Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection

Abstract

T cell Ig and mucin domain (Tim) 3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes infection. Analysis of wild-type (WT) mice infected with L. monocytogenes revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to L. monocytogenes by WT and Tim-3 knockout (KO) mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide Ag ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to L. monocytogenes infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection.

FIGURE 1

CD8 T cells responding to LM infection express Tim-3. Wild-type mice were infected with attLM-OVA and longitudinal analysis of CD8 T cell responses was performed. Splenocytes were harvested on the indicated days postinfection and analyzed as shown. (A) Tim-3 expression on CD8T cells relative to samples stained with isotype control antibody (Iso). For naïve mice, data for total CD8 T cells are shown; for infected mice (days 7, 14 and 50; day 50 + 6), data for activated (Thy1.2⁺CD8α^loCD11a^hi) CD8T cells are shown. Numbers in the upper right corner of histograms are the mean frequencies of Tim-3⁺ cells (n ≥ 4 data points) and the standard error between data points. (B) Surface marker expression by activated CD8T cells. Upper panel: expression of KLRG1 and CD127; lower panels: Tim-3 expression by KLRG1⁺CD127⁻ and KLRG1⁻ CD127⁺ cells within the activated CD8T cell pool. (C) Frequencies of Tim-3⁺ cells within the KLRG1⁺CD127⁻ and KLRG1⁻ CD127⁺ subsets of activated CD8T cells. Shown for each symbol are the average and standard error of at least 4 data points. (D) CD107a or IFN-γ expression by CD8T cells stimulated with OVAp ex vivo. Histograms show the total CD8 T cell population (Thy1.2⁺CD8α⁺CD11a^hi) present in the samples. All data shown are representative of results from at least 2 independent experiments.

FIGURE 2

Tim-3 KO mice have impaired primary CD8 T cell responses to LM infection. Wild-type (WT) and Tim-3 KO (KO) mice were infected with attLM-OVA. Peripheral blood samples or spleens were obtained on the indicated days postinfection and analyzed. (A) Longitudinal analysis of the frequencies of activated (Thy1.2⁺CD8α^loCD11a^hi) CD8T cells in peripheral blood. (B) Total numbers of activated CD8T cells recovered from spleens. (C) Analysis of OVA tetramer+ CD8 T cells in splenocytes obtained on day 7 postinfection. (D) Total numbers of OVA tetramer⁺ CD8 T cells recovered from spleens. (E) IFN-γ and CD107a expression by CD8 T cells following ex vivo stimulation with OVAp. Assays were performed using splenocytes obtained on day 7 postinfection. (F) Total numbers of IFN-γ or CD107a-expressing CD8 T cells recovered from spleens as calculated from data represented in panel E. All data shown are representative of results from at least 2 independent experiments. For all graphs, symbols or bars represent the mean and standard error of 4 to 8 data points. * p≤ 0.05; **p≤0.01.

FIGURE 3

Decreased responses to LM by Tim-3 KO CD8 T cells within a wild-type host. (A) Outline of the co-adoptive transfer system used to assess responses by wild-type (WT) and Tim-3 KO (KO) OT-I CD8 T cells within a shared WT host. Samples containing 1000 WT and 1000 KO OT-I cells of the indicated Thy1 allotypes were prepared and injected into Thy1.1/1.1 hosts. The next day, hosts were infected with attLM-OVA (B) Frequencies of WT and Tim-3 KO OT-I cells in cell mixtures immediately prior to injection into hosts. (C) Tim-3 expression by splenic OT-I cells as determined on the indicated days postinfection. (D) Longitudinal analysis of Tim-3 expression by WT OT-I cells in spleen. Each symbol represents the mean and standard error of between 4 and 20 data points. (E) Expression of KLRG1 and CD127 by OT-I cells in splenocytes obtained on day 7 postinfection. (F, G) Expression of IFN-γ (panel F) or TNF (panel G) by OT-I cells in splenocytes following ex vivo stimulation with OVAp. Data were obtained from spleens isolated on day 6 postinfection. (H) Frequencies of IFN-γ ⁺ and TNF⁺ cells within the fractions of WT and Tim-3 KO OT-I cells detected in splenocytes. Frequencies were calculated from data represented in panels E and F. Each set of box and whiskers was generated from 12 data points; **p≤0.01. All data shown are representative of results from at least 2 independent experiments.

