Cross-reactive memory CD4+ T cells affect CD8+ T-cell responses to secondary dengue infections in mice 15. Therefore, JEV/WNV cross-reactive CD4+ T-cell epitopes may also play an important role in heterologous protection of JEV-immunized rodents from WNV infection 12. We investigated JEV/WNV cross-reactive CD4+ and CD8+ T-cell responses following primary JEV and WNV infection as a first step in elucidating the ALK activation role these cells may play in heterologous immunity. We characterized effector functions elicited by a previously identified immunodominant WNV NS4b CD8+ T-cell epitope and its JEV variant in both JEV- and WNV-infected mice and found that the homologous peptide variant to the immunizing

virus induced higher levels of cytotoxic activity and cytokine responses. However, there were striking virus-dependent differences in the quality of the response; the ratio of IFN-γ+ CD8+ T cells to IFN-γ+TNF-α+ CD8+ T cells was greater in JEV-infected mice compared with WNV-immunized mice. To further understand these differences, we compared epitope-specific CD8+ T-cell responses (cytokine profile, epitope hierarchy, phenotype) as well as the effect of virus

burden in mice buy CYC202 immunized with a low or high dose of pathogenic JEV and compared these responses to those seen in attenuated JEV and pathogenic WNV infection. To identify cross-reactive CD4+ and CD8+ T-cell epitopes, we stimulated splenocytes harvested on

day 7 from JEV SA14-14-2 immunized mice with peptide pools corresponding to each of the ten WNV proteins. We found that the JEV/WNV cross-reactive CD4+ T-cell IFN-γ responses, as assessed by intracellular cytokine staining, were mainly directed at peptides in the NS4b, NS2a, NS3 and E proteins (Supporting Information Fig. 1A and Supporting Information Table 1). In contrast, the majority of the JEV/WNV cross-reactive IFN-γ-producing CD8+ T cells was induced by a single peptide pool corresponding to the WNV NS4b protein. Deconvolution of the positive peptide pools identified three peptides, WNV NS1 A, WNV NS3 MycoClean Mycoplasma Removal Kit B and WNV NS4b209–226, which consistently induced the highest responses in splenocytes from JEV-immunized mice (Table 1). WNV NS3 B and WNV NS4b209–226 have previously been identified as epitopes in WNV-infected C57BL/6 mice 8, 9, 16. WNV NS1 A and WNV NS3 B and their corresponding truncations (NS1 A-1 and NS3 B-2) induced IFN-γ production by splenocytes from both H2-Db−/− and H2-Kb−/− mice, suggesting that these might be CD4+ T-cell epitopes. We confirmed that NS1 A-1 and NS3 B-2 are JEV-specific CD4+ T-cell epitopes that are cross-reactive to WNV by intracellular cytokine staining (Fig. 1A, Table 1). Stimulation with WNV NS4b209–226 and its truncations of splenocytes from JEV SA14-14-2-immunized H2-Kb−/−, but not from H2-Db−/− mice induced IFN-γ, confirming H2-Db restriction 7, 8.