Rapamycin Prevents and Breaks the Anti-CD3–Induced Tolerance in NOD Mice

OBJECTIVE—Non–Fc-binding anti-CD3–specific antibodies represent a promising therapy for preserving C-peptide produc-tion in subjects with recent-onset type 1 diabetes. However, the mechanisms by which anti-CD3 exerts its beneficial effect are still poorly understood, and it is questionable whether this therapeutic approach will prove durable with regard to its ability to impart metabolic preservation without additional actions designed to maintain immunological tolerance. We used the NOD mouse model to test whether rapamycin, a compound well-known for its immunomodulatory activity in mice and humans, could increase the therapeutic effectiveness of anti-CD3 treat-ment in type 1 diabetes. RESEARCH DESIGN AND METHODS—Rapamycin was ad-ministered to diabetic NOD mice simultaneously with anti-CD3 or to NOD mice cured by anti-CD3 therapy. The ability of thi

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

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

Transplant Tolerance to Pancreatic Islets Is Initiated in the Graft and Sustained in the Spleen

The immune system is comprised of several CD4(+) T regulatory (Treg) cell types, of which two, the Foxp3(+) Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg-cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3(+) Treg and Tr1 cells. Here, we show that Foxp3(+) Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4(+) CD25(−) T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long-term tolerance. Importantly, the role of Foxp3(+) Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long-term tolerance induced by Foxp3(+) Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen

Transplant Tolerance to Pancreatic Islets Is Initiated in the Graft and Sustained in the Spleen

The immune system is comprised of several CD4(+) T regulatory (Treg) cell types, of which two, the Foxp3(+) Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg-cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3(+) Treg and Tr1 cells. Here, we show that Foxp3(+) Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4(+) CD25(-) T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long-term tolerance. Importantly, the role of Foxp3(+) Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long-term tolerance induced by Foxp3(+) Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen

A Prevalent CXCR3+ Phenotype of Circulating Follicular Helper T Cells Indicates Humoral Dysregulation in Children with Down Syndrome

Patients with Down syndrome (DS) are characterized by increased susceptibility to autoimmunity and respiratory tract infections that are suggestive of humoral immunity impairment. Here, we sought to determine the follicular helper (Tfh) and follicular regulatory (Tfr) T cell profile in the blood of children with DS. Blood was collected from 24 children with DS, nine of which had autoimmune diseases. Children with DS showed skewed Tfh differentiation towards the CXCR3+ phenotype: Tfh1 and Tfh1/17 subsets were increased, while Tfh2 and Tfh17 subsets were reduced. While no differences in the percentage of Tfr cells were seen, the ratio of Tfh1 and CXCR3+PD-1+ subsets to Tfr cells was significantly increased in the affected children. The excessive polarization towards a CXCR3+ phenotype in children with DS suggests that re-calibration of Tfh subset skewing could potentially offer new therapeutic opportunities for these patients

Reduced PD-1 expression on circulating CXCR5+ and CXCR5- FOXP3+ Treg cells marks type 1 diabetes initiation in children

Autoantibodies (AAbs) are a hallmark of Type 1 diabetes (T1D). Alterations in the frequency and phenotype of follicular helper (Tfh) T cells have been previously documented in patients with type 1 diabetes (T1D), but the contribution of follicular Treg cells, which are responsible for suppressing AAb development, is less clear. Here, we investigated the frequency and activation status of follicular (CXCR5+) and CXCR5- Treg cells in the blood of children with new onset T1D and children with risk for developing T1D (AAb-positive) and compared them to AAb-negative controls. Blood CXCR5+ and CXCR5- Treg cells were higher in frequency children with new onset T1D and expressed reduced amounts of PD-1 as compared to controls. Interestingly, the proportion of circulating FOXP3+ Tregs expressing PD-1 was also reduced in AAb-positive at-risk children as compared to controls, suggesting its potential use as a biomarker of disease initiation