Urinary cell levels of mRNA for OX40, OX40L, PD-1, PD-L1, or PD-L2 and acute rejection of human renal allografts.
ABSTRACT: The positive costimulatory proteins OX40 and OX40L and negative regulatory proteins programmed death (PD)-1, PD ligand 1, and PD ligand 2 have emerged as significant regulators of acute rejection in experimental transplantation models.We obtained 21 urine specimens from 21 renal allograft recipients with graft dysfunction and biopsy-confirmed acute rejection and 25 specimens from 25 recipients with stable graft function and normal biopsy results (stable). Urinary cell levels of mRNAs were measured using real-time quantitative polymerase chain reaction assays, and the levels were correlated with allograft status and outcomes.Levels of OX40 mRNA (P<0.0001, Mann-Whitney test), OX40L mRNA (P=0.0004), and PD-1 mRNA (P=0.004), but not the mRNA levels of PD ligand 1 (P=0.08) or PD ligand 2 (P=0.20), were significantly higher in the urinary cells from the acute rejection group than the stable group. Receiver operating characteristic curve analysis demonstrated that acute rejection is predicted with a sensitivity of 95% and a specificity of 92% (area under the curve=0.98, 95% confidence interval 0.96-1.0, P<0.0001) using a combination of levels of mRNA for OX40, OX40L, PD-1, and levels of mRNA for the previously identified biomarker Foxp3. Within the acute rejection group, levels of mRNA for OX40 (P=0.0002), OX40L (P=0.0004), and Foxp3 (P=0.04) predicted acute rejection reversal, whereas only OX40 mRNA levels (P=0.04) predicted graft loss after acute rejection.A linear combination of urinary cell levels of mRNA for OX40, OX40L, PD-1, and Foxp3 was a strong predictor of acute rejection in human renal allograft biopsies. This prediction model should be validated using an independent cohort of renal allograft recipients.
Project description:OX40 is a member of the TNFR superfamily that has potent costimulatory properties. Although the impact of blockade of the OX40-OX40 ligand (OX40L) pathway has been well documented in models of autoimmune disease, its effect on the rejection of allografts is less well defined. In this article, we show that the alloantigen-mediated activation of naive and memory CD4(+) T cells results in the induction of OX40 expression and that blockade of OX40-OX40L interactions prevents skin allograft rejection mediated by either subset of T cells. Moreover, a blocking anti-OX40 had no effect on the activation and proliferation of T cells; rather, effector T cells failed to accumulate in peripheral lymph nodes and subsequently migrate to skin allografts. This was found to be the result of an enhanced degree of cell death among proliferating effector cells. In clear contrast, blockade of OX40-OX40L interactions at the time of exposure to alloantigen enhanced the ability of regulatory T cells to suppress T cell responses to alloantigen by supporting, rather than diminishing, regulatory T cell survival. These data show that OX40-OX40L signaling contributes to the evolution of the adaptive immune response to an allograft via the differential control of alloreactive effector and regulatory T cell survival. Moreover, these data serve to further highlight OX40 and OX40L as therapeutic targets to assist the induction of tolerance to allografts and self-Ags.
