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...

We identified B cell maturation antigen (BCMA) as a potential therapeutic target in 778 newly diagnosed and relapsed myeloma patients. We constructed an IgG-based BCMA-T cell bispecific antibody (EM801) and showed that it increased CD3 T cell/myeloma cell crosslinking, followed by CD4/CD8 T cell activation, and secretion of interferon-γ, granzyme B, and perforin. This effect is CD4 and CD8 T cell mediated. EM801 induced, at nanomolar concentrations, myeloma cell death by autologous T cells in 34 of 43 bone marrow aspirates, including those from high-risk patients and patients after multiple lines of treatment, tumor regression in six of nine mice in a myeloma xenograft model, and depletion of BCMA cells in cynomolgus monkeys. Pharmacokinetics and pharmacodynamics indicate weekly intravenous/subcutaneous administration.

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.

Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopro-, in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fc receptor cross-linking-induced degranulation in mast cells (IC 50 ϭ 2.9 nM), Fc␥ receptor engagement-mediated tumor necrosis factor ␣ production in monocytes (IC 50 ϭ 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC 50 ϭ 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and boneprotective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Memory T cells can be a significant barrier to the induction of transplant tolerance. However, the molecular pathways that can regulate memory T cell-mediated rejection are poorly defined. In the present study we tested the hypothesis that the novel alternative costimulatory molecules (i.e., ICOS, 4-1BB, OX40, or CD30) may play a critical role in memory T cell activation and memory T cell-mediated rejection. We found that memory T cells, generated by either homeostatic proliferation or donor Ag priming, induced prompt skin allograft rejection regardless of CD28/CD154 blockade. Phenotypic analysis showed that, in contrast to naive T cells, such memory T cells expressed high levels of OX40, 4-1BB, and ICOS on the cell surface. In a skin transplant model in which rejection was mediated by memory T cells, blocking the OX40/OX40 ligand pathway alone did not prolong the skin allograft survival, but blocking OX40 costimulation in combination with CD28/CD154 blockade induced long-term skin allograft survival, and 40% of the recipients accepted their skin allograft for >100 days. In contrast, blocking the ICOS/ICOS ligand and the 4-1BB/4-1BBL pathways alone or combined with CD28/CD154 blockade had no effect in preventing skin allograft rejection. OX40 blockade did not affect the homeostatic proliferation of T cells in vivo, but markedly inhibited the effector functions of memory T cells. Our data demonstrate that memory T cells resisting to CD28/CD154 blockade in transplant rejection are sensitive to OX40 blockade and suggest that OX40 is a key therapeutic target in memory T cell-mediated rejection.

T cell Ig mucin (Tim) molecules modulate CD4 + T cell responses. In keeping with the view that Tim-1 generates a stimulatory signal for CD4 + T cell activation, we hypothesized that an agonist Tim-1-specific mAb would intensify the CD4 + T cell-dependant allograft response. Unexpectedly, we determined that a particular Tim-1-specific mAb exerted reciprocal effects upon the commitment of alloactivated T cells to regulatory and effector phenotypes. Commitment to the Th1 and Th17 phenotypes was fostered, whereas commitment to the Treg phenotype was hindered. Moreover, ligation of Tim-1 in vitro effectively deprogrammed Tregs and thus produced Tregs unable to control T cell responses. Overall, the effects of the agonist Tim-1-specific mAb on the allograft response stemmed from enhanced expansion and survival of T effector cells; a capacity to deprogram natural Tregs; and inhibition of the conversion of naive CD4 + T cells into Tregs. The reciprocal effects of agonist Tim-1-specific mAbs upon effector T cells and Tregs serve to prevent allogeneic transplant tolerance.

The mechanism underlying apoptosis induced by proteasome inhibition in leukemic Jurkat and Namalwa cells was investigated in this study. The proteasome inhibitor lactacystin differentially regulated the protein levels of proapoptotic Bcl-2 family members and Bik was accumulated at the mitochondria. Bik overexpression sufficed to induce apoptosis in these cells. Detailed examination along the respiration chain showed that lactacystin compromised a step after complex III, and exogenous cytochrome c could overcome this compromise. Probably as a result, the succinate-stimulated generation of mitochondrial membrane potential was significantly diminished. Bcl-xL interacted with Bik in the cells, and Bcl-xL overexpression prevented cytochrome c leakage out of the mitochondria, corrected the mitochondrial membrane potential defect, and protected the cells from apoptosis. These results show that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members, Bik being one of them.

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4+ T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4+OX40+ cells consist of Foxp3+ Tregs and Foxp3− T effector/memory cells. The ALS-resistant CD4+OX40+ cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag−/− mice, but removal of Foxp3+ Tregs from the OX40+ cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3+ Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3+ Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3+ Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.