency in a glycosylation pathway (Huber, 19821. By restriction fragment length polymorphism, a family of X-linked genes located close to or at the xid locus has been mapped. It appears that mature normal B cells, but not xid B cells, transcribe gene products that are encoded in this region (Cohen et al., 19851. No defects in T-cell functions, such as cytotoxicity, graft rejection, or delayed-type hypersensitivity reactions, have been observed, but there are several studies suggesting that the help provided by T cells from xid mice is not optimal (cf. Phillips and Campbell, 19811. Agreement on this point has not been achieved. When the xid allele is backcrossed onto a C3H/HeN or a C3H/HeJ back- ground, the affected mice have a greater defect in B-cell function than do CBA/HN-xid mice. Responses to the mitogen Nocardia are diminished, and the frequency of sIg+ B cells, particularly in the lymph nodes, is greatly reduced. It has been proposed that those B cells present are nonresponsive to some of the T-cell-generated lymphokines. The major functional defect so far described (in addition to those already ascribed to xid) is a diminished B-cell response to type I antigens (Mona et al., 19831. Backcrossing the xid allele onto the NZB (Nakajima et al., 1979), NZB x NZW F~ (A. D. Steinberg et al., 1984), or BXSB (A. D. Steinberg et al., 1984) backgrounds results in a modification of the natural history of

HEREDITARY IMMUNODEFICIENCIES 87 autoimmune disease that is characteristic of these strains. All have reduced quantities of autoantibodies and increased life spans. Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally and can be maintained by brother x sister matings. Yaa (Y -Linked Autoimmune Accelerator) Genetics The Y chromosome linkage for acceleration of the autoimmune disease of male mice of the SB/Le and BXSB/Mp strains was first hypothesized from studies of reciprocal F1 hybrid males resulting from crosses to strains C57BL/6J, NZB/BlNJ, and SJL/J (Murphy and Roths, 19793. Conclusive evidence for this holandric inheritance of accelerated autoimmunity came from analysis of the Y-consomic stocks developed by transfer (backcross) of the Y chromosome of strain BXSB to strains C57BL/6J and NZB/BlNJ and the transfer of the normal Y chromosome of strain C57BL/6J to strain BXSB/Mp. The gene symbol Yaa (Y-linked autoimmune accelerator) has been assigned (Roths, 19871. Yaa acts synergistically with a presumptive autosomal autoimmune in- duction genes in strains SB/Le and BXSB/Mp. The development and ge- netics of these strains are described in the section on immunodeficient inbred strains (see page 961. Pathophysiology BXSB-Yaa males are generally 10-15 percent smaller than their female siblings prior to and at weaning. They develop moderate lymphadenopathy, which is most noticeable as enlargement of the cervical and axillary lymph nodes, by 3-4 months of age. The spleens are greatly enlarged and easily palpable. Greater than 60 percent of BXSB/Mp-Yaa males develop a profound generalized edema within a few days prior to death. BXSB/Mp-Yaa males survive to 4-6 months compared with 14-16 months for female BXSB siblings. They remain healthy until near the end of their short life span without evidence of infectious disease. Male BXSB mice bearing the normal Y

88 IMMUNODEFICIENT RODENTS chromosome of C57BL/6J (i.e., BXSB/Mp-+Yaa) live for greater than 24 months without evidence of autoimmune disease. Yaa operates independently of the sex hormonal milieu. For example, testes weights, serum testosterone levels, and secondary sex characteristics are not altered by Yaa (Roths, 19871. In addition, Eisenberg and Dixon (1980) have demonstrated that orchiectomy does not delay the early onset of fatal autoimmune disease of BXSB males. The moderate lymph node enlargement is due to proliferating lymphocytes and admixed plasma cells and histiocytes. Lymph node architecture is blurred. Immunoblasts are common and microvascular proliferation may occur. The enlarged spleens show moderate hyperplasia of white pulp and greatly in- creased erythropoiesis. The kidneys of BXSB/Mp-Yaa males show an acute or subacute exudative and proliferative glomerulonephritis and striking tu- bular involvement. Immune complex vascular disease is also common. Im- munofluorescence studies of kidneys of BXSB males reveal a heavy granular deposition of immunoglobulins in the glomerular capillary walls and mes- angium. Nearly all BXSB/Mp-Yaa males are clinically anemic (packed eryth- rocyte volumes of 30-33 percent at 6 months of age), with high titers of circulating erythrocyte autoantibody and elevated protoporphyrin. High titers of antinuclear and thymocyte-binding autoantibodies are also char- acteristic. Hyperimmunoglobulinemia (17 percent of total globulins) with ninefold elevations of the IgA and IgG1 isotypes has been described (Mur- phy and Roths, 1 978a) . There is an increase in the frequency and absolute numbers of immu- noglobulin-bearing (B-cell) lymphocytes in the lymph nodes by 4 months of age (Theofilopoulos et al., 19791. The Yaa gene appears to play a significant role in the abnormal resistance to tolerance induction to heterologous IgG (Izui and Masuda, 19841. Cell-mediated immune func- tions of BXSB/Mp-Yaa males are similar to those of nonautoimmune con- trol strains, but BXSB males have reduced reticuloendothelial function and lower bactericidal activity against Listeria sp. (Creighton et al., 19791. The Yaa mutation has been transferred by multiple backcross matings to C3H/HeJ, C57BL/6J, and SJL/J inbred strains. Compared with their con- somic controls, C3H/HeJ-Yaa and SJL/J-Yaa males do not develop autoim- mune manifestations. C57BL/6J-Yaa males, but not C57BL/6J- + Yaa males, develop antinuclear autoantibodies, have a fourfold increase of serum IgG2b, and have splenomegaly (58 percent increase in mass). The mean life span of C57BL/6J-Yaa males is 80 percent that of their consomic controls (23 versus 29 months) (Roths, 19871. The Yaa gene is capable of accelerating the appearance of the anti-DNA response induced by the Ipr gene (Pisetsky et al., 19851.

HEREDITARY IMMUNODEFICIENCIES 89 Husbandry Special husbandry procedures are not required. Reproduction BXSB/Mp females give birth to an average of five pups per litter, and in paired matings they will produce only two to three litters because of the early morbidity of males. S-Region-Linked Genes Controlling Murine C4 Genetics and Pathophysiology Mice have two C4-like molecules, each of which is encoded by a separate gene in the S region of the MHC (Shreffler, 19821. One glycoprotein, coded for by the sex-limited protein (Sip) locus, is structurally similar to human and guinea pig C4, is testosterone dependent in many strains, but has no hemolytic activity (Hobart, 19841. A second closely linked gene, C4 (for- merly designated Ss), encodes for the antigenic, structural, and functional homologue of human C4 (Ferreira et al., 19771. Mice expressing the H-2W7 haplotype have C4 that has only 30 percent of the functional complement activity of other strains (Atkinson et al., 19801. The alpha chain of this C4 is smaller in H-2W7 mice because of a difference in the carbohydrate content (Karp et al., 1982a,b). This genetic variation in glycosylation of C4 has been shown to directly affect hemolytic activity, because evidence for a plasma inhibitor or altered C4 stability is lacking (Atkinson et al., 19801. Recent evidence indicates that mice with the H-2W7 haplotype have four Slp genes, three of which appear to be C4-Slp recombinant genes (Nakayama et al., 19871. Synthesis of the products of these genes is not testosterone dependent, and at least two Sip genes of H-2W7 mice are transcribed (Ogata and Sepich, 19851. The relationship between products of these recombinant genes and the H-2W7-linked C4 gene product with faulty glycosylation is unclear. A quantitative difference in C4 is also controlled by a single autosomal gene with codominant expression. The original locus designation was Ss and the alleles were h (high levels) and I (low levels), that is. S5h and Ssi. Current nomenclature for the Ss locus is C4; however, the alleles described above have not appeared associated with the new nomenclature in the literature. ssi strains are all of the H-2k haplotype (Shreffler and Owen, 1963) and have 20-fold less functional C4 in the circulation. F1 hybrids have intermediate levels. No func- tional impairment in complement activation or resistance to infectious disease has been attributed to the ssi allele alone in mice that bear it. Another locus or gene cluster linked to the S region of mice controls the

90 IMMUNODEFICIENT RODENTS quantitative levels of complement components 1, 2, and 4. The data suggest that functional levels of at least these three components are controlled by a single gene or a small gene cluster within the H-2 complex (Goldman and Goldman, 1976~. In humans, a gene or closely linked gene cluster controls expression of factor H. C4-binding protein, and the C3b receptor. This gene is called the regulator of complement activation (RCA', and perhaps similar loci regulate the expression of multiple complement genes in mice (De Cor- doba and Rubinstein, 1986~. Husbandry Special husbandry procedures are not required. Reproduction No reproductive problems have been attributed to the various loci con- trolling the expression of the early components of complement described above. MICE WITH MULTIPLE MUTATIONS1 gld (Generalized Lymphoproliferative Disease); xid (X-Linked Immune Deficiency) C3H/HeJ-gld/gld xid mice have been developed to detains the effect of the xid gene on the development of the gld autoimmune lymphoproliferative phenotype (Seldin et al., 19871. A comparison of C3H-gldlgld and C3H- gldlgld xid males shows that the xid gene has no effect on the extent of lymphadenopathy or on the phenotype of the expanded T-cell subset (Ly-2-, Ly-1+, L3T4-, Ly-S+) of gld homozygotes. By contrast, the xid gene significantly reduces serum IgM and nearly abolishes the generation of both IgM and IgG anti-ssDNA and anti-dsDNA autoantibodies. Thus, the xid gene dramatically decreases B-cell manifestations without affecting T- cell abnormalities in gld homozygotes. [pr (I~ymphoproliferation); nu (Nude) The C57BL/6J-lpr/lpr nulnu double mutant strain has recently been de- veloped. The Ipr phenotype on the C57BL/6J background has been defined iThe husbandry and reproduction of any double mutation carrying nulnu are like those discussed in the narrative on nude mice (pas. 69-71 and Chapter 4).

HEREDITARY IMMUNODEFICIENCIES 91 (Mosbach-Ozmen et al., 1985a). Homozygosity at the nu locus prevents the development of Ipr-induced lymphadenopathy (a T-cell hyperplasia) (Mos- bach-Ozmen et al., 1985b). This finding is consistent with the inhibition of lymphadenopathy seen in neonatally thymectomized MRL/Mp-lpr/lpr mice (A. D. Steinberg et al., 1 9801. Ipr (Lymphoproliferation); xid (X-Linked Immune Deficiency) MRL/Mp-lpr/lpr xid mice have been developed (E. B. Steinberg et al., 19831. Double mutant Iprllprxid mice show the lymphadenopathy, expansion of a dull Ly-1 + T-cell population, impaired cellular proliferation in response to ConA, and diminished IL-2 production characteristic of homozygous Ipr mice. However, the double mutants do have a marked reduction in anti- ssDNA and anti-dsDNA autoantibody levels and serum gp70 immune com- plexes. At 6 months of age, histologically defined renal disease and pro- teinuria are markedly reduced in Iprllpr xid mice, and their life span is nearly double that of mice homozygous for the Ipr gene alone. Possibly, this is due to the effect of the xid mutation on the maturation of the B-cell subset necessary for high autoantibody production, which requires nonspecific T- cell helper factors (Fieser et al., 19841. Ipr (Lymphoproliferation); Yaa (Y-Linked Autoimmune Accelerator) The Y-linked autoimmune accelerator (Yaa) gene, originally identified in studies of strain BXSB, is capable of promoting or accelerating autoimmune disease induced by genes other than those resident in the BXSB autosomal genome. In C57BL/6J-lpr/lpr Yaa double mutant males, Ipr-induced lymph- adenopathy occurs earlier and is more extensive than in homozygous Ipr controls. Moreov-er, the survival time of Iprllpr Yaa mice is reduced to 9 months, compared with 15 months for males homozygous for Ipr alone (Roths, 19871. C57BL/6J-lpr/lpr Yaa males have markedly increased levels of IgG anti-DNA (8.2 1og2 titers) in comparison with Ipr homozygotes without Yaa (2.5 log2 titers). Serum IgM and IgG levels are 1.5- and 2.1-fold greater, respectively, in males with both mutations than in males homozygous for Ipr alone (Pisetsky et al., 19851. nu (Nu(le); bg (Beige) Comparisons between homozygous N:NIHS-nu/nu and double homozy- gous N:NIHS-bg/bg nulnu mice reveal that NK cell activity in double ho- mozygotes is significantly reduced compared with that in nude homozygotes and three times greater than that in beige homozygotes (Fodstad et al., 1984a).

92 IMMUNODEFICIENT RODENTS An evaluation of the double homozygotes as hosts for human tumor trans- plantation has shown no correlation between host NK cell activity and sub- cutaneous growth of various human (LOX, CEM, and K562) and murine (YAC- 1) tumor cells (Fodstad et al., 1 984b). In addition, low NK cell activity has not been associated with increased lung colony formation in a metastasis model in which intravenously injected human LOX or murine B16F10 mel- anoma cells are used. Thus, the data failed to support the idea that NK cells exert significant effects on tumor cells in viva, although they are known to be toxic to tumor cells in vitro. nu (Nude); xid (X-Linked Immune Deficiency) Mice with these mutations have no mature T cells and few or no mature B cells (Azar et al., 1980~. The result is a defect resembling severe combined immunodeficiency (Mona et al., 1982; Wortis et al., 19821. B-cell precursors are blocked at the pre-B-cell stage, at which point they express the cell surface antigen Lyb-5, have incompletely rearranged immunoglobulin genes, and are insensitive to transformation by A-MuLV (Karagogeos et al., 1986~. Grafts of thymus epithelium restore both T- and B-cell function, demon- strating that B cells expressing the xid mutation depend on T cells or their products to mature past the pre-B-cell stage (Karagogeos and Wortis, 19871. There are two explanations for this synergistic effect. One is that all B cells in xid mice are "crippled" and need the extra stimulation provided by T cells to reach maturity. The alternative is that normal B cells develop into two separate subpopulations, one of which (found in normal and xid mice) is T-cell dependent and the other (found in normal and nulnu mice) is T-cell independent. Histologic examination of 173 N:NIHS-nu/nu xidlxid female mice revealed a high incidence of both lymphosarcoma and ovarian granulosa cell tumors (Sadoff et al., 19881. nu (Nude); bg (Beige); xid' (X-L`inked Immune Deficiency) The simple mode of inheritance of these mutations provides a means of designing experimental systems that allow the study of the effects of these mutants either singly or in various combinations in the same experiment. The technique involves developing congenic strains of each mutant on the same genetic background. First, two of these mutations and finally all three are combined. The combination used depends on the purpose of the study. The immune system effects of the possible combinations are given in Table 2-2. This scheme meets all the criteria for good experimental design and is also flexible, because selected combinations can be used without compromising

