The influence of T cell Ig mucin-3 signaling on central nervous system autoimmune disease is determined by the effector function of the pathogenic T cells

Abstract

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS mediated by self-reactive, myelin-specific T cells. Both CD4(+) and CD8(+) T cells play important roles in the pathogenesis of MS. MS is studied using experimental autoimmune encephalomyelitis (EAE), an animal model mediated by myelin-specific T cells. T cell Ig mucin-3 (Tim-3) is a cell surface receptor expressed on CD4(+) IFN-γ-secreting Th1 cells, and triggering Tim-3 signaling ameliorated EAE by inducing death in pathogenic Th1 cells in vivo. This suggested that enhancing Tim-3 signaling might be beneficial in patients with MS. However, Tim-3 is also expressed on activated CD8(+) T cells, microglia, and dendritic cells, and the combined effect of manipulating Tim-3 signaling on these cell types during CNS autoimmunity is unknown. Furthermore, CD4(+) IL-17-secreting Th17 cells also play a role in MS, but do not express high levels of Tim-3. We investigated Tim-3 signaling in EAE models that include myelin-specific Th17, Th1, and CD8(+) T cells. We found that preventing Tim-3 signaling in CD4(+) T cells altered the inflammatory pattern in the CNS due to differential effects on Th1 versus Th17 cells. In contrast, preventing Tim-3 signaling during CD8(+) T cell-mediated EAE exacerbated disease. We also analyzed the importance of Tim-3 signaling in EAE in innate immune cells. Tim-3 signaling in dendritic cells and microglia did not affect the manifestation of EAE in these models. These results indicate that the therapeutic efficacy of targeting Tim-3 in EAE is dependent on the nature of the effector T cells contributing to the disease.

FIGURE 1

Tim-3 signaling determines the Th17:Th1 ratio among responding CD4⁺ T cells in vivo. (A) The number of total CD4⁺ T cells in the spleens of WT and Tim-3^−/− mice 7 d post-immunization with rMOG is shown. Splenocytes from immunized mice were counted, stained for CD4 and analyzed by flow cytometry. CD4⁺ T cell number was calculated as the total splenocyte number × % CD4⁺ cells. Error bars represent s.e.m. (n = 6). (B) The number of IFN-γ- (Th1) and IL-17-secreting (Th17) cells in the spleens of WT and Tim-3^−/− mice 7 d post-rMOG immunization is shown. The numbers of Th1 and Th17 cells were determined by ELISPOT using MOG_97–114 to stimulate the T cells. Error bars represent s.e.m. (n = 6), *p < 0.05. (C) The Th17:Th1 ratio observed in the spleens of Tim-3^−/− mice compared to WT mice after rMOG immunization is shown. The ratios were calculated from the data in (B); each dot represents an individual mouse. **p < 0.01. Data are representative of three independent experiments with 6 mice/group in each experiment.

FIGURE 2

Deficiency in Tim-3 signaling increases the incidence of classic EAE. (A) The numbers of WT and Tim-3^−/− mice exhibiting atypical or classic EAE following rMOG immunization are shown. Data are pooled from three independent experiments with a total of 30 mice/group. *p < 0.05. (B) The Th17:Th1 ratios of MOG_97–114-specific T cells isolated from the brains and spinal cords (SC) of WT and Tim-3^−/− mice are shown. The numbers of Th1 and Th17 cells were determined by ELISPOT within 1 d of disease onset. Each dot represents an individual mouse. *p < 0.05. Data are representative of three independent experiments using 5 mice/group in each experiment.

FIGURE 3

Lack of Tim-3 signaling in T cells alters the clinical signs of EAE without affecting disease severity or onset. (A) Disease course in WT recipients after adoptive transfer of WT or Tim-3^−/− CD4⁺ MOG_97–114-specificT cells. Disease incidence was 100% in both groups (20/20). (B) Average day of onset of EAE is shown for mice receiving WT or Tim-3^−/− cells. Error bars represent s.e.m. (n = 20). (C) Numbers of WT mice exhibiting atypical or classic EAE after adoptive transfer of CD4⁺ MOG_97–114-specific WT or Tim-3^−/− cells (n = 20). *p < 0.05. Data are pooled from three independent experiments.

