T-cell Depleted Bone Marrow Research Articles

Intestinal Immune Conditioning with Helminths Employs Host T Helper 2 (Th2) Pathway to Induce Mixed Chimerism and Regulate Graft-Versus-Host Disease

Mixed hematopoietic chimerism is proven to lead to immune tolerance after bone marrow transplant (BMT) and can reduce graft-vs-host disease (GVHD)-related mortality. It can be achieved with chemotherapeutic agents or strategies that block activation of donor or depletion of host T cells. Immune conditioning with helminthic commensals regulate the host immune system and promotes a transient mixed chimerism. Conditioning with helminths induces IL4 production via host Th2 pathway and regulates GVHD while preserving graft-versus-tumor effect. IL4 signals through IL4 receptor (IL4R) and STAT6. We hypothesize that induction of host Th2 pathway is critical for achieving mixed chimerism by intestinal immune conditioning Methods We evaluated the role of host Th2 signaling in the establishment of mixed chimerism in an MHC I/II major mismatch (H2b→H2d) model of BMT using total body irradiation (TBI). Three weeks after infection with mouse nematode h. polygyrus-bakeri (Hpb), GVHD was induced via delivery of T cell-depleted bone marrow (TCD-BM) and splenic T cells from uninfected WT C57BL/6 (MHC:H2b) donors into lethally-irradiated WT BALB/c, IL4 -/-, IL4Rα-/- and STAT6-/- recipients (MHC: H2d). Percentage of donor vs recipient T cells, GVHD-mediated inflammation in the colon and lung, and survival of mice were assessed Results In 3 models of disruption of the Th2 pathway, we demonstrated that host Th2 signaling is essential for establishing mixed chimerism. Helminth infection promotes mixed chimerism in WT BALB/c BMT recipients; recipient T cells constitute 3-5% of peripheral population at 6 days post-BMT in infected mice, and Fig 1 ). Helminths regulate GVHD-related inflammation in lungs and the colon of WT BALB/c recipients (p Fig 2 ). Helminth infection did not promote survival of Th2-/- BMT recipients; all mice died of lethal GVHD, in contrast to WT BALB/c BMT recipients (P Conclusions Immune conditioning with helminths promotes mixed chimerism after TBI and regulates lethal GVHD following BMT. The host Th2 pathway is critical for achieving this mixed chimerism and regulation of GVHD, and induction of CTLA4 is partially dependent on Th2 signaling.

Distinct Immune Regulatory Potential of Invariant Natural Killer T (iNKT) Cell Subsets: iNKT2 and iNKT17, but Not iNKT1, Protect from Graft-Versus-Host-Disease

Invariant natural killer T (iNKT) cells can potently inhibit graft-versus-host-disease (GVHD) in animal models through the production of Interleukin-4. Once considered a homogeneous population, it is now well established that murine iNKT cells differentiate during thymic development into three distinct sublineages, distinguished based on the expression of transcription factors and effector molecules: Th1-like iNKT (iNKT1) cells, Th2-like iNKT (iNKT2) cells, and Th17-like iNKT (iNKT17) cells. In this study, we investigated the immune regulatory potential of iNKT1, iNKT2 and iNKT17 cell subsets. In order to identify surface molecules whose expression would allow us to isolate viable iNKT1, iNKT2 and iNKT17 cells, we performed single cell RNA sequencing analysis on murine thymic iNKT cells isolated from FVB/N mice by flow cytometry based on PBS-57-CD1d-Tetramer staining. The results of this analysis together with previous knowledge allowed us to develop a sorting strategy to isolate iNKT1 (ICOS- PD1- CD27+ CD24-), iNKT2 (ICOS+ PD1+ CD27+ CD4+ CD24-), iNKT17 (ICOS+ PD1+ CD4- CD27- CD24-) cells, whose purity was confirmed by intra-nuclear staining for the transcription factors PLZF and RORgT. To assess the immune regulatory potential of the three iNKT sublineages, we employed a murine major histocompatibility complex (MHC)–mismatched bone marrow transplantation model. Lethally irradiated BALB/c (H-2Kd) mice were injected intravenously with T cell depleted-bone marrow (TCD-BM) cells and conventional CD4 and CD8 T cells (Tcon, 10e6 cells/mouse) from FVB/N (H-2kq) mice. Additionally, 5 × 10e4 sorted iNKT1, iNKT2 or iNKT17 cells from FVB/N donors were injected the same day. A significant survival benefit was observed when iNKT2 (p=0.0166) and iNKT17 (p=0.033) cells were adoptively transferred compared to mice that only received TCD-BM and Tcon, whereas there was no survival benefit in the group that received iNKT1 cells. In addition, body weight was improved in mice that received iNKT2 (day +41: p=0.009, day +49: p=0.005 and day +59: p= 0.005) or iNKT17 (day +59: p= 0.006) compared to mice that received iNKT1 cells. Clinical GVHD scores were also improved in mice that received iNKT2 (day +41: p= 0.012, day +51: p= 0.005) or iNKT17 (day +28: p=0.05, day +51: p=0.007) compared to mice that received iNKT1 cells. Interestingly, we found that even 1 × 10e4 iNKT2 (p= 0.008) and iNKT17 (p= 0.04) significantly suppressed GVHD. In summary, we demonstrate here that only iNKT2 and iNKT17 cells mitigated GVHD, whereas iNKT1 had no detectable immune regulatory potential. To our knowledge, this is the first study to show functional differences between iNKT sublineages, indicating that iNKT1, iNKT2 and iNKT17 cells have diverse functions and providing new biological insights that will be useful for developing iNKT cell-based cell therapies.

SatM Involved in Chronic Gvhd after Bone Marrow Transplantation.