HEREDITARY IMMUNODEFICIENCIES 93 TABLE 2-2 Cells Present in Mutant Combinations of bg, nu, and xid Mutant Locus Cells Present bg nu xid T B NK +I+ +I+ +I+ + + + bgibg + I + + I + + + - +I+ nulnu +I+ - + + + I + + I + xidlxid + - + bgibg nulnu + I + - + bgibg + I + xidlxid + - - + I + nulnu xidlxid - - + bgibg nulnu xidlxid the remainder of the immune system. As an example, Andriole et al. (1985), using some of the combinations given in Table 2-2, were able to demonstrate that NK cells function independently from lymphokine-activated killer (LAK) cells. nu (Nude); Dh (Dominant Hemimelia) Mice with the mutations nude and dominant hemimelia (nulnu Dhl + J are both athymic and asplenic. They are sometimes referred to as lasat mice (Lozzio, 1976), although this is not standardized nomenclature. The im- munologic deficits of nulnu +/ +, +/ + Dhl +, and nulnu Dhl + mice have been described and compared (Ikeda and Gershwin, 1978; Erickson and Gershwin, 19811. Some of the deficits in the double mutant (e.g., the his- tologic organization of lymph nodes) are predictable. Other aspects of im- mune function in these animals (e.g., the response to PHA, the numbers of circulating Thy-1.2-bearing cells, and the acceptance of tumor allografts) are modified differently in the absence of both the thymus and spleen than they are in the absence of either organ alone. The nulnu Dhl + mutant provides a model for investigating the functional relationship between the spleen, thymus, and bone marrow. Yaa (Y-Linked Autoimmune Accelerator); bg (Beige) An SB/Le-sa balsa bg male provided the aberrant Y-linked autoimmune accelerator gene (Yaa) in the creation of strain BXSB/Mp (see strain BXSB/Mp, page 961. In addition to developing the BXSB recombinant strain from off- spring of C57BL/6J x SB/Le-sa balsa bg Yaa F2 breeders, the segregating inbred strain SB/Le-bg/bg Yaa and bgl ~ Yaa was created. It became apparent that the SB/Le-bg/bg Yaa males had a retarded development and progression

94 IMMUNODEFICIENT RODENTS of the clinical phase of autoimmune disease seen in the SB/Le-bg/+ Yaa males. Mice with both genotypes had similarly high levels of ANA and serum IgG, lymphadenopathy, and splenomegaly with hemolytic anemia. However, bglbg Yaa males did not develop immune complex glomerulo- nephritis with nephrotic syndrome and survived twice as long (69 weeks' as bgl + Yaa males (35 weeks) (Roths and Murphy, 19821. In studies by Clark et al. (1982), the findings of decreased deposition of IgM in renal glomeruli, reduced LPS responsiveness, and reduced number of ThB+ spleen cells suggested that the bg gene could be providing a protective effect by acting directly on B cells. The role of NK cells, which are impaired in bgibg mice, has not been adequately explored. Yaa (Y-Linked Autoimmune Accelerator); aide (X-Linked Immune Deficiency) The X-linked immunodeficiency (xid) mutation has been shown to delay the expression of accelerated autoimmunity determined by the Yaa gene. Studies by Golding et al. (1983) have shown that CBA/Ca x BXSB/Mp F~- Yaa hybrid males, but not CBA/N x BXSB/Mp F~-xid Yaa hybrid males, have progressive autoimmune lymphoproliferative disease. These latter xid Yaa males are devoid of splenic B colonies, are unresponsive to trinitrophenol (TNP)-Ficoll, and do not develop splenomegaly or lymphadenopathy. At 10 months of age nearly all of the CBA/Ca x BXSB/Mp F~-Yaa males, but none of the age-matched CBA/N x BXSB/Mp F,-xid Yaa males, are anti- nuclear antibody positive. Smith et al. (1983) reported on the development of the congenic BXSB- xid strain. Their studies demonstrated that the xid mutation markedly reduced autoimmune disease in BXSB-Yaa males. The double mutants showed re- duced lymphoid hyperplasia, hypergammaglobulinemia, and autoantibody levels; less severe renal disease; and prolonged survival. They concluded that the autoimmune disease of BXSB/Mp-Yaa males is dependent on B-cell subset depletion in xid individuals. INBRED STRAINS OF MICE BSVR, BSVS Genetics In 1930 Webster developed, from Rockefeller Institute stock, lines of mice that were resistant (BR) and susceptible (BS) to Bacillus enteritidis (Sal- monella spp.) (Webster, 19331. Later tests with louping ill and St. Louis encephalitis viruses in these lines led to the development of virus-resistant and -susceptible derivatives (VR and VS) and thus to the strains BRVR,

HEREDITARY IMMUNODEFICIENCIES 95 BSVS, BRVS, and BSVR (Webster, 19371. Recent evidence indicates that the bulk of the susceptibility of the BSVS mouse to Salmonella is controlled by a single autosomal locus unrelated to H-2, Igh-C, or Hbb (Briles et al., 1977, 19811. Using F1 hybrids, O'Brien et al. (1981) have provided evidence that the genetic defect in this strain is probably due to Itys. Thus, many of the differences in the BR and BS strains are probably due to genetic differ- ences at the I'y locus. Interestingly, the BSVR mouse has been found to be much more susceptible to salmonella infection than is the BSVS mouse (Benjamin and Briles, 19821. This added susceptibility is probably due to an additional non-I'y gene or genes. It is not known whether this gene has any relationship to the viral resistance or susceptibility traits of this group of mouse strains. The BRVR and BRVS strains are now extinct and will not be discussed further. Pathophysiology BSVR and BSVS differ in their susceptibility to experimental autoimmune encephalomyelitis (Olitsky and Lee, 1953) and thyroiditis (Rose et al. 19731. A defect in thymus-dependent antibody responses in BSVS mice has been suggested (Briles et al., 19791. Streptococcal group A carbohydrate (GAC), when injected as a component of a killed streptococcal vaccine, is a T-cell- dependent antigen (Briles et al., 19821. BSVS mice make a poor immune response to GAC, apparently because of regulation by genes linked to the H-2 and Igh-C loci, as well as to an additional gene or genes (Briles et al., 19771. It was originally thought that the poor response of BSVS mice to T-cell- dependent antigens might account for the susceptibility of this strain to Sal- monella infection (Briles et al., 19791. However, in 1981 Briles et al. dem- onstrated that a Salmonella resistance gene in A/J mice (presumably Itys) rendered these mice resistant to Salmonella infection without conferring the high T-cell-dependent immune responsiveness of A/J mice. The mechanism by which the Ity locus affects the susceptibility to S. typhimurium is in dispute. It has been claimed that the locus regulates both the efficiency with which S. typhimurium are killed in macrophages in vitro (Lissner et al., 1983) and their rate of multiplication within the host (Hor- maeche, 19801. It seems likely that the latter claim correctly represents the . . . lI1 VlVO ClrCUmStaIlCeS. Survival of mice following infection with S. typhimurium is thought to be largely the result of the activation of macrophages by either LPS or T cells, so that they can more effectively kill intracellular S. typhimurium (Collins and Mackaness, 1968; O'Brien et al., 19801. The findings that the effects of the I~ locus can be observed in nulnu mice (lacking T cells) (O'Brien

96 IMMUNODEFICIENT RODENTS and Metcalf, 1982) and Lpsd mice (macrophages unresponsive to LPS) (Briles et al., 1986) indicate that the effect of I'y on resistance to Salmonella is not by the modulation of either of these macrophage activation mechanisms. Furthermore, S. typhimurium with full virulence properties, but lacking a functional aroA gene (which is necessary for the synthesis of aromatic com- pounds that are absent in vivo), fails to show significant growth in vivo (Hoiseth and Stocker, 1981). When this organism is injected into a panel of BXD recombinant inbred mice, the number of aroA Salmonella recoverable from the mice after several days is totally unrelated to the IN type of the mice, a finding that indicates that the Iffy locus does not effect killing in vivo (Benjamin and Briles, 1982). This conclusion has been confirmed by infecting mice with S. typhimurium carrying a plasmid that fails to replicate at 37°C. The dilution of the plasmid in the Salmonella taken from infected mice allows the rate of in vivo growth of the Salmonella to be calculated. The total recovery of the plasmid from the mice provides an unbiased index of killing. The data obtained by this procedure make it clear that the major effect of the alleles of the Ity locus in vivo is to modulate Salmonella growth (Benjamin et al., 19871. Husbandry Special procedures are not required for maintaining BSVR and BSVS mice. Reproduction BSVS mice reproduce very poorly when they are raised under conventional conditions. The major problem is high neonatal mortality. Necropsy exam- ination of offspring reveals the absence of milk in the stomach. Such ob- servations are consistent with murine coronavirus (mouse hepatitis virus) infection. In fact, when this strain is reared under pathogen-free conditions, the mice have larger litters and neonatal survival is much improved. The frequency of stillborn litters increases with age in BSVR females. Therefore, it is recommended that they be bred as soon as they reach sexual maturity (D. E. Briles, University of Alabama, Birmingham, unpublished data). BXSB/Mp Females Genetics BXSB/Mp is a recombinant inbred strain of mice derived from a cross between a C57BL/6J female and a SB/Le male. The inbred strain SB/Le is homozygous for the linked mutations satin (say and beige (bg). Both the sa

HEREDITARY IMMUNODEFICIENCIES 97 :: ~ : : The BXSB mouse, a recombinant inbred strain with an agouti coat color. Females develop autoimmune disease during their second year of life. Males carry the Y-linked autoimmune accelerator (Yaa) gene and develop autoimmune disease in their first year of life. Photograph courtesy of Fred W. Quimby, New York State College of Veterinary Medicine, Cornell University, Ithaca. New York. and bg genes were removed from the developing BXSB strain by negative selection. Initial aging studies demonstrated a major sex difference in life span (Murphy and Roths, 1978a). Accelerated autoimmune disease in males is due to the presence of the Y-linked gene Yaa described previously (see page 871. SB/Le females also develop autoimmune disease. Presumably, both the Y-linked autoimmune accelerator gene (Yaa) and an autosomal gene or genes responsible for induction of autoimmunity in BXSB/Mp mice were derived from strain SB/Le. Pa thophys to logy Female BXSB/Mp mice develop lymphoid hyperplasia, splenomegaly, and Coombs'-positive hemolytic anemia; however, the age at onset is two to three times greater than that of their male siblings. The serologic and im- munologic abnormalities characteristic of male BXSB-Yaa mice by 5 months of age also occur in older (greater than 12 months of age) BXSB females. BXSB females survive to a mean age of 15 months. At necropsy the common findings are severe, chronic immune complex glomerulonephritis and vas- cular disease. Husbandry Special husbandry procedures are not required.

98 IMMUNODEFICIENT RODENTS Reproduction BXSB/Mp females give birth to an average of five pups per litter and in paired matings will produce only two to three litters because of the early morbidity of males. DBA/2Ha Genetics DBA/2Ha was established as a subline of DBA/2 in 1933 (Bailey, 19791. Pathophysioltogy B cells of the DBA/2Ha strain fail to respond to some T-cell-replacing factors (Tominaga et al., 1980~. The precise nature of the defect and its genetics is uncertain. Takatsu and Hamaoka (1982) concluded that an X- linked recessive allele was responsible for a failure to respond to activation factors produced by activated T cells and the T-cell hybridoma BlSlK12. It is likely that the factor in question is B-cell growth factor type 2 (IL-6) (Takatsu et al., 19851. Antibody against the putative receptor has been pre- pared (Tominaga et al., 1980~. Sidman et al. (1986a), using a different assay system, concluded that the defect caused a lack of response to y-interferon and to two other B-cell maturation factors (Bmf). They also reached the conclusion that the genes mapped to autosomal loci (Bmir-1 on chromosome 4 and Bmfr-2 on chromosome 91. It remains unresolved whether the differ- ences in these experiments are technical or are due to a segregation of genes within the DBA/2 lineages. Affected mice do not appear to be susceptible to overt infection or to have rat . . Immune c .enclencles. Husbandry Special husbandry procedures are not required. Reproduction These animals reproduce normally. MRL/Mp Genetics The MRL/Mp strain was developed as a by-product of a series of crosses involving AKR/J, C57BL/6J, C3H/Di, and LG/J mice for the purpose of

HEREDITARY I3,IMUNODEFICIENCIES 99 creating a compatible genetic background for maintaining the mutation achon- droplasia (en). It is estimated that 75 percent of the MRL/Mp genome is derived from strain LG/J. The mutant gene lymphoproliferation (Ipr) arose during the development of this inbred strain (see page 59) (Murphy and Roths, 1977~. Nearly all MRL/Mp mice have ANAs by 5 months of age. By contrast, no ANAs have been detected in LG/J mice as old as 24 months. To examine the genetics of this phenomenon, F1 and F2 hybrid matings and F1 backcross matings to MRL/Mp were performed; and the offspring were assayed for ANAs at 4 months of age. The data from the F1 and F2 hybrid matings suggest that the MRL strain contributed a single autosomal recessive gene. However, the F1 backcross to MRL produced a greater than expected fre- quency of ANA-positive offspring. More work must be done to determine whether a single gene causes the development of ANA in MRL/Mp mice (Roths, 1987~. Pathophysiology MRL/Mp mice develop clinically defined autoimmune disease without lym- phoid hyperplasia. By 4-5 months of age the sera of 94 percent of MRL/Mp mice have ANAs. These mice also spontaneously produce anti-DNA and anti-ribonucleoprotein (RNP) autoantibodies, and anti-Sin is found in 35 percent of males and 45 percent of females (Billings et al., 1982~. Retroviral gp70 immune complexes are found in MRL/Mp females over 1 year of age (Izui et al., 19791. Nearly all MRL/Mp mice have severe chronic glomer- ulonephritis resembling the lupuslike renal disease of NZB x NZW Fit hybrid females. MRL/Mp mice have extremely elevated levels of urinary protein after 14 months of age (Kelley and Roths, 19851. Degenerative arterial disease and polyarteritis are also common. MRL/Mp mice show widespread inflam- matory infiltrates involving cerebral vessels and meninges but not the choroid plexus (Alexander et al., 19839. At autopsy, one-half have malignant tumors, one-third of which are reticulum cell neoplasms. The mean life span of MRL females and males is 74 and 92 weeks, respectively (Murphy, 19811. Early exposure to physiologic levels of estrogen modifies humoral components of the disease in both MRL + / + and MRL-lprllpr mice (Brick et al., 19889. Husband~ry MRL/Mp mice are large and appear to be healthy during their breeding period. Although special husbandry procedures are not required, these ani- mals are maintained at the Jackson Laboratory, Bar Harbor, Maine, on a diet of pelleted 96W chow (21.9 percent protein, 7.2 percent fat).

100 IMMUNODEFICIENT RODENTS Reproduction MRL/Mp mice are excellent breeders. In one study of reproductive per- formance, 37 breeding pairs produced an average of 4.5 litters per pair, with an average of 6.2 pups per litter and 96 percent of the pups surviving to weaning (Murphy and Roths, 1978c). NOD (Non-Obese Diabetic) Genetics NOD (non-obese diabetic) is an inbred strain of mice derived from ICR/Jcl mice by selection for spontaneous development of insulin-dependent diabetes (Ida) (Making et al., 19801. A polygenic basis for diabetes suscep- tibility in the NOD strain has been established by outcrossing to the related, diabetes-resistant inbred strain NON (non-obese normal), which was sepa- rated by Makino from the NOD line at the sixth generation of inbreeding. For diabetes to occur following outcrossing to NON and backcrossing to NOD, a minimum of three recessive diabetogenic genes must be inherited in the homozygous state from NOD (Prochazka et al., 19871. The first recessive diabetogenic locus, observed initially by Hattori et al. (1986) in mice outcrossed to C3H and backcrossed to NOD, is tightly linked to the MHC on chromosome 17 and has been provisionally designated Idd-l (Letter et al., 19861. It has not yet been definitively shown, however, that this locus is within the MHC. A unique I-A,(3 locus (Acha-Orbea and McDevitt, 1987) and a failure to express an l-E gene product (Nishimoto et al., 1987) have also been proposed. The second diabetogenic locus, Idd-2, is on chromosome 9 (Prochazka et al., 1987), about 15 cM centromeric to the Thy-llAlp-1 marker loci (M. Prochazka, D.V. Serreze, S. M. Worthen, and E. H. Leiter, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). The third locus has not yet been mapped but can be shown to segregate in a second backcross (BC2) to NOD using diabetes-free BC 1 individuals typed for hom- ozygosity for the NOD alleles at marker loci linked to Idd-1 and Idd-2. In addition to these recessive genes, a gene or genes underlying T-lymphocyte proliferation is inherited from NOD in a dominant fashion. Segregation anal- ysis using a NOD.NON-H-2 congenic stock has shown that the increased percentage of T lymphocytes in NODis not MHC linked (M. Prochazka, D.V. Serreze, S. M. Worthen, and E. H. Leiter, The Jackson Laboratory, Bar nar~or, Ma~ne, unpublished data). In an outcross of NOD to C57BL/lOJ followed by a backcross to NOD, Wicker et al. (1987) found that the de- velopment of diabetes and insulitis is under partially overlapping but distinct genetic control, with the initiation of insulitis determined by a single gene 7 . . , ~.

