allo-BMT Recipients Research Articles

Initiation of Acute Graft-Versus-Host Disease By Angiogenesis

Angiogenesis and inflammation are two closely related processes and inhibition of angiogenesis can ameliorate inflammatory diseases by reducing the recruitment of tissue infiltrating leukocytes. However, there is limited evidence on initial mechanisms of both processes and it is unknown if angiogenesis contributes to initiation of inflammation or is a mere consequence of inflammation.Recent research showed that graft-versus-host disease (GVHD) is associated to angiogenesis making the endothelium a potential new therapeutic target during allogeneic stem cell transplantation (bone marrow transplantation, allo-BMT).We studied kinetics and quantity of angiogenesis and leukocyte infiltration in murine acute GVHD models (LP/J→B6, B6→Balb/c, B6→BDF and 129S2/Sv→B6) by assessing CD31, CD4, CD8, CD11b by immune staining and FACS analysis. In GVHD target organs colon, skin and liver, we found significant increased vascular density and endothelial cell (EC) number but no leukocyte infiltration as early as day+2 after BMT (Fig. 1a,b). Leukocyte infiltration occurred secondary to angiogenesis at day+7 or later. No angiogenesis and inflammation occurred in the non-acute GVHD target organs joints, cardiac and skeletal muscle (Fig. 1c) suggesting a previously unrecognized role of the endothelium in GVHD organ tropism. Similar analyses in a standard model for inflammatory bowel disease, the 3% dextran sulfate sodium-induced (DSS) colitis, confirmed that angiogenesis preceded leukocyte infiltration in colon (d+1 vs. d+3) implicating that the significance of our results surpasses the field of transplantation biology.We aimed at identifying pathways that are relevant for initiation of angiogenesis during GVHD and first investigated the Vegfa/VEGFR1+2 axis. Based on our previous observation demonstrating a reduction of GVHD due to inhibition of neovascularization by anti-VE-cadherin antibodies (Penack et al, JNCI 2010), we performed therapeutic interventions with monoclonal blocking antibodies. Anti-VEGFR1+2 (DC101+MFI, Imclone) and anti-Vegfa (B20-4.1.1, Genentech) did not lead to GVHD reduction (Fig. 1d). Furthermore, we found no consistent upregulation of Vegfa and VEGFR2 expression levels in GVHD target organs during initiation of GVHD or later time points (Fig. 1e) suggesting that the Vefga/VEGFR2 pathway is not a major mechanism for initiation of angiogenesis in GVHD target organs.We next assessed known endothelial activation signs and performed qPCR analysis of adhesion molecules Icam1, Vcam1, E- and P-selectin (Fif. 1f) as well as immune staining of MHC-II (Fig. 1g). We confirmed previous knowledge that endothelial cells upregulate adhesion molecules and MHC-II during established GVHD. In sharp contrast, adhesion molecules and MHC-II were downregulated or not changed during the initiation of angiogenesis at day+2. This suggests that alternative pathways are important during initial angiogenesis in early GVHD.To identify alternative pathways during initial angiogenesis in an unbiased approach, we performed microarray analyses of FACS-sorted colon ECs (Fig. 2a) and LC-MS/MS proteome analyses of MACS-isolated liver ECs (Fig. 2c) at day+2 after allo-BMT vs. syn-BMT. Strikingly, we detected substantial metabolic and cytoskeletal changes in ECs of allo-BMT recipients (Fig. 2b,d). Recently, metabolic processes have been shown to be associated with cytoskeletal remodeling during EC migration and angiogenesis (Uebelhoer et al., J Vasc Res 2016). Accordingly, measuring single-cell mechanical characteristics with Real-Time deformabiliy cytometry (Otto et al., Nature Methods 2015) revealed profoundly higher deformation (D = 0.0547 ± 0.0011) and overall softening of ECs from allo-BMT recipients (Fig. 2e) indicating to enhanced EC migration and proliferation.We demonstrate that angiogenesis initiates GVHD in target organs and is restricted to target organs. Our findings implicate that endothelial biology plays a major role in GVHD organ tropism and disease development. We revealed novel genes and proteins regulating migration and proliferation of EC in initial angiogenesis during GVHD. Our study amends the knowledge on the interplay between the vasculature and inflammation opening a new window to develop therapeutic strategies targeting the endothelium. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Checkpoint Inhibitor Therapy and Graft Versus Host Disease in Allogeneic Bone Marrow Transplant Recipients of Haploidentical and Matched Products with Post-Transplant Cyclophosphamide.

