Clinical Graft-versus-host Disease Research Articles

Helper T-Cell Differentiation in Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapeutic option for many malignant diseases. However, the efficacy of allo-HSCT is limited by the occurrence of destructive graft-versus-host disease (GVHD). Since allogeneic T cells arethe driving force in the development of GVHD, their activation, proliferation, and differentiation are key factors to understanding GVHD pathogenesis. This review focuses on one critical aspect: the differentiation and function of helper T (Th) cells in acute GVHD. We first summarize well-established subsets including Th1, Th2, Th17, and T-regulatory cells; their flexibility, plasticity, and epigenetic modification; and newly identified subsets including Th9, Th22, and T follicular helper cells. Next, we extensively discuss preclinical findings of Th-cell lineages in GVHD: the networks of transcription factors involved in differentiation, the cytokine and signaling requirements for development, the reciprocal differentiation features, and the regulation of microRNAs on T-cell differentiation. Finally, we briefly summarize the recent findings on the roles of T-cell subsets in clinical GVHD and ongoing strategies to modify T-cell differentiation for controlling GVHD in patients. We believe further exploration and understanding of the immunobiology of T-cell differentiation in GVHD will expand therapeutic options for the continuing success of allo-HSCT.

Mouse Flk-1+Sca-1- Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) represent a powerful tool in regenerative medicine because of their differentiation and migration capacities. Moreover, the immunomodulatory ability of MSCs may be used to develop therapies for the treatment of autoimmune diseases. In this study, we isolated Flk-1Sca-1 MSCs from bone marrow (bMSCs). Next, we studied their biological characteristics and immunologic functions. We also investigated their effects on graft-versus-host disease (GVHD) associated with allogeneic bone marrow transplantation in mice. Flk-1Sca-1 bMSCs were able to differentiate into fat and cartilage cells, indicating that the isolated cells had stem cell properties. They could also suppress alloantigen-induced T cell proliferation in vitro in a dose-dependent manner. Infusion of bMSCs into allogeneic bone marrow-transplanted mice alleviated the lethal GVHD that occurred in control recipient mice. There was significantly lower mortality among the recipients of the Flk-1Sca-1 bMSCs that also ameliorated the clinical symptoms and GVHD histopathology. Beneficial effects on GVHD by Flk-1Sca-1 bMSCs were also observed when MSCs were engineered to express anti-inflammatory cytokines IL-4 and IL-10 and decrease expression of proinflammatory cytokines IFN-γ, TNF-α, and IL-2. Flk-1Sca-1 bMSCs have stem cell properties and can efficiently ameliorate the GVHD associated with allogeneic bone marrow transplantation in mice.

Defining The Mechanism Involved In The Inhibition Of GvHD By Azacytidine In Vivo Through The Use Of FoxP3 Diphtheria Toxin Receptor (Foxp3DTR) Donor T Cells

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired Graft vs. Leukemia (GvL) effect is frequently accompanied by Graft vs. Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression (Choi et al 2010) or, 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells (Sánchez-Abarca et al. 2010).To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT) (Kim et al. 2007). To induce GvHD, either 1x107 panT or 1x107 Treg-depleted panT cells obtained from Foxp3DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16, +18, +20, (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery.Mice transplanted with panT cells and injected with PBS (n=10) or DT (n=10) (without azaC) developed severe GvHD and died by day ∼26 post HSCT. In sharp contrast, 90% of mice (n=10) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC+DT (n=10) resulted in significantly prolonged survival compared to the PBS treated mice (p=<0.001) but in contrast to the azaC group, most failed to survive beyond day 60 (p=<0.001, Fig 1a). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=<0.001), CD3+ T cells (p=<0.001), and B220+ B cells (p=0.04) than the azaC+DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. In separate studies, mice transplanted with panT cells depleted of natural Tregs upfront (using anti-CD25 antibody), lost weight, developed GvHD and failed to survive, even when treated with azaC alone (Fig. 1b). These results suggest that the GvHD sparing effect of azaC is dependent on both the conversion of CD4+ T effectors to Tregs and the presence of natural Tregs, and eliminating this population with DT results in greater GvHD and reduced survival. Further experiments are ongoing to determine the relative importance of both natural Tregs and azaC generated Tregs to prevent GvHD in this mouse model. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

BB02 Improves Therapeutical Effectiveness Of Extracorporeal Photopheresis With 8-MOP In a Murine Model Of Graft-Versus-Host Disease

IntroductionExtracorporeal photopheresis (ECP) is a cell-based immunomodulatory therapy involving the separation of peripheral blood autologous mononuclear cells followed by ex-vivo administration of 8-methoxypsoralen (8-MOP) and UVA radiation before reinfusion. ECP is efficient for the treatment of multiple diseases mediated by unregulated T cell populations, such as cutaneous T cell lymphoma, autoinmune diseases or graft-versus-host disease (GVHD), the major complication after allogeneic bone marrow transplantation. Our aim in the present work was to compare the therapeutic effectiveness of 8-MOP with other two new compounds (BB01 and BB02) in a experimental murine model of GVHD. MethodsMurine GVHD was induced after transplanting bone marrow cells and splenocytes from donor Balb/c mice into C57Bl6J recipients previously conditioned with a lethal dose of 10 Gy split into two doses of 5 Gy spaced 24 hours apart. To investigate the therapeutic effectiveness of ECP with either 8-MOP, BB01 or BB02, splenocytes from separate cohorts of C57Bl6J with GVHD were isolated 12 days after transplantation, incubated with the different compounds, irradiated with UVA light and injected intravenously once a week for four weeks. Survival after transplantation was monitored daily and clinical GVHD was graded using a previously described score analyzing weight loss, posture (hunching), activity, skin integrity and fur texture (Cooke et al, 1996). Mice of each group were evaluated and graded from 0 to 2 for each criterion, obtaining a clinical index by summation of the five criteria scores (maximum index=10). ResultsMice treated weekly with BB02 showed a significant higher survival than those treated with 8-MOP (p=0,038), while BB01 had a similar effect to that of 8-MOP. Mice treated with either compound improved their clinical GVHD score compared to untreated mice group, being significantly lower with BB02 than with BB01 and 8-MOP (p=0,023). ConclusionsBB02 was more efficient than 8-MOP in the reversal of murine GVHD, while BB01 showed the same therapeutic effectiveness than 8-MOP. AcknowledgmentsWork financed by the Spanish Ministry of Science and Innovation (Ref: BFU2010-19599) and the Spanish Net of Cell Therapy (TerCel) from Institute of Health Carlos III. Disclosures:No relevant conflicts of interest to declare.

