Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.

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Despite the increasing use of humanized mouse models to study new approaches of graft-versus-host disease (GVHD) prevention, the pathogenesis of xenogeneic GVHD (xGVHD) in these models remains misunderstood. The aim of this study is to describe this pathogenesis in NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice infused with human PBMCs and to assess the impact of the expression of HLA-A0201 by NSG mice cells (NSG-HLA-A2/HHD mice) on xGVHD and graft-versus-leukemia (GvL) effects, by taking advantage of next-generation technologies. We found that T cells recovered from NSG mice after transplantation had upregulated expression of genes involved in cell proliferation, as well as in TCR, co-stimulatory, IL-2/STAT5, mTOR and Aurora kinase A pathways. T cells had mainly an effector memory or an effector phenotype and exhibited a Th1/Tc1-skewed differentiation. TCRβ repertoire diversity was markedly lower both in the spleen and lungs (a xGVHD target organ) than at infusion. There was no correlation between the frequencies of specific clonotypes at baseline and in transplanted mice. Finally, expression of HLA-A0201 by NSG mice led to more severe xGVHD and enhanced GvL effects toward HLA-A2+ leukemic cells. Altogether our data demonstrate that the pathogenesis of xGVHD shares important features with human GVHD and that NSG-HLA-A2/HHD mice could serve as better model to study GVHD and GvL effects.

The online version of this article has a supplementary Appendix. BackgroundLong-term immune recovery in older patients given hematopoietic cell transplantation after non-myeloablative conditioning remains poorly understood. This prompted us to investigate long-term lymphocyte reconstitution and thymic function in 80 patients given allogeneic peripheral blood stem cells after non-myeloablative conditioning. Design and MethodsMedian age at transplant was 57 years (range 10-71). Conditioning regimen consisted of 2 Gy total body irradiation (TBI) with (n=46) or without (n=20) added fludarabine, 4 Gy TBI with fludarabine (n=6), or cyclophosphamide plus fludarabine (n=8). Stem cell sources were unmanipulated (n=56), CD8-depleted (n=19), or CD34-selected (n=5) peripheral blood stem cells. Immune recovery was assessed by signal-joint T-cell receptor excision circle quantification and flow cytometry. ResultsSignal-joint T-cell receptor excision circle levels increased from day 100 to one and two years after transplantation in patients under 50 years of age (n=23; P=0.02 and P=0.04, respectively), and in those aged 51-60 years (n=35; P=0.17 and P=0.06, respectively), but not in patients aged over 60 (n=22; P=0.3 and P=0.3, respectively). Similarly, CD4 + CD45RA + (naïve) T-cell counts increased from day 100 to one and two years after transplantation in patients aged 50 years and under 50 (P=0.002 and P=0.02, respectively), and in those aged 51-60 (P=0.4 and P=0.001, respectively), but less so in patients aged over 60 (P=0.3 and P=0.06, respectively). In multivariate analyses, older patient age (P<0.001), extensive chronic GVHD (P<0.001), and prior (resolved) extensive chronic graft-versus-host disease (P=0.008) were associated with low signal-joint T-cell receptor excision circle levels one year or more after HCT. ConclusionsIn summary, our data suggest that thymic neo-generation of T cells occurred from day 100 onwards in patients under 60 while signal-joint T-cell receptor excision circle levels remained low for patients aged over 60. Further, chronic graft-versus-host disease had a dramatic impact on thymic function, as observed previously in patients given grafts after myeloablative conditioning.Key words: thymus, hematopoietic cell transplantation, non-myeloablative, graft-versus-host disease, immunity, age. Hannon M, Dutrieux J, Humblet-Baron S, Seidel L, Cheynier R, Willems E, Gothot A, Vanbellinghen J-F, Geenen V, Sandmaier BM, Storb R, Beguin Y, and Citation: Castermans E,

The demethylating agent 5-azacytidine (AZA) has proven its efficacy in the treatment of myelodysplastic syndrome and acute myeloid leukemia. In addition, AZA can demethylate intron 1 () leading to the generation of regulatory T cells (Treg). Here, we investigated the impact of AZA on xenogeneic graft-vs.-host disease (xGVHD) and graft-vs.-leukemia effects in a humanized murine model of transplantation (human PBMCs-infused NSG mice), and described the impact of the drug on human T cells . We observed that AZA improved both survival and xGVHD scores. Further, AZA significantly decreased human T-cell proliferation as well as IFNγ and TNF-α serum levels, and reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells. In addition, AZA significantly increased Treg frequency through hypomethylation of as well as increased Treg proliferation. The latter was subsequent to higher STAT5 signaling in Treg from AZA-treated mice, which resulted from higher IL-2 secretion by conventional T cells from AZA-treated mice itself secondary to demethylation of the IL-2 gene promoter by AZA. Importantly, Tregs harvested from AZA-treated mice were suppressive and stable over time since they persisted at high frequency in secondary transplant experiments. Finally, graft-vs.-leukemia effects (assessed by growth inhibition of THP-1 cells, transfected to express the luciferase gene) were not abrogated by AZA. In summary, our data demonstrate that AZA prevents xGVHD without abrogating graft-vs.-leukemia effects. These findings could serve as basis for further studies of GVHD prevention by AZA in acute myeloid leukemia patients offered an allogeneic transplantation.