Background: Concerns have been raised whether immune checkpoint inhibitor therapy in the alloBMT setting will result in graft versus host disease (GvHD) and transplant related mortality (TRM). We report our experience with a variety of checkpoint inhibitors used before or after allogeneic bone marrow transplantation (alloBMT). Our series comprises patients who received T cell-replete hematopoietic stem cells from HLA-haploidentical or -matched donors and is limited to those treated with post-transplant cyclophosphamide (PTCy) as primary GvHD prophylaxis.Patient selection: We retrospectively reviewed the records of alloBMT recipients who received PTCy and received checkpoint inhibitor therapy before or after alloBMT. GvHD was assessed using the CIBMTR GVHD index.Results:Eleven patients received checkpoint inhibitor therapy prior to alloBMT: anti-PD-1: Nivolumab n=6, anti-CTLA4: Ipilimumab n=8 (3 patients received both nivolumab and ipilimumab). These patients received a median of 4 (range 1 - 18) cycles of therapy. The median interval from last checkpoint inhibitor treatment to day of transplant was 43 (range 18-302) days. All patients received nonmyeloablative conditioning; 6 received partially mismatched allografts (5 were HLA haploidentical). Four patients developed Grade II aGvHD: Three patients who had received partially mismatched allografts (haplo-2, 9/10 unrelated-1) experienced stage 3 cutaneous GvHD only; one patient who received a 10/10 unrelated donor allograft developed stage 3 cutaneous GvHD with stage 1 liver involvement. Three patients were on immunosuppression when GvHD developed, the fourth patient with cutaneous and liver GvHD had been taken off tacrolimus on day 68 due to concerns of graft failure. GvHD resolved with treatment in each case. None of these patients developed chronic GvHD and none have died [median follow-up of 0.66 (range 0.91 - 2.0) years post alloBMT].Nine patients received checkpoint therapy following alloBMT: anti-PD-1: Pembrolizumab n = 1, Nivolumab n= 6, anti-CTLA4: Ipilimumab n= 3 (one patient received nivolumab and ipilimumab). Eight patients had received nonmyeloablative conditioning; 5 received haploidentical allografts. Six received treatment for relapse of their hematologic malignancy, 1 for relapsed pediatric sarcoma, and 2 for newly diagnosed lung cancer. The median time to initiation of checkpoint inhibitor therapy was 1.2 (range: 0.8 - 5.8) years post alloBMT. Patients received a median of 5 (range 1 - 24) cycles of therapy. There was 1 case of Grade II aGvHD; stage 3 cutaneous GvHD when DLI from a 10/10 matched unrelated donor was given for relapsed disease after ipilimumab. This resulted in GvHD which was not accompanied by the desired graft-vs-leukemia effect. There were no other cases of acute or chronic GvHD in this group. There were 4 tumor-related deaths: pediatric sarcoma (1), lung cancer (1), and AML (2). The median follow-up for this group is 2 years (range 0.85 - 8.0) post alloBMT.Conclusions: In this small series, the incidence and severity of GvHD seen in patients who received checkpoint inhibitors was similar to that seen in patients treated with PTCy as GvHD prophylaxis without checkpoint inhibitors. GvHD was seen in patients treated with checkpoint inhibitors prior to alloBMT, but was generally mild and readily controlled and there were no associated deaths. In patients treated with checkpoint inhibitors after alloBMT, the only case of GvHD occurred after the patient received DLI. We caution that use of checkpoint inhibitors in closer temporal proximity to transplant might well be associated with increased risk of GvHD or severity of GvHD. [Display omitted] DisclosuresBorrello:WindMIL Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding. Wagner-Johnston:Seattle Genetics: Research Funding. Smith:Celgene: Consultancy, Other: member of DSMB.

Modulation of Immune Checkpoints and Graft-versus-Leukemia in Allogeneic Transplants by Antagonizing Vasoactive Intestinal Peptide Signaling.

The goal of allogeneic bone marrow transplantation (allo-BMT) is elimination of leukemia cells through the graft-versus-leukemia (GvL) activity of donor cells, while limiting graft-versus-host disease (GvHD). Immune checkpoint pathways regulate GvL and GvHD activities, but blocking antibodies or genetic inactivation of these pathways can cause lethal GVHD. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide that regulates coinhibitory pathways; its role in allo-BMT has not been studied. We found VIP transiently expressed in donor NK, NK-T, dendritic cells, and T cells after allo transplant, as well as host leukocytes. A peptide antagonist of VIP signaling (VIPhyb) increased T-cell proliferation in vitro and reduced IL10 expression in donor T cells. Treatment of allo-BMT recipients with VIPhyb, or transplanting donor grafts lacking VIP (VIP-KO), activated donor T-cells in lymphoid organs, reduced T-cell homing to GvHD target organs, and enhanced GvL without increasing GvHD in multiple allo-BMT models. Genetic or ex vivo depletion of donor NK cells or CD8+ T cells from allografts abrogated the VIPhyb-enhanced GvL activity. VIPhyb treatment led to downregulation of PD-1 and PD-L1 expression on donor immune cells, increased effector molecule expression, and expanded oligoclonal CD8+ T cells that protected secondary allo transplant recipients from leukemia. Blocking VIP signaling thus represents a novel pharmacologic approach to separate GvL from GvHD and enhance adaptive T-cell responses to leukemia-associated antigens in allo-BMT. Cancer Res; 76(23); 6802-15. ©2016 AACR.

