The molecular mechanisms of acute lung injury resulting in inflammation and fibrosis are not well established. Here we investigate the roles of the IL-1 receptor 1 (IL-1R1) and the common adaptor for Toll/IL-1R signal transduction, MyD88, in this process using a murine model of acute pulmonary injury. Bleomycin insult results in expression of neutrophil and lymphocyte chemotactic factors, chronic inflammation, remodeling, and fibrosis. We demonstrate that these end points were attenuated in the lungs of IL-1R1-and MyD88-deficient mice. Further, in bone marrow chimera experiments, bleomycin-induced inflammation required primarily MyD88 signaling from radioresistant resident cells. Exogenous rIL-1β recapitulated a high degree of bleomycin-induced lung pathology, and specific blockade of IL-1R1 by IL-1 receptor antagonist dramatically reduced bleomycin-induced inflammation. Finally, we found that lung IL-1β production and inflammation in response to bleomycin required ASC, an inflammasome adaptor molecule. In conclusion, bleomycin-induced lung pathology required the inflammasome and IL-1R1/MyD88 signaling, and IL-1 represented a critical effector of pathology and therapeutic target of chronic lung inflammation and fibrosis.

Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the late development of chronic rejection. Blocking the CD28-mediated T cell costimulation pathway is a non toxic alternative immunosuppression strategy that is currently achieved by blockade of CD80/86, the counter receptors for CD28 on antigenpresenting cells.. However interaction of CD80/86 with CTLA-4 is required for immune regulation. Therefore CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and favor immune regulation. By using monovalent antibodies, we identified true CD28 antagonists inducing a CTLA-4-dependent decreased T cell function compatible with regulatory T cell (Treg) suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood regulatory T cells, induced molecular signatures of immune regulation and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 favors CTLA-4-dependent immune regulation and promotes allograft survival.

No abstract

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.

These authors contributed equally to the senior authorship of this study.Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA-4; PD-L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti-CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti-CD28 Fab' antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid-free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios-negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Tregspecific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)-low or CNI-free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI-sparing strategy to B7 antagonists for kidney transplant recipients.

FR104 is a monovalent pegylated Fab' Ab, antagonist of CD28, under development for treatment of transplant rejection and autoimmune diseases. In contrast to CD80/86 antagonists (CTLA4-Ig), FR104 selectively blunts CD28 costimulation while sparing CTLA-4 and PD-L1 coinhibitory signals. In the present work, FR104 has been evaluated in a first-in-human study to evaluate the safety, pharmacokinetics, pharmacodynamics, and potency of i.v. administrations in healthy subjects. Sixty-four subjects were randomly assigned to four single ascending dose groups, two double dose groups and four single ascending dose groups challenged with keyhole limpet hemocyanin. Subjects were followed up over a maximum of 113 d. Overall, the pharmacokinetics of FR104 after a single and double infusions was approximately linear at doses ≥0.200 mg/kg. CD28 receptor occupancy by FR104 was saturated at the first sampling time point (0.5 h) at doses above 0.02 mg/kg and returned to 50% in a dose-dependent manner, by day 15 (0.020 mg/kg) to 85 (1.500 mg/kg). FR104 was well tolerated, with no evidence of cytokine-release syndrome and no impact on blood lymphocyte subsets. Inhibition of anti-keyhole limpet hemocyanin Ab response was dose-dependent in FR104 recipients and was already apparent at a dose of 0.02 mg/kg. Abs to FR104 were detected in 22/46 (48%) of FR104 recipients and only 1/46 (2.2%) was detected during drug exposure. In conclusion, selective blockade of CD28 with FR104 was safe and well tolerated at the doses tested. The observed immunosuppressive activity indicated that FR104 has potential to show clinical activity in the treatment of immune-mediated diseases.

Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.

Cancer (INCA MDSCAN PRT-K15-136 grant), and the Regional Ligue Contre le Cancer 44. We thank the staff of the Humanized Rodent Platform and MicroPICell Cellular and Tissue Imaging