Tolar, Jakub

Abstract Front-line pharmaceutical interventions for treating acute graft-versus-host disease (GVHD) are not uniformly effective and have toxic side effects. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent in vitro and in vivo immunosuppressive functions. Clinical translation of in vitro–generated MDSCs has been limited because of requirements for multiple, high infusion doses, the relatively low yield from peripheral blood–sourced MDSCs (PB-MDSCs), and inconsistent product quality. To circumvent these obstacles, we developed a methodology to generate MDSCs using human induced pluripotent stem cell (iPSC)–derived CD34+ cells. Compared with PB-MDSCs, iPSC-derived MDSCs (iMDSCs) shared similar morphology, phenotype, and suppressive function. We found that the CD14+ iMDSC subset possessed the highest suppressor function. In previous studies, we reported that MDSCs transferred into mice with GVHD lost suppressor function because of inflammasome activation and immature myeloid cell maturation. In striking contrast to human PB-MDSCs, we show herein that iMDSCs retained 95% of suppressor function in vitro despite exposure to lipopolysaccharide (LPS) plus adenosine triphosphate (ATP), which are stimuli that activate the inflammasome via danger-associated molecular patterns released during early posttransplant conditioning and GVHD-induced injury. In an in vivo xenogenic GVHD model with PB mononuclear cells, iMDSCs significantly increased recipient survival without loss of antileukemia effects. iMDSC RNA sequencing and gene knockdown studies revealed that the maintenance of the purine metabolizing enzyme, phosphoglycerate dehydrogenase, during LPS plus ATP treatment, was linked to iMDSC inflammasome resistance. Taken together, these findings provide a platform for translating in vitro–generated, off-the-shelf iMDSCs into the clinic for suppressing a spectrum of adverse immune responses, including GVHD.

Hematopoietic cell homing after hematopoietic cell transplant (HCT) is governed by several pathways involving marrow niche cells that are evoked after pre-HCT conditioning. To understand the factors that play a role in homing, we performed expression analysis on the zebrafish marrow niche cells following conditioning. We determined that the non-collagenous protein extracellular matrix related protein dermatopontin (Dpt) was upregulated seven-fold in response to irradiation. Studies in mice revealed DPT induction both with radiation and lipopolysaccharide exposure. Interestingly, we found that co-incubation of zebrafish or murine hematopoietic cells with rDPT impedes hematopoietic stem and progenitor cell homing by 50% and 86%, respectively. Similarly, this translated into a 24% reduction in long term engraftment (versus control, p = 0.01). We found DPT to interact with VLA-4 and block hematopoietic - endothelial cell adhesion and transendothelial migration. Finally, a DPT knockout mouse displayed a 60% increase in homing of hematopoietic cells versus wildtype (p = 0.03) with slight improvement in long-term LSK-SLAM engraftment (2-fold, p = 0.04). These data show that the extracellular matrix (ECM)-related protein DPT increases with radiation and transiently impedes the transendothelial migration of hematopoietic cells to the marrow.

Abstract Abstract 3009FN2 Objective: To determine toxicity and efficacy of adding fludarabine (FLU) to a regimen of total body irradiation (TBI), anti-thymocyte globulin (ATG) and cyclophosphamide (CY), with de-escalation of the CY dose. The goal is to minimize CY-related toxicities while preserving (or improving) engraftment and survival in patients with SAA receiving an unrelated donor marrow transplant. Patients and Methods: Since May 2006, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), sponsored by the NHLBI and NCI, has conducted a prospective Phase I/II clinical trial of unrelated donor marrow transplantation in SAA (BMT CTN 0301). Patients with SAA are eligible if they are < 65 years, have adequate organ function, and have an available unrelated marrow donor matched 7 of 8 HLA-A, B, C, DRB1 loci. Patients with Fanconi anemia and other marrow failure syndromes are excluded. All patients receive TBI 200 cGy, ATG (either thymoglobulin: 3 mg/kg IV or ATGAM 30 mg/kg IV daily × 3, days –4 to –2), FLU (30 mg/m2 IV daily × 4, days –5 to –2). The Phase I portion of the trial sequentially tested four CY dose levels: 150 mg/kg (administered days –4 to –2); 100 mg/kg (days –3 to –2); 50 mg/kg (day –2); and, 0 mg/kg, and allowed enrollment of up to six patients at each CY dose level unless toxicity or graft failure boundaries were crossed. In the Phase II portion of the trial, patients enroll onto the optimal CY dose level, which is chosen using adaptive Bayesian criteria to rank desirability of the CY doses. All patients are followed for two years after transplantation. Early stopping guidelines are used to monitor for graft failure and early transplant-related mortality through day 100. Results: Twenty-one patients accrued to the Phase I portion of the trial. CY dose level 0 mg/kg was closed after all three enrolled patients developed secondary graft failure. All received a second allograft. One patient remains alive at 23 months after the first transplantation. The other two died from adult respiratory distress syndrome (ARDS) at 114 days after the first transplant and idiopathic pneumonia at 200 days after the first transplant. Review of Phase I results for the other three dose levels suggested CY dose 150 mg/kg as the optimal dose level for Phase II testing. However, after an additional eight patients were treated at this dose, the level was closed to further accrual because of excess toxicity. Seven of the 14 patients receiving 150 mg/kg of CY (and 7 of the last 8 enrolled) died. Causes of death were cardiac/pulmonary/multi-organ failure (n=4), ARDS (n=2), and parainfluenza virus type 3 pneumonia (n=1). Bayesian evaluation of the two remaining dose levels indicates very similar desirability scores and accrual continues at both the CY 100 mg/kg and 50 mg/kg levels. As of July 15, 2011, a total of 61 patients have been enrolled, 17 on the two closed levels, 33 on the 100 mg/kg level and 11 on the 50 mg/kg level. Conclusions: Early analysis of this trial, made necessary by these unexpected severe adverse events, revealed two important findings among patients receiving low-dose TBI, ATG, and FLU at the doses outlined: 1) CY dose 0 mg/kg is associated with higher than expected graft failure; and, 2) CY dose 150 mg/kg is associated with excess transplant-related toxicity. Neither CY dose level should be tested further in the context of the regimen used in this trial. To date, the two intermediate CY dose levels (100 mg/kg and 50 mg/kg) have not crossed the graft failure or fatality stopping boundaries and accrual is in progress. Disclosures: Pulsipher: Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees.