Key role of macrophages in tolerance induction via T regulatory type 1 (Tr1) cells.

T regulatory type 1 (Tr1) cells are a class of regulatory T cells (Tregs ) participating in peripheral tolerance, hence the rationale behind their testing in clinical trials in different disease settings. One of their applications is tolerance induction to allogeneic islets for long-term diabetes-free survival. Currently the cellular and molecular mechanisms that promote Tr1-cell induction in vivo remain poorly understood. We employed a mouse model of transplant tolerance where treatment with granulocyte colony-stimulating factor (G-CSF)/rapamycin induces permanent engraftment of allogeneic pancreatic islets in C57BL/6 mice via Tr1 cells. The innate composition of graft and spleen cells in tolerant mice was analyzed by flow cytometry. Graft phagocytic cells were co-cultured with CD4+ T cells in vitro to test their ability to induce Tr1-cell induction. Graft phagocytic cells were depleted in vivo at different time-points during G-CSF/rapamycin treatment, to identify their role in Tr1-cell induction and consequently in graft survival. In the spleen, the site of Tr1-cell induction, no differences in the frequencies of macrophages or dendritic cells (DC) were observed. In the graft, the site of antigen uptake, a high proportion of macrophages and not DC was detected in tolerant but not in rejecting mice. Graft-infiltrating macrophages of G-CSF/rapamycin-treated mice had an M2 phenotype, characterized by higher CD206 expression and interleukin (IL)-10 production, whereas splenic macrophages only had an increased CD206 expression. Graft-infiltrating cells from G-CSF/rapamycin-treated mice-induced Tr1-cell expansion in vitro. Furthermore, Tr1-cell induction was perturbed upon in-vivo depletion of phagocytic cells, early and not late during treatment, leading to graft loss suggesting that macrophages play a key role in tolerance induction mediated by Tr1 cells. Taken together, in this mouse model of Tr1-cell induced tolerance to allogeneic islets, M2 macrophages infiltrating the graft upon G-CSF/rapamycin treatment are key for Tr1-cell induction. This work provides mechanistic insight into pharmacologically induced Tr1-cell expansion in vivo in this stringent model of allogeneic transplantation.

[CAR-T cells: Lymphocytes that express a chimeric antigen receptor]. / CAR-T cells : lymphocytes exprimant un récepteur chimérique à l'antigène.

CAR-T cells are genetically modified human lymphocytes and gene therapy medicinal products. They are developed to treat cancers that express a membrane antigen targeted by the CAR. The FDA approved the two first-in-class medicinal products in 2017 and EMA in August 2018; both are autologous CAR-T cells targeting CD19 that is expressed at the surface of normal B-cells throughout their differentiation, and on B-cell lymphoid malignancies. Clinical efficacy was demonstrated for B-cell acute lymphoblastic leukemias, non-Hodgkin's lymphoma and chronic lymphocytic leukemia, although the marketing authorizations are less liberal in terms of indications. Manufacturing of these personalized treatments necessitates that a novel organization and supply chain be set in place, to ensure product preservation, patient safety and compliance with complex regulatory requirements. Side effects are commensurate with clinical efficacy and can be life-threatening: proper management imposes tight coordination between various specialists, particularly between hematologists and intensive care practitioners. High pricing for these treatments is part of a long-term trend for increasing costs of innovations in hematology and oncology; it questions the ability of healthcare systems to sustain their reimbursement.

Deleterious effect of CTLA4-Ig on a Treg-dependent transplant model.

Blockade of the B7:CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, results from the belatacept phase III clinical trial demonstrated a higher rejection rate when compared to cyclosporine, raising concern about potential deleterious effects of this agent. In this study, we investigated the consequences of B7:CD28 blockade by hCTLA4Ig on regulator T cell (Treg) generation in different major histocompatibility complex (MHC) mismatch transplant models. Administration of hCTLA4Ig significantly decreased the amount of Tregs in B6 WT animals and this effect was predominant in thymus-induced Tregs (Helios(+) ). Although hCTLA4Ig prevented rejection in a fully allogeneic mismatch model, it accelerated rejection in a MHC class-II mismatch model (MST = 26, p < 0.0001), in which long-term allograft survival is dependent on Tregs. This accelerated rejection was associated with a marked reduction in thymus-induced Tregs and led to a higher effector/regulatory T-cell ratio in secondary lymphoid organs and in the allograft. This study confirms the importance of the B7:CD28 pathway in Treg homeostasis in an in vivo transplant model and suggests that hCTLA4Ig therapy may be deleterious in circumstances where engraftment is dependent on Tregs.

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

Determination of optimal incubation time for the production of acute phase cytokines ex vivo by peripheral blood mononuclear cells from renal transplant recipients.

Levels of acute phase cytokines secreted ex vivo by peripheral blood mononuclear cells (PBMCs) have been shown to be associated with clinical conditions or histologic lesions in renal transplant recipients. One of the limiting factors for the potential use of this assay as a diagnostic tool is the incubation time needed to measure adequate cytokine levels. Here, we validated that shorter time periods than the usual 48 h are sufficient for the production of acute phase cytokines. Cytokine levels were measured with the Luminex platform. We observed that, in contrast to cytokines associated with adaptive immunity, cytokines such as IL-1ß, IL-6 and TNF-α are measurable as early as 2 h following incubation at a concentration of 1.5 million PBMC/150 µL. Levels obtained in the 2 h cultures have good correlations with the levels obtained after 48 h of culture for IL-1ß and TNF-α (R=0.79, P=0.004 and R=0.92, P<0.001 respectively). We conclude that same-day incubation of PBMCs and measurement of these cytokines following blood collection in transplant recipients is feasible. It provides a rationale for further studies using shorter incubation times for ex vivo cellular assay measuring acute phase cytokine levels.

Improved function of circulating angiogenic cells is evident in type 1 diabetic islet-transplanted patients.

Circulating angiogenic cells (CACs) are vascular-committed bone marrow-derived cells that are dysfunctional in type 1 diabetes (T1D). Here we studied whether restoration of normoglycemia following islet transplantation is associated with better CAC function. We carried out a cross-sectional study of 18 T1D patients, 14 insulin-independent islet-transplanted patients (ITA) and 14 healthy controls (C) evaluating in vivo and in vitro CACs viability and function. We found that the percentage of CACs in vivo did not differ among the three groups while the number of CAC colonies obtained from T1D, but not from ITA, was reduced compared to C (C = 7.3 ± 1.9, T1D = 0.9 ± 0.4 and ITA = 4.7 ± 1.9; p < 0.05 T1D vs. all). In vitro CAC migration/differentiation were similar, while in vivo an improved angiogenic ability of ITA compared to T1D was shown (capillary density: C = 93.5 ± 22.1, T1D = 19.2 ± 2.8 and ITA = 44.0 ± 10.5, p < 0.05 T1D vs. all). Increased apoptosis and lesser IL-8 secretion were evident in CACs obtained from T1D compared to C and ITA. in vitro addition of anti-hIL-8 reduced the number of colonies obtained from C. Finally, T1D, but not ITA, had a lower endothelial-dependent dilatation (EDD) compared with C. These data suggest that CAC function is altered in T1D and may be improved after islet transplantation.

Sickle cell disease: the Lebanese experience.

Sickle cell disease (SCD), the commonest single gene disorder worldwide, is an inherited disease that has different clinical and hematological manifestations in different populations. The objective of this study is to describe the characteristics of the Lebanese SCD population. This was a retrospective study that included information on 387 patients with either sickle cell anemia (SS) or sickle beta-thalassemia (ST). The mean (+/-SD) age was 17.9 years (+/-12.5), and the mean (+/-SD) follow-up was 9.3 +/- 6.9 years. Fifty percent of the patients were males and SS/ST distribution was 3 : 1. The disease was clustered in two geographic areas in North and South Lebanon. Nearly, all patients were Muslims and 56% were the offspring of consanguineous parents. The prevalence of splenomegaly beyond 6 years of age among SS patients was 28.9%. The prevalence rates of stroke, leg ulcers and priapism were 4.1%, 1.4%, and 0.8%, respectively. Comparing the SS and the ST patients, there were no statistically significant differences in the prevalence of all clinical manifestations except for splenomegaly (SS: 28.9%, ST: 54.9%, P-value < 0.001) and splenectomy (SS: 16.1%, ST: 35.7%, P-value < 0.001). In contrast to Northern American populations and similar to some Mediterranean populations, Lebanese SCD patients have a higher prevalence of persistent splenomegaly. The relatively low incidence of thrombotic complications deserves further investigation. The study's limitations include those of any other retrospective study and the fact that not all Lebanese centers caring for inherited hemoglobin disorders were included. However, the results of this first large scale national survey indicate that preventive efforts should target the Northern and Southern regions of Lebanon to decrease the number of new off springs afflicted with this disease similar to what has been successfully achieved with Thalassemia, another hemoglobinopathy that is highly prevalent in the country.