PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Abstract

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8(+) T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8(+) T cells and increased on memory CD8(+) T cells according to antigen specificity. Memory CD8(+) T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8(+) T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8(+) T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8(+) T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8(+) T cell numbers, but possibly not all functions in vivo.

Figure 1.

PD-1 is highly expressed on HIV-specific CD8⁺ T cells. (A) Representative histograms depicting PD-1, PD-L1, and PD-L2 expression in monocytes, mDCs, and pDCs from one subject tested. Cell preparations from at least three donors were tested. (B) The polychromatic flow cytometry gating scheme for identification of cell populations is shown. Histograms depict the PD-1 expression in HIV- and CMV-specific CD8⁺ T cells from the same sample. Memory subsets identified by CD27 and CD45RO staining of total CD8⁺ T cells are presented. (C) Pooled data showing the percentage of CD8⁺ T cells expressing PD-1⁺ phenotype in total CD8⁺ T cells from healthy donors (n = 7) and HIV⁺ individuals (n = 17), HIV-specific (n = 11), CMV-specific (n = 12), and EBV-specific (n = 5) CD8⁺ T cells from HIV⁺ individuals, and VV-specific CD8⁺ T cells from vaccinated healthy donors (n = 3). PD-1 expression in memory subsets of total CD8⁺ T cells from HIV⁺ and healthy donors is also shown. Horizontal lines depict mean values. The p values were calculated using Student's t test.

Figure 2.

PD-1 expression is independent of the maturational status of HIV- and CMV-specific CD8⁺ T cells. (A) Representative flow cytometry showing the distribution of PD-1⁺ and PD-1⁻ HIV- and CMV-specific CD8⁺ T cells in memory CD8⁺ T cell subsets. Cells were gated as in Fig. 1. The distribution of total CD8⁺ T cells is also shown. (B) Pooled data showing the percentage of CD8⁺ T cells in memory populations for PD-1⁺ and PD-1⁻ subsets of HIV-specific (n = 8) and CMV-specific (n = 11) CD8⁺ T cells from HIV-infected individuals. The p values were calculated using Student's t test.

Figure 3.

No direct association between PD-1 expression and cytokine production by virus-specific CD8⁺ T cells. (A) Pooled data depicting the percentage of antigen-specific CD8⁺ T cells that produce IFN-γ (left), TNF-α (middle), or IL-2 (right) after stimulation with HIV Gag peptides, CMV pp65 peptides, or VV. Cells from HIV⁺ (n = 7), CMV⁺ (n = 10), and healthy individuals immunized with modified vaccinia virus Ankara (n = 25) were tested. (B) PBMCs from an HIV and CMV coinfected subject were stimulated with A2-Gag or A2-CMV peptides, and epitope-specific CD8⁺ T cells were gated according to their expression of tetramer and/or production of cytokine, and the production of cytokine in the PD-1⁺ and PD-1⁻ subsets was assessed. The left panel shows the PD-1 staining of CD8⁺ cells to demonstrate how the gating of PD-1⁺ and PD-1⁻ cells was chosen. (C) Pooled data showing the percentage of nonfunctional virus-specific CD8⁺ T cells from three HIV-infected individuals off antiretroviral therapy [(tetramer⁺cytokine⁻) × 100/(tetramer⁺cytokine⁻) + (tetramer⁺cytokine⁺)] at the different peptide concentrations for both PD-1⁺ and PD-1⁻ compartments are shown. (D) Flow cytometry showing the production of IFN-γ, TNF-α, or IL-2 from CD8⁺ T cells after stimulation with Gag or CMV peptides for 6 h in the absence or presence of anti–human PD-1 antibody (left). Pooled data are shown on the right. Samples from three HIV⁺ individuals were tested.

Figure 4.

PD-1–PD-L1 manipulation alters virus-specific CD8⁺ T cell proliferation and cytokine production. (A) Representative histograms depicting the CFSE profile of CD8⁺ T cells from an HIV⁺ donor. Pooled data showing the fold change of the percentage of CFSE low cells under treatment with aPD-L1 or aPD-1 antibody (right). The percentage of cells that divided in the absence of antibody treatment was assigned a value of 1. The ratio of the percentage in the presence and absence of antibody treatment for each response was calculated. The fold change for antigen-specific CD8⁺ (those that diluted CFSE in response to antigen; • and ◯) and gated antigen-specific tetramer⁺ CD8⁺ T cells (□ and ▪) is shown. The p values were calculated using Wilcoxon's paired t test. (B) Flow cytometry showing the CD8⁺ T cells secreting IFN-γ and TNF-α after stimulation with Gag peptides in the absence or presence of anti–PD-1 or anti–PD-L1 antibodies. Cells cultured for 6 d were restimulated with Gag peptides for the last 6 h (left). Pooled data showing the percentage of CD8⁺ T cells producing IFN-γ and TNF-α under these conditions (right). At least four patients were tested for each treatment.

Figure 5.

PD-1 regulates the in vitro survival of virus-specific CD8⁺ T cells. (A) Representative histograms depicting the annexin V positivity in total, PD-1⁺ and PD-1⁻ CD8⁺ T cells from an HIV⁺ donor under spontaneous or CD95/Fas-induced apoptosis. (B) Pooled data showing the percentage of annexin V⁺ cells in PD-1⁺ and PD-1⁻ subsets of total (n = 8), HIV- (n = 6), and CMV-specific (n = 5) CD8⁺ T cells from HIV⁺ individuals. (C) Annexin V positivity in PD-1⁺ versus PD-1⁻ CD8⁺ T cells in memory subsets defined by CD27 and CD45RO markers. (D) PBMCs were cultured in the absence or presence of plate-bound anti–PD-1, and the percentage of annexin V⁺ cells was determined for CD8⁺ T cell subsets with different levels of PD-1 expression. Flow cytometry showing the different levels of spontaneous and anti–PD-1–induced apoptosis according to the level of PD-1 expression (high, medium/high, medium/low, and low) in total, Gag- and CMV-specific CD8⁺ T cells from the same HIV⁺ donor.