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...

Several transplant patients maintain stable kidney graft function in the absence of immunosuppression. Here we compared the characteristics of their peripheral B cells to that of others who had stable graft function but were under pharmacologic immunosuppression, to patients with chronic rejection and to healthy volunteers. In drug-free long-term graft function (DF) there was a significant increase in both absolute cell number and frequency of total B cells; particularly activated, memory and early memory B cells. These increased B-cell numbers were associated with a significantly enriched transcriptional B-cell profile. Costimulatory/migratory molecules (B7-2/CD80, CD40, and CD62L) were upregulated in B cells; particularly in memory CD19(+)IgD(-)CD38(+/-)CD27(+) B cells in these patients. Their purified B cells, however, responded normally to a polyclonal stimulation and did not have cytokine polarization. This phenotype was associated with the following specific characteristics which include an inhibitory signal (decreased FcgammaRIIA/FcgammaRIIB ratio); a preventive signal of hyperactive B-cell response (an increase in BANK1, which negatively modulates CD40-mediated AKT activation); an increased number of B cells expressing CD1d and CD5; an increased BAFF-R/BAFF ratio that could explain why these patients have more peripheral B cells; and a specific autoantibody profile. Thus, our findings show that patients with DF have a particular blood B-cell phenotype that may contribute to the maintenance of long-term graft function.

Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing ␤ cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to ␤ cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional ␤ cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with ␣1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to ␤ cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of ␤ cells expands in AAT-treated diabetic NOD mice.autoimmunity ͉ type 1 diabetes ͉ inflammation A destructive T cell-dependent autoimmune process directed against insulin-producing ␤ cells causes type 1 diabetes mellitus (T1DM) in humans and the nonobese diabetic (NOD) mouse model (1, 2). Although many therapeutic interventions, including viral-mediated gene transfer of human ␣1-antitrypsin (AAT) (3), can prevent T1DM or resolve the T cell-rich, ␤ cell-invasive insulitis lesion in prediabetic hosts, surprisingly few therapies have succeeded in restoring long-term drug-free euglycemia and immune tolerance to ␤ cells in overtly diabetic NOD mice (4-8). Although each of these successful therapies directly targets T cells, each bears an element that may dampen proinflammatory responses or their consequences upon target tissues.Inflammatory cytokines direct the commitment of antigenactivated CD4 ϩ T cells to specific effector or Foxp3ϩ regulatory phenotypes (9-11). In addition, islets are sensitive to proinflammatory cytokines (12-15). Adverse inflammation in muscle and fat causes faulty insulin signaling and insulin resistance (16) in type 2 diabetes mellitus (13) and, as recently shown, T1DM (7,17). Hence, we have tested the hypothesis that treatment with AAT, an acutephase reactant with known antiinflammatory and antiapoptotic effects (18-21) including effects on islets (21,22), is effective in NOD mice with overt new-onset T1DM. In short, we are probing the hypothesis that inflammatory mechanisms trigger T1DM. Results AAT Does Not Inhibit T Cell Activation.Purified carboxyfluorescein diacetate succinmidyl ester (CFSE)-labeled C57BL/6 mouse T cells were stimulated with plate-bound anti-CD3 plus soluble anti-CD28 mAbs. AAT did not impair T cell proliferation or acquisition of an ...

Regulatory T (T reg ) cells are indispensable for maintaining peripheral tolerance, whereas T helper (Th)1 and Th17 cells induce inflammation and tissue destruction. Using Foxp3-GFP knock-in mice, we report a novel regulatory role for B cell subsets in influencing the differentiation of T reg versus Th1/Th17 cells. Peritoneal B1 cells strongly promoted T cell proliferation and cytokine secretion when presenting nominal or allogeneic antigens, as compared to conventional follicular B (B2) cells. However, peritoneal B1 cells largely failed to convert naive Foxp3 -CD4 + T cells into Foxp3 + T reg cells in the presence of TGF-b and IL-2, in marked contrast to conventional B2 cells, which excelled in T reg conversion. Interestingly, under the same T reg conversion conditions, peritoneal B1 cells preferentially promoted Th1 and Th17 cell differentiation. Blockade of CD86 but not CD80 costimulation markedly enhanced T reg cell induction by B1 cells. Thus, B cell antigen presentation function is inversely correlated with de novo T reg cell induction for these B cell subsets. Our findings suggest that B1 and B2 cell subsets play distinct roles in immune regulation by promoting reciprocal differentiation of T cell lineages.

