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Clinical Trial
. 2012;8(8):e1002840.
doi: 10.1371/journal.ppat.1002840. Epub 2012 Aug 16.

CD160 and PD-1 co-expression on HIV-specific CD8 T cells defines a subset with advanced dysfunction

Yoav Peretz  1 , Zhong He, Yu Shi, Bader Yassine-Diab, Jean-Philippe Goulet, Rebeka Bordi, Ali Filali-Mouhim, Jean-Baptiste Loubert, Mohamed El-Far, Franck P Dupuy, Mohamed Rachid Boulassel, Cécile Tremblay, Jean-Pierre Routy, Nicole Bernard, Robert Balderas, Elias K Haddad, Rafick-Pierre Sékaly
Affiliations
Clinical Trial

CD160 and PD-1 co-expression on HIV-specific CD8 T cells defines a subset with advanced dysfunction

Yoav Peretz et al. PLoS Pathog. 2012.

Abstract

Chronic viral infections lead to persistent CD8 T cell activation and functional exhaustion. Expression of programmed cell death-1 (PD-1) has been associated to CD8 T cell dysfunction in HIV infection. Herein we report that another negative regulator of T cell activation, CD160, was also upregulated on HIV-specific CD8 T lymphocytes mostly during the chronic phase of infection. CD8 T cells that expressed CD160 or PD-1 were still functional whereas co-expression of CD160 and PD-1 on CD8 T cells defined a novel subset with all the characteristics of functionally exhausted T cells. Blocking the interaction of CD160 with HVEM, its natural ligand, increased HIV-specific CD8 T cell proliferation and cytokine production. Transcriptional profiling showed that CD160(-)PD-1(+)CD8 T cells encompassed a subset of CD8(+) T cells with activated transcriptional programs, while CD160(+)PD-1(+) T cells encompassed primarily CD8(+) T cells with an exhausted phenotype. The transcriptional profile of CD160(+)PD-1(+) T cells showed the downregulation of the NFκB transcriptional node and the upregulation of several inhibitors of T cell survival and function. Overall, we show that CD160 and PD-1 expressing subsets allow differentiating between activated and exhausted CD8 T cells further reinforcing the notion that restoration of function will require multipronged approaches that target several negative regulators.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CD160 and PD-1 expression defines 4 distinct subsets of HIV-specific CD8 T cells.
(A) Representative flow cytometry plot of CD160 and PD-1 co-expression on total, CMV and HIV-specific CD8 T cells. (B) Scatter plots represent the median frequency of 4 distinct subsets of CD8 T cells based on CD160 and PD-1 expression (CD160−PD-1−; CD160−PD-1+; CD160+PD-1−; CD160+PD-1+) from 7 HIV-1 uninfected and 38 HIV-infected subjects separated into four groups: 7 during acute/early infection (AHI), 9 chronic progressors (CHI), 12 successfully treated(ST) and 10 Elite controllers (ECs). PBMCs were labelled with fluorochrome conjugated αCD3, αCD8, αPD-1,αCD160 and HLA class I-matched tetramers (see materials and methods). Dying cells were eliminated with an amine-reactive viability dye. Blue and red dots represent CMV and HIV-specific CD8 T cells, respectively. P-values were determined by Mann Whitney and unpaired t tests.
Figure 2
Figure 2. The frequency of HIV-specific CD8 T cells with a CD160+PD-1+ phenotype increases with disease progression.
(A) Representative flow cytometry plots of CMV and HIV-specific CD8 T cells (A*02 CMV and A*03 Gag) detected during AHI (<3 months) and CHI (>6 months) within the same individual. Frequencies of 4 distinct CD8 T cell subsets expressing CD160 and/or PD-1 were measured at both time points. Figures represent the frequency of CD160 and/or PD-1 expressing subsets over time for (B) HIV and (C) CMV/EBV-specific responses in 4 HIV-infected subjects. P-values were determined by the Wilcoxon matched pairs test.
Figure 3
Figure 3. Co-expression of CD160 and PD-1 identifies a fully exhausted antigen-specific CD8 T cell.
PBMCs were isolated from HIV viremic individuals (n = 14) and stimulated with (A) SEB, (B) CMV and (C) HIV-1 peptides and analyzed by multiparametric flow cytometry for CD160, PD-1, IFNγ, TNFα and CD107a expression. HIV and CMV-specific CD8 T cells were identified using PE-conjugated tetramers. Dying cells were eliminated with an amine-reactive viability dye. # symbols represent statistically significant comparisons with the DP subset. P-values were determined by the Wilcoxon matched pairs test. ##represents a P value<0.005 and # represents a P value<0.05.
Figure 4
Figure 4. Blocking the interaction between CD160 and HVEM enhances CMV and HIV-specific CD8 T cell proliferation and cytokine production.
HIV-infected individuals(n = 11) were stimulated with HLA-restricted CMV and HIV peptides in the presence of blocking antibodies to HVEM and/or PD-L1. Dying cells were eliminated with an amine-reactive viability dye and PBMCs were stained at day 6 with HLA class I matched-tetramers and mAbs to CD3 and CD8. (A) Representative flow cytometry plots of an HIV-infected patient stimulated for 6 days with a CMV and HIV peptide in the presence of isotype, αPD-L1 and/or αHVEM blocking antibodies. Scatter plots represent the median fold increase in (B) CMV and (C) HIV-specific proliferation (Tetramer+/CFSElow) compared to the isotype control. Each dot represents a CMV or HIV tetramer-specific response. P-values were determined by the Wilcoxon matched pairs test.
Figure 5
Figure 5. CD160+PD-1+CD8 T cells represent a distinct subset with a unique transcriptional profile.
PBMCs were stained with 7AAD (cell viability dye), αCD3, αCD8, αPD-1, αCD160 and total CD8 T cells were sorted using a FACS ARIA based on CD160 and PD-1 expression. (A) Heatmap illustrating the differentially expressed genes (39 genes; p<0.05) between DP and SP-PD-1 sorted subsets. (B) Network analysis of significantly inferred genes and predicted targets. Node colors indicate fold change of gene expression between ex-vivo sorted CD160+PD-1+ and CD160−PD-1+ CD8 T cells sorted from 4 HIV viremic patients. The different shapes indicate genes in the different functional categories (see legend). (C) Histogram and scatter plot showing the MFI of KIR2DL2 and KIR2DL3 expression on DP and SP-PD-1 subsets in 6 HIV viremic patients.
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