GVHD In Mice Research Articles

Inhibition of histone methylation arrests ongoing graft-versus-host disease in mice by selectively inducing apoptosis of alloreactive effector T cells

AbstractHistone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell–mediated inflammatory disorders in a broad context.

Enrichment of Donor Plasmacytoid Dendritic Cells in Bone Marrow Allografts Enhances Graft Vs. Leukemia Activity and Th1 Polarization of Donor T Cells

Abstract 4701 Background:Allogeneic hematopoietic stem cell (HSC) transplant can cure patients with high risk and relapsed acute leukemia via a graft versus leukemia (GvL) effect. In our previous studies, depletion of CD11b+ cells (containing CD11b+ dendritic cells, CD11b+ NK cells, and myeloid suppressor progenitor cells) from donor bone marrow (BM) improved immune reconstitution and enhanced GvL effects without increased rates of GvHD in mice (Li, et al. BBMT 2004). We also have transplanted combinations of FACS purified HSC, donor T cells, and CD11b- dendritic cells (DC) and found similar improvement in GvL activity associated with donor T cells polarized towards a Th1 phenotype (Li, et al. JI 2009). In contrast, grafts containing FACS purified HSC, donor spleen T cells, and CD11b+ DC did not have significant GvL activity and donor T cells were polarized towards a Th2 phenotype after transplant. The objective of this study was to determine if enriching the relative numbers of donor plasmacytoid dendritic cells by selectively depleting CD11b+ DC from BM would yield similar enhancement of the GvL activity of donor T-cells as transplants of purified populations of HSC, CD11b-DC, and T cells in tumor-bearing mice. Methods:Selective depletion of CD11b+ DC from the BM allograft was achieved by FACS sorting and selectively removing the CD11b+ DC that comprised ∼1% of the BM. The CD11b+ DC-depleted graft thus contained ∼99% of all nucleated cells in the BM. Control undepleted BM grafts were also stained and sorted using only light scatter gates. To study the immunological effects of specific CD11b+ DC depletion, lethally irradiated B10.BR or BA.B10 recipients were transplanted with 3 × 10E6 CD11b+ DC FACS-depleted or undepleted BM cells and 1×10E6 spleen T cells from C57BL/6J or BA donors. Mice received 500,000 luciferase-positive LBRM cells (a T cell lymphoma) i.v. 1 day prior to transplant. Recipient mice were monitored for survival, weight change, and GvHD score (based on weight change, activity, posture, fur texture, and skin condition) throughout the duration of the experiment and for donor cell engraftment at days 30, 60, and 100 post transplant. Donor T cells were recovered from transplant recipients on days 3 and 10 post transplant and were examined for proliferation, Th1/Th2 polarization by flow cytometry, and ELISA measured serum cytokines. Results:BMT recipients of CD11b+ DC-depleted BM had higher survival than recipients of undepleted BM in tumor-bearing mice (p<0.05) (Figure 1). T cell chimerism at day 100 was > 95% for all mice, regardless of CD11b+ DC-depletion from the BM allograft. The level of GvHD in recipient mice did not differ significantly between groups. Donor T cell proliferation was increased on day 3 post transplant in recipients of CD11b+ DC-depleted BM (p< 0.05). On day 10 post transplant, serum IFN-g levels were increased in recipients of CD11b+ DC-depleted BM compared with undepleted BM(p<0.05). On day 10 post transplant, recipients of CD11b+ DC-depleted BM had significantly higher numbers of TNF-alpha producing donor CD8 T cells compared with donor CD8 T cells from recipients of undepleted BM (p<0.05) (Figure 2). [Display omitted] [Display omitted] Conclusions:Transplantation of BM allografts enriched for plasmacytoid DC by selective depletion of CD11b+ DC increased GvL activity of donor T cells without increasing GvHD. These data suggest that donor BM CD11b+ DC inhibit donor T-cell proliferation, Th1 polarization, and limit GvL activity. Disclosures:No relevant conflicts of interest to declare.

Reduced GVHD in C3-Deficient Mice Is Associated with a Decrease in Donor Th1/Th17 Polarization and Recipient Th1- Driven Dendritic Cell Activation

Abstract 308Graft-vs-host disease (GVHD) is an alloimmune response after allogeneic hematopoietic stem transplantation (HSCT) mediated by donor T cells against antigen presented by recipient dendritic cells. Our studies with a murine model of GVHD indicate that the complement system regulates the alloimmune response leading to acute GVHD.We used the disparity in MHC class I and II antigens between BALB/c (H-2d) as donors and either wild-type (WT) or complement deficient C57BL/6 (H-2b) as recipients. We found that mice deficient in the central component of the complement system (C3−/−) had significantly lower GVHD-related mortality and morbidity compared to WT recipient mice. Within 8 weeks after BMT, 80% of WT recipient mice and only 25% of C3−/− mice died (p=0.0008, n=20 in each group). While WT mice showed a moderate to severe GVHD in the skin, intestine, liver, lung and kidney, C3−/− mice had mild changes in these organs, reflected in a significantly lower GVHD scores compared to WT mice.Donor T cells proliferation is a critical step in development of GVHD. Therefore, we analyzed donor-derived T cells in C3−/− mice 7 days post-transplant, and found a significantly lower number of CD4+ and CD8+ T cells in spleen, lymph node and Peyer’s patches compared to those in WT mice. Complement deficiency not only affected the number of T cells but also their polarization. In the spleen and lymph nodes of C3−/−mice, we found a significantly lower number of IFNg-producing T cells and Th17+IFNg+ cells compared to WT recipients, consistent with a reduced Th1 and Th17+ differentiation of donor T cells in C3−/− recipient mice.Since the interaction between recipient-derived dendritic cells (DCs) and donor T cells is one of the initial events in the pathogenesis of the alloimmune response in GVHD, we analyzed both lymphoid and nonlymphoid DCs in the C3−/− recipient mice. Murine lymphoid DCs are divided into CD8a+ and CD8a− subsets that stimulate Th1 and Th2 cells, respectively. We detected a significantly lower number of CD8a+ (Th1-driven) and higher number of CD8a− (Th2-driven) DCs in the spleen of C3−/− recipient compared to WT mice. Additionally, the number of CD8a+ DCs was significant decreased in lymph node of C3−/− mice. In the nonlymphoid tissues, CD103+ DCs are developmentally and functionally related to the CD8a+ DCs. We studied DCs in the lung, liver, intestine and skin of recipient mice and found a significant reduction in the number of CD103+ DCs in the lung of C3−/− compared to WT recipient mice. Thus, C3 deficiency is associated with a decrease in Th1-driven DCs in both lymphoid (CD8a+) and nonlymphoid organs (CD103+) resulting in a reduced donor Th1 differentiation in C3−/− recipient mice.In the present study, we show for the first time that complement plays a role in GVHD. Our results are consistent with the findings of previous studies showing that C3 production by DCs in allograft is essential for maturation of DCs, effective antigen presentation to alloreactive T cells, and the development of Th1 response. We demonstrated that a similar effect of complement after BMT might be important in regulating Th1-driven DC activation and donor Th1/Th17differentiation, and in determining the severity of GVHD. Our study improves the understanding of the molecular mechanisms of GVHD and provides a rationale for using complement inhibitors as a novel potential therapeutic tool in GVHD. Disclosures:No relevant conflicts of interest to declare.