Background We investigated the ability of clinical‐grade enriched human regulatory T cells (Treg) to attenuate experimental xenogeneic graft‐versus‐host disease (GVHD) induced by peripheral blood mononuclear cells (PBMNCs; autologous to Treg) infusion in NSG mice, as well as verified their inability to induce xenogeneic GVHD when infused alone. Study Design and Methods Human Treg were isolated from peripheral blood apheresis products with a cell separation system (CliniMACS, Miltenyi Biotec GmbH) using a two‐step procedure (simultaneous CD8 and CD19 depletion followed by CD25‐positive selection) in six independent experiments with six different healthy volunteer donors. Sublethally (2.5 Gy) irradiated NSG mice were given 2 × 106 cytapheresis (PBMNC) product cells intravenously (IV) without (PBMNC group) or with 1 × 106 Treg (PBMNC + Treg group), while other NSG mice received 2 × 106 enriched Treg alone (also in IV; Treg group). Results The first five procedures were successful at obtaining a relatively pure Treg population (defined as >50%), while the sixth procedure, due to a technical problem, was not (Treg purity, 42%). Treg cotransfusion significantly delayed death from xenogeneic GVHD in the first five experiments, (p < 0.0001) but not in the sixth experiment. Importantly, none of the mice given enriched Treg alone (Treg group) experienced clinical signs of GVHD, while, interestingly, the CD4+ cells found in these mice 26 days after transplantation were mainly conventional T cells (median CD25+FoxP3+ cells among human CD4+ total cells were only 2.1, 3.1, and 12.2% in spleen, marrow, and blood, respectively). Conclusions Infusion of clinical‐grade enriched Treg delayed the occurrence of xenogeneic GVHD without inducing toxicity in this murine model.

Mesenchymal stromal cells (MSCs) have potent immunomodulatory properties that make them an attractive tool against graft- vs.-host disease (GVHD). However, despite promising results in phase I/II studies, bone marrow (BM-) derived MSCs failed to demonstrate their superiority over placebo in the sole phase III trial reported thus far. MSCs from different tissue origins display different characteristics, but their therapeutic benefits have never been directly compared in GVHD. Here, we compared the impact of BM-, umbilical cord (UC-), and adipose-tissue (AT-) derived MSCs on T-cell function in vitro and assessed their efficacy for the treatment of GVHD induced by injection of human peripheral blood mononuclear cells in NOD-scid IL-2Rγ null HLA-A2/HHD mice. In vitro , resting BM- and AT-MSCs were more potent than UC-MSCs to inhibit lymphocyte proliferation, whereas UC- and AT-MSCs induced a higher regulatory T-cell (CD4 + CD25 + FoxP3 + )/T helper 17 ratio. Interestingly, AT-MSCs and UC-MSCs activated the coagulation pathway at a higher level than BM-MSCs. In vivo , AT-MSC infusions were complicated by sudden death in 4 of 16 animals, precluding an analysis of their efficacy. Intravenous MSC infusions (UC- or BM- combined) failed to significantly increase overall survival (OS) in an analysis combining data from 80 mice (hazard ratio [HR] = 0.59, 95% confidence interval [CI] 0.32–1.08, P = 0.087). In a sensitivity analysis we also compared OS in control vs. each MSC group separately. The results for the BM-MSC vs. control comparison was HR = 0.63 (95% CI 0.30–1.34, P = 0.24) while the figures for the UC-MSC vs. control comparison was HR = 0.56 (95% CI 0.28–1.10, P = 0.09). Altogether, these results suggest that MSCs from various origins have different effects on immune cells in vitro and in vivo . However, none significantly prevented death from GVHD. Finally, our data suggest that the safety profile of AT-MSC and UC-MSC need to be closely monitored given their pro-coagulant activities in vitro .

BackgroundPre-transplant infusion of rabbit anti-T cell globulin (ATG) is increasingly used as prevention of graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (PBSCT). However, the precise impact of pre-transplant ATG on immune recovery after PBSCT is still poorly documented.MethodsIn the current study, we compared immune recovery after myeloablative PBSCT in 65 patients who either received (n = 37) or did not (n = 28) pre-transplant ATG-Fresenius (ATG-F). Detailed phenotypes of circulating T, B, natural killer (NK) and invariant NKT (iNKT) cells were analyzed by multicolor flow cytometry at serial time-points from day 40 to day 365 after transplantation. Thymic function was also assessed by sjTREC quantification. Serious infectious events were collected up to 2 years post-transplantation.ResultsPre-transplant ATG-F had a prolonged (for at least up to 1-year) and selective negative impact on the T-cell pool, while it did not impair the recovery of B, NK nor iNKT cells. Among T cells, ATG-F selectively compromised the recovery of naïve CD4+, central memory CD4+ and naïve CD8+ cells, while it spared effector memory T and regulatory T cells. Levels of sjTRECs were similar in both cohorts at 1-year after PBSCT, suggesting that ATG-F unlikely impaired thymopoiesis at long-term after PBSCT. Finally, the incidence and rate of serious infections were similar in both groups, while ATG-F patients had a lower incidence of grade II-IV acute graft-versus-host disease.ConclusionsPre-transplant ATG-F induces long-lasting modulation of the circulating T-cell pool after myeloablative PBSCT, that may participate in preventing graft-versus-host disease without deeply compromising anti-pathogen defenses.