Ectopic CD4+ t cell epitopes in variant proteins: Structural influences on immunodominance and phenotype

CD4+ T cells are the master regulators of all immune responses, dictating the tone and magnitude of the subsequent immune response after each infection or vaccination. While the breadth of the contributions helper T cells make has long been acknowledged, very little is known about the processes required to induce naive CD4+ T cell to differentiate into the multitude of mature, effector subsets necessary for a fully functioning immune system. We have previously shown that protein three dimensional structure has a profound influence on the immunodominance of CD4+ T cell responses following immunization with a variety of antigens. We more recently found a potential link between the flexibility of the epitope locations within the three dimensional antigen structure and the elicitation of either Th1 or Th2 effector phenotypes. The most flexible epitope locations induced larger IFNgamma:IL-4 ratios (Th1:Th2) than more rigid epitopes locations, in which the IFNgamma:IL-4 ratio skewed towards the decrease of IFNgamma production by the effector cells and a shift towards production of IL-2 and IL-4. In order to fully test the hypothesis that more flexible epitope locations tended towards IFNgamma-specialization, we engineered ectopic CD4+ T cell epitopes into varying structural environments within HIV-1 gp120JR-FL. By using the ectopic epitope 2W1S, we were able to look at epitope-specific CD4+ T cells and determine cytokines produced as a result of different structural placements. Overall, we were able to successfully express and purify two structural variants, T38 as a variant with 2W1S in a flexible location and T39 as a variant with 2W1S in a more rigid location. We found that following immunization of mice with both structural variants, there was no evidence of differences in cytokine-specialization between the two variants or rates of antigen presentation of the ectopic epitope. We also used previously made structural variants with an ectopic HEL epitope inserted into the mobile loop of T4 Hsp10 gp31 to test the phenomenon. Ultimately, no differences in the IFNgamma:IL-4 ratios were seen between the two gp31 variants, further confirming that the cytokine-specialization was not induced by either of our variant models. We will continue to increase the breadth of epitope placements in our structure variants to probe the effect structure has on CD4+ T cell effector phenotypes.