Route of delivery influences biodistribution of human bone marrow-derived mesenchymal stromal cells following experimental bone marrow transplantation

Mesenchymal stromal cells (MSCs) have shown promise as treatment for graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (alloBMT). Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs) were injected via carotid artery (IA) or tail vein (TV) into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI) using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [(99m)Tc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole-body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments.

TLR5 Agonist Recombinant Flagellin, CBLB502 Increases Anti-CMV Immunity By Increasing T-Bet and Eomes Expressing T Cells after MCMV Infection

We have previously shown that highly purified cGMP grade recombinant flagellin, CBLB502, a TLR5 agonist, protected allo-HSCT recipients from GvHD without compromising anti-CMV immunity of donor T cells in vivo. A single prophylactic dose (25mg/mouse i.p) of CBLB502 in wild type C57BL/6 (B6) mice also enhanced anti-CMV immunity of NK cells against CMV. CMV is a potential opportunistic infectious agent and most often causes severe life-threatening clinical complications in immunocompromized patients, particularly in cancer patients treated with allogenic bone marrow transplantation (allo-BMT). A mechanism by which CBLB502 protected host against CMV infection has not been well investigated. Transcription factors ROR gammaT, T-bet and EOMES are known to control immune responses of innate and adaptive immunity in vivo. In this study, we have explored the transcription factors ROR gammaT, T-bet and EOMES expressing NK and T cells in CBLB502-treated and MCMV-infected B6 mice. Total numbers of spleen cells increased by 1.7-fold within 48 hours of CBLB502 treatment (day 0 MCMV infection) and by 2.3-fold on day 3 after MCMV infection compared with PBS-treated MCMV-infected mice. T-bet expressing CD8+ T cells (not CD4+ T cells) increased significantly on day 0 of mCMV infection, 48hrs after CBLB502 treatment (CD4, p=0.1; CD8, p<0.001) and both CD4+ and CD8+ T cells increased significantly on day 3 (CD4, p<0.001; CD8, p<0.01) after MCMV infection. CBLB502 treatment had no effect on the ROR gammaT expression on T cells. Interestingly, although EOMES expressing CD4+ and CD8+ T cells increased significantly on day 0 (48 hrs after CBLB502 treatment) (CD4, p<0.01; CD8, p<0.01) and day 3 (CD4, p<0.001; CD8, p<0.001) in the spleens of CBLB502-treated mice compared with the PBS-treated mice, EOMES expression on CMV-specific tetramer+ CD8+ T cells rapidly decreased by day 10 after infection in both CBLB502 and PBS treated mice. However, >90% of CMV-specific CD8+ T cells had persistent expression of T-bet even after 30 days post CMV infection. In contrast, CBLB502 treatment did not have a significant effect on the numbers of ROR gammaT, T-bet and EOMES expressing NK cells compared with PBS-treated mice. These data suggest that 1) CBLB502-induced anti-CMV immunity of T cells is persistently maintained through increased T-bet expression but not by EOMES expression; and 2) CBLB502-induced anti-viral immune regulation is not maintained through transcription factor ROR gammaT. 3) CBLB502-induced increased anti-CMV immunity of NK cells is independent of transcription factors ROR gammaT, T-bet and EOMES. Taken together, these data support the early peri-transplant use of TLR5 agonists as a novel method to enhance antigen-specific anti-viral immunity in recipients of allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T Cell Activation By Plasmacytoid Dendritic Cells Is Augmented By Inhibition of Vasoactive Intestinal Polypeptide Signaling: Implications for Cellular Immunotherapy