HEREDITARY IMMUNODEFICIENCIES 101 unlinked to the MHC. However, the essential role of Idd-1 in diabetes patho- genesis is indicated by the finding that no diabetes and practically no insulitis could be observed in NOD.NON-H-2b congenic homozygous mice produced at the fifth backcross (M. Prochazka, D. V. Serreze, S. M. Worsen, and E. H. Leiter, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). A further indication of the overlapping control of insulitis by MHC-linked genes has been provided by a transgenic mouse study. NOD mice do not express I-E surface antigen. C57BL/6-Ead transgenic mice, which do express I-E surface antigen, were mated to NOD mice, and the F1 progeny that expressed I-E surface antigen were backcrossed with NOD to produce off- spring differing in I-A and I-E phenotypes (and segregating for other Idd loci). It was found that the expression of I-E surface antigen in backcrossed mice homozygous for the NOD I-A marker gene prevented the appearance of insulitis at 9 weeks of age (Nishimoto et al., 19871. To assess the effect of this transgene on diabetes development, it must be injected directly into NOD zygotes since outcrossing NOD with C57BL/6J strains produces very low diabetes incidence at BC1 (L. Herberg, Diabetes Institute, Dusseldorf, Federal Republic of Germany, personal communication to E. H. Letter, 19889. The introduction of single mutant genes associated with glucose in- tolerance syndromes in mice (db, oh, Ail on the NOD background produced transitional hyperplasia of islets followed by insulitis, B-cell atrophy, and severe diabetes characteristic of NOD (Nishimura and Miyamoto, 1987~. The genetic, pathologic, and therapeutic implications of diabetes in NOD mice have been reviewed (Tochino, 19871. Pathophysiology Clinical features of the diabetes syndrome in NOD mice are quite similar to human type I diabetes, including abrupt onset between 90 and 120 days of age (equivalent to early adolescence in humans), ketonuria, glycosuria, hyperglycemia, hypercholesterolemia, polydipsia, polyuria, and polyphagia. An autoimmune etiopathogenesis is indicated by the infiltration of T lym- phocytes into degenerating pancreatic islets (insulitis), which is detectable shortly after weaning (Miyazaki et al., 19851; production of spontaneous autoantibodies against islet antigens (Kanazawa et al., 19841; and lymphoid tissues unusually enriched for T lymphocytes. Heavy lymphoid cell aggre- gations are also found in the salivary (submandibular) gland in both sexes (Making et al., 19851. Females develop diabetes at an earlier age and with a higher frequency than males. Ovariectomy reduces and orchiectomy increases the incidence of diabetes (Making et al., 19811. The incidence of diabetes in females is approximately 80 percent in most colonies; the incidence in males varies between 10 and 70 percent. The wide variation in incidence among males

102 IMMUNODEFICIENT RODENTS is thought to be due to environmental factors, including environmental path- ogens, that can modulate disease expression. This has been confirmed by using NOD males from a colony in which the incidence of diabetes in males was below 10 percent. Producing these animals under germfree conditions caused the incidence to rise to 70 percent (Suzuki et al., 19871. While germfree conditions accelerate diabetes, a variety of systemic immunosti- mulatory treatments, including viral infection (Oldstone, 1988) and injection of bacterial cell wall preparations (Toyota et al., 1986), prevent diabetes. Apparently, NOD mice are capable of generating immunoregulatory protec- tive mechanisms against autoreactive cells when their immune systems are stimulated by antigen challenges in the environment. NOD-nu/nu mice do not develop diabetes unless they are reconstituted with splenocytes from euthymic littermates and concomitantly treated with IL-2, indicating a requirement for T lymphocytes to mediate pathogenesis (Making et al., 19861. Moreover, transferring splenocytes from older NOD mice into irradiated 7-week-old recipients accelerates the development of diabetes in the younger animals (Wicker et al., 19861. Using this syngeneic transfer paradigm, others have shown that both L3T4+ and Ly-2+ T-cell subsets are necessary to mediate subcell destruction (Bendelac et al., 1987; Miller et al., 19881. Accordingly, treatment of prediabetic NOD mice with a wide variety of immunosuppressive reagents that deplete T-cell subsets prevents the development of overt diabetes. For example, Shizuru et al. (1988) have shown that sustained treatment of NOD mice with monoclonal antibody directed against the L3T4 determinant of murine Th cells halts the progression of diabetes and, in some cases, leads to long-term reversal of the disease after therapy is discontinued. NOD bone marrow cells can transfer diabetes to irradiated diabetes- resistant F1 hybrids, showing that the polygenes controlling diabetes sus- ceptibility must be expressed in marrow-derived effecter cells (Serreze et al., 1988c; Wicker et al., 19881. NOD mice exhibit a spectrum of im- munoregulatory defects. Both monocyte and NK cell functions are im- paired (Kataoka et al., 1983; Serreze and Leiter, 1988), and T cells generated in a syngeneic MLR fail to induce suppression of an MLR (Serreze and Leiter, 19881. The defective ability of antigen-presenting cells to activate T-suppressor inducer cells has been associated with cytokine deficiencies, including both decreased endoxin-stimulated IL-1 production from NOD macrophages and decreased endogenous production of IL-2 from T lym- phocytes in syngeneic MLRs (Serreze and Leiter, 1988~. Functional T-suppressor inducer blast cells can be elicited in vitro by exposure to ConA, IL-1, or IL-2 (Serreze and Leiter, 19881. NOD mice treated in vivo with low doses of IL-2 not only exhibit a normal syngeneic MLR but also show markedly reduced diabetes incidence (Serreze et al., 1988a). NOD/L: mice that are free of the diabetic syndrome by 1 year of age are prone to develop follicle center cell lymphomas.

HEREDITARYIMAIUNODEFICIENCIES 103 Husbandry Insulin treatment is required to maintain diabetic mice; without insulin they survive only 1-2 months after diagnosis. Diabetes is diagnosed by determination of an elevated (nonfasting) blood or plasma glucose level. This determination can be made by measuring blood glucose directly or by screen- ing for glycosuria using Tes-Tape(~) (Eli Lilly & Cob. Glycosuria, as read by the Tes-Tape(~), usually denotes a plasma glucose of 300 mg/dl. Large numbers of mice can be easily screened by this method. Diet appears to be extremely important in the expression of diabetes. Feeding NOD mice Pregestimil(~ (a nonallergenic infant formula containing a casein hydrolysate profile of amino acids) does not support the same level of diabetes as does a semidefined diet containing a broad spectrum of in- gredients (Elliott et al., 19881. At the Jackson Laboratory, Bar Harbor, Maine, a high incidence of diabetes in both males (~50 percent) and females (~90 percent) is obtained by maintaining the colony on a diet of pelleted Old Guilford 96W, a complex formulation containing wheat germ, ground wheat, soybean meal, and brewer's yeast as well as milk proteins. Feeding the chemically defined diet AIN-76 to mice from the same colony delays the development of diabetes by 3-4 months. The addition of 25 percent (w/w) of Old Guilford 96 to AIN-76 will reestablish the expected onset of diabetes (D. L. Coleman, The Jackson Laboratory, Bar Harbor, Maine, personal communication to E. H. Leiter, 19881. The presence of immunosuppressive compounds in the complex grain-containing diets is suspected. Reproduction NOD mice are maintained by brother x sister mating. They have an excitable disposition but breed well, even though they have been inbred for more than 40 generations. Siblings bred before the development of overt diabetes can usually produce two large (9-14 pups) litters in which nearly all the pups survive to weaning. Breeders can be protected from developing diabetes by a single injection of complete Freund's adjuvent (B. Singh, Department of Immunology, University of Alberta, Edmonton, Alberta, Can- ada, unpublished data). NON (Non-Obese Normal) Genetics Non-obese nodal (NON) mice were separated from the NOD line at the sixth generation of inbreeding (Making et al., 19851. Paradoxically, NON progenitors were initially selected for a high fasting blood glucose level with the goal of producing a spontaneously diabetic model, while the progenitors

104 IMMUNODEFICIENT RODENTS of the NOD strain were initially selected for a normal fasting blood glucose level to provide a nondiabetic control. However, as described in the preceding section, spontaneous autoimmune diabetes developed in the NOD line, and NON mice, despite the strain name, are neither normal nor nonobese. Pathophysiology NON males exhibit impaired glucose tolerance after weaning (Tochino et al., 1983) and become obese (50 g) by 20 weeks of age (E. H. Letter, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). Although islet morphology and Q-cell granulation remain normal, the strain is characterized by the development of severe kidney lesions (Tochino et al., 19831. NON/L: mice also exhibit anomalies in their immune systems. T-lymphocyte number and function are normal in young NON/L: mice, but T lymphocytopenia and lymphopenia, coupled with declining T-cell mitogen responses, develop in these mice as they age (Letter et al., 19861. NON/L: mice are similar to NOD/L: mice in that they exhibit a defective syngeneic MLR, but unlike NOD/L: mice, which lack functional NK cells, NON/L: mice exhibit normal NK cell activity levels (D. V. Serreze and E. H. Leiter, The Jackson Lab- oratory, Bar Harbor, Maine, unpublished data). The unique features of the MHC of NON mice and another related strain, CTS, have been described recently (Ikegami et al., 1988~. Although NON/L: mice are distinguished from NOD/L: mice by the absence of insulitis, leukocytic infiltrates of the submandibular gland are a histopathologic characteristic shared by both strains. Serum autoantibodies against insulin and p73, an endogenous retroviral protein, have been detected by enzyme-linked immunosorbent assay (ELISA) in NON/L: mice, although titers are considerably lower than those in NOD/L: mice (Serreze et al., 1 988b). Husbanclry Special husbandry procedures are not required. Reprodluction These animals reproduce normally; however, litter sizes are smaller (5-8 pups) than those of NOD mice (9-14 pups). NZB (New Zealand Black) Genetics The NZB strain was produced by inbreeding from an outbred mouse colony and selecting for black coat color (Bielschowsky et al., 19561. The NZB

HEREDITARY IMMUNODEFICIENCIES 105 ~ ~ ~ I, OF ~ ' '''' ' NZB, NZW, and BWF~ hybrid mice. NZB mice (right center, black coat) develop hemolytic anemia; NZW (nght, white coat) and BWF~ hybrid mice (left and left center, agouti coats) develop a systemic lupus erythematosus-like disease. Photograph courtesy of Fred W. Quimby, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York. strain and the NZB x NZW Fat hybrid strain have been used as prototype strains for the study of spontaneous autoimmune disease. Studies with these strains were designed to elucidate the genetic basis of autoimmunity and have demonstrated complex polygenic inheritance (Theofilopoulos and Dixon, 19851. NZB mice develop autoimmune hemolytic anemia at an early age (Bielschowsky et al., 1959) and, in addition, have elevated levels of immunoglobulin, anti-DNA antibodies, anti-thymocyte antibodies, and circulating immune complexes (Quimby and Schwartz, 1982; Theofilo- poulos and Dixon, 1985~. Genes that contribute to these abnormalities in mice have been postulated: three genes (Aia-l, Aia-2, Aem-l ~ controlling anti- ery~rocyte antibody production, two genes (Nta-l, Ntm-l) controlling anti- thymocyte antibody production, four genes (Ass-l, Ass-2, Ass-3, Ass4 ~ controlling production of anti-ssDNA, four genes (Ads-l, Ads-2, Ads-3, Ads4) controlling production of anti-dsDNA, one gene (Imh-l) controlling elevated Igloo, and two genes (Agp-l, Agp-3) controlling levels of anti-gp70 circulating immune complexes. The proposed genotype of the NZB strain is Aia-l+, Aia-2+, Aem-l-, Nta-l+, Ntm-l+, Ass-l+, Ass-2+, Ass-3-, Ass4-, Ads-1+, Ads-2 +, Ads-3 -, Ads-4-, Imn-1 +, Agp-1+, Agp-3 -. Four of these genes, Ads-l, Ass-l, Agp-l, and Nta-l, are linked to the H-24 haplotype (MHC class I genes) on chromosome 17 (Shirai et al., 19841.

106 IMMUNODEFICIENT RODENTS Pathophysiology The most consistent abnormality seen in NZB mice is Coombs'-positive hemolytic anemia. Anti-erythrocyte antibodies appear as early as 3 months of age and reach an incidence of 100 percent by 12-15 months of age. Initially, these antibodies are of the immunoglobulin IgG class, in contrast to those observed later in life, which are of the IgM class. Hemolytic anemia usually develops 5 months following the appearance of autoantibodies and is not gender specific. Anemia is associated with retic- ulocytosis and reduced erythrocyte survival time. Splenomegaly is present as a result of erythrocyte sequestration, increased hematopoiesis, and lym- phoid hyperplasia. Both males and females have an average life span of 16.6 months (Eastcott et al., 19831. Andrews et al. (1978) reported a mortality of 90 percent by 23 months. Lymphoproliferative lesions resulting in hyperplasia of spleen, lymph nodes, bone marrow, thymus, lung, kidney, liver, and salivary glands are consistent features of the disease process in NZB mice. Two phases of lymphoproli- feration are seen. Between 3 and 11 months of age, the white pulp of the spleen and both cortical and medullary regions of the lymph nodes are char- acterized by enlarged lymphoid follicles containing multiple germinal centers. Later in life a second phase of lymphoproliferation occurs; this is charac- terized by extreme plasma cell hyperplasia in lymphoid tissue throughout the body. An increased incidence of lymphoma has been reported (East, 19701. The thymus is characterized by hyperplasia with follicular aggregates of lymphocytes and mast cells in the medulla. There is premature thymic in- volution in which degeneration and vacuolization of epithelial cells are con- sistent features (Andrews et al., 19781. An impressive early decline in thymulin levels has been reported (Bach et al., 1973~. NZB mice begin to produce anti-DNA antibodies by 2 months of age. High anti-ssDNA titers are observed in 54 percent of NZB mice by 9 months of age; however, only 10 percent have a significant titer of anti-dsDNA antibodies at this time (Andrews et al., 19781. Natural thymocytotoxic an- tibodies (NTAs) are made by all NZB mice by 7 months of age (Maruyama et al., 19801. This autoantibody is cytotoxic for thymocytes and T cells and reacts with brain tissue. NTAs are known to react with the Thy-1 complex as well as with a T-cell differentiation antigen expressed during the early stage of T-cell maturation (Surh et al., 19871. T cells with suppressor cell function appear to have the highest density of NTA-reactive antigen on their surface (Shirai et al., 1978), and NTA has been postulated in one laboratory as the cause of the T-cell abnormalities (Shirai et al., 1 9721. However, Taurog et al. (1981) demonstrated that the T-cell defects seen in NZB mice are also seen in NZB.CB/N-xid mice, which do not produce NTA, suggesting a primary T-cell defect in the NZB strain. A primary T-cell defect was also