Noninvasive Prediction of Acute Graft-Verus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation by Circulating miRNA Profiling

Abstract 311 Background:Acute graft-versus-host disease (aGVHD) results in significant morbidity and mortality and remains the main complication of alloHSCT. Noninvasive, diagnostic and prognostic tests for aGVHD are currently lacking but essential to predict GVHD and to improve the safety and accessibility of alloHSCT. We hypothesized that the prospective analysis of miRNA expression profile in the plasma of allografted patients could allow for the detection of specific miRNAs with predictive role for aGVHD. Methods:After informed consent, we collected plasma samples from 10 healthy donors and 22 patients (median age: 59 and 41 years) who received unmanipulated alloHSCT (18 from Matched Unrelated Donors and 4 from HLA-matched siblings). Blood samples were collected weekly after HSCT and patients were monitored to assess aGVHD onset. MicroRNAs were isolated from the plasma and the miRNA expression profile examined using a quantitative PCR-method (TaqMan® Human microRNA Cards, Applied Biosystems). The results obtained were subsequently validated with specific miRNA Single Assays (Applied Biosystems). To verify whether the miRNAs emerged from the human studies represent markers of aGVHD and provide information regarding the involvement of specific target organs, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GVHD cohort, n=22) or with BM cells only (negative control, n=18). Syngeneic transplants (B6àB6, n=6 were also included. Mice were characterized for GVHD onset by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 9, 14 and 18 post transplant and GVHD confirmed by histology and scored according to Foley et al, 2008. MiRNAs expression profile have been characterized in the plasma, skin, liver, colon and lymphocytes of GVHD and non-GVHD control cohorts Results:Three of 22 patients developed intestinal GVHD (grade 2) and 9 of 22 patients developed cutaneous GVHD (grade 2–3). By comparing the circulating miRNAs expression profiles of GVHD patients and non GVHD patients, we identified a group of 8 differentially expressed miRNAs (miR-136, 194, 203, 367, 148b, 196b, 26a, 340) (p<0.05). On day 14 post transplant mice in the GVHD cohort developed GVHD as confirmed by organ evaluation (GVHD score: 14–15 out of 20) and weight loss while the control group had no GVHD (GVHD score 0–1 out of 20). The analysis of circulating miRNAs in mice supported the results in humans: the 8 circulating microRNA signature could clearly predict those subjects developing GVHD (area under the ROC curve ≥ 0.75). Of note, pathway enrichment analysis performed using DIANA-mirPath software on the gene targets predicted by microT-4.0, indicate that these miRNAs regulate critical pathways of GVHD pathogenesis (TGFbeta and cytokine signaling, T and B cell differentiation and proliferation, immune response). The analysis of the miRNAs expression profiles of the lymphocytes and of histologically confirmed skin, liver and colon biopsies of GVHD mice highlighted the presence of several deregulated miRNAs when compared to control samples. Pathway enrichment analysis indicated that the differentially expressed miRNAs mainly target genes involved in the TGFbeta and Wnt signaling pathways as well as in the cytoskeleton rearrangements. Of interest, when analysing the expression of the 8 circulating miRNAs able to discriminate GVHD samples from controls, in the organs of mice developing GVHD, miR-203 is also abundant in the skin and colon, miR-367 in the colon and miR-136 in the lymphocytes. These findings strengthen a role for these miRNAs in the modulation of aGVHD and suggest that the presence of plasma miRNAs is linked to the specific organs target of this pathological process. Conclusions:Considering the noninvasive characteristics of plasma sampling and the reproducible and easy detection of miRNAs, our results indicate that circulating miRNAs might represent a promising tool for the early diagnosis of aGVHD thus enhancing therapeutic success and increasing life expectancy of allografted patients. In addition the miRNA profiling of the target organs and lymphocytes of GVHD mice, allowed the identification of several deregulated genes that might play a role in the modulation of aGVHD and warrant further investigations. Disclosures:No relevant conflicts of interest to declare.

Plasmacytoid Dendritic Cells (PDC) and Th17 Immune Response Contribution in Gut Acute Graft-Versus-Host-Disease (GVHD)

Abstract 2968 Background.Acute GVHD after allogeneic stem cell transplantation (allo-SCT) is an exaggerated immune response against alloantigens involving dysregulation of inflammatory cytokine cascades. Previous studies established an important role of Th1 cells in acute GVHD pathophysiology. However, the identification of proinflammatory Th17 cells which contribute to autoimmune diseases pathophysiology, raised the issue of the role of Th17 cells in human acute GVHD. Indeed, the contribution of Th17 cells in acute GVHD was assessed in GVHD mouse models with conflicting results. In addition, the role of the PDC subset (the professional type I IFN-secreting cells), which play an important role in triggering Th17-related cytokines and autoimmune diseases, is not yet established in the acute GVHD setting. This report investigated the role of Th17 cells and their interaction with PDC in gastrointestinal (GI) biopsies taken from patients with or without acute GVHD. Patients and Methods.Studies described in this report were performed in a single centre series of 21 patients who underwent allo-SCT for different hematological malignancies (n=19) and severe aplastic anemia (n=2). The median age of patients was 53 years (range, 16–69). The stem cell source was PBSCs in 19 cases (85%), CB in 2 cases and BM in one case. Ten patients received transplant from a matched-related donor, and 11 patients from a matched-unrelated donor. A reduced-intensity conditioning regimen was used in the majority of cases (n=19; 90%) Immunohistochemistry was performed on deparaffinized tissues sections using an indirect immunoperoxydase method. A quantitative evaluation of antigens expression was performed by counting the number of positive cells in the whole biopsy at 200 magnifications for each sample. Results.In this cohort, based on standard pathology criteria, 16 patients had a histologically proven gastrointestinal acute GVHD. In all cases, biopsies were taken before initiation of systemic corticosteroid therapy. The remaining 5 patients did not have histological signs of acute GVHD (and did not develop clinical signs of acute GVHD) and thus, were used as controls. In order to identify the Th17 cell population, biopsies were tested for expression of the CD161 and CCR6 markers, and ROR-gamma-t, the key transcription factor that orchestrates the differentiation of Th17 cells. Significantly higher numbers of ROR-gamma-t+ and CD161+ cells were counted in the intestinal mucosa of patients with acute GVHD compared with intestinal mucosa of patients without acute GVHD, mainly found in the lamina propria but also in the epithelium of altered glands (p=0.016 and p=0.009 for ROR-gamma-t and CD161 expression respectively).Given the role of PDCs in triggering Th17-related cytokines, we sought next to determine the proportion of PDCs in intestinal biopsies from these same patients. This analysis showed a significant increase of CD123+ PDCs in the intestinal mucosa of patients with acute GVHD compared with mucosa of patients without acute GVHD (p=0.017). Moreover, we observed a significant correlation between the number of CD123+ PDCs and ROR-gamma-t or CD161 expressing cells in the intestinal mucosa of acute GVHD patients, highlighting the link between PDC and Th17 cells. Conclusion.The current study shed some light on the role of Th17 cells in the context of gastro-intestinal acute GVHD. Using well-established specific markers, we show that Th17 cells infiltrate intestinal biopsies from patients with acute GVHD. In addition, Th17 infiltration was paralleled by the infiltration of PDCs, suggesting a potential new pathophysiological link between PDCs and Th17 response in the context of gastro-intestinal acute GVHD. This is consistent with studies showing that PDCs can drive the differentiation of Th17 cells. Functional analyses are currently ongoing. Although the exact mechanism that links type I IFN production to PDC-mediated Th17 responses is still unclear in acute GVHD, these data raise the prospect of future innovative approaches to optimize immunosuppression regimens for the treatment or prophylaxis of acute GVHD by targeting PDCs and the Th17 response. Disclosures:No relevant conflicts of interest to declare.

