The factors that determine CD4؉ T cell (T CD4 ؉) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the T CD4 ؉ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV-and VACV-specific T CD4؉ at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from T CD4؉ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific T CD4؉ as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on T CD4 ؉ responses and memory. IMPORTANCE Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted T CD4؉ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific T CD4؉, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of T CD4 ؉ responses at both acute and memory time points, with VACV-specific T CD4 ؉ memory being notably compromised. These data offer insight into the impact of epitope-extrinsic factors on the resulting T CD4 ؉ responses.

We have developed a carefully-designed transgenic model to learn how expression of an autoantigen influences the degree and nature of tolerance. The surrogate antigen, directed to thyroid by a tissue-specific promoter, consists of a cell-surface glycoprotein (human interleukin-2 receptor α chain, Tac) to which well-defined B cell, CD8+ T cell, and CD4+ T cell epitopes have been appended in order to assess all three lymphocyte subset responses. A LoxP-flanked stop cassette was inserted between the tissue-specific promoter and the transgene open reading frame (ORF) to allow for conditional expression. To avoid positional and copy number effects in traditional transgenesis, the transgene was targeted to HPRT locus with a single-copy insertion. Here we show that embryonic but not adult expression of Tac transgene results in a profound epitope-specific CD8 T cell tolerance. However, prolonged expression of postnatal antigen is able to facilitate tolerance induction. Moreover, the ability to induce Tac-specific immune tolerance correlates with the loss of susceptibility to rejection of Tac-expressing tumor cells. These findings provide direct evidence of dynamic tolerance induction to normal or tumor antigens.

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