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. Author manuscript; available in PMC: 2025 Nov 15.
Published in final edited form as: J Immunol. 2024 Nov 15;213(10):1488–1498. doi: 10.4049/jimmunol.2400119

Tim-3 is required for Treg-mediated promotion of T cell exhaustion and viral persistence during chronic LCMV infection

Hector M Nieves-Rosado *,†,‡, Hridesh Banerjee *, Angela Gocher-Demske *, Priyanka Manandhar *,‡, Isha Mehta *,§, Ogechukwu Ezenwa *,§, Bingxian Xie *,, Ben Murter *,‡, Jishnu Das *,§, Dario AA Vignali *, Greg M Delgoffe *,, Lawrence P Kane *,1
PMCID: PMC11671103  NIHMSID: NIHMS2023092  PMID: 39345172

Abstract

Expression of Tim-3 is upregulated on regulatory T cells (Treg) 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 anti-viral immune responses. However, the role of Tim-3+ Treg during persistent viral infections has not been fully defined. We employed an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) murine model to dissect the role of Tim-3 on Treg during chronic LCMV infection. Tim-3 Treg KO 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 Treg 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.

Keywords: tim-3, treg, lcmv

Introduction

Regulatory T cells (Treg), a subset of CD4+ T cells, are critical mediators of immune tolerance under homeostatic conditions. Treg can also modulate the balance between immunity and tolerance during infection. The importance of Treg in T cell-mediated viral clearance is highlighted by a report showing that transient in vivo ablation of Treg during chronic LCMV infection enhances virus-specific T cell responses (1). This report served as a proof-of-concept that the suppressive activity of Treg might contribute to chronic viral infection. Consistent with such a model, in people with untreated chronic hepatitis B virus (HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV) there is an increase in the peripheral Treg frequency which also correlates with higher viral load, and a decrease in virus-specific T cell responses (2–6). Ex vivo depletion of human Treg improves viral-specific T cell responses to HCV (2, 4), HBV (3) and HIV (5, 7, 8). Altogether, these studies suggest that the presence of Treg limits effective anti-viral immunity in certain chronic viral infections.

T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a transmembrane protein that can be either constitutively or inducibly expressed on multiple different immune cell types, including Treg. In murine and human chronic infections, the expression of Tim-3 by CD8+ effector T cells is associated with poor control of viral burden and impaired anti-viral immune responses (9–11). Nevertheless, the biological and clinical relevance of Tim-3 on Treg during chronic infection remains unknown. Data from our group and others show that Tim-3+ Treg have increased expression of CD39, PD-1, CTLA-4, ICOS, Ki67 and CD44 in comparison to Tim-3− Treg (12–17). More recently, our group and others showed that in people with HIV on anti-retroviral therapy (PWH-ART) there is an increase in the frequency of peripheral Tim-3+ Treg that is not normalized by anti-retroviral therapy (ART) (18, 19). By contrast, a small subset of people with HIV known as long-term non-progressors, who have low viral load, high CD4+ T cell counts, and are ART-naïve, do not exhibit increased peripheral Tim-3+ Treg frequency (19). We recently reported that Tim-3+ Treg have a robust suppressive phenotype compared to Tim-3− Treg from the same individual (18). In addition, we showed that anti-Tim-3 monoclonal antibody (mAb) reversed the in vitro suppression capacity of Treg in PWH-ART. Thus, Tim-3+ Treg may represent a target for the treatment of chronic inflammation and viral persistence.

Based on these findings, we hypothesized that Tim-3 expression on Treg promotes viral persistence and limits anti-viral T cell responses during chronic infection. Employing conditional Treg-specific Tim-3 KO mice, we demonstrate for the first time that deletion of a single surface protein in Treg is sufficient to limit LCMV viral persistence. Deficiency of Tim-3 in Treg also led to an increase in the activation phenotype and function of virus-specific T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Lack of Tim-3+ Treg reduced morbidity, boosted T cell function, and limited the development of T cell exhaustion. Therefore, our data revealed that Tim-3 expression in Treg contributes to LCMV viral persistence, the development of T cell exhaustion, and poor virus-specific T cell function.

Materials and Methods

Mice and Cre induction

To evaluate the specific contribution of Tim-3 deletion in Treg during chronic LCMV infection we employed a conditional Treg-specific Tim-3 loss-of-function mouse model, as previously reported (12). Havcr2flox mice were generated by the University of Pittsburgh Department of Immunology Transgenic and Gene Targeting (TGT) core. Briefly, Crispr/Cas9 technology was applied for inserting LoxP sites on either side of the exon 4 of the Havcr2 gene in C57BL/6J zygotes. Deletion of exon 4 by Cre-mediated recombination results in splicing of exon 3 into exon 5 which results in a premature stop codon 20 base pairs into exon 5, before the transmembrane domain. A premature stop codon before the last exon leads to nonsense mediated mRNA decay resulting in the absence of transcript and lack of expression of a potential soluble truncated protein. Finally, incorporation of the LoxP sites was confirmed by PCR followed by sequencing of the targeted region. Foxp3eGFP-Cre-ERT2 mice were then crossed with Havcr2flox/flox mice to generate mice homozygous for both genetic modifications. FoxP3eGFP-Cre-ERT2 mice had been previously backcrossed to C57BL/6 mice for at least ten generations.