Background GVHD is a result of immune attack of host tissues, such as the skin, gut, liver, and lung, by various immune cells in hematopoietic stem cell transplantation (HSCT). Chronic GVHD (cGVHD) is the main cause of late death and morbidity after allogeneic HSCT. Fibrosis constitutes the end stage of the inflammatory process in cGVHD leading to major morbidity. Recently, segregated-nucleus-containing atypical monocytes (SatM) that involved in lung fibrosis has been identified. We hypothesized that SatM involved in tissue fibrosis of chronic GVHD after bone marrow transplantation. Materials and Methods Lung cGVHD model: B10.BR mice received were conditioned with Cyclophosphamide (120mg/kg/day, intraperitoneally, day-3 and -2) and TBI (8.3Gy, day-1), followed by infusion of 10•∼106 C57BL/6j T cell–depleted BM (TCD-BM) plus 60000 purified splenic T cell. Respiratory function test and flowcytometry of BALF were analyzed at BMT day56. Sclerodermatous cGVHD model: BALB/c mice received a single dose of 5.8 Gy x-ray total body irradiation. Recipient mice were injected with 2•∼106 purified splenic T cells and 8•∼106 TCD-BM cells from B10.D2 donors. Donor cells were injected intravenously into the recipients on day 0. The SatM in the skin and the spleen are analyzed by flow cytometry at BMT day28. Results Compared to syngeneic group of the lung cGVHD model, allogeneic recipient developed fibrosis around the bronchi. In the respiratory function test, allogeneic recipient showed higher respiratory resistance. No difference was observed in the number of splenic monocytes by flowcytometry. However, Ly6clow monocyte, Ly6c+ monocyte and SatM increased in BALF of allogeneic recipient. In the sclerodermatous cGVHD model, SatM increased in ear of allogeneic recipient but not in spleen ( Figure 1 ). Discussion In this study, SatM observed in tissues targeted by cGVHD, but not in spleen. This result may indicate that SatM involved in fibrosis of cGVHD model. To demonstrate that SatM causes the fibrosis of cGVHD, we currently establish cGVHD model with SatM deficiency mouse (Cebpb−/− mouse).

Tracking T Cell Activation By OX40 ImmunoPET: A Novel Strategy for Imaging of Graft Versus Host Disease

Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior noninvasive imaging strategies are critically needed. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F FDG) has largely been confounding, mainly due to its low specificity. Monitoring T cell activation and expansion using Tcell targeted PET tracers seems a more promising approach. We recently reported a novel immunoPET tracer (64CuDOTAmAbOX40) that enables noninvasive imaging of activated T cells expressing the cell surface activation marker OX40. In the present work, we evaluated the utility of this immunoPET strategy to image activated T cells in a major MHCmismatch mouse model of acute GvHD. Lethally irradiated Balb/C (H2Kd) recipients received 5 × 10e6 Tcell depleted bone marrow (BM) cells intravenously (i.v.) with or without 10e6 CD4 and CD8 T cells from C57BL/6 (H2Kb) mice. AntiOX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (average ratio of 1.4 DOTAs per mAb) and radiolabeled with 64CuCl2 (final specific activity 10 15μCi/μg, radiochemical purity >99%). Mice were tail vein injected with 64CuDOTAmAbOX40 (100 μCi, i.v.) at day 7 after HCT and PETCT performed 24h after injection. In vivo OX40ImmunoPET revealed increased radiotracer uptake in spleen, mesenteric LN and abdominal region of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64CuDOTAmAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Immediately following PETCT radioactivity was measured in dissected weighed tissues using a gammacounter. Biodistribution analysis confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01), small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). In conclusion, the OX40 immunoPET tracer enabled specific imaging of OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64CuDOTAmAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting. Graft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior noninvasive imaging strategies are critically needed. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F FDG) has largely been confounding, mainly due to its low specificity. Monitoring T cell activation and expansion using Tcell targeted PET tracers seems a more promising approach. We recently reported a novel immunoPET tracer (64CuDOTAmAbOX40) that enables noninvasive imaging of activated T cells expressing the cell surface activation marker OX40. In the present work, we evaluated the utility of this immunoPET strategy to image activated T cells in a major MHCmismatch mouse model of acute GvHD. Lethally irradiated Balb/C (H2Kd) recipients received 5 × 10e6 Tcell depleted bone marrow (BM) cells intravenously (i.v.) with or without 10e6 CD4 and CD8 T cells from C57BL/6 (H2Kb) mice. AntiOX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (average ratio of 1.4 DOTAs per mAb) and radiolabeled with 64CuCl2 (final specific activity 10 15μCi/μg, radiochemical purity >99%). Mice were tail vein injected with 64CuDOTAmAbOX40 (100 μCi, i.v.) at day 7 after HCT and PETCT performed 24h after injection. In vivo OX40ImmunoPET revealed increased radiotracer uptake in spleen, mesenteric LN and abdominal region of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64CuDOTAmAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Immediately following PETCT radioactivity was measured in dissected weighed tissues using a gammacounter. Biodistribution analysis confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01), small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). In conclusion, the OX40 immunoPET tracer enabled specific imaging of OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64CuDOTAmAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting. Figure 1.

PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD.Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores.Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients.Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vasoactive Intestinal Peptide Synthesis in Transplant Recipients Critically Regulate Donor T Cell Alloreactivity and Graft-Versus-Host Disease

Introduction: Vasoactive Intestinal peptide (VIP) is a neuropeptide expressed by neural and lymphoid cells with pleiotropic effects on brain, immune, pulmonary and GI systems. The potent immunomodulatory activity of VIP make it an attractive therapeutic target to suppresses immune responses in conditions of deleterious inflammation. In allogeneic bone marrow transplantation (allo-BMT), graft-versus-host disease (GvHD) is mediated by activated alloreactive T cells that also upregulate a variety of co-inhibitory pathway molecules, including VIP expression on T cells. It is unclear whether VIP production by host cells in the transplant recipient influence the severity of GvHD and donor T cell activation. We tested the hypothesis that lack of VIP production by recipient cells would enhance donor T cell activation and increase GvHD in allo-BMT recipients. Methods and Results: We examined the effect of endogenous VIP signaling in host tissues in modulating GvHD in a murine allo-BMT model, in which C57BL/6 wildtype (WT) and VIP-knock-out (VIP-KO) mice received 11Gy irradiation on day -1 and were transplanted i.v. on day 0 with 5x106 T cell depleted (TCD) bone marrow (BM) plus 0, 1x106 or 3x106 splenocytes (SP) from MHC mis-matched B10.BR donors. All three groups of WT recipients had better survival than VIP-KO recipients. VIP-KO mice transplanted with TCD BM alone had 33.3% survival at day 75 compared with 73.3% survival of WT recipients. Chimerism studies on day 20 post-BMT showed equivalent levels of donor chimerism within each group that received the same dose of donor SP. WT and VIP-KO recipients received BM only showed significantly lower donor chimerism compared to either WT or VIP-KO recipients received additional SP (p<0.001), indicating increased mortality in recipients transplanted with BM only was primarily due to early graft rejection. The addition of donor SP to the graft resulted in significantly more GvHD-related mortality in VIP-KO mice compared with WT recipients, with 80% vs 100% day 75 survival among recipients of 1x106 SP (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3x106 donor SP. WT and VIP-KO mice treated with lethal doses of irradiation without allo-BMT showed no survival difference, suggesting VIP-KO mice are not intrinsically more susceptible to irradiation. To determine how the absence of VIP synthesis by VIP-KO host cells modulates immune effector mechanisms that contributes to increased GvHD, we analyzed donor-derived lymphocytes in the spleen of transplant recipients received 5M TCD BM plus 3 × 106 SP from WT B10BR donors following a time course, with flow cytometry. WT recipients had significantly increased levels of Lag3+ CD4 (p<0.001) on D6, PD1+ CD8 (p<0.01) on D13, Tim3+ CD4 (p<0.05) on D19, and increased levels of Tim3+ CD4 (p<0.001), PD1+ CD4 (p<0.05), and PD1+ CD8 (p<0.001) on D25 post allo-BMT compared to VIP-KO recipients. In addition, VIP-KO recipients had significantly higher expression of selected Th1 and Th17 cytokines. We observed significantly higher levels of IFN-γ+ CD4 (p<0.001) and IL2+ CD4 (p<0.01) on D4, significantly higher levels of TNF-α+ CD4 (p<0.05) and TNF-α+ CD8 (p<0.001) on D19, and significantly increased TNF-α+ CD8 (p<0.05) on D25 post allo-BMT in VIP-KO compared to WT recipients. Consistent with the significant elevation of IL17 in CD4 and CD8 (p<0.001) donor T cells of VIP-KO mice on D19, increased transcriptional factor RORγt in VIP-KO mice were detected in both CD4 (p<0.0001) and CD8 (p<0.0001) donor T cells. Transplant experiments using radiation chimeric recipients in which either the hematopoietic or non-hematopoietic compartment lacked expression of VIP further demonstrated that VIP production in non-hematopoietic cells is the key factor in limiting the GvHD activity of donor T cells (Fig 1). Finally, immunofluorescent imaging of transgenic mice in which GFP is regulated by the VIP promoter showed VIP production is highly expressed in efferent neurons innervating the lungs, and that host expression of VIP in lung tissues continues for at least 15 days post-transplant (Fig 2). Conclusion: These data suggest local production of VIP in the lung may be a key factor in the control of alloreactive donor T cells and subsequent GVHD in epithelial target organs. Furthermore, local production of VIP by host neurons surrounding lung alveoli suggests a novel pathway for pharmacological modulation of allo-reactive T cells and control of GVHD. DisclosuresWaller:Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Kalytera: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celldex: Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership.

Focused Regulatory T Cell Repertoires Are Indicative of Successful GvHD Control in Experimental and Clinical Stem Cell Transplantation

Acute Graft versus Host Disease (aGvHD) remains a major complication and leading cause of mortality after allogeneic stem cell or bone marrow transplantation (BMT). Current strategies for treatment are still based on unspecific immunosuppressive therapy. Over the last decade, there have been major advances in the field of adoptive immunotherapy using regulatory CD4+CD25+Foxp3+ T cells (Treg cells). Nonetheless, not much is known about the exact mechanisms of Treg-mediated suppression, and even less about the importance of T cell receptor (TCR) specificity and its diversity on the functionality of Tregs.We hypothesized that an optimal Treg TCR repertoire is necessary for successful prevention of aGvHD. To test this hypothesis, we sequenced the TCR repertoire of 8 patients who were diagnosed with aGvHD on day 30 post transplantation and compared it with the TCR repertoire of nine GvHD-free patients. Analysis of GvHD-free patients on day 30 (and 100 days-follow up) revealed a lower TCR diversity when compared to the patients suffering from GvHD. A more detailed analysis of the TCR repertoire showed that in patients without GvHD, fewer clonotypes were needed to comprise 50% of the whole repertoire as compared with samples from patients with GvHD (Figure 1A). Thus, expansion of protective clones indicates their potent immunosuppressive capabilities.Next, we employed a well-described murine model of allogeneic BMT (BL/6–>Balb/c) with co-injection of Tregs. Recipient Balb/c mice transplanted in this fashion were previously shown to be protected from aGvHD. However, the mechanisms involved in this Treg-mediated protection are not fully understood. Therefore, Tregs were FACS sorted from B6.Cg-Foxp3tm1Mal/J mice based on their Foxp3 expression. Recipient mice were transplanted with T-cell depleted bone marrow and a mixture of conventional T cells (Tconv) and Tregs in 1:1 ratio. Transferred Tregs were re-sorted on day 7 and day 14 from secondary lymphoid organs based on the congenic marker Thy 1.1 and Foxp3 expression. Using this model, we investigated the kinetics of the Treg TCR repertoire early after BMT in 5 independent experiments. We found a consistently similar narrowing of the repertoire and clonal expansion in mice protected from GvHD (Figure 1B). Diversity analysis using inverse Simpson Index also confirmed our findings. These data further support the notion that a clonal expansion of Tregs is necessary for an optimal immunosuppression of an allogeneic response, both in human and in mice. To test the functionality and phenotype of such expanded Tregs, they were re-sorted from BMT-recipient mice 14 days after transplantation. These Tregs were expanded using α-CD3 and α-CD28 antibodies and were functionality tested in an in vitro Treg suppression assay. Re-sorted Tregs after expansion showed expression of established Treg surface and intracellular markers such as Helios, CD25, GITR and CTLA-4. For the suppression assay, responder CD4 Tconv were stained with a proliferation tracking dye eFluor670 and stimulated in vitro with CD3 and CD28 beads in the presence of different ratios of re-sorted and expanded, or polyclonaly activated Tregs as the control. Allo-specific ex vivo Tregs exhibited a superior suppressive potential when compared with polyclonaly activated Tregs in vitro.Taken together, our current study highlights the importance of specific Treg driven allo-response in GvHD prevention. Further studies are needed, particularly in larger patient cohorts to confirm these findings. However, we propose that this approach might lead to identification and subsequent use of specific Treg clones with high immunosuppressive capacity for the prevention of aGvHD. [Display omitted] DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:abbvie: Consultancy; BMS: Consultancy; Roche: Consultancy; Amgen: Consultancy.

