Comprehensive analysis of the immunomodulatory effects of rapamycin on human T cells in graft-versus-host disease prophylaxis.

Graft-versus-host disease (GVHD) is a major cause of toxicity after allogeneic hematopoietic cell transplantation (allo-HCT). While rapamycin (RAPA) is commonly used in GVHD prophylaxis in combination with a calcineurin inhibitor (CNI), the understanding of its mechanism of action on human T cells is still incomplete. Here, we performed an extensive analysis of RAPA effects on human T cells in a humanized mouse model of GVHD, in ex-vivo T cell cultures and in patients given RAPA plus tacrolimus as GVHD prophylaxis after nonmyeloablative allo-HCT. We demonstrate that RAPA mitigates GVHD by decreasing T cell engraftment and differentiation, inhibiting CD8+ T cell activation and increasing the long-term IL-2 secretion, thereby supporting regulatory T cell (Treg) proliferation. In contrast, graft-versus-leukemia effects were not abrogated, as RAPA-treated T cells had increased resistance to apoptosis and retained their effector function and proliferative capacity upon re-stimulation. Importantly, we found that RAPA impact on Treg and CD8+ T cells was closely dependent upon IL-2 signaling and that therapeutic options interfering with IL-2, such as calcineurin inhibitors, antagonize the IL-2-dependent promotion of Treg mediated by RAPA. Our results suggest that RAPA immunological efficacy could be improved in combination with drugs having possible synergistic effects such as the hypomethylating agent 5-azacytidine.

In Vitro Th17-Polarized Human CD4+ T Cells Exacerbate Xenogeneic Graft-versus-Host Disease.

Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. The role of Th17 cells in its pathophysiology remains a matter of debate. In this study, we assessed whether enrichment of human peripheral blood mononuclear cells (PBMCs) with in vitro Th17-polarized CD4+ T cells would exacerbate xenogeneic GVHD (xGVHD) into NOD-scid IL-2Rγ null (NSG) mice. Naive human CD4+ T cells were stimulated under Th17-skewing conditions for 8 to 10 days and then coinjected in NSG mice with fresh PBMCs from the same donor. We observed that Th17-polarized cells engrafted and migrated toward xGVHD target organs. They also acquired a double-expressing IL-17A+IFNγ+ profile in vivo. Importantly, cotransfer of Th17-polarized cells (1 × 106) with PBMCs (1 × 106) exacerbated xGVHD compared with transplantation of PBMCs alone (2 × 106). Furthermore, PBMC cotransfer with Th17-polarized cells was more potent for xGVHD induction than cotransfer with naive CD4+ T cells stimulated in nonpolarizing conditions (Th0 cells, 1 × 106 + 1 × 106 PBMCs) or with Th1-polarized cells (1 × 106 + 1 × 106 PBMCs). In summary, our results suggest that human Th17-polarized cells can cooperate with PBMCs and be pathogenic in the NSG xGVHD model.

Infusion of mesenchymal stromal cells after deceased liver transplantation: A phase I-II, open-label, clinical study.

