HCV infection is a highly significant clinical problem, representing one of the most frequent causes of liver failure worldwide. The experimental evidence implicating CD8+
T cells as pivotal in host defense against this common infection is compelling, including the demonstration, first in chimpanzees (7
) and then in humans (8
), that mutational escape predates the development of persistence. One major criticism of the mutational escape hypothesis has been that, although it is a highly mutable RNA virus, HCV is unlikely to evade the multispecific CTL responses observed in some chronically infected individuals (5
). Furthermore, HCV infection is characterized by virus-specific CTLs that exhibit phenotypic changes consistent with early stages of differentiation with functional impairment or anergy (13
). Upregulation of inhibitory receptors on exhausted T cells is an important mechanism of T cell dysfunction during chronic viral infections (13
), and the repertoire of known negative regulators expressed on T lymphocytes has expanded considerably in the past few years.
Numerous groups have studied the role of PD-1, an ITIM-containing inhibitory receptor expressed on activated T cells that binds 2 known ligands (PD-L1 [B7-H1] and PD-L2 [B7-DC]; ref. 34
), in mediating the T cell hyporesponsiveness in HCV. These studies have found that PD-1 expression is significantly increased in chronic infection (35
), is further enriched in the hepatic compartment (35
), and predicts lack of response to combination antiviral therapy (39
). Whether PD-1 expression on HCV-specific CTLs predicts spontaneous virologic outcome in acute infection remains controversial, with one group demonstrating that PD-1 levels do not differ significantly between those who clear infection and those who do not in the acute or chronic phase of infection (40
), but another showing that PD-1 expression inversely correlates with viral clearance independent of viral level (14
). Regardless of the reason for these differences, it is notable that the overlap in PD-1 expression was considerable in the acute→resolved versus acute→chronic patient groups.
Our prior results indicate that Tim-3 expression may play an important pathogenic role in patients with longstanding chronic HCV infection (17
), correlating with a dysfunctional and senescent phenotype (CD127lo
). The first important finding of the current study was that acute HCV infection differentially affected the expression of Tim-3 on bulk CD4+
T cells: subjects who cleared HCV did not demonstrate increased Tim-3 expression on CD4+
T cells, whereas, regardless of whether viral infection was spontaneously controlled or became persistent, CD8+
T cells demonstrated upregulated Tim-3 expression that did not normalize until late after spontaneous recovery. It is hypothesized that Tim-3–expressing CD4+
T cells may not provide the T cell help in the earliest stages of infection that determines whether CTL effectors develop into long-lived TEM
cells, conferring immune protection. Provision of adequate CD4+
T cell help via production of cytokines (41
) or by assisting professional antigen-presenting cells via CD40/CD40L-mediated activation (42
) is a prerequisite for the generation of effective CTL memory and development of protective immunity to HCV. We found that Tim-3+
T cells had impaired secretion of IL-2 (Supplemental Figure 6) compared with their Tim-3–
counterparts, supporting this hypothesis and potentially identifying a useful marker to predict viral persistence. As shown in Figure B, at all time points studied, patients who developed persistent HCV infection demonstrated higher frequencies of PD-1+
HCV-specific CTLs. Furthermore, PD-1+
T cells were predominantly of the TCM
phenotype, whereas TEM
and EMRA cells were typically negative for coexpression.
Wherry and others (20
) have proposed a hierarchal model of CTL exhaustion that follows a pattern of progressive loss of function: decreased IL-2 and TNF-α secretion, followed by loss of IFN-γ production. Exhaustion culminates in loss of all effector functions, including cytolytic activity, especially if epitope presentation to T cells is high as would be expected in the liver. Moreover, the ligands for PD-1 (PD-L1/PD-L2; ref. 37
) and Tim-3 (galectin-9; ref. 45
) are highly expressed in HCV-infected livers. The liver compartment contained the highest frequency of PD-1hi
HCV-specific CTLs and the lowest frequency of PD-1lo
CTLs (Figure D). Furthermore, our ex vivo data demonstrate that the extent to which Tim-3 and PD-1 are dually expressed on HCV-specific CTLs correlates with the spectrum of T cell functional impairment, persistence versus spontaneous eradication of viral infection, and the tissue microenvironment. Interestingly, a recent study in murine LCMV has demonstrated that dual expression of these inhibitory receptors is associated in vivo with development of chronic viral infection (28
). Taken together, these results extend a paradigm in which increasing expression of PD-1 and Tim-3 correlates with progressive exhaustion of T cells (Figure ).
Gradient of function and exhaustion in HCV-specific T cells according to expression of Tim-3 and PD-1.
Whether blockade of the PD-1 pathway leads mostly to a qualitative improvement in the polyfunctionality of CTLs or a quantitative increase in virus-specific CTLs by proliferative expansion remains open to question (18
). Recent work suggests that only a subset of exhausted CTLs can be functionally restored with PD-1/PD-L blockade (21
), and our current results suggest a novel role for Tim-3 in demarcating particularly exhausted viral-specific PD-1+
T cells. PD-1/PD-L1 blockade has been shown by others to enhance cytotoxicity only at the population level as a result of increased proliferation (46
), but not at the single-cell level in exhausted cells (R. Ahmed, unpublished observation). In keeping with these results, we did not find that PD-1/PD-L1 blockade consistently enhanced cytotoxicity of HCV-specific CTLs, as assessed by CD107a secretion or killing of HepG2 cells expressing cognate peptide. However, we demonstrated for the first time to our knowledge that significant cytotoxic activity was more frequently detected in T cell cultures (including intrahepatic CTLs) with anti–Tim-3 blockade, which sometimes worked additively with anti–PD-L blockade. The fact that Tim-3 blockade more consistently enhanced cytotoxicity of HCV-specific CTLs indicates that Tim-3 and PD-1 are associated with distinct steps that mediate functional exhaustion in T cells. Work is ongoing to examine the transcriptional profile of T cells treated with anti–Tim-3 or anti–PD-L antibodies in order to define mechanistic differences.
In summary, our findings demonstrate that early accumulation of PD-1+
T cells is associated with functional impairment, and consequently with development of persistent HCV. The present study provides a basis for improving current therapies by simultaneous blockade of multiple inhibitory pathways that could result in additive efficacy without excessive toxicity (18