Donor Cells In Peripheral Blood Research Articles

ASXL1 Truncating Mutations Drive Leukemic Resistance to T Cell Attack

Restoration of graft-versus leukemia activity underlies the efficacy of donor lymphocyte infusion (DLI), a curative immunotherapy for leukemic relapse following allogeneic stem cell transplant (SCT). We found that while specific exhausted T cell subsets defined DLI response, no pattern of T cell dysfunction appeared in nonresponders (NRs) [PMID: 34758319]. We hypothesized that leukemic molecular pathways define DLI resistance (DLIr). We performed whole exome sequencing on bone marrow samples collected before and after DLI from 11 patients (pts) with relapsed chronic myeloid leukemia (CML) at Dana-Farber Cancer Institute. We used MuTect2 to discover somatic alterations using genomic DNA from recipient-derived mesenchymal stromal cells and donor peripheral blood cells as germline sources. The burden of nonsynonymous mutations and mutational spectra was similar between responders (Rs) and non-responders (NRs). Known non-Philadelphia (Ph) chromosome cancer drivers were found in 0/7 Rs and 4/4 NRs (RUNX1 (1/4), IKZF1 (2/4), and truncating exon 12 ASXL1 mutations (ASXL1 m) (3/4)). We profiled 6 more DLI-treated pts with relapsed CML from the MD Anderson Cancer Center with a targeted mutation panel that identified ASXL1 m in 4/5 NRs. Altogether, we found ASXL1 m in 78% (7/9) of NRs and 0% (0/10) of Rs (p=7e-4, two-sided Fisher's test), suggesting ASXL1 m as a driver of DLIr. To discover molecular pathways of DLIr, we interrogated unpublished non T-cell single cell (sc)RNA-seq data in our DLI cohort (8 R, 6 NR). Leukemic cells were identified for patients (n=8) with sex-mismatched SCT by male sex-defining genes, which correlated with Ph chromosomal positivity (Pearson correlation = 0.85). These cells were primarily confined to the myeloid population, so we performed a scRNA myeloid reclustering (138,152 cells) with Harmony batch correction and identified 5 populations enriched in R vs NR; 4 clusters in NR, and 1 monocyte cluster in R (p=0.039, 0.005, 0.038, 0.043, and 0.025, respectively). These NR clusters were enriched in progenitor cell identities, notably the LSC17 [PMID: 27926740] signature (p<2e-16). Interrogation of immune pathways in the LSC cluster showed downregulation of HLA-I and multiple antigen processing/presentation (APP) genes in NRs (p<2e-16), while HLA-II remained unchanged. Thus, ASXL1 m may mediate immune escape through suppression of HLA-I expression and antigen presentation. To directly test this hypothesis, we CRISPR corrected the endogenous ASXL1 m (Y591*) in a BCR-ABL-driven leukemic cell line (K562). This wildtype reversion (ASXL1 c) increased HLA-I expression at the RNA (RNA-seq; HLA-B/C, log2foldchange= -4.77, q=7e-42) and protein level (flow cytometry; 49.0% vs 1.62%, q=2e-4); here too, HLA-II expression remained unaffected by ASXL1 mutation status. Gene-set enrichment analysis also showed increased APP expression in ASXL1 c K562 (NES=-1.75, q=0.01). We then sought to identify whether correction of ASXL1m acted through known regulators of HLA-I. Treatment with IFNɣ or EPZ011989, an inhibitor of enhancer of zeste-homolog 2 (EZH2), resulted in greater HLA-I in K562 ASXL1 c (85.0% vs 50.2% and 89.5% vs 39.3%, respectively, q<0.001) suggesting that ASXL1 cacts orthogonally to IFNɣ or EZH2. ASXL1 c also mediated increased T-cell cytotoxicity towards K562 ASXL1 c vs ASXL1 m without (43% vs 16%, q=2e-4) and with IFNɣ pre-treatment (71% vs 34%, q=1e-6) at 10:1 target to effector ratios. Finally, to study other disease contexts, we examined published sc chromatin accessibility and scRNA-seq data from a chronic myelomonocytic leukemia cohort [PMID: 35301312] and an acute myeloid leukemia cohort [PMID:32649887], respectively. We found both chromatin and transcriptional states enriched in ASXL1m samples to have decreased accessibility and expression, respectively, of HLA-I and APP genes (p<2E-16). In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion. Specifically, we link DLIr to ASXL1 m that suppresses transcription of HLA-I and antigen presentation machinery in myeloid diseases; moreover, its correction restores anti-leukemic CD8+ T-cell cytotoxicity. Ongoing studies will examine additional clinical contexts and the epigenetic mechanisms by which ASXL1m regulates HLA-I expression.

Preconditioning Single High-Dose Palifermin Alters the Posttransplant Inflammatory Cytokine Profile

Palifermin is a truncated form of human recombinant keratinocyte growth factor (KGF) that binds to the FGF receptor 2b expressed in epithelia including that of the epidermis, oral and GI mucosa, and thymus. In animal models, the cytoprotective and regenerative effects of KGF showed efficacy in controlling acute and chronic graft-versus-host disease (A/C GVHD), but these were not demonstrated in subsequent clinical studies using the FDA-approved dosing schedule for oral mucositis prevention (60 µg/kg/day for 3 consecutive days). The current study aims to investigate alterations in immune cell reconstitution and cytokine expression among patients (n=31) participating in the ongoing prospective open-label phase 1/2 trial NCT02356159. This trial evaluates the incidence of severe CGVHD (primary objective of phase 2) following the addition of a single high dose of palifermin to GVHD prophylaxis with TMS (tacrolimus, methotrexate, sirolimus). Four different dose levels of palifermin (DL1: 180 µg/kg; DL2: 360 µg/kg; DL3: 540 µg/kg; DL4: 720 µg/kg) were tested on day −7 in the phase 1 part of the trial, with the recommended phase 2 dose being 720 µg/kg. All patients received cyclophosphamide (total 4.8 g/m 2) and fludarabine (total 120 mg/m 2) as conditioning on days −6 to −3 followed by infusion of an 8/8 HLA-matched unrelated donor peripheral blood cell graft on day 0. Results of correlative studies were compared with patients of the identical TMS treatment arm of the NCT00520130 study without palifermin (n=31). In the DL4 group, a previous interim analysis revealed no cases of classic (day +100) AGVHD II-IV in 16 patients (P=0.014), whereas the TMS group showed a 22.6% (95% CI: 9.8% to 38.6%) incidence (Schulz et al. Cell Ther Transplant. 2023;29(2):S258). T, B and NK cells were routinely measured by flow cytometry at days +14, +28, +60, +100, +180, +365, +730. The V-PLEX human cytokine 36-plex kit and U-PLEX assays of human BAFF, CXCL9 and IL1RL1/ST2 (all MSD, Rockville, MD) were used to measure cytokine levels in plasma at days −8, 0, +14, +28, +60, +100. The measures at various time points were compared between two treatment groups using multiple Mann-Whitney tests, and the false discovery rate approach was applied with Q=0.05. Comparison between the four DL groups and the TMS group was performed using the Kruskal-Wallis H-test and Dunn's post-hoc test, with a significance threshold set at P<0.05. The statistical analyses were conducted using GraphPad Prism 9. In comparison to patients receiving TMS only, those treated with KGF exhibited reduced NK cell counts at day +28 (Q=0.0155) and increased B cell counts from day +60 (Q<0.01) onwards. However, there were no differences in the absolute numbers of CD3+, CD4+, and CD8+ T cells between patients from both trials. Additionally, no apparent dose-response relationship was observed. KGF-treated patients showed significantly reduced plasma levels of proinflammatory cytokines, including TNF-α at day 0, TNF-β at days 0, +14, +28, IL-12/IL23p40 at days 0 and +14, and CXCL9 at days 0 and +14, compared to patients receiving TMS only. Notably, levels of homeostatic IL-7, previously linked to AGVHD, were significantly lower in KGF-treated patients from day +28 (TMS vs. DL4, P=0.002) to +100. There was a trend towards lower levels of IL-22 at days 0 and +14 (both unadjusted P<0.05, Q>0.05) in KGF-treated patients. Other proinflammatory biomarkers showed increased levels in KGF-treated patients: IL-15 at day 0 (Q=0.0121), BAFF at days +14 and +28 (both Q<0.01), and ST2 at day 0 and day +14 (both Q<0.01). As reported previously by others, patients who developed classic or late onset AGVHD II-IV without prior relapse up to day +180 had higher ST2 levels at day +14 compared to patients without AGVHD II-IV (P=0.03). Here, this association was mainly observed in patients from the TMS and DL1 groups (6/8 tested patients with any AGVHD II-IV). The administration of a single high dose of palifermin (KGF) prior to conditioning treatment resulted in a notably distinct cytokine profile, characterized by a reduction in several GVHD associated pro-inflammatory cytokines, consistent with KGF's proposed cytoprotective effect. Further investigations focusing on GI microbiome and immune cell subsets are needed to elucidate the consequences of cytokine alterations induced by KGF on immune cell reconstitution. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

