HIV-1-specific IFN-gamma/IL-2-secreting CD8 T cells support CD4-independent proliferation of HIV-1-specific CD8 T cells

Abstract

Functional and phenotypic characterization of virus-specific CD8 T cells against cytomegalovirus, Epstein-Barr virus, influenza (flu), and HIV-1 were performed on the basis of the ability of CD8 T cells to secrete IFN-gamma and IL-2, to proliferate, and to express CD45RA and CCR7. Two functional distinct populations of CD8 T cells were identified: (i) dual IFN-gamma/IL-2-secreting cells and (ii) single IFN-gamma-secreting cells. Virus-specific IFN-gamma/IL-2-secreting CD8 T cells were CD45RA-CCR7-, whereas single IFN-gamma CD8 T cells were either CD45RA-CCR7- or CD45RA+CCR7-. The proportion of virus-specific IFN-gamma/IL-2-secreting CD8 T cells correlated with that of proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell proliferation. Substantial proliferation of virus-specific CD8 T cells (including HIV-1-specific CD8 T cells) was also observed in CD4 T cell-depleted populations or after stimulation with MHC class I tetramer-peptide complexes. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation. These results indicate that IFN-gamma/IL-2-secreting CD8 T cells may promote antigen-specific proliferation of CD8 T cells even in the absence of helper CD4 T cells.

Fig. 1.

Analysis of different virus-specific IFN-γ- and IL-2-secreting CD8 T cells after stimulation with single peptides. (A) Distribution of IFN-γ- and IL-2-secreting virus-specific CD8 T cells. Cells were stimulated with single peptides. One representative profile is shown for HIV-1-, CMV-, EBV-, or flu-specific CD8 T cell responses. The cluster of events shown in red corresponds to the responder CD8 T cells, i.e., secreting IFN-γ or IL-2, and the blue clusters correspond to the nonresponder cells. (B) Cumulative data on the percentage (mean ± SE) of IFN-γ/IL-2-secreting cells within the different virus-specific CD8 T cell responses. (C) Cumulative data on the proportion (mean ± SE) of IL-2-secreting cells within IFN-γ-secreting CD8 T cells. ^*, P < 0.05.

Fig. 2.

Analysis of HIV-1-specific IFN-γ- and IL-2-secreting CD8 T cells in progressors and LTNPs after stimulation with peptide pools. Flow cytometry profiles of IFN-γ- and IL-2-secreting HIV-1-specific CD8 T cells of progressor 2113 (A) and three different LTNPs (B) after stimulation of blood mononuclear cells with different peptide pools spanning gag, pol, and nef proteins.

Fig. 3.

IFN-γ- and IL-2-secreting CD8 T cells in different populations defined by CD45RA and CCR7. Shown is the distribution of IFN-γ- and IL-2-secreting CD8 T cells in different populations defined by CD45RA and CCR7. (A) Cells of LTNP 2073 were stimulated with different peptide pools spanning gag, pol, and nef proteins. (B) Cells of subjects 205 and 35 were stimulated with CMV or flu peptides, respectively.

Fig. 4.

Virus-specific CD8 T cell proliferation after stimulation with single peptides or peptide pools. (A) CFSE-labeled cells of HIV-negative donors 248 and 359 were stimulated with CMV-, flu-, or EBV-derived peptides. Profiles of proliferating cells, i.e., CFSE low cells, are gated on CD8 T cells. (B) HIV-1-specific CD8 T cell proliferation in HIV-1 progressors after stimulation with different HIV-1 peptide pools or SEB. (C) HIV-1-specific CD8 T cell proliferation in LTNPs after stimulation with different HIV-1 peptide pools. (D) Correlation between the proportion of IL-2-secreting and -proliferating virus-specific CD8 T cells.

Fig. 5.

Virus-specific CD8 T cell proliferation after stimulation with HLA class I tetramers. (A) Blood mononuclear cells of HIV-negative donors 172 and 180 were stimulated with A2-flu or -CMV tetramers, respectively. Flow cytometry profiles of proliferating CD8 (Left) and CD4 (Right) T cells are shown. (B) Blood mononuclear cells of progressor 2056 were stimulated with an A2-pol tetramer and cultured in the absence or presence of 10% of exogenous IL-2.

Fig. 6.

Virus-specific CD8 T cell proliferation in CD4-depleted cells or after neutralization of IL-2. (A) CD8 T cell proliferation was evaluated in CD4 T cell-depleted populations stimulated with HIV-1-derived peptide. The purity of the sorted CD4^- T cell populations was 99%. (B) Inhibition of virus-specific CD8 T cell proliferation with anti-IL-2 Ab. Cells of subject 180 were stimulated with an A2-restricted CMV tetramer and cultured in the presence of anti-IL-2 or isotype control Abs.