HEREDITARY IMMUNODEFICIENCIE5 107 proposed by Laskin et al. (1986) based on cell mixing experiments and by Miller and Calkins (1988), who demonstrated an active role for T cells in promoting an in vitro autoantibody response against erythrocytes. A primary B-cell defect has also been clearly demonstrated in NZB mice. This hyperactivity of B cells is manifested by high levels of IgG immuno- globulins by 3 months of age (Andrews et al., 1978), increased number and augmented secretion of IgM by B cells (Manny et al., 1979), and the pro- duction of numerous autoantibodies (Quimby and Schwartz, 19821. Defective clonal inactivation of autoreactive B cells has been proposed to account for the increased autoantibodies seen in NZB mice (Cowdery et al., 1987~. However, Cantor et al. (1978) provided evidence that there was impaired feedback regulation of antibody synthesis because of an abnormally func- tioning Ly-123+ T-cell subset. At least one B-cell defect is known to reside in a Lyb-5 + subpopulation of B lymphocytes (Ly-1 + B cell) characterized by the normal allele of the xid mutant gene (A. D. Steinberg et al., 19824. This Ly-1 + B cell is increased in young NZB mice, is responsible for much of the autoantibody produced, and has unusual oncogene and receptor gene expression (A. D. Steinberg et al., 1987; Wolfsy and Chiang, 19871. NZB splenic B cells, which are rich in Ly-1 + B cells, are capable of transferring autoantibody production into NZB-xid mutant mice (Ishigatsubo et al., 1 987), which again suggests that a primary defect resides in a subpopulation of NZB B cells. However, others have demonstrated that autoantibodies and autoim- mune disease can occur independent of B-cell hyperactivity (putative Ly-1 + B cells) in NZB mice backcrossed to other strains (Datta et al., 1982; Eastcott et al., 19831. Furthermore, abnormal B cells are observed in athymic (nude) mice (Ohsugi and Gershwin, 19791. The B lymphocytes of NZB mice appear to be more resistant to induction of immunologic tolerance relative to those of other mice. It has been shown that NZB splenic B cells require a higher epitope density of the trinitrophenyl hapten on a protein carrier to induce unresponsiveness. The mechanism for this increased B-cell resistance to induction of unresponsiveness was independent of T cells, macrophages, or a Fc~y receptor defect (Goldings, 19881. The expression of infectious xenotropic retrovirus in NZB mice is con- trolled by two genes, Nzv-1 and Nzv-2 (Datta and Schwartz, 1976, 19771. In addition to high levels of gp70 envelope antigen in the blood, NZB mice also have high levels of anti-gp70 immune complexes in their serum (Andrews et al, 19789. However, the incidence of glomerulonephritis in NZB mice is low (Knight and Adams, 19781. Datta et al. (1978b) have shown that the genes controlling virus expression are independent of the development of autoantibodies or glomerulonephritis. Transplantation of bone marrow from autoimmune-resistant mice to NZB mice corrects all the abnormalities in immunity and prevents the development of autoimmune disease. Likewise, transplantation of bone marrow from NZB

108 IMMUNODEFICIENT RODENTS mice to young autoimmune-resistant mice results in both immunologic ab- normalities and autoimmune disease (Akizuki et al., 1978; Jyonouchi et al., 19811. Husbandry Spontaneous infection of NZB mice with certain murine viruses has been shown to modify the course of the autoimmune disease (Tonietti et al., 19701; therefore, this strain should be maintained in a pathogen-free environment. Reproduction This strain breeds normally NZB x NZW Fit Hybrids The NZW (New Zealand White) strain was produced by W. H. Hall in 1952 by inbreeding from an outbred mouse colony and selecting for white coat color (Talal, 19831. Although mice of this strain have relatively normal life spans, they do develop anti-DNA antibodies, high serum levels of re- troviral gp70 antigen, and nephritis later in life (Kelley and Winkelstein, 19801. NZB x NZW Fat hybrids (hereafter called BWF~) develop a systemic lupus erythematosus (SLE)-like syndrome. It has been proposed that the NZB parent contributes the dominant Ads-1 and Ads-2 genes controlling anti- dsDNA production, and the NZW parent contributes the dominant Ads-3 and Ads-4 genes, which are modifier genes. Similarly, the NZW parent contrib- utes two dominant genes, Ass-3 and Ass-4, which enhance the effect of Ass- l and Ass-2 in the production of anti-ssDNA antibodies. The Agp-3 gene of NZW intensifies the effect of the NZB Agp-1 gene controlling anti-gp70 circulating immune complexes. However, the NZW Aem-1 gene suppresses the activity of the NZB Aia-1 gene responsible for anti-erythrocyte antibody production. Finally, a dominant trait coding for lupus nephritis, Lpn-l, is modified by two additional genes, Lpn-2 and Lpn-3, both of which are donated by the NZW partner. The genes Ass-3, Ads-3, Agp-3, and Lpn-2 are linked to the H-2- haplotype of NZW (Shirai et al., 1984, 1987; Bearer et al., 19861. The net result is that BWF~ hybrids have an intensified pro- duction of anti-dsDNA antibodies, anti-ssDNA antibodies, and circulating immune complexes; an increased susceptibility to lupus nephritis; and a decreased production of anti-erythrocyte antibody when compared with NZB. Although evidence exists for each of the proposed loci listed above, there is still disagreement concerning the precise assignment of genetic loci to the

HEREDITARY IMMUNODEFICIENCIES 109 autoimmune phenotype (Kotzin and Palmer, 1987~. Recently Kotzin et al. (1985) demonstrated a large deletion (8.8-kilobase segment) in the DNA containing C,l31, DI32 and Jib cluster encoding the T-cell ~ chain; this has been confirmed recently (Theofilopoulos, 19861. The functional significance of this deletion in the BWF~ hybrid is unknown. Pa thop hys lo logy BWF~ mice develop a disease characterized by high levels of antibodies directed toward nucleic acid antigens, progressive immune complex glo- merulonephritis, and a marked enhancement of the disease in females. As early as 2 months of age, ANA can be detected in some BWF~ mice. By 12 months of age all BWF~ hybrids have detectable levels of ANA (Andrews et al., 1978; Quimby and Schwartz, 19821. The anti-dsDNA antibodies have nephritogenic properties and appear to be principally responsible for the immune complexes deposited in the glomerulus (Lambert and Dixon, 19681. Unique subsets of T and B lymphocytes are found in BWF~ mice that are responsible for the production of pathogenic (cationic) IgG anti-DNA (Datta et al., 19871. Hybridomas secreting anti-dsDNA antibodies derived from BWF~ mice injected with phosphocholine were isolated, and two DNA sequences asso- ciated with the rearranged immunoglobulin heavy- and light-chain genes were compared with the VH gerrnline sequences of normal BALB/c and C57BL/10 mice as well as the germline BWF~ gene. All 11 hybridomas binding dsDNA used the same VH11 germline gene and 9 used the same heavy-chain VDJ and light-chain VJ combinations. Five of these hybridomas were used for comparative purposes, and the nucleotide sequences encoding the heavy-chain variable regions differed from those of the germline by 6 to 16 base changes, indicating extensive somatic diversification. The authors speculated that the large number of base substitutions and the IgG2a subclass strongly suggest that T cells affected both the proliferation and differentiation of the B cells that produce these autoantibodies in vivo (Behar and Scharff, 1988). Independently, Eilat et al. (1988) compared the nucleotide sequences of rearranged heavy- and light-chain genes encoding spontaneous natural anti- DNA autoantibodies from different BWF~ mice. They found that the H chains of two anti-DNA antibodies had VH segments belonging to two different VH gene families, but both had similar D segments and J sequences. Furthermore, one of these IgG anti-DNA antibodies had a heavy-chain V region encoded by the VH11 germline nucleotide sequence. In fact, the VH nucleotide se- quence of the autoantibody differed from the germline VH11 sequence by four nucleotides, three of which occurred in the complementarily-determining regions (CDRs). These authors suggest that this relationship between a germ

110 IMMUNODEFICIENT RODENTS line gene and an expressed gene, with a concentration of nucleotide changes in the CDRs, is characteristic of an antigen-selected somatic mutation and that the actual autoantigen drives this process. Analysis of the rearranged genes encoding spontaneous anti-RNA anti- bodies derived from BWF~ mice demonstrated that there are closely related VH gene sequences between the two antibodies but no similarity to the VH segment of the anti-DNA antibodies (Eilat et al., 19881. Renal changes include the "lumpy-bumpy" deposits of IgG, antigen, and complement along the glomerular capillary basement membrane, as detected by immunofluorescence microscopy. This process begins between 3 and 6 months of age, causes significant proteinuria by 6-7 months of age, and is responsible for chronic renal insufficiency and death in females by 12-14 months of age and in males by 19 months of age (Andrews et al., 1978; Knight and Adams, 19781. In addition, anti-gp70 complexes can be dem- onstrated in damaged glomeruli (Dixon et al., 19691. The deposition of antigen-antibody complexes in medium and small ar- teries of BWF~ mice, especially in the heart, appears to lead to thrombotic and obliterative vascular changes and myocardial infarction (Accinni and Dixon, 19791. BWF~ hybrids have a generalized lymphoid hyperplasia involving the spleen and all lymph nodes and an increased incidence of lymphoma (East et al., 19671. Newborn hybrids have thymic medullary epithelial cell hy- perplasia, increased Hassall's corpuscles, and abundant lymphoid tissue; however, severe thymic cortical atrophy occurs early in life (Andrews et al., 19781. In young (1- to 4-month-old) BWF~ mice, the numbers and function of lymphocytes appear to be normal. In contrast to mice of other strains, the proliferative response of lymphocytes from young BWF~ hybrids to mitogens and allogeneic cells in vitro and their ability to induce tumor rejection appears to be enhanced (Evans et al., 1968; Gazdar et al., 19711. Likewise, young BWF~ mice make an augmented antibody response to nearly all antigens and are refractory to tolerance induction (Goldings et al., 19801. Older BWF~ hybrids show marked deficiencies in cell-mediated immunity (CMI). This abnormality is characterized by decreased lymphocyte prolif- eration to mitogenic stimuli, impaired graft-versus-host disease, impaired skin graft rejection, and a decreased cytotoxic response of spleen cells after alloimmunization (Howie and Simpson, 19761. These abnormalities might be due to the production of NTA (Shirai and Mellors, 1971) or abnormal differentiation of T-cell subpopulations due to lowered levels of thymic hu- moral factor (Bach et al., 19731. Old BWF~ mice have an expanded popu- lation of L3T4-/Ly-2- Thy-1 + cells that can induce pathogenic autoantibody (Datta et al., 19871. The augmented antibody response to antigens, increased levels of IgM

HEREDITARY IMMUNODEFICIENCIES 111 and IgG in blood, and production of many autoantibodies suggest hyperac- tivity of the B-cell system. This hyperactivity of B cells occurs late in fetal life and is present at birth (Jyonouchi and Kincade, 19841. Studies by Manny et al. (1979) suggest that these B cells have a reduced number of -chain surface receptors when compared with y-chain receptors and that the indi- vidual B cells are capable of synthesizing greater amounts of IgM antibody. Convincing support for a primary B-cell defect responsible for autoimmunity in BWF~ mice was provided by Gershwin et al. (1980), who observed all of the features of autoimmunity and autoimmune disease in nude BWF~ hybrid mice. The defect of BWF1 hybrid mice, like that of the NZB parent, probably resides in the bone marrow stem cell, because the immune disorders are preventable by early bone marrow transplantation (Ikehara et al., 19871. Bone marrow transplantation decreases the concentration of autoantibodies and circulating immune complexes, abrogates the development of morpho- logic abnormalities in the kidney and thymus, and restores cell-mediated immune function in old BWF~ mice (Ikehara et al., 19871. As a result, bone marrow transplants from autoimmune-resistant donors prevent the expression of autoimmune disease in BWF~ hybrids, which suggests that the etiopath- ogenesis of autoimmune disease lies in a primary stem cell defect. A defective production of the Boone IL-2 has also been described in BWF~ hybrid mice (Altman et al., 1981), and elevated levels of type II interferon (~-interferon) have been implicated in the pathogenesis of the disease in BWF~ mice (Engleman et al., 19811. The greater incidence and earlier onset of autoimmune disease in female BWF~ hybrids suggest a role for sex hormones in the pathogenesis of murine lupus erythematosus. Indeed, when male BWF~ mice are castrated, they develop the female pattern of the disease. Likewise, androgen administration greatly prolongs the life of female BWF~ hybrids (Raveche et al., 19791. A variety of therapeutic measures has proven to be successful in dimin- ishing the severity of BWF~ lupus. Among these are immunosuppressive drugs (Jones and Harris, 1985; Dueymes et al., 1986), protein and caloric restriction (Fernandes et al., 1976; Johnson et al., 1986), a ConA-induced soluble suppressor (Krakauer et al., 1976), antibodies against y-interferon (Jacob et al., 1987), prostaglandin E~ (Zurier, 1982), ribavirin (Klassen et al., 1977), and an eicosapentenoic acid (fish oil)-supplemented diet (Prickett et al., 1981~. Husbandry While certain infectious agents have been shown to induce autoantibodies and immune complex disease in norma-l mice (Schulman et al., 1964; Barnes and Tuffrey, 1967; Dixon et al., 1969), other infectious agents have been

112 IMMUNODEFICIENT RODENTS shown to inhibit autoantibodies and ameliorate disease in NZB and BWF~ hybrid mice (Oldstone and Dixon, 19721. Engleman et al. ~ 1981 ~ proposed that virus-induced type 1 interferon is responsible for the accelerated au- toimmune disease seen in some BWF~ hybrids. Therefore, it appears prudent to maintain this hybrid strain in a pathogen-free environment. Rabin (1985) reported a significant difference in the survival of BWF~ hybrids associated with the cage type. Mice held in wire mesh cages lived considerably longer that F1 hybrids housed in solid-bottom cages. Reproduction Both parental strains breed normally; therefore, production of F1 hybrids from these parental strains requires no special practices. NZB x SWR Fit or SWR x NZB F. Hybrids Genetics Hybrid mice derived from the mating of mice of the autoimmune-prone NZB and normal SWR strains (hereafter called SNIP have been shown to develop severe early-onset glomerulonephritis and circulating anti-DNA an- tibodies (Datta et al., 1978a,b). Female SNIP mice produce nephritogenic autoantibodies encoded by the SWR parent. Immunoregulatory defects in- clude two subpopulations of helper T cells capable of inducing nephritogenic anti-DNA antibodies from SNF~, but not SWR, B cells. Genes found on chromosomes 6 and 17 of the normal SWR parent interact with NZB-derived genes, leading to the development of accelerated and severe nephritis in the SNF~ mouse (Ghatak et al., 19871. Pathophysiology The NZB parent of the SNIP hybrid spontaneously develops autoimmune hemolytic anemia, expresses high levels of retroviruses and retroviral gp70 antigen, and infrequently develops glomerulonephritis (see page 1044. The SWR parent, by contrast, does not express retroviruses, has very low serum gp70 levels, and does not develop autoimmune disease or circulating auto- antibodies (Datta et al., 1978a,b; Eastcott et al., 19831. Female SNIP mice develop high serum levels of anti-DNA antibodies and lethal glomerulone- phritis by 1 year of age (Datta et al., 1978a; Eastcott et al., 19831. Since severe glomerulonephritis can occur in SNIP females in the absence of glo- merular deposits of gp70, much attention has focused on the nature of cir- culating and kidney-bound anti-DNA antibodies in this strain. Analysis of monoclonal anti-DNA antibodies derived from SNF~ and NZB