Sel1L, an Endoplasmic Reticulum Associated Degradation Adaptor, Regulates CD8+ Tcell Fate

CD8+ T cells are critical players in protective immunity against intracellular pathogens and cancer. Upon activation with cognate antigen, CD8+ T cells undergo marked increase in protein synthesis and significant proteome remodeling as they clonally expand and acquire effector function. However, little is known about the mechanisms by which CD8+ T cells maintain protein homeostasis (proteostasis) during T cell differentiation and which proteostatic pathways regulate T cell fate. Proteostasis is maintained through two main pathways: ER-associated degradation (ERAD) and the unfolded protein response (UPR). While UPR mediators have been implicated in CD8+ T cell immune responses, nothing is known about the role of ERAD in CD8+ T cell immunity. To interrogate proteostasis following CD8+ T cell activation in vivo, we utilized the murine model of acute viral infection LCMV-Armstrong (LCMV) to evaluate endoplasmic reticulum (ER) stress and proteostatic pathway activation over the course of initial effector differentiation. We found that endogenous LCMV-specific CD8+ T cells experience dynamic ER stress as measured by an increase in ER size [fold change (F.C.) ER Tracker median fluorescence intensity (MFI) relative to D0: D5 7.48± 4.91 *p<0.05; D8: 0.86±1.78, n.s., one-way ANOVA Dunnett's multiple comparisons test] and protein aggregation (Proteostat F.C. relative to D0: D5 1.95± 0.24 *p<0.05; D8 1.6 ±0.11, n.s.) over the course of infection with a peak at day 5 post-infection (p.i.), largely resolving by day 8 p.i. Furthermore, we assessed activation of the UPR pathways and found induction of UPR mediators XBP1s (F.C. relative to D0 D5: 3.96 ± 1.88 **p<0.01 D8 1.60±0.36 n.s.) and CHOP (F.C. relative to D0: D5 12.47±0.32 ***p<0.001; D8 2.58±1.5, n.s.) in LCMV-specific CD8+ T cells at the highest levels on day 5 p.i., which largely resolved by day 8 p.i.. Together, these data suggest that activated virus-specific CD8+ T cells experience dynamic ER stress and upregulation of proteostatic pathways during the course of CD8+ T cell differentiation. To test the cell-intrinsic role of ERAD in CD8+ T cell immune responses, we utilized TCR-transgenic CD8+ T cells that were specific for LCMV (termed P14 cells) and were deficient in Sel1L, a critical adaptor of the ERAD complex that recognizes misfolded proteins in ER and recruits them to be retro-translocated to cytosol for proteasomal degradation. We adoptively transferred Sel1L-deficient P14 cells mixed in a 1:1 ratio with wildtype (WT) P14 cells into cogenically disparate mice that were subsequently infected with LCMV. By day 15 p.i., Sel1L-deficient P14 represented only 29.50% of donor cells in peripheral blood, a fraction which was reduced to 7.5% by day 45 p.i.. These data suggest that Sel1L/ERAD is required for CD8+ T cell survival and memory formation following acute viral infection. To investigate the mechanism by which Sel1L/ERAD regulated CD8+T cell fate, we performed RNA-sequencing on WT and Sel1L-deficient P14 cells at day 8 p.i.. Pathway analysis revealed a reduction in pathways corresponding to T-cell metabolism, specifically 'mitochondrial respiratory chain' (FDR 1.59E-03) and 'oxidative phosphorylation'(FDR 2.45E-08), as well as an increase in apoptotic terms. Furthermore, immune signature analysis suggested Sel1L-deficient P14 cells lack memory precursors CD8+ T cells due to enrichment in immune signature of KLRG1hi effector CD8+ T cells (FDR 1.34E-18). Functional validation by flow cytometry revealed that Sel1L-deficient P14 cells are significantly reduced in their ability to generate memory precursor CD8+ T cells (9.05%±3.84 vs. 6.12%±4.28 ***p<0.001, paired t-test) compared to WT on day 8 p.i. and that D6 in-vitro activated Sel1L-deficient CD8+ T cells exhibit reduced mitochondrial membrane potential relative to WT (TMRM MFI FC relative to WT 0.39±0.29 *p<0.05, unpaired t-test) Our studies demonstrate that virus-specific CD8+ T cells experience dynamic ER stress in vivo during an acute viral infection and that Sel1L/ERAD is required for antigen-specific CD8+ T cell persistence following an infection. Ongoing mechanistic studies will help determine how Sel1L/ERAD promotes CD8+ T cell survival and persistence.

A Novel Bispecific T Cell Engager (UMG2-BTCE) Targeting CD1a-CD3ε Is Effective Against Cortical-Derived Acute Lymphoblastic Leukemia