Fatty Acid Metabolism Provides a Potential Therapeutic Target To Treat Graft-Versus-Host Disease

The metabolism of lymphocytes activated in vivo remains poorly understood. Previous work has demonstrated that T cells activated during graft-versus-host disease (GVHD) adopt metabolic profiles distinct from other cell types. We hypothesized that a deeper understanding of allogeneic T cell metabolism, followed by exploitation of metabolic differences, might allow selective elimination of pathogenic T cells while preserving normal immune responses following bone marrow transplantation (BMT). We tested this hypothesis by evaluating fatty acid (FA) metabolism in proliferating donor T cells during a parent into F1 model of GVHD (C57Bl/6 into B6D2F1). Compared to naive donor T cells, T cells from allogeneic recipients increased FA transport (44.3 ±6.9% vs. 0.7 ±0.1%, p=0.003) and up-regulated a regulator of oxidative metabolism, PGC-1α (0.9 ±0.2 vs. 0.03 ±0.01, p=0.005). These changes were present in T cells recovered from both the liver and the spleen. Allogeneic T cells also up-regulated mRNA and protein levels for FA oxidation enzymes (e.g. CPT1a) and oxidized more fatty acids ex vivo. We confirmed these changes in a second, minor histocompatibility model of GVHD, where we observed significantly increased levels of FA transport, PGC-1α, and FA oxidation enzymes.To identify potential therapeutic targets selective for alloreactive T cells, we compared the metabolic profile of allogeneic T cells to the profile observed in T cells following acute activation in vivo. We injected OT-I T cells into naïve C57Bl/6 mice, challenged mice one day later with ovalbumin-bearing dendritic cells, and harvested T cells 7 days after immunization. OT-I T cells proliferated robustly to cellular immunization, but did not increase FA transport or levels of PGC-1α (Figure 1A,B). We then tested the ability of inhibitors of FA oxidation (targeting CPT1a) to selectively eliminate allogeneic T cells during GVHD. A single dose of etomoxir decreased the total number of donor T cells in allogeneic recipients by 35% and eight doses over 14 days significantly improved clinical GVHD scores (Figure 1C). Treatment with a second CPT1a inhibitor improved post-transplant survival (78% vs. 50%, inhibitor vs. PBS respectively). Importantly, CPT1a inhibition did not diminish the number of regulatory T cells or the number of T cells reconstituting via homeostatic proliferation following syngeneic BMT. In total, these data demonstrate that allogeneic T cells increase FA metabolism during GVHD and that this phenotype differentiates allogeneic cells from T cells responding to acute activation or proliferating during homeostatic reconstitution. This study challenges the paradigm of effector lymphocyte metabolism in vivo and we conclude that inhibition of FA oxidation may be a selective way to eliminate pathogenic T cells causing immune-mediated disease. [Display omitted] Disclosures:Ferrara:University of Michigan: Patent for GVHD Biomarkers, Patent for GVHD Biomarkers Patents & Royalties.

Graft-Versus-Host-Disease Recruits Both Monocyte and Pre-Conventional Dendritic Cell-Derived Tissue Antigen Presenting Cells Independent Of CCR2 and CCR6

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and in promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs, including dendritic cells (DCs), in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells, suggesting they have a role in local GVHD reactions. Donor-derived tissue APCs (t-APCs), including tissue-DCs (t-DCs) could activate donor T cells through indirect or cross-presentation of host antigens, produce chemokines that recruit other effectors, and elaborate inflammatory mediators or suppressors of inflammation. We first characterized t-DC subsets in the skin and bowel of GVHD-affected mice. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) BM with or without CD4+ and CD8+ T cells to induce GVHD and analyzed mononuclear cells from skin and bowel approximately 4 weeks post transplant. In skin, both main dermal DC populations (CD11b+ and CD103+) were significantly increased in GVHD mice as compared to BM alone controls, though the ratios of CD11b+: CD103+ DCs were similar. In the bowel lamina propria, the ratios of CD11b+CD103- to CD11b+CD103+ were increased in GVHD mice in the colon but were similar to that in BM alone controls in the small bowel. We next studied the roles of CCR6 and CCR2 in the recruitment of donor-derived APCs to skin and bowel. We transplanted mice with CCR6-/- BM in competition with wild type (wt) BM and found that the contribution of each to skin and bowel APCs matched their contributions to myeloid hematopoiesis in BM, spleen and blood, indicating that CCR6 is not required. To study the role of CCR2 we first compared mice transplanted with either wt or CCR2-/- BM with wt T cells. Despite having a profound reduction in blood monocytes, all skin and bowel t-APC subsets were present in CCR2-/- recipients, indicating that CCR2 is not required for t-APC recruitment in contrast to its role in many other models of inflammation. However, CD103+ DCs were more prevalent relative to CD11b+ DCs, consistent with a pre-cDC origin. Despite monocytopenia, recipients of CCR2-/- BM developed clinical GVHD; histology data is being analyzed and will be presented. To better define the contributions of CCR2 to t-APC recruitment and to determine monocyte versus pre-cDC origin of t-DCs, we transplanted mice with CCR2-/- BM in competition with wt BM and compared ratios of BM and blood precursors (pre-cDCs and monocytes) to t-DC ratios. For CD103+ DCs, wt/KO ratios matched the ratios of general myeloid hematopoiesis and pre-cDCs, indicating a pre-cDC origin. For CD11b+CD103- DCs, the ratio of wt/KO matched that in blood monocytes. We further subsetted CD11b+ t-DCs based on the expression of Ly6C, MAR1, CD64 and CD24, used to differentiate pre-cDC from mono-derived DCs in other organs, and did not identify any population with wt/KO ratios that did not match that of the general CD11b+ DC population, suggesting that most if not all CD11b+ t-DCs are of monocyte origin. Experiments are underway examining the role of CX3CR1 in t-APC recruitment and these data will be presented. Disclosures: No relevant conflicts of interest to declare.