Intestinal Immune Conditioning with Helminths Employs Host T Helper 2 (Th2) Pathway to Induce Mixed Chimerism and Regulate Graft-Versus-Host Disease

BackgroundAchieving mixed chimerism suppresses donor T cell alloreactivity and reduces graft-versus-host disease (GVHD)-related mortality after bone marrow transplantation (BMT). Mixed hematopoietic chimerism is the only condition proven to lead to immune tolerance after transplantation. It can be achieved with protocols utilizing chemotherapeutic agents or strategies that block T cell activation or deplete host T cells. Selective irradiation of the thymus or lymphoid organs can also lead to mixed hematopoietic chimerism. After total lymphocyte irradiation (TLI), mixed chimerism is mediated by host iNKT lymphocytes and the T helper 2 (Th2) cytokine IL4 generated by those cells. IL4 signals through IL4 receptor (IL4R) and STAT6. After a toxic myeloablative pretransplant regimen, total body irradiation (TBI), intestinal immune conditioning with helminthic commensals regulates the host immune system and promotes a transient mixed chimerism. Conditioning with helminths also induces IL4 production by host cells and regulates GVHD while preserving the graft-versus-tumor effect. We hypothesize that induction of host Th2 pathway is critical for achieving mixed chimerism by intestinal immune conditioningMethodsWe evaluated the role of recipient Th2 signaling in the establishment of mixed chimerism in an MHC I/II major mismatch (H2b→H2d) model that utilizes TBI. Three weeks after infection with the mouse nematode, Heligmosomoides polygyrus-bakeri (Hpb), GVHD was induced by the delivery of T cell-depleted bone marrow (TCD-BM) and splenic T cells from uninfected WT C57BL/6 (MHC:H2b) donors into lethally-irradiated WT BALB/c, interleukin 4 (IL4) -/-, IL4Rα-/- and STAT6-/- recipients (MHC: H2d). Cellular composition for donor vs recipient cells, GVHD-mediated inflammation in end-organs (colon and lung), and survival of mice in each setting were assessedResultsIn 3 models of disruption of the Th2 pathway, we demonstrated that Th2 signaling in the host is essential for the establishment of mixed chimerism. Helminth infection promotes mixed chimerism in WT BALB/c BMT recipients; recipient T cells constitute 3-5% of peripheral population at 6 days post-BMT, whereas recipient T cells constitute <1-2% of population in uninfected BMT recipients (p<0.001). By contrast, when recipient cell IL4, IL4Rα or STAT6 is silenced, the percentage of recipient T cells remains negligible (<1% of total T cell population) in uninfected or Hpb-infected BMT recipients (p: NS between uninfected and Hpb-infected) (fig 1). Hpb infection promotes chimerism in mesenteric lymph nodes (MLN) of iNKT-deficient (Jα18-/-; MHC: H2d) BMT recipients of C57BL/6 donors (p<0.05 between uninfected and Hpb-infected Jα18-/- BMT recipients). Although helminths regulate GVHD-related inflammation in lungs and the colon of WT BALB/c recipients (p<0.05 between uninfected and Hpb-infected WT BALB/c BMT recipients), they fail to do so in these organs in IL4-, STAT6- or IL4Rα-deficient BMT recipients (p: NS between uninfected and Hpb-infected BMT recipients for each Th2 gene deficient group)(fig 2). Moreover, helminth infection did not promote survival of Th2-/- BMT recipients, where all mice died of lethal GVHD, although helminth infection promotes survival in WT BALB/c BMT recipients (P<0.05 between Hpb-infected WT BMT recipients and each other group). Because tolerance of alloreactive cells in mixed chimerism occurs with the induction of CTLA4 on T cells (a check point inhibitor and critical immune regulatory protein in GVHD), we also explored CTLA4 expression on peripheral T cells and found that helminth-induced CTLA4 production on splenic T cells was dependent on expression of IL4 and STAT6. In IL4-/- or STAT6-/- mice, there was no increase in the percentage of CTLA4+ T cells after Hpb infection (p: NS between uninfected and Hpb-infected BMT recipients for each Th2 gene deficient group), whereas helminth infection increased 3-fold the percentage of CTLA4+ T cells (p<0.05 between uninfected and Hpb-infected WT BALB/c mice)ConclusionsIntestinal immune conditioning with helminths promotes mixed chimerism after toxic, host cell depleting myeloablative TBI and regulates lethal GVHD following BMT. Host cell Th2 pathway is critical for achieving mixed chimerism. Helminths stimulate CTLA4 - a cellular regulatory protein known to promote mixed chimerism after BMT- in a Th2 dependent manner [Display omitted] DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding.

