Programmed Death-1 Expression on Epstein Barr Virus Specific CD8+ T Cells Varies by Stage of Infection, Epitope Specificity, and T-Cell Receptor Usage

BACKGROUND: Programmed Death-1 (PD-1) is an inhibitory member of the CD28 family of molecules expressed on CD8+ T cells in response to antigenic stimulation. To better understand the role of PD-1 in antiviral immunity we examined the expression of PD-1 on Epstein-Barr virus (EBV) epitope-specific CD8+ T cells during acute infectious mononucleosis (AIM) and convalescence. METHODOLOGY/PRINCIPAL FINDINGS: Using flow cytometry, we observed higher frequencies of EBV-specific CD8+ T cells and higher intensity of PD-1 expression on EBV-specific CD8+ T cells during AIM than during convalescence. PD-1 expression during AIM directly correlated with viral load and with the subsequent degree of CD8+ T cell contraction in convalescence. Consistent differences in PD-1 expression were observed between CD8+ T cells with specificity for two different EBV lytic antigen epitopes. Similar differences were observed in the degree to which PD-1 was upregulated on these epitope-specific CD8+ T cells following peptide stimulation in vitro. EBV epitope-specific CD8+ T cell proliferative responses to peptide stimulation were diminished during AIM regardless of PD-1 expression and were unaffected by blocking PD-1 interactions with PD-L1. Significant variability in PD-1 expression was observed on EBV epitope-specific CD8+ T cell subsets defined by V-beta usage. CONCLUSIONS/SIGNIFICANCE: These observations suggest that PD-1 expression is not only dependent on the degree of antigen presentation, but also on undefined characteristics of the responding cell that segregate with epitope specificity and V-beta usage

Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART.

Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25μg/mL) or nivolumab (5 or 1.25μg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency

PD-1 expression on CD8+ T cells during AIM: correlations with viral load and contraction at convalescence.

<p><b>A</b>. Left panel: Viral load (log transformed) and the corresponding PD-1 MFI of CD8+ T cells are plotted (Spearman r = 0.698; p = 0.008). Right panel: Viral load (log transformed) and the corresponding PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) are plotted (Spearman r = 0.170; p = 0.578 and r = 0.560; p = 0.046, respectively). <b>B</b>. Left panel: PD-1 MFI of CD8+ T cells during AIM and the corresponding fold-change in CD8+ T cells (log-transformed) are plotted (Spearman r = 0.955; p<0.0001). The fold-change in CD8+ T cells is the ratio of the cell #/ml during AIM over the cell #/ml during convalescence. Right panel: PD-1 MFI of A2-BMLF-1 (solid squares, dotted line) and A2-BRLF-1 cells (open circles, solid line) during AIM and the corresponding fold-change in each of these subsets (log-transformed) are plotted (Spearman r = 0.646; p = 0.037, and r = 0.764; p = 0.009, respectively). Lines of best fit are shown for each combination of variables.</p

Differential PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells at AIM and convalescence.

<p>PD-1 expression on A2-BMLF-1 and A2-BRLF-1 CD8+ T cells during AIM (upper panels) and convalescence (lower panels). Representative dot plots demonstrate differences in PD-1 percent positivity, and histograms showing differences in distribution and PD-1 MFI of these EBV-specific CD8+ T cells. Histograms overlay the two subsets on a background of the total CD8+ T cell population.</p

EBV-specific CD8+ T cells and viral load during AIM and convalescence.

<p><b>A</b>. Absolute values (cells per ml) of CD8+ T cells, and three different EBV-specific subsets (lytic and latent antigen specific) measured in samples from individuals during AIM (within the first two weeks after presentation with symptoms) and convalescence (CONV; 6–12 months after presentation with symptoms). A2-BMLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BMLF-1, GLCTLVAML; A2-BRLF-1 CD8+ T cells: specific for the HLA-A*0201 restricted epitope of BRLF-1, YVLDHLIVV; A2-EBNA-3c CD8+ T cells: specific for the HLA-A*0201 restricted epitope of EBNA-3c, LLDFVRFMGV. When no EBV-specific CD8+ T cells were detected with these reagents, a value of 100 cells per milliliter was assigned (less than half the lowest detected value). <b>B</b>. Viral load (EBV copies per million B cells) during AIM and convalescence. Lines define paired results for each individual. All paired comparisons indicated by brackets differ significantly by the Wilcoxon signed rank test; * p<0.05, ** p<0.01, *** p<0.001.</p

Analysis of CD127 and PD-1 expression on CD8+ T cells.