Prevention of GVHD while sparing GVL effect by targeting Th1 and Th17 transcription factor T-bet and RORγt in mice

AbstractAllogeneic hematopoietic cell transplantation (HCT) is effective therapy for hematologic malignancies through T cell–mediated GVL effects. However, HCT benefits are frequently offset by the destructive GVHD, which is also induced by donor T cells. Naive Th can differentiate into Th1 and Th17 subsets and both can mediate GVHD after adoptive transfer into an allogeneic host. Here we tested the hypothesis that blockade of Th1 and Th17 differentiation is required to prevent GVHD in mice. T cells with combined targeted disruption of T-bet and RORγt have defective differentiation toward Th1 and Th17 and skewed differentiation toward Th2 and regulatory phenotypes, and caused ameliorated GVHD in a major MHC-mismatched model of HCT. GVL effects mediated by granzyme-positive CD8 T cells were largely preserved despite T-bet and RORγt deficiency. These data indicate that GVHD can be prevented by targeting Th1 and Th17 transcription factors without offsetting GVL activity.

All-Trans Retinoic Acid Promotes TGF-β-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus

BackgroundIt has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4+Foxp3+ regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.Methodology/Principal FindingsAddition of atRA to naïve CD4+CD25− cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3+ iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4+ cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4+ cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4+ cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4+ cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.Conclusions/SignificanceWe have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Early expression of plasma CCL8 closely correlates with survival rate of acute graft-vs.-host disease in mice

To elucidate the significance of early expression of CC-chemokine ligand motif 8(CCL8) in mice with graft-vs.-host disease (GVHD), we investigated its induction mechanisms and correlation with overall survival rate in GVHD mice. Plasma CCL8 increases on day 5 of allogeneic transplantation, when signs of GVHD are barely detectable. Increase of allogeneic splenocytes in grafts exacerbates GVHD and leads to upregulation of plasma CCL8 on day 5. Overall survival is the gold standard in determining the severity of acute GVHD in mice, but the absence of clinical and/or pathological manifestations in the early phase make it difficult to estimate vital outcomes at this stage of allogeneic marrow transplantation. After lethal irradiation, BALB/c mice received bone marrow transplantation from C57BL/6 mice. Survival rate was monitored and clinical and pathological scores of GVHD were examined. Coculture of BALB/c-derived dendritic cells and C57BL/6-derived splenocytes was performed. CCL8 was measured by immunoassay. The plasma CCL8 level at day 5 of transplantation was closely correlated with survival rate and clinical/pathological scores on day 14. Invitro study revealed that the BALB/c-derived dendritic cells expressed CCL8 upon stimulation of C57BL/6 CD4(+) T cells by cell interactions through major histocompatibility complex class II molecules. These investigations indicate that early and preclinical expression of CCL8 in plasma predicts overall survival of GVHD mice. Together with an involvement of allo-recognition in CCL8 expression, it suggests that CCL8 plays an important role in GVHD pathology.

Single Dose Administration of ECP Treated Cells Prior to Transplantation Significantly Increases Survival in a MHC-Mismatched Model of Acute GvHD

Extracorporeal photophoresis (ECP) has shown promise as a therapeutic modality in treating steroid-resistant chronic and acute GvHD. The way ECP mediates its effect has not been clearly elucidated, but some of the proposed mechanisms include impaired maturation of dendritic cells and reduced antigen presentation induced by apoptotic cells, as well as increased regulatory T (Treg) cells. We have developed a pre-emptive murine model of ECP to inhibit the initiation phase of acute GvHD and improve survival. Apoptosis was induced by ECP using 8-Methoxypsoralen and UVA light (UVAR light set, Therakos). Two days prior to bone marrow transplantation (BMT), 10^7 syngeneic (BalbC; H2-d) or allogeneic (C57BL6; H2-b) splenocytes treated with ECP were infused into BalbC recipients. To induce acute GvHD mice received 0.75x10^6 conventional T cells (Tcon) and 5x10^6 T cell depleted bone marrow from C57BL6 (H2-b). Animals were followed for survival, and GvHD was scored using standard criteria. In some experiments, Tcon were derived from luciferase-transgenic animals, allowing for quantification of T cell proliferation using bioluminescent imaging (BLI). A single dose of pre-emptive ECP treated splenocytes led to a significant survival benefit (median survival 27 versus 52 days, p < 0.0001). Re-isolation experiments on day 7 after BMT showed an increase in host Treg in peripheral and mesenteric lymph nodes, whereas donor Treg were similar in both groups. However, Tcon proliferation was not significantly impacted, as quantified by BLI. Using pulsed BrdU labeling no proliferation of host Treg was seen after transplantation. Also, intracellular staining and analysis of serum cytokine levels to characterize Th1/Th2 polarization of T cells revealed no differences at day 7. Importantly, preliminary experiments showed no alteration in graft versus tumor effect. In summary, a single dose of apoptotic cells prior to transplantation led to a significant survival benefit. Whether this effect is related to the relative sparing of host-Tregs remains to be determined. However, the mechanism does not appear to be related to reduced T cell proliferation. Other avenues to be explored include further studies of cellular mechanism, e.g. maturation status of host APC, as well as evaluating multiple administration doses to further improve outcome. Pre-emptive ECP of host cells prior to BMT may be a clinically relevant strategy to ameliorate acute GvHD.

Manipulating the Bioenergetics of Alloreactive T Cells Causes Their Selective Apoptosis and Arrests Graft-Versus-Host Disease

Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.

HDAC11 Inhibits T-Cell Response to Alloantigens and Reduces Gvhd In Mice.

