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.

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.

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.

Neuronal surface antibody-mediated encephalopathy as manifestation of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Although it remained controversial for a long time, central nervous system (CNS) involvement of graft-versus-host disease (GVHD) is now becoming recognized as a real nosological entity. Previous case reports have suggested heterogeneous clinical presentations and it is not excluded that the whole spectrum of manifestations has not yet been fully described. Here, we report the case of a 58-year-old man with chronic GVHD who developed a rapidly ingravescent encephalopathy. There was no evidence for CNS immune-mediated lesions on conventional imaging nor for cellular infiltration in the cerebrospinal fluid. Serum analyses revealed the presence of anti-neuronal antibodies directed against anti-contactin-associated protein 2 (anti-Caspr2), a protein associated with voltage-gated potassium neuronal channels. Functional imaging with 2-deoxy-2-[fluorine-18] fluoro- d-glucose integrated with computed tomography (18F-FDG PET-CT) demonstrated diffuse cortical and subcortical hypometabolism. The patient was treated with a combination of immunosuppressive agents (corticosteroids, cyclophosphamide and rituximab) and progressively recovered normal neurocognitive functions. Taken together, these data suggest that CNS-GVHD may manifest as a reversible antibody-mediated functional encephalopathy. This report suggests for the first time the interest of screening for anti-neuronal antibodies and functional imaging with brain 18F-FDG PET-CT in diagnosing this severe complication of allogeneic hematopoietic cell transplantation (alloHSCT).

Bim regulates the survival and suppressive capability of CD8+ FOXP3+ regulatory T cells during murine GVHD.

CD8+ Foxp3+ T cells (Tregs) are a potent regulatory population whose functional and ontological similarities to CD4+ Fox3+ T cells have not been well delineated. Using an experimental model of graft-versus-host disease (GVHD), we observed that CD8+ Tregs were significantly less potent than CD4+ Tregs for the suppression of GVHD. To define the mechanistic basis for this observation, we examined the T-cell repertoire and the transcriptional profile of in vivo-derived CD4+ and CD8+ Tregs that emerged early during this disease. Polyclonal and alloantigen-induced CD8+ Tregs had repertoire diversity that was similar to that of conventional CD8+ T cells, indicating that a restricted repertoire was not the proximate cause of decreased suppression. Transcriptional profiling revealed that CD8+ Tregs possessed a canonical Treg transcriptional signature that was similar to that observed in CD4+ Tregs, yet distinct from conventional CD8+ T cells. Pathway analysis, however, demonstrated that CD8+ Tregs had differential gene expression in pathways involved in cell death and survival. This was further confirmed by detailed mRNA sequence analysis and protein expression studies, which demonstrated that CD8+ Tregs had increased expression of Bim and reduced expression of Mcl-1. Transplantation with CD8+ Foxp3+ Bim-/- Tregs resulted in prolonged Treg survival and reduced GVHD lethality compared with wild-type CD8+ Tregs, providing functional confirmation that increased expression of Bim was responsible for reduced in vivo efficacy. Thus, Bim regulates the survival and suppressive capability of CD8+ Tregs, which may have implications for their use in regulatory T-cell therapy.

Host interleukin 6 production regulates inflammation but not tryptophan metabolism in the brain during murine GVHD.

Graft-versus-host disease (GVHD) induces pathological damage in peripheral target organs leading to well-characterized, organ-specific clinical manifestations. Patients with GVHD, however, can also have behavioral alterations that affect overall cognitive function, but the extent to which GVHD alters inflammatory and biochemical pathways in the brain remain poorly understood. In the current study, we employed complementary murine GVHD models to demonstrate that alloreactive donor T cells accumulate in the brain and affect a proinflammatory cytokine milieu that is associated with specific behavioral abnormalities. Host IL-6 was identified as a pivotal cytokine mediator, as was host indoleamine 2,3-dioxygenase (IDO-1), which was upregulated in GVHD in an IL-6-dependent manner in microglial cells and was accompanied by dysregulated tryptophan metabolism in the dorsal raphe nucleus and prefrontal cortex. Blockade of the IL-6 signaling pathway significantly reduced donor T cell accumulation, inflammatory cytokine gene expression, and host microglial cell expansion, but did not reverse GVHD-induced tryptophan metabolite dysregulation. Thus, these results indicate that inhibition of IL-6 signaling attenuates neuroinflammation, but does not reverse all of the metabolic abnormalities in the brain during GVHD, which may also have implications for the treatment of neurotoxicity occurring after other T cell-based immune therapies with IL-6-directed approaches.

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.

A colitogenic memory CD4+ T cell population mediates gastrointestinal graft-versus-host disease.

Damage to the gastrointestinal tract is a major cause of morbidity and mortality in graft-versus-host disease (GVHD) and is attributable to T cell-mediated inflammation. In this work, we identified a unique CD4+ T cell population that constitutively expresses the ß2 integrin CD11c and displays a biased central memory phenotype and memory T cell transcriptional profile, innate-like properties, and increased expression of the gut-homing molecules α4ß7 and CCR9. Using several complementary murine GVHD models, we determined that adoptive transfer and early accumulation of ß2 integrin-expressing CD4+ T cells in the gastrointestinal tract initiated Th1-mediated proinflammatory cytokine production, augmented pathological damage in the colon, and increased mortality. The pathogenic effect of this CD4+ T cell population critically depended on coexpression of the IL-23 receptor, which was required for maximal inflammatory effects. Non-Foxp3-expressing CD4+ T cells produced IL-10, which regulated colonic inflammation and attenuated lethality in the absence of functional CD4+Foxp3+ T cells. Thus, the coordinate expression of CD11c and the IL-23 receptor defines an IL-10-regulated, colitogenic memory CD4+ T cell subset that is poised to initiate inflammation when there is loss of tolerance and breakdown of mucosal barriers.

Blockade of interleukin-27 signaling reduces GVHD in mice by augmenting Treg reconstitution and stabilizing Foxp3 expression.

Reestablishment of competent regulatory pathways has emerged as a strategy to reduce the severity of graft-versus-host disease (GVHD), and recalibrate the effector and regulatory arms of the immune system. However, clinically feasible, cost-effective strategies that do not require extensive ex vivo cellular manipulation have remained elusive. In the current study, we demonstrate that inhibition of the interleukin-27p28 (IL-27p28) signaling pathway through antibody blockade or genetic ablation prevented lethal GVHD in multiple murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cell-derived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology.

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.

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.

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.

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.

Imatinib and nilotinib inhibit hematopoietic progenitor cell growth, but do not prevent adhesion, migration and engraftment of human cord blood CD34+ cells.

BACKGROUND: The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34(+) cells. DESIGN AND METHODS: After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice. RESULTS: TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34(+) cell cycle entry. Adhesion of CD34(+) cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT. CONCLUSIONS: These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.