Close To Clinics: Acute Gvhd In a Novel Chemotherapy Based Minor Mismatch Transplantation Model

GVHD (graft-vs-host disease) is one of the major complications after allogeneic hematopoietic stem cell transplantation leading to substantial morbidity and mortality. Several murine models are used to investigate this systemic inflammatory disease in vivo and to identify possible targets for clinical treatment. However, current well-established murine GVHD models have their clinical limitations as they are based on major histocompatibility mismatches and only lethal irradiation as conditioning. Development of new treatment options would be forwarded by the availability of a more clinically relevant murine model.To reflect the clinical situation of transplantation and GVHD, we established a new murine GVHD model which is based on Chemotherapy conditioning and MHC-matched bone marrow transplantation (BMT). In LP/J -> C57BL/6 mouse model, our chemotherapy transplantation protocol resulted in stable full donor chimerism and development of acute GVHD.C57BL/6 recipients were conditioned with a 7-day busulfan-cyclophosphamide Chemotherapy protocol. Mice received for 5 days 20 mg/kg/day busulfan intraperitoneally (i.p.) and the last three days additionally 100 mg/kg/day cyclophosphamide. After a two-day resting phase, recipients were transplanted with 1.5 x 107 bone marrow and 2 x 106 splenic T cells from LP/J donors. Recipients showed good engraftment with already 80 % engraftment in peripheral blood at day 15 after BMT. 90 % donor chimerism in peripheral blood and bone marrow could be shown already at day 30 after BMT.LP/J and C75BL/6 mice share the same haplotype (H2b) with only mismatches at different minor histocompatibility loci. As in clinical BMT, GVHD still arises after MHC-matched transplantation because of multiple minor mismatches. We could show in our LP -> C57BL/6 model typical features of acute GVHD. Allogeneic transplanted animals showed significant decreased survival compared to syngeneic transplanted animals (Figure 1A). GVHD animals exhibited GVHD-typical weight loss (Figure 1B) and a significant increased clinical GVHD index consisting of five clinical parameters: weight loss, posture, activity, fur and skin (Figure 1C). Our model showed a strong skin phenotype, especially between day 15 and 25 after BMT extensive scurf spread over the whole body combined with fur loss. Pronounced weight loss and highest clinical GVHD index were monitored between day 15 and 25, implicating this period as acute phase of GVHD. Histopathologic analysis of GVHD organs (large bowel, liver and skin) confirmed acute GVHD symptoms in our model as histopathological scores after Lerner (1974,Transplant Proc 6:367-371) were significantly increased in GVHD animals compared to BMT recipients without GVHD (Figure 1D). [Display omitted] We next characterized systemic inflammation in our model by determining the number of circulating inflammatory cells in peripheral blood at different time points by flow cytometry. CD8+ T cells were significantly increased in GVHD animals in acute phase (day 15 after BMT), whereas CD4+ T cells were elevated in late phase (day 50-60 after BMT). These data suggests, that in our model two inflammatory phases occur, an early acute CD8-driven and a late CD4-driven phase.Finally, we could demonstrate GVHD-typical T cell infiltration in GHVD target organs in acute phase (Figure 2). In large and small bowel, liver and skin CD4 and CD8 infiltration was significantly elevated in GVHD animals, however, CD8 infiltration was more prominent. We conclude that CD8 T cells play a prominent role in acute phase of GVHD in our model. [Display omitted] In summary, we could establish a novel chemotherapy based minor mismatch GVHD model which shows good engraftment, typical features of acute GVHD and systemic and target organ specific inflammation. Our murine GVHD model closely resembles the clinical situation of MHC-matched stem cell transplantation and may help to better understand pathogenic mechanisms in GVHD and develop new treatment options. Disclosures:No relevant conflicts of interest to declare.

JAK1/2 Inhibition Suppresses T Cell Proliferation and Inflammatory Cytokine Production In An Allogeneic Setting and Ameliorates Graft-Versus-Host Disease (GvHD) In a Murine GvHD Model

