Recipients Of Bone Marrow Cells Research Articles

CLECL1+ CLL Cells: A Key Subset That Drives CLL Survival and Proliferation and Shapes a Th2-Biased Tumor Microenvironment

Chronic lymphocytic leukemia (CLL) cells depend on elements in the tissue microenvironment (TME) that produce factors such as IL-4 to promote leukemic B cell survival and growth. CLECL1, a C-type lectin-like protein, is a T cell co-stimulatory molecule originally identified on dendritic cells, but also expressed on normal B and myeloid cells. Interactions of CLECL1 with naïve T cells (Tn) lead to Th2 cell development and production of IL-4. While IL-4 can have pro- and anti- tumor activities, a role for CLECL1 in tumorigenesis has not been tested. Here we studied the role of CLECL1 in CLL. Flow cytometry analyses of CD19+CD5+ cells from 7 normal people and 77 patients with CLL revealed significantly higher CLECL1 levels on CLL cells (CLL vs Normal = 10.1% vs 3.9%, P = 0.01). CLECL1 was found most in the fraction of leukemic clones enriched in recently divided cells (PF; CXCR4DimCD5Bright) and least in the resting fraction (RF; CXCR4BrightCD5Dim) (PF vs RF = 20% vs 0.4%, P < 0.001). Importantly, high levels of CLECL1 in the PF correlated with shorter time to first treatment (P = 0.01). Notably, effective treatment with ibrutinib significantly reduced CLECL1+ cells (pre vs post treatment: 9.1% vs 1.8%, P = 0.01), and these levels increased when patients developed ibrutinib resistance (sensitive vs resistance = 1.8% vs 7.5%, P < 0.05). Flow cytometry analyses also indicated that, compared to CLECL1- cells, CLECL1+ CLL cells expressed higher levels of other cell interaction molecules such as CD40 and CD80, as well as activation markers, CD23, CD27, CD69, CD71 and CD72. Consistent with this, CLECL1+ PF cells were mainly in the G2/M phase of the cell cycle and express AID. In contrast, CLECL1- PF cells were AID- and chiefly in G1/S phases or undergoing apoptosis. Single cell RNAseq analyses of CLL PF versus RF cells corroborated that the expression of CLECL1 is [1] restricted to the PF, [2] associated with genes expressed in G2/M, and [3] correlated with IL-4-related gene expression. Together, these data suggest CLECL1+ CLL cells are activated cells generated from CLECL1- cells. Indeed, stimulation of CLECL1- cells in vitro revealed that anti-IgM+IL-4 converted CLECL1- to CLECL1+ cells. In CLL patient lymph nodes, CLECL1+ cells expressed Ki67 and co-localized with T cells. Thus, we questioned the abilities of CLECL1+ and CLECL1- cells to interact with T cells and to affect T cell growth and cytokine production. Co-culture of unseparated autologous T cells with CLECL1+, but not CLECL1- CLL cells significantly enhanced proliferation of T cells stimulated through T-cell receptors (CLECL1+ vs CLECL1-: 12.9% vs 2.8%, P = 0.02) and induced increased production of IL-4 (CLECL1+ vs CLECL1-: 15% vs 7%, P < 0.05). Contrary to published results that recombinant CLECL1 (rCLECL1) only binds to healthy Tn, we identified direct binding of rCLECL1 to both Tn and T memory (Tmem) cells in CLL patients. Moreover, when Tn and Tmem cells were incubated with rCLECL1, both T cell subsets responded with enhanced proliferation and IL-4 production. Subsequently, we tried to recapitulate the above in vitro findings in vivo, using patient derived xenografts. Injection of NSG mice with autologous CD3+ T cells plus CLECL1+ or CLECL1- PF cells led to significantly higher numbers of CLL B and T cells in the spleens of recipient mice (CLECL1+ vs CLECL1- for B cells: 508,800 vs 37,000; P = 0.03; for T cells: 273,700 vs 47,000; P = 0.03). Additionally, Th2 cells outgrew Th1 T cells (CLECL1+ vs CLECL1- for Th2 cells: 60.1% vs 12%; P < 0.05; for Th1 cells: 20.1% vs 1.8%; N.S). The same results were seen when we injected CLECL1+ or CLECL1- CLL PF cells plus autologous Tn or Tmem cells into NSG mice, as only CLECL1+ PF cells stimulated Tn or Tmem cells to expand and produce IL-4. In turn, these CLECL1 stimulated T cells facilitated the growth of CLL cells in recipient spleen and bone marrow. Overall, these ex vivo, in vitro, and in vivo results indicate that CLL cells that express CLECL1, specifically those in the recently-divided fraction, engage both Tn and Tmem cells to promote T cell growth and IL-4 production. Consequently, the CLECL1-shaped Th2 TME facilitates the survival and proliferation of tumor cells. Therefore, despite the relatively small numbers, CLECL1+ cells in the PF of CLL clones are potential key targets for therapy. Eliminating CLECL1+ CLL cells or blocking their promotion of IL-4 production could improve treatment, especially for those patients with CLL resistant to ibrutinib.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors

Regeneration of functional B lymphopoiesis from pluripotent stem cells (PSCs) is challenging, and reliable methods have not been developed. Here, we unveiled the guiding role of three essential factors, Lhx2, Hoxa9, and Runx1, the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source. In the presence of Lhx2, Hoxa9, and Runx1 expression, PSC-derived induced hematopoietic progenitors (iHPCs) immediately gave rise to pro/pre-B cells in recipient bone marrow, which were able to further differentiate into entire B cell lineages, including innate B-1a, B-1b, and marginal zone B cells, as well as adaptive follicular B cells. In particular, the regenerative B cells produced adaptive humoral immune responses, sustained antigen-specific antibody production, and formed immune memory in response to antigen challenges. The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach, which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

Recipient bone marrow-derived IL-17 receptor A-positive cells drive allograft fibrosis in a mouse intrapulmonary tracheal transplantation model

IL-17A is implicated in the pathogenesis of chronic lung allograft dysfunction, which limits survival after lung transplantation. While many cells express the IL-17 receptor A (IL-17RA) which is the main receptor for IL-17A, the cellular targets of IL-17A in development of post-transplant fibrosis are unknown. The purpose of this study was to determine whether IL-17RA expression by donor or recipient structural or bone marrow (BM) cells is required for the development of allograft fibrosis in a mouse intrapulmonary tracheal transplantation (IPTT) model. BM chimeras were generated using C57BL/6 and IL-17RA-knockout mice. After engraftment, allogeneic IPTTs were performed using the chimeric and BALB/c mice as donors or recipients. This allowed us to assess the effect of IL-17RA deficiency in recipient BM, recipient structural, donor BM, or donor structural compartments separately. Tracheal grafts, the surrounding lung, and mediastinal lymph nodes were assessed 28 days after IPTT. Only recipient BM IL-17RA deficiency resulted in attenuation of tracheal graft obliteration. In the setting of recipient BM IL-17RA deficiency, T cells and neutrophils were decreased in mediastinal lymph nodes. Additionally, recipient BM IL-17RA deficiency was associated with increased B220+PNAd+ lymphoid aggregates, consistent with tertiary lymphoid organs, in proximity to the tracheal allograft. In this IPTT model, recipient BM-derived cells appear to be the primary targets of IL-17RA signaling during fibrotic obliteration of the tracheal allograft.

CRISPR-Cas9 Genome Editing of γ-Globin Promoters in Human Hematopoietic Stem Cells to Induce Erythrocyte Fetal Hemoglobin for Treatment of β-Hemoglobinopathies

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P &lt; 0.0001. Figure Disclosures Metais: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

Downregulation of Mir-142 Promotes Leukemia Growth in Philadelphia Chromosome-Positive (Ph+) Acute Lymphoblastic Leukemia (ALL): A Possible Novel Therapeutic Target?

