Type 1 regulatory T (Tr1) cells are an inducible subset of CD4 + Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4 + T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP + LV-IL-10-transduced human CD4 + T (CD4 LV-IL-10 ) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4 LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4 LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4 + T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.

OBJECTIVEIn type 1 diabetes, allogeneic pancreatic islet transplant restores insulin production, but life-threatening immunosuppression is required to avoid graft rejection. Induction of antigen (Ag)–specific tolerance by cell therapy with regulatory T-cells (Tregs) represents an attractive alternative approach but its therapeutic efficacy in islet transplant remains to be determined. Among the different subsets of CD4+ Tregs, the T inducible regulatory type 1 (Tr1) cells can be generated from naive T-cells in the presence of interleukin-10 (IL-10) and represent one promising therapeutic choice. This study was designed to define the efficacy of Tr1-cell therapy in preclinical models of islet transplant.RESEARCH DESIGN AND METHODSNon–Ag-specific polyclonal Tr1 cells and donor Ag-specific Tr1 cells were transferred, in the absence of any pharmacological treatment, in two distinct mouse models of islet transplant. The two models differed in their therapeutic stringency, based on the mean rejection time of untreated mice that underwent a transplant.RESULTSTransfer of polyclonal Tr1 cells engendered graft tolerance only in the nonstringent mouse model. Conversely, cell therapy with Ag-specific Tr1 cells induced an IL-10–dependent tolerance in the stringent mouse model of islet transplant. The therapeutic advantage of Ag-specific Tr1 cells over polyclonal Tr1 cells was due to their donor Ag specificity.CONCLUSIONSThese results demonstrate that Tr1-cell therapy leads to tolerance in settings of islet transplant and that its therapeutic efficacy is highly dependent on the antigen specificity of these cells.

The online version of this article has a Supplementary Appendix. BackgroundThe clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3 + Tregulatory cells may contain interleukin-17-producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3 + T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy. Design and MethodsT-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability. ResultsWe found that CD4+ T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a "Th17-favorable" environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed. ConclusionsThese data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.Key words: T-regulatory cells, ex vivo expansion, rapamycin, cell therapy, T-regulatory-cell stability. + T regulatory cells. Haematologica 2011;96(9):1357-1365

BackgroundA large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3+Treg and IL-10–producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell–associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent.Methodology/Principal FindingsDiabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3+Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4+IL-10+IL-4− T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4+IL-10+IL-4− T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4+IL-10+IL-4− T cells.Conclusions/SignificanceThe anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic.

The immune system is comprised of several CD4 þ T regulatory (Treg) cell types, of which two, the Foxp3 þ 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 þ Treg and Tr1 cells. Here, we show that Foxp3 þ Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4 þ CD25 À 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 þ 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 þ Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen.Key words: Graft, spleen, T regulatory cells, transplant tolerance Abbreviations: 2TT mice, mice comprising two Tregcell types; ENGR-2TT mice, engrafted mice analyzed 30 days after transplant; TOL-2TT mice, tolerant mice analyzed 150 days after transplant and after donor cell boost; Fir/Tiger mice, double reporter mice Foxp3-RFP and Il10-eGFP.