Graft-versus-host-disease (GvHD) constitutes a severe complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). In GvHD, tissue damage is mediated by pro-inflammatory cytokines. Cytokine responses are mediated by activated Janus kinases (JAKs). We hypothesized that JAK1/2 inhibition might reduce T effector cell responses and inflammatory cytokine production in an allogeneic system, thereby ameliorating GvHD. We established an allogeneic (major mismatch) cell culture system using naive BALB/c CD4+ CD62Lhigh T cells that were co-cultured with C57BL/6 (B6) bone marrow derived dendritic cells (DC). JAK 1/2 signaling was specifically blocked using the small molecule inhibitor Ruxolitinib (INCB018424). By using a 3H Thymidine proliferation assay, we found that Ruxolitinib was able to inhibit the proliferation of allogeneic CD4+ effector T cells. In our intracellular cytokine staining experiments Ruxolitinib was able to impair the differentiation of naïve T cells into IFN-gamma and IL17A-producing T effector cells. Both cytokines – IFN-gamma and IL-17A – have been linked to aggravated courses of GvHD severity.In vivo administration of Ruxolitinib signficantly reduced lethal GvHD after allo-HSCT in a murine major mismatch model. BALB/c recipient mice were irradiated and received C57BL/6 (B6) T cell depleted bone marrow along with CD4+ and CD8+ T cells. Animals that were treated with an oral gavage of Ruxolitinib twice daily displayed significantly lower serum levels of IFN-gamma, TNF-alpha, IL-10 and IL-12p70, lower histological and clinical GvHD scores and improved overall survival compared to the control group.By using luciferase-positive (luc+) CD4+ and CD8+ T cells, we were able to visualize and quantify T cell migration after GvHD induction. The recipient mice received luc+ T cells along with conventional T cell depleted bone marrow. We were able to detect the luc+ T cell signal with a bioluminescence imaging system. At day 9 after allogeneic bone marrow transplantation, migration of donor T cells to GvHD target sites was significantly reduced in Ruxolitinib treated mice compared to control mice.In summary, our data demonstrate that suppression of inflammatory cytokine responses by JAK1/2 inhibitors modulates T effector cell responses in an allogeneic setting and improves survival in a murine major mismatch GvHD model. These observations suggest that JAK1/2 inhibition might be used for the treatment of GvHD following allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

NKT cells and graft-versus-host disease-review

NKT cells (nature killer T cells), as a regulatory cellular compartment in the immune system, express cell surface markers of T cells and NK cells. It secretes a variety of cytokines that stimulate specific antigens. Through regulating the balance of Th1/Th2, the NKT cells play an important role in prevention and treatment of graft-versus-host disease (GVHD). Its antitumor and anti-infectious effects serve as a basis of its application in allogeneic hematopoietic stem cell transplantation. A better understanding of the biological and immunological features of NKT cell, as well as its specific immune regulatory mechanisms, will further justify the rationales of using NKT cells in the management of GVHD for patients. In this review, the biologic properties, classification, differentiation and development, immune activation of NKT cells as well as the NKT cells and GVHD including the related mechanisms of prevention and treatment of GVHD with NKT cells, NKT cells and tumors, NKT cells and infection, and NKT cells and clinical GVHD are summarized.

Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation

Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared.

Low Paneth cell numbers at onset of gastrointestinal graft-versus-host disease identify patients at high risk for nonrelapse mortality

AbstractAcute graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is an often lethal complication of allogeneic hematopoietic cell transplant. Clinical severity correlates with outcomes, but histopathologic grading is primarily used to confirm the clinical diagnosis. One barrier to using histopathologic grading to predict clinical outcomes is inter-grader variability among transplant centers. Recent experimental models have shown that the loss of Paneth cells, which are located in the small intestine and help regulate the GI microbiome by secreting antimicrobial peptides, correlates with clinical GVHD severity. Because Paneth cells are easy to identify and quantify by light microscopy, we evaluated the mean number of Paneth cells per high-powered field (hpf) in 116 duodenal biopsies obtained at diagnosis of GI GVHD at 2 different centers with their clinical outcomes. Paneth cell counts were reproducible between centers (r2 = 0.81; P < .0001). Lower numbers of Paneth cells at diagnosis correlated with clinically more severe GI GVHD (P < .0001) and less likelihood of response to GVHD treatment (P < .0001). A threshold of 4 Paneth cells per hpf stratified patients according to nonrelapse mortality (28% vs 56%; P = .004). We conclude that the enumeration of duodenal Paneth cells is a readily available index of disease severity that provides important information regarding GVHD prognosis.

Fluoxetine Reduces Murine Graft-Versus-Host Disease by Induction of T cell Immunosuppression

Serotonin reuptake inhibitors (SRIs) are widely used drugs in the treatment of depression and anxiety disorders. Although SRIs are generally regarded as safe drugs with relatively few side effects, literature suggests that high concentrations of SRIs may alter immune function. We investigated whether high-dose treatment with fluoxetine was able to suppress acute graft-versus-host disease (GvHD) in a MHC-matched, minor histocompatibility antigen mismatched murine bone marrow transplantation model. We found that high doses fluoxetine induce a significant reduction of clinical symptoms and increase survival of these animals. The amelioration of clinical GvHD was accompanied by a reduced expansion of alloreactive T cells. We further analyzed the direct in vitro effect of six SRIs on the viability and proliferation of human T cells and found an anti-proliferative and pro-apoptotic effect that was significantly larger in activated than in resting T cells. We discuss these results in the light of potential future exploration of SRIs as a novel class of T cell immunosuppressive drugs.

Dissecting graft‐versus‐leukemia from graft‐versus‐host‐disease using novel strategies

The intrinsic anti-leukemic effect of allogeneic hematopoietic cell transplantation (HCT) is dependent on genetic disparity between donor and recipient, intimately associated with graft-versus-host disease (GVHD), and mediated by lymphocytes contained in or derived from the donor hematopoietic cell graft. Three decades of intense effort have not identified clinical strategies that can reliably separate the graft-versus-leukemia (GVL) effect from the alloimmune reaction that drives clinical GVHD. For patients who require HCT and for whom two or more human leukocyte antigen (HLA)-A, -B, -C, and -DRB1-matched donor candidates can be identified, consideration of donor and recipient genotype at additional genetic loci both within and outside the major histocompatibility complex may offer the possibility of selecting the donor [candidate(s)] that poses the lowest probability of GVHD and the highest probability of a potent GVL effect. Strategies for engineering conventional donor lymphocyte infusion also hold promise for prevention or improved treatment of post-transplant relapse. The brightest prospects for selectively enhancing the anti-leukemic efficacy of allogeneic HCT, however, are likely to be interventions that are designed to enhance specific antitumor immunity via vaccination or adoptive cell transfer, rather than those that attempt to exploit donor alloreactivity against the host. Adoptive transfer of donor-derived T cells genetically modified for tumor-specific reactivity, in particular, has the potential to transform the practice of allogeneic HCT by selectively enhancing antitumor immunity without causing GVHD.

