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the peptide to raise CD8 CTLs against the peptide. This approach was based on earlier work with HIV peptides in mice, using peptide-pulsed spleen cells; however, researchers found that they could also use peripheral blood mononuclear cells (PBMCs). Both contain about 1 or 2 percent dendritic cells. Peptide can bind to the MHC Class I molecules on these dendritic cells without going through the intracellular processing pathway. When the cells are washed, irradiated, and reinjected intravenously, they induced CD8 CTLs and specific lysis.

The key cell in this process is the dendritic cell. Researchers found that immunizing with 100,000 purified dendritic cells produced as much or more specific lysis as 8 million peptide-pulsed spleen cells. This is consistent with the ratio of one or two dendritic cells per 100 spleen cells.

Previous experiments had shown that HIV peptide 18, part of the V3 loop of the envelope protein, was a major target for CTLs in BALB/c mice. Other researchers had successfully demonstrated that this same peptide-pulsed spleen cell immunization technique could protect against a mouse tumor that was expressing HIV gp160 (made tumorigenic by cotransfecting with ras). Based on this, investigators decided to use the same approach with peptides corresponding to mutations of p53, specifically a mutation called T1272, that came from a human lung carcinoma.

Researchers immunized BALB/c mice with peptides corresponding to this mutation and were able to induce CTLs that would kill targets. Using various truncated peptides, they mapped the minimal epitope to a tenmer that contained the point mutation (a cystine to tyrosine). Interestingly, this epitope avoids the differences between human p53 and mouse p53, and the induced CTLs are specific only for the mutation difference—they do not kill controls or targets pulsed with wild-type peptide.

In other words, the cystine-to-tyrosine mutation created a new antigenic determinant that was not present in natural p53. This peptide is presented by KFD, whose binding motif involves a tyrosine at position 2. Since natural p53 doesn’t bind to KFD, and isn’t recognized by the CTLs, there should be no danger of inducing autoimmune disease if the subject were a human with cancer.

The key question was whether endogenously expressed mutant p53 would be processed and presented in such a way, and in sufficient quantity, to be killed by the induced CTLs. Researchers transfected mouse tumor cells with mutant p53 and found that they were killed, whereas untransfected cells were killed only if peptide was added to the culture. In addition, the level of mutant p53 expressed by the transfected cells was at the low end of what is found in natural human and murine tumors. This indicated that the tumor antigen was not produced by overexpression of p53 and would in fact work at the levels found in natural tumors.

Researchers concluded from these findings that endogenously expressed mutant tumor suppressor oncogene product p53 can serve as a target antigen for CD8 CTLs, and that such CTLs can be induced by peptide immunization. They chose this method of immunization in part to avoid the need to attach a helper



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