Operationally tolerant patients (TOL) display a higher number of blood B cells and transcriptional B cell signature. As they rarely develop an allo-immune response, they could display an abnormal B cell differentiation. We used an in vitro culture system to explore T-dependent differentiation of B cells into plasma cells. B cell phenotype, apoptosis, proliferation, cytokine, immunoglobulin production and markers of differentiation were followed in blood of these patients. Tolerant recipients show a higher frequency of CD20 þ CD24 hi CD38 hi transitional and CD20 þ CD38 lo CD24 lo na€ ıve B cells compared to patients with stable graft function, correlating with a decreased frequency of CD20 À CD38 þ CD138 þ differentiated plasma cells, suggestive of abnormal B cell differentiation. B cells from TOL proliferate normally but produce more IL-10. In addition, B cells from tolerant recipients exhibit a defective expression of factors of the end step of differentiation into plasma cells and show a higher propensity for cell death apoptosis compared to patients with stable graft function. This in vitro profile is consistent with down-regulation of B cell differentiation genes and anti-apoptotic B cell genes in these patients in vivo. These data suggest that a balance between B cells producing IL-10 and a deficiency in plasma cells may encourage an environment favorable to the tolerance maintenance.

IntroductionIn the peripheral lymphoid tissues of normal mice and humans, 1% to 5% of ␣␤ T-cell receptor-positive (TCR ϩ ) T cells are CD4 Ϫ CD8 Ϫ (double-negative [DN]) T cells. 1,2 MRL/Mpj-lpr/lpr mice have a mutant Fas gene and a massive lymphadenopathy consisting of an age-related accumulation of DN ␣␤TCR ϩ T cells. 3,4 The DN T cells have been shown to possess the capacity to regulate auto-and alloimmune responses and induce immune tolerance. [5][6][7][8] However, the origin of peripheral DN T cells is still unclear. The heterogeneity of DN T-ells in the expression of surface markers suggests that several maturation/differentiation pathways may exist. In murine models, several studies have demonstrated that DN ␣␤ TCR ϩ T cells can be derived directly from CD8 ϩ T cells. [9][10][11][12] Other studies suggest that DN ␣␤ TCR ϩ natural killer T cells (NKT cells) arise extrathymically from bone marrow (BM). 13 More recently, Ford et al reported that DN T regulatory cells can develop outside the thymus, but not from mature CD8 ϩ T-cell precursors. 14 However, a differentiation pathway of peripheral DN T cells from CD4 ϩ T cells was not identified.In this report, we monitored CD4 expression during CD4 ϩ T-cell proliferation and differentiation and identified a new pathway for the generation of a DN regulatory T-cell subset. This pathway uncovered a new intrinsic homeostatic mechanism that regulates the magnitude of immune responses to alloantigen both in vitro and in vivo. Our observations will permit the development of novel, cell-based, therapeutic approaches for the prevention of allograft rejection and for the treatment of autoimmune diseases. Materials and methodsMice Male C57BL/6 (H-2 b ), C57BL/6 congenic for CD45.1, C57BL/6 TEa TCR-transgenic, C57BL/6 perforin gene knock-out (KO), C57BL/6 RAG Ϫ/Ϫ , DBA/2 (H-2 d ), C3H (H-2 k ), and B6D2F1 (H-2 b/d ) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Foxp3 gfp knock-in C57BL/6 mice were provided by Dr Wenda Gao (Boston, MA). 15 All mice were maintained in the animal facilities of Harvard Institutes of Medicine. Reagents and antibodiesRecombinant mouse interleukin-2 (IL-2), IL-4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Biosource (Camarillo, CA). CD4 ϩ T-cell enrichment column, T-cell enrichment column, and recombinant mouse IL-15 were obtained from R&D Systems (Minneapolis, MN). Fluorochrome-conjugated antibodies to mouse CD3, CD4, CD8, CD25, CD28, CD40, CD44, CD45.1, CD69, CD86, Ter119, B220, CD11b, CD11c, Gr1, NK1.1, TCR␤, TCR␥␦, and isotype controls were obtained from eBioscience (San Diego, CA). Annexin V-PE was purchased from BD Pharmingen (San Diego, CA). CD4 ϩ CD25 ϩ regulatory T cell (Treg) isolation kits, anti-PE microbeads, and magnetic bead separation columns were obtained from Miltenyi Biotec (Auburn, CA). Mitomycin C was obtained from Sigma (St Louis, MO).Purification of CD4 ؉ , CD4 ؉ CD25 ؉ , CD4 ؉ CD25 ؊ , and CD4 ؊ CD8 ؊ DN T cellsSingle-cell suspensions were prepared from the spleens and...

Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4 + CD25 2 T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4 + CD25 2 effector T cell response in a dosedependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB + B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21 + T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

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.