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Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCRαβ heterodimer selection and resultant diversity is unclear. The D b PA 224 –specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) β-length of 6 aa. Despite these restrictions, D b PA 224 -specific BV29 + T cells use a wide array of unique CDR3β sequences. Structural characterization of a single, TRBV29 + D b P A224 -specific TCRαβ-pMHCI complex demonstrated that CDR3α amino acid side chains made specific peptide interactions, but the CDR3β main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in Vβ-region use. In support of this hypothesis, retrovirus expression of the D b PA 224 -specific TCRVα-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRVα paired with a diversity of CDR3βs in the context of a preferred TCRVβ spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity.

When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at leastin vitro. Such impairment of MHC class II (MHC II) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs maturedin vivocould still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activationin vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II–peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections.

Immunodominance hierarchies are a substantial, but poorly understood, characteristic of CD8+T cell-mediated immunity. Factors influencing the differential responses to the influenza A virus nucleoprotein (NP366–374) and acid polymerase (PA224–233) peptides presented by H2Dbhave been analyzed by disabling (N5→ Q substitution) these peptides in their native configuration, then expressing them in the viral neuraminidase protein. This strategy of shifting epitopes within the same viral context resulted in an apparent equalization of DbNP366[epitope consisting of viral nucleoprotein (NP) amino acid residues 366–374 complexed with the H2DbMHC class I glycoprotein] and DbPA224(H2Db+PA224–233) epitope abundance after direct infectionin vitroand induced reproducible changes in the magnitude of the DbNP366- and DbPA224-specific T cell subsets generated after infection of mice. Comparison of DbNP366- and DbPA224-specific CD8+T cell responses induced from the native configuration and from the viral neuraminidase stalk demonstrated that the size of both primary and secondary responses is influenced by relative epitope levels and that, at least after secondary challenge, the magnitude of responses is also determined by CD8+T cell precursor frequency. Thus, this immunodominance hierarchy is a direct function of antigen dose and T cell numbers.

Much of the CD8+T cell response in H2bmice with influenza pneumonia is directed at the nucleoprotein366-374(NP366) and acid polymerase224-233(PA224) peptides presented by the H2DbMHC class I glycoprotein. These DbNP366- and DbPA224-specific T cell populations are readily analyzed by staining with tetrameric complexes of MHC+peptide (tetramers) or by cytokine production subsequent toin vitrostimulation with the cognate peptides. The DbPA224-specific CD8+effector T cells make more tumor necrosis factor (TNF) α than the comparable CD8+DbNP366+set, a difference reflected in the greater sensitivity of the CD8+DbPA224+population to TNF receptor (TNFR) 2-mediated apoptosis under conditions ofin vitroculture. Freshly isolated CD8+DbNP366+and CD8+DbPA224+T cells from influenza-infected TNFR2-/-mice produce higher levels of IFN-γ and TNF-α afterin vitrostimulation with peptide, although the avidity of the T cell receptor-epitope interaction does not change. Increased numbers of both CD8+DbPA224+and CD8+DbNP366+T cells were recovered from the lungs (but not the spleens) of secondarily challenged TNFR2-/-mice, a pattern that correlates with the profiles of TNFR expression in the TNFR2+/+controls. Thus, it seems that TNFR2-mediated editing of influenza-specific CD8+T cells functions to limit the numbers of effectors that have localized to the site of pathology in the lung but does not modify the size of the less activated responder T cell populations in the spleen. Therefore, the massive difference in magnitude for the secondary, although not the primary, response to these DbNP366and DbPA224epitopes cannot be considered to reflect differential TNFR2-mediated T cell editing.