Mice were bred in-house under SPF conditions and used at 6–8 weeks. All experiments were age- and sex-matched. All animal procedures were conducted in accordance with NIH and University of Pittsburgh IACUC guidelines. For inducing Cre-mediated Tim-3 deletion, tamoxifen was resuspended in sunflower oil and administered daily for 5 days intraperitoneally (1 mg/day). After the last dose, mice were left to rest for two days, and were used for experiments on day 7 after tamoxifen was initiated. Validation of Tim-3 deletion is shown in Fig. S2A–B.

Acute and chronic LCMV infection

LCMV Cl-13 was obtained from Rafi Ahmed, Emory University, and propagated as described previously (20). For chronic infections, mice were infected with 2×106 PFU LCMV Cl-13 i.v. at day 0 and analyzed at the indicated time points post-infection. For acute infections, mice were infected with 2×105 PFU, intraperitoneally.

Flow cytometry and staining

Spleen single cell suspensions were stained with Ghost DyeTM Violet 510 (Cat no. 13–0870-T500; Tonbo biosciences) and FC block for 20 mins. Samples were washed and stained for several surface markers using the following monoclonal antibodies/clones: Tim-3 (RMT3–23, BioLegend), CD4 (GK1.5, BD Biosciences), CD8 (53-6.7, BD Biosciences), CD44 (IM7, BioLegend), ICOS (7E.17G9, ThermoFisher), PD-1 (RMP1–14, ThermoFisher), CD39 (Duha59, BioLegend), CD25 (PC61, BioLegend), KLRG-1 (2F1, ThermoFisher), PD-L1 (10F.9G2, Cytek Biosciences), CD45.1 (A20, BD Biosciences), CD45.2 (104, BD Biosciences), CD11b (M1/70 BD Biosciences), Ly6G (1A8-Ly6g, ThermoFisher), MHCII (M5/114.15.2, BioLegend), CD24 (M1/69, Tonbo Biosciences), F4/80 (BM8, ThermoFisher). After performing extracellular staining, samples were fixed and permeabilized using a FoxP3 staining kit (eBioscience), and stained for intracellular transcription factors, cytokines, and phosphorylated proteins; FoxP3 (FJK-16s, ThermoFisher), CTLA-4 (UC10–4B9, BioLegend), IL-10 (JES5–16E3, BioLegend), Bcl-2 (BCL/10C4, ThermoFisher), Ki67 (B56, BD Biosciences), pSTAT3 (Y705) (4/P-STAT3, BD Biosciences), pSTAT5 (Y694) (47/Stat5 (pY694) BD Biosciences), TNF (MP6-XT22, BioLegend), IFNγ (XMG1.2, ThermoFisher), GzmB (NGZB, ThermoFisher). H-2Db-gp33 tetramers were obtained from the NIH tetramer core. Flow cytometry gating strategy for analyzing Treg (Fig. S4A), gp33-specific T cells (Fig. S4B), P14 T cells (Fig. S4C), and innate immune cells (Fig. S4D). Flow cytometry data was obtained using a Cytek Aurora spectral flow cytometer and results were analyzed using FlowJo (version 10.8.1).

Quantification of viral titers

Viral titer was measured from infected tissue after RNA extraction using TRIzol LS reagent, followed by RNA-to-cDNA conversion, and qPCR for the GP protein. Viral copy number was calculated by comparing CT values with a plasmid standard curve, as previously described (21). The VERO cell plaque assay was used to confirm the results obtained using the qPCR method in a selected group of samples, as previously reported (22).

In vitro T cell stimulation with gp33 peptide

For assessing T cell functional capacity, 5×106 splenocytes from infected mice were stimulated with 100 ng/mL LCMV gp33 peptide (AnaSpec) for 5 h at 37°C in complete RPMI with bovine growth serum (BGS; HyClone). Cells were simultaneously treated with a protein transport inhibitor containing monensin (BD GolgiPlug, Cat. no. 554724), following the manufacturer’s instructions, to improve cytokine detection by flow cytometry. Surface and intracellular staining was performed as described above.

P14 adoptive transfer

Splenic live CD45.1 congenically marked P14 cells were stained, as described above, and FACS-sorted from a female CD45.1/CD45.2 P14 mouse. Sorted P14 cells were then transferred into tamoxifen-pretreated congenically-distinct WT and Tim-3 Treg KO mice (1,500 P14/mouse, i.v.). One day after transfer, mice were infected with LCMV Cl-13 and analyzed at indicated timepoints.

