Functional impairment of HIV-specific CD4+ T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4+ and CD8+ T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4+ T cells. To investigate the role of PD-1 in HIV-associated CD4+ T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-γ-producing HIV-specific CD4+ T cells compared with total or CMV-specific CD4+ T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4+ T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4+ T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4+ T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4+ T cells.

Elevated expression of inhibitory receptors on virus-specific T cells has been implicated as a mechanism by which viruses evade host immune surveillance. Blockade of these pathways during chronic infection leads to increased T cell function and improved immune control of viral replication. To explore the association between costimulatory receptors and HIV replication, we examined the expression of programmed death 1 (PD-1), CTLA-4, T cell Ig domain and mucin domain 3 (TIM-3), and CD28 on HIV-specific CD4+ T cells from HIV-infected subjects. Greater than 30% of HIV-specific CD4+ T cells from untreated subjects coexpressed PD-1, CTLA-4, and TIM-3, whereas <2% of CMV- or varicella-zoster virus-specific CD4+ T cells expressed all three receptors. Coexpression of all three inhibitory receptors on HIV-specific CD4+ T cells was more strongly correlated with viral load compared with the expression of each receptor individually. Suppression of HIV replication with antiretroviral therapy was associated with decreased expression of all three inhibitory receptors on HIV-specific CD4+ T cells. Surprisingly, a high percentage of HIV-specific CD4+ T cells that expressed inhibitory receptors also coexpressed CD28. In vitro blockade of PD-1 binding concurrent with stimulation through CD28 synergistically increased HIV-specific CD4+ T cell proliferation to a greater extent than did either alone. These findings indicate that HIV-specific CD4+ T cell responses during chronic infection are regulated by complex patterns of coexpressed inhibitory receptors and that the synergistic effect of inhibitory receptor blockade and stimulation of costimulatory receptors could be used for therapeutic augmentation of HIV-specific CD4+ T cell function.

The Programmed Death-1 (PD-1) pathway limits the function of virus-specific T cells during chronic infection. We have previously shown that blockade of the PD-1 pathway increases HIV-1 associated T cell function in vitro. However the effect of PD-1 blockade on HIV-1 disease progression in vivo has not been examined. As in humans, HIV-1 infected humanized Balb/c-Rag2−/−gc−/− (Rag-hu) mice express elevated levels of PD-1 on T cells during chronic infection. To examine the effect of PD-1 blockade on disease progression, Rag-hu mice with chronic HIV-1 infection were treated with a blocking monoclonal antibody (mab) directed against PD-L1, the ligand for PD-1. PD-L1 treated Rag-hu mice exhibited a progressive decrease in the HIV-1 plasma viral load with a 7 fold decrease by day 7, 20 fold by day 14, 178 fold by day 21 and 269 fold by day 28 post initiation of treatment. By day 7 the percentage of CD4+ T cells was statistically higher in the treated compared with the untreated group and this trend was sustained throughout the 28 day treatment period. Moreover, there was a strong inverse correlation between plasma viral load and the percentage of both CD4+ (r= −0.66; P<0.0001) and CD8+ (r=−0.64; P<0.0001) T cells in the treated mice but not the untreated mice. This study provides “proof of concept” that humanized mice can be used to examine the effects of immunotherapeutic interventions on HIV-1 infection. Furthermore, these data demonstrate for the first time that blockade of the PD-1 pathway reduces HIV-1 viral loads.

HIV-1-specific interferon gamma (IFN-g)-producing DP T cells were identified at a median frequency of 0.48% compared with 1.08% and 0.02% for CD8 and CD4 SP cells, respectively, in response to pooled HIV-1 peptides. HIV-1- specific DP T cells exhibited polyfunctionality with characteristics of both CD4 and CD8 SP T cells, including coproduction of IFN-gamma and IL-2 and expression of cytolytic-associated lysosomal-associated membrane protein. No differences in frequencies of unstimulated DP T cells were observed in early compared with chronic infection. However, chronic infection was associated with higher frequencies of HIV-specific, IFN-gamma-producing DP T cells and higher fractions of effector memory and lysosomal-associated membrane protein expression among these cells, suggesting an effect of cumulative viral antigen burden on DP T-cell function.

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CD4+ T cell dysfunction in subjects with chronic HIV infection is in part due to an imbalance of costimulatory and coinhibitory receptors. We report that virus-specific CD4+ T cells expressing 4-1BB (CD137) or OX40 (CD134) produced more IL-2 than cells lacking these costimulatory receptors (P<0.05) and that 4-1BB was expressed at a lower level on HIV− than CMV-specific IFN-γ and IL-2 producing CD4+ T cells (P<0.0001 and P<0.01, respectively). Suppression of viral replication with antiretroviral therapy was associated with increased 4-1BB expression on HIV− and CMV-specific IL-2 producing CD4+ T cells (P<0.05 and P<0.01, respectively) and the percentage of IL-2 producing HIV-specific CD4+ T cells that expressed 4-1BB was inversely correlated with HIV plasma viral load (r=−0.75, P=0.007). These findings indicate that the loss of 4-1BB on HIVspecific CD4+ T cells is associated with viral replication and that it may contribute to reduced IL-2 production observed during chronic infection.