HEREDITARY IMMUNODEFICIENCIE5 113 mice demonstrated that most SNF~-derived anti-DNA antibodies were of the IgG class and were cationic, compared with NZB-derived anti-DNA anti- bodies, which were mostly of the IgM class and were anionic (Gavalchin et al., 19851. Among SNF~-derived anti-DNA antibodies are a set of highly cationic, IgG2b anti-DNA antibodies containing the SWR allotype and heavy- chain V regions responsible for the charge characteristics (Gavalchin et al., 19851. Since cationic IgG anti-DNA antibodies are selectively deposited in the glomerular lesions of mice with lupus nephritis (Dang and Harbeck, 1984), it was proposed that these anti-DNA antibodies were responsible for glomerulonephritis in SNIP mice. Gavalchin et al. (1987) developed a library of 15 anti-idiotypic antibodies prepared by immunizing rabbits with 15 monoclonal anti-DNA antibodies derived from NZB or SNIP mice. Using these anti-idiotypic antibodies, they identified 10 cross-reactive idiotype (CRI) families among the 65 monoclonal anti-DNA antibodies. Five CRI families were restructured to cationic anti- DNA antibodies exclusively of SNIP origin and encoded by genes derived from the SWR parent (determined by allotype). These cationic anti-DNA CRI families were grouped into an interrelated cluster, IdS64, which was prominently represented in the kidneys of SNIP mice with early nephritis (Gavalchin and Datta, 19871. Additional anti-DNA antibodies deposited in the kidneys of SNF~ mice were identified as a second interrelated cluster, Id512, in the CRI family. These autoantibodies were not restricted to a particular charge or allotype. Both IdS64 and IdS 12 antibodies could be found in the serum of old SNIP mice but not in the serum of either parental strain, suggesting that these nephritogenic idiotypes were dormant in the NZB and SWR parents and became deregulated and expanded in SNIP hybrids (Gav- alchin and Datta, 19871. Studies directed at elucidating an immunoregulatory defect present in SNIP mice showed that Th cells were essential for inducing B cells to produce highly cationic, IgG anti-DNA antibodies in vitro (Datta et al., 19871. These Th cells appear in the spleens of SNIP mice just before they begin to develop lupus nephritis and comprise two immunophenotypes, that is, L3T4 +, Lyt-2-, Thy-1 +, Ig- (CD4+/CD8-) and L3T4-, Lyt-1 +, Lyt-2-, Thy-1 +, Ig - (CD4 - /CD - ). In addition, B cells capable of secreting the highly cationic anti-DNA antibodies are present only in older animals and are an expand- ed clone (Datta et al., 19871. IL-2-dependent CD4+/CD8- and CD4-/CD8- T-cell lines were derived from nephritic SNF~ mice. While some cell lines of each T-cell phenotype can induce nephritogenic anti-DNA antibodies from SNF~ B cells, the CD4 + T-cell lines are highly autoreactive in culture. These CD4+ cell lines could not induce cationic IgG anti-DNA antibodies from B cells derived from either the SWR or the NZB parents, suggesting that the parental strains may be deficient in select B cells committed to the production of nephritogenic anti-DNA antibodies (Saints and Datta, 19881.

114 IMMUNODEFICIENT RODENTS PN (Palmerston North) Genetics Inbred PN mice trace their ancestry to albino mice purchased from a pet store in New Zealand in 1948. From Palmerston North Hospital, Palmerston North, New Zealand, these "TOO" outbred mice were sent to Massey Uni- versity, then to Glaxo New Zealand Ltd., and eventually were returned to Palmerston North Hospital, where they were renamed PN. In 1964, under the direction of R. D. Wigley, inbreeding was begun. Selection for ANA positivity was carried out during the first three generations. The primary breeding line was named PN/n A. A second breeding line, PN/n B. was established after nine generations of inbreeding of PN/n A. PN/n A (at F28) and PN/n B (at F23) were imported into the United States in 1974 by S. E. Walker (subline code Sw). The PN/n A strain was arbitrarily discarded in 1975 (Walker et al., 19781. The PN strain carries the MHC haplotype H-2q. These mice are C-4 high, Sip negative, and G7 (C4d) negative (Schultz et al., 19821. In preliminary studies of inheritance of autoimmunity, PN/Sw mice crossed with the non- autoimmune DBA/2J strain produced offspring that did not develop prolif- erative glomerulonephritis or vasculitis. It was concluded that the autoimmune disease in PN mice has a recessive mode of inheritance. Pathophysiology PN mice are considered to be a model for SLE. The mean life span of female PN/Sw mice is 1 1 .6 months. Males survive for an additional 4 months. Walker et al. (1978) determined that the most common causes of death are renal disease and vasculitis. Glomerulonephritis was present in 74 percent of autopsied mice. Mesangial thickening, hypercellularity, fibrinoid necrosis, and crescent formation were found. Perivascular infiltrates of lymphoid cells were found in nearly all kidneys of autopsied PN mice. Arteritis, especially prominent in lymphoid organs and spleen, was found in the majority of autopsied mice. A 13 percent incidence of lymphoma was described. In mice studied histologically from 1 to 21 months of age, hyperplastic lymph nodes were a common finding. Thymuses of mice 1 to 5 months of age were also hyperplastic. PN/Sw x NZB/Sw F~ or NZB/Sw x PN/Sw F~ hybrid females develop anti-DNA antibodies and die prematurely (mean age, 43 weeks) with vas- culitis, renal disease, and lymphomas. In contrast, hybrid males have di- vergent patterns of mortality. PN/Sw x NZB/Sw F~ males have a mean longevity of 67 weeks; NZB/Sw x PN/Sw F~ males have a mean longevity of 104 weeks (Walker, et al., 19861.

HEREDITARY IMMUNODEFICIENCIES 115 Unique among animal models of SLE is the finding of ANA or anti-DNA antibodies in some newborn PN/Sw mice. Anti-DNA antibodies are detected in serum of 51 percent of mice between 2 weeks and 2 months of age. At 1 year of age, all female and 78 percent of male PN/Sw mice have anti- DNA antibodies. Glomerular deposits of IgG 1, IgM, IgA, and C3 are detected (Walker et al., 19781. All strains of autoimmune mice synthesize antihistone antibodies. In PN mice, the antihistone antibodies recognize preferentially HI and H2B; this pattern of antihistone antibody reaction is seen in human SLE (Costa and Monier, 19861. One interesting finding that differentiates the PN strain from other murine models of SLE is that their spleen and thymus cells do not express high levels of MuLV-related gp70 and cannot be induced to produce infectious ecotropic or xenotropic MuLVs (Davidson, 19821. Walker and Hewett (1984) have described modest reductions in respon- siveness of spleen cells to PHA and ConA in female, but not in male, PN/Sw mice after 24 weeks of age. Davidson (1982) has shown that 4- to 6-month-old PN mice show a marked increase in levels of serum IgG1, IgG2b, and IgA. PN mice also have an increase in spontaneous dinitrophenol (DNP)-specific PFCs but a decrease in PFC response to SRBCs. Even before severe disease symptoms are exhibited, the spleens of 2- to 3-month-old PN mice have a reduced frequency of detectable sIg-positive cells. Studies of primary splenic PFC responses to thymus-dependent and thy- mus-independent antigens have shown that PN/Sw mice have defective in vivo IgG responses to thymus-dependent antigens. Defective production of IgG-specific plaques is evident as early as 3 weeks of age, is not influenced by aging to 43 weeks, and is not corrected by increasing the antigenic challenge 10-fold. Analyses of IgG subclasses show that there is a shift away from the expected predominance of IgG1-specific PFC (Walker, 19881. Husbandry Special husbandry procedures are not required. Reproduction These mice breed normally. SAM-P (Senescence Accelerated Mouse) Genetics Inbred SAM strains were established from several pairs of AKR/J mice by continuous selection for short-lived litters. Two strains, SAM-P/1 and

1 16 IMMUNODEFICIENT RODENTS SAM-P/2, were selected as being prone to early senescence. The SAM-P strains differ from AKR/J mice by two biochemical and two lymphocytic markers; however, both SAM-P and AKRIJ are H-2Kk, Ik, Dk, suggesting that there was either an accidental crossing of AKR parents with other strains or that genetic polymorphisms were introduced into the SAM-P strains (Tak- eda et al., 1 98 1 ). Pathophysiology SAM-P/1 and SAM-P/2 strains have a mean life span of 9 months when they are raised under conventional conditions (Hosokawa et al., 1987a). Postnatally they grow normally until about 6 months of age, at which time they develop a severe loss of physical activity, alopecia, coarse skin, per- iophthalmic lesions, lordokyphosis of the spine, cataracts, and osteoporosis. In one strain, SAM-P/1, spontaneous age-associated amyloidosis is charac- teristic, and a unique amyloid fibril protein has been isolated from the livers of SAM-P/1 mice (Higuchi et al., 19861. Both SAM-P/1 and SAM-P/2 strains demonstrate markedly diminished antibody-for~ning capacity to T-cell-independent (DNP-Ficoll) and T-cell- dependent (SRBC) antigens compared with control AKR/J and C3H/He mice (Hosokawa et al., 1987a). In addition, NK cell activity also shows an early onset (2 months of age) of regression and a sharp decline from the level in control mice. In contrast, allospecific cytotoxic T-lymphocyte (CTL) re- sponses from SAM-P strains remain at normal (control) levels until 6 months of age. The cellular site of the defect in antibody response to T-cell-dependent (TD) antigen was investigated with a cell culture system. The Th-cell activity for the antibody response to TD antigen was impaired, but no evidence could be found for suppressor factors, abno~al adherent cells, or B cells (in the TD antigen assay). In contrast, the MLR and delayed-type hypersensitivity reactions were normal (Hosokawa et al., 1987b). These results suggest that two subsets of Th cells exist in SAM-P mice, one providing defective help for B-cell differentiation and the other providing normal activity for cell- mediated responses. It is still unclear whether the defect in T-cell function of SAM-P mice is controlled by a genetic defect expressed in the immune system or whether immunity is influenced by a genetic alteration in the ;n~croenv~ronment. Husbandry SAM-P strains develop clinical illness at 6 months of age and die by 9 months when they are maintained in a conventional facility. No attempts at barrier maintenance of SPF SAM-P mice have been reported.

HEREDITARY IMMUNODEFICIENCIES 1 17 Reproduction No special breeding practices have been reported. Sale/] Genetics The SJL/J strain was derived from noninbred Swiss Webster mice. Murphy (1963) described the appearance of lymphomas resembling those of Hodg- kin's disease in this strain. Histologically, these lymphomas were classified as type B reticulum-cell sarcomas (RCSs) (Dunn, 1954; reviewed in Ponzio et al., 19869. The genetic basis for the development of RCSs in SJL/J mice is unknown. A single autosomal dominant gene, Rcs-l, that suppresses the appearance of spontaneous RCSs in SJL/J mice has been described in the A.SW strain, and Rcs-1 has been shown to be distinct from H-2 and from the genes affecting MuLV, that is, Fv-4, Cv, and Fgv-1 (Bubbers, 19841. Neither the major histocompatibility locus, which is H-2s in SJL/J mice, nor the Igh- 1 locus, which is Igh-l b in SJL/J mice, appears to affect the incidence of RCS (Bubbers, 1983; E. B. Jacobson, Merck Institute, Rahway, New Jersey, unpublished data). A study comparing the congenic strains SJL/J and SJL/J-lpr/ + has shown that the appearance of RCS is greatly accelerated in SJL/J-lpr/+ mice (Morse et al., 19851. While SJLlJ-bglbg mice have an incidence of RCS similar to that seen in the SJL/J strain, the appearance of these tumors is slightly accelerated in the mutant (J. B. Roths, The Jackson Laboratory, Bar Harbor, Maine, unpublished data). SJL/J mice produce low levels of IgE because of a strong tendency to develop IgE-specif~c suppressor cells (Watanabe et al., 1976; Itaya and Ovary, 19791. This suppression is controlled by a single autosomal recessive gene that is not linked to either the H-2 or the Igh locus. Two genes have been described in SJL/J mice that mediate resistance to the demyelinating effects of the JHM strain of MHV (Stohlman et al., 1980~. One of these genes is dominant (Rhv-l ); the other is recessive (rhv-2~. Neither of these is linked to the H-2 or Igh locus (Stohlman and Frelinger, 19781. In contrast, SJL/J mice are much more susceptible than are mice of other inbred strains to demyelinating disease induced by Theiler's murine enceph- alomyelitis virus (Lipton and Dal Canto, 19791. The inheritance of this susceptibility is recessive and is dependent on the H-2D locus and one or more unlinked loci that contribute to susceptibility in a manner consistent with a gene dosage model (Lipton and Melvold, 1984; Clatch et al., 1985; Rodriguez et al., 19861. SJL mice are highly susceptible to the induction of acute experimental allergic encephalomyelitis (EAE) by immunization with allogeneic spinal

118 IMMUNODEFICIENT RODENTS cord homogenate in adjuvant, and they later develop a relapsing form of EAE (Brown and McFarlin, 19811. Susceptibility to EAE is inherited as a dominant trait. One gene controlling sensitivity has been mapped to the H-2K region (Bernard, 19761. Other important genes regulating sensitivity to EAE are the recessive gene for vasoactive amine hypersensitivity and the dominant gene governing induction of histamine sensitivity by Bordetella pertussis (Linthicum and Frelinger, 19821. Both are expressed by SJL/J mice. It has recently been discovered that in the SJL/J strain (Behlke et al., 1986), as well as in some known autoimmune strains (Singer et al., 1986), there is a deletion of a large portion of the V,3T-cell receptor genes, resulting in a skewed Via gene repertoire. In addition, a subpopulation of T cells from SJL mice (approximately 10 percent of peripheral T cells) expresses the recently described VT-cell receptor allele V~317a, which is associated with responsiveness to I-E (class II MHC) surface antigens (Kappler et al., 19871. SJL/J mice have very low levels of circulating Al immunoglobulin compared with other strains. This appears to be due to a transcriptional defect affecting the level of Al synthesis, rather than to the numbers of B cells capable of A 1 production (Sanchez et al., 1 9851. Pathophysiology At the age of 12.5-13.5 months, the incidence of RCS is approximately 90 percent in males and females, whether they are virgins or retired breeders (Murphy, 19791. Primary RCSs are thought to arise in germinal centers of Peyer's patches and mesenteric lymph nodes (Siegler and Rich, 1968) and to spread to other lymph nodes, spleen, liver, and ovaries (Haran-Ghera et al., 19731. The primary lesions contain mainly lymphocytes, as well as histiocytes, eosinophils, plasma cells, and Reed-Sternberg-like cells (Mc- Intire and Law, 1967; Murphy, 1969; Kumar, 19831. RCSs are most likely of B-cell origin, because intravenously injected RCS cells exhibit typical B- cell homing patterns (Pattengale and Taylor, 1983), many of the cells found within primary lesions contain cytoplasmic immunoglobulin (Taylor, 1976), the surface properties of RCS cells are consistent with a B-cell derivation (Beisel and Lerman, 1 98 1; Kincade et al., 1 98 1; Scheid et al., 1 98 1 ), and primary RCSs fail to arise in mice treated with goat antimouse -chain antiserum from birth (Katz et al., 19801. The appearance of spontaneous RCSs is often accompanied by abnor- malities in serum immunoglobulins. Paraproteins of the IgG1, IgG2a, and IgG2b isotypes are found in sera from SJL/J mice greater than 6 months of age (Wanebo et al., 19661. Although the appearance of these paraproteins is temporally correlated with the appearance of primary RCSs in SJL/J mice, it is unclear whether the paraproteins are a product of RCSs or are formed by the host in response to RCSs. It is possible that B-cell hyperstimulation