<p><b>A</b>. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells that are CD127+ in AIM and convalescence (mean and SEM). <b>B</b>. Percentages of A2-BMLF-1, A2-BRLF-1, and total CD8+ T cells subsets that are PD-1 high or PD-1 negative that are also CD127+ (in AIM and convalescence). Brackets indicate paired comparisons that differ significantly, Wilcoxon signed rank test, *p<0.05.</p

PD-1 expression on EBV-specific CD8+ T cells at AIM and convalescence.

<p><b>A</b>. The percentages of EBV-specific CD8+ T cells that expressed PD-1 are shown. Two lytic (A2-BMLF-1 and A2-BRLF-1) and two latent (A2-EBNA-3c and B7-EBNA-3a CD8+ T cells) antigen specific CD8+ T cell subsets are shown. Lines define paired results for each individual. <b>B</b>. The median fluorescence index (MFI) of PD-1 expression for these EBV-specific CD8+ T cells is shown. All assays were performed with the PD-1 antibody conjugated to PE. The MFI was normalized as described in methods. Lines define paired results for each individual. All paired comparisons indicated by brackets differ significantly by the Wilcoxon signed rank test; * p<0.05, ** p<0.01, *** p<0.001.</p

High level PD-1 expression on CD8+ T cells during AIM and convalescence.

<p><b>A</b>. Representative CD8+ T cell plots of a sample obtained during AIM demonstrating the gating strategy to define PD-1 high, dim and negative populations. <b>B</b>. Percentages of PD-1 high, dim, or negative CD8+ T cells in AIM and convalescence. <b>C</b>. Percentages of PD-1 high, dim, or negative A2-BMLF-1 and A2-BRLF-1 CD8+ T cells in AIM and convalescence. Lines define paired results for each individual. Brackets indicate that differences between selected comparisons are significant (Wilcoxon signed rank test; *p<0.05, ** p<0.01, *** p<0.001).</p

Proliferation and PD-1 expression on CD8+ T cells responding to peptide stimulation.

<p><b>A</b>. Three representative examples of proliferation upon stimulation with HLA-A*0201-restricted BMLF-1 and BRLF-1 peptides. Upper and middle rows: PBMC from an individual at presentation with AIM and in convalescence; lower row: PBMC from an individual with chronic infection. Left panels (baseline prior to peptide stimulation) show PD-1 expression of the indicated tetramer positive cells (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on that of CD8+ T cells (grey solid). Middle panels show CD8+ T cells that proliferate (CFSE dilution) in response to the indicated peptides (A2 BMLF-1 black line; A2 BRLF-1 grey line) superimposed on unstimulated controls (grey solid). Right panels show the upregulation of PD-1 on CD8+ T cells responding to the indicated peptides superimposed on unstimulated CD8+ T cells. Marked regions on histograms depicting PD-1 expression indicate high levels of expression with corresponding percentages of A2 BMLF-1 and A2-BRLF-1 CD8+ T cells. Numbers in text boxes are the percentages of CD8+ T cells that are positive for each tetramer at baseline (left panels), or responding to peptide post-stimulation (CFSE low; middle panels). <b>B</b>. Upregulation of PD-1 on cells responding to stimulation with A2-BMLF-1 and A2-BRLF-1 peptides. Results shown are a mixture of chronic and convalescent samples in three separate assays. To demonstrate the extent of PD-1 upregulation observed, PD-1 MFI is expressed as a ratio of the MFI of cells that responded to peptide, relative to the MFI in control assays without peptide. Median PD-1 MFI ratio values for A2-BMLF-1 and A2-BRLF-1 were 8.1 and 2.8, respectively (Wilcoxon signed rank test; p = 0.06). Solid and open symbols demonstrate paired values for A2-BMLF-1 and A2-BRLF-1 for each sample.</p