Tracking T Cell Activation By OX40 Immuno-PET: A Novel Strategy for Imaging of Graft Versus Host Disease

BACKGROUNDGraft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior non-invasive imaging strategies specifically detecting early GvHD are critically needed to improve clinical care of HCT recipients. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F-FDG) have largely been confounding, mainly due to their low specificity. Monitoring T cell activation and expansion using T-cell targeted PET tracers seems a more promising approach (Ronald et al., Cancer Res 77(11) 2893, 2017). We recently reported a novel immuno-PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive imaging of activated murine T cells expressing the cell surface activation marker OX40 (Alam et al., JCI 128(6) 2569, 2018). In the present work, we evaluated the utility of this immuno-PET strategy to image activated T cells in a major MHC-mismatch mouse model of acute GvHD.METHODSBalb/C (H-2Kd) recipients were irradiated with 8.8 Gy and on the same day received intravenously (i.v.) 5 x 10e6 T-cell depleted bone marrow (BM) cells with or without 1 x 10e6 CD4 and CD8 T cells positively selected from C57BL/6 (H-2Kb) mice. Severity of GvHD was assessed by clinical GvHD scoring. Flow cytometry of lymphoid organs from BM control and GvHD mice was performed at day 7 after HCT to determine OX40 protein expression on immune cells. For imaging studies, anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (an average ratio of 1.4 DOTAs per mAb was obtained) and subsequently radiolabeled with 64CuCl2 (final specific activity 10-15μCi/μg and radiochemical purity >99%). Mice were tail-vein injected with 64Cu-DOTA-mAbOX40 (100 µCi, i.v.) at day 7 after HCT and PET-CT imaging performed 24 hours after injection. Immediately following PET-CT mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter.RESULTSFlow cytometry analysis of OX40 expression in lymphoid organs isolated at day 7 after HCT revealed significantly higher proportions and absolute numbers of OX40 expressing cells in the spleen and cervical lymph nodes (LN) isolated from mice that received BM + T cells (GvHD group) compared with mice having received BM cells alone (p<0.05). In vivo OX40-ImmunoPET performed at day 8 after HCT revealed increased radiotracer uptake in spleen (p < 0.0001), mesenteric LN (p < 0.01) and the abdominal region (p < 0.001) of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64Cu-DOTA-mAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Biodistribution analysis using gamma counting of tissues confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01) and GvHD target organs e.g. small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). Importantly, outcome analysis of GvHD mice receiving tracer doses of OX40 mAb at day 7 after HCT did not reveal any significant worsening of GvHD in terms of survival, body weight loss or GvHD score, compared with mice receiving the appropriate isotype control, supporting the safety of this OX40-targeted imaging approach.CONCLUSIONThe OX40 immuno-PET tracer enabled specific imaging of alloreactive OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64Cu-DOTA-mAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting.FIGURE 1. 64Cu-DOTA-AbOX40 PET-CT imaging in a mouse model of acute GvHD. (A) Representative day 8 64Cu-DOTA-AbOX40 PET-CT images in BM controls or GvHD group. H, heart (including cardiac muscle and blood); Li, liver; Sp, spleen; Bl, blood vessels and venous sinuses; BM, bone marrow; Ab, abdomen. (B) Quantitative region of interest PET image analysis of indicated organs in BM controls (n=12, blue filled boxes) or GvHD mice (n=12, red filled boxes). Outliers are represented as dots. [Mann-Whitney test , ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05]. [Display omitted] DisclosuresGambhir:CellSight Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Flt3L Treatment of Bone Marrow Donors Increases Graft Plasmacytoid Dendritic Cell Content and Improves Allogeneic Transplantation Outcomes.

A higher number of donor plasmacytoid dendritic cells (pDCs) is associated with increased survival and reduced graft-versus-host disease (GVHD) in human recipients of unrelated donor bone marrow (BM) grafts, but not granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood grafts. We show that in murine models, donor BM pDCs are associated with increased survival and decreased GVHD compared with G-CSF-mobilized pDCs. To increase the content of pDCs in BM grafts, we studied the effect of FMS-like tyrosine kinase 3 ligand (Flt3L) treatment of murine BM donors on transplantation outcomes. Flt3L treatment (300 μg/kg/day) resulted in a schedule-dependent increase in the content of pDCs in the BM. Mice treated on days -4 and -1 had a >5-fold increase in pDC content without significant changes in numbers of HSCs, T cells, B cells, and natural killer cells in the BM graft. In an MHC-mismatched murine transplant model, recipients of Flt3L-treated T cell-depleted (TCD) BM (TCD F-BM) and cytokine-untreated T cells had increased survival and decreased GVHD scores with fewer Th1 and Th17 polarized T cells post-transplantation compared with recipients of equivalent numbers of untreated donor TCD BM and T cells. Gene array analyses of pDCs from Flt3L-treated human and murine donors showed up-regulation of adaptive immune pathways and immunoregulatory checkpoints compared with pDCs from untreated BM donors. Transplantation of TCD F-BM plus T cells resulted in no loss of the graft-versus-leukemia (GVL) effect compared with grafts from untreated donors in 2 murine GVL models. Thus, Flt3L treatment of BM donors is a novel method for increasing the pDC content in allografts, improving survival, and decreasing GVHD without diminishing the GVL effect.

Anti-CD3 Antibody Ameliorates Transfusion-Associated Graft-Versus-Host Disease in a Chemotherapy-Based Mouse Model With Busulfan and Fludarabine

ABSTRACT To establish a transfusion-associated graft-versus-host disease (TA-GVHD) mouse model with busulfan and fludarabine for effective treatment evaluation. BALB/c (H-2d) mice were injected with busulfan (15 mg/kg) and fludarabine (30 mg/kg) twice a day for 4 days. The mice were transfused with 10 6 T cell-depleted bone marrow (TCD-BM )and cells in different groups 3 days after chemotherapy: syngeneic BALB/c, MHC minor mismatch DBA/2 (H-2d), or MHC major mismatch C57BL/6(H2-b). Recipient BALB/c mice were injected with either blood only or blood+splenocyte. TA-GVHD was monitored in terms of body weight loss, clinical scores, and survival. Dexamethasone (50 mg/kg), cyclophosphamide (50 mg/kg), cyclosporine A (30 mg/kg), and anti-CD3 (1 mg/kg) were injected to each group to examine the treatments. Blood transfusion alone is insufficient to induce TA-GVHD in a chemotherapy-based mouse model. A MHC-mismatched TA-GVHD model can be induced by splenocyte and blood transfusion. This MHC-mismatched TA-GVHD model was resistant to dexamethasone treatment. Treatment based on anti-CD3 monoclonal antibody slightly ameliorated TA-GVHD. Treatment effectiveness was associated with T-cell depletion following activation by anti-CD3. Busulfan and fludarabine chemotherapy regimen can be used to establish a TA-GVHD mouse model. Anti-CD3 monoclonal antibody is a potential alternative to treat TA-GVHD.