The Philadelphia (Ph) chromosome or t(9;22) results in the generation of a fusion gene, namely BCR/ABL1, which encodes a chimeric protein with aberrant tyrosine kinase activity that drives leukemia cell growth and survival. This molecular/cytogenetic aberration occurs in ~20%-30% of ALL cases and confers poor prognosis. Ph+ ALL patients (pts) are often referred for allogeneic hematopoietic stem cell transplantation (alloHCT), although more recently BCR-ABL-specific tyrosine-kinase inhibitors (TKIs) and immunotherapeutic approaches seemingly induced long-term remission in some patients. Nevertheless, it is still a challenge to determine which Ph+ ALL of the pts could be treated more conservatively without alloHCT. Thus identification of new prognostic biomarkers and/or therapeutic targets may be helpful. Regulation of short non-coding microRNAs(miRNAs) associated with initiation and progression of acute leukemia has been reported. miR-142(both miR-142-3p and miR-142-5p) is expressed at a relatively high level in hematopoietic tissue, and plays a role in myeloid lineage differentiation. In fact, low miR-142-3p expression was associated with myeloid differentiation failure, and miR-142 mutations was reported to promote acute myeloid leukemia (AML). More recently, Kramer et al demonstrated a role of miR-142 in lymphopoiesis by showing that miR-142 deficiency impaired B cell production in a miR-142 knock-out(ko) mouse model (Blood. 2015).Here, we first investigated if miR-142 levels were altered in ALL pts. Analysis of a publically available miRNA expression dataset(GSE23024) showed lower level of miR-142-3p, but not miR-142-5p in Ph+ ALL pts(n=10) vs. healthy donors(n=7;p=0.0093); while no significant differences were observed in Ph- pre-B ALL pts(n=61) vs. healthy donors (n=7). In ALL Tg(P190-BCR/ABL) transgenic mice(Ph+ ALL; Nature. 1990), we found bone marrow (BM) miR-142-3p level to be ~2.3-fold lower than those in the wild-type (wt) controls(p=0.036). Compared to wt mice, Ph+ ALL mice showed significantly lower miR-142-3p level in all the immunophenotypically identified BM lymphoid subpopulations, including progenitor B (pro-B, B220+CD19+CD43+IgM-,~19.1-fold lower,p<0.0001), precursor B (pre-B, B220+CD19+CD43-IgM-,~9.7-fold lower, p<0.0001), and other immature B (B220lowCD19+CD43-IgM+, ~2.4-fold lower, p<0.001) cells, except for mature B (B220highCD19+CD43-IgM+) cells. Ph+ ALL mice exhibited a miR-142-3p gradient expression pattern following the lymphoid differentiation hierarchy, with the lowest levels found in the pro-B and pre-B populations. These results prompted us to hypothesize that, loss of miR-142 may contribute to primitive B cell expansion possibly due to B cell differentiation blockage in Ph+ ALL mice. To prove this, we crossed miR-142 double knock-out (d-ko)mice with Ph+ ALL mice to generate miR-142(ko)Tg(P190-BCR/ABL) mice. Homozygous miR-142(d-ko)Tg(P190-BCR/ABL) mice were not viable due to an overly aggressive leukemia phenotype. Heterozygous miR-142(wt/ko)Tg(P190-BCR/ABL) mice had evidence of more rapid expansion of pro-B cells in blood(PB; 47.9% vs. 9.8%, p<0.0001), BM (48.2% vs. 13.2%, p<0.01)and spleen(32.3%vs. 4.4%, p<0.01) at 6 weeks old and a significantly reduced survival(median survival 44 vs.80 days, p<0.0001), compared to miR-142(wt/wt)Tg(P190-BCR/ABL) controls. BM cells (CD45.2) from miR-142(wt/ko)Tg(P190-BCR/ABL) mice (n=4) or miR-142(wt/wt)Tg(P190-BCR/ABL) mice (n=5) were then transplanted into congenic CD45.1 recipient mice (n=18 and n=15 respectively).Recipients of BM cells from miR-142(wt/ko)Tg(P190-BCR/ABL) donors showed increased engraftment (94% vs. 77% in PB at 4 weeks, p<0.0001) and significantly reduced survival(median survival 25 vs. 49 days, p<0.0001), as compared with recipients of BM cells from miR-142(wt/wt)P190-BCR-ABL mice. Finally, upon ex vitro exposure to the TKI nilotinib (5uM for 48 hours), miR-142(wt/ko)Tg(P190-BCR/ABL) BM cells showed reduced apoptosis (7.0% vs.37.5% vs p<0.05) and increased cell viability (66% vs.16.2%, p<0.05) compared with miR-142 (wt/wt)Tg(P190-BCR/ABL) BM cells. In vivo treatment studies with TKI treatment are ongoing and data will be presented at the meeting. In conclusion, miR-142 downregulation promotes rapid Ph+ ALL growth likely by contributing to a blockage of B cell differentiation, and may mediate resistance to TKIs. DisclosuresStein:Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau. Jin:The National Natural Science Foundation of China: Research Funding; College of Medicine, Zhejiang University: Employment.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of Mitochondrial Vulnerability in Acute Myeloid Leukemia

BACKGROUND60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs. AIMWe aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL). RESULTSWe identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients. CONCLUSIONSWe uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients. DisclosuresNo relevant conflicts of interest to declare.

