Systematic identification of immunodominant CD8+ T-cell responses to influenza A virus in HLA-A2 individuals

Abstract

Immunodominant T-cell responses are important for virus clearance. However, the identification of immunodominant T-cell peptide + HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for immunodominant CD8(+) T-cell specificities. The analysis targeted healthy HLA-A2(+) donors to allow comparison with responses to the well-studied influenza matrix protein 1 epitope. Although influenza matrix protein 1 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three of eight, whereas for the other five, prominent CD8(+) T-cell responses tended to focus on various peptides from the influenza nucleoprotein that were not presented by HLA-A2. Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8(+) T-cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity.

Fig. 1.

The T-cell epitope screening protocol.

Fig. 2.

Most immunodominant T cells are NP-specific. Polyclonal T-cell lines were raised using IAV-infected PBMCs. After 12–15 d, these lines were tested for their reactivity to autologous BLCLs infected (MOI = 10) with single rVVs encoding individual IAV proteins. The total IAV responses stimulated by IAV-infected, autologous BLCLs are shown in black bars for easier comparison. A–H correspond to donors 1–8, and the donor HLA alleles are shown in Insets.

Fig. 3.

Mapping immunodominant NP epitopes. The same IAV-specific T-cell lines used in Fig. 2 derived from five donors with either higher or equivalent CD8⁺ T-cell responses to NP than to M1 were also screened for their specific response to the 81 overlapping 18mer NP peptides at a final concentration around 1 μM in an ICS assay. The identified 18mer sequences are shown, and the subsequently identified minimal epitopes are bold (Figs. 1 and 2). A–E correspond to donors 1–5.

Fig. 4.

Various HLA molecules present immunodominant NP peptides. (A) The 13mer peptides within the NP209–237 18mers (donor 2) were screened, with the 18mer results being shown as open bars (i). Three 13mer overlapping peptides were titrated to compare their activity (ii), and HLA-matched BLCLs were used to identify the HLA presenting the NP219–231 13mer (iii and iv). (B) The 13mer peptides within NP133–150 (donor 4) were screened as in A (B, i). The minimal NP_140–150 peptide and its amino acid-truncated and -extended variants were tested under serum-free conditions (ii), and the HLA restriction profile for NP_140–150 was determined using HLA matched BLCLs (iii, iv). (C) The 13mer peptides within the NP331–348 18mer (donor 5) were screened as in A (C, i). Four 13mers were titrated to determine their activity (ii); single amino acid-truncated and -extended peptides of NP_336–344 were tested under serum-free conditions (iii), and the HLA restrictions of 13mer containing NP_336–344 were analyzed as in A (iii and iv).

Fig. 5.

Ex vivo CTLp analysis for two immunodominant T-cell responses. The M58, NP_140–150 (A and B) and NP_336–344 (C and D) peptides were used to stimulate purified CD8⁺ T cells from donors 4 and 5, respectively, for 5 h in the presence of Brefeldin A in an ICS (A and C) or an overnight ELISpot assay (B and D).