Caterina Riillo*and Daniele Caracciolo*equally contributed to the work.Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and orphan hematological disease derived from malignant transformation of thymic T cell precursors. At present, the prognosis of relapsed/refractory patients remains poor. While immunotherapy has significantly improved the outcome of B cell acute lymphoblastic leukemia (B-ALL), the lack of tumor-restricted T cell antigens hampers its efficacy in T-ALL. (Caracciolo D, Riillo C, et al. Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia. J Immunother Cancer. 2021) Therefore, the development of novel immune-therapeutics is eagerly awaited.CD1a is a cell surface glycoprotein restricted to cortical T-ALL subtype expressed only by cortical thymocytes and Langerhans's cells among human healthy tissue and might represent a valuable therapeutic target for the treatment of T-ALL. On this basis, we develop an asymmetric monovalent 2 +1 bispecific T cell engager (BTCE) derived from a novel humanized UMG2 mAb directed against an original CD1a epitope selectively expressed by cortical T-ALL cells.Methods.To evaluate if UMG2 recognizes a specific CD1a epitope, HEK293T cell line, negative for CD1a expression, has been transfected with a plasmid encoding for CD1a (HEK293T/CD1a) and with a negative control vector and UMG2 reactivity has been evaluated by flow cytometry. A competitive binding assay between UMG2 and commercially available CD1a antibodies was performed. The UMG2 expression profile was evaluated on healthy donor peripheral blood cells and on a panel of cortical T-ALL cell lines. To develop a UMG2 targeting immunotherapeutic construct with a limited unspecific T cell activation, an asymmetric 2+1 UMG2-CD3 bispecific T cell engager (BTCE) was generated using knobs into holes technology. UMG2-CD3 BTCE in vitro T cell-mediated activity was evaluated on HEK293T CD1a antigen-negative cell line, on HEK293T/CD1a, on patient-derived and T-ALL cell lines, co-cultured with healthy donors derived peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at 10:1 E:T ratio.T cell activation, degranulation, proliferation, and pro-inflammatory cytokine secretion were assessed by flow cytometry on primary blasts and on T-ALL cells with effector lymphocytes.To evaluate UMG2-CD3 BTCE anti-tumor activity against CD1a expressing T-ALL cell line in vivo, Hu-PBMCs NSG mouse model was generated, and tumor growth was assessed by fluorescent imaging probe.ResultsUMG2 mAb recognizes a previously uncharacterized CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs. While a strong UMG2 reactivity is observed on both patient-derived samples and T-ALL cells, no binding is found on normal blood cells, indicating the tumor-restricted pattern of reactivity of UMG2.UMG2-CD3 BTCE specifically binds CD1a on leukemic cells and activates CD3ε downstream signaling pathway on T lymphocytes, as demonstrated by the concentration-dependent increase of T cell proliferation, cytotoxic degranulation (CD107a), expression of cell surface activation markers (CD25, CD69), and pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ).UMG2-CD3 BTCE mediates strong and concentration-dependent specific T cell re-directed cytotoxicity only on CD1a expressing leukemic cells in the presence of T lymphocytes. Minimal UMG2-CD3 BTCE residual anti-tumor activity is observed in CD4/CD8 depleted and CD56 enriched lymphocytes, while CD56 depleted and Fc-blocked PBMCs are able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-BTCE could not recruit monocytes and NK cells through Fc-FcyR interaction by reducing the risk of immune-mediated adverse events. Most importantly, in an in vivo of immune-humanized NSG mice engrafted with human T-ALL cells, UMG2-BTCE significantly inhibits tumor growth translating into the survival advantage of treated animals.Conclusion: Taken together, all these results provide a framework for the clinical development of UMG2-CD3 BTCE potentially offering a novel therapeutic path for cortical-derived T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Reduced Autoimmune Cytopenias after Cord Blood Transplant in Pediatric Patients with Nonmalignant Disease Conditioned without Serotherapy

Introduction Autoimmune cytopenias (AIC) are a known complication of hematopoietic stem cell transplant that can lead to increased morbidity and mortality. Higher rates of AIC have been reported in patients transplanted for nonmalignant conditions ranging from 19.5%-56% with the highest rate reported in very young infants (≤3 months) with non-malignant diseases who had undergone unrelated cord blood transplants following ablative conditioning with serotherapy. Objectives Evaluate incidence of AIC in non-malignant pediatric patients receiving an umbilical cord blood transplant conditioned with a fully ablative regimen without serotherapy. Methods We performed a retrospective chart review of 42 pediatric patients receiving umbilical cord blood transplants (UCBT) for nonmalignant disease from 2009-2017. Indications included: SCID (30), IPEX (2), LAD (1), WAS (1), CGD (1), metabolic disorders (3), HLH (1), and other hematologic disorders (3). All patients received Busulfan, Cyclophosphamide, and Fludarabine without serotherapy. AIC included: Autoimmune hemolytic anemia, defined as clinically significant hemolysis (a drop in hemoglobin >2 g/dL, reticulocytosis) and positive direct antiglobulin test (DAT); Immune thrombocytopenia purpura, defined as presence of antiplatelet antibodies with new thrombocytopenia; and autoimmune neutropenia, defined as presence of anti-neutrophil antibodies and neutropenia Results All patients engrafted after receiving a cord blood unit with a median TNC of 15 × 107 kg (range, 5.1 – 26.4). The median time to neutrophil and platelet recovery were 18 days (range,6-30d) and 35 days (range,22-85d), respectively. All but one evaluable patient achieved full donor chimerism (defined as > 95% donor cells in peripheral blood by day +42). The overall incidence of AIC was 11.9% (n=5/42). For patients transplanted at age ≤3 months old, only 1 patient developed AIC (n=1/16). AIC included: AIHA (n=2), Evan's syndrome (n=2) and AIHA in combination with autoimmune neutropenia (n=1).There was no evidence of isolated ITP. Of note, one patient with leaky SCID who developed AIHA had a history of AIHA prior to transplant. Average time of onset of AICs post-transplant was 4.6 months (range 3.9-5.4 months). Four of the patients who had AICs had leaky SCID. The remaining patient had IPEX. All patients with AIC received UCB matched at 8 and 9 alleles. Mixed donor chimerism was noted in 3 patients at time of development of AIC. Conclusion We have observed decreased rates of AIC after UCBT in pediatric patients with nonmalignant diseases, particularly in patients transplanted at ≤ 3 months age, which compared favorably to previous reports. We hypothesize that omission of serotherapy after UCBT allows for early T cell recovery perhaps allowing for expansion of regulatory T cells and decreased rates of autoimmunity.

Excellent Engraftment with Reduced Treatment Related Mortality for Young Pediatric Patients Using Umbilical Cord Blood Transplantation (UCBT) Conditioned without Serotherapy

Introduction There is no consensus regarding the best donor for children undergoing stem cell transplantation in the absence of a matched related donor (MRD), especially for patients with nonmalignant diseases. The incidence of infections, GvHD, and graft failure remain major problems. Objectives Evaluate outcome for patients undergoing UCBT conditioned without serotherapy, which is as an attractive option for these patients because of immediate availability, lower incidence of GvHD, and high probability of adequate cell dose for young patients. Methods From 2008-2019, we performed UCBT in 84 children, median age 10 mo. (range, 2–108 mo.) with: ALL (13), AML (15), MDS (5), SCID (36), IPEX (2), LAD (1), WAS (1), CGD (1), metabolic disorders (4), HLH (2), and hematologic disorders (4). Eleven patients (30%) with malignancies had minimal residual disease present before UCBT. Five patients with AML had chloromas present prior to UCBT. 26 patients with immune deficiencies had persistent infections at transplant. All patients were enrolled on prospective clinical trials. Pts with AML/ MDS/ or nonmalignant disorders received busulfan, cyclophosphamide, and fludarabine; pts with ALL received TBI (12Gy), cyclophosphamide, and fludarabine. No patient was treated with serotherapy. All cord blood units were matched 5 or 6/6 HLA antigens at A, B and allele at DR. Results Infused cord blood contained median TNC 15 × 107 kg (range, 5.1 – 26.4). All evaluable patients engrafted by day 42. The median time to neutrophil and platelet recovery were 18 days (range, 6-42d) and 35 days (range,22-85d), respectively. All evaluable patients achieved full donor chimerism (defined as > 95% donor cells in peripheral blood by day +42). The overall survival (OS) at 2 years was 75% with a median follow up of 5 years (range: 0.5 – 11yr). The OS for patients with SCID was 92% (33/36) at 2 years with engraftment of all lineages. The cumulative incidence of aGvHD grade II-IV by day 100 was 9% (n=8) with only 3 patients having grade IV. No pt developed cGvHD. Twenty-one patients died, 13/21, 62% died from disease relapse or disease progression. Treatment related mortality occurred for 7 patients; severe GvHD (n=2) and MOF (n=5). No infection related mortality was seen. Conclusion We conclude that lacking a MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children. Our experience highlights minimal treatment related mortality, rapid engraftment, resolutions of antecedent infections, and a low incidence of GvHD. These results translate in a better quality of life for these young patients.