Introduction:Data from clinical allogeneic bone marrow transplant (allo-BMT) and pre-clinical murine models of allo-BMT have shown that donor plasmacytoid dendritic cells (pDC) have important roles in regulating graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activities of donor T cells. Using murine models of allo-BMT we have previously shown that 1) donor pDCs induce Th1 polarization of donor T cells and augment the GvL activity of T cells; and 2) the addition of pDC to grafts composed of purified T cells and HSC limited the subsequent development of GvHD. VIP is an immunosuppressive neuropeptide that regulates adaptive immune responses. We reasoned that VIP signaling may regulate activation of allo-specific T-cells, and the VIP pathway may be target for regulating GvHD and GvL in allo-BMT.Methods:To explore the mechanisms by which pDC and VIP signaling regulate T cell activation we used: 1) transgenic mice expressing GFP under the control of the VIP promoter to measure VIP expression in vivo in allo-BMT recipients; 2) one-way mixed lymphocyte reaction (MLR) to measure the proliferative response of transgenic luciferase positive T cells in response to allo-antigens via bioluminescence imaging (BLI); and 3) a model system of indirect presentation of allo-peptides derived from a H2-Ab MHC class II molecule by pDC to transgenic T cells expressing the TEa TCR. The effect of blocking vasoactive intestinal polypeptide (VIP) signaling during activation of allo-reactive T cells was assessed by using VIP-KO cells and by the addition of VIP peptide or a peptide antagonist of VIP (VIPhyb) to the one-way MLR. T cell proliferation and activation was measured by flow cytometry.Results:Analysis of VIP expression in donor pDC in murine models of allo-BMT showed >100-fold induction of VIP promoter activity in donor pDC and donor T cells during the first two weeks post-transplant, indicating that VIP expression in donor pDC may regulate T cell activation.Addition of endogenous native VIP suppressed T cell proliferation in one-way MLR but was reversed by addition of a 10x concentration of the VIP antagonist peptide (Figure 1B). Furthermore, adding 3 uM of the VIP antagonist to the MLR cultured significantly enhanced T cell proliferation. TEa peptide-primed T cells cultures with peptide-primed pDC from VIP knock-out mice had increased proliferation and expressed more of the activation markers CD69 and CD71 compared with T cells cultured with VIP-WT pDCs. Comparing pDC purified from marrow versus spleen, we found significantly more proliferation in T cells cultured with bone marrow VIP-KO pDCs than splenic VIP-KO pDCs, indicating that the less mature marrow pDC have greater antigen presenting ability than the more mature splenic pDC.Conclusion:These data suggest that 1) VIP is produced by donor pDC early after allo-BMT; 2) VIP inhibits T cell allo-proliferation in one-way MLR's; and 3) blocking VIP signaling using donor cells that cannot produce VIP or through the use of pharmacological inhibitors of VIP can augment activation and proliferation of T cells in response to indirect antigen presentation.The present findings support studies of VIP antagonist in allo-BMT to augment the GvL activity of T cells through indirect antigen presentation. Future studies include using the BLI analysis of MLR to determine the effect of novel drugs on T cell proliferation.**Jingxia Li and Reema Panjwani were equal contributors to this abstract. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNA profiling in peripheral blood, buccal swabs, hair follicles and semen from a patient following allogeneic hematopoietic stem cells transplantation.

Allogeneic peripheral blood stem cells transplantation (allo-PBSCT) or allogeneic bone marrow transplantation (allo-BMT) have been widely used to treat patients exhibiting certain severe illnesses. However, previous studies have shown that the biological materials of allo-PBSCT or allo-BMT recipients may not constitute credible materials for personal identification. In the present study, four types of commonly used samples were collected from a male individual following gender-matched allo-BMT. Autosomal short tandem repeat (STR) and Y-STR markers analysis, based on polymerase chain reaction, were used to evaluate the chimerism status. The results showed that the blood sample were all donor type, the buccal swab sample were mixed chimerism, and the sperm and hair follicle samples maintained a recipient origin of 100%. In conclusion, identical results were obtained by the two methods and it was confirmed that DNA extracted from hair follicles and sperm can be used as a reference for the pre-transplant genotype DNA profile of the recipient in the gender-match allo-BMT or -PBSCT.

Recombinant TLR5 agonist CBLB502 promotes NK cell-mediated anti-CMV immunity in mice.

Prior work using allogeneic bone marrow transplantation (allo-BMT) models showed that peritransplant administration of flagellin, a toll-like receptor 5 (TLR5) agonist protected murine allo-BMT recipients from CMV infection while limiting graft-vs-host disease (GvHD). However, the mechanism by which flagellin-TLR5 interaction promotes anti-CMV immunity was not defined. Here, we investigated the anti-CMV immunity of NK cells in C57BL/6 (B6) mice treated with a highly purified cGMP grade recombinant flagellin variant CBLB502 (rflagellin) followed by murine CMV (mCMV) infection. A single dose of rflagellin administered to mice between 48 to 72 hours prior to MCMV infection resulted in optimal protection from mCMV lethality. Anti-mCMV immunity in rflagellin-treated mice correlated with a significantly reduced liver viral load and increased numbers of Ly49H+ and Ly49D+ activated cytotoxic NK cells. Additionally, the increased anti-mCMV immunity of NK cells was directly correlated with increased numbers of IFN-γ, granzyme B- and CD107a producing NK cells following mCMV infection. rFlagellin-induced anti-mCMV immunity was TLR5-dependent as rflagellin-treated TLR5 KO mice had ∼10-fold increased liver viral load compared with rflagellin-treated WT B6 mice. However, the increased anti-mCMV immunity of NK cells in rflagellin-treated mice is regulated indirectly as mouse NK cells do not express TLR5. Collectively, these data suggest that rflagellin treatment indirectly leads to activation of NK cells, which may be an important adjunct benefit of administering rflagellin in allo-BMT recipients.