BACKGROUND & AIMS: Mesenchymal stromal cell (MSC) infusion could be a means to establish tolerance in solid organ recipients. The aim of this prospective, controlled, phase I study was to evaluate the feasibility, safety and tolerability of a single infusion of MSCs in liver transplant recipients. METHODS: Ten liver transplant recipients under standard immunosuppression received 1.5-3×106/kg third-party unrelated MSCs on postoperative day 3±2, and were prospectively compared to a control group of ten liver transplant recipients. As primary endpoints, MSC infusion toxicity was evaluated, and infectious and cancerous complications were prospectively recorded until month 12 in both groups. As secondary endpoints, rejection rate, month-6 graft biopsies, and peripheral blood lymphocyte phenotyping were compared. Progressive immunosuppression weaning was attempted from month 6 to 12 in MSC recipients. RESULTS: No variation in vital parameters or cytokine release syndrome could be detected during and after MSC infusion. No patient developed impairment of organ functions (including liver graft function) following MSC infusion. No increased rate of opportunistic infection or de novo cancer was detected. As secondary endpoints, there was no difference in overall rates of rejection or graft survival. Month-6 biopsies did not demonstrate a difference between groups in the evaluation of rejection according to the Banff criteria, in the fibrosis score or in immunohistochemistry (including Tregs). No difference in peripheral blood lymphocyte typing could be detected. The immunosuppression weaning in MSC recipients was not successful. CONCLUSIONS: No side effect of MSC infusion at day 3 after liver transplant could be detected, but this infusion did not promote tolerance. This study opens the way for further MSC or Treg-based trials in liver transplant recipients. LAY SUMMARY: Therapy with mesenchymal stromal cells (MSCs) has been proposed as a means to improve results of solid organ transplantation. One of the potential MSC role could be to induce tolerance after liver transplantation, i.e. allowing the cessation of several medications with severe side effects. This study is the first-in-man use of MSC therapy in ten liver transplant recipients. This study did not show toxicity after a single MSC infusion but it was not sufficient to allow withdrawal of immunosuppression. CLINICAL TRIAL REGISTRATION NUMBER: Eudract: # 2011-001822-81, ClinicalTrials.gov: # NCT 01429038.

Impact of the immunomodulating peptide thymosin alpha 1 on multiple myeloma and immune recovery after hematopoietic stem cell transplantation.

Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells in the bone marrow and causes several immune alterations in patients. Thymosin α1 (Tα1) is a thymic peptide that has been associated with immuno-stimulating properties. In addition, this peptide exerts anti-tumor effects in several cancer types. Beneficial effects of Tα1 administration have also been shown on immune reconstitution after hematopoietic stem cell transplantation (HSCT), a current treatment modality in hematological malignancies including MM. In this study, we observed a slight reduction in the proliferation of murine and human MM cell lines in the presence of Tα1 in vitro. However, using two immunocompetent murine MM models (5TGM1 and MOPC315.BM), we did not observe any impact of Tα1 administration on MM development in vivo. Furthermore, no beneficial effects of Tα1 treatment were observed on lymphocyte immune reconstitution after transfusion of human hematopoietic stem cells into immunodeficient mice. In conclusion, despite direct effects of Tα1 on human MM cell line proliferation in vitro, Tα1 did not exert anti-myeloma effects in vivo in the two murine models tested. Moreover, Tα1 failed to improve immune recovery in a xenogeneic HSCT model.

Infusion of clinical-grade enriched regulatory T cells delays experimental xenogeneic graft-versus-host disease.

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 × 10(6) cytapheresis (PBMNC) product cells intravenously (IV) without (PBMNC group) or with 1 × 10(6) Treg (PBMNC + Treg group), while other NSG mice received 2 × 10(6) 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.

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease.

BACKGROUND AIMS: Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects. METHODS: The ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed. RESULTS: Injection of 200 × 10(6) human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10(6) MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10(6) human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10(6) MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. CONCLUSIONS: Injection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.

Thymic recovery after allogeneic hematopoietic cell transplantation with non-myeloablative conditioning is limited to patients younger than 60 years of age.

BACKGROUND: Long-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 METHODS: Median 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. RESULTS: Signal-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. CONCLUSIONS: In 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.

Comparison of Mesenchymal Stromal Cells From Different Origins for the Treatment of Graft-vs.-Host-Disease in a Humanized Mouse 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-2Rγnull 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.

Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice.

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. TCRß 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.

Reconstitution of adaptive immunity after umbilical cord blood transplantation: impact on infectious complications.