Project description:Background:Anti-tumoral immunotherapy of anti-program death-1/program death-ligand 1 (PD-1/PD-L1) immune checkpoint therapy demonstrated promising efficacy and tolerability in patients with lung cancer. Apart from inhibitory checkpoints, OX40, the co-stimulatory receptor related to T cell priming and proliferation, was valued identically. In this study, the relationship between OX40/OX40L expressed on tumor infiltrating lymphocytes (TILs), PD-1/PD-L1 and other immunological factors, as well as its role serving as the potential prognostic biomarker, were analyzed in NSCLC. Methods:We investigated the relationship between OX40/OX40L, PD-1/PD-L1 and TILs in surgical samples from 139 patients with NSCLC by immunohistochemistry (IHC). Factors related to OX40/OX40L expression were analyzed by logistic regression and multi-linear regression. Cox analysis was also performed to find the influencing factors. Survival analysis was conducted in order to testify its role in predicting patients' prognosis. Results:The TILs OX40, OX40L expression were negatively correlated with the PD-1/PD-L1 expression, respectively. PD-1 expression was negatively correlated with the TILs OX40 expression [R=0.250, (P=0.003)], it was also negatively correlated with the TILs OX40L expression [R=0.386, (P=0.0001)]. PD-1 expression was positively correlated with TILs grades and negatively correlated with the TILs OX40L expression in multiple linear model [R=0.531, (X1, 95% CI: 3.552-8.176, P=0.0001; X2, 95% CI: 0.216-0.683), (P=0.0001)]. The expression of TILs OX40 varied significantly among tumor OX40 and OX40L, PD-1, PD-L1, TILs and pathology types. Tumor OX40L expression, TILs OX40L expression, PD-1 expression, PD-L1 expression and TILs were considered as risk factors for TILs OX40 expression. The staging and TILs OX40L were considered as risk factors for overall survival (OS) while stage and gender were risk factors for recurrence-free survival (RFS). The low-expression of OX40 was related to longer RFS, OS and better prognosis. Conclusions:OX40 plays a pivotal role in NSCLC, which was closely correlated with immunological factors, RFS and prognosis.
Project description:Tregs are impaired in human systemic lupus erythematosus (SLE) and contribute to effector T cell activation. However, the mechanisms responsible for the Treg deficiency in SLE remain unclear. We hypothesized that the OX40L/OX40 axis is implicated in Treg and regulatory follicular helper T (Tfr) cell dysfunction in human SLE. OX40L/OX40 axis engagement on Tregs and Tfr cells not only specifically impaired their ability to regulate effector T cell proliferation, but also their ability to suppress T follicular helper (Tfh) cell-dependent B cell activation and immunoglobulin secretion. Antigen-presenting cells from patients with active SLE mediated Treg dysfunction in an OX40L-dependent manner, and OX40L-expressing cells colocalized with Foxp3+ cells in active SLE skin lesions. Engagement of the OX40L/OX40 axis resulted in Foxp3 downregulation in Tregs, and expression in SLE Tregs correlated with the proportion of circulating OX40L-expressing myeloid DCs. These data support that OX40L/OX40 signals are implicated in Treg dysfunction in human SLE. Thus, blocking the OX40L/OX40 axis appears to be a promising therapeutic strategy.
Project description:The co-inhibitory receptor Programmed Death-1 (PD-1) curtails immune responses and prevent autoimmunity, however, tumors exploit this pathway to escape from immune destruction. The co-stimulatory receptor OX40 is upregulated on T cells following activation and increases their clonal expansion, survival and cytokine production when engaged. Although antagonistic anti-PD-1 or agonistic anti-OX40 antibodies can promote the rejection of several murine tumors, some poorly immunogenic tumors were refractory to this treatment. In the present study, we evaluated the antitumor effects and mechanisms of combinatorial PD-1 blockade and OX40 triggering in a murine ID8 ovarian cancer model. Although individual anti-PD-1 or OX40 mAb treatment was ineffective in tumor protection against 10-day established ID8 tumor, combined anti-PD-1/OX40 mAb treatment markedly inhibited tumor outgrowth with 60% of mice tumor free 90 days after tumor inoculation. Tumor protection was associated with a systemic immune response with memory and antigen specificity and required CD4(+) cells and CD8(+) T cells. The anti-PD-1/OX40 mAb treatment increased CD4(+) and CD8(+) cells and decreased immunosuppressive CD4(+)FoxP3(+) regulatory T (Treg) cells and CD11b(+)Gr-1(+) myeloid suppressor cells (MDSC), giving rise to significantly higher ratios of both effector CD4(+) and CD8(+) cells to Treg and MDSC in peritoneal cavity; Quantitative RT-PCR data further demonstrated the induction of a local immunostimulatory milieu by anti-PD-1/OX40 mAb treatment. The splenic CD8(+) T cells from combined mAb treated mice produced high levels of IFN-? upon tumor antigen stimulation and exhibited antigen-specific cytolytic activity. To our knowledge, this is the first study testing the antitumor effects of combined anti-PD-1/OX40 mAb in a murine ovarian cancer model, and our results provide a rationale for clinical trials evaluating ovarian cancer immunotherapy using this combination of mAb.