AbstractAbstract 1466 Background:Histone acetyltransferases and histone deacetylases (HDAC) regulate gene expression through acetylation-deacetylation of histones. HDACs are the target of a family of compounds known as HDAC inhibitors, which have been shown to suppress pro-inflammatory cytokines and reduce acute graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT) in mice. However, the role of individual HDAC members in the development of GVHD is not clear. Recently, HDAC11, the newest member of the HDAC family has emerged as an important transcriptional regulator of inflammatory responses in antigen-presenting cells (APCs)1. Here, we evaluated the role of HDAC11 on APCs and T cells in the allogeneic BMT setting in mice with genetic disruption of HDAC11. Method:Proliferation of wild-type (WT) and HDAC11 knock-out (KO) T cells in response to allogeneic antigens was compared by [H3] thymidine incorporation assay. Using the same method, we also tested the antigen presentation ability of WT and HDAC11 KO APCs. For in vivo studies, we used a clinical relevant mouse model of BMT: C57BL/6 (B6) ® BALB/c. To evaluate the role of HDAC11 in the function of T cells and APCs, WT and KO mice on B6 background were used as donors and recipients, respectively. Recipient survival was monitored daily and GVHD symptom was evaluated at least twice a week. HDAC11 KO mice were supplied by Merck and Co., Inc. Results:In vitro, HDAC11 KO T cells proliferated stronger than WT T cells under the stimulation of allogeneic APCs. Recipients of HDAC11 KO T cells lost significantly more body weight (p < 0.05), and died significantly sooner than those of WT T cells (p < 0.01). The pathologic score of KO recipients was higher than that of WT recipients in each of GVHD target organs including lung, liver, small intestine and colon. Mechanistically, we found that there were significantly more total and IFNγ-producing donor T cells in the recipients of KO cells than those of WT cells (p < 0.05). Collectively, HDAC11 KO T cells have higher activity in response to alloantigens in vitro and induced more severe GVHD in vivo compared to WT T cells. In contrast, KO and WT APCs had a similar ability to stimulate allogeneic T cells in vitro, and no significant difference in GVHD development was observed in WT or KO recipients after allogeneic BMT. Conclusion:HDAC11 negatively regulates T-cell function, but has no significant effect on APC function. This finding provides a rationale to promote T-cell immunity or tolerance by inhibiting or enhancing HDAC11, respectively.1 Villagra et al. Nature Immunology, 10:92-100, 2009. Disclosures:No relevant conflicts of interest to declare.

Effect of the HDAC Inhibitor SAHA on the Immunmodulatory Properties of MSC and Tumor Stroma Cells.

AbstractAbstract 2592Histone deacetylase inhibitors (HDACi), like suberoylanilide hydroxamic acid (SAHA), are new drugs in oncologic treatment protocols. Histone deacetylases together with histone acetyltransferases (HAT) regulate the acetylation status of distinct lysine residues in histones. Not only do they affect the plasticity of the chromosomes and thereby have a direct impact on gene expression, but also regulate several non-histone-proteins via hyperacetylation. Among these are proteins involved in gene expression like transcription factors. SAHA is a pan HDACi and displayed antiproliferative effects on transformed cells in vitro and in vivo. SAHA was the first HDACi that was approved for treatment of refractory cutaneous T cell lymphoma. Interestingly, SAHA was reported to reduce the production of the proinflammatory cytokines TNFα, IFNγ, IL-1β and IL-12 at nanomolar concentrations, at which cell proliferation is not yet affected. Proinflammatory cytokines play a central role in the pathogenesis of acute GvHD. Indeed, it has been reported that SAHA reduced GvHD in mice while the important graft-versus-leukemia-effect (GvL) was not impaired.A cellular tool for treatment of acute GvHD after allogenic stem cell transplantation are multipotent mesenchymal stromal cells (MSC). Their immunosuppressive effects are conducted by an altered cytokine profile of peripheral blood mononuclear cells (PBMC). We analysed the effect of SAHA on T cells directly as well as on MSC. Both cell types tolerated low micromolar doses of SAHA well. Morphology and immunophenotype of MSC was not considerably affected. For the treatment of GvHD a synergistic or additive effect of SAHA and MSC on immune effector cells would be desirable. Instead, we found that SAHA reduced the suppressive effects of MSC on PBMC.Therefore, we focused to a closely related cell type under investigation in our lab, tumor stromal cells (TStrC). In previous studies, we found that tumor stromal cells (TStrC) also impaired T cell and NK cell function. As TStrC share several properties with MSC, we hypothesized that in this particular setting SAHA may facilitate tumorimmunological responses by interfering with the immune suppression exerted by TStrC. We isolated TStrC from neuroblastoma patients and analysed the effect of SAHA on these TStrC including cytokine patterns. In supernatants from TStrC cultures, we were not able to detect any of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-12p70, IL-15, IL-17, TNFα, RANTES and IFNγ. Comparable to MSC, TStrC reduced the production of the proinflammatory cytokines IL-12p70, TNFα and IFNγ and the anti-inflammatory IL-10 in PBMC. In contrast, the level of IL-1β was markedly increased. Low micromolar concentrations of SAHA did neither affect the morphology and immune phenotype of TStrC nor impaired their proliferation. Pre-incubation of TStrC with SAHA resulted in an attenuated immune suppression by TStrC. Untreated and solvent-treated TStrC inhibited the proliferation of PBMC stimulated by IL-2/OKT3. PBMC co-cultivated with SAHA-treated TStrC showed a significantly enhanced, but not fully restored proliferation in a concentration-dependend manner.Functional assays showed that NK cell cytotoxicity against the leukemia cell line K562 was impaired by TStrC. The activating NK cell receptors NKp44 and NKp46 were downregulated while the inhibitory receptor NKG2A remained unaffected. Pre-incubation of TStrC with SAHA partially restored NK cell cytotoxicity. In multiplex analyses, we investigated the cytokine profiles of TStrC and SAHA-treated TStrC monocultures as well as co-cultures of PBMC with TStrC and previously SAHA-treated TStrC. Control and SAHA-treated TStrC did not produce any of the cytokines IL-1β, IL-2, IL-4, IL-10, IL-12p70, IL-15, IL-17, TNFα, RANTES or IFNγ at a detectable level. Co-incubation of PBMC with SAHA-treated TStrC changed their cytokine profile as compared to co-incubation with untreated TStrC. PBMC produced higher levels of the cytokines IL-10, TNFα and IFNγ when co-cultivated with SAHA-treated TStrC. The secretion of IL-1β was also increased. Taken together, these findings suggest that SAHA may modify the tumor microenvironment at low micromolar concentrations to allow for more efficient anti-tumor immune responses. Disclosures:No relevant conflicts of interest to declare.

The Novel Combination of Sirolimus and Bortezomib Prevents Graft-Versus-Host Disease but Mantains the Graft-Versus-Leukemia Effect After Allogeneic Transplantation.