HEREDITARY IMMUNODEFICIENCIES 119 in SJL/J mice is an important factor in the pathogenesis of both phenomena. They could also be two independent characteristics of these mice. Also related may be the high incidence of spontaneous amyloidosis in SJL mice (Schein- berg et al., 1976~. The role of ecotropic MuLV in the etiology of spontaneous RCSs in SJL/J mice has not been clearly defined (DeRossi et al., 19831. Spontaneous RCS development is a thymus-dependent process (Katz et al., 19811. This is of interest in light of the unusual thymic abnormalities in aging SJL/J mice. SJL/J mice have a secondary increase in thymic weight after 7 months of age that appears to be caused by an influx of surface immunoglobulin-positive B cells (Ben Yaakov and Haran-Ghera, 1975) that peak in SJL/J mice between 6 and 12 months of age prior to RCS devel- opment. Although the tendency toward B-cell proliferation in SJL/J mice might contribute to this penetration of B cells into the thymus, there is also a contribution of the thymic environment through a leaky blood-thymus barrier (Claesson et al., 1978~. Although SJL/J mice are sensitive to tolerance induction at birth, by 2 months of age, unlike other strains, they become quite resistant to tolerance induction by antigens such as aggregate-free rabbit gamma-globulin, human gamma-globulin, and bovine serum albumin (Fujiwara and Cinader, 1974a; Owens and Bonavida, 19761. The presence of endogenously activated mac- rophages (Crowle and May, 1978) seems to play an important role in this resistance to tolerance induction (Fujiwara and Cinader, 1974b). There is an age-dependent decline in thymic and peripheral suppressor T cells (Nakano and Cinader 1980; D. A. Clark et al., 1981) that also appears to contribute to the tolerance resistance and to a tendency to develop autoantibodies (Vla- dutiu and Rose, 1971; Bentwich et al., 1972; Bernard, 1976; Owens and Bonavida, 1976; Cooke and Hutchings, 19841. SJL/J mice are able to produce a Th-cell factor that acts on B cells in a T-cell-independent manner to promote immunoglobulin secretion earlier in life than in the A/J or C57BL/6J strains (Matsuzawa and Cinader, 1982~. Production of this factor reaches a maximum by 10-12 weeks of age (as opposed to 20-30 weeks for the A/J and C57 BL/6J strains). Although the early onset of Th-cell factor production is correlated with the age-dependent decline in the generation of suppressor cells, attempts to establish a causal relationship between the two phenomena have failed (Amagai et al., 19821. Ten- to twelve-month-old SJL/J mice (both normal and obviously tumor nearing) exhibit abnormalities in their abilities to mount graft-versus-host responses, to develop delayed-type hypersensitivity reactions, and to reject skin allografts (Haran-Ghera et al., 19731. Normal SJL/J mice have low levels of endogenous NK cell activity and are extremely resistant to NK cell induction by interferon (IFN), poly(I-C), and Corynebacterium parvam (Fitz- gerald and Ponzio, 1981; Kaminsky et al., 19831. SJLlJ-nu/nu mice have elevated levels of endogenous NK cell activity compared with those in

120 IMMUNODEFICIENT RODENTS SJLlJ-nul + or SJL/J + / + mice, and their NK cells are responsive to IFN (Kaminsky et al., 1985J. It has been shown that resistance to central nervous system challenge with the JHM strain of MHV is a function of adherent cells, indicating that endogenously activated macrophages might be an important aspect of the resistance of SJL/J mice to the induction of demyelinating disease by the JHM strain of MHV (Stohlman et al., 19801. Pathophysiologic aspects of the high sensitivity of the SJL/J mouse and some of its F1 hybrids to autoimmune diseases such as EAE include H-2s- linked recognition by T cells of the autoantigenfs) in question. A second important factor is the tendency of mice such as SJL/J, which have a high vasoactive amine sensitivity and are suseptible to the Bordetella pertussis- induced increased histamine sensitivity, to develop leakiness of their blood- organ barriers (Teuscher, 19851. In the case of EAE, enhanced vascular permeability in brain tissue has been observed after B. pertussis administra- tion to SJL/J x BALB/c Fit hybrids, and the induction of EAE might be inhibited by vasoactive amine antagonists (Linthicum et al., 19821. Thus, SJL/J mice might be a good model for autoimmune disease, based on the strain's high lymphokine production and macrophage activity, decreased suppressor cell activity, high vasoactive amine sensitivity, and a skewed Via T-cell receptor gene repertoire. Husband~ry SJL/J and some of its F1 hybrid males become extremely aggressive after 8 weeks of age. Fighting is severe enough to require that older males be caged singly (Crispers, 19731. Alternatively, the nasal installation of zinc chloride solution is known to disrupt olfactory sensitivity to pheromones and has proven effective in preventing fighting among mice (Alberta, 19741. No other special procedures are necessary. Reprod~uction SJL/J mice breed well and have relatively large litters (average of 7.9 pups per litter) (Crispers, 19791; however, only about half survive to weaning. SL`/Ni Genetics The SL/Ni strain, which should not be confused with the Sl (Steel) mu- tation, was developed in 1970 by Y. Nishizuka (Aichi Cancer Center Re- search Institute, Nagoya, Japan) from the lymphoma-prone SL strain (Nishizuka

HEREDITARY IMMUNODEFICIENCIES 121 et al., 19751. The SL/Ni strain exhibits a decreased incidence of lymphoma. Two related substrains are also available. One retains the high incidence of nonthymic lymphoma; the other possesses a dominant epistatic gene, Slvr-l, that selectively restricts the expression of endogenous ecotropic virus (Hiai et al., 19821. Pa thophys to logy SL/Ni mice show a high frequency of spontaneous arteritis and glomer- ulonephritis (Kyogoku, 1977, 1980; Nose et al., 198 11. These lesions have histological features similar to those seen in other murine models of SLE, such as the NZB x NZW Fit hybrid and the MRL/Mp inbred strain. The vascular lesions (segmental fibrinoid arteritis) histologically resemble those of human polyarteritis nodosa (Kyogoku et al., 1981~. The most frequently affected vessels are medium- to small-sized arteries of the ovary, uterus, parotids, kidney, spleen, and pancreas (Miyazawa et al., 19871. Approxi- mately 30-50 percent of SL/Ni mice manifest acute and chronic arteritis beginning at 9 months of age. The incidence is higher in females than in males, and nearly all multiparous females develop arteritis (Nishizuka, 19799. Most SL/Ni mice develop renal glomerular disease starting at 5-6 months of age (Yoshiki et al., 19791. Granular deposition of gp70 immune complexes is found in vessel walls and along glomerular capillary loops and mesangium (Yoshiki et al., 1979; Miyazawa et al., 19871. The renal disease has a progressive and protracted clinical course and affects females earlier than males. C-type MuLV particles have been found in abundance around the smooth muscle cells of the vessels, and budding from the membranes of these cells has been observed (Nose et al., 1981; Miyazawa et al., 19871. A decrease in suppressor T-cell activity (Matsumoto, 1979), a reduced in vivo anti-SRBC PFC response (Matsumoto, 1979), and production of anti-gp70 antibodies (Miyazawa et al., 1987) are associated with the onset of disease. SL/Ni mice also produce anti-ssDNA autoantibodies resulting in large concentrations of circulating immune com- plexes (Kyogoku, 19801. Husband~ry SL/Ni mice are extremely susceptible to Sendai virus infection. Breeding and maintenance in isolators or under SPF conditions is recommended. ReprodFuction SL/Ni mice are perpetuated by brother x sister mating. Females have a tendency to become infertile, because their vascular inflammatory lesions

122 IMMUNODEFICIENT RODENTS affect the uterine and ovarian arteries; therefore, they are bred as soon as they reach sexual maturity (M. Kyogoku, Department of Pathology, Tohoku University School of Medicine, Sendai, Japan, unpublished data). OUTBRED MICE SWAN (Swiss Antinuclear) Genetics The SWAN mouse stock was derived from a pair of Gif:S (Swiss) mice that were positive for ANA. Following an initial three generations of in- breeding, the SWAN stock was maintained by random matings as a closed colony (Monier and Robert, 19741. Pa thophys to logy The hallmark of SWAN mice is the development of ANA. Monier et al. (1971) determined that 100 percent of SWAN mice tested after 8 months of age were positive for ANA. Females are more precocious in the development of ANA than are males. Both neonatal thymectomy and treatment with Freund's adjuvant accelerate the age of onset of ANA positivity. SWAN mice are reported to have a decreased lymphoproliferative response to PHA, and the peak primary antibody response to SRBCs is delayed from the time of immunization and is reduced in magnitude (Monier and Robert, 19741. The studies of Blaineau et al. (1978) indicate that the autoimmune pathology of SWAN mice occurs in the absence of xenotropic-type C-virus production. SWAN mice, like NZB and BWF~ hybrid mice, are reported to have a premature decline in secretion of circulating facteur thymique serique (FTS) (Bach et al., 19801. SWAN mice have histopathologic features similar to those of human SLE. By 8 months of age, all SWAN mice have glomerulonephritis with immu- noglobulin deposits in their glomeruli. Perivascular lymphoid infiltrates, mes- angial thickening, hyalinization, thickened basement membrane, and dilation of the renal tubules are commonly observed. Amyloidosis is also frequently found (Monier et al., 19711. Histopathologic abnormalities at the dermo- epidermal junction are also seen (Monier and Sepetjian, 19751. The life span of these mice is unknown. Husbandry Special husbandry procedures are not required.

HEREDITARY IMMUNODEFICIENCIES 123 Reproduction SWAN mice are maintained by random breeding. RAT MUTANTS ia (Incisor Absent) Genetics Incisor absent Vial is one of three autosomal recessive, osteopetrotic mu- tations in the rat (see also op. page 124, and tl, page 128~. Although the chromosomal locations of these three mutations are not known, it has been shown that they are not allelic (Mousier et al., 1976~. The mutation ia arose spontaneously in a stock of unknown genetic makeup (Greep, 1941~. Older literature used in (incisorless) as the gene symbol (Castle and King, 19441. Pathophysiology Osteopetrotic rats are smaller than their normal littermates. Teeth fail to erupt and incisors are absent. Although the teeth do not erupt, they continue to grow. Consequently, large odontomatous masses develop in the jaws, frequently causing facial distortion (Schour et al., 1949; Marks, 1976a). Unlike the mouse, osteopetrotic mutations in the rat are not associated with pigmentation defects. In all three mutants, skeletal growth is abnormal; long bones have no flared ends or marrow cavities. In ialia rats, bone resorption is reduced by about 30 percent compared to normal littermates (Marks, 19731. Homozygotes can be identified on the day after birth by radiographic examination, which reveals a clawlike appearance of the distal humerus and generalized radiopacity of the long bones (Marks, 19781. Marrow spaces begin to develop during the second postnatal week (Marks, 1976a) and approach normal proportions during the third month. However, the shape of the bones remains abnormal, and metaphyseal bone continues to be markedly sclerotic throughout life (Marks, 1976a, 19811. The skeletal defect in ialia rats appears to be cellular, not humoral (Nyberg and Marks, 19754. Osteoclasts in ia homozygotes are more numerous than in their normal littermates (C. R. Marks et al., 1 984; Miller and Marks, 1 982), have elevated levels of acid phosphatase (Marks, 1973; Ek-Rylander et al., 1989), and lack cytoplasmic vacuoles and ruffled borders (Marks, 19731. Osteoclasts can produce enzymes for resorption but cannot deliver them efficiently to the bone surfaces because of the lack of ruffled borders. Serum calcium levels in ia homozygotes are normal; however, there is a poor response to the administration of exogenous parathyroid hormone (Marks,

124 IMMUNODEFICIENT RODENTS 1973, 1977). Phosphate levels are reduced, at least up to 40 days postpartum (Kenny et al., 19581. Serum levels of 1,25 dihydroxyvitamin D are elevated, and levels of the 24,25 dihydroxy metabolite are reduced (Zerwekh et al., 1987). The defect in iamb rats is curable by transplantation of bone marrow, thymus, or spleen cells from normal animals (Milhaud et al., 1975; Marks, 1976b, 1978; Marks and Schneider, 1978; Miller and Marks, 19821. Skeletal sclerosis can be induced by the transplantation of mutant spleen cells into immunosuppressed normal littermates (Marks, 1976b). The cell-mediated response of ia homozygotes to oxazolone stimulation is normal for both the skin and lymph node blastogenesis assays (Schneider, 1978~. Husbandry The ia mutation is not lethal; however, soft diets are essential to compensate for the lack of incisors. The husbandry of this mutant has been reviewed (Marks, 19871. Homozygotes have a normal life span with delayed sexual maturity. Reproduction There is no evidence that the ia mutation impairs reproductivity; however, to ensure larger, better-cared-for litters, breeding heterozygous females with homozygous males is suggested. Op (Osteopetrosis) Genetics Osteopetrosis (op) is an autosomal recessive mutation that arose sponta- neously in a random-bred stock carrying the mutation fatty (fa) (Mousier et al., 19731. It is not allelic to ia (page 123) and It (page 128), the two other genes that confer osteopetrosis in the rat. The op gene is epistatic to ia (Mousier et al., 19741. Pathophysiollogy The op mutation is lethal; maximum life span has been reported to be 116 days (Milhaud et al., 19771. Skeletal manifestations are similar to those in ia homozygotes, except that there is no delayed development of marrow spaces. Bones contain numerous, large extracellular lipoid inclusions. There are reduced numbers of osteoclasts (Marks and Popoff, in press), and those present exhibit pleomorphic shapes and have reduced levels of tartrate

HEREDITARY IMMUNODEFICIENCIES 125 resistant ATPase (Ek-Rylander et al., 1989). Therefore resorption is reduced by a deficiency of both cells and enzymes. Bone resorption is not stimulated by transplantation of compatible normal thymic grafts, indicating that the defect lies in myeloid rather than in thymic regulation of osteoclast function (Nisbet et al., 1983~. Serum levels of 1,25 dihydroxyvitamin D are elevated, and levels of the 24,24 dihydroxy metabolite are reduced (Zerwekh et al., 19871. Milhaud et al. (1977) reported a progressive loss of responsiveness of thymic and splenic cells toward both T- and B-cell mitogens. Evans et al. (1985) could not confirm a decreased responsiveness for spleen cells; how- ever, the response of thymic and lymph node T cells to PHA was lower in oplop rats over 12 days of age than in normal littermates of the same age. Osteopetrotic rats under 10 days of age did not show this deficit. Since osteopetrotic obliteration of the marrow cavities precedes the T-cell defi- ciency, the investigators suggest that this deficiency is secondary to the skeletal derangements. Transplantation of bone marrow from normal littermates cures the disease (Milhaud et al., 1 9751. Adoptive transfer of the disease has not been reported. Husbandry Because they have no incisors, these animals must be fed soft diets. It is preferable that they be maintained in a pathogen-free environment. The hus- bandry of this mutant has been reviewed (Marks, 19871. Reproduction The op mutation is maintained by breeding heterozygotes, which reproduce satisfactorily. It is possible to breed homozygotes after thymus transplanta- tion. rnu (Rowett Nude); rnuN (nznu, New Zealand Nude) Genetics Nude (mu) is an autosomal recessive mutation that has not yet been mapped. It was first found in 1953 in an outbred colony of hooded rats at the Rowett Research Institute, Aberdeen, Scotland; however, its biomedical significance was not recognized. In 1977 athymic nude rats were again observed in this colony and were subsequently distributed (May et al., 19771. A second nude mutation in the rat arose in a colony of outbred albino rats at Victoria University, Wellington, New Zealand (Berridge et al., 1979) and was given the name New Zealand nude, rnuN (synonyms, nznu, rnuNZ). The two mu