Project description:We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3- effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-? leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-?-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40-/-, and PKC-?-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-? status. Although PKC-? is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.
Project description:A recipient usually rejects a transplanted organ and thus needs immunosuppressive treatments to prevent rejection. Achieving long-term allograft survival without continuous global immunosuppression is highly desirable in transplantation as long-term immunosuppression causes various side effects. Therefore, it is necessary to search for medicine with potentially less side effects. Traditional Chinese medicine PSORI-CM01 (Yin Xie Ling), a formula with seven natural herbs, has been used to treat patients with psoriasis. Here, we investigated a "sharpened" formula, PSORI-CM02 consisting of only five herbs from PSORI-CM01: Curcumae rhizoma, Radix paeoniae rubra, Rhizoma smilacis glabrae, Mume fructus, and Sarcandrae herba. We examined whether or not PSORI-CM02 would suppress alloimmunity and found that PSORI-CM02 significantly inhibited murine skin allograft rejection and reduced graft-infiltration of CD3+ T cells. Interestingly, omitting any single herbal component rendered the whole formula ineffective in suppression, indicating that these herbal components exert their effects cooperatively as a whole. Moreover, PSORI-CM02 increased CD8+CD122+PD-1+ Treg frequency with CD4+FoxP3+ Tregs remaining unchanged in recipient mice, whereas CsA reduced CD4+FoxP3+ Treg frequency. PSORI-CM02 also hindered CD11c+ DC maturation posttransplantation. Importantly, PSORI-CM02-induced CD8+CD122+PD-1+ Tregs were more potent in suppression of allograft rejection in Rag-/- mice than control Tregs. On the other hand, PSORI-CM02 suppressed T cell proliferation in vitro and reduced their phosphorylation of P70S6K and P50/P65, suggesting that it inhibits both mTOR and NF?B signaling pathways. It also increased IL-10 production while reducing IFN? level in the supernatant of activated T cells co-cultured with CD8+CD122+PD-1+ Tregs. Furthermore, HPLC fingerprinting ruled out that PSORI-CM02 contained CsA or rapamycin. PSORI-CM02 also did not cause any illness and toxic injury in recipient mice. Thus, we demonstrate that PSORI-CM02 formula suppresses allograft rejection without toxicity.
Project description:The T cell co-stimulatory molecule OX40 and its cognate ligand OX40L have attracted broad research interest as a therapeutic target in T cell-mediated diseases. Accumulating preclinical evidence highlights the therapeutic efficacy of both agonist and blockade of the OX40-OX40L interaction. Despite this progress, many questions about the immuno-modulator roles of OX40 on T cell function remain unanswered. In this review we summarize the impact of the OX40-OX40L interaction on T cell subsets, including Th1, Th2, Th9, Th17, Th22, Treg, Tfh, and CD8+ T cells, to gain a comprehensive understanding of anti-OX40 mAb-based therapies. The potential therapeutic application of the OX40-OX40L interaction in autoimmunity diseases and cancer immunotherapy are further discussed; OX40-OX40L blockade may ameliorate autoantigen-specific T cell responses and reduce immune activity in autoimmunity diseases. We also explore the rationale of targeting OX40-OX40L interactions in cancer immunotherapy. Ligation of OX40 with targeted agonist anti-OX40 mAbs conveys activating signals to T cells. When combined with other therapeutic treatments, such as anti-PD-1 or anti-CTLA-4 blockade, cytokines, chemotherapy, or radiotherapy, the anti-tumor activity of agonist anti-OX40 treatment will be further enhanced. These data collectively suggest great potential for OX40-mediated therapies.
