Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig-and mucin-domaincontaining molecule-3 (Tim-3) is well known to negatively regulate T-cell responses, but its role in CD8 T-cell exhaustion during chronic infection in vivo remains unclear. In this study, we document coregulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic lymphocytic choriomeningitis virus infection. Whereas Tim-3 was only transiently expressed by CD8 T cells after acute infection, virus-specific CD8 T cells retained high Tim-3 expression throughout chronic infection. The majority (approximately 65% to 80%) of lymphocytic choriomeningitis virus-specific CD8 T cells in lymphoid and nonlymphoid organs coexpressed Tim-3 and PD-1. This coexpression of Tim-3 and PD-1 was associated with more severe CD8 T-cell exhaustion in terms of proliferation and secretion of effector cytokines such as IFN-γ, TNF-α, and IL-2. Interestingly, CD8 T cells expressing both inhibitory receptors also produced the suppressive cytokine IL-10. Most importantly, combined blockade of Tim-3 and PD-1 pathways in vivo synergistically improved CD8 T cell responses and viral control in chronically infected mice. Taken together, our study defines a parameter for determining the severity of CD8 T cell dysfunction and for identifying virus-specific CD8 T cells that produce IL-10, and shows that targeting both PD-1 and Tim-3 is an effective immune strategy for treating chronic viral infections.

SUMMARY Functionally exhausted T cells express high levels of the PD-1 inhibitory receptor, and therapies that block PD-1 signaling show promise for resolving chronic viral infections and cancer. Using human and murine systems of acute and chronic viral infections we analyzed epigenetic regulation of PD-1 expression during CD8 T cell differentiation. During acute infection, naïve to effector CD8 T cell differentiation was accompanied by a transient loss of DNA methylation of the Pdcd1 locus that was directly coupled to the duration and strength of TCR signaling. Further differentiation into functional memory cells coincided with Pdcd1 remethylation providing an adapted program for regulation of PD-1 expression. In contrast, the Pdcd1 regulatory region was completely demethylated in exhausted CD8 T cells and remained unmethylated even when virus titers decreased. This lack of DNA remethylation leaves the Pdcd1 locus poised for rapid expression, potentially providing a signal for premature termination of antiviral functions.

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8 + T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8 + T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8 + T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

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