The balance between positive and negative signals delivered by costimulatory molecules to T cells appears to be critical for the ultimate fate of cellular immune responses (13
). Recent data (10
) suggest that manipulation of T cell costimulatory pathways may present a novel approach for enhancing and restoring virus-specific CD8+
T cell responses, especially in the context of a chronic infection like HIV. We report here on the role of PD-1, a negative costimulatory receptor of T cells, as a regulator of virus-specific CD8+
T cells in HIV infection. To understand how PD-1 can affect the function of CD8+
T cells, it is critical to identify which cells provide the ligand(s) for PD-1. Our data indicate that mDCs and monocytes may use the PD-1–PD-L system to regulate adaptive antiviral immunity. Our findings in this context are very preliminary and much more needs to be investigated. For instance, the relative kinetics of costimulatory molecule expression on mDCs and monocytes after activation and the impact of the “positive” and “negative” signals delivered by these molecules to responding T cells remains to be elucidated. In addition, it will be important to determine whether there is redundancy between the various costimulatory signals affecting CD8+
T cells or if they act independently by stimulating separate intracellular pathways after initiation of virus-specific CD8+
T cell responses.
We found remarkably high expression of PD-1 on HIV-specific CD8+
T cells. The frequency of PD-1 expression on the different virus-specific CD8+
T cells (HIV = EBV > CMV > VV) is consistent with PD-1 regulation according to antigen stimulation. Although it is unlikely that the level of antigen in chronic EBV infection reaches that which occurs in HIV infection, it has been shown that EBV is continuously shed into saliva by induction of the lytic cycle as B cells differentiate into plasma cells, thereby chronically stimulating lytic cycle antigen-specific CD8+
T cells (44
). The mechanism by which CD8+
T cells control HIV and EBV may differ. Therefore, it may not follow that expression of PD-1 on EBV- and HIV-specific CD8+
T cells will similarly impact CD8-mediated control of these two different virus infections. Further experiments are needed to clarify the relative role of PD-1 in regulation of EBV-specific CD8+
T cell responses and compare it to the regulation of HIV-specific responses. In summary, although we cannot conclude that chronic antigen stimulation is the sole factor determining PD-1 expression, our data reveal that HIV-specific CD8+
T cells, because of their high expression of PD-1, may be vulnerable to negative signals delivered by PD-1, potentially leading to functional consequences in vivo.
Despite their differential expression of PD-1, no difference in IFN-γ production was found between HIV-, CMV-, and EBV-specific CD8+ T cells. On the other hand, PD-1 expression is associated with significantly lower ability of HIV-specific CD8+ T cells to produce TNF-α and even lower production of IL-2. This is in agreement with the finding that PD-1–PD-L1 blockage has a substantial impact on LCMV-specific CD8+ T cells producing both IFN-γ and TNF-α, whereas it has only a slight effect on single IFN-γ producers. However, we found that PD-1− and PD-1+ antigen-specific CD8+ T cells were equally able to produce cytokines upon antigen stimulation, indicating that PD-1 expression has no direct effect on cytokine production. This was further supported by our finding that ligation of PD-1 during antigen stimulation had no effect on cytokine production by virus-specific CD8+ T cells. Collectively these data clearly demonstrate that PD-1 has no direct effect upon the immediate ability of antigen-specific CD8+ T cells to produce IFN-γ, TNF-α, or IL-2.
Manipulation of the PD-1–PD-L system was found to alter the proliferation of virus-specific CD8+ T cells. This was accompanied by altered percentages of CD8+ T cells producing cytokines. The change in proliferation could result from an altered ability of these cells to either survive or divide. Importantly, we found no relationship between PD-1 expression and the degree of change in proliferative capacity after manipulation of the PD-1–PD-L1 axis. In fact, in some instances where the expression of PD-1 was very high on HIV-specific CD8+ T cells, only minor effects of PD-1 ligation on proliferation were observed. Therefore, although PD-1 has a demonstrable effect on the ability of virus-specific (CMV and HIV) CD8+ T cells to proliferate, it is not the sole factor regulating this function. This is not surprising as other factors (i.e., TCR activation threshold, relative expression of other costimulatory molecules, or levels of adaptor proteins mediating the intracellular signaling delivered by PD-1) could also contribute to the ability of PD-1 ligation to affect proliferative capacity.
At least two interventions have now proven successful in vitro in restoring the proliferation of HIV-specific CD8+
T cells: the addition of IL-2 (or CD4+
T cells producing IL-2) and the manipulation of costimulatory pathways such as PD-1. This raises the question of whether these two different manipulations affect proliferation through overlapping intracellular mechanisms. In addition, whether they can act in a synergistic mode remains to be elucidated. Since IL-2 cannot overcome the proliferative defect in CD57+
T cells (45
), it is of particular interest to examine whether manipulation of PD-1–induced pathways could specifically restore their proliferative capacity.