A Preclinical Canine Model of in Utero Hematopoietic Cell Transplantation–Induced Graft Vs. Host Disease.

AbstractAbstract 3002 Introduction:In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allo-engraftment and associated donor-specific tolerance (DST) without toxic conditioning. To evaluate IUHCT in a clinically relevant large animal model, we have developed the haploidentical canine model of IUHCT. Recent optimization of the model using a CD3 depleted graft has achieved consistent and stable levels of mixed hematopoietic chimerism (average > 10%) without graft versus host disease (GVHD). However, since GVHD represents the greatest theoretical risk of clinical IUHCT, it is important to identify the phenotype and requirements for IUHCT-induced GVHD in the canine model prior to clinical application. In the current study, we attempted to establish a threshold dose of CD3+ cells required for IUHCT-induced GVHD in the haploidentical canine model by increasing the absolute percentage of CD3+ cells in the donor inoculum and we characterized the GVHD phenotype observed. Methods:43 fetuses of 9 pregnant bitches underwent ultrasound-guided intracardiac injection of maternal BM cells at 37.5–43 days gestational age (GA). In our large animal model, bone marrow yields, cellularity and litter size differ between dams. Due to these factors and because the targeted gestational age range coincides with an exponential growth phase for the dog fetus, standardization of cell dose per weight was not possible. Following MACS CD3 depletion, graft composition was standardized by adding back the CD3+ fraction to achieve donor CD3 content of 1%, 3%, 7.5%, and 16% (whole bone marrow [WBM] positive control) (Table 1). Live born puppies were monitored daily for signs of GVHD, including skin lesions, diarrhea, hematochezia, jaundice, weight loss and lethargy. Chimerism was measured by a PCR-based VNTR assay using commercially available primers. Results:Though prenatal survival of 75% was not significantly different than baseline (76%) and pups were not runted at birth, acute GVHD was observed postnatally in all ten live born recipients of either 7.5% CD3 cells or WBM, with onset ranging from 1–14 days of age. Recipients of grafts containing 1% and 3% CD3 cells showed no clinical evidence of GVHD.Weight loss was the earliest and most consistent clinical feature of IUHCT-induced GVHD in our model. Various affected animals manifested additional symptoms in all GVHD target organs, including increased respiratory rate/effort, hepatic dysfunction, bloody diarrhea and skin lesions. The phenotype was severe and all ten ultimately required euthanasia between 2 and 21 days of age.Necropsies performed during acute GVHD revealed significant erythematous and/or ulcerated skin change, hepatosplenomegaly, and punctate hemorrhage throughout the gastrointestinal tract and lung. Histology (H&E) demonstrated lymphocytic infiltrates and focal necrosis in target tissues as well as effacement of the dermal-epidermal junction and villous surfaces. Canalicular bile stasis was the most uniform and characteristic microscopic finding irrespective of hepatic functional status.Chimerism levels in pups who survived to birth were compared based upon CD3 content of the donor graft (Table 1). Increasing CD3 percentage trended toward an increase in chimerism, though only the WBM group reached statistical significance (p=.043). Conclusions:In the preclinical haploidentical canine model of IUHCT the threshold for induction of GVHD appears to be between 3% and 7.5% graft CD3+cell content. Interestingly, the absolute dose of CD3+ cells/kg fetal weight does not entirely correlate with the occurrence of GVHD, suggesting that the composition of the graft rather than the absolute T-cell content may determine the likelihood of GVHD. Given the complete absence of GVHD at a CD3 dose of 1% and the excellent engraftment achieved, it appears that there is a safe therapeutic index for achievement of chimerism without GVHD. When IUHCT-induced GVHD occurs, it is severe and clinically similar to postnatal GVHD. Notably, clinical GVHD only occurred after birth, suggesting suppressive mechanisms in utero. Additional studies are needed to further optimize the graft for support of engraftment without GVHD.Table 1DosenGA at IUHCTCD3 cells/kgCD34 cells/kgMean ChimerismGVHD1%2039–41.37–2.72 × 10 (9)5.8–58.2 × 10 (8)12.1%N3%4381.26 × 10 (9)9.8 × 10 (8)7.3%N7.5%341.52.26 × 10 (9)7.0 × 10 (8)17.7%Y16% (WBM)7432.0 × 10 (9)5.1 × 10 (8)20.3%Y Disclosures:No relevant conflicts of interest to declare.

Use of FoxP3 Diptheria Toxin Receptor (Foxp3DTR) Donor T Cells to Define the Mechanism Involved in the Inhibition of GvHD by Azacytidine in Vivo