T cell killing assay

To measure the functional activity of gp33-specific CD8+ T cells, we employed the xCELLigence system, which allows for real-time and label-free monitoring of cell viability. Briefly, this technology uses microtiter plates embedded with gold microelectrodes to noninvasively measure the viability of target cells attached to the plate by using electrical impedance as the readout (23). To test T cell functional capacity, we plated 20,000 B16-gp33 melanoma cells (target cells) per well on an E-Plate View 96 (Cat. no. 3000-601-020, Agilent) and added RPMI1640 supplemented with 10% fetal bovine serum. Cell cultures were maintained at 37°C and 5% CO2. After eight hours, target cells were 100% confluent, at which time effector T cells were added to the culture. For effector T cells, we sorted live CD44hiCD62Llo CD8+ T cells from WT and Tim-3 Treg KO mice at 8 days post-infection. As a negative control, we sorted CD44hiCD62Llo CD8+ T cells from an uninfected WT mouse. A total of 60,000 Teff were added into each well to achieve a 3:1 ratio (Teff:Target) and the assay ran uninterrupted for 25 hours, after T cells were added. Light field microscopy images at a 10x magnification were taken with each electrical impedance readout every 15 min. Finally, the percent cytolysis was calculated by the xCELLigence system using the following formula: percentage of cytolysis = ((cell index, no effectors – cell index, plus effectors)/cell index, no effectors) × 100.

Gene expression analysis and bulk RNA sequencing

After initial QC and adapter trimming, sequence data was quantified using Kallisto (24) to obtain transcript level abundances, using mm10 (UCSC) as the reference genome. Post-quantification, differentially expressed genes and transcripts between the WT and KO mice were identified using Sleuth (25). Significant differentially expressed transcripts were defined using a q-value (Benjamini-Hochberg adjusted p-value) threshold of <0.2. Over-representation analysis was performed using the clusterProfiler (26, 27) tool to identify enrichment of the genes in specific pathways described in Kyoto Encyclopedia of Genes and Genomes (KEGG) (28) and Reactome (29) databases. RNAseq data are available under GEO accession number GSE254211.

Statistical analysis

We based the sample size on data obtained from previous work with the LCMV model (21, 30) and performed a power calculation. Based on that, we determined that a sample size of 8 mice each (For a two-group experiment) is sufficient to detect an effect size of 2.8 (with 10% standard deviations), with an alpha value of 0.05 and power of 0.95. Each such in vivo experiment was performed at least twice. Two-way comparisons were analyzed using student’s t-test. P values <0.05 were considered significant for all tests. All statistical analyses were performed using GraphPad Prism (version 9.4.1).

Results

Tim-3 expression in Treg during acute and chronic LCMV infection

To examine the role of Tim-3 in Treg we first evaluated the changes in the frequency of Treg and Tim-3+ Treg during acute (Armstrong) vs. chronic (Clone-13) LCMV infection. As expected, Treg frequency decreased at the early stage of chronic infection, and then became persistently elevated (Fig. S1A). By contrast, during acute infection the frequency of total Treg remained unaltered at day five and decreased at later time points, compared to uninfected mice (Fig. S1A). We next assessed the phenotype of these Treg. Our data revealed a significant increase in the frequency of Tim-3+ Treg at five-, eight-, and fifteen-days post-infection with LCMV Cl-13 (Fig. 1A). We also observed an increase in Tim-3+ Treg frequency on day five post-infection with LCMV-Arm, relative to uninfected mice, which normalized to 2.5% by day eight (Fig. 1A). Despite the changes in the frequency of Treg and Tim-3+ Treg during acute and chronic infection, we did not detect any significant differences in the proportion of CD4+FoxP3− conventional T cells (Tconv) (Fig. S1B) nor in Tim-3+ Tconv T cells (Fig. S1C). Therefore, these data indicate that the percentage of Tim-3+ Treg increases in conditions of high viral load and that these cells persist during chronic infection.

Figure 1:

Figure 1:

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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.

Tim-3 expression in Treg is associated with a suppressive phenotype

Next, we characterized the phenotype of Tim-3+ Treg at fifteen days post-infection, the peak of Tim-3+ Treg frequency during LCMV Cl-13 infection. Strong Treg immunosuppression has been associated with an increase in Treg proliferation and survival. We found that the frequency of Ki67+ cells (indicative of active proliferation) was elevated in Tim-3+ Treg compared to Tim-3− Treg (Fig. 1B). These cells also showed a higher frequency of KLRG-1+ Treg (Fig. S1D), a marker of short-lived effector T cells, and downregulation in the expression of Bcl-2 (Fig. S1E), an anti-apoptotic molecule. Thus, the phenotype of Tim-3+ Treg is consistent with these cells having an enhanced immunosuppressive capacity and perhaps an accelerated turnover.

Flow cytometry analysis also revealed upregulation in the expression of several activation and effector markers like CD44, CTLA-4, ICOS, PD-1, and CD39 on Tim-3+ Treg (Fig. 1C). In addition, we found elevated effector Treg signaling in Tim-3+ Treg, as indicated by increases in pSTAT3 (suggesting IL-10 signaling) and pSTAT5 (suggesting IL-2 signaling) (Fig. 1D–E). Altogether, these findings demonstrate that Tim-3 expression correlates with a robust immunosuppressive phenotype in Treg during chronic viral infection.