Microbial metabolites modulate GI mucosal damage from graft versus host disease (GHVD). (MUC4P.850)

Abstract The microbiome of the GI tract is known to affect GI mucosal responses and regulate disease processes. GI tract damage is the major cause for graft versus host disease (GVHD) mortality. Studies have shown the microbiome affects the course of allogeneic bone marrow transplant and modulates GVHD. However, little is known about the microbial metabolites crucial for achieving the effects of the microbiome on immune responses, specifically GVHD. We examined the metabolome of naïve mice and murine recipients of syngeneic (B6→B6) and allogeneic (B6→BALB/c) BMT, in an unbiased way. We made a surprising observation that short chain fatty acids (SCFA), including butyrate, were altered in the intestinal epithelium but not in the luminal contents of allo-BMT recipients. Butyrate, a known metabolite of the microbiome was previously shown to have HDAC inhibitor (HDACi) and salutary effects on the intestinal epithelium. Additionally, recent data showed systemic chemical inhibition of HDAC protects GI epithelium from GVHD severity. The decrease of butyrate in the epithelial cells of the small intestine (P&amp;lt;0.03) suggested poor uptake of butyrate following allo-BMT. Consistent with this notion, we found that treatment of epithelial cells with butyrate confers protection from irradiation and decreases allo-induced cell death. Our results suggest that the microbiome regulates the damage from allogeneic GVHD due to the reduction in the tissue level of the SCFA-metabolite and HDACi, butyrate.

Absence of Post-Transplantation Lymphoproliferative Disorder after Allogeneic Blood or Marrow Transplantation Using Post-Transplantation Cyclophosphamide as Graft-versus-Host Disease Prophylaxis

Immunosuppressive regimens that effectively prevent graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (allo-BMT) have been associated with an increased incidence of post-transplantation lymphoproliferative disorder (PTLD) in the first year after transplantation. We evaluated the incidence of PTLD associated with the use of high-dose post-transplantation cyclophosphamide (PTCy) as GVHD prophylaxis. Between 2000 and 2011, a total of 785 adult allo-BMT recipients were given PTCy as GVHD prophylaxis at the Johns Hopkins Hospital, including 313 patients who received PTCy as sole GVHD prophylaxis. HLA-haploidentical or unrelated donor graft transplantation was performed in 526 patients (67%). No cases of PTLD occurred during the first year after allo-BMT in this series. PTLD is a rare occurrence after allo-BMT using PTCy, even in high-risk alternative donor transplantations.

T Cell–Specific Notch Inhibition Blocks Graft-versus-Host Disease by Inducing a Hyporesponsive Program in Alloreactive CD4+ and CD8+ T Cells

Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4(+) T cells in mice. To elucidate the mechanisms of Notch action in GVHD and its role in CD8(+) T cells, we studied the effects of Notch inhibition in alloreactive CD4(+) and CD8(+) T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4(+) or CD8(+) T cells. Both CD4(+) and CD8(+) Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased IFN-γ production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited decreased Ras/MAPK and NF-κB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb, and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4(+) and CD8(+) T cells that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD.

Pharmacological inhibition of VIP signaling enhances antiviral immunity and improves survival in murine cytomegalovirus-infected allogeneic bone marrow transplant recipients

AbstractCytomegalovirus (CMV) infection following allogeneic bone marrow transplant (allo-BMT) is controlled by donor-derived cellular immunity. Vasoactive intestinal peptide (VIP) suppresses Th1 immunity. We hypothesized that blocking VIP-signaling would enhance anti-CMV immunity in murine recipients of allo-BMT. Recipients were transplanted with bone marrow (BM) and T-cells from major histocompatibility complex (MHC)–mismatched VIP-knockout (KO) or wild-type donors, and treated with 7 daily subcutaneous injections of VIPhyb (peptidic VIP-antagonist) or phosphate-buffered saline (PBS). Genetic and pharmacological blockade of VIP-signaling protected allo-BMT recipients from lethal murine CMV (mCMV) infection, improving survival without increasing graft-versus-host disease. Mice treated with VIPhyb or transplanted with VIP-KO allografts had significantly lower viral loads, increased numbers of mCMV-M45-peptide-MHC-tetramer+ CD8+ T-cells, with lower PD-1 expression, and enhanced primary and secondary cellular immune responses after mCMV infection than did PBS-treated mice. These results demonstrate that administration of a VIP antagonist after allo-BMT is a promising safely therapeutic approach to enhance antiviral cellular immunity.

Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

AbstractAbstract 255 Background:Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods:To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results:Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection.In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures:No relevant conflicts of interest to declare.

Intestinal Microbiota in Bone Marrow Transplantation

Abstract SCI-51Regulators of the intestinal flora include diet, antibiotics, and importantly, intestinal inflammation. As a result, the cause-effect relationships between intestinal inflammation and changes in microbiota have been difficult to define. The success of allogeneic bone marrow transplantation (allo BMT), a standard therapy for conditions including hematopoietic malignancies and inherited hematopoietic disorders, is limited by graft-versus-host disease (GVHD) morbidity and mortality. With GVHD, vigorous activation of donor immune cells, most importantly T cells, leads to damage of the skin, liver, hematopoietic system, and gut. The major sources of immune activation are histocompatibility complex differences between donor and recipient. Combinations of chemotherapy and radiation also contribute, as damage to the intestinal epithelium results in systemic exposure to microbial products normally sequestered in the intestinal lumen. We have demonstrated in murine and human recipients of allo BMT that intestinal inflammation secondary to GVHD is associated with major shifts in the composition of the intestinal microbiota. The microbiota, in turn, can modulate the severity of intestinal inflammation. In mouse models of GVHD we observed loss of overall diversity and expansion of Lactobacillales and loss of Clostridiales. Eliminating Lactobacillales from the flora of mice prior to BMT aggravated GVHD, while reintroducing the predominant species of Lactobacillus mediated significant protection against GVHD. We then characterized the gut flora of patients during onset of intestinal inflammation due to GVHD and found patterns mirroring those in mice. We also identified increased microbial chaos soon after allo BMT as a potential risk factor for subsequent GVHD. Together, these data demonstrate regulation of flora by intestinal inflammation, and suggest that flora manipulation may reduce intestinal inflammation and improve outcomes for allo BMT recipients. Disclosures:No relevant conflicts of interest to declare.