AbstractAbstract 4113Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the primary treatment for high risk and relapsed hematological malignancies. However, the desired graft versus leukemia (GvL) effect is frequently accompanied by Graft versus Host Disease (GvHD) in which donor lymphocytes target host tissues, often resulting in significant morbidity and mortality. We and others have shown that the hypomethylating agent, 5-azacytidine (azaC), can mitigate GvHD in both murine preclinical transplant models and in human clinical trials while maintaining a robust GvL effect. Two mechanisms have been proposed to explain how azaC reduces GvHD: 1) the generation of suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) through the in vivo conversion of alloreactive donor T effectors (FoxP3-) into suppressive regulatory T cells (Tregs, CD4+CD25+Foxp3+) via the pharmacologic hypomethylation of the Foxp3 promoter resulting in enhanced gene expression or,1 2) the direct anti-proliferative and pro-apoptotic effects of azaC on allogeneic T cells.2To assess the importance of azaC-generated Tregs in ameliorating GvHD we obtained Foxp3DTR mice in which Tregs can be selectively ablated (>97%) following the administration of diphtheria toxin (DT).3 To induce GvHD, 1×107 panT cells obtained from Foxp3 DTR mice were injected (CD45.2+) via the lateral tail vein on day 11 post allo-HSCT (CD45.1+) into allogeneic recipients (Balb/c). Transplanted mice were treated with PBS (carrier) or azaC (2 mg/kg) (i.p.) on days +15, +17, +19, and +21, followed by injection (i.p.) of DT or PBS on days +16 +18 +20 (DT 10 μg/kg) and +22 (DT 50 μg/kg) to deplete azaC-induced Tregs. Complete depletion of Tregs by DT was confirmed by flow cytometery.Mice injected with PBS (n=5) or DT (n=5) (without azaC) developed severe GvHD and died by day 26 post HSCT. In sharp contrast, 100% of mice (n=5) treated with azaC alone survived >100 days, displayed no visible GvHD related symptoms, and gained weight at a similar rate as T cell depleted bone marrow controls. The treatment of mice with azaC +DT (n=5) resulted in significantly prolonged survival compared to the PBS treated mice (p=0.003) but in contrast to the azaC group, failed to survive beyond day 60 (p=0.003). Furthermore, the azaC alone group had higher percentages of Tregs (CD4+/CD25+/FoxP3+, p=0.001), CD3+ T cells (p=0.002), and B220+ B cells (p=0.02) than the azaC +DT group (consistent with less clinical GvHD) and less gastrointestinal GvHD when graded by an independent pathologist. These results suggest that BOTH pharmacologic conversion of alloreactive FoxP3- donor T cells to FoxP3-expressing Tregs and direct toxicity to allogeneic T cells by azaC represent important mechanisms involved in the amelioration of GvHD by azaC in vivo. Disclosures:No relevant conflicts of interest to declare.

Lentiviral-mediated ho-1 gene transduced bone mesenchymal stem cells protects against acute graft-Versus-host disease in vitro and vivo

Abstract 4675 Objective:Bone mesenchymal stem cells (BMSCs) possessing immunoregulatory activities have been evaluated in the treatment of graft-versus-host disease (GVHD). In this study, mice's heme oxygenase 1 (HO-1) was transduced into mice's bone marrow-derived mesenchymal stem cells (mBMSCs), we assessed the immuno-suppressive capacity of lentiviral vector transduced BMSCs expressing HO-1 in BALB/c mice aGVHD model, and the immuno-regulatory effect of mBMSCs on alleviating acute GVHD in vivo was measured to provide laboratory data for gene therapy for aGVHD which used mBMSCs as vehicles. Methods:We cloned mice's HO-1 cDNA from mice's bone marrow and constructed recombinant lentivirus vectors (Lentivirus-V5-D-TOPO-HO-1-EGFP/Lentivirus–V 5-D-TOPO-EGFP) which titer was 1×1011 pfu/mL. These mBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and the mRNA level of the neural ganglioside GD2 gene which is a surface marker for the identification of MSCs by RT-PCR. Then recombinant vectors were transferred into mBMSCs, and the expression of EGFP and HO-1 were detected by fluorescence microscope, RT-PCR and Western blot respectively. The immunsuppressive capacity of HO-1 over-expressing mBMSCs was investigated using transwell assay in vitro. And before infusion, the homing of MSC was detected by Flow cytometry. In addition, we established BALB/c mice's aGVHD model after Allo-HSCT, the recombinant vectors tranfected mBMSCs and primary mBMSCs were injected into the BALB/c mice aGVHD model in tail vein, respectively. Four groups were separated in vivo test (Group A: aGVHD control; Group B: aGVHD model injected in mBMSCs; Group C: aGVHD model injected in mBMSCs transfected with EGFP; Group D: aGVHD model injected in mBMSCs transfected with HO-1 gene.) The survival, body weight and clinical score of GVHD in transplanted mice were monitored to evaluate the severity of aGVHD. The aGVHD targeted organ, such as Liver, intestine and lung from mice in each group were obtained for histological examination and pathological score. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ and TNF-γ were also determined using a Cytometric Bead Array. Results:We cloned mice HO-1 gene from mice's bone marrow and constructed the recombinant adenovirus vectors (Lentivirus-V5-D-TOPO-HO-1- EGFP/Lentivirus-V5-D-TOPO-EGFP) successfully. mBMSCs was separated and identified successfully. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of HO-1 in gene-transfected group cells. It clearly showed that mBMSCs in Group D can migrate more through the polycarbonate filter toward bone marrow micro-environment in the lower chamber than Group A, B, C in vitro, while homing of Group D showed similar trend compared with others groups. In the mice GVHD model, treatment with HO-1 over-expressing BMSCs significantly decreased the mortality rate and attenuated clinical and pathological GVHD scores, and volume of spleen was not more obviously enlarged than others groups. Moreover, compared with control groups, the plasma IL-2, IL-6, IFN-γ and TNF-γ levels in recipients infused with HO-1 over-expressing BMSCs were significantly decreased, while those of IL-4 and IL-10 were increased. Conclusion:Lentiviral vectors carrying the HO-1 were successfully used to transduce mBMSCs. In our report, mBMSCs transferred with lentiviral vectors expressed strongerly immunoregulatory activities to alleviate aGVHD. Not only did HO-1 enhance that migration of mBMSCs, but also strengthen homing of MSCs. In vivo experiments, the evidence of survival rate, body weight, clinical score and pathological score fully proved that HO-1-transduced BMSCs effectively controlled the occurrence of mice's aGVHD following allogeneic BM transplantation, and HO-1 may be a potential target to overcome aGVHD in vivo. Disclosures:Ma:National Natural Science Foundation of China: Research Funding. Li:National Natural Science Foundation of China: Research Funding. Fang:National Natural Science Foundation of China: Research Funding. Chen:National Natural Science Foundation of China: Research Funding. Sun:National Natural Science Foundation of China: Research Funding.

