The alloimmune response against fully MHC-mismatched allografts, compared with immune responses to nominal antigens, entails an unusually large clonal size of alloreactive T cells. Thus, induction of peripheral allograft tolerance established in the absence of immune system ablation and reconstitution is a challenging task in transplantation. Here, we determined whether a reduction in the mass of alloreactive T cells due to apoptosis is an essential initial step for induction of stable allograft tolerance with non-lymphoablative therapy. Blocking both CD28-B7 and CD40-CD40 ligand interactions (co-stimulation blockade) inhibited proliferation of alloreactive T cells in vivo while allowing cell cycle-dependent T-cell apoptosis of proliferating T cells, with permanent engraftment of cardiac allografts but not skin allografts. Treatment with rapamycin plus co-stimulation blockade resulted in massive apoptosis of alloreactive T cells and produced stable skin allograft tolerance, a very stringent test of allograft tolerance. In contrast, treatment with cyclosporine A and co-stimulation blockade abolished T-cell proliferation and apoptosis, as well as the induction of stable allograft tolerance. Our data indicate that induction of T-cell apoptosis and peripheral allograft tolerance is prevented by blocking both signal 1 and signal 2 of T-cell activation.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.

OX40 is a recently identified T-cell co IntroductionT cells with regulatory properties are critical to the induction of self-tolerance and acquired tolerance. 1,2 Among the cell types that exhibit potent suppressor functions, the CD4 ϩ Foxp3 ϩ regulatory T cells (Tregs) are particularly important, as deficiency or functional impairment of this cell type often leads to the development of autoimmunity and the failure to establish acquired tolerance, 3,4 albeit other regulatory cell types also contribute to tolerance via different mechanisms. 5 The CD4 ϩ Foxp3 ϩ Tregs are not a uniform cell type. Depending on the origin of these cells, the CD4 ϩ Foxp3 ϩ T cells can be divided into those that are developed in the thymus (natural Tregs) and those that are induced in the periphery (induced Tregs). 6 Natural Foxp3 ϩ Tregs are selected and matured in the thymus, and then exported to the periphery where they suppress potentially cytopathic T cells. 7 It is well known that lineage commitment of the natural Foxp3 ϩ Tregs requires Foxp3, 8,9 and their survival and expansion demand the presence of IL-2 and expression of IL-2 receptors. 10 However, some activated T effector cells can be converted to Foxp3 ϩ Tregs in the periphery and such induced Foxp3 ϩ Tregs also act as potent suppressor cells. 11,12 From a therapeutic point of view, therapies that can preserve or expand the Foxp3 ϩ Tregs and at the same time inhibit cytopathic T effector cells would be highly desirable in the induction of transplant tolerance or in the treatment of autoimmune diseases.Phenotypically, Foxp3 ϩ Tregs and activated T effector cells often express similar cell surface molecules. For example, both cell types express CD25, CD28, CD154, GITR, CTLA-4, and others, although the functions of such molecules are not always the same in both cell types. 4 Recently, it has been shown that the CD4 ϩ CD25 ϩ Tregs constitutively express OX40 (also called CD134), 13 a new costimulatory molecule that belongs to the TNF-R superfamily. 14 Also, T effector cells, though they do not express OX40 at resting state, can readily express OX40 upon activation, 13 and OX40 engagement delivers a potent costimulatory signal to T effector cells. 15 The recent finding that deliberately stimulating OX40 in vivo can break tolerance to peptide antigens 16 and that blocking OX40 costimulation can enable allograft survival in stringent transplant models 17 suggests that the impact of OX40 signaling on a regulatory type of immune response is likely to be profound. However, very little is known about the role of OX40 in regulating the Foxp3 ϩ Tregs. There are 2 reports in the literature suggesting that OX40 may be capable of modifying the suppressor functions of Tregs, but the findings appear to be contradictory. 18,19 As OX40, like CD25, can be expressed by both Foxp3 ϩ Tregs and activated T effector cells, partition of such functionally distinct T-cell subsets in the initial studies based solely on the CD25 marker has obvious limitations. Moreover, activated T effector cells, which...

The mechanisms regulating T helper 9 (TH9) cells and TH9-mediated diseases remain poorly defined. Here, we demonstrate that the receptor OX40 (Tnfrsf4) is a powerful inducer of TH9 cells in vitro and TH9-dependent airway inflammation in vivo. Under TGF-β based polarizing conditions, OX40 ligation eliminated production of induced regulatory T cells and TH17 cells, and divertedCD4+Foxp3− T cells to a TH9 phenotype. Mechanistically, OX40 activated the ubiquitin ligase TRAF6, which triggered the induction of NF-kB-inducing kinase (NIK) in CD4+ T cells and the non-canonical NF-kB pathway which subsequently lead toTH9 generation. Thus, our study identifies a previously unknown mechanism of TH9 induction and may have important clinical implications in allergic inflammation.

