Tim-3 Is Required for Regulatory T Cell-Mediated Promotion of T Cell Exhaustion and Viral Persistence during Chronic Lymphocytic Choriomeningitis Virus Infection

Abstract

Expression of T cell Ig and mucin domain-containing protein 3 (Tim-3) is upregulated on regulatory T cells (Tregs) during chronic viral infections. In several murine and human chronic infections, the expression of Tim-3 is associated with poor control of viral burden and impaired antiviral immune responses. However, the role of Tim-3+ Tregs during persistent viral infections has not been fully defined. We employed an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg knockout) murine model to dissect the role of Tim-3 on Tregs during chronic lymphocytic choriomeningitis virus infection. Tim-3 Treg knockout mice exhibited a decrease in morbidity, a more potent virus-specific T cell response, and a significant decrease in viral burden. These mice also had a reduction in the frequency of PD-1+Tim-3+ and PD-1+Tox+ gp33-specific exhausted CD8+ T cells. Our findings demonstrate that modulation of a single surface protein on Tregs can lead to a reduction in viral burden, limit T cell exhaustion, and enhance gp33-specific T cell response. These studies may help to identify Tim-3-directed therapies for the management of persistent infections and cancer.

Figure 1:

Tim-3⁺ Treg exhibit a robust immunosuppressive phenotype during chronic LCMV infection. WT splenic Treg (CD4⁺Foxp3⁺) were analyzed by flow cytometry during a 30-day LCMV Armstrong (acute) and LCMV Cl-13 infection (chronic). (A) Frequency of Tim-3⁺ Treg during acute and chronic LCMV infection compared to uninfected mice. (B) Proportion of Ki67⁺ in Tim-3⁻ versus Tim-3⁺ Treg in LCMV Cl-13 at 15 days post-infection. (C) Median fluorescent intensity (MFI) of several immunosuppressive molecules in Tim-3⁻ versus Tim-3⁺ Treg in LCMV Cl-13 at 15 days post-infection. (D) Levels of pSTAT3 and IL-10 in Tim-3⁻ versus Tim-3⁺ Treg in LCMV Cl-13 at 15 days post-infection. (E) Levels of pSTAT5 and CD25 in Tim-3⁻ versus Tim-3⁺ Treg in LCMV Cl-13 at 15 days post-infection. WT (n=6). Each dot represents a biological replicate. Graphs show mean +/− SEM. Student’s t-test analysis. P-values less than 0.05 were considered significant and included in the graphs.

Figure 2:. Tim-3^Treg KO mice have reduced morbidity, decreased viral burden, and increased effector T cell response late during LCMV chronic infection.

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by a 30-day LCMV Cl-13 infection. Splenic cells were analyzed at 30 days post-infection by flow cytometry. (A) Cre-induction and infection timeline. (B) Weight loss curve, WT (n=8), KO (n=8). (C) LCMV viral copies in 50 ng of total tissue RNA from liver and kidney. (D) Total splenocyte count. (E) CD8 to Treg ratio. (F) CD4⁺FoxP3⁺ Treg frequency gated on CD4⁺CD8⁻ T cells. (G) Frequency of CD44^hiCD62L^lo effector CD8⁺CD4⁻ T cells. (H) Total cell count of gp33⁺ CD8⁺ T cells. (I) Frequency of gp33⁺ CD8⁺ T cells. For C-I, WT (n=7–9), Tim-3 Treg KO (n=7–9). Graphs show mean +/− SEM. Two-way ANOVA repeated measures analysis for B, and Student’s t-test for C-I. Each in vivo experiment was performed at least twice. P-values less than 0.05 were considered significant and included in the graphs.

Figure 3:. Inducible deletion of Tim-3 in Treg limits the development of Tex in gp33-specific T cells late during LCMV chronic infection.

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by a 30-day LCMV Cl-13 infection. Splenic cells were analyzed by flow cytometry at 30 days post-infection. (A) Frequency of Tim-3⁻PD-1⁺ (PD-1^int) and Tim-3⁺PD-1⁺ (DP, double positive) CD44^hi CD8⁺ T cells. (B) Frequency of PD-1⁺Tox⁺ in CD44^hi CD8⁺ T cells. (C) Frequency of PD-1^int and DP gp33⁺ CD8⁺ T cells. (D) Frequency of PD-1⁺Tox⁺ in gp33⁺ CD8⁺ T cells. WT (–8), Tim-3^Treg KO (n=6–8). Each dot represents an independent biological replicate. Graphs show mean +/− SEM. Student’s t-test analysis. Each in vivo experiment was performed at least twice. P-values less than 0.05 were considered significant and included in the graphs.