Excellent Outcomes for Pediatric Non-Malignant Diseases Using Umbilical Cord Blood Transplantation (UCBT) Conditioned without Serotherapy in the Absence of a Matched Related Donor

Introduction There is no consensus about the best donor for children with non-malignant disorders in the absence of a matched related donor (MRD). Objectives Evaluate UCBT as an attractive option for these patients because of prompt availability, lower risk of GvHD, and increased cell dose at young ages. Methods From 2008-2017, we performed UCBT in 42 children, median age 5 mo. (range, 2–108 mo.) with: SCID (30), IPEX (2), LAD (1), WAS (1), CGD (1), metabolic disorders (3), HLH (1), and other hematologic disorders (3). 26 patients had persistent infections prior to transplant: PJP (5), fungal (1), RSV (4), Parainfluenza 3 (4), VZV (1), Norovirus (1), CMV (4), Rhinovirus (2), bacterial (4); with 9 patients having pneumonia (20%), and 7 (17%) patients requiring mechanical ventilation prior to transplantation. Thirty-four percent of patients were 6/6 HLA antigen matched, and 66% were one HLA antigen mismatched. All patients were enrolled on a prospective clinical trial using fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. The median TNC was 15 × 107 kg (range, 5.1 – 26.4). Results The median time to neutrophil and platelet recovery were 18 days (range,6-30d) and 35 days (range,22-85d), respectively. All but one evaluable patient achieved full donor chimerism (defined as > 95% donor cells in peripheral blood by day +42). The overall survival (OS) at 2 years was 90% (95% CI:77-96%) with a median follow up of 4 years (range: 0.5 – 8.5yr). The OS for patients with SCID was 93.3% (28/30) at 2 years with engraftment of all lineages. Four patients died, from severe GvHD (n=2) and MOF (n=2). The cumulative incidence of aGvHD grade II-IV by day 100 was 16% (n=7) with only 2 (IV). No cGvHD has been seen. All but three patients developed engraftment syndrome at a median time of 19 days, (range: 6-46) successfully treated with steroids. All patients with viral infections at the time of transplant cleared infection at a median time of 54 days (range, 44-91). ELISpot analysis after resolution of infections showed T cell responses against the pertinent viruses. The median absolute CD3 (x10^6/L) counts by day 42 and 60 were 361 and 733; respectively. The median absolute CD3CD4 counts by day 42 and 60, were 296, and 506; respectively. The median absolute CD3CD8 absolute count by day 42 and 60, were 71 and 114; respectively. IVIg was discontinued at a median 138 days; (range: 52-769d). Median time to begin immunizations was 11 months, (range: 8-26). All evaluable patients have had correction of their immune or metabolic defect including adequate B cell function in SCID patients. Conclusion We conclude that in the absence of a MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in children with non-malignant disorders, producing rapid engraftment, prompt functional immune-reconstitution, and a low incidence of GvHD.

Specific Bone Marrow Mesenchymal Subsets in Patients with Myelodysplastic Syndromes Harbor Molecular Perturbations That Alter the Dynamics of Competition between Pre-Leukemic Clones and Normal Cells

Myelodysplastic syndromes (MDS) are clonal disorders of aberrant hematopoietic stem cells. The role of the hematopoietic microenvironment (HMEV) in the initiation and maintenance of MDS remains unclear, though model systems have provided strong evidence for the potential of specific mesenchymal subsets, mostly osteoprogenitor cells, within the HMEV as a driver of the disease. Distinct behavior of various models emphasizes the importance of analyzing specific and highly defined populations rather than bulk mesenchymal cells when investigating patient-derived bone marrow (BM) samples.We have developed a platform that allows us to prospectively isolate specific human BM mesenchymal subsets with a comparable transcriptional profile to mouse osteoprogenitor cells that have been shown to drive hematological malignancies when perturbed (Raaijmakers et al., 2010; Dong et al, 2016). Using a combination of three cell surface markers (CD271, CD146, CD106) that were previously shown to prospectively enrich for mesenchymal cells in human BM, we isolated and performed transcriptional and functional analysis of four distinct subsets within the non-hematopoietic (CD45, CD235), non-endothelial (CD31) compartment of the human BM. The four populations are labeled 1A (CD271+CD106-), 1B (CD271+CD106+), 2A (CD271+CD146+CD106-), and 2B (CD271+CD146+CD106+) (Figure 1A). Comparative analysis of differentially expressed gene sets based on RNA-seq within the human and mouse mesenchymal populations identified population 2B to be the most similar to osterix (OSX)-labeled mouse osteoprogenitor cells, whereas population 1A was more similar to osteocalcin (OCN)-labeled mouse mature osteoblasts. In animal models, genetic perturbations specifically in OSX but not OCN labeled cells resulted in hematological malignancy. Based on that observation, we hypothesized that population 2B is the cell subset most likely to harbor molecular perturbations in the BM of MDS patients. We analyzed populations 2A, 2B, 1(1A+1B) and another population 3 which is devoid of colony forming cells and encompasses all CD271-CD146- non hematopoietic stromal cells in 16 human MDS patients. BM from 11 patients undergoing hip replacement surgeries was used as a source of age- and gender-matched normal controls.Despite the heterogeneity of the disease, principal component analysis revealed that the transcriptional profile of normal samples clearly separated from MDS patients mostly in populations 2B and 1 (Figure 1B). Functional annotation of the differentially expressed genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) demonstrated minimal overlap of enriched terms between the four populations. Thus our data demonstrate transcriptionally distinct subsets of BM mesenchymal cells and identify signatures of transcriptional deregulation which differentiate these cell subsets in MDS patients from normal individuals.Osteopontin, an extracellular matrix protein expressed by osteoblasts among other cells, was the most significantly differentially expressed gene in population 2B of MDS patients.To study the effect of the gene expression perturbations on MDS evolution in vivo, we created a chimeric model where BM cells expressing the Nup98/HoxD13 (NHD13) translocation were transplanted in competition with wild type cells in wild type (WT) or osteopontin knock out (Opn-KO) recipients. Animals were conditioned using immunotoxin to avoid the damaging effect of irradiation on the microenvironment. Following chimerism of donor cells in peripheral blood over 24 weeks demonstrated a competitive advantage of NHD13 cells in the Opn-KO recipients leading to a significant difference in survival when compared to NHD13 cells transplanted in wild type mice (Figure 1C, D).Altogether, our data provide evidence that specific mesenchymal progenitor subsets in the BM of MDS patients harbor molecular perturbations that contribute to the competitive advantage of MDS over normal hematopoietic cells. DisclosuresSykes:Clear Creek Bio: Equity Ownership, Other: Co-founder.