Natural killer (NK) cells accumulate at the maternal-fetal interface in large numbers, but their exact roles in successful pregnancy remain poorly defined. Here, we provide evidence that T H 17 cells and local inflammation can occur at the maternal-fetal interface during natural allogenic pregnancies. We found that decidual NK cells promote immune tolerance and successful pregnancy by dampening inflammatory T H 17 cells via IFN-γ secreted by the CD56 bright CD27 + NK subset. This NK-cell-mediated regulatory response is lost in patients who experience recurrent spontaneous abortions, which results in a prominent T H 17 response and extensive local inflammation. This local inflammatory response further affects the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data identify NK cells as key regulatory cells at the maternal-fetal interface by suppressing T H 17-mediated local inflammation.regulatory NK cells | fetomaternal tolerance D uring pregnancy, allogeneic fetal cells invade the maternal decidua but they are protected from the maternal immune system. This invasion of extraembryonic trophoblasts does not harm gestation during normal pregnancy; it establishes tolerance at the maternal-fetal interface (1), (2), although the mechanism of such tolerance is not clear. In addition, inflammatory responses induced by a variety of mechanisms can result in embryo loss, but mild inflammation can be effectively controlled through regulatory mechanisms to maintain successful pregnancy (3). Thus, suppression of strong inflammatory responses is essential to ensure normal pregnancy (4, 5), although the mechanisms involved in regulating local inflammation without compromising overall maternal immunity during a successful pregnancy remain unknown.Multiple mechanisms are potentially involved in promoting immune tolerance during pregnancy. For example, T H 2 cytokine polarization (6-9), the expression of the Fas ligand on trophoblast cells (10), and the inhibition of complement activation (11) are crucial for ensuring tolerance at the maternal-fetal interface. In addition, a delicate balance exists between inhibitory (PD-L1, Stat3, and TGF-β1) and stimulatory (CD80 and CD86) signals during the establishment of immune privilege (12-18). Furthermore, studies have shown that galectin-1 (19) and indoleamine 2,3-dioxygenase (20) play pivotal roles in maternal-fetal tolerance. Several types of immune cells, such as CD4 + CD25 + regulatory T cells, are also essential in the generation of maternal-fetal tolerance in mice and humans (7,(21)(22)(23)(24). Furthermore, natural killer (NK) T cells and immature dendritic cells have been reported to promote the expansion of Treg cells that confer protection of the fetus (19).Despite considerable progress, many questions remain unanswered. The most striking feature at the maternal-fetal interface is the accumulation of NK cells, which account for ∼60-90% of immune cells in the decidua in humans during early pregnancy (25)(26)(27)(28)(29) and a...

Natural killer (NK) cells are programmed to kill target cells without prior antigen priming. Because of their potent cytolytic activities, NK cells are one of the key cell types involved in dismantling allografts. However, in certain transplant models, NK cells also express potent immunoregulatory properties that promote tolerance induction. The precise mechanism for such striking dichotomy remains unknown. In the present study, we showed in a skin transplant model that the skin allografts contain a subset of antigen-presenting cells (APCs) that can home to the recipient mice. We also showed that such graft-derived APCs are usually destroyed by the host NK cells. But in the absence of NK cells, donor APCs can survive and then migrate to the host lymphoid and extralymphoid sites where they directly stimulate the activation of alloreactive T cells. T cells activated in the absence of NK cells are more resistant to costimulatory blockade treatment, and under such conditions stable skin allograft survival is difficult to achieve. Our study identified a novel role for NK cells in regulating T cell priming in transplant models, and may have important clinical implications in tolerance induction.

Interleukin (IL)-2 and IL-15 are redundant in stimulating T-cell proliferation in vitro. Their precise role in vivo in governing T-cell expansion and T-cell homeostasis is less clear. Each may have distinct functions and regulate distinct aspects of T-cell activation. The functional receptors for IL-2 and IL-15 consist of a private alpha-chain, which defines the binding specificity for IL-2 or IL-15, and shared IL-2 receptor beta- and gamma-chains. The gamma-chain is also a critical signaling component of IL-4, IL-7 and IL-9 receptors. Thus, the gamma-chain is called the common gamma or gamma-c. As these receptor subunits can be expressed individually or in various combinations resulting in the formation of receptors with different affinities, distinct signaling capabilities or both, we hypothesized that differential expression of IL-2 and IL-15 receptor subunits on cycling T cells in vivo may direct activated T cells to respond to IL-2 or IL-15, thereby regulating the homeostasis of T-cell response in vivo. By observing in vivo T-cell divisions and expression of IL-2 and IL-15 receptor subunits, we demonstrate that IL-15 is a critical growth factor in initiating T cell divisions in vivo, whereas IL-2 limits continued T-cell expansion via downregulation of the gamma-c expression. Decreased gamma-c expression on cycling T cells reduced sustained Bcl-2 expression and rendered cells susceptible to apoptotic cell death. Our study provides data that IL-2 and IL-15 regulate distinct aspects of primary T-cell expansion in vivo.

Leptin has direct effects not only on neuroendocrine function and metabolism, but also on T cell-mediated immunity. We report in this study that leptin receptor (ObR) is expressed on resting normal mouse CD4+, CD8+, B cells, and monocyte/macrophages. ObR expression is up-regulated following cell activation, but with different kinetics, in different lymphocyte subsets. Leptin binding to ObR results in increased STAT-3 activation in T cells, with a different activation pattern in resting vs anti-CD3 Ab stimulated T cells. Leptin also promotes lymphocyte survival in vitro by suppressing Fas-mediated apoptosis. B lymphocytes appear to be more susceptible to the antiapoptotic effects of leptin, and they show higher surface expression of ObR, compared with T cells. Moreover, CD4+ T cells isolated from ObR-deficient mice displayed a reduced proliferative response, compared with normal controls. Furthermore, ObR/STAT-3-mediated signaling in T lymphocytes is decreased in the diet-induced obese mouse model of obesity and leptin resistance. In summary, our findings show that the ObR is expressed on normal mouse lymphocyte subsets, that leptin plays a role in lymphocyte survival, and that leptin alters the ObR/STAT-3-mediated signaling in T cells. Taken together, our data further support the notion that nutritional status acting via leptin-dependent mechanisms may alter the nature and vigor of the immune response.