Figure 4.. Tim-3 Treg KO mice exhibit an enhanced gp33-specific T cell response early during chronic infection:

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by an 8-day LCMV Cl-13 infection. (A) Cre-induction and infection timeline. (B) Total splenocyte count. (C) LCMV viral copies in 50 ng of total tissue RNA from liver, and kidney. (D) Total CD4⁺CD8⁻ and CD4⁻CD8⁺ T cell count from spleen. (E) CD4⁺FoxP3⁺Treg cell count from spleen. (F) CD8 to Treg ratio in spleen. (G) CD4⁺FoxP3⁺Treg frequency gated on CD4⁺CD8⁻. (H) Frequency of CD44^hiCD62L^lo effector CD8⁺CD4⁻ T cells. (I) Total cell count of gp33⁺ CD8 T cells. (J) Frequency of gp33⁺ CD8⁺CD4⁻ T cells. (K) Expression of CD39 in total Treg. WT (n=6–8) KO (n=6–8) LCMV-infected mice. Graphs show mean +/− SEM. Student’s t-test analysis. Each in vivo experiment was performed at least twice. P-values less than 0.05 were considered significant and included in the graphs.

Figure 5.. Inducible Treg-specific Tim-3 deletion limits the development of Tex in gp33-specific T cells early during chronic infection:

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by 8-day LCMV Cl-13 infection for (A-D). Similarly, WT and Tim-3^Treg KO mice for E-F received adoptively transferred WT P14 T cells before infection. For all experiments, splenic cells were analyzed by flow cytometry at 8 days post-infection. (A) Frequency of Tim-3⁻PD-1⁺ (PD-1^int) and Tim-3⁺PD-1⁺ (DP-“double positive”) CD44^hi CD8⁺ T cells. (B) Frequency of PD-1⁺Tox⁺ in CD44^hi CD8⁺ T cells. (C) Frequency of PD-1^int and DP gp33⁺ CD8⁺ T cells. (D) Frequency of PD-1⁺Tox⁺ in gp33-specific CD8⁺ T cells. (E) Frequencies of PD-1^int and DP in adoptively transferred WT P14 T cells. (F) Frequency of Tox⁺ gated on adoptively transferred WT P14 T cells. WT (–8), KO (n=6–8). Each dot represents an independent biological replicate. Graphs show mean +/− SEM, based on Student’s t-test. Each in vivo experiment was performed at least twice. P-values less than 0.05 were considered significant and included in the graphs.

Figure 6.. Tim-3 on Treg limits T cell function in gp33-specific T cells early during chronic infection.

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by LCMV Cl-13 infection (A-D). For E-F, WT and Tim-3^Treg KO mice received adoptively transferred WT P14 T cells before infection. For all experiments cells were analyzed by flow cytometry at 8 days post-infection. (A) Light microscopy images displaying cell killing of B16.gp33 target cells by CD44^hiCD8⁺ T cells at a 3:1 effector:target ratio. (B) Cytolysis of B16.gp33 cells during 25 hr incubation. (C-D) Frequency of TNF⁺ and IFNγ⁺ (C) and Gzmb expression (D) in gp33⁺ CD8⁺ T cells after in vitro gp33 peptide simulation for 5 hours. (E) Frequency of TNF⁺ and IFNγ⁺ in adoptively transferred WT P14. (F) Gzmb expression in adoptively transferred WT P14. Single-cell suspensions were stimulated in vitro with gp33 peptide. WT (–8), KO (n=6–8). Each dot represents an independent biological replicate. Graphs show mean +/− SEM. Student’s t-test. Each in vivo experiment was performed at least twice. P-values less than 0.05 were considered significant and included in the graphs.

Figure 7:. Treg-specific Tim-3 deletion alters the transcriptional profile of specific major WT P14 Tex subsets early during chronic infection.

WT and Tim-3^Treg KO mice were treated with tamoxifen for 5 days, followed by adoptive transfer of WT P14 and an 8-day LCMV Cl-13 infection. Splenic lymphocytes were analyzed by flow cytometry at 8 days post-infection. (A) Experimental timeline and the specific P14 Tex subsets that were FACS-sorted for bulk-RNA seq analysis. P14 Tex subsets sequenced, PD-1⁻Tim-3⁻ (DN “double negative”), PD-1⁺Tim-3⁻ (PD-1^int), and PD-1⁺Tim-3⁺ (DP “double positive”). (B) Principal component analysis for Tex subsets from WT and Tim-3^Treg KO mice. (C) Heatmap of selected significantly differentiated genes (p-value <0.05, and p-adjusted <0.02) in DN, PD-1^int, and DP. WT (n=3), KO (n=3). For (B), each dot represents a biological replicate.