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Hematopoietic cell transplantation (HCT) is the only readily available cure for many life-threatening pediatric nonmalignant diseases (NMD), but most patients lack a matched related donor and are at higher risk for graft-versus-host disease (GVHD). Use of abatacept (Aba) to target donor T-cell activation has been safe and effective in preventing GVHD after unrelated donor (URD) HCT for malignant diseases (Aba2 trial). Our primary objective was to evaluate the tolerability of Aba added to standard GVHD prophylaxis (cyclosporine and mycophenolate mofetil) in pediatric patients with NMD undergoing URD HCT. In this single-arm, single-center phase 1 trial, 10 patients receiving reduced intensity or nonmyeloablative conditioning underwent URD HCT. Immune reconstitution was assessed longitudinally via flow cytometry and compared to pediatric patients on Aba2. Nine patients successfully engrafted, with 1 primary graft rejection in the setting of inadequate cell dose; secondary graft rejection occurred in 1 patient with concurrent cytomegalovirus viremia. Two deaths occurred, both unrelated to Aba. One patient developed probable posttransplant lymphoproliferative disease, responsive to rituximab and immune suppression withdrawal. No patients developed severe acute or chronic GVHD, and 8 patients were off systemic immunosuppression at 1 year. Immune reconstitution did not appear to be impacted by Aba, and preservation of naïve relative to effector memory CD4+ T cells was seen akin to Aba2. Thus, 4 doses of Aba were deemed tolerable in pediatric patients with NMD following URD HCT, with encouraging preliminary efficacy and supportive immune correlatives in this NMD cohort.

During acute Graft-versus-Host Disease (aGVHD), donor T cells invade host tissues and cause multi-organ damage. However, the organ-specific drivers of pathogenic alloimmunity are still poorly understood. To uncover both the universal and organ-specific mechanisms driving T cell-mediated damage in target tissues during aGVHD, we analyzed the flow cytometric phenotype as well as transcriptomic signatures of T cells in healthy control animals, in autologous hematopoietic cell transplant (HCT) controls, and during aGVHD after allogeneic HCT, using our established non-human primate aGVHD model. In this study we focused on 2 aGVHD target organs: the liver and lungs. Using both supervised and unsupervised flow cytometric analysis, we discovered that, during aGVHD, both liver- and lung-infiltrating T cells demonstrated similar phenotypes dominated by mature effector-memory CD8 T cells. In contrast, transcriptomic analysis revealed organ-specific gene expression programming. Through Weighted Gene Co-expression Network Analysis (WGCNA), differential expression (DE) analysis, and immune pathway analysis, we identified that the lung aGVHD T cell transcriptomic program was specifically associated with extracellular matrix-remodeling and chemotaxis pathways. In contrast, in liver aGVHD, we found enrichment for metabolic stress responses and cytokine signaling pathways. These results suggest that at the transcriptomic level, T cells infiltrating different aGVHD-target organs are uniquely programmed and could be amenable to organ-specific therapeutic targeting. Supported by grants from the NIH (F31HL1562880)

Abstract 2550 We have developed a novel, MHC-defined rhesus macaque model of total body irradiation-based haploidentical hematopoietic stem cell transplantation. This model has permitted us, for the first time, to perform a rigorous study of the cellular and molecular basis of uncontrolled primate GvHD, and to evaluate the efficacy of a novel, clinically-relevant T cell costimulation blockade-based immunosuppressive regimen to control this disease. We have found that after unprophylaxed haploidentical transplant, severe GvHD developed, which was characterized by rapid clinical decline, and widespread T-cell infiltration and organ damage, with histopathologic evidence of disease in the lungs, the liver, and the GI tract. Mechanistic analysis revealed activation as well as possible counter-regulation, with rapid, CD8-predominant T-cell expansion and accumulation of both CD8+ and CD4+ granzyme B+ effector cells as well as FoxP3pos/CD27high/CD25pos/CD127low CD4+ T-cells. In addition, CD8+ cells downregulated CD127 and BCl-2 and upregulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GvHD-specific secretion of IL-1Ra, IL-18, and CCL4. The combination of CD40/CD28 costimulation blockade (using a monoclonal antibody against CD40 and the CTLA4Ig fusion protein) and mTOR inhibition with sirolimus (Costimulation Blockade and Sirolimus, “CoBS”) resulted in striking protection against GvHD. Thus, at the 30-day primary end-point, CoBS-treated recipients demonstrated 100% survival compared to no survival in untreated recipients. Long-term analysis revealed that CoBS treatment resulted in mean survival increasing from 11.6 to 62 days (p These results demonstrate the utility of this novel primate model to provide mechanistic insights into the molecular and cellular basis of GvHD as well as to allow a rigorous evaluation of novel, clinically-relevant immunosuppression strategies. Our results with CoBS suggest that while significant disease control was accomplished, the CD28-negative CD8+ T cell compartment was relatively resistant to CD28/CD40 costimulation blockade and sirolimus, and that adjuvant treatments targeting this subpopulation may be needed for full disease control, especially after high-risk, T cell replete haploidentical transplantation. Disclosures: No relevant conflicts of interest to declare.