Specific Immunomodulation of Alloantibody Responses

Introduction Antibody (Ab) mediated rejection of transplanted organs presents a formidable barrier to solid organ transplantation. We have previously shown that intravenous (i.v.) exposure to MHC-mismatched donor peripheral blood cells using a novel, mild conditioning protocol results in B-cell tolerance with transient T-cell unresponsiveness. Here we show that B-cell tolerance is stable and allo-Ab cannot be induced even after loss of chimerism and repeated exposure to donor cells without immunosuppression. Materials and Methods MGH miniature swine conditioned with CD3-immunotoxin, low-dose (100cGy) total body irradiation (TBI), and a short, 45 day course of cyclosporine A, received cells i.v. from a haplo- or fully MHC-mismatched donor. Weekly immunophenotyping of peripheral blood was performed. Hematopoietic cells from donor and third-party MHC-mismatched pigs were injected subcutaneously (s.c.) +/- complete Freund’s adjuvant (CFA) monthly, starting 60 days following conditioning. Control animals with and without conditioning, but without donor HCT, were challenged in the same manner. The draining lymph nodes were analyzed by Histology and bulk RNAseq. Allo-Ab was measured by flow cytometry and complement dependent Ab mediated cellular cytotoxicity assay. Results and Discussion Phenotypic analysis of recipient cells during conditioning revealed that most cell populations, including CD4+ Foxp3+ Treg cells, were only partially depleted and quickly recovered to baseline. Allo-Ab responses were not detected even after repeated s.c. injections of donor cells. The addition of CFA provoked a transient, weak IgM response suggestive of an extrafollicular B-cell response with no germinal center development. Control animals consistently developed Allo-Ab responses against donor cells. Histologic analysis of draining lymph nodes shows similar morphology and presence of active Germinal centers in both groups. However, preliminary analysis reveals an abundance of FoxP3+ cells in tolerant animals. This suggests that donor cells trigger a germinal center reaction in tolerant animals that may be controlled by memory T-follicular regulatory cells. Further histologic and transcriptome analysis of draining LN cells are currently underway and may yield additional insights into the mechanical underpinnings. Conclusion Stable B-cell tolerance to donor antigens across fully MHC-mismatched barriers can be induced by a novel, mild conditioning protocol that does not result in prolonged T-cell depletion. We are currently investigating the mechanism of this phenomenon. Understanding immunomodulatory mechanisms involved in controlling B-cell responses to transplantation antigens could lead to significant advances in improving transplantation outcomes.

Inhibition of Both Activated p38 Mapkα and mTORC1 Potentiates the Effect of SR1 on Promoting Hematopoietic Reconstitution of Ex Vivo Expanded Human Umbilical Cord Blood Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) transplantation is a curative treatment for various hematological disorders. However, its use has been limited in part by the difficulty to obtain sufficient numbers of transplantable HSCs, especially from human umbilical cord bloods (hUCBs). Ex vivo expansion of cord blood HSCs can potentially mitigate the shortfall if the expanded HSCs can maintain their stemness for long-term hematopoietic reconstitution.Our previous studies have shown that some hUCB HSCs lose their "stemness" during ex vivo expansion in part due to induction of cellular senescence via p38MAPKα(p38) or mTORC1 activation and inhibition of p38 and mTOR C1 promotes HSCs expansion (Ann Hematol. 2012; 91:813-823; Transplantation. 2014; 97:20-29). Recently, it was shown that StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AHR), could significantly promote ex vivo expansion of HSCs, primarily via inhibition of HSCs differentiation. Therefore, we investigated whether the combination of SR1 with LY2228820 (LY) and rapamycin (Rapa) to inhibit p38 and mTOR C1, respectively, can enhance ex vivo expansion of hUBC HSCs by inhibiting HSCs differentiation and senescence.Next, we confirmed that co-inhibition of activated p38 and mTORC1 promotes SR1 induced HSCs expansion ex vivo through inhibition of senescence.Our combined culture system in 7 days led to a 16.2-fold increase for CD34+CD90+ subpopulation and a 6.1-fold increase for CD34+CD45RA- subpopulation compared to input cells (Blood,126(23):381-381) .To determine the hematopoietic reconstitution ability of the progeny of hUCB CD34+ cells expanded with inhibitors of p38, mTORC1, and AHR, we transplanted 1000 to 30000 uncultured hUCB CD34+ cells or a fraction of the final culture equivalent to 1000 to 10000 starting hUCB CD34+ cells into sub-lethally irradiated 6 to 10-week-old NOD-Prkdcscid Il2rgnull (NOG) mice. Donor cell engraftment was analyzed at 8 and 13 weeks after the transplantation. The results showed that the progeny of 10,000 hUCB CD34+ cells cultured with LY, Rapa and SR1 produced significant greater donor cell engraftment in the recipients’ peripheral blood compared with the same number of un-cultured hUCB CD34+ cells (2.19-fold, p<0.01), the progeny of 10,000 hUCB CD34+ cells cultured with vehicle (2.76-fold, p<0.01), or the progeny of 10,000 hUCB CD34+ cells cultured with SR1 alone (1.72-fold, p<0.05). The similar results were also found in the recipients’ bone marrow and spleen. In addition, the progeny of 3,000 hUCB CD34+ cells cultured with LY, Rapa and SR1 generated the levels of peripheral blood donor cell engraftment that similar to these produced by 30,000 un-cultured hUCB CD34+ cells and the progeny of 10,000 hUCB CD34+ cells cultured with SR1 alone. More importantly, the hUCB CD34+ cells cultured with LY, Rapa and SR1 retained multi-lineage differentiation potential as they were able to produce human B cells, myeloid cells, red blood cell progenitors, megakaryocytes, and NK cells in the recipients’ bone marrow. Furthermore, hUCB CD34+ cells cultured with LY, Rapa and SR1 produced the highest levels of human HSCs (CD34+CD38-/CD34+CD90+/CD34+CD45RA- cells) engraftment compared to all other cells.Collectively, these findings suggest that the combined inhibition of p38, mTORC1 and AHR is more effective in promoting the expansion and hematopoietic reconstitution of hUBC HSCs than inhibition of p38, mTORC1 or ARH alone probably via inhibition of HSCs senescence and differentiation. This new strategy may alleviate the limitation of hUBC for transplantation in adult patients. DisclosuresNo relevant conflicts of interest to declare.

The Intravenous Route of Injection Optimizes Engraftment and Survival in the Murine Model of In Utero Hematopoietic Cell Transplantation

In utero hematopoietic cell transplantation (IUHCT) has the potential to treat a number of congenital hematologic disorders. Clinical application is limited by low levels of donor engraftment. Techniques that optimize donor cell delivery to the fetal liver (FL), the hematopoietic organ at the time of IUHCT, have the potential to enhance engraftment and the clinical success of IUHCT. We compared the 3 clinically applicable routes of injection (intravenous [i.v.], intraperitoneal [i.p.], and intrahepatic [i.h.]) and assessed short- and long-term donor cell engraftment and fetal survival in the murine model of IUHCT. We hypothesized that the i.v. route would promote direct donor cell homing to the FL, resulting in increased engraftment and allowing for larger injectate volumes without increased fetal mortality. We demonstrate that the i.v. route results in (1) rapid diffuse donor cell population of the FL compared with delayed diffuse engraftment after the i.p. and i.h. routes; (2) higher FL and spleen engraftment at early prenatal time points; (3) enhanced stable long-term peripheral blood donor cell engraftment; and (4) improved survival at higher injectate volumes, allowing for higher donor cell doses and increased long-term engraftment. These findings support the use of an i.v. route for clinical protocols of IUHCT.

The Use of Eicosanoids to Enhance Donor Cell Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)

IntroductionIn utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE2) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment.MethodsTen million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2Kb) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2Kd) via the vitelline vein. Donor cells were treated with vehicle, PGE2, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE2 and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM.ResultsPGE2 pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p < 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE2 treatment. PGE2 treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p<0.05) and decreased donor cell anti-caspase 3 staining (9.3±1.2% vs. 64.1±10.6%; p<0.05) at 96 hours post-IUHCT. These findings of increased FL/FS engraftment and increased survival at 72 to 96 hours post IUHCT following donor cell treatment with PGE2 translated into significantly increased long-term donor cell engraftment up to 6 months of age. Similar to PGE2, ex vivo treatment of donor cells with 11,12-EET or 14,15-EET resulted in enhanced long-term allogeneic donor cell engraftment (Figure 1). Balanced multilineage engraftment was seen for all treatments suggesting enhanced engraftment at the level of the HSC. No deleterious effects were observed on murine growth or overall health.ConclusionsThe ex vivo treatment of donor cells with eicosanoids including PGE2, 11,12-EET, and 14,15 EET prior to IUHCT results in enhanced long-term multilineage allogeneic donor cell engraftment. Detailed studies of early homing to fetal hematopoietic organs and survival at 96 hours following IUHCT of PGE2 treated donor cells suggest the mechanism of enhanced engraftment is mainly do to a pro-survival effect of PGE2 and related eicosanoids. This work represents a novel application of these eicosanoids in an allogeneic model of IUHCT and highlights their potential to assist in future clinical applications of IUHCT. [Display omitted] DisclosuresZon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Umbilical Cord Blood Transplantation Conditioned without Serotherapy Is an Excellent Curative Alternative for Pediatric Non-Malignant Diseases

Stem cell transplantation for patients with non-malignant disorders is often challenging, with frequent failure to engraft. In the absence of an HLA-identical source, there is no consensus about the best alternative hemopoietic stem cell donor for patients who lack a matched sibling. Although cord blood transplant has been a recent option for these patients, an increased risk of viral infections and the potential for failed engraftment have remained problematic and associated with significant mortality. We therefore studied 25 children who underwent UCBT transplant at our institution for non-malignant disorders between 2007 and 2014. Their median age was 8 months (range, 2–66 months) an their diagnoses were: SCID (n=17), IPEX (n=2), LAD (n=1), Wiskott-Aldrich syndrome (n=1), Krabbe (n=1), mannosidosis (n=1), HLH (n=1), and Diamond-Blackfan anemia (n=1). Five patients had persistent viral infections prior to transplant: RSV (n=2), Parainfluenza 3 (n=1), VZV (n=1) and Norovirus (n=1), and 7 (30%) patients had required mechanical ventilation prior to transplantation. Eight of 25 patients were 6/6 HLA antigen matched with their cord stem cell donor, and 17 were one HLA antigen mismatched. All patients participated in a prospective trial consisting of myeloablative conditioning using busulfan, cyclophosphamide and fludarabine without the use of serotherapy. The median total nucleated cell dose in the transplant was 10x107 TNC/kg (range, 5.9–25.4). The median time to neutrophil recovery was 19 days (range,8-30) and the median time to platelet recovery was 38 days (range, 26-127). All but one evaluable patient achieved full donor chimerism (defined as > 95% donor cells in peripheral blood) by day +42. The exception was a patient with Krabbe’s disease who is nonetheless 60% donor chimeric at 6 months post-transplant with normalization of enzyme levels. No severe aGVHD or cGVHD has occurred and only one patient developed grade II aGVHD (skin). All except the patient with HLH (who died of multiorgan failure) are alive with a median follow up of 2 years (range, 10 days–5 years) and a 3-yr OS by Kaplan Meier analysis of 96% (95%CI: 73%-99%). All patients with viral infections at the time of transplant cleared the infection at a median time of 51 days (range, 44-54). ELISPOT analyses of peripheral blood at the time that infections responded showed significant T cell responses against the pertinent viruses. All evaluable patients have normalization of their immune or metabolic defect including adequate B cell function in SCID patients. Hence, UCBT after full intensity conditioning without serotherapy for pediatric non-malignant diseases can produce rapid functional engraftment and immune-reconstitution in the absence of significant GVHD, leading to excellent overall survival.

Defining Engraftment Potential within the Lineage Positive Population in Murine Marrow

The study of highly purified hematopoietic stem cells (HSCs) has dominated the field of hematopoietic stem cell biology. It is widely believed that the true stem cell population lies within the Lineage negative (Lin-) population, further sub-fractionated using positive and negative selection for surface markers such as c-Kit, Sca-1, CD150, CD41, CD48, and CD34. It is research on these highly purified subsets of HSCs that forms the foundation for almost all our knowledge of HSCs, and has led to the dogma that marrow stem cells are quiescent with a stable phenotype and therefore can be purified to near homogeneity. In contrast, we have shown that a large percentage of long-term multi-lineage marrow repopulating cells in whole bone marrow (WBM) are actively cycling, that these cycling stem cells are lost during conventional HSC isolation, and that they can be found, in part, within the discarded Lineage positive (Lin+) population. Here we present data further characterizing the stem cell potential in the Lin+ fraction. We incubated WBM from B6.SJL mice with fluorescently tagged antibodies directed against TER119, B220, or T-cell markers (CD3, CD4, CD8), isolated the distinct Lin+ subsets by FACS, and then competitively engrafted each Lin+ subset into lethally irradiated C57BL/6 host mice. Donor chimerism and lineage specificity of donor cells in peripheral blood were analyzed by flow cytometry at 3 months. Although classically considered devoid of stem cell activity, we found that, when competed against equal numbers of C57BL/6 WBM, the TER119+ and B220+ B6.SJL donor cells contributed to 33% and 13% of the peripheral blood chimerism, respectively. In both cases, the engraftment was multi-lineage. When 70,000 T cell marker+ donor cells were competed with 300,000 C57BL/6 WBM, the donor cells contributed up to 1.6% of the peripheral blood multi-lineage chimerism. Given the size of the Lin+ fraction in WBM, such chimerism indicates a significant stem cell potential within this typically discarded population. Further time-points, secondary transplants and limited dilution studies are in progress to further define the prevalence and potency of this stem cell population.We have been testing mechanisms governing the loss of this stem cell population during HSC purification. First, we have previously shown that bulk Lin+ engraftment potential is due to cycling stem cells. We hypothesize that fluctuations in surface epitope expression with cell cycle transit render this population difficult to isolate with antibody-mediated strategies that rely on stable epitope expression. To begin testing this, we tracked the fluctuation of stem cell markers on Lin- cells in vitro. We isolated Lin- cells that were also negative for the stem cell markers c-Kit and Sca-1, placed them in liquid culture and, 18 hours later, re-assessed for stem cell marker expression by flow cytometry. We found that, although initially stem cell marker negative, up to 6%, 14%, and 2% of the Lin-/stem cell marker negative cells became positive for c-Kit alone, Sca-1 alone, or both c-Kit and Sca-1 expression, respectively. We are currently testing this population for a correlation between gain of c-Kit- and Sca-1 expression and stem cell function. Second, it is possible that there is a distinct subset of HSCs that are positive for both Lin+ markers and stem cell markers with stable stem cell capacity and that these distinct stem cells are thrown out in the process of lineage depletion. To begin testing this hypothesis, we have simultaneously stained WBM with antibodies directed against the Lin+ markers and conventional stem cell markers. Our preliminary data indicate that each Lin+ fraction tested to date has a subpopulation that is also positive for c-Kit and Sca-1. For example, 21% of CD3+ cells, 6.2% of CD4+ cells, 2.26% of CD8+ cells, 0.5% of B220+, and 0.45% of TER119+ cells express both c-Kit and Sca-1. We suspect these two populations have distinct functional phenotypes and experiments characterizing the molecular phenotype and engraftment capacity of these subpopulations are ongoing. In sum, our data indicate that stem cell purification skews isolation towards a small population of quiescent stem cells, underrepresenting a potentially large pool of actively cycling HSCs that are found within the Lin+ fraction. These data underscore the need to re-evaluate the total hematopoietic stem cell potential in marrow on a population level. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Mechanistic Studies Investigating Neutrophil and Lymphoid Cell Depletion By IMGN529, a CD37-Targeting Antibody-Drug Conjugate (ADC)

