IntroductionNatural killer (NK) cells comprise 5% to 20% of human peripheral blood lymphoid cells and are a critical component of the immune system, providing protection against viral infections and contributing to tumor immune surveillance. NK-cell activity is regulated by an intricate balance of signals transmitted by inhibitory and activating receptors. 1,2 Functionally distinct NK-cell subsets can be defined based on the level of CD56 and CD16 coexpression. 3 CD56 bright CD16 Ϫ NK cells produce abundant IFN-␥ in response to stimulation with interleukin (IL)-12 and proliferate robustly when cultured in IL-2, whereas CD56 dim CD16 ϩ NK cells are more cytolytic and produce significant amounts of cytokine when their activating receptors are engaged. 4 CD56 dim CD16 ϩ NK cells are considered mature NK cells and are differentiated from the immature CD56 bright CD16 -NK-cell subset. This is further supported by recent data demonstrating the dynamics of expression of the killer immunoglobulin-like receptors (KIR), CD57, CD94, and CD62L expression on the CD56 dim CD16 ϩ NK cells as they mature from CD56 bright CD16 -NK-cell precursors. [5][6][7][8][9] T-cell immunoglobulin-and mucin domain-containing (Tim)-3 is a member of Tim family of receptors of which there are 3 in humans (Tim-1, Tim-3, and Tim-4). 10 These molecules are involved in diverse metabolic and immunoregulatory processes. 11 Tim-3 is a type I transmembrane protein that contains no defined signaling motifs in its cytoplasmic domain, but it has been implicated both in activation and inhibition of immune responses 12,13 and in the induction of apoptosis of Tim-3-bearing cells through interactions with galectin-9. 14 Tim-3 is expressed on CD4 ϩ T cells, dendritic cells, monocytes, 15-17 CD8 ϩ T cells, 18,19 and NK cells. 20 In a comparison of lymphocyte populations in healthy human subjects, the highest transcription of the gene encoding Tim-3 was observed in NK cells. 21 There is evidence that engagement of Tim-3 on mouse T cells with the ligand galectin-9 promotes aggregation, leading to the death of T-helper 1 cells and the selective loss of interferon (IFN)-␥-producing T cells. 14 On human T cells, the expression of Tim-3 regulates cell proliferation and IFN-␥ secretion. 19,21,22 We and others have observed that increased amounts of Tim-3 on T cells during HIV, hepatitis C virus, and other chronic viral infections correlated with T-cell dysfunction, suggesting that Tim-3 is part of a negative regulatory pathway. 19,[23][24][25] In this study, we investigated the expression of Tim-3 on human NK cells and its regulation by cytokines, and we provide evidence for the role of Tim-3 in the restraint of NK cell-mediated cytotoxicity in healthy individuals. Methods Primary cells and cell linesPeripheral blood mononuclear cells (PBMCs) of healthy individuals were obtained from the Stanford Blood Bank. Cord blood PMBCs were obtained The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, ...

HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G 1 -phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at ؊82 and ؊69 relative to the transcription start site. We also found that a TATA element at ؊46 and two overlapping consensus Sp1 sites at ؊60 and ؊55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.Butyrate is one of the most abundant short chain fatty acids in the large intestine, generated by bacterial fermentation of dietary fibers (1). Butyrate shows potent effects on growth arrest and differentiation in vitro in various malignant tumor cell lines, such as breast cancer cells, hepatoma cells, and others (2-5). In colorectal cancer cells, butyrate inhibits cell growth and induces differentiation marker proteins such as alkaline phosphatase and carcinoembryonic antigen (6 -9). Furthermore, butyrate arrests the cell cycle progression at the G 1 phase (9) and decreases c-myc oncogene expression in human colon cancer cell lines (9, 10). However, the precise mechanism of growth suppression by butyrate in colon cancer cells has not been clarified. WAF1/Cip1 protein potently inhibits the various G 1 cyclindependent kinases activities (11-13) by suppressing the phosphorylation of retinoblastoma (RB) protein, thereby supposedly inhibiting the G 1 -S phase transition (11,14). Besides its role as a kinase inhibitor, it has been reported recently that WAF1/ Cip1 at low doses assembles kinase complexes and promotes a kinase activity (15). Furthermore, the transcription of the WAF1/Cip1 gene is directly activated by wild-type p53 protein (16). Thus, WAF1/Cip1 could play a key role as a downstream mediator of the p53-induced cell growth arrest.Several studies have already shown the p53-independent induction of WAF1/Cip1 by serum, transforming growth factor ␤, and other differentiation-inducers (17)(18)(19)(20). In addition, butyrate has been reported to induce WAF1/Cip1 mRNA independently of p53 during differentiation of hematopoietic cells, hepatoma cells, and colon cancer cells in vitro (18,21). Butyrate can also dephosphorylate the retinoblastoma protein in mouse fibroblasts (22). To investigate the mechanism of butyrateinduced growth arrest, we used a human colon cancer cell line WiDr harboring a point mutation in p53 at codon 273 (23) and examined the effect of butyrate on t...

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