T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is a T

T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is a T helper cell (Th)1-connected cell surface TAE684 area molecule that regulates Th1 reactions and promotes tolerance in mice but its expression and function in human being T cells is unknown. by costimulatory blockade. Finally reduction of TIM3 on ex vivo CD4+ T cells using small interfering (si)RNA enhanced proliferation and IFN-γ secretion directly demonstrating that TIM3 expression on human T cells regulates proliferation and IFN-γ secretion. Failure to up-regulate T cell expression of TIM3 in inflammatory sites may represent a novel intrinsic defect that contributes to the pathogenesis of MS and other human autoimmune diseases. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) believed to TAE684 be initiated and mediated by autoreactive T cells directed against myelin antigens (1). It has been hypothesized that Th1 cells secreting proinflammatory cytokines including IFN-γ and TNF-α contribute to the pathogenesis of MS whereas Th2 cells secreting IL-4 IL-5 and IL-13 ameliorate the inflammatory process and disease (2). Transcription factors and cell surface molecules have been identified that are tightly associated with Th1 or Th2 cells (3 4 Although it does not promote Th1 differentiation T cell immunoglobulin- and mucin domain-containing molecule (TIM)3 is selectively expressed on fully differentiated Th1 but not Th2 cells (5). More recently engagement of TIM3 with TIM3 ligand has been shown to regulate both the function of Th1 cells and the ability to induce tolerance as blockade of the TIM3 pathway accelerates diabetes in the nonobese diabetes (NOD) mice model of diabetes and prevents the acquisition of transplantation tolerance induced by costimulatory blockade (6). Furthermore TIM3-deficient mice are refractory to induction of high dose tolerance in experimental autoimmune encephalomyelitis (EAE) (7). Here we explored the mechanism for increased IFN-γ secretion by CD4+ T TAE684 cells in the cerebrospinal fluid (CSF) of patients with MS postulating that alterations in the TIM3 pathway may underlie the enhanced IFN-γ secretion observed in patients with the disease. We generated 104 independent T cell clones from the CSF of MS patients and control subjects; cytokine secretion was measured and quantitative RT-PCR was used to analyze the specificity and stability of transcription factors that dictate Th1 and Th2 programs. Paradoxically MS CSF clones express higher levels of IFN-γ and they express lower levels of messenger RNA (mRNA) for both T-bet and TIM3. In vitro polarization experiments with CSF clones from MS TAE684 and control subjects demonstrate robust enhancement of IFN-γ secretion but a relative inability to up-regulate TIM3 in MS CSF clones. Mouse monoclonal antibody to CaMKIV. The product of this gene belongs to the serine/threonine protein kinase family, and to the Ca(2+)/calmodulin-dependent protein kinase subfamily. This enzyme is a multifunctionalserine/threonine protein kinase with limited tissue distribution, that has been implicated intranscriptional regulation in lymphocytes, neurons and male germ cells. Using small interfering (si)RNA to reduce TIM3 expression on CD4+ T cells and IFN-γ secretion when TIM3 expression was inhibited providing a mechanistic link between our observations of lower TIM3 levels and elevated IFN-γ secretion among MS CSF clones. These results are the first human data to demonstrate that TIM3 expression regulates T cell secretion of IFN-γ. Moreover these data suggest that TIM3 may critically regulate self-reactive Th1 cells and maintenance of tolerance in human autoimmune disease. RESULTS AND DISCUSSION Stability and specificity of markers of human Th1 cells We generated a total of 104 independent CD4+ T cell clones from the CSF of patients with MS (= 6) and control subjects (= 4) and examined their cytokine profiles using ELISA after stimulation with anti-CD3 monoclonal antibody. We selected several Th1 and Th2 clones from healthy subjects and examined mRNA expression levels of Th1- and Th2-associated molecules using quantitative RT-PCR. Levels of TIM3 and T-bet in T cell clones isolated from control topics were indicated at considerably higher amounts in Th1 clones whereas GATA-3 was considerably raised in Th2 cells (Fig. 1 A). Degrees of STAT1 and STAT4 tended to become TAE684 higher on Th1 clones however TAE684 the differences weren’t statistically considerable (Fig. 1 A rather than depicted). We verified that TIM3 and T-bet are differentially indicated on representative Th1 or Th2 clones differentiated from PBMCs of healthful topics (Fig. 1 B). Shape 1. Differential manifestation of T-bet TIM3 and GATA-3 among Th1.