CD37 is a surface antigen widely expressed on malignant B cells in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In normal tissues, CD37 expression is restricted to lymphoid tissues and blood cells, with high levels of expression on B lymphocytes and low levels on non-B lymphoid and myeloid cells. IMGN529 is a CD37-targeting ADC currently in a Phase I clinical study in adult patients with relapsed or refractory NHL (NCT01534715). This ADC uniquely combines the intrinsic pro-apoptotic and immune effector activities of its anti-CD37 antibody component with the potent cytotoxic mechanism provided by targeted delivery of its maytansinoid payload, DM1. In the Phase I study, IMGN529 has demonstrated early evidence of clinical activity. A reduction in lymphocyte counts was also observed in the majority of patients after dosing, consistent with the proposed mechanism of action of a CD37-targeted therapy. However, in the initial dose-escalation phase, some patients experienced transient, early-onset neutropenia.To investigate the potential mechanisms of this transient neutropenia observed in patients, different pre-clinical models were considered and utilized to recapitulate clinical findings. In vitro studies with peripheral blood cells from normal human donors demonstrated that incubation with IMGN529 for 1 hour or 24 hours resulted in significant B-cell depletion with no apparent neutrophil depletion detected, similar to observations after rituximab treatment. In contrast, alemtuzumab treatment in vitro resulted in both B-cell and neutrophil depletion. This is consistent with the high level of CD37 expression on target B cells and the relatively low CD37 expression level on other blood cells. Analysis of cytokine release by normal human donor peripheral blood cells incubated with IMGN529 revealed increased levels of IL-8, CCL2 (MCP-1) and CCL4 (MIP-1β), but not IL-6 or TNF, to a similar extent as rituximab but less pronounced than alemtuzumab. An anti-murine CD37 antibody was identified to enable in vivo studies in a murine model and characterize CD37 expression on murine blood cells. Similar to the expression profile of CD37 in human peripheral blood cells, CD37 expression on murine peripheral blood cells was highest in B cells, with much lower expression seen on T cells and granulocytes. In vivo activity of the anti-muCD37 antibody and the corresponding ADC, with the same SMCC-DM1 linker-payload combination as IMGN529, was evaluated to discern antibody and payload-mediated events in comparison to the classic cytotoxic cyclophosphamide (CPA). Treatment of C57/B6 mice with 1-10 mg/kg of anti-muCD37 antibody or anti-muCD37 ADC resulted in a significant decrease in absolute lymphocyte counts (ALC) lasting greater than 7 days and a transient decrease in absolute neutrophil counts (ANC) lasting 1-2 days. A non-targeted control SMCC-DM1 ADC had no effect on ALC or ANC counts, suggesting the decrease is a CD37-mediated effect. In contrast, treatment with CPA resulted in an ALC decrease with similar kinetics but a more pronounced ANC decline. No impact on bone marrow lymphocyte, myeloid or erythroid precursor cell counts was observed in response to the anti-muCD37 antibody or anti-muCD37 ADC, whereas CPA treatment caused reduced cellularity with a decrease in the percentage of mature myeloid precursors and neutrophils in bone marrow. Elevated levels of CCL2 and CCL4 chemokines were detected in mouse plasma after anti-muCD37 ADC treatment, which may contribute to a redistribution of circulating neutrophils into peripheral tissues. Studies are currently underway to assess neutrophil distribution in murine tissues post anti-muCD37 ADC treatment.Current preclinical studies provide no clear evidence for direct IMGN529-mediated depletion of normal human neutrophils in the context of B-cell depletion in vitro. In vivo studies with an anti-muCD37 ADC recapitulate transient peripheral lymphopenia and neutropenia with no impact on bone marrow precursors observed, indicative of a different mechanism than classic chemotherapy-induced bone marrow myelosuppression. These preliminary results suggest a role for chemokine-mediated neutrophil redistribution following CD37 engagement, which is the subject of further studies. DisclosuresDeckert:ImmunoGen, Inc.: Employment, Equity Ownership. Ponte:ImmunoGen, Inc.: Employment, Equity Ownership. Coccia:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Chicklas:ImmunoGen, Inc.: Employment, Equity Ownership. Yi:ImmunoGen, Inc.: Employment, Equity Ownership. Watkins:ImmunoGen, Inc.: Employment, Equity Ownership. Ruiz-Soto:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership; sanofi: Employment. Lutz:ImmunoGen, Inc.: Employment, Equity Ownership.

269 : TSLP driven expansion and activation of type 2 innate lymphoid cells is augmented by IL-25 and IL-33

TSLP, IL-25 and IL-33 are primarily epithelial derived cytokines which act on common immune cell populations and individually promote pro-inflammatory responses generally with a T H 2 bias. Type 2 innate lymphocytes (ILC2s) are a newly identified population of lineage negative innate immune cells which are present in a number of inflammatory disease settings capable of producing high levels of type 2 cytokines in response to stimulation with the IL-25, IL-33 and most recently TSLP. Previously we observed that in combination TSLP, IL-25 and IL-33 induced multiple pro-inflammatory factors in human and mouse CD4 + T-cell cultures dominated by high levels of IL-5 and IL-13 production provoking the hypothesis that these responses may be due to the expansion of ILC2 cells in these cultures. In this study we demonstrate TSLP in combination with IL-25 and IL-33 is capable of driving the expansion of ILC2 cells from normal donor peripheral blood cells including PBMC, negatively selected untouched CD4 + T-cell cultures, and sorted ILC2s. Expansion of ILC2 cells occurred in response to TSLP in combination with either IL-25 or IL-33 but was most abundant when all three cytokines were used in combination. ILC2 expansion was dependent upon the presence of TSLP as neither IL-25 nor IL-33 alone or in combination was capable of inducing a similar response. ILC2s expanded by TSLP, IL-25 and IL-33 produced high levels of IL-5 and IL-13 and expressed high surface levels of the TSLPR. The specificity of the responses were demonstrated utilizing CD4 + T-cell cultures from receptor deficient mice and cytokine specific inhibitors. Additionally sorting CD4 + T-cells to >99% purity abolished the observed ILC2 induction. ILC2s represent an additional population of innate immune cells which may have a profound influence on both developing and established immune responses in inflammatory settings where IL-25, IL-33 and TSLP are released.

