Human cytotoxic T-lymphocyte repertoire to influenza A viruses

Abstract

The murine CD8(+) cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8(+) CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4(+) cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.

Figures

FIG. 1

(A) Influenza A virus subtype-cross-reactive lysis in bulk culture (donor 1). PBMC were stimulated with A/PR/8/34 virus in vitro on days 0 and 7 and tested on day 14 at an E-T ratio of 60:1. (B) Recognition of influenza A virus proteins in a bulk culture CTL assay (donor 1). The same bulk culture was tested for specific lysis of influenza viral proteins expressed in vaccinia virus (Vac.) constructs infected in BLCL at an E-T ratio of 60:1. Lysis of targets infected with wild-type vaccinia virus was subtracted from lysis of recombinant vaccinia virus-infected targets.

FIG. 2

MHC restriction of cell lines determined by using panels of partially HLA-matched allogeneic targets infected with vaccinia virus recombinants expressing either an influenza virus protein or a specific peptide. Lysis of targets infected with wild-type vaccinia virus was subtracted from lysis of recombinant vaccinia virus-infected targets. (A) Bulk culture cells tested against targets loaded with the NP aa 174 to 184 peptide. (B) Cell line 10-1B7 tested against vaccinia virus PB2-infected targets. (C) Cell line 1-2F8 tested against vaccinia virus PB1-infected targets. (D) Cell line 4-30E11 tested against vaccinia virus M1-infected targets. (E) Cell line 10-1G5 tested against vaccinia virus HA-infected targets. The E-T ratio was 10:1, except for panel A, for which the E-T ratio was 30:1.

FIG. 3

Mapping of CTL epitopes by using peptides. The epitopes recognized by cell line 10-1C4 from donor 1 (A), 3E5 from donor 2 (B), and 124 (C) and 77 (D) from donor 3 were mapped by using peptide-pulsed BLCL targets at a concentration of 25 μg/ml. The E-T ratios were 10:1 (A and B), 7.5:1 (C), and 15:1 (D).

FIG. 4

Dose response of peptide-pulsed target cell lysis. Cell line 10-1C4 from donor 1 (A), 3E5 from donor 2 (B), and 124 (C) and 77 (D) from donor 3 recognized target cells pulsed with various peptide concentrations. The E-T ratios were 10:1 (A and B) and 15:1 (C and D).