126 IMMUNODEFICIENT RODENTS rations are allelic (Festing et al., 1978), and breeding data indicate that rnu is dominant to rnuN (H. J. Hedrich, Central Institute for Laboratory Animal Breeding, Hannover, Federal Republic of Germany, unpublished data). Both alleles have been backcrossed onto a variety of inbred strains, including DA, F344, LEW, PVG, and WAG. Pathophysiology Both rnulrnu and rnuN/rnuN rats possess only rudimentary thymic tissue (Fossum et al., 1980; Vos et al., 1980), which becomes cystic during on- togeny, as in nude mice (Groscurth et al., 19741. There are, however, dif- ferences in immunologic responsiveness between the two mutations. Unfortunately, these differences have not been worked out in detail. These mutants are also dissimilar with respect to the degree of hairlessness. Homozygous rnuN rats have no hair; homozygous rnu rats have cyclic hair growth, sometimes producing a pelage that covers the entire body. The degree of hairlessness is not due to a genetic variation in the strain on which the genes are carried (Festing, 1981~; the same phenotypic differences are present when rnu and rnuN are backcrossed onto identical inbred strains. Under conventional conditions, Rowett nude rats survive for approximately 9 months, whereas New Zealand nude rats rarely survive for longer than 4 months. Life expectancy appears to be genetically determined and not pri- marily a result of the microbiological status of the environment (H. J. Hed- rich, Central Institute for Laboratory Animal Breeding, Hannover, Federal Republic of Germany, unpublished data). In both nude mutations, lymphocytes are severely depleted in the thymus- dependent paracortical areas of all lymph nodes, and cortical germinal centers are sometimes absent. The thymus-dependent splenic periarteriolar sheaths are variably depleted of lymphocytes, and Peyer's patches are smaller than normal. Total leukocyte counts are nearly normal, lymphocyte counts are significantly lower, and neutrophil counts are elevated (Brooks et al., 1980; Fossum et al., 1980; Vos et al., 19801. In rnu homozygotes, serum im- munoglobulins are nearly no~al, although some variations are present: IgM, IgD, and IgG2b are normal; IgA, IgGl, and IgG2 are slightly elevated; and IgG2a is lower. Antigen retention by follicular dendritic cells (OX2+) is similar in young homozygous rnulrnu rats and heterozygous rnul+ rats passively infused with antibody. However, during active immunization, fol- licular dendritic cells of rnulrnu rats fail to retain antigen, suggesting that antigen retention is dependent on specific antibody and not on the presence of thymus or functional T cells (Mjaaland and Fossum, 19871. NK cell activity is markedly increased in these mutants, as it is in nude mice. B cells are relatively, but not absolutely, increased in lymphoid organs and the thoracic duct. Thymocytes and T lymphocytes are reduced

HEREDITARY IMMUNODEFICIENCIES 127 in these sites, the most dramatic reduction being in the thoracic duct. Measurable numbers of T lymphocytes, identified by using the monoclonal antibody OX19, are present in the spleen, lymph nodes, and peripheral blood. Less than 2 percent of the cells are OX19+ in rnulrnu and rnuNlrnuN rats that are less than 8 weeks old. Whereas New Zealand nude rats show no increase in T-cell marker expression, Rowett nude rats older than 6 months of age have about 35 percent of lymph node cells that are OX19+ . No age-associated appearance of OX19+ cells has been observed in heterozygous controls. Homozygous rnu rats are capable of responding to PHA or ConA, although the response is reduced. This responsiveness also is age dependent and can be altered by microbiological status. For example, spleen cells from germfree or SPF rnulrnu rats (younger than 16 weeks of age) respond, whereas spleen cells from conventionally main- tained animals are unresponsive (H. J. Hedrich, Central Institute for Lab- oratory Animal Breeding, Hannover, Federal Republic of Germany, unpublished data). Some rnulrnu rats resist systemic and regional graft- versus-host disease (Rolstad et al., 19831. Mixed lymphocyte culture (MLC) reactivity is absent in young rnulrnu rats but can be demonstrated in adult animals. Lymph node cells from rnulrnu rats older than 6 months of age mount a clear-cut proliferative response against a stimulatory cell pool comprising different MHC haplotypes (Wonigeit et al., 19871. The re- sponses, however, do not reach the levels observed in euthymic controls, which indicates a lower frequency of alloreactive cells in athymic animals. Measurable amounts of IL-2 are produced by these T cells, which indicates that the cells are most likely T helper cells. Furthermore, cytotoxic T-cell activity can be induced in rnulrnu rats (Schwinzer et al., 19871. In ad- dition, aged Rowett nude rats show allograft rejection in vivo. A random pattern of rejection can be observed, which indicates a marked difference in the individual clonal repertoire (Hedrich et al., 19871. The reports on rejection or acceptance of no~al xenogeneic skin are somewhat contradictory (Hedrich et al., 19871. An association between the success of xenogeneic tumor grafts and the age of the recipients has been clearly shown (Maruo et al., 19821; rnulrnu rats should not be older than 8 weeks of age when grafted. Because of the delayed and imperfect onset of T-cell function, these mutants are good models for investigating the thymus-independent ma- turation of T-cell precursors to functional effector cells in vitro as well . . as 1n VlVO. There is little information on the response of nude rats to typical rat pathogens. Unlike the nude mouse, nude rats "clear" experimental Lis- teria monocytogenes infection, although at a slower than normal rate. NK cell activity can be stimulated with bacille Calmette-Guerin (BCG) (an attenuated strain of Mycobacterium bovis) and Corynebacterium parvum,

128 IMMUNODEFICIENT RODENTS and this activity appears to be normal. Experimental Sendai virus infection causes respiratory distress, loss of bronchial epithelium, and interstitial pneumonia. Antigen persists for at least 32 days postinfection (Carthew and Sparrow, 1980; Sparrow, 19801; however, the ultimate outcome of the infection is unknown, and no circulating antibody has been detected. Chronic respiratory disease (Mycoplasma pulmonis), conjunctivitis, and periorbital abscesses have been reported as problems. Tyzzer's disease (Bacillus piliformis) can destroy a colony; young rnulrnu rats are very susceptible and die within 2-3 weeks after exposure (Thunert et al., 1985~. Thus, these animals can serve as sentinels. Although Pneumocystis carinii is not a specific pathogen for rats, nude rats are very susceptible to this parasite and spontaneously develop a severe pneumonitis when exposed to the organism (Ziefer et al., 19841. Husbandry Most husbandry practices include precautions similar to those used for nude mice, including use of cesarean derivation, barrier maintenance, limited access, filter hoods, laminar-flow devices, isolators, and autoclaved diet. These precautions appear to be warranted (Festing, 19811. Both nude mu- tations appear to be more sensitive to exogenous factors (microbial and physical environment) on an inbred than on an outbred background. Reproduction Both sexes of rnuirnu rats are fertile, but homozygous females have dif- ficulties in raising their young. The most profitable matings are usually between heterozygous (rnul + ~ females and homozygous (rnuirnu) nude males. Such matings produce 36 percent rnuirnu offspring, which suggests that in utero loss occurs. Heterozygous matings produce the anticipated 25 percent homozygous nude pups. Under germfree and certain SPF conditions, ho- mozygous (rnuirnu) females litter approximately 0. 16 young per female per week and raise approximately 0.05 young per female per week. New Zealand nude rats are more difficult to breed. Inbred background appears to affect the productivity of nude offspring. tZ (Toothless) Genetics Toothless (tl) is an autosomal recessive mutation that arose spontaneously in a partially inbred colony of Osborne-Mendel-derived rats (Cotton and Gaines, 19741.

HEREDITARY IMMUNODEFICIENCIES 129 Pathophysiology Homozygous tl rats can be distinguished from their normal littermates by 10 days of age by their smaller size, short snout, and lack of incisors. Periorbital incrustation, possibly caused by defective lacrimal ducts, is com- mon. Bones are thickened and lack marrow cavities. Osteoclasts are rare (Cotton and Gaines, 1974; Marks, 1977), and those present are small and have greatly reduced concentrations of acid hydrolases (Seifert et al., 1988~. Toothless rats are hypophosphatemic and mildly hypocalcemic (Seifert et al., 1988) and respond poorly to exogenous parathyroid hormone (Marks, 19779. Peritoneal macrophages in tlltl rats are decreased 100-fold compared to normal littermates (Wiktor-Jedrzejczak et al., 1981~. In vitro responsiveness of spleen cells to T- and B-cell mitogens exceeds that of normal littermates and appears to be a function primarily of the adherent cell population (Wiktor- Jedrzejczak et al., 19811. Toothless rats are not cured by transplantation of normal bone marrow or spleen cells (Marks, 1977), and skeletal sclerosis cannot be induced by transplantation of mutant spleen cells into normal animals (S. C. Marks, Jr., University of Massachusetts Medical School, unpublished data). Husbandry The it mutation is not lethal; however, a soft diet is essential to compensate for the lack of incisors. The husbandry of this mutant has been reviewed (Marks, 19871. SPF conditions are recommended. Reproduction Homozygotes breed poorly or not at all. The mutation should be maintained by heterozygous matings. C4 Deficiency Genetics A single-component deficiency for the fourth component of complement has been reported in Wistar rats (Arroyave et al., 19741. The mode of inheritance was reported to be autosomal recessive, with heterozygotes ex- pressing 50 percent of normal levels of C4. The gene encoding for the complement fraction C4 has been mapped to the right of RT1.B in the rat MHC (Watters et al., 19871.

130 IMMUNODEFICIENT RODENTS Pathophysiology Arroyave et al. (1977) found that total hemolytic complement activity (CHso) in these animals was 20 percent of normal. It could be restored to normal by the addition of purified human C4. No C4 inhibitor was found in the sera of affected rats that were capable of responding to the injection of normal rat serum by producing anti-C4 antibodies. Although both female and male rats were deficient, a significantly higher number of males were affected. Husbandry Special husbandry procedures are not required. Reproduction Although no precise mechanism has been identified, the reproductive ef- ficiency of this colony is lower than expected (small litter size). INBRED STRAINS OF RATS BB/Wor anti Other Sublines Genetics The BB Eformerly called BB (BioBreeding) Wistar] rat develops an acute form of spontaneous juvenile insulin-dependent diabetes mellitus (IDDM) resembling human diabetes mellitus type I. The development of this model has been reviewed (Chappel and Chappel, 19831. The gene conferring sus- ceptibility to diabetes has been shown to be associated with the MHC. Spe- cifically, there is a requirement for the RTlU haplotype or a gene in close linkage with the gene coding for this haplotype (Colle et al., 19811. Non- MHC genes are also involved in the susceptibility to diabetes in this strain (Jackson et al., 1 9841. Several features of the syndrome suggest the involvement of the class II u antigens (RTl.B/DU) in the pathogenesis of IDDM (Goldner-Sauve et al., 19861. This is supported by the data of Buse et al. ~ 1984), which are indicative of a restriction fragment length polymorphism (RFLP) in class II MHC genes. On the other hand, Kastern et al. (1984) have demonstrated by RFLP analysis that a BB/Wor-derived subline lacks a major proportion of class I MHC genes. Furthermore, Wonigeit has found that BB/Wor-derived rats express the uv4 haplotype defined by a variation in atypical class I antigens (RT 1. CUV4) (K. Wonigeit, Klinik fur Abdominal- und Transplantationschirurgie der Med

HEREDITARY IMMUNODEFICIENCIES 131 izinischen Hochschule, Hannover, Federal Republic of Germany, personal communication to H. J. Hedrich, 19884. The presence of the RTl U allele is not in itself sufficient to result in clinical manifestation of the disease. Additional factors are required for overt dia- betes; a susceptibility for the development of insular, periductular, or in- traacinar lymphocytic infiltration in the pancreas has been proposed. This susceptibility is thought to be coded for by the dominant gene Pli (pancreatic lymphocytic infiltration), which segregates independently of RT1 (Colle et al. 19834. Overt IDDM is strongly associated with a genetically controlled depression of circulating T lymphocytes (Guttmann et al., 19831. The nature of the T lymphopenia gene (1) is not yet known, but it has not been linked to the MHC. Whether the expression of the I gene is fortuitous or obligatory for IDDM needs to be verified. Herold et al. (in press) have reported on the results of a cross between an inbred (F25) diabetes-prone BB substrain with an incidence of IDDM of greater than 90 percent and an inbred (F25) diabetes- resistant BB substrain with no diabetes and no lymphopenia. The F1 offspring were normal, but in the F2 generation, the overall incidences of diabetes and lymphopenia were 30 and 27 percent, respectively. Lymphopenia was present in 76 percent of the diabetic rats but in only 5 percent of the nondiabetic animals. Furthermore, the diabetes occurred earlier in nonlymphopenic than in lymphopenic rats. Like et al. (1986b) have demonstrated that diabetes occurs among diabetes-resistant control lines (WA, WB, and WC) without lymphopenia. Several groups are inbreeding BB rats originating from the outbred BioBreeding Wistar colony. Various lines exist with a variable degree of inbreeding and differences in the genetic background, except for the genes determining IDDM (RTl U and Pli, with or without 11. Selective breeding has resulted in a 60-80 percent penetrance in affected substrains. Normoglycemic sublines (diabetes-resistant) with less than 1 percent incidence of IDDM are available. Recently, Guberski et al. (in press) demonstrated that diabetes- resistant BB/Wor rats have a geneks) controlling the age of onset of diabetes. However, the genetic basis of the disease needs further investigation. Pathophysiology The disease is characterized by insulitis (mononuclear cell infiltration of the pancreas), which causes destruction of the islets of Langerhans (Seemayer et al., 19831. Acute clinical signs, including hyperglycemia (greater than 300 mg/dl), nonmeasurable insulin levels, ketoacidosis, hyperglucagonemia, polydipsia, and polyuria, develop between 6 weeks and 6 months of age (average age at manifestation is 3 months). The rats survive only if insulin is administered daily (Chappel and Chappel, 19831.