In comparison with allogeneic stem cell transplantation (alloHSCT) with other stem cell sources, umbilical cord blood transplantation (UCBT) was traditionally associated with increased risk of infections, particularly during the first 3 months after transplantation. Longitudinal studies of immune monitoring reported peculiar patterns of T- and B-cell recovery in the peripheral blood of UCB recipients during the first months post-transplantation. Overall, current data suggest delayed reconstitution of naive and memory CD4+ and CD8+ T-cell pools after UCBT. This is particularly true for adult recipients and for patients who received in vivo T-cell depleting approaches before the transplantation. Such delayed T-cell recovery may increase susceptibility of UCB recipients for developing opportunistic infections and viral reactivations. Regarding B-cell recovery, UCBT was associated with accelerated B-lymphopoiesis. Recent studies also reported evidence for faster functional memory B-cell recovery in UCB recipients. In this article, we briefly review T- and B-cell reconstitution after alloHSCT, with emphasis on peculiarities observed after UCBT. We further put these data in lines with risks of infections after UCBT.

Azacytidine prevents experimental xenogeneic graft-versus-host disease without abrogating graft-versus-leukemia effects.

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 FOXP3 intron 1 (FOXP3i1) 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 in vivo. 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 FOXP3i1 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.

Limited Impact of Imatinib in a Murine Model of Sclerodermatous Chronic Graft-versus-Host Disease.

BACKGROUND: Sclerodermatous chronic Graft-versus-Host Disease (scl-cGVHD) is one of the most severe form of cGVHD. The Platelet-derived Grotwth Factor (PDGF) and the Transforming Growth Factor-ß (TGF-ß) play a significant role in the fibrosing process occurring in scl-cGVHD. This prompted us to assess the impact of the PDGF-r and c-Abl tyrosine kinase inhibitor imatinib on scl-cGVHD. METHODS: To assess the impact of imatinib on T cell subset proliferation in vivo, Balb/cJ recipient mice were lethally (7 Gy) irradiated and then injected with 10x106 bone marrow cells from B10.D2 mice on day 0. Fourteen days later, 70x106 carboxyfluorescein succinimidyl ester (CFSE)-labeled splenocytes from B10.D2 mice were infused and imatinib or sterile water was administered for 5 days. To induce severe scl-cGVHD, Balb/cJ mice were injected i.v. with 10.106 bone marrow cells and 70.106 splenocytes from B10.D2 donor mice after 7 Gy irradiation. Mice were then given sterile water or imatinib from day +7 after transplantation to the end of the experiment (day +52). RESULTS: Imatinib decreased the proliferation of total T cells (P = 0.02), CD8+ T cells (P = 0.01), and of regulatory T cells (Tregs) (P = 0.02) in the spleen. In the severe scl-cGVHD model, imatinib-treated mice had significantly lower levels of PDGF-r phosphorylation than control mice on day 29 after transplantation (P = 0.008). However, scl-cGVHD scores were similar between vehicle- and imatinib-treated mice during the whole experiment, while there was a suggestion for less weight loss in imatinib-treated mice that reached statistical significance at day +52 following transplantation (P = 0.02). CONCLUSIONS: Imatinib had a limited impact in murine scl-cGVHD despite significant inhibition of PDGF-r.

Novel approaches for preventing acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (alloHSCT) offers potential curative treatment for a wide range of malignant and nonmalignant hematological disorders. However, its success may be limited by post-transplant acute graft-versus-host disease (aGVHD), a systemic syndrome in which donor's immune cells attack healthy tissues in the immunocompromised host. aGVHD is one of the main causes of morbidity and mortality after alloHSCT. Despite standard GVHD prophylaxis regimens, aGVHD still develops in approximately 40-60% of alloHSCT recipients. AREAS COVERED: In this review, after a brief summary of current knowledge on the pathogenesis of aGVHD, the authors review the current combination of a calcineurin inhibitor with an antimetabolite with or without added anti-thymocyte globulin (ATG) and emerging strategies for GVHD prevention. EXPERT OPINION: A new understanding of the involvement of cytokines, intracellular signaling pathways, epigenetics and immunoregulatory cells in GVHD pathogenesis will lead to new standards for aGVHD prophylaxis allowing better prevention of severe aGVHD without affecting graft-versus-tumor effects.

Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease.

