We found that treatment with antiCTim-3 alone had little or no effect and treatment with antiCPD-L1 alone showed a pattern toward delayed tumor growth, but this varied between experiments and did not reach statistical significance (Fig. IL-2, TNF, and IFN-. We further find that combined targeting of the Tim-3 and PD-1 pathways is more effective in controlling tumor growth than targeting either pathway alone. The importance of the immune system in protection against cancer was originally proposed in the theory of cancer immunosurveillance. This theory holds that the immune system can recognize cancerous cells as they arise and can mount both innate and adaptive immune responses to eliminate them. In support of cancer immunosurveillance is the fact that both immunodeficient or immunosuppressed patients and experimental animals are more susceptible to tumor development (for reviews see Dunn et al., 2004; Zitvogel et al., 2006; Swann and Smyth, 2007). Counter to the role of the immune system in staving off cancer is the ability of tumors to escape the immune system by engendering a state of immunosuppression (for review see Zitvogel et al., 2006). One example of a mechanism of immunosuppression present in tumor-bearing hosts is the promotion of T cell dysfunction or exhaustion. T cell exhaustion explains a state of T cell dysfunction that was initially observed during chronic lymphocytic choriomeningitis computer virus (LCMV) contamination in mice (Zajac et al., 1998). Exhausted T cells fail BPTES to proliferate and exert effector functions such as cytotoxicity and cytokine secretion in response to antigen stimulation. Further studies identified that exhausted T cells are characterized by sustained expression of the inhibitory molecule PD-1 (programmed cell death 1) and that blockade of PD-1 and PD-L1 (PD-1 ligand) interactions can reverse T cell exhaustion and restore antigen-specific T cell responses in LCMV-infected mice (Barber et al., 2006). T cell exhaustion also occurs during chronic infections in humans (for review see Klenerman and Hill, 2005). CD8+ T cells in humans chronically infected with HIV (Day et al., 2006; Petrovas et al., 2006; Trautmann et al., 2006), hepatitis B computer virus (Boettler et al., 2006), and hepatitis C computer virus (HCV; Urbani et al., 2006) express BPTES high levels of PD-1, and blocking of PD-1CPD-L interactions can restore T cell function in vitro. Several lines of evidence also implicate the PD-1CPD-L pathway in T cell exhaustion in cancer. First, PD-1 expression is found on tumor-infiltrating CD8+ T cells in multiple solid tumors (Blank et al., 2006; Ahmadzadeh et al., 2009; Gehring et al., 2009) and on antigen-specific CD8+ T cells in hosts with nonsolid tumors (Yamamoto et al., 2008; Mumprecht et al., 2009). Second, these PD-1+ T cells are dysfunctional. Third, PD-L1 is usually expressed at high levels in several different cancers (Latchman et al., 2001; Dong et al., 2002; Brown et al., 2003), and high expression of PD-L1 on tumors is usually strongly associated with poor prognosis (Thompson et al., 2006). Fourth, interference with PD-1CPD-L signaling, either through antibody blockade or PD-1 deficiency, has been shown to improve clinical outcome and restore functional T cell responses in several cancers (Blank et al., 2006; Yamamoto et al., 2008; Mumprecht et al., 2009; Zhang et al., 2009). However, targeting the PD-1CPD-L1 pathway does not always result in reversal of T cell exhaustion (Blackburn et al., 2008; Gehring et al., 2009) and PD-1 expression is not usually associated with exhausted phenotype (Petrovas et al., 2006; Fourcade et al., 2009), indicating that other molecules are likely involved in T cell exhaustion. A recent study in BPTES patients with HIV has shown that the immune regulator T cell immunoglobulin mucin (TIM) 3 is usually up-regulated on exhausted CD8+ T cells (Jones et al., 2008). Tim-3 is usually a molecule originally identified as being selectively expressed on IFN-Csecreting Th1 and Tc1 cells (Monney et al., 2002). Conversation of Tim-3 with its Rabbit Polyclonal to KR1_HHV11 ligand, galectin-9, triggers cell death in Tim-3+ T cells. Thus, both Tim-3 BPTES and PD-1 can function as unfavorable regulators of T cell responses. In HIV patients, TIM-3 and PD-1 mark distinct populations of exhausted cells, with cells positive for both PD-1 and TIM-3 comprising the smallest fraction (Jones et al., 2008) of CD8+ T cells. Similarly, another group has shown that TIM-3 is usually up-regulated on exhausted T cells in patients with HCV (Golden-Mason et al., 2009). In this case, BPTES cells that coexpress TIM-3 and PD-1 are the most abundant fraction among HCV-specific CD8+ T cells. In both studies, blocking TIM-3 restored T cell proliferation and enhanced cytokine production. Because targeting the PD-1CPD-L pathway alone does not result in complete restoration of T cell function (Blackburn et al., 2008), and in some cancers targeting the PD-1CPD-L pathway.