The Role of TIM-3 in Adaptive Immunity

T cell immunoglobulin mucin-3 (TIM-3) is an immune modulatory molecule that functions in both adaptive and innate immunity. In the adaptive immune system, TIM-3 was originally identified as a molecule selectively expressed on interferon gamma (IFN-γ) producing cluster of differentiation (CD)-4+ and CD8+ T cells and was subsequently found to be upregulated in T cells that become dysfunctional or exhausted in chronic infection or cancer. In the innate immune system, expression of TIM-3 is also found on natural killer (NK) cells, dendritic cells, macrophages and monocytes. Clinically, up-regulation of TIM-3 on NK cells, as well as on monocytes and macrophages, correlates with worsening disease in settings of chronic infection and cancer. TIM-3’s co-expression with Programmed Cell Death-1 (PD-1) on exhausted tumor infiltrating lymphocytes (TILs) and NK cells, together with enhanced anti-tumor efficacy upon co-blockade of TIM-3 and PD-1 in several pre-clinical syngeneic tumor models, make it an attractive target for immune-therapy. The specific aims of this thesis will investigate the functional role of TIM-3 in CD8+ T cells using gene knockout or pharmacological inhibition studies. First, we determined the impact of loss of TIM-3 on tumor control and TIL function using TIM-3 knock-out (KO) mice in comparison with PD-1 KO, TIM-3 and PD-1 double (dKO) or wild type (WT) mice. Utilizing syngeneic murine models of cancer we found that loss of TIM-3 alone was not sufficient to provide tumor control, however, in the background of PD-1 null, TIM-3 loss resulted in a significant increase in lifespan, increased CD8+ tumor infiltrating lymphocytes (TILs) and decreased T regulatory cells compared to WT and PD-1 alone. Overall, there were relatively distinct gene expression signatures from CD8+ TILs isolated from TIM-3 KO and PD1 KO mice bearing MC38 tumors. There were, however, some notable exceptions which include similarities in upregulation of phosphoinositide 3-kinase (PI3K) and B cell receptor signaling pathways. Interestingly, the CD8+ TIL’s isolated from TIM-3 KO mice did not share any overlapping gene expression pathway changes when compared to dKO. However, dKO and PD1 KO CD8+ TILs shared downregulation of the cell control and nucleotide excision repair (NER)pathways. Second, using pharmacologic blockade, we determined the effect of TIM-3 inhibition on CD8+ T cells functionality and gene expression profiles utilizing a well characterized function-blocking anti-human TIM-3 monoclonal antibody JNJ-64158120 (JnJ-120). We describe for the first time that JnJ-120 enhances IFN-γ and tumor necrosis factor alpha (TNF-α) production, promotesCD8+ T cell proliferation, and increases gene expression of T cell activation markers such asinterleukin-2 receptor (IL-2R) and gene associated with IFN-γ regulation, such as (interferon regulatory factor) IRF-4. Together, these findings suggest that TIM-3 functions largely through a pathway that is non-redundant with PD-1 and that targeting both enhances anti-tumor efficacy through activation of T cell responses. These data provide rationale to support the combined targeting of TIM-3 with PD-1 agents in the clinic