Effect of IL-22 on intestinal stem cells, GVHD-related tissue damage, and GVL.

6539 Background: Intestinal graft vs. host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (allo-BMT). Strategies to limit GVHD by selective promotion of epithelial regeneration in the absence of immunosuppression are largely unknown. Methods: We investigated the role of IL-22 in allo-BMT by utilizing wild type and IL-22 knockout (KO) mice as donors or recipients in allo-BMT. Results: We found that administration of an anti-IL-22 neutralizing antibody to allo-BMT recipients during the first month post-BMT led to increased GVHD mortality, indicating that IL-22 functioned to reduce GVHD severity post-BMT. In contrast, use of IL-22 KO donor T cells did not impair their ability to eliminate A20 lymphoma cells upon tumor challenge, thus indicating that IL-22 was not essential for donor T cells to mediate graft vs. lymphoma (GVL) responses. BMT with WT donors and recipients indicated that IL-22 levels in small and large intestine were increased after BMT and after radiation injury (RI) without BMT. IL-22 upregulation after RI was dependent on the presence of IL-23p40. Although intestinal IL-22 levels were increased after T cell-depleted (TCD) BMT, intestinal IL-22 was reduced by GVHD, as IL-22 production was mediated by host-derived innate lymphoid cells (ILC) that were eliminated by GVHD. Furthermore, host-derived IL-22 was critical for reduction of GVHD morbidity, mortality, and intestinal pathology. GVHD in IL-22 KO mice led to increased apoptosis in epithelial crypts where the intestinal epithelial stem/progenitor cell niche is located. Immunohistochemistry and immunofluorescence demonstrated IL-22 receptor expression on intestinal stem cells (ISC) and progenitors. Allo-BMT in Lgr5-LacZ reporter mice indicated that ISC were targeted by GVHD, and GVHD in IL-22 KO mice led to dramatic ISC depletion. Conclusions: IL-22 is critical for protection of host epithelium during GVHD and critical for protection of ISC, and it does not contribute to donor T cell GVL responses. These findings may have broad relevance for protection of ISC and intestinal epithelium in clinical GVHD and other inflammatory intestinal diseases, and may be useful for clinical separation of pathologic GVH and therapeutic GVL responses.

Increased Susceptibility to Fungal Infection in Recipients with GVHD Is Not Due to Impaired Pathogen Clearance and Can Be Prevented by Co-Transfer of Donor CD4+CD25+ Regulatory T Cells