AbstractAbstract 3738Graft-versus-host disease (GVHD) represents a major challenge and the main cause of morbidity and mortality after allogeneic transplantation. Using the standard GVHD prophylaxis based on a calcineurin inhibitor plus methotrexate (MTX). The incidence of acute GVHD is in the range of 30–60%, so that new strategies are required in order to decrease GVHD without hampering GVL. Sirolimus, an mTOR inhibitor, allows to decrease the risk of GVHD and increases the number of Treg after transplantation. Unfortunately, its use may increase the risk of microangiopathy and, moreover, its combination with calcineurin inhibitors blocks the development of Treg. Bortezomib is a proteosome inhibitor and blocks the nuclear translocation and activation of NF-kB. It induces depletion of alloreactive T and allows the expansion of T-cells with suppressive properties. Accordingly, both drugs could favour the development of a tolerogeneic immune response after transplantation. In the current study we have analyzed the synergistic effect of sirolimus together with bortezomib. Bortezomib 100 nM plus sirolimus 5nM synergistically inhibited T-cell activation as assessed by the expression of CD25, production of IFNg and expression of CD40L as well as proliferation assessed as expression of PKH. Remain vibility, these effects could not be attributed to a decreased viability of T-cells, as assessed by 7-AAD, at the concentrations evaluated. As compared to each drug alone, the combination significantly decreased the production of Th1 cytokines (IFNg, IL-2 and TNF) while regarding TH2 cytokines, only IL-6 significantly decreased upon combining both drugs. Concerning the mechanisms involved in this synergistic effect, the combination of both drugs resulted in an inhibition of the Akt and Erk ½ phophorylation, thus indicating that sirolimus inhibit pathways which could allow T-cells to escape from the effect of bortezomib at the doses used in the current experiment. In order to confirm in vivo the synergistic effect of sirolimus and bortezomib, a GVHD mouse model (C57/BL6-Balb/c) was carried out. Mice receiving both drugs (Bortezomib 1μg/day intravenous on days 0, +1, +2 postransplantat and sirolimus 0.25mg/Kg intra-peritoneal on days 0 to 12) has a significantly lower incidence of GVHD and longer survival as compared to each drug alone. We also wanted to evaluate whether the immunosuppressive effect of the combination was unspecific or, by contrast, it allowed induce specific immune tolerance against host but maintaining the immune response against other antigens. For this purpose a haematopoietic cells of Balb/c mice which were C57/BL6 complete chimeras were infused to NOD-SCID mice. While none of the donors had developed GVHD after transplantation plus sirolimus and bortezomib postransplant, the NOD-SCID mice succumbed due to GVHD. Furthermore, we infused WEHI cells to BALB/c after total body irradiation and we observed that, while all BALB/c mice receiving WEHI plus C57BL/6 donor BM cells died due to leukemic infiltration, none of those receiving WEHI cells plus C57BL/6 donor BM cells plus splenocytes (and GVHD prophylaxis with sirolimus and bortezomib) did develop leukemic infiltration, thus confirming that, using this approach, we were able to separate GVHD and GVL effect. In conclusion, the current study shows a potent synergistic effect between sirolimus and bortezomib in vitro and in vivo which prevent GVHD while maintaining GVL. Disclosures:Cañizo:CELGENE: Membership on an entity's Board of Directors or advisory committees. San Miguel:JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees; CELGENE: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; MILLENNIUM: Membership on an entity's Board of Directors or advisory committees. Off Label Use: sirolimus and bortezomib are not approved for use in prophylaxis of graft versus host disease.

Adenosine A2A Receptor Agonist Mediated Prevention of Graft-Versus Host Disease In the Mouse Is Associated with An Increase of T-Regulatory Cells In Target Organs.

AbstractAbstract 3744Graft versus host disease (GVHD) is a serious complication of allogenic hematopoietic stem cell transplantation (HSCT) affecting primarily the liver, intestine, skin and lymphoid organs. T-regulatory cells (Tregs) are a subset of T cells that are characterized by CD4+CD25high and express the Foxp3 transcription factor. There is increasing recognition that Tregs likely are important in preventing development, reducing the severity and/or mediating resolution of GVHD. It has been reported that giving donor Tregs will suppress development of GVHD in a mouse model; however it is not clear whether the immunologic suppression by Tregs occurs in the target organs. Agonists of the Gs-coupled Adenosine A2A receptor (A2AR) agonists have been demonstrated to terminate inflammation and improve survival in solid organ transplant ischemia models. We previously have reported that the A2AR specific agonist, ATL146e, also can decrease the incidence and severity of GVHD as well as improve survival of mice in a GVHD transplant mouse model. In this same GVHD mouse model, ATL146e treatment increases the number of donor derived CD4+CD25highFoxp3+ Tregs. In order to further understand the role of the agonist in GVHD abrogation we performed studies looking at A2AR agonist mediated appearance of Tregs in the target organs affected by GVHD in this model. Using a parental into irradiated F1 offspring transplant model (C57BL/6J [B6, H-2b] -> B6D2F1/J [BDF1, H-2b/d]) we can induce GVHD as manifested by weight loss and mortality in 100% of mice by infusing an additional 10 million donor T cells into mice previously engrafted with 10 million bone marrow donor cells using 850cGy conditioning. We administered the A2AR specific agonists, ATL1223 or ATL370, or a PBS control by osmotic mini pumps resulting in continuous subcutaneous infusion for 14 days starting one day before the donor T cell infusion. Mice that received only the donor bone marrow transplant and not the additional donor T cells did not develop GVHD and served as an additional control. We collected ear and colon biopsies from the transplanted mice to assess these target organs (skin and gut) usually affected by GVHD in this model. Tissue was obtained, placed in 10% formalin and paraffin embedded and stained by immunohistochemistry (IHC) for intracellular FoxP3 by incubating overnight with 1:200 mouse anti-mouse/human/rat—FoxP3 IgG. The slides were washed and then incubated with anti-mouse IgG conjugated to Tetramethylrhodamine (TRITC). We then counted the number of Foxp3+ cells per high power field. We also examined whether specific agonist activation of A2AR can increase the number of Tregs in vitro by culturing CD4+ CD25− cells in the presence of either TGF-beta and/or the A2AR specific agonists. As in our previous studies with the specific A2AR agonist ATL146e, the agonists ATL1223 and ATL370 also were effective at preventing the acute GVHD syndrome of weight loss and mortality seen in the PBS treated controls. Treatment with these agonists resulted in an increase in CD4+CD25+FoxP3+ Tregs in the spleen compared to the PBS treated group at days 14 to 20 after HSCT. In addition we were able to document significant increases in the number of Foxp3 positive T lymphocytes infiltrating skin and colon compared to the PBS treated group. The number of Foxp3+ T cells per high power field was 14 ± 6 in the control and 64 ± 14 in the A2AR agonist treated mice (p = 0.0281). Finally from our in vitro data we also observed a 4.2 to 5.4 fold increase in the number of Foxp3+ Tregs in vitro after A2AR activation compared to TGF beta treatment only. Thus we have confirmed that the specific activation of A2AR inhibits acute GVHD through the increase of donor-derived CD4+ CD25+ FoxP3+ immunosuppressive T regulatory cells in vivo and in vitro. Furthermore, our histological observation suggests the possibility that increased Tregs by treatment with A2AR specific agonist is responsible for suppressing the immune response in GVHD target tissues. Therefore, our observation provides additional mechanistic basis for the anti-inflammatory capacity of A2AR agonist in acute GVHD. Additional studies are ongoing to further elucidate the mechanism by which A2AR specific agonist increases Tregs. Disclosures:No relevant conflicts of interest to declare.