BACKGROUND: Previous studies have demonstrated that regulatory T cells (Tregs) play a protective role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Tregs constitutively express the gene of the transcription factor Foxp3 whose CNS2 region is heavily methylated in conventional CD4(+) T cells (CD4(+)Tconvs) but demethylated in Tregs. METHODS: Here, we assessed the impact of azacytidine (AZA) on cGVHD in a well-established murine model of sclerodermic cGVHD (B10.D2 (H-2d) → BALB/cJ (H-2d)). RESULTS: The administration of AZA every 48 h from day +10 to day +30 at the dose of 0.5 mg/kg or 2 mg/kg mitigated chronic GVHD. Further, AZA-treated mice exhibited higher blood and thymic Treg frequencies on day +35, as well as higher demethylation levels of the Foxp3 enhancer and the IL-2 promoter in splenocytes at day +52. Interestingly, Tregs from AZA-treated mice expressed more frequently the activation marker CD103 on day +52. AZA-treated mice had also lower counts of CD4(+)Tconvs and CD8(+) T cells from day +21 to day +35 after transplantation, as well as a lower proportion of CD4(+)Tconvs expressing the Ki67 antigen on day +21 demonstrating an anti-proliferating effect of the drug on T cells. CONCLUSIONS: Our results indicate that AZA prevented sclerodermic cGVHD in a well-established murine model of cGVHD. These data might serve as the basis for a pilot study of AZA administration for cGVHD prevention in patients at high risk for cGVHD.

Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets.

BACKGROUND: Regulatory T cells (Tregs) are key players in immune tolerance. They express the transcription factor FOXP3 and are dependent of the STAT5 signaling for their homeostasis. So far, the study of phosphorylated epitopes by flow cytometry required treating the cells with methanol, which is harmful for several epitopes. METHODS: Here we assessed whether the PerFix EXPOSE reagent kit (PFE)(Beckman Coulter) allowed monitoring the phosphorylation level of STAT5 in Treg subpopulations together with complex immunophenotyping. Results observed with the PFE kit were compared to those observed without cell permeabilization for surface markers, with paraformaldehyde permeabilization for non-phosphorylated intracellular epitopes, and with methanol-based permeabilization for phosphoSTAT5 staining. RESULTS: In human PBMCs, the PFE kit allowed the detection of surface antigens, FOXP3, KI67 and phosphoSTAT5 in similar proportions to what was observed without permeabilization (for surface antigens), or with PFA or methanol permeabilizations for FOXP3/KI67 and phosphoSTAT5, respectively. Comparable observations were made with murine splenocytes. Further, the PFE kit allowed determining the response of different human and murine Treg subsets to IL-2. It also allowed demonstrating that human Treg subsets with the highest levels of phosphoSTAT5 had also the highest suppressive activity in vitro, and that anti-thymocyte glogulin (ATG) induced Treg independently of the STAT5 pathway, both in vitro and in vivo. CONCLUSIONS: We have validated a multicolor staining method that allows monitoring phosphoSTAT5 levels in Treg subsets. This staining could be useful to monitor responses of various Treg subsets to IL-2 therapy.

Impact of Pre-Transplant Anti-T Cell Globulin (ATG) on Immune Recovery after Myeloablative Allogeneic Peripheral Blood Stem Cell Transplantation.

BACKGROUND: Pre-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. METHODS: In 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. RESULTS: Pre-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. CONCLUSIONS: Pre-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.

Immune Recovery after Allogeneic Hematopoietic Stem Cell Transplantation Following Flu-TBI versus TLI-ATG Conditioning.