Recipient-Derived BAFF and Alloantigen Synergistically Activate B Cells in Murine Chronic Gvhd

Increased B cell-activating factor (BAFF) and aberrant B cell survival and activation are associated with chronic graft versus host disease (cGVHD) in patients. Whether excessive BAFF production has a pathologic role in the development of cGVHD after allogeneic hematopoietic stem cell transplantation (allo-HCT) remains unknown. Herein, we address this hypothesis by employing BAFF knockout (KO) and BAFF overexpressing (Tg) donor mice in the major MHC mismatched C57BL/6 (B6)-to-BALB/c cGVHD model (Wu et al J Immunol 2013;191:488), except we increased the T cell depleted bone marrow (TCDBM) dose to 1x107 per recipient to improve engraftment. BALB/c recipients receiving TCDBM plus 1x106 splenocytes (Sp) are the disease group, and those receiving TCDBM only are controls. After verifying cGVHD phenotypic manifestations (including longevity, weight loss, eye and lung findings), we examined whether cGVHD mice had the increased BAFF and B cell findings as occur in human cGVHD (Sarantopoulos et al, Blood 2009; 113: 3865). We observed that cGVHD mice had higher BAFF/B cell ratios compared to controls (Fig. 1A, n=10 each). We fully characterized the peripheral B cell compartment in these mice, and found that the relative composition of B cell subsets was significantly altered in diseased mice. CD93 cell surface expression has been used in mice to define B cells that emigrate from BM and circulate through secondary lymphoid organs. In cGVHD mice, we found a significant increase in the relative proportion of CD93+ B cells and a significant decrease in the proportion of CD93- B cells (Fig1B). Also, a significant increase in the frequency of cells positive for the germinal center and activation marker GL7 was found only within CD93- B cell subset of cGVHD mice (Fig 1C). When stimulated via BCR ex vivo, this GL7+ mature B cell subset had significantly increased Syk and BLNK activation, measured by phosphoflow (n=5, p=0.008). We next aimed to determine whether high BAFF alone or high BAFF and alloantigen together lead to altered B cell homeostasis and to the promotion of GL7+ BCR-activated B cells. C57BL/6 (B6) BAFF Tg TCDBM cells were used as donor to afford excessive and persistent BAFF levels after HCT in either syngeneic or BALB/c recipients. After B6 BAFF Tg TCDBM to BALB/c HCT, body weight decreased significantly. By contrast, B6 BAFF Tg TCDBM to B6 syngeneic HCT recipients remained healthy (Fig.1D, left panel,*p=0.004). Notably, the frequency of the CD93-GL7+ B cell subset was increased in BALB/c mice that received B6 BAFF Tg TCDBM only, compared to recipients of syngeneic BAFF Tg or WT TCDBM only (Fig.1D, right panel, p=0.008). Thus, both BAFF and alloantigen are required for B cell activation after HCT. Together, our data also suggest that GL7+ B cells identify aberrantly BCR-activated B cells in murine cGVHD, arising in the setting of BAFF-driven altered B cell homeostasis.While polymorphisms in either donor or recipient BAFF genes are associated with human GVHD (Clark et al Blood 2011; 118: 1140), the source of excess BAFF after allo-HCT remains unknown. We addressed whether pathologic BAFF was derived from donor or recipient hematopoietic cells using TCDBM from B6 BAFF KO mice vs WT as donor cells for HCT to BALB/c recipients. Consistent with the known requirement of recipient BAFF for recovery of the B cell compartment after syngeneic transplant (Gorelik et al JEM 2003; 198:937), recovery of a donor peripheral B cell pool was not significantly different between groups. Remarkably, plasma BAFF levels were also not different between BALB/c recipients of B6 BAFF KO TCDBM +Sp vs recipients of WT TCDBM +Sp, suggesting that radioresistant recipient cells produced BAFF in cGVHD. Chronic GVHD-associated weight loss was also not different between groups (Fig.1E, representative of 3 separate experiments). We found that BAFF production in the engrafted KO recipient BM was uniformly low. Thus, we have now determined that non-BM, radioresistant recipient cells are the principal source of soluble pathologic BAFF in murine cGVHD. Further characterization of BAFF-producing cells is underway in order to identify therapeutic targets.In summary, we have now demonstrated that recipient-derived BAFF has a mechanistic role in aberrant activation of B cells in cGVHD. Our findings will lead to the development of anti-BAFF and BCR targeting agents for patients.This work was supported the National Institutes of Health (NHLBI R01 HL 129061-01). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Next Generation Veto Cells for Non-Myeloablative Haploidentical HSCT: Combining Anti-Viral and Graft Facilitating Activity

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm).When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance.Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2).The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3).In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection.*E.B.L and N.O.G contributed equally [Display omitted] [Display omitted] [Display omitted] DisclosuresBachar Lustig:Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

NaïVe T Cell Depletion of PBSC Grafts Results in Very Low Rates of Chronic Gvhd and High Survival

