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Pathogen-induced immune responses prevent the establishment of transplantation tolerance in experimental animal models. Whether this occurs in humans as well remains unclear. The development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection allows us to address this question. We conducted a clinical trial of immunosuppression withdrawal in HCV-infected adult liver recipients to elucidate (i) the mechanisms through which allograft tolerance can be established in the presence of an ongoing inflammatory response and (ii) whether anti-HCV heterologous immune responses influence this phenomenon. Of 34 enrolled liver recipients, drug withdrawal was successful in 17 patients (50%). Tolerance was associated with intrahepatic overexpression of type I interferon and immunoregulatory genes and with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8(+) T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced proinflammatory gene expression and the breadth of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data suggest that in humans, persistent viral infections exert immunoregulatory effects that could contribute to the restraining of alloimmune responses, and do not necessarily preclude the development of allograft tolerance.

Introduction Most cases of acquired idiopathic severe aplastic anemia (SAA) are immune mediated. Following an insult which is likely to be viral, there is an increase in auto-reactive oligoclonal T-cells that result in apoptotic death of stem and progenitor cells, due to lack or absence of regulatory T-cells (Tregs) and a pro-inflammatory environment with clonal expansion of Th1 cells and increased Th17 cells. We have previously shown a correlation between low Treg numbers and disease severity, and that Tregs are not only reduced in number but are also unable to effectively suppress T effectors (Teff)1. The only curative treatment option for SAA is hematopoietic stem cell transplantation (HSCT) but this option is not accessible to everyone because of lack of donor or unfitness for the procedure. Moreover, patients who fail standard immunosuppressive treatment (ATG and Cyclosporine A) have limited alternative therapeutic options. The aim of this study was to investigate the expandability of Tregs in AA, their subsequent function and stability following expansion. Such data may provide the rationale for a novel future approach to treatment of refractory SAA. Methods and results IL-2 sensitivity assay One of the hallmarks of response to IL-2 by Tregs is STAT5 phosphorylation. To evaluate AA Tregs response to IL-2, freshly isolated cells from 6 AA patients were cultured in the presence of IL-2 (ranging from 0.1 to 1000 IU/ml). STAT5 phosphorylation was assessed by intracellular staining for pSTAT5 after 15 and 30 minutes. Tregs from AA patients have shown an increase in STAT5 phosphorylation similar to 2 healthy donors (HD) Tregs, confirming their responsiveness to IL-2. Tregs expansion Peripheral blood mononuclear cells (PBMCs) were obtained from six AA patients and eight HDs and enriched for Tregs using a magnetic beads regulatory T cells isolation kit according to the manufacturer instructions. Tregs were then stimulated with anti CD3/CD28 beads (1:1 ratio) and high dose IL-2 (1,000 IU/ml) for four weeks in a "Treg friendly" culture with all-trans retinoic acid 2 mM and rapamycin 100 nM. After four weeks of culture, expanded Tregs were then 'rested' for 5 days with decreasing doses of IL-2. Tregs from AA patients were expanded in a comparable rate to HD Tregs, with a median fold increase of 33 (range 29-149) compared to 21 fold increase (range 8-36) in HD. There was no significant difference between AA and HDs in terms of fold increase after 4 weeks. The expanded Tregs demonstrated more than 90% FoxP3+ expression in both AA and HDs. To assess IL-2 dependency of the expanded Tregs, Tregs were gradually deprived of IL-2 following expansion and STAT5 phosphorylation evaluated in both AA and HDs Tregs. Although expanded AA Tregs showed slightly lower pSTAT5 level following IL-2 deprivation, they responded to low dose IL-2 and pSTAT5 returned to similar level as in HDs. Expanded Tregs function and methylation status of TSDR To assess the suppressive activity of expanded Tregs, Teff were stained with a fluorescent proliferation dye (CellTrace Violet, Life Technologies) and co-cultured with autologous expanded Tregs (1:1 ratio) for 5 days in the presence of anti CD3/CD28 beads (Teff:Treg:beads = 20:20:1). The expanded Tregs from AA patients suppressed the proliferation of both autologous and allogeneic CD4+ T effectors (43% versus 5%, respectively, p = 0.009) and the suppressive function of expanded AA Tregs was not significantly different from expanded HD Tregs. In order to assess the stability of our Tregs we investigated the methylation status of 15 CpG sites within the Foxp3 Treg-specific demethylated region (TSDR) by amplicon sequencing of bisulfite treated DNA on an Illumina MiSeq instrument. The TSDR CpG sites in expanded Tregs from HDs and AA patients were found to be >98% unmethylated (no significant difference between AA and HD), which confirms the stability of expanded Tregs. Conclusions Our data show that Tregs from AA patients can be expanded efficiently ex vivo. Importantly, expanded Tregs are functional and stable with similar phenotypic characteristics to HD Tregs. These results may lead to the development of a novel treatment option for patients who fail/relapse after standard immunosuppression and for those who are not eligible for HSCT. References 1. Kordasti, S., et al. Functional characterization of CD4+ T cells in aplastic anemia. Blood119, 2033-2043 (2012). Disclosures Mufti: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.