Graft-Versus-Host Disease Alters Intestinal Microbial Ecology by Inhibiting Production of Enteric Defensins

AbstractAbstract 244Bacterial infection is a serious complication of bone marrow transplantation (BMT). Intestinal GVHD, in particular, significantly enhances the risk for Gram-negative septicemia. The majority of the intestinal microbiome of the hosts consists of non-cultivable obligate anaerobes, while the Gram-negative bacteria such as Escherichia coli (E. coli) make up a small proportion of the microflora. Thus, it remains unclear why Gram-negative septicemia is dominant in intestinal GVHD, while the role of systemic immunosuppression and use of antibiotics is well-appreciated. We evaluated gut flora changes in the course of GVHD in mouse models of BMT without giving antibiotic or immunosuppresive drugs. Lethally irradiated B6D2F1 (H-2b/d) or B6C3F1 (H-2b/k) mice were injected with 5 × 106 T-cell depleted BM alone (non-GVHD controls) or with 2 × 106 T cells (GVHD group) from MHC-mismatched B6 (H-2b) donors on day 0. Intestinal microflora was identified by using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene libraries constructed from each sample of gut contents. It consisted of approximately 80% of obligate anaerobe, and 20% of anaerobe such as Lactobacilli and Clostridia with very few E. coli before BMT. After BMT, this diversity of gut flora was preserved in non-GVHD controls (Table). In contrast, GVHD mice showed a marked increase of E. coli with a significant decrease in the members of obligate anaerobe. This was associated with dissemination of E. coli to the mesenteric lymph nodes (mLNs) and liver, with elevated serum levels of lipopolysaccharide (LPS). Such a loss of diversity of gut flora with a flora shift towards E. coli was significantly associated with morbidity and mortality of GVHD. Numbers of T-RFLP peaks that indicate diversity of intestinal flora were inversely correlated with GVHD clinical scores (p<0.001). A degree of E. coli proportion has significant correlation with GVHD clinical scores (p<0.001) and GVHD mortality (80% in high E. coli group vs. 0% in low E. coli group). We then investigated the underlying mechanisms of the disruption of intestinal ecology in GVHD. Paneth cell derived α-defensins are essential regulators of intestinal microbial ecology. We therefore hypothesized that Paneth cell damage in GVHD inhibited production of enteric defensins and disrupted intestinal ecology. Immunohistochemistry for lysozyme that marks Paneth cells showed significant reduction of Paneth cells in GVHD and quantitative real-time PCR analysis showed dramatically reduced expression of enteric defensins including Defa21, Defa1, Defa4, Defa5, and Defcr-rs1 (Table). These results suggest that Paneth cell injury in GVHD could lead to lower expression of enteric defensins and a shift of gut flora from commensal microorganisms towards widespread prevelance of gram-negative bacteria in the intestinal microbiome, and the subsequent high risk for the development of life threatening Gram-negative septicemia. LPS derived from Gram-negative bacteria plays an important role in amplifying systemic GVHD. Thus, such an alteration of intestinal ecology may be related to exaggeration of systemic GVHD. These results thus uncover the previously unrecognized role of the intrinsic antimicrobial peptides and the crosstalk between hosts and the intestinal microbes in the pathogenesis of GVHD and infection after allogeneic BMT. Disclosures:No relevant conflicts of interest to declare.

Adoptive Transfer of Natural Killer (NK) Cells to Prevent Gvhd and Enhance GVT Effects After Allogeneic Hematopoietic Cell Transplantation (HCT): The Timing of Donor NK Cell Infusions Critically Impacts Transplant Outcome.

Abstract 786Murine models have shown infusions of donor NK cells from hematopoietic stem cell transplant donors can prevent GVHD while simultaneously mediating a graft-vs-tumor (GVT) effect. Reduction of GVHD by alloreactive NK cells can be mediated indirectly through the eradication of host antigen presenting cells (APC) or directly by NK cell killing of alloreactive T cells. To assess how the timing of NK cell administration impacts these effects, donor NK cells from B10.d2 (H-2d) mice (1-2×106 cells) were infused into MHC-matched BALB/c (H-2d) recipients following lethal irradiation (950cGy) at one of the following time-points: 1) two days prior to a T cell replete (TR) HCT to target host APC or 2) at the time of a TR-HCT or 3) five days following a TR-HCT to target both host APC and in vivo primed alloreactive donor T cells. We also evaluated whether donor NK cells given on the day of a T cell deplete (TD) HCT could be used to prevent GVHD in mice that subsequently received a delayed donor lymphocyte infusion (DLI) given four days following HCT.Administration of donor NK cells two days prior to allogeneic TR-HCT did not result in a reduction of GVHD (figure). In contrast, the administration of NK cells given either at the time of a TR-HCT or five days following a TR-HCT reduced the incidence of GVHD (GVHD incidence 70% (p=0.2) and 40% (p=0.01) respectively) compared to controls that did not receive NK cells following a TR-HCT (GVHD incidence 100%). Similarly, mice that received a TD-HCT followed by a DLI on day four had a significantly lower incidence of GVHD when NK cells were infused on the day of transplantation compared to controls that did not receive donor NK cells (GVHD incidence 40% vs 100% respectively, p=0.01). Using bioluminescence imaging, we next investigated the impact of the timing of NK cell infusions on GVT effects in tumor bearing mice. Luciferase transduced RENCA tumors were injected intravenously into BALB/c mice ten days prior to allogeneic HCT. Tumor progression was significantly delayed in recipients of a TR-HCT when NK cells were infused on day five compared to when NK cells were infused two days prior to or at the same day of a TR-HCT (tumor doubling times 22.9 days, 7.6 days and 8.5 days respectively; p=0.03) (figure). This delay in tumor progression correlated with a significant improvement in overall survival; recipients of a TR-HCT given NK cells on day five had significantly longer survival compared to recipients of TR-HCT that did not receive NK cells (median survival 54±14 days vs 44±9; p=0.008), whereas infusion of NK cells prior to or concomitant with a TR-HCT did not significantly prolong survival (median survival 49±4 days and 50±12 days respectively; p=0.23). A comparable delay in tumor progression and longer survival was observed in mice that received a TD-HCT followed by a DLI on day four when NK cells were infused at the time of transplant compared to controls not receiving NK cells (tumor doubling time 19.7 days vs 7.2 days and survival 62±16 days vs. 50±9 days respectively; p=0.07). [Display omitted] In conclusion, these results show that the timing of adoptive donor NK cell transfer has a critical impact on the ability of NK cells to prevent GVHD and enhance GVT effects following both T-cell replete and T-cell depleted allogeneic HCT. Following a TR-HCT, a delayed add-back of NK cells maximizes GVHD reducing and anti-tumor effects of adoptively transferred donor NK cells. Disclosures:No relevant conflicts of interest to declare.

Developing a Therapeutic Anti - Dendritic Cell Antibody to Prevent Graft Versus Host Disease.