BackgroundGraft-versus-host disease (GVHD) frequently causes morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) as a result of organ damage and infections. In HLA-identical HCT, GVHD results from recognition by donor T cells of minor histocompatibility (H) antigens on recipient tissues. Complete T cell depletion (TCD) of donor hematopoietic cell products is more effective than pharmacologic immunosuppression for preventing GVHD, but is complicated by delayed immune reconstitution and consequent life-threatening infections.Approaches to HCT which preferentially deplete the T cells that primarily cause GVHD and preserve pathogen-specific T cells may improve HCT outcomes.Mature CD3+ CD8+ and CD3+ CD4+ T cells can be classified into CD45RA+ CD62L+ naïve (TN) and CD45RO+ memory (TM) subsets, the latter of which includes effector memory (TEM) and central memory (TCM) cells. Murine studies in which allogeneic TCD bone marrow (BM) is transplanted with purified T cells from individual T cell subsets to irradiated minor H antigen disparate recipients have demonstrated that the most severe GVHD results from transplanting T cells of the TN subset. Purified TCM causes mild GVHD and TEM do not cause detectable GVHD and can transfer immunity to pathogens.In vitro studies have similarly demonstrated that human donor CD8+ T cells specific for recipient minor H antigens are found predominantly within the TN cell subset, suggesting selective TN cell depletion may alter the GVHD incidence and/or severity in human HCT.Methods and resultsWe developed an effective process for engineering human peripheral blood stem cell (PBSC) grafts that depletes CD45RA+ TN cells and retains CD34+ stem cells and functional CD45RO+ TM cells specific for a broad range of opportunistic pathogens (Bleakley BBMT 2014). We are conducting clinical trials to evaluate the selective depletion of TN cells from HLA-matched allogeneic PBSC grafts for the prevention of GVHD in patients with acute leukemia, the first of which has been published (Bleakley JCI 2015, N=35). Seventy patients have now been treated on three consecutive phase II trials. The median age was 34 years (1-56 years), 56% of patients had a diagnosis of ALL, 46% had previously relapsed or had detectable disease (MRD or relapse) at the time of HCT, and 23% had unrelated donor (URD) grafts. Intensive myeloablative, TBI-containing (13.2Gy) conditioning was used for 63 patients, whilst 7 patients received a medium intensity ’midi’ preparative regimen, including 4Gy of TBI. The TN-depletion procedure was successfully performed on URD PBSC products shipped overnight from donor centers throughout the US, as well as on MRD PBSC collected at our centers. Reliable engraftment with high-level donor chimerism was observed in recipients of ’midi’ as well as intensive myeloablative conditioning. The 2-year estimates of overall survival, disease-free survival, survival free of relapse and chronic GVHD (CRFS) and survival free of relapse, grade II-IV acute GVHD, and chronic GVHD (GRFS) are 79%, 73%, 69% and 63% respectively. Median follow-up among survivors is 26 months. The frequency and severity of chronic GVHD is remarkably low (5%) compared to historical rates of 40-60% chronic GVHD in HLA-matched PBSC transplantation with conventional calcineurin inhibitor-based immunosuppression. Relapse and non-relapse mortality (NRM) are acceptably low at 19% and 8%, respectively. No NRM occurred in patients <40 years. Updated results will be presented.ConclusionsThe outcomes of recipients of TN-depleted PBSC grafts compare very favorably to published results of HCT for patients with acute leukemia. For example, the 69% incidence of CRFS at 2 years in TN-depleted recipients compares with reported 2-year GRFS rates of 37% and 17% in recipients of allogeneic PBSC from HLA-matched related donors with or without ATG (Kroger et al. NEJM 2016). Our results suggest that TN-depletion of PBSC grafts may reduce the risk of chronic GVHD without negatively impacting other important HCT outcomes. DisclosuresRiddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria.

Inhibition of HIF Prolyl Hydroxylases Mitigate Gut Graft-Versus-Host Disease

The etiology of graft-versus-host disease (GVHD) is rooted in the alloreactive response of donor T cells present in the hematopoietic graft resulting in the destruction of the patient's tissues, particularly the gastrointestinal tract. Gut GVHD affects up to 50% of patients, is a leading cause of death, and has overlapping features with inflammatory bowel disease (IBD). Increased gut permeability, alterations of the gut microbiome and intestinal stem cell niche damage predispose the gut to the local and systemic effects of GVHD, and is exacerbated by the inability of the gut to adequately regenerate.Severe shifts in metabolism and reduced oxygen (O2) availability in the inflamed gut are major underlying factors in the pathogenesis of IBD. Two related transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, originally discovered as master regulators of the adaptive response to hypoxia, have been recently shown to be gut protective and promote mucosal healing in response to injury. Using a MHC mismatched B10.BR→B6 bone marrow transplant (BMT) model, we previously found that loss of intestinal epithelial (IE) HIF-1α or HIF-2α worsened survival compared to wild-type mice and exhibited increased GVHD-induced-histopathology. Thus, we hypothesized that HIF-1 and HIF-2 protects and repairs the gut from conditioning and GVHD-related damage.HIF-1 and HIF-2 are heterodimers consisting of an O2-labile HIF-1α or HIF-2α subunit respectively and a constitutively expressed HIF-1β subunit. The recent discovery that iron-dependent prolyl hydroxylase enzymes (PHD1-3) can trigger the O2-dependent proteasomal degradation of the HIF-1α subunit has led to the development of pan-PHD inhibitors (PHDi) that activate HIF-1 and HIF-2. PHDi such as dimethyloxallyl glycine (DMOG) and AKB-4924 have been shown to attenuate colitis and radiation-induced gut toxicity in animal models. We thus sought to determine whether PHDi could also ameliorate gut GVHD in the B10.BR→B6 BMT model. B6 mice were lethally irradiated (10Gy, split dose) and transplanted with 5x106 T-cell depleted bone marrow (BM) cells from B10.BR donors with 2x106 enriched T cells from spleens and lymph nodes (BM+T). B6 mice transplanted with only T cell depleted BM cells served as our non-GVHD control group. B6 mice were treated with AKB-4924 (AKB, 5 mg/kg) or vehicle (β-cyclodextrin) via intraperitoneal delivery, beginning 1 day prior to BMT and for 6 consecutive days post-BMT. Treatment with AKB prevented significant weight loss, compared to vehicle-treated mice, 7 days post-BMT (n=6, p<.002). Histopathologic assessment of the jejunum of AKB treated mice after 7 days revealed fewer apoptotic cells and an increased number of Paneth and goblet cells compared to vehicle-treated mice. Immunohistochemical staining for lysozyme showed that AKB prevented GVHD-induced Paneth cell ablation compared to their vehicle BMT counterpart (2.8 Lyz+ cells/crypt vs 0.84 Lyz+ cells/crypt, n=3, p<.003). Additionally, we evaluated the effects of AKB on T cell alloreactivity. In mixed lymphocyte reaction Elispot assays, AKB and DMOG, reduced the alloreactive interferon (IFN)-γ response in B10.BR anti-B6 (p<0.001) and MHC matched, minor antigen-mismatched B6 anti-BALB.B cultures (p<0.05). Furthermore, AKB reduced the number of gut-infiltrating CD3+ T cells compared to vehicle-treated BMT mice (39 vs 155.1 CD3+ cells/high-power field, n=3, p<.0003). Our results provide the framework to validate the therapeutic potential of PHDi as an intestinal regenerative strategy in mitigating GVHD. DisclosuresPeters:Aerpio Therapeutics: Employment, Equity Ownership.