FIGURE 4

Tim-3 signaling induces death in activated CD8⁺ T cells. (A) Flow cytometric analysis of Tim-3 expression on naïve 8.8 T cells and 8.8 T cells stimulated once or twice with MBP_79–87 peptide in vitro. Data are gated on CD8⁺ T cells and are representative of three experiments. (B) Flow cytometric analysis of Annexin V and 7-AAD expression on 8.8 T cells that were stimulated with MBP_79–87 peptide for 7 d and then cultured overnight with increasing concentrations of galectin-9. The numbers above the plots indicate the concentration of galectin-9 (μg/ml) added to the culture. Data are gated on CD8⁺ T cells and are representative of three independent experiments. (C) Naïve and previously activated 8.8 T cells were incubated with increasing concentrations of galectin-9 overnight and the number of live cells was determined in each culture by counting cells stained with trypan blue (n = 5). *p < 0.05. Data are representative of three independent experiments.

FIGURE 5

Severity and onset of CD8⁺ T cell-mediated EAE is influenced by Tim-3 signaling. (A) Disease course of CD8⁺ T cell-mediated EAE induced by adoptive transfer of activated CD8⁺ 8.8 T cells into recipients that were treated every other day with either 100 μg Tim-3 blocking Ab or control Ab (rat IgG1, κ). The percent weight loss relative to d 0 is shown (n = 5). *p < 0.05, **p < 0.01. Data are representative of three experiments with 5 mice/group in each experiment. (B) Disease course of CD8⁺ T cell-mediated EAE induced in Tim-3^+/+ and Tim-3^/– 8.8 mice by vaccinia virus infection. Error bars represent s.e.m (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative of three experiments with 5 mice/group in each experiment. (C) The numbers of CD8⁺ T cells in the CNS of Tim-3^+/+ and Tim-3^−/− 8.8 mice 7 d post-vaccinia virus infection are shown. The numbers were determined by staining CNS mononuclear cells with Abs specific for CD8 and Vβ8. Each dot represents an individual mouse. *p < 0.05. (D) The mean fluorescence intensity (MFI) for IFN-γ intracellular staining is shown for CD8⁺ T cells isolated from the CNS of Tim-3^+/+ and Tim-3^−/− 8.8 mice 7 d post-vaccinia infection. Error bars represent s.e.m. (n = 5). Data are representative of 6 mice from three independent experiments.

FIGURE 6

Lack of Tim-3 expression on innate immune cells does not influence the effector function of myelin-specific CD4⁺ and CD8⁺ T cells primed in vitro and manifestation of EAE. (A) Flow cytometric analyses of CD4⁺ MOG_97–114-specific TCR transgenic T cells that were incubated with sorted WT (top) or Tim-3^−/−(bottom) splenic DCs, MOG_97–114 peptide and LPS for 7 d. Data are gated on CD4⁺ T cells. (B) Flow cytometric analysis of MBP-specific CD8⁺ 8.8 T cells that were incubated with either sorted WT (top) or Tim-3^−/−(bottom) splenic DCs in the absence (left) or presence (right) of MBP_79–87 peptide for 7 d. Data are gated on CD8⁺ T cells. Data are representative of 6 mice analyzed from three independent experiments for (A) and (B). (C) The number of indicated BMC recipients exhibiting classic or atypical EAE after adoptive transfer of activated WT MOG_97–114-specific CD4⁺ T cells is shown (n = 16). (D) Disease course of EAE in the indicated BMC recipients following adoptive transfer of activated CD8⁺ 8.8 T cells. The percent weight loss relative to d 0 is shown; differences in weights between the two groups of mice were not statistically significant. Error bars represent s.e.m. (n = 5). Data are representative of three independent experiments with 5 mice/group in each experiment. (E) The number of mice with classic or atypical EAE is shown for WT or Tim-3^−/− recipients of activated WT MOG_97–114-specific CD4⁺ T cells (n = 10). (F) Disease course of EAE in WT or Tim-3^−/− recipients of activated CD8⁺ 8.8 T cells. Error bars represent s.e.m. (n = 4). Data are representative of three experiments with 4 mice/group in each experiment.