Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

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.

OX40 expressed on activated T cells is known to be an important costimulatory molecule on T cell activation in vitro. However, the in vivo functional significance of the interaction between OX40 and its ligand, OX40L, is still unclear. To investigate the role of OX40L during in vivo immune responses, we generated OX40L-deficient mice and a blocking anti-OX40L monoclonal antibody, MGP34. OX40L expression was demonstrated on splenic B cells after CD40 and anti-immunoglobulin (Ig)M stimulation, while only CD40 ligation was capable of inducing OX40L on dendritic cells. OX40L-deficient and MGP34-treated mice engendered apparent suppression of the recall reaction of T cells primed with both protein antigens and alloantigens and a significant reduction in keyhole limpet hemocyanin–specific IgG production. The impaired T cell priming was also accompanied by a concomitant reduction of both T helper type 1 (Th1) and Th2 cytokines. Furthermore, antigen-presenting cells (APCs) derived from the mutant mice revealed an impaired intrinsic APC function, demonstrating the importance of OX40L in both the priming and effector phases of T cell activation. Collectively, these results provide convincing evidence that OX40L, expressed on APCs, plays a critical role in antigen-specific T cell responses in vivo.

The OX40 (CD134) molecule is induced primarily during T cell activation and, as we show in this study, is also expressed on CD25+CD4+ regulatory T (Treg) cells. A necessary role for OX40 in the development and homeostasis of Treg cells can be inferred from the reduced numbers of the cells present in the spleens of OX40-deficient mice, and their elevated numbers in the spleens of mice that overexpress the OX40 ligand (OX40L). The homeostatic proliferation of Treg cells following transfer into lymphopenic mice was also found to be potentiated by the OX40-OX40L interaction. Suppression of T cell responses by Treg cells was significantly impaired in the absence of OX40, indicating that, in addition to its homeostatic functions, OX40 contributes to efficient Treg-mediated suppression. However, despite this, we found that CD25−CD4+ T cells became insensitive to Treg-mediated suppression when they were exposed to OX40L-expressing cells, or when they were treated with an agonistic OX40-specific mAb. OX40 signaling could also abrogate the disease-preventing activity of Treg cells in an experimental model of inflammatory bowel disease. Thus, although the data reveal important roles for OX40 signaling in Treg cell development, homeostasis, and suppressive activity, they also show that OX40 signals can oppose Treg-mediated suppression when they are delivered directly to Ag-engaged naive T cells.