Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8þ T cells and infiltration of T-regulatory cells (T reg ). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T reg infiltration and CD8þ T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T reg against CD8 þ T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T reg cell infiltration and CD8 þ T-cell apoptosis was noted. TGF-b signaling blockade diminished apoptosis of

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TIM-3 functions to enforce CD8+ T cell exhaustion, a dysfunctional state associated with the tolerization of tumor microenvironment. Here we report apoptosis of IFN-γ competent TIM-3+ population of tumor-infiltrating CD8+ T cells in colon cancer. In humans suffering from colorectal cancer, TIM-3+ population is higher in cancer tissue-resident relative to peripheral blood CD8+ T cells. Both the TIM-3+ and TIM-3- cancer tissue-resident CD8+ T cells secrete IFN-γ of comparable levels, although apoptotic cells are more in TIM-3+ compared to TIM-3- population. In mouse CT26 colon tumor model, majority of tumor-infiltrating CD8+ T cells express TIM-3 and execute cytolysis function with higher effector cytokine secretion and apoptosis in TIM-3+ compared to TIM-3- population. The tumor cells secrete galectin-9, which increases apoptosis of tumor-infiltrating CD8+ T cells. Galectin-9/TIM-3 signaling blockade with anti-TIM-3 antibody reduces the apoptosis and in addition, inhibits tumor growth in mice. The blockade increases therapeutic efficacy of cyclophosphamide to treat tumor in mice as well. These results reveal a previously unexplored role of TIM-3 on tumor-infiltrating CD8+ T cells in vivo.

Activated T regulatory (T reg ) cells are potent suppressors that mediate immune tolerance. We investigated the relationship between activated T reg cells and the progression of human colon cancer. We designed a cross-sectional study of CD4 1 Foxp3 1 T cells from peripheral blood, primary tumor and nontumor colon tissue of 42 patients with colon cancer and correlated the percentages of different subgroups of T reg cells with colon cancer stage. The phenotypes, cytokine-release patterns and suppression ability of these T reg cells were analyzed. We found that T reg cells increased significantly in both peripheral blood and cancer tissue. In addition, the T reg cells expressed significantly lower levels of CCR7, CD62L and CD45RA in comparison to normal volunteers. Further dividing T reg cells into subgroups based on Foxp3 and CD45RA expression revealed that both activated T reg cells (Foxp3 hi CD45RA 2 ) and nonsuppressive T reg cells (Foxp3 lo CD45RA 2 ), but not resting T reg cells (Foxp3 low CD45RA 1 ), increased in the peripheral blood and cancer tissue of patients with colon cancer. Only the activated T reg cells expressed significantly higher levels of tumor necrosis factor receptor 2 and cytotoxic T-cell antigen-4. Activated T reg cells, however, secreted significantly lower levels of effector cytokines (interleukin-2, tumor necrosis factor-a and interferon-c) than did resting T reg cells and nonsuppressive cells upon ex vivo stimulation. Activated, but not resting, T reg cells in cancer tissue correlated with tumor metastases. In summary, we confirmed that activated T reg cells are a distinct subgroup with effector memory phenotype and fully functional regulatory activity against human colorectal cancer immunity.CD4 þ Foxp3 þ Regulatory T cells (T reg cells) are essential to maintain self-tolerance and immune hemostasis. 1,2 They play important roles in a variety of human diseases including autoimmune disease, chronic infection and cancers. 3-5 A large body of evidence suggests that T reg cells are one of the major players for tumor immune suppression and the main obstacle to successful tumor immunotherapy. 3,[6][7][8] The prevalence of T reg cells significantly increases in the human peripheral blood and/or tumors of multiple types of cancers. 9-12 These cells are recruited to tumor sites, where they suppress antitumor cytotoxic responses. 3,[13][14][15] Animal models show that inhibiting T reg cells improves tumor immunity or enhances tumor vaccine therapeutic effects. 16,17 In human colon cancer, studies have revealed that CD4 þ CD25 þ Foxp3 þ T reg cells are increased in peripheral blood mononuclear cells (PBMCs) and draining lymph nodes. These T reg cells were capable of suppression on antigen-specific CD4 þ T cells. Surgical removal of colon cancer reduces the T reg cell population and restores antigen-specific CD4 þ activity. 18,19 However, it is believed that nonspecific suppression of T reg cells might be dangerous, as it may produce an overwhelming autoimmune response.Human CD4 þ Foxp3 þ T reg ce...