1B and D), implying that as priming with DbPA224 normally stimulates a much broader spectrum of TCRβ than is the case for DbNP366, this diversity could help to ensure the integrity of the response when TCRα is limiting. Interestingly, the DbNP366>DbPA224 CH5424802 in vivo immunodominance hierarchy recognized for secondary responses to wt influenza A viruses in H2b mice 21 was no longer maintained in A7 transgenics (Fig. 1E–H). Similar to

the primary response (Fig. 1A–D), the DbNPCD8+ sets recovered from the spleen (Fig. 1E and G) and BAL (Fig. 1F and H) of the secondarily infected A7 mice were reduced in magnitude (p<0.05) compared with those from the B6 controls. A possible explanation is that some of the DbNP366-specific TCRβ that pair with this irrelevant Vα chain may not form TCR that can be efficiently recruited after secondary challenge, leading to the A7 DbNPCD8+ and DbPACD8+ recall responses being of comparable magnitude. The next question was whether limiting the available TCRαβ pairs by confining the response to an “irrelevant” TCRα in any way reflects that such “aberrantly selected” TCRαβ use a different

mode of pMHC-I recognition. We made sequential alanine (A) substitutions at different positions within NP366–374 and PA224–233 peptides, excluding the anchor residues (p5, p9 for NP366; p5, p10 for selleck compound PA224). These mutant NP366 and PA224 peptides were used to probe CD8+ T-cell responsiveness as determined by IFN-γ production. The recognition profiles of polyclonal DbNPCD8+ and DbPACD8+ T cells obtained from mice primed-and-boosted with influenza viruses (H1N1 then H3N2) were modified by different aa substitutions (Fig. 2A and B). Following infection of A7 mice, p6M and p4E were critical for TCR recognition by DbNPCD8+ T cells (Fig. 2A), whereas p6F and p7R were important for DbPACD8+ T cells (Fig. 2B). These SPTBN5 profiles were identical to those found previously for B6 mice (Fig. 2) 17, 22. It thus seems that the Vα2-constrained and DbNP366- and DbPA224-specific TCR recognize the same features within the antigenic peptides, raising the question of whether the DbNP366 and DbPA224 epitopes are recognized by the same CDR3β clonotypes in A7 and wt B6

animals. Furthermore, these peptide recognition data suggest that the Vβ-chain may play a dominant role in antigen recognition for both DbNPCD8+ and DbPACD8+ T-cell responses. Alternatively, for the Vα chain to participate in pMHC-I recognition, it might be expected not to have any structural features that would interfere with pMHC-I docking in A7 mice. To determine the clonal composition defined by TCRβ diversity, the DbNP366-specific CD8+ T cells were first analyzed for Vβ usage by staining with a panel of anti-Vβ mAb. In B6 mice, Vβ8.3 consistently accounts for an average of 42.8% 14, 23 of the DbNPCD8+ response, with Vβ4 24 being subdominant (∼13%). This strong Vβ8.3 bias was no longer apparent for the A7 mice (Fig. 3A). However, Vβ4 emerged for DbNP366-specific T cells from spleen (51.4%; range 18.