Introduction: While bone marrow transplantation (BMT) offers curative therapy for many malignant and non-malignant hematologic diseases, it remains plagued by high morbidity and mortality, most prominently through the complications of graft-versus-host disease (GvHD). The field of GvHD research has been limited by a small number of clinically relevant model systems, which, until now, have focused on murine and canine models. However, many new human therapeutics, including those directed at T cell costimulation pathways, do not cross-react with targets in these model systems, limiting their utility to fully evaluate the new generation biologic anti-GvHD therapies. Methods: We have developed a fully pedigreed and MHC-typed rhesus macaque bone marrow transplant system, with which to model GvHD and its prevention. This model relies upon microsatellite typing of both autosomal chromosomes to create genetically-verified family trees and microsatellite-and allele-specific PCR-based MHC typing, with which to identify MHC haplotypes within each family group. By this methodology, full and half-sibling pairs can be identified with either full MHC matching, one MHC haplotype matching or full MHC disparity. This represents a significant advance compared to previous primate haplo-studies, which relied upon parent-offspring transplants, as it optimizes animal availability and colony stability as well as permiting the rigorous evaluation of the degree of MHC disparity on the non-shared chromosome 6. For the current study, we have concentrated on MHC haplo-identical BMT. Our preparative regimen consists of TBI (8.00 Gy with lung shielding to 6.00 Gy), which is performed on the day of transplant with hematopoietic stem cells. After transplant, donor engraftment is measured by microsatellite analysis, from both whole blood and from flow cytometrically sorted T and B cell populations. GvHD is graded using standard clinical grading scales. The immune phenotype after transplant is also measured by cell- and serum-based flow cytometric analyses. Results: Three animals have so far been studied. The first animal served as a control for TBI-based preparation. This animal exhibited both neutropenia (nadir = 0.015 × 103/uL at 12 days) and thrombocytopenia (nadir = 2 × 103/uL at 12 days) and prolonged anemia (hgb=5.8 g/dL on the day of sacrifice). This animal was sacrificed at day 29 due to prolonged pancytopenia. The second animal was transplanted with haploidentical hematopoietic stem cells (4.47 × 108 total nucleated cells/kg and 1.10 × 108 CD3+ T cells/kg) and was treated with only rapamycin for immunosuppression. He exhibited profuse diarrhea and necrotic skin changes within 8 days of transplant, coincident with early engraftment and was sacrificed at day 14. A full histologic analysis to determine the cause of these lesions (and the histopathologic extent of GvHD) is currently underway. The third animal was transplanted with haploidentical donor bone marrow (1.1 × 109 total nucleated cells/kg and 6.9 × 107 CD3+ T cells/kg), but was given T cell costimulation blockade with abatacept (targeting the CD28/B7 pathway) and an anti-CD40 antibody (targeting the CD40/CD154 pathway) in addition to rapamycin. Abatacept is currently clinically approved for treatment of rheumatoid arthritis, but has not been added to clinical GvHD regimens, largely due to the paucity of translational efficacy data. This animal is currently >43 days post-transplant, with full donor chimerism (including 100% T and B cell chimerism), without signs of GvHD: he has no rash, no diarrhea and is stable clinically. Additional animals are now being added to the study, to confirm these initial results. Conclusions: We have established a robust non-human primate model of GvHD using pedigreed and MHC-typed rhesus macaques. We have used this model to begin to test the efficacy of a novel agent combination, capable of preventing the onset and complications of acute GvHD. Preliminary results suggest that CD28- and CD40-directed costimulation blockade may be active agents for the prevention of GvHD. A large scale analysis of their efficacy and immune consequences is currently underway.