The Prolyl Hydroxylase Inhibitor Dimethyl Oxalyl Glycine Decreases the Severity of Acute Lung Injury After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 3006 Background:Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges, with acute lung injury being associated with significant morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that the PHI DMOG (Dimethyl oxalyl glycine) may reduce lung injury evolving after murine allogeneic HCT. Methods:14 week old BALB/c were conditioned with 750 cGy single dose TBI, followed by transplantation of 5×10E6 bone marrow cells and 4×10E6 splenocytes from either syngeneic BALB/c or allogeneic C57BL/6 donors. DMOG in PBS was given to both syngeneic and allogeneic recipients at 8mg per mice ip on alternate days for the first two weeks post-transplant and then twice a week till day 50, while syngeneic and allogeneic controls received PBS only. Animals were monitored for clinical GVHD and survival. At day 50 post HCT, gut, liver and lung pathology as well as BAL fluid T cell numbers were assessed by using a scoring system quantifying severity and extent of parenchymal and peribronchial/perivascular infiltrates and flow cytometry, respectively. In addition, T cell expansion was analyzed both in vivo in the spleen on day +7 and in vitro by mixed lymphocyte reaction (MLR). Results:Allogeneic HCT resulted in severe lung pathology in PBS treated animals (mean 118.8±47.19), but was significantly reduced when DMOG was given (36.0±17.99; p < 0.05), and absent in both PBS and DMOG treated syngeneic controls. Consistently, BAL CD4+ and CD8+ T cells in DMOG recipients were decreased by 53.7% and 68.4%, respectively, when compared to PBS controls. Decreased pulmonary injury was associated with a trend towards better overall survival by day 50 (allogeneic DMOG: 59.2% vs allogeneic PBS 40.7%) and all syngeneic recipients survived. GVHD related injury to liver and GI tract at time of analysis was not different between allogeneic groups. Alloreactive proliferative T cell responses in vitro were suppressed in the presence of DMOG at a concentration of 1mM (cpm after 96h: mean 29316±875.9 vs 41735±3085; p < 0.01). In vivo, splenic T cell expansion of CD4+ but not CD8+ T cells was reduced at day +7 as well (p < 0.05). Interestingly, when compared to PBS treated controls reduced mortality of DMOG treated allogeneic HCT recipients could already be seen soon after HCT (day +10: 26.0% vs 11.1%) in association with decreased CD4+ T cell proliferation, while lung injury in this model this early is rather marginal. Conclusion:DMOG treatment decreases the severity of acute noninfectious lung injury and improves overall survival after allogeneic HCT. While impaired early T cell responses likely contribute to improvement of both endpoints, altered recruitment of CD8+ T cells to the lung seems to uniquely affect the development of pulmonary but not gastrointestinal and hepatic injury at later time points. Current studies are ongoing to delineate the effects of DMOG on T cell proliferation and recruitment to different target organs, and to define the role of this novel approach in the prevention and treatment of HCT related complications. Disclosures:No relevant conflicts of interest to declare.

A Critical Role of MyD88 Signaling in Donor T Cells for Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia Effect (GVL) After Experimental Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

AbstractAbstract 1893The therapeutic potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT) relies on the graft-versus-leukemia effect (GVL) to eradicate residual tumor cells by immunologic mechanisms. However, graft-versus-host disease (GVHD) remains the major toxicity of allo-HSCT. Alloreactive donor T cells are important effector cells in the development of GVHD, and proinflammatory cytokines enhance the generation of donor antihost cytotoxic function. Myeloid differentiation factor (MyD88) is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the Toll-like receptor (TLR) family. TLR engagement on professional antigen-presenting cells induces their maturation, resulting in optimal T-cell activation. However, recent advances indicate that the adjuvant effects of certain TLR agonists may also be attributed to the activation of TLRs and MyD88 directly in T cells. Both CD4 and CD8 T cells express functional TLRs. It remains to be defined whether direct TLR signaling on donor T cells is critical for GVHD or GVL activity.We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) experimental allo-HSCT model, which differs at major and minor histocompatibility loci, to address the role of donor T cell MyD88 signaling on GVHD and GVL. Lethally irradiated recipient mice were transplanted TCD-BM (5 × 106) together with either wild-type (WT) or MyD88 knock out (KO) mice spleen T cells (1 × 106) on day 0 and then host-type P815 mastocytoma or L1210 leukemia (H-2d) cells were injected either intravenously (3 × 103) or subcutaneously (1 × 106) on day 1 to generate a GVHD/GVL model. First of all, clinical GVHD scores were comparable between recipients of WT T cells and MyD88 KO T cells. At 70 days post-allo-HSCT, 50 % of allogeneic recipients of WT T cells died due to severe GVHD, but necropsy showed no evidence of tumor. In contrast, 83.5% of those of MyD88 KO T cells died with gross evidence of tumors (P<.05). Moreover, subcutaneous tumors in the allogeneic recipients receiving MyD88 KO T cells exhibited markedly increased growth in vivo compared to those receiving WT T cells (tumor volume on day 41, 15205.6 vs. 373.9 mm3, P<.01). GVHD mortality is critically dependent on donor CD4 T cells in this donor/recipient strain combination (B6 → B6D2F1) and CD8 T cells that mediate cytotoxicity are more potent effectors of GVL. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two T cell types was examined; apoptosis of CD8 T cells in recipients of MyD88 KO T cells was significantly enhanced compared to those of WT T cell recipients (P<.01) whereas apoptosis of CD4 T cells was comparable between two groups. Resultingly, the percentages of CD8 T cells in recipients of MyD88 KO T cells were significantly lower (P<.01). We next examined the effects of MyD88 signaling in donor T cells on cytolytic activity to host antigens. Splenocytes harvested from WT mice showed stronger cytolytic activity against P815 targets compared to those from MyD88 KO mice (P<.01). After allogeneic mixed leukocyte reaction, responder T cells from MyD88 KO mice showed markedly reduced IFN-γ, MCP-1 and IL-17A production with a significant augmentation in IL-10 secretion. We further evaluated the effect of T-cell MyD88 deficiency on GVL mediated by the intensity of total body irradiation (TBI) conditioning (1300 vs. 900 cGy, Exp Hematol 2011; 39: 1018–29). Enhanced GVL in the allogeneic recipients receiving 1300 cGy TBI was not shown in the recipients of MyD88 KO T cells.In summary, these results highlight a critical role for MyD88 signaling in T-cell activation and cytotoxicity, offering the opportunity for improving GVL activity by targeting TLR-MyD88 signaling within donor T cells. Furthermore, these data demonstrated that MyD88 deficiency in T cells can impair cytolytic function or subsequent GVL activity of CD8 T cells without significant change in the severity of CD4-dependent GVHD. This difference is attributed to the fact that MyD88 deficiency in T cells causes an enhanced apoptosis of donor CD8 T cells but not donor CD4 T cells in vivo after HSCT. Disclosures:No relevant conflicts of interest to declare.

Donor Treatment with α1 Anti-Trypsin (AAT) Mitigates Gvhd and Increases Survival While Sparing GVL Effects

Abstract 1889 Background:Graft-versus-host disease (GVHD) is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). While cytolytic effector function of donor-derived CD8+ cytotoxic T lymphocytes (CTLs) is critical for the elimination of the underlying malignant disease, the goal is to achieve this graft-versus-leukemia (GVL) effect without inducing GVHD. We and others showed in murine models that administration of AAT in the peri-transplant period decreased GVHD incidence and mortality. Concurrently, levels of pro-inflammatory cytokines were suppressed compared to albumin-treated recipients. Since both GVHD and GVL effects are initiated by donor cells, we asked whether donor treatment with AAT would modify GVHD or GVL effects in recipients. Of concern was the observation that AAT enhanced expression of heme oxygenase-1 (HMOX-1) which, via generation of carbon monoxide, enables the transcription factor Nrf2 to bind to the promoters of IL10 and IL1Ra, which might interfere with GVL activity. Methods:We determined the effect of donor AAT treatment on GVHD and GVL in two murine transplant models: C3H/SW(H-2Kbc) into C57Bl/6(H-2Kb) (minor mismatch), and C57Bl/6(H-2Kb) into BALB/C (H-2Kd) (major mismatch). Four regimens of donor and recipient treatment were studied: 1) Donor albumin /recipient albumin; 2) donor albumin/recipient AAT; 3) donor AAT/recipient albumin; 4) donor AAT /recipient AAT. AAT and albumin were given to donors at 3 μg/ dose every 72 hours for two weeks before cell harvesting. Recipients received 3 μ/dose every 72 hours following total body irradiation with 800 cGy. On day 4, recipients were injected with 5 × 106 T cell-depleted bone marrow (TCD-BM) plus 5 × 105 CD4+/CD8+ splenic T cells to induce acute GVHD. To test our hypothesis that donor AAT treatment would interfere with the GVL effect, BALB/C (H-2Kd) recipients were injected i.v. with 20 × 103 A-20 lymphoma cells containing a luciferase reporter construct (A20-luc/yfp; H-2Kd), following TBI. The infusion of TCD-BM and T cells from C57Bl/6(H-2Kb) donors was delayed until day 4 to allow the tumor to home and get established. Results:RT-PCR analysis of donor BM and T cells prior to transplantation showed up-regulation of HMOX-1 and Nrf2 (10 and 15 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively, compared to albumin treated donors). In contrast, TNFa, was down-regulated (7 log2 decrease in comparison to albumin-treated donors) in both unsorted bone marrow and in CD8+/CD4+spleen cells from AAT pre-treated donors. FACS analysis revealed a 4-fold increase in frequency of CD56+NK cells, and a 5-fold increase in CD4+/CD24+FoxP3+ Treg cells, associated with a 3 fold increase in CD8+/CD205+ APCs/ DC in comparison to albumin treated donors. Mice transplanted with cells from AAT-treated donors showed lower clinical GVHD scores and decreased mortality (Figure 1) in comparison to mice transplanted from albumin-treated donors. BALB/C (H-2Kd) animals transplanted from AAT-treated donors showed significantly lower A-20 tumor signal (luciferase) intensity (over the first 3 weeks post-HCT) than did mice transplanted from albumin-treated donors (n=6, mean 4.8 × 106 (AAT) versus,7 × 106 (albumin) photons/sec/mouse, P =.03). Summary and conclusions:These data suggest that pre-transplant in vivo AAT exposure of donor cells results in protection against acute GVHD. Tumor growth was not increased in mice transplanted from AAT treated donors and, in fact, appeared to be suppressed, presumably related to a significant increase in NK cells. Further studies on donor pre-treatment or direct administration of AAT to the transplant recipients are warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acute graft vs host disease (GVHD) induced by in utero hematopoietic cell transplantation is reversed by a regulatory response resulting in a potent graft vs hematopoietic effect without clinical GVHD

Acute graft vs host disease (GVHD) induced by in utero hematopoietic cell transplantation is reversed by a regulatory response resulting in a potent graft vs hematopoietic effect without clinical GVHD