The evolution of T-cell depletion in haploidentical stem-cell transplantation.

T-cell depletion (TCD) can prevent the onset of graft-versus-host disease (GvHD) in animal models of bone marrow transplantation; this manipulation enabled the successful application in the 1980s of T-cell depleted bone marrow (BM) for the treatment of babies with severe combined immune deficiency (SCID). However, in leukaemia patients, implementation of T-cell depletion has been more difficult, especially due to high rate of graft-rejection, leukaemia relapse and delayed immune reconstitution. These hurdles were gradually overcome by modifying the cell composition of the graft, and by reducing the toxicities associated with conditioning protocols. Although no 'gold standard' TCD method exists, T-cell depletion in its modern forms could offer clinical benefit, even for patients with a matched sibling donor.

Alternative donor hematopoietic cell transplantation for Fanconi anemia

AbstractHistorically, alternative donor hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) patients resulted in excessive morbidity and mortality. To improve outcomes, we made sequential changes to the HCT conditioning regimen. A total of 130 FA patients (median age, 9.0 years; range, 1-48) underwent alternative donor HCT at the University of Minnesota between 1995 and 2012. All patients received cyclophosphamide (CY), single fraction total body irradiation (TBI), and antithymocyte globulin (ATG) with or without fludarabine (FLU), followed by T-cell–depleted bone marrow or unmanipulated umbilical cord blood transplantation. The addition of FLU enhanced engraftment 3-fold. The incidence of grades 2-4 acute and chronic graft-versus-host disease was 20% and 10%, respectively. Severe toxicity was highest in patients >10 years of age or those with a history of opportunistic infections or transfusions before HCT. Mortality was lowest in patients without a history of opportunistic infection or transfusions and who received conditioning with TBI 300 cGy, CY, FLU, and ATG. These patients had a probability of survival of 94% at 5 years. Alternative donor HCT is now associated with excellent survival for patients without prior opportunistic infections or transfusions and should be considered for all FA patients after the onset of marrow failure. These studies were registered at http://www.clinicaltrials.gov as NCT00005898, NCT00167206, and NCT00352976.

Impaired expansion of CD11b+Gr-1+ cells from MyD88-deficient donor bone marrow cells aggravates graft-versus-host disease (TRAN2P.966)

Abstract Myeloid differentiation 88 (MyD88), an adaptor molecule in TLR-associated signaling, has recently been known to play a role in expansion/activation of CD11b+Gr-1+ myeloid-derived suppressor cells. To understand role of MyD88 expressed by donor-originating myeloid cells in GVHD development, we investigated dynamics of T cell-depleted bone marrow (TCD-BM) cells and gene-expressions of CD11b+Gr-1+ cells originated from Myd88-/- donor mice after transplantation into allogeneic BALB.B hosts with WT T cells. The allogeneic recipients of Myd88-/- TCD-BM and WT T cells showed aggravated GVHD, with significant loss of CD11b+Gr-1+ population, compared with BALB.B recipients of WT TCD-BM and T cells. Impaired expansion of Myd88-/- TCD-BM cells in BALB.B GVHD hosts was confirmed via in vivo tracking analysis. However, their expansion was intact in syngeneic C57BL/6 recipients, indicating that impaired CD11b+Gr-1+ cell expansion from Myd88-/- TCD-BM cells was specific to GVHD circumstance. In transcriptome analysis, CD11b+Gr-1+ cells of MyD88-/- TCD-BM origin exhibited higher expression of genes related to apoptosis and antigen-presentation than did those of WT TCD-BM origin. These results suggest that impaired expansion of the CD11b+Gr-1+ cells from Myd88-/- TCD-BM cells in allogeneic hosts is related to enhanced apoptosis in combination with enhanced differentiation into DC-like cells in the presence of activated allo-reactive T cells, and provide insight into the control of GVHD.

Immunosuppressive mechanisms of human bone marrow derived mesenchymal stromal cells in BALB/c host graft versus host disease murine models.

BackgroundMesenchymal stromal cells (MSCs) are proven to have immunosuppressive functions via various mechanisms. These mechanisms were demonstrated by administering bone marrow derived human MSCs (hMSCs) to graft versus host disease (GVHD) murine models.MethodsBALB/c host mice were irradiated prior to receiving C57BL/6 donor T cell depleted bone marrow (TCDBM) cells (negative control) and donor CD4+ T lymphocyte with (treatment group) or without hMSCs (positive control). The presence of hMSCs in target tissues and lymphoid organs was documented by using in vivo imaging and measuring the expression of EphB2 and ephrin-B2 by RTqPCR. Survival rate and GVHD score were also monitored. Tissue sections were obtained for histopathologic analysis. Flow cytometry was used to document donor T cell alloreactivity and expression of CCR5, CXCR3 and CCR7. ELISA was utilized to determine levels of proinflammatory cytokines, RANTES (CCL5) and phosphorylated STAT 5A/B. RTqPCR was performed to quantify expression of CCL3 and CXCL9. Western blotting was performed to qualitatively measure iNOS expression.ResultsSurvival rate and GVHD score improved with hMSC treatment. Pathologic changes of GVHD were abrogated. Documentation of suppression of RANTES, CCL3, CXCL9, CCR5 and CXCR3 with simultaneous decrease of donor T cell alloreactivity was demonstrated 6 days after transplantation, along with reduction of levels of inflammatory cytokines, suppression of STAT 5A/B phosphorylation, increased expression of CCR7 and increased production of nitrous oxide by hMSCs. Documentation of homing of hMSCs to lymphoid organs and target tissues was also performed.ConclusionsThese mechanisms contribute to the current understanding of MSC mechanisms of immunosuppression and forms a comprehensive picture of how they exert immunosuppression in an in vivo model of immune dysregulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-015-0007-0) contains supplementary material, which is available to authorized users.