Abstract 3554Poster Board III-491 IntroductionHost and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent. MethodsRAM83 specificity and function was tested by flow cytometry and allogeneic MLRs. It was administered as a single dose on day -1 of murine syngeneic and allo HSCT. Human and mouse single chain variable fragment (scFv) phage libraries were panned on recombinant human CD83 extracellular domain. After screening for specificity and affinity, the clones were reformatted to human IgG1 and expressed by transfected CHO cells. The purified mAb were tested for their ability to block a mixed leucocyte reaction (MLR). ResultsLin-,Sca1+,Kit+ murine stem or progenitor cells were CD83-ve. RAM83 treated mice receiving syngeneic HSCT survived >35days (normal day 14 WBC). Full MHC mismatched (2× 60mg/kg cyclophosphamide + 2× 500cGy conditioned), B6 [H-2b] into BALB/c [H-2d]) recipient mice treated with a single dose of RAM83 developed GVHD later and survived longer (mean 26.9 days ±3.3) compared to controls (RAneg 12.3±1.3days, p=0.001; Nil antibody 7.7±1.5 days, p=0.0008). Daily high dose (50mg/kg) cyclosporin A delayed GVHD by a mean 18.2±3.3 days. Four phage clones that bind to human CD83 have been reformatted. Of the two tested to date, one mouse/human chimeric mAb has lost binding affinity, probably as a result of immunoglobulin variable region glycosylation. The other reformatted fully human anti-CD83 (3C12) mAb blocked a MLR, by an ADCC mechanism. ConclusionsmAb targeting of activated DC is a promising novel approach to immunosuppression. The 3C12 clone and others will be subjected to preclinical testing in the xenogeneic SCID mouse model. The human mAb, which prevents GVHD without impairing the desired graft versus leukaemia effect will be developed for phase 1 clinical trials in clinical alloHSCT. Disclosures:Hart:CRC-BT: Consultancy.

A Novel Function of CD26-Mediated Costimulation in the Cytotoxic Activity of Human CD8+ T Cells in Xenogeneic Chronic Graft-Versus-Host Disease (cGVHD) and GVL Mice Model.

Abstract 3548Poster Board III-485CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain, capable of cleaving amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. CD26 activity is dependent on cell type and the microenvironment, which influence its multiple biological roles. CD26 plays an important role in immunology, autoimmunity, diabetes and cancer. The role of CD26 in immune regulation has been extensively characterized, with our recent findings elucidating its linkage with signaling pathways and structures involved in T cells activation as well as antigen presenting cell (APC)-T-cell interaction. CD26 is preferentially expressed on human CD4+ memory T cells and CD4+CD45RO+CD26+ T cells exhibit response maximally to the recall antigen tetanus toxoid. Human T-helper 1 (Th1) cells display a higher expression of CD26 and are much more sensitive to CD26-mediated costimulation than human Th2 cells. In contrast, the role of CD26 in human CD8+ T cells still remains to be elucidated, while CD8+ T cells express CD26 as well as CD4+ T cells. In this study, we studied the effector function of CD26-mediated costimulation of CD8+ T cells. In comparison with CD28-medaited costimulation, which is one of representative T cell costimulatory pathway, CD26-mediated costimulation in CD8+ T cells showed delayed proliferation than that of CD28 stimuatlion, but finally expanded to a similar extent. The secretion of inflammatory cytokines, TNF-alpha and IFN-gamma, was strongly induced after CD26-mediated costimulation. In contrast, the secretion of IL-2 and IL-5 was significantly less as compared with CD28-mediated costimulation. Finally, we showed that the expression of Granzyme B, one of the major effector molecules in the cytotoxic activity of CD8+ T cells, was markedly enhanced by CD26-mediated costimulation in dose dependent manner of anti-CD26 monoclonal antibody stimulation. Moreover, with CD26-mediated costimulation, CD8+ T cells were observed to kill target cells in a Granzyme B-dependent manner. Next, we examined CD26-dependent organ injury by human T cells using xenogeneic chronic GVHD (x-cGVHD) mice model. About 6 weeks after transplantation of human peripheral blood mononuclear cells (PBMC) into NOG-Scid mice, mice develop loss pf hair, loss of weight, and human CD8+ lymphocytes infiltration into liver, lung, and salivary glands, which exhibit pathological changes of cGVHD. Administration of anti-human CD26 mAb decreased onset of x-cGVHD, while human lymphocytes were grafted successfully. Finally, we examined whether GVL effect is exerted via CD26-mediated costimulation, using x-cGVHD mice model in which P388 leukemic cells were grafted subcutaneously. In this tumor-graft model, P388 cells were not grafted in mice with onset of x-cGVHD, which indicated that GVL effect was exerted by human lymphocytes. On the other hand, P388 cells were also diminished in mice with decreased x-cGVHD by administration of anti-CD26 mAb. Taken together, our data strongly suggest that CD26-mediated costimulation in CD8+ T cells deeply involves in the pathology of cGVHD. The present study offers a novel notion that CD26 may become one of therapeutic targets of GVHD without impairment of GVL effect. Disclosures:Yamada:Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees. Dang:Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Morimoto:Y's Therapeutics: Patents & Royalties, a board member.

Prognostic Significance of Complement System Activation After Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract 1166Poster Board I-188 Introduction:The complement system consists of several serum proteins and cell membrane receptors that can be activated by three possible pathways leading to the production of pro-inflammatory factors such as the anaphylatoxins C3a and C5a and the formation of the membrane attack complex. The complement system has been implicated in the pathophysiology of several immune diseases and we previously reported that it was activated after allogeneic haematopoietic stem cell transplantation (HSCT) both in humans and mice. It's activation after allogeneic HSCT following a myeloablative conditioning could predict the development of gastrointestinal (GI) GVHD in humans. We now present updated data with long term follow up on an enlarged series of patients who did or did not activate complement after allogeneic HSCT. We also discuss the preliminary results of the inhibition of complement activation on experimental models of GVHD. Materials and methods:Complement activation was determined by measurment of complement proteins before and once a week up to 3 months after allogeneic HSCT in 34 patients allografted for diverse haematological malignancies in our institution following conventional myeloablative conditioning. Results were correlated to the clinical evolution of allogeneic HSCT. Preclinically, inhibition of mouse complement activation by C1 inhibitor esterase or an anti-C5 monoclonal antibody was tested in a parent (C57BL/6, H-2b) to F1 [(C57BL/6xDBA2), H-bd] GVHD mouse model. Lethally irradiated (9.75 to 11 Gy) mice reconstituted with either syngeneic or allogeneic bone marrow cells and splenocytes (107 and 13 to 20 ×106 cells/recipient, respectively) were treated with complement inhibitors between day-7 to Day 21 or day 1 to Day 28 post-HSCT. Clinical Results:Fifteen (44%) patients showed an activation of the classical pathway, as defined by a decrease of C3 and C4 proteins below normal values and of at least 50% of their pre-HSCT levels. Activation occurred during the 4 first weeks following HSCT in 11 patients, between 6 and 8 weeks in 2 cases and later, after withdrawal of immunosuppression, in 2 patients. Pre-transplant characteristics (age at transplant, sex, underlying haematological disease, conditioning regimen, use of ATG, type of donor and CMV risk) of patients who showed an activation of complement were comparable to those who did not. We confirmed that activation of complement was significantly associated with acute GI GVHD (80 % in the activated group versus 5.3% in the non activated, p=0.0028) and occurred from 1 to 3 weeks before the appearance of clinical GVHD. Patients in the activated group had a significantly increased incidence of capillary leak syndrome concomitant to the conditioning toxicity or GVHD (66.7% in the activated group vs 10.5% in the non activated, p=0.019). There was no significant difference between the groups in terms of skin or hepatic acute GVHD, TMA, VOD and chronic GVHD. In a landmark analysis by day 100 post-HSCT, overall survival was significantly impaired in the group of patients with complement activation (p=0.008) because of increased toxicity related mortality (p=0.02) and relapse (p=0.01) risks in comparison to the patients without complement activation. Preclinical Results:Complement inhibitors capable of blocking the first pathway (C1 inhibitor esterase) or terminal complement activation (anti-C5 mAb) were used in a mouse model of GVHD. These inhibitors were administrated before and/or during the phase of activation of complement which starts on day 4 post-HSCT. However, GVHD clinical signs and mortality did not appear to be improved following complement inhibitor administration in this mouse model. Conclusion:Activation of complement after allogeneic HSCT following a myeloablative conditioning appears as a predictive marker of acute GI GVHD and a pejorative prognostic factor of transplant outcome. However, inhibition of complement activation in a mouse experimental model of allogeneic HSCT did not allow the control of GVHD. It is not clear, therefore, that this mouse model is useful as a model of human GVHD. Disclosures:No relevant conflicts of interest to declare.