PURPOSE: A conditioning regimen for allogeneic hematopoietic cell transplantation (HCT) combining total lymphoid irradiation (TLI) plus anti-thymocyte globulin (ATG) has been developed to induce graft-versus-tumor effects without graft-versus-host disease (GVHD). EXPERIMENTAL DESIGN: We compared immune recovery in 53 patients included in a phase II randomized study comparing nonmyeloablative HCT following either fludarabine plus 2 Gy total body irradiation (TBI arm, n = 28) or 8 Gy TLI plus ATG (TLI arm, n = 25). RESULTS: In comparison with TBI patients, TLI patients had a similarly low 6-month incidence of grade II-IV acute GVHD, a lower incidence of moderate/severe chronic GVHD (P = 0.02), a higher incidence of CMV reactivation (P < 0.001), and a higher incidence of relapse (P = 0.01). While recovery of total CD8(+) T cells was similar in the two groups, with median CD8(+) T-cell counts reaching the normal values 40 to 60 days after allo-HCT, TLI patients had lower percentages of naïve CD8 T cells. Median CD4(+) T-cell counts did not reach the lower limit of normal values the first year after allo-HCT in the two groups. Furthermore, CD4(+) T-cell counts were significantly lower in TLI than in TBI patients the first 6 months after transplantation. Interestingly, while median absolute regulatory T-cell (Treg) counts were comparable in TBI and TLI patients, Treg/naïve CD4(+) T-cell ratios were significantly higher in TLI than in TBI patients the 2 first years after transplantation. CONCLUSIONS: Immune recovery differs substantially between these two conditioning regimens, possibly explaining the different clinical outcomes observed (NCT00603954).

Monoclonal antibodies against GARP/TGF-ß1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo.

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-ß1 (TGF-ß1). On the Treg cell surface, TGF-ß1 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-ß1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-ß1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-ß1 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.

Thinking out of the box--new approaches to controlling GVHD.

Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic cell transplantation (allo-HCT). Despite major advances in the understanding of GVHD pathogenesis, standard GVHD prophylaxis regimens continue to be based on the combination of a calcineurin inhibitor with an antimetabolite, while first line treatments still rely on high-dose corticosteroids. Further, no second line treatment has emerged thus far in acute or chronic GVHD patients who failed to respond with corticosteroid treatment. After briefly reviewing current standards of GVHD prevention and treatment, this article will discuss recent approaches that might change GVHD prophylaxis/treatment for decades to come, with a special focus on recently developed immunoregulatory strategies based on infusion of mesenchymal stromal or regulatory T-cells, or injection of low-dose interleukin-2.

Establishment of a murine graft-versus-myeloma model using allogeneic stem cell transplantation.

BACKGROUND: Multiple myeloma (MM) is a malignant plasma cell disorder with poor long-term survival and high recurrence rates. Despite evidence of graft-versus-myeloma (GvM) effects, the use of allogeneic hematopoietic stem cell transplantation (allo-SCT) remains controversial in MM. In the current study, we investigated the anti-myeloma effects of allo-SCT from B10.D2 mice into MHC-matched myeloma-bearing Balb/cJ mice, with concomitant development of chronic graft-versus-host disease (GvHD). METHODS AND RESULTS: Balb/cJ mice were injected intravenously with luciferase-transfected MOPC315.BM cells, and received an allogeneic (B10.D2 donor) or autologous (Balb/cJ donor) transplant 30 days later. We observed a GvM effect in 94% of the allogeneic transplanted mice, as the luciferase signal completely disappeared after transplantation, whereas all the autologous transplanted mice showed myeloma progression. Lower serum paraprotein levels and lower myeloma infiltration in bone marrow and spleen in the allogeneic setting confirmed the observed GvM effect. In addition, the treated mice also displayed chronic GvHD symptoms. In vivo and in vitro data suggested the involvement of effector memory CD4 and CD8 T cells associated with the GvM response. The essential role of CD8 T cells was demonstrated in vivo where CD8 T-cell depletion of the graft resulted in reduced GvM effects. Finally, TCR Vß spectratyping analysis identified Vß families within CD4 and CD8 T cells, which were associated with both GvM effects and GvHD, whereas other Vß families within CD4 T cells were associated exclusively with either GvM or GvHD responses. CONCLUSIONS: We successfully established an immunocompetent murine model of graft-versus-myeloma. This is the first murine GvM model using immunocompetent mice that develop MM which closely resembles human MM disease and that are treated after disease establishment with an allo-SCT. Importantly, using TCR Vß spectratyping, we also demonstrated the presence of GvM unique responses potentially associated with the curative capacity of this immunotherapeutic approach.