To evaluate whether Tim-3+ Treg are derived from the thymus or induced in the periphery upon viral infection and persistence, we assessed the expression of Helios in Treg during LCMV Cl-13 infection. Helios, a member of the Ikaros family, is expressed early in the development of all thymic-derived Treg but is absent in peripherally induced Treg (31). Consistent with FoxP3+ Treg being thymically derived, we found that Tim-3+ Treg have higher expression of Helios (Fig. S1F) than Tim-3− Treg. We also noted a significant increase in the expression of FoxP3 in Tim-3+ Treg compared to Tim-3− Treg, suggesting that expression of Tim-3 enhances, or is at least associated with, Treg stability (Fig. S1G).

We next assessed the kinetics of the phenotypic changes in Treg during chronic LCMV infection. Thus, there was a significant increase in the proportion of Tim-3+ Treg with high levels of the proliferation marker Ki67+, while this metric remained unchanged in Tim-3− Treg (Fig. S1H). Expression of Tim-3 also marked Treg with a more profound change in the upregulation of CD44 and loss of CD62L, i.e. activated/effector Treg (Fig. S1I); these Treg also had the highest proportion of the effector/activation Treg markers ICOS (Fig. S1J) and PD-1 (Fig. S1K). Taken together, these data suggest that Tim-3+ Treg are poised for a stronger response than Tim-3− Treg during chronic infection, at least based on the expression of commonly used activation markers.

Tim-3+ Treg control viral persistence in chronic infection

To better understand the function of Tim-3+ Treg during chronic LCMV infection, we used conditional Treg-specific Tim-3 KO mice previously described by our group, breeding mice carrying a floxed exon 4 of Havcr2 (encoding Tim-3) with mice expressing a FoxP3-driven, tamoxifen-inducible, Cre (FoxP3eGFP-Cre-ERT2). These mice will be referred to as Tim-3Treg KO; mice expressing the Cre alone will be referred to as Cre-only or WT (12). These mice were treated with tamoxifen and infected with LCMV Cl-13 (experimental outline in Fig. 2A). Validation of the efficiency of Tim-3 KO after tamoxifen administration, with or without infection is shown in Fig. S2A–B. Thus, we observed that Tim-3Treg KO mice lost less weight and had a significantly faster recovery in weight than WT mice throughout the course of infection (Fig. 2B). Tim-3Treg KO mice also recovered fully to their starting weight.

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

Figure 2:

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WT and Tim-3Treg 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 CD44hiCD62Llo 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.

Next, we assessed (by qPCR) the viral burden in the liver and kidney on day 30 post-infection. Dramatically, we found that Tim-3Treg KO mice had a significant reduction of about 500-fold in viral copies in the liver and 100-fold in the kidney, compared with WT mice (Fig. 2C). Kidney viral titers at d30 were validated by plaque assay, for which 6/7 of the Tim-3 Treg KO kidney samples were below the limit of detection (Fig. S2C). In contrast, these mice did not show a difference in viral burden at day fifteen post-infection (Fig. S2D). Tim-3Treg KO mice were also mostly protected from severe lymphopenia (Fig. 2D) and had an increase in the CD8:Treg ratio (Fig. 2E). Treg-specific Tim-3 deletion also led to a decrease in the frequency of total Treg (Fig. 2F), and an elevated frequency of effector T cells in the spleen (Fig. 2G). Based on tetramer staining, Tim-3 Treg KO mice had an increase in the total number of gp33-specific T cells (Fig. 2H) compared to WT mice, although there was no change in the frequency of gp33-specific CD8+ T cells (Fig. 2I).

Tim-3 expression in Treg promotes Tex late during chronic infection

The development of exhausted T cells (Tex) among the pool of virus-specific T cells impairs the clearance of certain chronic viral infections, including LCMV (32, 33). To test the changes in Tex cells in Tim-3Treg KO mice during chronic LCMV infection, we focused on the expression of PD-1 and Tim-3, well-described markers of T cell dysfunction during persistent viral infection (34–37). We found that late during chronic infection (d30), antigen-experienced T cells (CD44hiCD8+) in Tim-3+ Treg KO mice exhibited a significant decrease in the frequency of terminally exhausted (PD-1+Tim-3+) T cells (Fig. 3A). We also measured the expression of Tox, a transcription factor required for the development of dysfunctional T cells (38). Thus, antigen-experienced CD8+ T cells from Tim-3Treg KO mice also had a lower proportion of PD-1+Tox+ T cells (Fig. 3B). These findings were recapitulated by Tex analysis of gp33-specific T cells. Thus, gp33-specific T cells also showed a decrease in the frequency of terminally exhausted PD-1+Tim-3+ (Fig. 3C) and PD-1+Tox+ T cells (Fig. 3D). Taken together, these data suggest that the expression of Tim-3 by Treg contributes to the development of gp33-specific Tex during the later stages of chronic infection, perhaps due to changes in the levels of virus.

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

Figure 3:

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WT and Tim-3Treg 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-1int) and Tim-3+PD-1+ (DP, double positive) CD44hi CD8+ T cells. (B) Frequency of PD-1+Tox+ in CD44hi CD8+ T cells. (C) Frequency of PD-1int and DP gp33+ CD8+ T cells. (D) Frequency of PD-1+Tox+ in gp33+ CD8+ T cells. WT (6–8), Tim-3Treg 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.