Liposomal KRN7000 (RGI-2001) Synergizes with Low-Dose Rapamycin in Preventing GvHD by promoting expansion of naturally-Occurring CD4+FOXP3+ Regulatory T Cells.

Abstract 4491 IntroductionRGI-2001 is a liposomal formulation of KRN7000 (a synthetic derivative of a-galactosylceramide). We previously demonstrated that RGI-2001 potently reduces GvHD lethality in a mouse model by inducing host-specific tolerance through the expansion of donor-derived CD4+Foxp3+ Regulatory T cells (Tregs) (ASH 2008 Poster III-582). In the present study, we examined if the combination with low-dose rapamycin (LD-RAPA) may further improve the efficacy of RGI-2001. In addition, the origin of Tregs induced by RGI-2001 was investigated. MethodsA fully MHC mismatched GvHD mouse model using lethally-irradiated Balb/c (H-2d) recipients transplanted with C57BL/6 (H-2b) T cell depleted bone marrow cells with total spleen cells were used. In some experiments, Foxp3-GFP reporter mice (C57BL/6 background) were used as the donor. RGI-2001 (0.01 ug/kg to 1 ug/kg) was given on day 0 via single intravenous administration. LD-RAPA (0.1 mg/kg) was given daily from day 0 to day 14 intraperitoneally. Treg frequency was determined on day 15 post-BMT by flow cytometry either by using an antibody for Foxp3 or by detecting GFP as a surrogate marker for Foxp3. ResultsFirst, the pharmacological dose induction of Tregs by RGI-2001 was investigated. Day 15 post-BMT analysis of mice treated with RGI-2001 demonstrated a dose-dependent increase in the % CD4+Foxp3+ Treg cells in the spleen, lymph nodes, and bone marrow. As shown in the Table, frequencies of CD4+Foxp3+ Tregs in the spleen increased in a dose dependent manner, from 3.0 ± 1.4 % in untreated mice to 14.9 ± 5.8% in 1 ug/kg RGI-2001 treated mice. The total Treg cell counts in the spleen also increased in a dose dependent manner with 1 ug/kg RGI-2001 consisting of 4.2×10e5 Tregs as compared with 0.3×10e5 Tregs in untreated mice. The frequency of CD8+ T cell population decreased in a dose dependent manner, suggesting an inverse relationship with frequencies of Tregs. When LD-RAPA was combined, a robust synergy between the two compounds was noted. The frequency of Treg in mice that received combination treatment was significantly higher than that in each monotherapy group (p = .0005 vs. RGI-2001 only, p< .0001 vs. LD-RAPA only). Absolute Treg counts in the spleen were markedly increased in mice received the combination treatment. In three independent experiments, Tregs in combination treatment group were consistently ∼3-fold higher than that in RGI-2001 monotherapy group and ∼9-fold higher than that in LD-RAPA monotherapy group. In contrast, CD8+ T cell frequencies decreased in mice treated with LD-RAPA containing regimen. Efficacy in survival was also improved in mice that received the combination treatment with 80-100% survival over 100 days.Table 1:Cellular composition of Day 15 post-BMT spleen in mice treated by RGI-2001 with or without LD-RAPA.RGI-2001-0.01 ug/kg0.1 ug/kg1 ug/kg-0.1 ug/kgRAPA----0.1 mg/kg0.1 mg/kg%Foxp3+/CD4+3.0 ± 1.4 (n=15)6.9 ± 3.0 (n=17)11.9 ± 4.1 (n=9)14.9 ± 5.8 (n=11)6.2 ± 3.1 (n=12)19.8 ± 4.1 (n=10)%CD8+/CD3+12.3 ± 1.4 (n=9)11.2 ± 4.0 (n=17)9.8 ± 4.7 (n=14)8.5 ± 4.2% (n=11)7.6 ± 2.3% (n=8)6.7 ± 2.6% (n=8)Next, the origin of the Tregs induced by RGI-2001 was investigated using the Foxp3-GFP reporter mice as the spleen cell donor. While 2.5×10e6 total spleen cells from Foxp3-GFP mice gave rise to 7.2 ± 2.4 % Tregs (n= 7) when treated with RGI-2001 (1 ug/kg), equivalent number of GFP+ Treg-depleted spleen cells gave rise to 1.4 ± 0.3 % Tregs (n=5, p < .0006). Likewise, when mice were treated with the combination of RGI-2001 (1 ug/kg) and LD-RAPA, Treg frequencies in mice that received total spleen cells and GFP+ Treg -depleted spleen cells were 17.8 ± 3.8 % (n=7) and 1.5 ± 0.4 % (n=7), respectively (p < .0001). These results together suggest that the majority of Tregs induced by RGI-2001 with or without LD-RAPA were derived from the pre-existing naturally-occurring Treg population, and the contribution of de-novo Tregs was minimal in the GvHD setting. ConclusionsRGI-2001 prevented GvHD by promoting the expansion of naturally-occurring Tregs. LD-RAPA synergistically enhanced the efficacy of RGI-2001 by significantly increasing Treg expansion while suppressing effector T cells. Disclosures:REGiMMUNE employees (OD, AL, JL, TT), Founder (YI), and consultant (RN). YI and RN are stockholders. Disclosures:No relevant conflicts of interest to declare.