transgenic mouse, which had only one particular T-cell receptor, to control for this diversity. This “mixing” experiment is described below.
Researchers removed peripheral T-cells from the spleens and the mesenteric and inguinal lymph nodes of transgenic mice and polarized them by stimulating with concanavalin A (conA) in the presence of recombinant IL-2, IFN-gamma, and anti-IL-4 (to generate Th-1) or in the presence of recombinant IL-4 and anti-IFN-gamma (to generate Th-2). The resulting T-cells had a high level of expression of D-beta-4 TCR, up-regulated the proper activation markers (CD44 and CD25), and down-regulated CD62L (also called L-select). After stimulating with islet cells and culturing for about a week, researchers transferred the cells into perinatal (5 to 7 days old) NOD mice and waited 14 to 28 days to see if insulitis and diabetes developed.
The results indicated that within 3 or 4 days after transfer, there is infiltraion of the islets by both Th-1 and Th-2 cells, with resulting peri-insulitis and insulitis. However, only the Th-1 recipients go on to develop diabetes. This suggests that, at some point in the future, it may be possible to regulate diabetes by forcing a Th-2 response, even when that cell carries the TCR for the as-yet-unknown antigen in the beta granules of the islets. Unfortunately, other experiments indicate that Th-2 does not provide this same protection when Th-1 is also present, even in small amounts.
Researchers are now conducting further experiments to test the hypothesis suggested above, namely that the real differentiation event is the loss of the beta chain of the IL-12 receptor. If it were possible to insert this receptor early on, it should be possible to generate a Th-0 cell that responds to IL-12 and makes IFN-gamma while still producing IL-4. It may be that the Th-2 cells produced in the mixing experiment have the IL-12 receptor and revert to Th-0 in an IL-12-producing environment, thereby preventing the desired suppression. Hence, researchers are trying to produce very heavily polarized Th-1 and Th-2 cells with no high-level expression of the receptor to see if this pattern will hold. Other possibilities for negative regulation of T-cell development include blocking the CD40 ligands or blocking the CD28 and B7 pathway, which may be important in the development of Th-1-type cells. Another is a soluble IL-12 receptor that might also switch the balance, even if the default is toward a Th-1 response.
In response to questions from the audience, Dr. Katz added the following:
It may be possible in the future to overcome genetic predispositions by choosing the correct immunogenic peptide, with correct affinity, to induce a strong Th-1 or Th-2 response. At present, however, researchers don’t know the antigens and peptides, and can’t answer questions about affinity.
The antigen in question copurifies with beta granule fraction by normal fractionation.
Researchers are conducting experiments to see whether they can activate a Th-2 population by immunization while blocking Th-1 cells, but they have no data as yet.