Tim-3 dampens the CD8+ T cell response early in chronic infection

The significant decrease in viral burden, along with the enhanced gp33-specific response and decrease in Tex late during chronic infection, motivated us to evaluate whether these changes are established in the early phase of chronic infection. We focused our analysis on day eight post-infection, as this is a time point at which there is partial T cell exhaustion (Fig. 4A). Our data revealed that at this early stage of infection, Tim-3Treg KO mice were protected from severe lymphopenia (Fig. 4B). However, Tim-3Treg KO mice did not have a decrease in viral burden by qPCR, at this time point (Fig. 4C). Tim-3Treg KO mice did have a selective increase in total CD8+ T cell count, with no change in the number of CD4+ T cells (Fig. 4D). Deletion of Tim-3 on Treg also did not change the number of total Treg at this early timepoint (Fig. 4E), rather the increase in the CD8:Treg ratio was driven mainly by the expansion of CD8+ T cells (Fig. 4F). Tim-3Treg KO mice did show a reduction in Treg frequency (Fig. 4G), with a corresponding elevated frequency of effector T cells (Fig. 4H). Furthermore, Tim-3Treg KO mice had increases in both total cell count and the frequency of gp33-specific CD8+ T cells (Fig. 4I–J). Finally, although we do not currently have the means to track Tim-3 “wannabe” Treg, we assessed the total Treg pool to determine whether selective loss of Tim-3 from these cells led to a corresponding loss of effector Treg. Indeed, total Treg in Tim-3Treg KO mice contained a smaller population of CD39+ Treg (Fig. 4K) but maintained normal levels of CD25 and PD-1 (Fig. S3A), compared to WT Treg.

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

Figure 4.

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WT and Tim-3Treg 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 CD44hiCD62Llo 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.

One of the consequences of wholesale Treg depletion is upregulation of the inhibitory ligand PD-L1 on innate immune cells and other infected cells (1). Therefore, we carried out a broad characterization of the innate immune compartment early during chronic infection of WT vs. Tim-3Treg KO mice. Interestingly, Tim-3Treg KO mice had a modest but significant reduction in the frequency of dendritic cells (DCs), and these cells also expressed lower levels of PD-L1 (Fig. S3 B–C). Additional innate populations examined, including monocytes, neutrophils, and macrophages, exhibited no difference in their proportions or in PD-L1 expression in Tim-3Treg KO vs. WT mice (Fig. S3 B–C). None of the innate cells that we examined exhibited a change in the expression of the costimulatory molecule CD80 (Fig. S3D) but there was an increase in CD86 expression by DC and monocytes (Fig. S3E). Altogether, these findings suggest that Tim-3+ Treg limit the functional capacity of gp33-specific T cells in part by promoting the upregulation of PD-L1 and limiting the co-stimulation potential of certain innate cells early during chronic infection.

Tim-3+ Treg promote T cell exhaustion early in chronic infection

We next aimed to determine the changes in exhausted T cells (Tex) early during chronic infection, at day eight after infection. Thus, antigen-experienced T cells did not show any difference in PD-1 and Tim-3 frequencies at day eight post-infection (Fig. 5A). Similarly, there was no detectable change in the proportion of Tox-expressing cells in this population (Fig. 5B). By contrast, Tim-3Treg KO mice had significant reductions in LCMV-specific PD-1+Tim-3+ (Fig. 5C) and PD-1+Tox+ gp33+ T cells (Fig. 5D), as well as an increase in the stem-like PD-1+Tim-3− gp33+ T cells (Fig. 5C). These findings suggest that Tim-3 expression byTreg promotes the early development of T cell exhaustion and limits the generation of the stem-like PD-1int population.

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

Figure 5.

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WT and Tim-3Treg KO mice were treated with tamoxifen for 5 days, followed by 8-day LCMV Cl-13 infection for (A-D). Similarly, WT and Tim-3Treg 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-1int) and Tim-3+PD-1+ (DP-“double positive”) CD44hi CD8+ T cells. (B) Frequency of PD-1+Tox+ in CD44hi CD8+ T cells. (C) Frequency of PD-1int and DP gp33+ CD8+ T cells. (D) Frequency of PD-1+Tox+ in gp33-specific CD8+ T cells. (E) Frequencies of PD-1int and DP in adoptively transferred WT P14 T cells. (F) Frequency of Tox+ gated on adoptively transferred WT P14 T cells. WT (6–8), 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.

Next, we sought to extend our results by evaluating the effects of Tim-3+ Treg on gp33-specific T cells with a fixed TCR, using adoptive transfer of WT P14 T cells into WT and Tim-3Treg KO mice. Reminiscent of our observations with polyclonal LCMV-specific T cells, there was a significant reduction in the frequencies of terminally exhausted PD-1+ Tim-3+ (Fig. 5E) and PD-1+Tox+ P14 T cells (Fig. 5F) in Tim-3Treg KO mice early during chronic infection. However, in contrast to polyclonal gp33-specific CD8+ T cells, there was no difference in the proportion of transferred P14 T cells with a PD-1+Tim-3− phenotype (Fig. 5E). Nonetheless, overall, these data suggest that selective deletion of Tim-3 from Treg is sufficient to limit the development of terminally exhausted WT P14 T cells.