Abstract 822Opportunistic fungal infections - in particular invasive aspergillosis (IA) – are still a cause of considerable morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). Susceptibility is particularly high during neutropenia early after allo-BMT. However, this period is usually only temporary and nowadays largely manageable by prophylactic anti-fungal medication. Graft-versus-host disease (GVHD) represents another frequent, life-threatening complication after BMT. It is mediated by mature alloreactive donor T cells and leads to dysregulated pro-inflammatory cytokine secretion and target organ destruction. GVHD ≥ grade II has repeatedly been identified as independent risk factor for late onset IA. However, due to the simultaneous pharmacologic treatment of patients with GVHD, the contribution of GVHD itself to course and severity of a fungal infection is so far incompletely understood. To answer this question, we employed an MHC-mismatched murine allo-BMT model (C57BL/6 into BALB/c), in which lethally irradiated recipients received 2.5 × 106 BM cells either alone (control group without GVHD) or together with 1 × 106 splenocytes (development of GVHD within 28 d after BMT [clinical GHVD score on average 4.0 (range: 3–5.5)]). On d 28 after BMT half of the recipients within both groups were infected with Aspergillus fumigatus (A.f.) conidia (strain D141; 0.5 × 106 i.v.) and all animals were subsequently monitored for survival. Whereas all non-infected and 60% of infected BM control animals survived the observation period of 30 d, all infected recipients with GVHD died within 10 d after infection. As expected, 90% of uninfected recipients with GVHD also died within 30 d, however, animals of this group survived significantly longer than their infected counterparts (p=0.017 for infected vs. uninfected GVHD group and p=0.004 for infected GVHD versus infected BM control group; n=5–9). Comparable results were obtained after i.t. infection of recipients with and without GVHD. To analyze pathogen clearance, BMT recipients with or without GVHD were infected, sacrificed 1 or 2 d later and CFU determined in lung (after i.t. or i.v. infection) as well as spleen, liver, kidney and brain (after i.v. infection). CFU decreased rapidly and comparably in all organs and in both recipient groups, indicating that pathogen clearance is not impaired in allo-BMT recipients with GVHD. However, leukocytes re-isolated from animals with GVHD reacted much stronger to in vitro stimulation with live germinated A.f. conidia than cells from BM controls, as indicated by a significantly increased release of TNF (10951 ± 4673 pg/ml vs 862 ± 256 pg/ml from splenocytes of GVHD and BM control animals, respectively; p= 0.045, n=4–5 animals/group; 7640 ± 3524 pg/ml vs. 2556 ± 620 pg/ml from liver leukocytes of GVHD and BM control animals, respectively; p=0.02, n=3 independent experiments). Similar results were obtained for IL-6 and IFN-γ. These data suggest that an unrestrained and dysregulated immune response to the fungus occurs in animals with GVHD. To further investigate this hypothesis, we transplanted an additional group with donor CD4+CD25+ Treg cells (0.25 × 106/mouse), BM cells (2.5 × 106) and splenocytes (1 × 106), infected the mice with A.f. conidia and compared their survival rate to that of infected GVHD animals. Interestingly, whereas again all mice without Treg cell transfer (GVHD group) died after infection, 60% of the animals that received Treg cells survived the observation period of 30 d (p=0.011; n=10). Importantly, Treg cell transfer did not interfere with pathogen clearance. The in vitro immune response to live A.f. conidia of lymphocytes re-isolated from Treg recipients, however, as measured by TNF secretion, was significantly reduced and resembled that of BM controls (TNF from liver leukocytes in pg/ml: BM control: 1693 ± 374; GVHD: 8280 ± 3561; Treg: 1921 ± 642; p=0.02 BM vs GVHD and GVHD vs Treg; n=3 independent experiments).In summary, our data indicate that an uncontrolled anti-fungal immune response rather than an impaired ability to deal with the pathogen underlies the increased mortality observed in murine allo-BMT recipients with GVHD. Furthermore, we show that co-transferred CD4+CD25+ donor Treg cells do not interfere with pathogen clearance but confine the anti-fungal immune response and thereby increase survival of infected animals. Disclosures:No relevant conflicts of interest to declare.

Over-Expression of TRAIL on Donor T Cells Enhances GVT and Suppresses Gvhd Via Elimination of Alloreactive T Cells and Host APC

Abstract 817Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p<0.01, 100% survival in TRAIL+ T cell group) (Fig 1A, also shown at the annual meeting last year). Precursor (pre)T cells have the benefit of regenerating the T cell compartment without causing GVHD and being available for “off the shelf” use. We generated TRAIL+ preT cells from transduced B6 hematopoietic stem cells and expanded them using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ preT cells into syngeneic-transplanted BALB/c mice could reconstitute the T cell compartment with TRAIL-expressing T cells and caused enhanced antitumor activity (p<0.05) compared to mock (GFP)-transduced controls.Interestingly, in addition to enhanced GVT, the recipients treated with TRAIL+ T cells had significantly less GVHD lethality and morbidity (Fig1B). This was observed across multiple GVHD models (B6 ^ CBF1, B6^ BALB/c and B10.BR^ B6). To explore the factors contributing to TRAIL-mediated suppression of GVHD, we used animals deficient in DR5 (DR5ko) in our models of GVHD. We found that GVHD suppressive effects of TRAIL were lost when hosts were DR5ko or when DR5ko TRAIL+ T cells were adoptively transferred indicating that TRAIL+ T cells suppress GVHD by targeting both host and donor compartments. We observed a higher DR5 expression in host MHC-IIhi antigen presenting cells (APC) following total body irradiation, suggesting that TRAIL+ donor T cells could potently eliminate host APC, resulting in less GVHD. Further, on transferring wild type T cells into irradiated hosts, we found that alloreactive CD25+ T cells had a significantly higher DR5 expression compared to CD25− T cells. This indicates that TRAIL+ T cells can specifically target the alloreactive CD25+ T cells in order to suppress GVHD.Collectively, our data demonstrate that donor T cells genetically engineered to express TRAIL can enhance GVT effects and suppress the development of lethal GVHD in recipients of allo-HSCT. Our data suggests that his suppression of GVHD is mediated by the elimination the alloreactive donor T cells and the elimination of GVHD-promoting residual APC. Furthermore, we demonstrated that allogeneic ex vivo generated preT cells expressing TRAIL could mediate a strong protection against tumor challenge in syngeneic HSCT recipients. TRAIL over-expression thus represents a potential off the shelf approach to enhancing GVT in both allogeneic and autologous transplantation. Despite elimination of alloreactive donor T cells, TRAIL+ T cells demonstrated enhanced GVT by directly targeting DR5+ tumors in the absence of alloreactivity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Dasatinib as Salvage Therapy for Steroid Refractory and Imatinib Resistant or Intolerant Sclerotic Chronic Graft-versus-Host Disease

