A Mutation in the HLA-B^*2705-Restricted NP_383-391 Epitope Affects the Human Influenza A Virus-Specific Cytotoxic T-Lymphocyte Response In Vitro

IFN-γ expression and lysis by CD3⁺ CD8⁺ cells after stimulation of PBMC with influenza virus A/NL/94-384G or A/NL/94-384R. (A to H) PBMC from an HLA-A^*0201- and HLA-B^*2705-positive donor, expanded after stimulation with influenza virus A/NL/94-384G (A, C, E, and G) or A/NL/94-384R (B, D, F, and H), were restimulated with BLCL pulsed with the M1_58-66 epitope (C and D), the NP_174-184 epitope (E and F), or the NP_383-391 epitope (G and H). Restimulation with untreated cells was used as a negative control (A and B). Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the percentages of IFN-γ⁺ cells (IFN-γ-PE) within the CD8⁺-T-cell population. FITC, fluorescein isothiocyanate. (Bottom panels) CTL specific for the M1_58-66 epitope (▴), the NP_174-184 epitope (○), and the NP_383-391 epitope (•) were also detected by a ⁵¹Cr release assay with peptide-pulsed BLCL as target cells and PBMC cultures stimulated with A/NL/94-384G (left) or A/NL/94-384R (right). Untreated cells were included as negative controls (□). Effector cells were added at different E/T ratios as indicated, and specific lysis was calculated.

Percentages of virus-specific CD8⁺ T cells in PBMC cultures stimulated in vitro. The percentages of IFN-γ⁺ CD8⁺ T cells in PBMC from donor 2 were determined after stimulation in vitro with influenza virus A/NL/94-384G (left) or A/NL/94-384R (right), without or with the HLA-B^*2705-restricted NP_383-391 epitope, respectively. Expanded cells were restimulated with autologous BLCL that were infected with influenza virus A/NL/94-384G or A/NL/94-384R or that were incubated with peptide NP_383-391 (SRYWAIRTR), rNP-HK, or rNP-NL. Virus-specific CTL were visualized after staining with MAbs specific for CD3, CD8, and IFN-γ. The data represent the proportions of CD3⁺ CD8⁺ IFN-γ⁺ cells in total PBMC cultures. These values were calculated as the product of the percentage of IFN-γ⁺ cells in the CD3⁺ CD8⁺ fraction multiplied by the percentage of CD8⁺ T cells in the PBMC culture. Error bars indicate standard deviations.

Reduction of the in vitro CTL response through loss of the NP_383-391 epitope. (A) Reduction (Δ) in the percentages of virus-specific IFN-γ⁺ CD8⁺ T cells in PBMC cultures stimulated with influenza virus A/NL/94-384G or A/NL/94-384R for all five donors. Reduction was calculated by subtracting the percentage of IFN-γ⁺ CD8⁺ T cells after restimulation with influenza virus A/NL/94-384G from the percentage of IFN-γ⁺ CD8⁺ T cells after restimulation with influenza virus A/NL/94-384R. The percentage of IFN-γ⁺ CD8⁺ T cells in PBMC cultures was determined as indicated in the legend to Fig. 3. (B) Reduction expressed as the difference in the percentages of virus-specific IFN-γ⁺ T cells within the CD3⁺ CD8⁺ fraction of PBMC cultures, as measured by flow cytometry. (C) Reduction in the percentages of virus-specific IFN-γ⁺ CD8⁺ T cells in PBMC cultures calculated as relative reduction as follows: 100 − [(percentage of IFN-γ⁺ CD8⁺ T cells after restimulation with influenza virus A/NL/94-384G × 100)/percentage of IFN-γ⁺ CD8⁺ T cells after restimulation with influenza virus A/NL/94-384R]. In all three panels, the average is shown as a horizontal bar. Numbers refer to the donors listed in Table 2.