Tim-3+ Treg restrict T cell function early in chronic infection

The improved gp33-specific T cell response observed at eight days post-infection in Tim-3Treg KO mice led us to further assess whether these cells were more functionally capable of killing gp33+ target cells and producing inflammatory cytokines. To test this, we cultured flow-sorted CD44hiCD8+ antigen-experienced polyclonal T cells with B16.gp33 target cells, a murine melanoma cell line that expresses gp33. Representative images showed a notable decrease in the confluency of B16.gp33 target cells adhered to the cell culture plate 25 hours after adding antigen-experienced T cells (Fig. 6A). Quantification revealed an increase in the cytotoxicity of antigen-experienced T cells from Tim-3Treg KO mice compared to WT mice (Fig. 6B). These data suggest that Treg-specific Tim-3 deletion enhances the functional capacity of gp33-specific T cells early during chronic infection.

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

Figure 6.

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WT and Tim-3Treg KO mice were treated with tamoxifen for 5 days, followed by LCMV Cl-13 infection (A-D). For E-F, WT and Tim-3Treg 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 CD44hiCD8+ 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 (6–8), 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.

Additionally, in vitro re-stimulation of CD8+ cells with gp33 peptide revealed that antigen-experienced T cells from Tim-3Treg KO mice had an elevated frequency of TNF−IFNγ+ and TNF+IFNγ+ T cells eight days post-infection (Fig. 6C) and a small increase in granzyme B (Gzmb) expression (Fig. 6D). To confirm these results, we adoptively transferred congenically marked WT P14 cells into either WT or Tim-3Treg KO mice, followed by LCMV Cl-13 infection, and harvested these cells from the spleen at eight days post-infection. Following in vitro gp33 peptide stimulation, P14 recovered from Tim-3Treg KO mice had an increase in the frequency of TNF+IFNγ+ cells (Fig. 6E) relative to WT mice. However, the level of Gzmb expression of P14 T cells transferred into Tim-3Treg KO mice was unchanged, compared to P14 cells transferred into WT mice (Fig. 6F). Overall, these findings support a model that Treg-specific Tim-3 deletion contributes to enhancing the effector functional capacity of LCMV-specific CD8+ T cells. Similarly, the lower level of Treg-mediated suppression in Tim-3Treg KO mice is sufficient to preserve the functional capacity of transferred WT P14 cells early during chronic LCMV infection. Nonetheless, there may be differences in the sensitivity of individual T cell clones to this effect.

Transcriptional profile of Tex subsets

Next, we wanted to determine if there are differences in the transcriptional identity of exhausted T cells when comparing the same Tex subsets between WT and Tim-3Treg KO mice. Thus, we performed bulk RNA sequencing (bulk RNA-seq) of adoptively transferred WT P14 T cells into WT and Tim-3 Treg KO mice eight days post-infection (Fig. 7A). We sorted the P14 cells into three Tex subsets: PD-1−Tim-3− (DN, double negative); PD-1+Tim-3− (PD-1int, PD-1 intermediate); and PD-1+Tim-3+ (DP, double positive). Principal component analysis revealed obvious divergence between the three P14 Tex subsets from WT vs. Tim-3Treg KO mice (Fig. 7B). This analysis suggests that Tim-3Treg KO drives (directly or indirectly) the development of Tex subsets with distinct transcriptional profiles during chronic infection.

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

Figure 7:

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WT and Tim-3Treg 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-1int), and PD-1+Tim-3+ (DP “double positive”). (B) Principal component analysis for Tex subsets from WT and Tim-3Treg KO mice. (C) Heatmap of selected significantly differentiated genes (p-value <0.05, and p-adjusted <0.02) in DN, PD-1int, and DP. WT (n=3), KO (n=3). For (B), each dot represents a biological replicate.

Differential gene expression analysis of these RNAseq data demonstrated a reduction in the transcript abundance of Tox in all three Tex groups from Tim-3Treg KO mice, compared to WT mice (Fig. 7C). DN P14 T cells that had been transferred into Tim-3Treg KO mice had elevated levels of transcripts encoding costimulatory molecules like Cd226 and markers of effector T cells such as Id2, Gzma, and an increase in genes involved in T cell lymph node egress like Cx3cr1 and S1pr5 (Fig. 7C). Analysis of the PD-1int (precursor or stem-like exhausted) P14 population from Tim-3Treg KO mice revealed a decrease in the inhibitory molecule Ctla4, along with an increase of the costimulatory molecule Cd86 (Fig. 7C). These cells also had an increase in genes associated with effector function (Fasl, Gzmb, Gzma, Id2, Klrg1, Tbx21 (T-bet)) and in genes encoding proteins that promote trafficking into the circulation (Cx3cr1 and S1pr1) (Fig. 7C). Finally, terminally exhausted DP P14 T cells from Tim-3Treg KO mice showed lower expression of Lag3, in addition to elevated gene sets associated with T cell activation like Gzmk, Gzmm, Id2 and Klrg1 (Fig. 7C). This population of DP P14 T cells also exhibited higher expression of genes important for migration, although they also upregulated Cd69, which antagonizes the function of S1pr1 (39–41) (Fig. 7C). Thus, these results demonstrate that deletion of Tim-3 in Treg alters the identity of gp33-specific T cells at different stages of T cell exhaustion during chronic infection.