Project description:The OX40-OX40L protein-protein interaction (PPI) is an important cell-surface signalling co-stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small-molecules. Here, we describe the identification of a small-molecule OX40 modulator and confirm its partial agonist character.Cell-free screening assays were developed and used to identify OX40-OX40L inhibitors. Modified versions of this assay were used to elucidate the binding partner and the binding nature of active compounds. OX40-transfected sensor cells with NF-?B reporters were constructed and used to confirm and characterize activity and specificity. Immunomodulatory activity and partial agonist nature were further confirmed by ex vivo?T-cell polarization assays.Several compounds that concentration-dependently affected OX40-OX40L were identified. Cell assays indicated that they were partial agonists with low micromolar potency and adequate selectivity. Under polarizing conditions based on TGF-?, the most promising compound mimicked the effect of an agonistic anti-OX40 antibody in suppressing regulatory T-cell generation and diverting CD4(+) CD62L(+) Foxp3(-) cells to TH 9 phenotype in vitro.We identified, to our knowledge, the first small-molecule compounds able to interfere with OX40-OX40L binding and, more importantly, to act as partial agonists of OX40. This is particularly interesting, as small-molecule agonism or activation of PPIs is considered unusually challenging and there are only few known examples. These results provide proof-of-principle evidence for the feasibility of small-molecule modulation of the OX40-OX40L interaction and for the existence of partial agonists for TNFRSF-PPIs.
Project description:We report that polyclonal CD8regs generated in one week ex-vivo with anti-CD3/28 beads and cytokines rapidly developed suppressive activity in vitro sustained by TGF-?. In immunodeficient mice, these CD8regs demonstrated a markedly protective, IL-10 dependent activity against a xeno-GVHD. They expressed IL-2R?/?, Foxp3, TNFR2, and the negative co-stimulatory receptors CTLA-4, PD-1, PD-L1 and Tim-3. Suppressive activity in vitro correlated better with TNFR2 and PD-L1 than Foxp3. Blocking studies suggested that TNF enhanced PD-L1 expression and the suppressive activity of the CD8regs generated. Unlike other polyclonal CD4 and CD8 Tregs, these CD8regs preferentially targeted allogeneic T cells, but they lacked cytotoxic activity against them even after sensitization. Unlike CD4regs, these CD8regs could produce IL-2 and proliferate while inhibiting target cells. If these CD8regs can persist in foreign hosts without impairing immune surveillance, they could serve as a practical remission-inducing product for the treatment of autoimmune diseases, graft-versus-host disease, and allograft rejection.
Project description:Animal studies have suggested a potential role for regulatory T cells (Tregs) in allograft tolerance, but these FOXP3+ cells seem to be an inherent component of acute rejection (AR) in human recipients of renal transplants. The balance between regulatory cells and effector/cytotoxic cells may determine graft outcome; this balance has not been described for chronic allograft injury. We investigated the expression of key regulatory, effector, and cytotoxic transcripts (i.e., FOXP3, T-bet, and granzyme B, respectively) in the grafts and peripheral blood of long-term-surviving renal transplant patients. We found that, whereas neither intragraft nor peripheral blood FOXP3 or T-bet mRNA could distinguish between rejection and nonrejection status, granzyme B (GrzB) mRNA could: It was significantly increased in the graft and significantly decreased in the peripheral blood of patients with chronic antibody-mediated rejection (CAMR). Quantifying peripheral blood GrzB mRNA demonstrated potential to aid in the noninvasive diagnosis of CAMR. In summary, these data affirm GrzB as a marker not only for AR but also for CAMR. In addition, we identified several previously unreported clinical or demographic factors influencing regulatory/effector/cytotoxic profiles in the peripheral blood, highlighting the necessity to consider confounding variables when considering the use of potential biomarkers, such as FOXP3, for diagnosis or prognosis in kidney transplantation.