Our data indicate that the primary mechanism by which PD-1 affects CD8+
T cell function is by regulating the ability of these cells to survive. This is in agreement with the originally described role of PD-1 as an apoptotic factor (24
) and the reduced survival that characterizes virus-specific CD8+
T cells under conditions of chronic antigen stimulation (46
). We can speculate on how PD-1 expression could affect HIV-specific CD8+
T cell survival. It is possible that stimulation through PD-1 can direct cells into a cell cycle resting state, as has been described for the PD-1–PD-L2 interaction (21
). We found that lack of PD-1 expression is associated with similar levels of spontaneous and CD95/Fas-induced apoptosis, whereas CD95/Fas-induced apoptosis is greatly augmented in CD8+
T cells that express PD-1, indicating that there may be cross-talk between the signals induced by these two receptors. Previously published data have shown that the Fas–FasL interaction impacts PD-L1–induced apoptosis of activated T cells (25
). Although no direct link between PD-1 and CD95/Fas was described in that work (25
), the possibility that PD-1 could prime (especially under conditions of chronic stimulation) CD8+
T cells to undergo CD95/Fas-induced apoptosis cannot be excluded. Therefore, clarification of the intracellular mechanism(s) governing the proapoptotic function of PD-1 is of particular interest. Furthermore, the role of such a function on CD4+
T cell survival in HIV infection would significantly add to our understanding of HIV pathogenesis.
A clear conclusion from our results is that the absolute level of PD-1 expression is a major determinant of spontaneous apoptosis and sensitivity to PD-1 ligation. We conclude this despite our observation that PD-1− HIV-specific CD8+ T cells are often more susceptible to apoptosis than PD-1+ CMV-specific CD8+ T cells (). Although it is known that sensitivity to apoptosis is also affected by other factors—specifically the level of T cell activation (defined by CD38 expression; unpublished data) and maturational state, which we have shown is independent of PD-1 expression (, A and B and )—our data indicate that PD-1 is a primary determinant of apoptosis sensitivity over and above these other factors. We conclude this because, within any population of CD8+ T cells (defined by activation, maturation, or antigen specificity), the PD-1+ population is more sensitive to apoptosis than the PD-1− population. In addition, although we have described PD-1 expression as either positive or negative in most of our data, expression really represents a continuum, with high expression being associated with greater impact upon PD-1–regulated functions (i.e., apoptosis; ). When CD8+ T cells of different antigen specificities, but similar levels of PD-1 expression, are analyzed, they exhibit similar levels of spontaneous apoptosis and sensitivity to PD-1 ligation. In addition, cells with moderate to low expression of PD-1 have low levels of spontaneous apoptosis and are not affected by PD-1 ligation. What this means is that within any population of antigen-specific CD8+ T cells, it is the absolute level of PD-1 that primarily dictates the rate of spontaneous apoptosis and sensitivity to PD-1 ligation. Therefore, what is unique about HIV-specific CD8+ T cells is their high level of PD-1 expression leading to a profound (but potentially reversible) survival defect. Although the proliferative capacity of a CD8+ T cell is determined by more than just an ability to resist apoptosis, it is not difficult to visualize how the level of PD-1 and associated sensitivity to apoptosis would impact on the ability of a cell to proliferate.
Overall, our data demonstrate that PD-1 is preferentially expressed on CD8+ T cells specific for chronic viruses, and that PD-1 interaction with its ligands can regulate the ability of these virus-specific CD8+ T cells to survive and proliferate. Therefore, manipulation of this axis may lead to at least partial restoration of antigen-specific cell numbers and function in chronic viral infections such as HIV. It is important to remember that our data do not support the ability of PD-1 manipulation to restore all of the T cell functions that define functional “exhaustion.” For instance, we have no evidence that PD-1 blockade will restore absent cytokine functions, and may only affect CD8+ T cell proliferation to the degree possible in the context of other as yet undetermined defects in HIV-specific CD8+ T cells. Therefore, although our data identify PD-1 as a potential therapeutic target for restoring functional capacity of HIV-specific CD8+ T cell responses, it may not be capable of fully restoring function. In addition, it should be appreciated that the PD-1–PD-L1 axis likely evolved to attenuate potentially harmful CD8+ T cell responses to both self-antigens and chronic pathogens. Given that many non–HIV-specific CD8+ T cells express PD-1 (), it is likely that interventions to release all CD8+ T cells from PD-1–mediated suppression will have untoward effects.