132 IMMUNODEFICIENT RODENTS The characteristics of IDDM development in BB rats are suggestive of an autoimmune etiology. The development of overt hyperglycemia can be sup- pressed by immune modulatory techniques, including antilymphocyte serum, cyclosporine A, neonatal thymectomy, or injections of monoclonal antibodies specific to NK cells or CD4 + T lymphocytes (Like et al., 1 979, 1 98 1 , 1 986a; Stiller et al., 19831. Furthermore, it is possible to prevent the manifestation of diabetes mellitus by the application of antibodies directed against class II MHC gene products (Boitard et al., 1985), by bone marrow transplantation (Naji et al., 1983), or by transfusion of whole blood from a nondiabetic subline without prior immunosuppression of the recipient (Rossini et al., 19851. The disease can be adaptively transferred to young, nonaffected, diabetes-prone rats or to immunosuppressed histocompatible rats by using ConA-activated spleen cells from acutely diabetic donors (Koevary et al., 19839. In addition to anti-class II MHC antibodies, it has been shown that multiple autoantibodies react with parietal cells of the gastric mucosa, thyroid colloid, thyroid follicular cells, skeletal muscle, smooth muscle, and nuclear protein (Dyrberg et al., 1982; Elder et al., 1982; Baekkeskov et al., 19841. A Hashimoto-like thyroiditis has also been demonstrated (Sternthal et al., 19811. The presence of autoantibodies directed against lymphocytes is controversial (Dyrberg et al., 1982; Elder et al., 19821. Diabetes-prone rats that develop diabetes mellitus always exhibit a severe impairment of the T-cell system. Both the helper/inducer and the cytotoxic/ suppressor T-cell subsets in peripheral blood and lymphoid tissues are pro- foundly reduced (Bellgrau et al., 1982; Colle et al., 1983; Elder and MacLaren, 19831. An inversion of the ratio of helper/inducer T cells (CD4) to cytotoxic/ suppressor T cells (CD8) occurs in all BB rats by about 60 days of age (Prud'homme et al., 19841. Woda et al. (1986) have shown that diabetes- prone BB rats lack the classic cytotoxic/suppressor T-lymphocyte subset (CD8~. Furthermore, Greiner et al. (1986b) have demonstrated that RT7.1 + T cells are depleted in diabetes-prone rats and that no RT6.1 + T cells are detected in their peripheral tissues. Functional analysis of lymphopenic animals shows a severe deficiency of all T-cell functions analyzed (Jackson et al., 1981; Elder and MacLaren, 1983), depressed skin allograft rejection across MHC and non-MHC barriers (Bellgrau et al., 1982; Kloting et al., 1984), defective proliferative responses to mitogens and to allogeneic cells in MLC (Rossini et al., 1983; Prud'homme et al., 1984), and defective cytotoxic T-cell function as measured by cell- mediated lympholysis (Prud'homme et al., 19881. The number of B cells and serum immunoglobulin levels are no1lllal, and the number of PMNs is increased, apparently in response to various chronic respiratory infections. The in vitro analysis of T-cell functions suggests that most BB sublines

HEREDITARY IMMUNODEFICIENCIES 133 exhibit immunologically incompetent T cells, which result from a postthymic or peripherally acquired maturational defect (Elder and MacLaren, 19831. It is not clear whether the mechanism of ,Bcell destruction and its pre- vention are the same in lymphopenic and nonlymphopenic (less than 3 percent of diabetic-resistant lines) animals (Like et al., 1986b). Butler et al. (1988) suggest that a deficiency in suppressor cell activity results in an unchecked effecter cell-induced ,Bcell cytotoxicity. NK cells or effecter cells that do not display the classical cytotoxic/suppressor T-cell phenotype might also be involved (Greiner, 19861. Guberski et al. (1988) were able to create an obese animal model of autoimmune diabetes mellitus by crossing Zucker female rats that were het- erozygous for the gene fatty (fal+) to male diabetic BB/Wor (+/+) rats. F1 hybrid females were backcrossed to BB/Wor diabetic males, and diabetic backcross males and females were mated. This latter mating fixed the re- cessive diabetes genes. Progeny from this mating were bred back to F1 hybrids, and progeny were selected that were genotypically diabetic and carriers of the fa gene. These rats served as parents for later inbreeding, which resulted in a new diabetic rat, BBZ/Wor. The BB/Wor diabetic rat resembles the lean BBZ/Wor rat (fal+ or +/+~: both are nonobese; both have insulitis, comparable rates of diabetes, thyroiditis, similar islet cell pathology, and ketosis; and both require insulin therapy. Obese BBZ/Wor rats (falfa) are heavier and have prominent ,~cell hyperplasia and degran- ulation. The obese BBZ/Wor rats are of the RTl U haplotype, are lymphopenic, and have an incidence of diabetes comparable to those of lean BBZ/Wor and BB/Wor rats. The obese BBZ/Wor rats, however, do not require exogenous insulin, most likely because of incomplete destruction of the pancreatic ,l3 cells (Guberski et al., 1 9881. Husbandry Maintaining BB rats is difficult because of their extreme susceptibility to opportunistic infections (Elder and MacLaren, 19831. It is therefore advisable to maintain these animals under laminar-flow, SPF, or gnotobiotic conditions. Gnotobiotic BB rats have the same incidence of diabetes as conventionally housed BB rats. All animals must be weighed several times a week; when weight decreases or fails to increase, the urine should be tested for glucose and ketones using Tes-Tape~ (Eli Lilly & Co., Indianapolis, Ind.) and Ketostix~ (Miles Di- agnostics, Elkhart, Ind.~. When glucosuria is found, plasma glucose should be determined. Once glucose levels are above 250 mg/dl, insulin must be administered daily. The dose is determined by the amount of glucosuria and is usually between 1 and 2.5 units.

134 IMMUNODEFICIENT RODENTS Reproduction Diabetes in BB/Wor rats is associated with reduced fertility. In males, this appears to be associated with a primary disorder of Leydig's cells, which precedes changes in seminiferous tubules (Murray et al., 1983~. Breeding efficiency in the high-incidence, diabetes-prone lines can be improved by administration of cyclosporine A, which delays the onset of the syndrome (Like et al., 1984), or whole-blood transfusions, which prevent the occur- rence of hyperglycemia (Rossini et al., 19831. Gross anatomical malformations observed in the offspring of diabetic BB rats are exencephaly, dysmaturity of the ossification process in cranial and long bones, and lumbosacral dysgenesis. Pups born to diabetic dams dem- onstrate significant delays in all aspects of motor development during the preweaning period. Control of maternal diabetes (regulation of blood glucose and prevention of ketosis) during pregnancy results in increased litter and fetal size, decreased perinatal mortality, and a significant reduction in the incidence of congenital malformation (Brownscheidle et al., 19831. LOU/C LOU/C rats spontaneously produce an IgG autoantibody that binds and neutralizes rat beta interferon (De Maeyer-Guignard et al., 19841. The titer of this autoantibody increases with age. This strain was selected for its high spontaneous incidence of myeloma, which is first seen at 8 months of age, and it has been postulated that the anti-IFN antibodies contribute to this disease (De Maeyer-Guignard et al., 19841. No studies have been conducted to investigate whether there is an increased susceptibility to infectious agents. GUINEA PIG MUTANTS C2 Deficiency Genetics Deficiency of the second component of complement (C2) in guinea pigs was the first C2 deficiency reported in an animal other than humans (Hammer et al., 19811. The gene for the defect behaves like a rare, silent allele of C2 that is MHC linked and inherited in the same way as normal C2 variants, that is, as an autosomal codominant trait. This mode of inheritance is similar to that in humans (BiKer-Suermann et al., 19811. The C2-deficient allele (C2°) is linked to C4S~ and BfF allotypes within the guinea pig MHC.

HEREDITARYIMMUNODEFICIENCIES 135 Pa thophys to logy C2 deficiency is characterized by a total absence of the second component of complement, as measured by hemolytic C2 and antigenic C2 protein assays. It is the most frequently seen complement deficiency in humans, with an incidence approaching 1 percent (Pettier, 19821. Macrophages in C2-deficient guinea pigs synthesize an abnormal C2-like protein rather than functionally active C2 (Goldberger et al., 19821. C2-deficient guinea pigs show no unique susceptibility to infectious dis- eases. Changes in the vascular permeability of skin induced by injection of the Cls subcomponent of the first component of complement are absent in C2-deficient guinea pigs but are present in C4-deficient guinea pigs, which suggests that the permeability agent is derived from C2 itself (Strang et al., 1986~. The C2-deficient guinea pig is unable to make a good antibody to the T-cell-dependent antigen, bacteriophage 4'X174. The antibody responses are characterized by low titers, failure to detect amplification of the secondary response, and no IgM to IgG switch (Ochs et al., 19861. Humans with C2 deficiency have a high incidence of SLE, discoid lupus, and Henoch-Schon- lein purpura. The C2 deficiency is more common in women than in men, and the incidence of lupuslike syndromes is more common in C2-deficient women than in C2-deficient men. C2-deficient guinea pigs have hypergam- maglobulinemia, anti-DNP antibodies, and rheumatoid factor; however, overt autoimmune disease is not seen (Bottger et al., 1986a). This guinea pig model might be a good model for studying the relationship between C2 deficiency and SLE in humans. Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally. C3 Deficiency Genetics The defect in the third component of complement (C3) arose spontaneously in strain 2 guinea pigs, was expressed in an autosomal codominant fashion, and was not linked to the MHC or to the gene controlling expression of the C3a receptor (Burger et al., 19861.

136 IMMUNODEFICIENT RODENTS Pa thop hys lo logy Homozygous C3-deficient (C3D) guinea pigs have a functional C3 titer and antigenic activity that is only 6 percent of normal. Serum from these animals has a markedly reduced hemolytic complement and bacteriocidal activity in vitro, and homozygous deficient animals have impaired antibody synthesis to the T-cell-dependent antigen, bacteriophage X174 (Bottger et al., 1986b). In addition, C3D guinea pigs have a defect in isotype switching from IgM to IgG. These in vivo abnormalities in humoral immunity are shared with guinea pigs with deficiencies of C2 or C4 (Bottger et al., 19851. However, in contrast to C2D and C4D guinea pigs, C3D guinea pigs do not have elevated IgM levels, nor do they have circulating immune complexes (Bottger et al., 1986b). Macrophages and hepatocytes from guinea pigs with homozygous C3D synthesize and secrete C3 in normal amounts. When ana- lyzed by sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis and immunoblotting, the product of C3D hepatocytes appeared normal; how- ever, it was greatly reduced in the serum of animals containing the deficiency. The catabolism of normal guinea pig C3 was not elevated in C3D guinea pigs; however, enhanced proteolysis of a defective C3 molecule in C3D animals could not be excluded (Auerbach et al., 19851. Husbandry C3D guinea pigs, unlike humans with a similar deficiency, do not appear to have an increased susceptibility to infectious agents or immune complex disease (Alper and Rosen, 1984; Bottger et al., 1986b). Reproduction No problems have been reported in the breeding of C3D guinea pigs. C4 Deficiency Genetics Deficiency of the fourth component of complement (C4) in guinea pigs is inherited as an autosomal recessive trait with full penetrance (Hyde, 1923; Ellman et al., 1970), resembling the inheritance of C4 deficiency in humans (Ochs et al., 1977; Schaller et al., 19771. Heterozygotes express intermediate levels of C4 (Ellman et al., 19701. It is thought that the deficiency arose from a mutation of a C4-F allele (Shevach et al., 19761.

HEREDITARY IMMUNODEFICIENCIES 137 Pathophysiology C4 deficiency was first discovered in a strain of guinea pigs whose serum did not lyse antibody-sensitized horse erythrocytes (Moore, 19191. The defect was discovered independently in a colony of outbred Hartley guinea pigs at the National Institutes of Health (Ellman et al., 19701. Guinea pigs with a C4 deficiency are unable to activate complement by the classical pathway (Hammer et al., 1981), because C4 is critical for the development of both C3 and CS convertase (Muller-Eberhard, 1975J. This model has been used extensively to demonstrate the amino acid sequence of pro-C4 (the precursor to C41. The structure, function, and quan- titation of guinea pig C4 have recently been reviewed (Quimby and Dil- lingham, 19881. The discovery of C4 deficiency in guinea pigs provided a good model in which to study the activities of the classical and alternative pathways of complement activation (Frank et al., 19711. The genetic control and biosynthesis of C4 have also been studied in the C4-deficient guinea pig (Colten and Frank, 1972; Colten, 19831. Recent experiments with cell-free biosynthetic systems have shown that guinea pig C4 is synthesized as the single-stranded precursor pro-C4 (200,000 dalton), which, prior to its release from the polysome, is converted to the three-chain C4 molecule (Hall and Colten, 19771. Hall and Colten (1977) failed to detect the presence of pro-C4 in hepatocytes of C4-deficient guinea pigs, and Colten (1983) has hypothesized, based on hybridoma studies, that the deficiency is due to a posttranscriptional defect in the processing of C4 precursor RNA to mature C4 messenger RNA. The C4-deficient guinea pig has also been used to evaluate the role of the classical pathway during various infections (Diamond et al., 1974; Gelfand et al., 19784. Early studies showed that animals with suspected C4 deficiency were more susceptible to Salmonella (formerly Bacillus) choleraesuis infec- tion than were normal animals (Moore, 19191. C4-deficient guinea pigs are more susceptible to the lethal effects of endotoxin injection (May et al., 19721. Resistance to the ixodid tick Dermacentor andersoni (Wikel, 1979) and to Candida albicans (Gelfand et al., 1978) and Cryptococcus neoformans (Diamond et al., 1974) infections is normal in C4-deficient animals. It can be inferred, therefore, that there is an intact alternative pathway of comple . . . . .. . ment act~vat~on ~n guinea pigs with t nese ~ntect~ons. Ellman et al. (1971) have demonstrated a slight but significant defect in the ability of C4-deficient guinea pigs to make an antibody response to DNP- bovine y-globulin. The antibody response to the T-cell-dependent antigen bacteriophage 4'X174 is clearly abnormal in C4-deficient guinea pigs. Not only is the IgM response depressed, but there is also no evidence for im- munologic memory (Ochs et al., 1978, 19861. C4-deficient guinea pigs have signs of polyclonally stimulated antibody synthesis, circulating rheumatoid

138 IMMUNODEFICIENT RODENTS factors, and anti-DNP antibodies, which suggests immune complex disease (Bottger et al., 1986a). Despite the latter observation, there have not been any particular problems associated with infectious agents in conventionally reared colonies (Peltier, 19821. Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally (Peltier, 19821. HAMSTER MUTANTS nu (Nude) Genetics Nude (nu) is a recessive mutation that arose spontaneously in the breeding colony of outbred Syrian hamsters at the Institut de Recherches Scientifiques sur le Cancer, Villejuif, France (Haddada et al.- 19821. Genetic studies involving these animals have not been conducted. Pa thophys to logy Nude hamsters, like nude mice and nude rats, are hairless and have only a rudimentary thymus. At 1 month of age, decreased numbers of mature lymphocytes are found in lymph nodes and the spleen. Spleen cells do not proliferate in response to the mitogens ConA, PHA, LPS, and protein A, and the level of serum IgG is reduced. In contrast to nude mice and nude rats, nude hamsters have no higher NK cell activity than do normal hamsters. Spontaneous tumors do not arise frequently; however, a study has shown that 1 of 11 animals developed an immunoblastic sarcoma in the mesenteric lymph node at 8 months of age (Loridon-Rosa et al., 19881. Injection of simian virus 40-transformed B-lymphoma cells induces tumor development in nude hamsters but not in immunocompetent controls (Loridon-Rosa et al., 1988). Husbandry No definitive studies have been performed to evaluate the susceptibility of nude hamsters to various infectious agents; however, animals housed under conventional conditions live 10-12 months and often develop skin disorders

HEREDITARY IMMUNODEFICIENCIES 139 after 8 months. Because the immunologic defect in nude hamsters appears to be similar to that of nude mice and rats, SPF conditions are recommended for their maintenance. Reproduction Female homozygous nude hamsters are fertile but are unable to feed their young; therefore, nude hamsters are maintained by mating homozygous nude males with heterozygous female siblings. C6 Deficiency Genetics The genetic control of the deficiency of the sixth component of complement (C6) in hamsters is incompletely understood. Pathophysiology C6 deficiency in hamsters was described by Yang and coworkers (1974~. Serum from affected hamsters was incapable of reconstituting C6-deficient rabbit or human sera but did reconstitute sera depleted of Clr, C2, and C4. The C6 deficiency was confirmed by titration of isolated complement com- ponents from normal and deficient hamster sera. C6-deficient hamster sera did not have altered immune adherence or phagocytic functions. Hamsters with the deficiency had a high incidence of proliferative enteritis (transmis- sible ileal hyperplasia). Husbandry Special husbandry procedures are not required. Reproduction These animals breed normally.

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This volume is an indispensable reference on the nature of immune defects in rodents and the special techniques necessary to maintain and breed them. The authors describe 64 inbred, hybrid, and mutant strains of rodents, each with some immune defect; explain mechanisms for ensuring genetic purity; and provide a standardized nomenclature for different varieties. Subsequent sections summarize and provide references on the genetics, pathophysiology, husbandry, and reproduction of each of the various strains as well as sound advice on planning for the selection, transportation, housing, and maintenance of these animals.

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