Odontoid infiltration and spinal compression in Farber Disease: reversal by haematopoietic stem cell transplantation

Farber disease (FD) is a lysosomal storage disorder caused by accumulation of ceramide in various organs and tissues, most notably the central nervous system, subcutaneous tissues and respiratory tract. We report a girl who developed major destructive bone involvement, which affected the odontoid process and produced spinal compression at 9 years of age. Bone involvement was proven histologically but resolved, as assessed by serial MRI scanning, following matched unrelated donor haematopoietic stem cell transplantation. This transplant resulted in only partial donor chimerism (less than 10 % donor cells in peripheral blood), yet this was sufficient to almost normalize acid ceramidase levels in leukocytes and to produce dramatic improvements in subcutaneous nodules and joint mobility as well as the beneficial effect on the involved bone. Unfortunately, the transplant was rejected after 2 years but the patient was rescued from an aplastic state by successful haploidentical peripheral blood stem cell transplantation and remained a full donor chimera without recurrence of the bone involvement and with steadily improving mobility at the age of 17 years. We describe an FD patient who presented with severe destruction of the odontoid by inflammatory tissue which was reversed after long-term control achieved by allogeneic hematopoietic stem cell transplantation. After extensive literature search, we believe that this is the first report of bony involvement in Farber disease.

Prostaglandin E2 Provides a Selective Donor Cell Competitive Advantage and Improves Long-Term Engraftment After in Utero Hematopoietic Cell Transplantation.

AbstractAbstract 2993 Introduction:Competition from the host hematopoietic compartment is the primary barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Prostaglandin E2 (PGE2) has been shown to increase donor cell homing, cell survival, and engraftment after postnatal HCT through increased CXCR4 and Survivin expression. We have previously shown that pre-incubation with Diprotin A, a CD26 inhibitor, confers a homing and engraftment advantage for donor cells after IUHCT. We hypothesize that pre-incubation of donor cells with PGE2 alone, or in combination with Diprotin A, will provide a selective donor cell homing and competitive advantage and enhance long-term engraftment after IUHCT. Methods:Balb/c pregnant mice underwent IUHCT with 1 × 107 B6GFP bone marrow (BM) cells, with or without pre-incubation with PGE2, Diprotin A, or PGE2 plus Diprotin A, by vitelline vein injection at E14. A 1μM per 1×106 BM cell concentration of dmPGE2, a long acting derivative, was used to treat the donor BM for two hours prior to IUHCT. In the combined therapy group, a 50 μM concentration of Diprotin A was used to treat the BM for an additional 15 minutes following PGE2 incubation. Homing to and engraftment in the fetal liver (FL) at early prenatal time points and frequency of donor cells in peripheral blood (PB) at 1 month and PB and BM at 4 months were assessed by flow cytometry. Analysis of donor cell cycling and cell survival was performed by in vitro BrdU cell labeling and anti-survivin and anti-caspase 3 antibody staining respectively. Results:Ex-vivo incubation of donor cells with PGE2 alone, Diprotin A, and PGE2 plus Diprotin A increased homing of allogeneic BM to the FL at 4 and 24 hours after IUHCT with the combined treatment group reaching statistical significance. Analysis of cell cycling of donor cells engrafted in the fetal liver at 96 hours after IUHCT demonstrated a 1.4 fold increase in the proportion of cells in the S and G2+M phases in PGE2 treated cells relative to non-treated BM controls (31.4% vs 21.2%) and a 1.2 fold increase over E18 FL controls (25.5%). A concurrent four fold increase in anti-survivin staining and a seven fold decrease in anti-caspase 3 staining were also seen in PGE2 treated cells engrafted in the fetal liver relative to non-PGE2 treated cells (anti-survivin: 19.8% vs. 4.75%. anti-caspase 3: 9.3% vs. 64.1%). Most importantly, a sustained increase in multi-lineage donor cell chimerism was observed (25–32%) in the PB and BM of recipients of PGE2 treated cells, as compared to controls, which demonstrated a decline from 13.4% to 4.9% over a 4 month period after IUHCT. Treatment with Diprotin A alone demonstrated a smaller increase in donor cell chimerism at 4 months after IUHCT. However, there was no statistical difference at 4 months between the PGE2 and PGE2 plus Diprotin A treatment groups (Figure 1). Conclusions:PGE2 exposure results in markedly improved long-term engraftment of donor cells. This improvement is associated with increased cell cycling, increased survivin expression and reduced apoptosis suggesting that PGE2 exposure confers a competitive advantage to donor hematopoietic stem cells that persists over time by increasing cell cycling and survival. These increases likely explain the long-term advantage of PGE2 treatment. While CD26 blockade conferred an improvement in early homing kinetics, the combination of PGE2 plus CD26 blockade did not result in higher long term engraftment than PGE2 alone. We conclude that PGE2 pre-incubation is a promising method to achieve sustained increased donor cell engraftment after IUHCT. [Display omitted] * p<0.05 Disclosures:No relevant conflicts of interest to declare.

Radiation rescue: mesenchymal stromal cells protect from lethal irradiation.

BackgroundSuccessful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals.Methods and FindingsMouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals.ConclusionsOur data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.

The Role of Human Leukocyte Antigen G in Inducing Immune Tolerance After Allogeneic Hematopoietic Stem Cell Transplantation.

Abstract 4478▪ Background and ObjectiveAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is now applied widely for the treatment of hematological or non-hematological malignancies, aplastic anemia and hereditary diseases. Recently, a protocol for haploidentical allo-HSCT that combines granulocyte-colony stimulating factor (G-CSF) primed bone marrow (G-BM) and peripheral blood stem cells (G-PBSC) without in vitro T-cell depletion received great success. But the mechanism of G-CSF inducing immunotolerance in haploidentical-HSCT has not yet been clarified. Human leucocyte antigen G (HLA-G) is a nonclassical HLA class I molecule, the tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. Because HLA-G closely related to immunotolerance, we investigate the role of HLA-G in inducing immune tolerance after allo-HSCT and the effects of G-CSF on the expression and secretion level of HLA-G. MethodsFlow cytometry was used to detect the expression of membrane-bound HLA-G (mHLA-G) on donor peripheral blood cells (PBC) or bone marrow (BM) cells. The levels of soluble HLA-G (sHLA-G) in the plasma and bone marrow fluid were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the expression and secretion level of HLA-G in bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) after G-CSF stimulated were detected by flow cytometry and ELISA, respectively; Separated T lymphocytes which expressed high level of HLA-G were cultured with allogeneic T lymphocytes and relative response index (RPI) was measured with MTT assay; Furthermore, separated T lymphocytes were cultured with allogeneic BMMCs and the levels of IFN-γ and IL-10 in culture supernatant were determined by ELISA. ResultsThe mean level of mHLA-G after G-CSF mobilization in the PBC or BM cells was significantly higher than that before G-CSF mobilization (P<0.05). The level of mHLA-G or sHLA-G in BM cells was higher than that in PBC after G-CSF mobilization (P<0.05). The level of mHLA-G or sHLA-G in BMMCs or PBSCs which were stimulated by G-CSF was higher than that of the controls (P<0.05), and the level of HLA-G in BMMCs was higher than that in PBSCs. HLA-G predominant expressed in CD3+ T cells; The results of allogeneic mixed lymphocyte culture revealed that immunological function of the separated T lymphocytes which expressed high level of HLA-G was inhibited (RPI: 54.3%). The separated T lymphocytes co-cultured with allogeneic BMMCs, the levels of IFN-γ and IL-10 in culture supernatant were significantly higher than the controls (P<0.05). ConclusionsHLA-G is rich in G-BM that might be interpret G-BM could induce better immunotolerance than G-PBSC. The G-CSF could regulate HLA-G expression and secretion directly. The mechanism of G-CSF inducing immunotolerance might be related to the inhibition of allogeneic T cell reactivity and the increase of IFN-γ and IL-10 secretion through HLA-G. Disclosures:No relevant conflicts of interest to declare.