Discussion

The goal of this study was to dissect the contribution of Tim-3+ Treg to viral persistence and T cell responses during chronic viral infection. We used the LCMV Cl-13 chronic infection model, which establishes a long-term systemic high viral load, develops T cell exhaustion, and is widely used to model chronic infections in humans. As expected, we observed that Tim-3 is upregulated in Treg during chronic LCMV infection. Strikingly, we found that conditional Treg-specific Tim-3 deletion was sufficient to ameliorate virus-specific T cell exhaustion and improve T cell function. Tim-3Treg KO mice also had greater viral clearance at later time points.

In LCMV Cl-13 infection, the increase in Tim-3 expression seems to be unique to Treg rather than Tconv. The kinetics for the proportion of total Tconv and Tim-3+ Tconv for acute and chronic LCMV infection did not show a notable difference compared to uninfected mice. Interestingly, this is also a phenotype that we have reported in people with HIV on anti-retroviral therapy (PWH-ART) (18). In addition, thymic development might be the source of the majority of Tim-3+ Treg that expand during chronic infection. In both infected and uninfected mice, splenic Tim-3+ Treg had elevated expression of Helios and FoxP3 in comparison to the Tim-3− Treg. Therefore, these data demonstrate that during chronic infection, there is an increase of thymically derived Tim-3+ Treg, but not in CD4+ Tconv.

Tim-3 expression by Treg may promote a robust but short-lived effector Treg phenotype during chronic infection. Similar to our previous findings in cancer (12) and HIV (18), Tim-3+ Treg upregulated important activation and immunosuppression markers such as CD25, CTLA-4 and IL-10 during LCMV Cl-13 infection. In addition, Tim-3+ Treg in PWH-ART have an increase in the frequency of Annexin V staining and active caspase-3, suggesting that these cells are prone to apoptosis (18). Interestingly, in chronic infection, these cells displayed a dramatic increase in expression of KLRG-1 and a decrease in Bcl-2. These findings indicate that Tim-3 expression identifies Treg with a strong effector phenotype, but which may also have a relatively short lifespan. Previous studies evaluating Tim-3+PD-1+ Treg found that the frequency of these cells increases at the peak of graft rejection and that ex vivo stimulation with galectin-9, a known ligand for Tim-3, induced cell death (17). In this same study, adoptive transfer of Tim-3+ Treg failed to prolong graft survival and these cells had elevated Annexin V staining at five days post-transfer. In addition, Tim-3 seems unlikely to drive Treg instability since Tim-3+ Treg maintained robust FoxP3 expression during infection. In conclusion, these findings demonstrate that Tim-3+ Treg possess a robust immunosuppressive phenotype during persistent LCMV infection.

In our experiments, deletion of Tim-3 in Treg led to rapid recovery from wasting disease that was independent of viral load during chronic infection. Previous work has shown that it is the CD4+ T cell compartment, and not necessarily the proinflammatory cytokines IFNγ and TNF, which promotes wasting disease (42). Our findings with Tim-3Treg KO mice indicate that Tim-3+ Treg are major contributors to morbidity during chronic infection. The fact that Tim-3Treg KO mice exhibited no change in the number of total Treg is consistent with the idea that Tim-3+ Treg are a key regulator of wasting disease.

Tim-3 has been described to have both inhibitory and stimulatory functions, depending on the cellular context (43–46). Our data demonstrating a positive association between Tim-3 expression and Treg effector status are more consistent with Tim-3 function on Treg falling under the latter category. This is also supported by a decrease in CD39, a major marker of immune suppression, in total Treg from Tim-3Treg KO mice compared to their WT counterpart. Thus, even at the points of infection when Tim-3 did not seem to affect Treg number, these cells exhibited phenotype indicative of reduced suppression. In addition, uninfected Tim-3Treg KO mice did not experience any signs of wasting disease during the 40 days following tamoxifen administration, suggesting that lack of Tim-3+ Treg is not enough to destabilize Treg and thus unleash the development of spontaneous autoimmunity. Consistent with these findings, germline deletion of Tim-3 also does not appear to lead to autoimmunity (47). Therefore, our study suggests that Tim-3 is not required for the development of all eTreg, including those that suppress self-reactivity in specific tissues. Alternatively, the duration of our experiments was possibly insufficient for development of autoimmunity and/or the genetic background of the mice (C57BL/6) was not conducive to the development of autoimmunity.

