SUMMARY Regulatory T cells (Treg cells) are critical mediators of self-tolerance, but they can also limit effective anti-tumor immunity. Although under homeostasis a small fraction of Treg cells in lymphoid organs express the putative checkpoint molecule Tim-3, this protein is expressed by a much larger proportion of tumor-infiltrating Treg cells. Using a mouse model that drives cell-type-specific inducible Tim-3 expression, we show that expression of Tim-3 by Treg cells is sufficient to drive Treg cells to a more effector-like phenotype, resulting in increases in suppressive activity, effector T cell exhaustion, and tumor growth. We also show that T-reg-cell-specific inducible deletion of Tim-3 enhances anti-tumor immunity. Enhancement of Treg cell function by Tim-3 is strongly correlated with increased expression of interleukin-10 (IL-10) and a shift to a more glycolytic metabolic phenotype. Our data demonstrate that Tim-3 + Treg cells may be a relevant therapeutic target cell type for the treatment of cancer.

Regulatory T cells (Treg) are critical mediators of self-tolerance but can also limit effective anti-tumor immunity. We and others previously reported that 40-60% percent of Treg-infiltrating head and neck cancer (HNC) and other tumors highly express Tim-3, compared with about 5% in lymphoid organs. Tumor-infiltrating Tim-3+ Treg also have enhanced suppressive function and display a more effector-like phenotype. Using a novel mouse model with cell type-specific Tim-3 expression, we show here that expression of Tim-3 by Treg is sufficient to drive Treg to a more effector-like phenotype, resulting in enhanced suppressive activity and increased tumor growth. These findings may help to reconcile previous reports that some Tim-3 antibodies enhance T cell responses in vivo, while expression of Tim-3 has a cell-intrinsic ability to enhance TCR signaling and T cell activation. Thus, we propose that Tim-3 regulates anti-tumor immunity at least in part through enhancement of Treg function. To our knowledge, this is the first example in which expression of a single co-stimulatory molecule is sufficient to drive differentiation of Treg in this manner.

There is a progressive TIM-3+ T reg increase in solid tumors as the tumor progress. W have found that knocking out the ability of T reg to induce TIM-3 lead to delayed progression of tumors in the TIM-3 conditional knockout mice upon both hot (MC38) and cold (B16F10) syngeneic tumor challenges. We are employing our conditional T reg specific TIM-3 Knockout mouse to study if presence or absence of TIM-3 on T reg cells within these tumor models. Preliminary data has shown that our conditional Tim3 knockout T reg cells are found at a much lower frequency, and have an altered suppressive phenotype within the microenvironment of these tumors. We are currently investigating the other changes in the different subsets of lymphoid compartment in tumors. These data suggest that altering the expression of TIM-3+ on T reg has the potential to act as an effective target to alter tumor microenvironment. Targeting TIM-3+ T reg may therefore be attractive model to study, along with other combination immunotherapy to increase the success in immunotherapy on patients who develop resistance to primary immunotherapy.

Regulatory T cells (T reg) are mediators effective anti-tumor immunity. We and others previously reported that 40–60% percent of T reg-infiltrating head and neck cancer (HNC) and other tumors highly express Tim-3, compared with about 5% in lymphoid organs. Tumor-infiltrating Tim-3+ T reg also have enhanced suppressive function. Using a novel mouse model with cell type-specific inducible Tim-3 expression, we show here that expression of Tim-3 by T reg is sufficient to drive Treg to a more effector-like phenotype, resulting in enhanced suppressive activity and increased tumor growth. TIM-3 +T reg also seem to be metabolically altered driven by active signaling in ERK and m-TOR pathway. We further see that T reg specific deletion of TIM-3 leads to decrease in T reg frequency in tumor infiltrating lymphocytes and delay in tumor progression. These findings suggest to a possibility of using Tim-3 as a target to alter the tumor progression and clearance highliting a qualitatively different role for TIM-3 in T reg cells. Thus, we propose that Tim-3 regulates anti-tumor immunity at least in part through enhancement of T reg function. To our knowledge, this is the first example in which expression of a single co-stimulatory molecule is sufficient to drive differentiation of T reg and alter immune response in this manner.

T cell (or transmembrane) immunoglobulin and mucin-domain containing protein-3 (Tim-3) is a cell-surface protein expressed during T cell exhaustion, a process that leads to a progressive loss of effector function due to chronic T cell receptor (TCR) stimulation. In humans, Tim-3 expression has also been found to be increased on Treg isolated from tumors and during chronic viral infection. However, the role of Tim-3 on Treg during chronic viral infection remains largely unknown. Our main hypothesis is that Tim-3 expression on Treg will increase their proliferation and suppressive capacity during LCMV infection. To test this, we infected C57/BL6 mice for 30 days with LCMV clone-13, a well-established model for chronic infection. We found that at 16 days post-infection there is an increase in the frequency and number of Tim-3+Foxp3+ Treg in the spleen. By flow cytometry we found that Tim3+ Treg have increased expression of CD44, Ki67, Helios, ICOS, KLRG-1, CD39 and CD25, compared with Tim-3− Treg. Therefore, our data suggest that Tim-3+ Treg have a phenotype associated with higher activation, proliferation and suppression during chronic infection. Understanding the contributions of Tim-3 to Treg activation and function may provide new insights into the role of Tim-3 during disease progression and response to therapy.