FIGURE 4

The ability of Tim-3 KO CD8 T cells to persist after LM infection is impaired. Wild-type (WT) and Tim-3 KO (KO) OT-I cells (1000 each) were injected into WT hosts. The next day, hosts were infected with attLM-OVA. OT-I cells were analyzed as indicated. (A, B) Frequencies of wild-type (WT) and Tim-3 KO (KO) OT-I cells in peripheral blood on day 8 (panel A) or day 20 (panel B) after infection with attLM-OVA. (C) Ratios between WT and Tim-3 KO OT-I cells in peripheral blood samples taken on the indicated days postinfection. (D) Frequencies of WT and Tim-3 KO OT-I cells in splenocytes obtained on the indicated days postinfection. (E) Ratios between WT and Tim-3 KO OT-I cells in splenocytes obtained on the indicated days postinfection. Filled circles in panels C and E represent the mean and standard error of values from 4 to 16 independent samples. Flow cytometric data shown are representative of results from at least 3 independent experiments.

FIGURE 5

Tim-3 KO CD8 T cells undergo less proliferation following LM infection. Wild-type (WT) and Tim-3 KO (KO) OT-I cells (1000 each) were injected into WT hosts. The next day, hosts were infected with attLM-OVA. Splenic OT-I cells were analyzed as indicated. (A) Expression of Bim by WT and Tim-3 KO OT-I cells as assessed on day 8 postinfection. (B) Mean fluorescence intensities (MFI) for Bim levels as determined on the indicated days postinfection. Bim expression was analyzed as shown in panel A. Each symbol represents the mean and standard error of 4 data points. (C) Levels of activated caspases 3 and 7 expressed by OT-I cells in splenocytes obtained on day 7 postinfection. (D) Frequencies of OT-I cells expressing activated caspases 3 and 7 as determined on the indicated days postinfection. (E) Levels of BrdU incorporated by OT- I cells in splenocytes as assessed 15 hrs after BrdU injection. Cells were obtained on day 7 postinfection. (F) Frequencies of BrdU⁺ splenic OT-I cells as determined on the indicated days postinfection. Each pair of connected symbols represents WT and Tim-3 KO OT-I cells within the same host. Data were pooled from 3 independent experiments. Each time point shows analysis of at least 8 mice. (G) Frequencies of BrdU⁺ cells in the KLRG⁺CD127⁻ and KLRG⁻ CD127⁺ subsets of splenic OT-I cells. Analysis was performed on day 7 postinfection. All data shown are representative of results from at least 2 independent experiments. * p≤ 0.05; **p≤0.01; ***p≤0.001.

FIGURE 6

Tim-3 KO mice have impaired secondary CD8 T cell responses to LM infection. Wild-type (WT) and Tim-3 KO (KO) mice were infected with virLM-OVA. Peripheral blood samples or spleens were taken on the indicated days after secondary infection and analyzed. (A) Longitudinal analysis of the frequencies of OVA tetramer⁺ CD8T cells in peripheral blood. (B) Total numbers of OVA tetramer-positive CD8T cells recovered from spleens taken on the indicated days postinfection. (C) IFN-γ expression by CD8 T cells stimulated with OVAp ex vivo. Data were obtained from splenocytes isolated on day 6 post-secondary infection. (D) Total numbers of IFN-γ⁺ CD8 T cells recovered from spleens as calculated from data represented in panel C. All data shown are representative of results from 2 independent experiments. For all graphs, symbols or bars represent the mean and standard error of 4 or 5 data points. * p≤ 0.05; **p≤0.01.

FIGURE 7

Decreased secondary responses to LM by Tim-3 KO CD8 T cells within a wild-type host. Samples containing a mixture of 70,000 WT and 70,000 Tim-3 KO OT-I memory cells were injected into WT hosts. The next day, hosts were infected with attLM-OVA. Responses by OT-I cells were analyzed as indicated. (A) Frequencies of WT and Tim-3 KO OT-I cells in cell mixtures immediately prior to injection into hosts. (B) Expression of KLRG1 and CD127 by OT-I cells immediately prior to injection into hosts. (C, D) Expression of Tim-3 (panel C) and KLRG1 and CD127 (panel D) by splenic OT-I cells as determined on day 6 postinfection. (E, F) Expression of IFN-γ (panel E) and TNF (panel F) by OT-I cells in splenocytes following ex vivo stimulation with OVAp. Data were obtained from splenocytes isolated on day 6 postinfection. (G) Frequencies of IFN-γ⁺ and TNF⁺ cells within the splenic WT and Tim-3 KO OT-I populations. Frequencies were calculated based on the data represented in panels E and F. Each set of box and whiskers was generated from 6 data points. (H) Frequencies of WT and KO OT-I cells in spleen on day 6 postinfection. (I) Frequencies of WT and KO cells in the total OT-I cell pool within spleens on day 6 postinfection. Frequencies were calculated based on the data represented in panel H. Each circle represents data obtained from an individual host mouse. All data were obtained from a single experiment involving multiple donors for memory cells; these were pooled and injected into host mice. **p≤0.001.