Programmed death-1 is a marker for abnormal distribution of naive/memory T cell subsets in HIV-1 infection

Abstract

Chronic activation of T cells is a hallmark of HIV-1 infection and plays an important role in disease progression. We previously showed that the engagement of the inhibitory receptor programmed death (PD)-1 on HIV-1-specific CD4(+) and CD8(+) T cells leads to their functional exhaustion in vitro. However, little is known about the impact of PD-1 expression on the turnover and maturation status of T cells during the course of the disease. In this study, we show that PD-1 is upregulated on all T cell subsets, including naive, central memory, and transitional memory T cells in HIV-1-infected subjects. PD-1 is expressed at similar levels on most CD4(+) T cells during the acute and the chronic phase of disease and identifies cells that have recently entered the cell cycle. In contrast, PD-1 expression is dramatically increased in CD8(+) T cells during the transition from acute to chronic infection, and this is associated with reduced levels of cell proliferation. The failure to downregulate expression of PD-1 in most T cells during chronic HIV-1 infection is associated with persistent alterations in the distribution of T cell subsets and is associated with impaired responses to IL-7. Our findings identify PD-1 as a marker for aberrant distribution of T cell subsets in HIV-1 infection.

Figure 1

Disruption of T cell subsets homeostasis in HIV-1 infection is specific to each disease stage. A. Seven-parameter flow cytometry gating strategy to identify CD4⁺ and CD8⁺ T cell subsets. T_N, T_CM, T_TM, T_EM and T_TD/T_E subsets are identified based on their CD45RA, CCR7 and CD27 expression. B. Distribution of blood CD4⁺ and CD8⁺ T cell subpopulations in acute-untreated (n=8; black symbol), chronic-untreated (n=13; red symbol), chronic-treated (n=15; blue symbol) and uninfected (n=15; green symbol) subjects. Each symbol represents one subject. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.

Figure 2

Differentiation toward senescent stages of all T cell subsets characterized by loss of CD28 expression and increased CD57 expression in HIV-1 infection. a. Percentage of ex-vivo CD28 expressing cells in CD4⁺ and CD8⁺ T cell subsets in HIV-1 infected individuals and controls. b. Percentage of ex-vivo CD57 expressing cells in CD4⁺ and CD8⁺ T cell subsets in HIV-1 infected individuals and controls. Each symbol represents one subject: acute-untreated (n=8; black symbol), chronic-untreated (n=13; red symbol), chronic-treated (n=15; blue symbol) and uninfected (n=15; green symbol) subjects. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.

Figure 3

Increased percentage of Ki67⁺ proliferating cells in CD4⁺ and CD8⁺ T cell subsets during HIV-1 infection. Bars represent mean values of acute-untreated (n=8; black bar), chronic-untreated (n=13; red bar) chronic-treated (n=15; blue bar) and uninfected (n=15; green bar) subjects. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.

Figure 4

PD-1 up regulation on all T cell subsets in both acute and chronic infection. Each symbol represents one subject: acute-untreated (n=8; black symbol), chronic-untreated (n=13; red symbol), chronic-treated (n=15; blue symbol) and uninfected (n=15; green symbol) subjects. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.

Figure 5

A. Concomitant expressions of Ki67 and PD-1 in CD8⁺ T cell subsets allow to discriminate between acute and chronic HIV-1 infection. Each symbol represents one subject: acute-untreated (n=8; black symbol), chronic-untreated (n=13; red symbol), chronic-treated (n=15; blue symbol) and uninfected (n=15; green symbol) subjects. B. Expression of PD-1 on Ki67⁺ cells versus Ki67⁻ cells in the CD8 compartment is much higher in chronic than in acute HIV-1 infection. Each bar represents one group of subjects: acute-untreated (n=8; black bar), chronic-untreated (n=13; red bar), chronic-treated (n=15; blue bar) and uninfected (n=15; green bar) subjects. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.

Figure 6

Baseline Bcl-2 and IL-7 induced Stat-5 phosphorylation in PD-1^high and PD-1^low CD45RA⁻ memory T cells. A. Representative dot plot analysis showing Bcl-2 expression in PD-1^high and PD-1^low CD45RA⁻ CD4⁺ or CD8⁺ T cells. B. Bcl-2 expression in PD-1^high and PD-1^low CD45RA⁻ CD4⁺ or CD8⁺ T cells of HIV-1 infected individuals and controls. Each symbol represents one subject: chronic-untreated (n=8; red symbol), and uninfected (n=5; green symbol) subjects. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001. C. Representative dot plot analysis showing pStat5a expression in PD-1^high and PD-1^low CD45RA⁻ CD4⁺ or CD8⁺ T cells. D. pStat5a expression in PD-1^high and PD-1^low CD45RA⁻ CD4⁺ or CD8⁺ T cells of HIV-1 infected individuals and controls. Each symbol represents one subject: chronic-untreated (n=8; red symbol), and uninfected (n=5; green symbol) subjects. Means are shown as horizontal bars. Statistical significance was determined using the Mann-Whitney U test. * p<0.05, ** p<0.001, *** p<0.0001.