Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection

Abstract

Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig- and mucin-domain-containing 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-gamma, TNF-alpha, 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.

Fig. 1.

Sustained coexpression of Tim-3 and PD-1 on antigen-specific CD8 T cells during chronic LCMV infection. Expression of Tim-3 and PD-1 on GP33-specific CD8 T cells during LCMV Arm (acute) or Clone-13 (chronic) infections was analyzed by multiparameter flow cytometry. (A) Intensity of Tim-3 expression on GP33-specific CD8 T cells during infection. (B) Representative data show the frequency of GP33-specific CD8 T cells expressing Tim-3 on day 8 and 30 after infection. (C) The frequency of GP33-specific CD8 T cell coexpressing Tim-3 and PD-1 is shown over time. (D) Representative data showing coexpression of PD-1 and Tim-3 by exhausted LCMV-specific CD8 T cells on day 30 after infection. Numbers in quadrants indicate percent of each subset. Error bars represent SEM. Data are representative of three independent experiments with three mice per experiment.

Fig. 2.

Coexpression of Tim-3 and PD-1 correlates with more severe exhaustion of LCMV-specific CD8 T cells during chronic infection. Functions of Tim3⁺PD1⁺ or Tim3⁻PD1⁺ CD8 T cells were analyzed using splenocytes at d 50 after infection. (A) The isolated Tim3⁺PD1⁺ or Tim3⁻PD1⁺ CD8 T cells were labeled with CFSE and stimulated with GP33 peptide or a pool of LCMV peptides for 3 d. Proliferation was determined by dilution of CFSE, and the number in flow cytometry plot indicates the frequency of proliferating cells. (B) Frequency of GP33- or GP276-specific CD8 T cells producing cytokine after stimulation for 5 h with GP33-41 or GP276-286 peptides. (C) Frequency of Tim3⁺PD1⁺, Tim3⁻PD1⁺, or Tim3⁻PD1⁻ CD8 T cells producing IL-10 was analyzed after stimulation for 5 h with the LCMV peptide. Data are representative of three independent experiments. Error bars represent SEM. LCMV peptide pool consists of GP33-41, GP276-286, GP70-77, GP92-101, NP166-175, NP205-212, NP235-249, and NP396-404.

Fig. 3.

In vivo blockade of Tim-3 and PD-1 pathways enhances virus-specific CD8 T-cell responses during chronic viral infection. Chronically infected C57BL/6 mice (80 d after infection) were treated every third day or every other day for 2 wk with αPDL1 or Tim3Ig, respectively. Frequency of GP33-specific CD8 T cells before and after treatment of individual mouse is shown in the blood. Total number of GP33-specific CD8 T cells in the indicated tissues at 2 wk after treatment. Data are representative of three independent experiments with five to six mice per group in each experiment.

Fig. 4.

Dual blockade of Tim-3 and PD-1 pathways enhances function in exhausted virus-specific CD8 T cells. (A) IFN-γ production and degranulation by CD8 T cells in treated mice at 2 wk after therapy. The percentage of IFN-γ⁺CD107⁺ CD8 T cells specific for each of the LCMV peptides are summarized. (B) Polyfunctional (TNF-α⁺IFN-γ⁺) CD8 T cells in treated mice at 2 wk after therapy. (C) The proliferation of antigen-specific CD8 T cell after dual blockade is shown as the percentage of Ki67⁺ on LCMV GP33-specific CD8 T cells. Data are representative of three independent experiments with five to six mice per group in each experiment. *P < 0.05; **P < 0.01.

Fig. 5.

Dual blockade of Tim-3 and PD-1 pathways enhances viral control. Viral titer was determined by plaque assay in the blood before and after treatment. Viral load in spleen, liver, and lung at 2 wk after treatment is shown. Data are representative of three independent experiments with five to six mice per group in each experiment. Error bars represent SEM.