Sclerotic chronic graft-versus-host disease (scGVHD) is a severe form of this disease that resembles systemic sclerosis and has limited and disappointing treatment options. Tyrosine kinase inhibitors (TKI) targeting up-regulated profibrotic pathways, such as imatinib mesylate, have been proposed as a potential therapeutic approach for patients with scGVHD. Dasatinib, a second-generation TKI with a well-established safety and efficacy profile in chronic myeloid leukemia patients, who are refractory or intolerant to imatinib, has also shown potent antifibrotic effects. We present here the first direct clinical evidence, from 3 patients treated in a small single-center series, suggesting that dasatinib can be a therapeutic option for patients with severe scGVHD resistant or intolerant to imatinib. All patients achieved partial response, with improvement in scGHVD target organs severity, joint mobility, lung impairment, and deep fibrotic lesions. This clinical response has remained stable or continued to improve after a median of 22 months (20-25) on dasatinib treatment, with very good tolerance. In addition, corticosteroids could be discontinued or significantly reduced in all patients. This clinical evidence suggests that dasatinib could be a safe and effective alternative for scGVHD patients refractory to corticosteroids and resistant or intolerant to imatinib. Based on these preliminary findings, and in order to address appropriate patient selection, time of intervention, and choice of drug, future larger studies should more formally establish the efficacy and safety of second-generation TKI for the treatment of scGVHD.

Ceacam1 Separates Graft-versus-Host-Disease from Graft-versus-Tumor Activity after Experimental Allogeneic Bone Marrow Transplantation

BackgroundAllogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models.MethodsWe studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1−/− T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25hi, CD62Llo). Additionally, Ceacam1−/− CD8 T cells had greater expression of the gut-trafficking integrin α4β7, though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1−/− recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1−/− mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1+ lymphoma model was improved in animals receiving Ceacam1−/− vs. control T cells.ConclusionsWe conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.

Abstract 4743: Genetically engineered allogeneic T Cells for BMT immunotherapy: Expression of TRAIL and PLZF selectively enhances GVT and abrogates GVHD

Abstract Efforts to improve graft-versus-tumor (GVT) activity of alloreactive donor T cells are limited by a concomitant rise in graft-versus-host disease (GVHD). We employed experimental allogeneic bone marrow transplantation (allo-BMT) models to assess two novel strategies, in which T lineage cells were genetically engineered to enhance selective effector functions. In addition to the transgenic expression of these molecules on mature donor T cells, we also used genetically engineered precursor T cells as an “off the shelf” adoptive cell therapy. One strategy relies on T cell cytolytic molecule TNF-Related Apoptosis Inducing Ligand (TRAIL), which induces apoptosis through death receptors (DR) 4 and 5 (only DR5 in mice). Certain tumor cells express high levels of DR5 making TRAIL an attractive candidate for engineering T cells to augment GVT. Mature T cells were transduced with a lentiviral TRAIL expression vector and adoptively transferred into lethally irradiated allogeneic bone marrow transplantation (allo-BMT) recipients, bearing LB27.4 tumors (B6 into CBF1+LB27.4). TRAIL+ T cells had enhanced antitumor effect compared to control-transduced T cells in vitro and upon transfer into tumor-bearing allo-BMT recipients (p&amp;lt;0.01). Interestingly, the recipients of TRAIL+ T cells had significantly less GVHD. We generated TRAIL+ precursor (pre)T cells using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ preT cells into syngeneic-transplanted BALB/c mice resulted in reconstitution with TRAIL+ T cells. When challenged with localized RENCA tumors, recipients of TRAIL+ preT cells had enhanced antitumor activity (p&amp;lt;0.05) compared to recipients of control-transduced preT cells. The second strategy explores the effect of overexpressing the transcription factor promyelocytic leukemia zinc finger (PLZF) in T cells. PLZF transgenic (tg) T cells have an effector/memory phenotype with innate-like characteristics. Following transfer, they cells display an altered cytokine profile with increased expression of TNF-alpha and decreased IFN-gamma. Using adoptive transfer of CFSE-labeled donor T cells we observed that fast proliferating allo-responsive PLZF+ T cells died after only a few cell cycles. Using PLZF Tg mice, we found that PLZF+ T cells cause less GVHD with intact GVT activity (p&amp;lt;0.001) against A20 lymphoma. We then transduced T cells with PLZF expression vector and confirmed that donor PLZF+ T cells have less GVHD and intact GVT activity. Our data suggest that adoptive therapy with genetically engineered TRAIL+ or PLZF+ T cells cause less GVHD while displaying intact or enhanced GVT activity. Furthermore, the “off the shelf” use of genetically enhanced preT cells represents a promising cell therapy strategy to enhance anti-tumor activity in both autologous and allo-BMT patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4743. doi:10.1158/1538-7445.AM2011-4743