Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression

Abstract

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

FIG. 1.

Degranulation activity and cytotoxic granules content of virus-specific CD8 T cells. (A) Flow cytometry profiles of the degranulation activity of CD8 T cells measured by CD107a mobilization after stimulation with Flu-, EBV-, CMV-, or HIV-1-derived peptides. (B) Cumulative data (means ± the SE) of the proportion of CD107a⁺ cells among IFN-γ-secreting CD8 T cells after Ag-specific stimulation. At least 600,000 live-gated events were acquired. With regard to the criteria of positivity of the ICS, the background in the unstimulated controls never exceeded 0.03%. An ICS to be considered positive had to have >0.05% cytokine-positive cells after subtraction of the background. (C) Perforin and GrmB, GrmA, and GrmK expression in virus-specific CD8 T cells. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) of a given virus-specific CD8 T-cell response versus all of the others. At least 600,000 live-gated events were acquired.

FIG. 2.

Correlation between CD127 expression, proliferation capacity, and IL-2 production. (A) CD8⁺ CD127⁻ and CD8⁺ CD127⁺ T cells were sorted (purity > 95%), stained with CFSE, and stimulated for 7 days with the CMV-derived peptide TPRVTGGGAM. Flow cytometry profiles show CFSE dilution in the two sorted populations after 7 days in vitro stimulation. Cumulative data of the percentage (means ± the SE) of proliferating (CFSE^low) CD8⁺ CD127⁻ and CD8⁺ CD127⁺ sorted T-cell populations after stimulation with CMV-, EBV-, or Flu-derived peptides. The data were obtained in six separate experiments. (B) Correlation between the percentage of CD127-expressing virus-specific CD8 T cells prior stimulation with the cognate peptides and the proliferation capacity (fold increase) of virus-specific CD8 T cells after 7 days of in vitro stimulation. The data were obtained by pooling 32 virus-specific CD8 T-cell stimulations. (C) Percentages of CD127 expression on Flu-, EBV-, CMV-, and HIV-1-specific CD8 T cells. (D) Flow cytometry profiles showing combinations of CD127 expression with IFN-γ- or IL-2-secreting CD8 T cells after stimulation with a CMV peptide (left panels). The right panel shows overlaid histograms of the distribution of IL-2-secreting CD8 T cells on CMV-specific (i.e., IFN-γ-secreting) CD127⁻ and CD127⁺ CD8 T cells. (E) Cumulative data on the distribution of IFN-γ- and/or IL-2-secreting CD8 T cells on CD127⁻ and CD127⁺ CD8 T cells. Of note, these data include HIV-1-specific CD8 T-cell responses from a patient treated at the time of the acute infection who underwent spontaneous treatment interruption and with an undetectable viremia (<5 HIV-1 RNA copies/μl of plasma) at the time of the analysis. Combinations of IFN-γ and IL-2 are shown on the x axis, whereas the percentage of virus-specific CD8 T cells is shown on the y axis. The pie charts summarize the data, and each slice corresponds to the proportion of virus-specific CD8 T cells positive for a certain combination of T-cell functions. In the pie charts, the blue line indicates functional subsets positive for IL-2.

FIG. 3.

Combination of CD127 and perforin expression, association with T-cell differentiation, and PD-1 and CD57 expression. (A) Representative flow cytometry profile and cumulative data showing that the combination of CD127 and perforin identified three distinct populations of CD8 T cells, i.e., CD127⁺ perforin⁻, CD127⁻ perforin⁻, and CD127⁻ perforin⁺. (B) Polychromatic flow cytometry assay combining CD8, perforin, and CD127, together with CD45RA and CCR7. Shown is the distribution of CD127 and perforin on the four populations defined by CD45RA and CCR7. This experiment is representative of five independent assays on five distinct subjects. (C) PD-1 expression on CD127⁺ perforin⁻, CD127⁻ perforin⁻, and CD127⁻ perforin⁺ CD8 T-cell populations. A representative histogram plot and cumulative data ( n = 7) are shown. PD-1 FMO control (for fluorescence minus one) was used to confirm the accuracy of PD-1 expression. (D) Cumulative data of PD-1 expression (MFI) on CD45RA⁺ CCR7⁺, CD45RA⁻ CCR7⁺, CD45RA⁻ CCR7⁻, and CD45RA⁺ CCR7⁻ CD8 T-cell populations (n = 8). (E) CD57 expression on CD127⁺ perforin⁻, CD127⁻ perforin⁻, and CD127⁻ perforin⁺ CD8 T-cell populations. A representative histogram plot and cumulative data (n = 7) are shown. MFI, mean fluorescence intensity.

FIG. 4.

Combination of CD127 and perforin expression, association with T-cell differentiation and CD57 expression on virus-specific CD8 T cells. (A) Representative examples and cumulative data of perforin and CD127 expression on Flu-, EBV-, CMV-, and HIV-1-specific CD8 T cells (identified by peptide-HLA tetramer complexes). (B) Inverse correlation between the percentages of perforin and CD127 expression on CMV-, EBV-, and Flu-specific CD8 T cells. (C) Inverse correlation between the percentage of CD127 expression and the percentage of specific lysis of virus-specific CD8 T-cell responses. (D) Polychromatic flow cytometry analysis combining perforin and CD127, together with CD45RA and CCR7, in virus-specific CD8 T cells. Representative examples of the distribution of CD127 and perforin on the populations defined by CD45RA and CCR7 in Flu-, EBV-, CMV-, and HIV-1-specific CD8 T-cell responses are shown. This experiment is representative of four independent experiments. (E) Representative example of CD57 expression on CMV-specific CD127⁺ perforin⁻, CD127⁻ perforin⁻, and CD127⁻ perforin⁺ CD8 T-cell populations.

FIG. 5.

Association between perforin expression and IL-2 secretion on CMV-specific CD8 T-cell responses. (A) Representative example of perforin expression and IFN-γ, TNF-α, and IL-2 secretion on CMV-specific CD8 T cells. (B) Analysis of perforin, IFN-γ, and TNF-α expression on IL-2-negative and IL-2-positive CMV-specific CD8 T-cell subsets. (C) Analysis of IL-2, IFN-γ,and TNF-α on perforin-negative and perforin-positive CMV-specific CD8 T-cell subsets. (D) Correlation between the proportion of CMV-specific CD8 T cells expressing perforin or secreting IL-2. In panels B and C, all of the possible combinations of the different markers are shown on the x axis, whereas the percentage of CMV-specific CD8 T cells is shown on the y axis. The pie charts summarize the data, and each slice corresponds to the proportion of CMV-specific CD8 T cells positive for a certain combination of T-cell functions.