A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro

Abstract

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP(383-391)]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP(383-391) epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using (51)Cr release assays with a T-cell clone specific for the NP(383-391) epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B*2705-positive target cells pulsed with the original NP(383-391) peptide. The proportion of virus-specific CD8+ gamma interferon (IFN-gamma)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-gamma staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B*2705. The proportion of virus-specific CD8+ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP(383-391) epitope is the most important HLA-B*2705-restricted epitope in the nucleoprotein of influenza A viruses.

FIG. 1.

Recognition of BLCL infected with recombinant influenza viruses by CTL clones. HLA-A^*0201- and HLA-B^*2705-positive BLCL were infected with influenza virus A/NL/94-384G (○) or A/NL/94-384R (•), pulsed with M1_58-66 (▵) or NP_383-391 (▴) peptide or left untreated (□), and used as target cells for CD8⁺-T-cell clones specific for the HLA-A^*0201-restricted M1_58-66 epitope (A) and the HLA-B^*2705-restricted NP_383-391 epitope (B) in a ⁵¹Cr release assay. CTL clones were added at different E/T ratios as indicated, and specific lysis was calculated.

FIG. 2.

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.

FIG. 3.

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.

FIG. 4.

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.

FIG. 5.

Recognition of BLCL infected with influenza viruses (A and B) or incubated with rNP (C and D) by in vitro-stimulated PBMC obtained from donor 2. PBMC expanded after stimulation with influenza virus A/NL/94-384G (A and C) or A/NL/94-384R (B and D) were used as effector cells in ⁵¹Cr release assays. Autologous BLCL infected with influenza virus A/NL/94-384G (○) or A/NL/94-384R (•) or BLCL incubated with rNP derived from influenza virus A/NL/18/94 (▵) or A/HK/2/68 (▴) were used as target cells. Untreated cells were included as negative controls (▪). The data represent the percentages of specific lysis at the indicated E/T ratios.