We also demonstrate that Tim-3+ Treg control viral persistence and gp33-specific responses during chronic infection. This finding may result from the improved gp33-specific T cell function and the reduction in the development of T cell exhaustion in Tim-3Treg KO mice. However, we did not assess the potential contribution of a simultaneous improvement in antibody-mediated clearance of virus. A previous study revealed that cessation of viremia in LCMV Cl-13 depends on the formation of viral envelop-specific antibodies, and that mice with impaired B cell responses fail to resolve chronic infection (48). This study also demonstrated that neutralizing antibody titers start to increase after day 40–50 post-infection. Therefore, while it is possible that improved B cell responses contribute to the decrease in viral burden in Tim-3Treg KO mice, the fact that we observed changes in viral burden at 10–20 days before the expected appearance of neutralizing antibodies, together with the early improved T cell responses, supports a model where most of the improved viral control is driven by T cells.

Early interactions between Tim-3+ Treg and DCs may also alter the priming phase of T cell activation and promote viral persistence. Our study revealed that Tim-3Treg KO mice led to a reduction in PD-L1 expression by DCs. PD-L1 is an important molecule in chronic infection, as blocking this inhibitory ligand reduces chronic LCMV viral burden (49, 50). Upregulation of PD-L1 by DCs and infected cells restricts clearance of a chronic LCMV infection after Treg ablation (1). Our findings also support the idea that Tim-3+ Treg limit the expression of CD86 in DCs and monocytes during infection, leading to a reduction in co-stimulatory capacity. Therefore, interactions of Tim-3+ Treg with DCs and monocytes may induce a tolerogenic state that dampens T cell responses and promotes viral persistence.

Conditional deletion of Tim-3 in Treg resulted in a decrease in exhausted T cells throughout chronic infection. This is a phenotype that we have also observed in terminally exhausted, tumor infiltrating, CD8+ T cells (12). Thus, Tim-3+ Treg limit anti-tumor immunity and promote tumor progression, another context where there is chronic antigen exposure of T cells. In viral chronic infection, we observed these changes in gp33-specific Tex subsets as early as eight days post-infection and they were even more pronounced later during infection. Interestingly, antigen-experienced T cells showed a significant difference in the proportion of T cell exhaustion markers (including both surface markers and Tox) only during late infection. These findings suggest that a decrease in the proportion of immunodominant gp33-specific Tex cells may contribute to the control of viral persistence in Tim-3Treg KO mice.

Our findings further suggest that Tim-3+ Treg may control viral persistence by promoting Tox-dependent gene programming in gp33-specific T cells. Bulk RNA-seq analysis of transferred P14 T cells early during the establishment of chronic infection revealed transcriptionally distinct profiles in PD-1−Tim-3− (DN), PD-1intTim-3− (PD-1int) and PD-1+Tim-3+ (DP) exhausted T cells in Tim-3Treg KO versus WT mice. Common changes observed in all three populations included upregulation of transcripts related to T cell activation and effector function. Surprisingly, the only major Tex-related molecule downregulated in all three populations from Tim-3Treg KO mice was Tox, a transcription factor that is required for the early commitment of dysfunctional T cells during chronic infection. In fact, a recent study demonstrated that enforced Tox expression is sufficient to induce a T cell exhaustion program (38). Therefore, early during chronic infection, Tim-3+ Treg may promote strong epigenetic and transcriptional changes driven by Tox in T cells which may promote exhaustion and thus limit viral clearance.

One limitation of this study is that we are currently not able to specifically track “ex-Tim-3+” Treg. The ability to track such cells would help address the question of whether they lose the effector phenotype after Tim-3 deletion and/or if Tim-3 is required for Treg survival and long-term maintenance. Nonetheless, based on previous work from our group, Treg from people with HIV have reduced suppressive capacity in the presence of a putative blocking mAb to Tim-3 (18), further supporting the model that Tim-3 is required for robust immunosuppressive responses. Finally, future studies are necessary to address whether changes in the spatial distribution of Tim-3+ Treg within tissues are needed to achieve such profound effects on viral persistence.

In summary, this work supports a model whereby Tim-3+ Treg control viral persistence by dampening gp33-specific T cell function and promoting the development of T cell exhaustion. Therefore, defining the mechanism by which Tim-3 functions on Treg may provide insights into new therapeutic avenues for the management of chronic infections.

Supplementary Material

1

Key Points:

  • There is a transient increase in Tim-3+ Treg during chronic LCMV infection

  • Tim-3+ Treg promote dysfunction of CD8+ T cells during chronic infection with LCMV

  • Treg-specific Tim-3 deficiency promotes clearance of a chronic LCMV infection

Acknowledgments

We would like to thank Dr. E. J. Wherry’s lab for kindly providing us with LCMV Cl-13 viral stock and BHK cells for further virus propagation in our lab. We thank the University of Pittsburgh Unified Flow Core for help with flow cytometry. This work benefitted from a Cytek Aurora CS Spectral Sorter funded by NIH (S10OD032265; PI L. Kane). Visual abstract created with BioRender.com. Author contributions: Conceptualization - LPK, HNR, HB; Investigation - LPK, HNR, HB, AGD, PM, BM, BX, GD, DV, IM, OE, JD; Funding acquisition and supervision - LPK; Writing - HNR, HB, LPK.

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