It seems that the M-to-A substitution at p6 in the NP366peptide created a novel peptide/MHC I complex (pMHCI) that is antigenically distinct from, but as immunogenic as, the WT epitope. production were comparable with those seen for the native response. Importantly, the overdominance profile characteristic of secondary DbNP366-specific clonal expansions was retained for the NPM6A mutant. The primary determinants of immunodominance in this endogenous, non-TCR-transgenic model of viral immunity are thus independent of TCR repertoire composition and diversity. These findings both FMK highlight the importance of effective antigen dose for T cell vaccination and/or immunotherapy FMK and demonstrate the feasibility of priming the memory T cell compartment with engineered viruses to protect against commonly selected mutants viral (or tumor) escape mutants. Keywords:avidity, viral escape, pMHC structure, TCR diversity, reverse genetics Virus-specific CD8+T cells play an essential role in limiting infectious process by killing virus-infected cells and/or producing proinflammatory cytokines (1). Because this CD8+T cell-mediated immunity tends to be directed at 1 or 2 2 immunodominant epitopes and a number of subdominant determinants (2), we need to identify the major factors controlling immunodominance if we are to develop optimized T cell vaccines and immunotherapy protocols. Influenza A virus infection of C57BL/6J (B6, H2b) mice provides a well-characterized natural [non-T cell receptor (TCR)-transgenic] system for dissecting CD8+T cell immunodominance hierarchies. Although there are at least 6 epitopes distributed between H2Dband H2Kb(3), the most prominent CD8+sets that emerge after an initial encounter with an influenza A virus are specific Rabbit polyclonal to beta defensin131 for H2Db-bound peptides from the viral nucleoprotein (DbNP366374) and acid polymerase (DbPA224233) proteins (4). These DbNP366+CD8+and DbPA224+CD8+populations reach essentially comparable sizes after primary infection, although the DbPA224+CD8+set peaks 12 days earlier (5), reflecting a higher nave T cell precursor frequency (6,7). After secondary challenge, the DbNP366+CD8+T cells are massively overdominant, constituting up to 80% of the total virus-specific CD8+response (8). This emphasis on DbNP366in the recall response has been attributed to differences in FMK the spectrum of antigen-presenting cell involvement (9), protein/peptide abundance, and T cell precursor frequency (7). TCR repertoires selected by DbNP366and DbPA224differ in both extent and character. Analysis of TCR CDR3 sequence variability and clonal prevalence shows that DbPA224recruits a predominantly private (specific for individual mice) and diverse range of TCR sequences (10), whereas CD8+T cell recognition of DbNP366is mediated via a narrower, public (conserved between individuals) TCR repertoire (11). Could selection of optimal public clones by DbNP366but not DbPA224explain the dramatic divergence in CD8+T cell response magnitude, especially after secondary challenge? To date, the role of TCR fine specificity and particular TCR clones in establishing immunodominance hierarchies remains unclear. The present analysis asks whether the limited, public TCR repertoire characteristic of the DbNP366+CD8+T cell response is a primary determinant of immunodominance. == Results == == Residues FMK Critical for TCR Recognition by DbNP366+CD8+T Cells. == To determine critical residues for recognition of the NP366peptide by H2Db-restricted CD8+T cells, single amino acid mutations were made at different positions within the viral NP366peptide, excluding the anchor amino acid (p5, p9). These included A substitutions (S2A, N3A, E4A, M6A, E7A, T8A), conserved (E4D, M6Q, E7D, T8S), and reverse charge (E4K, E7K) mutations. Mutant NP366peptides were used to probe CD8+T cells by intracellular cytokine secretion (ICS), a51Cr-release assay and stimulation of DbNP366+LacZ-inducible T cell hybridomas. The recognition profiles of polyclonal DbNP366+CD8+T cells obtained from mice infected with the WT virus were variably modified by different amino acid substitutions (Fig. 1AandB). p6M appeared to be critical for TCR recognition, as stimulation with 2 different amino acid substitutions (M6A, M6Q) resulted in a complete loss of peptide-induced IFN- production (Fig. 1A) and51Cr-mediated lysis (Fig. 1B). This finding is in accord with data showing that the emergence of viral escape mutants in mice transgenic for a DbNP366+TCR resulted from a p6M mutation (12). The p4E position was also important, while substitutions at p3, p7, and p8 led to a partial loss of TCR recognition (Fig. 1A). Stimulating 3 distinct.
It seems that the M-to-A substitution at p6 in the NP366peptide created a novel peptide/MHC I complex (pMHCI) that is antigenically distinct from, but as immunogenic as, the WT epitope