DNA damage induction in bone marrow cells of mice after farnesenes and 2,5-dimethylpyrazine sniffing

Background. Pheromones are an important regulatory link of synecological contacts in numerous animal species. Chemo-signaling participates in establishing of population social structure, it regulates different types of behavior, changes hormonal state and maturation rate, etc. It also can affect the genetic material expression and integrity.&#x0D; Material and methods. Groups of adult males of CBA/Lac/Sto/Rap strain were exposed to volatile chemosignals (mixture of - and -farnesenes or 2,5-dime thylpyrazine) for 2 or 24 hours. Bone marrow cells were prepared for single cell gel electrophoresis (comet assay test). Content of DNA in comet cells were analyzed. In case of 24 hours exposure bone marrow cells were fixed also for ana-telophase analysis.&#x0D; Results. It is shown that exposures with farnesenes or 2,5-DMP both damage genetic material of bone marrow cells. It also followed by induction of mitotic aberration frequency. Simultaneous exposure with all chemosignals does not increase damaging effect.&#x0D; Conclusion. Chemosignals which serve as stress-pheromones in mice decrease also the integrity of genetic material in bone marrow cells of recipients. It could be a mechanism of pheromonal impact on density and space-genetic structure of mouse populations.

Role of Graft-derived Graft-versus-Host T cells in Facilitating Multilineage Blood Chimerism after Human Intestinal Transplantation

Long-term multilineage chimerism often appears in blood following human intestinal transplantation (ITx), especially in multivisceral transplant (MVTx) recipients. Patients with donor T cell macrochimerism (>4%) in blood, frequently without graft-versus-host disease (GVHD), displayed less graft rejection and donor-specific antibody (DSA) production and slower graft T cell replacement by the recipient. To address the hypothesis that the balance of graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivity might underly these relationships, we analyzed the phenotype, repertoire and alloreactivity of donor- and recipient-derived T cells in the intestinal allograft, peripheral blood and bone marrow (BM) of ITx recipients. We identified GvH and HvG clones on the basis of high throughput TCRβ CDR3 sequencing from pre-Tx donor and recipient mixed lymphocyte reactions as we have described1. Enrichment of GvH compared to HvG clones in early intestinal biopsies and absence of Class I DSA were associated with donor T cell macrochimerism. Local expansion of GvH clones in the graft was likely driven by uniformly rapid replacement of recipient antigen presenting cells (APCs) within allografts early post-Tx. Cumulative frequency of GvH clones in blood early post-Tx was positively correlated with the peak level of donor T cell chimerism (Fig.1).Unlike donor ileal lymphocytes, which display a tissue-resident memory phenotype, long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype compared with recipient cells, and GvH clones were undetectable among these late circulating naïve donor-derived T cells. Enrichment of donor circulating naïve B cells and T cells and recurrent myeloid chimerism late post-Tx are consistent with our observation that donor-derived hematopoietic stem and progenitor cells are present in intestinal allografts and are detectable in the circulation of patients with macrochimerism. Multilineage chimerism might reflect a lymphohematopoietic GvH response (LGVHR) migrating from recipient circulation to the BM, making “space” for engraftment of progenitor cells from the graft. Indeed, 0.0356% of CD45+ cells in the BM of an MVTx patient on day 105 post-Tx were donor-derived, among which 72.9% were T cells and 1.37% were CD34+ progenitors. TCR sequencing of sorted donor cells in recipient BM identified donor-mappable clones, among CD4s of which 60% by frequency were GvH-reactive. GvH CD4 cells did not show marked enrichment in donor-mappable PBMCs on the same day. (Fig.2)Together, these data suggest that donor GvH-reactive T cells expanding within the graft in response to recipient APCs entering the graft migrate into the recipient circulation and BM, playing a key role in promoting and maintaining mixed chimerism. LGVHR without GVHD in ITx recipients suggests a novel approach to achieving intestinal allograft tolerance. 1. Zuber et al., 2016 Science Immunology. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Differential role of Id1 in MLL-AF9–driven leukemia based on cell of origin

AbstractInhibitor of DNA binding 1 (Id1) functions as an E protein inhibitor, and overexpression of Id1 is seen in acute myeloid leukemia (AML) patients. To define the effects of Id1 on leukemogenesis, we expressed MLL-AF9 in fetal liver (FL) cells or bone marrow (BM) cells isolated from wild-type, Id1−/−, p21−/−, or Id1−/−p21−/− mice, and transplanted them into syngeneic recipient mice. We found that although mice receiving MLL-AF9–transduced FL or BM cells develop AML, loss of Id1 significantly prolonged the median survival of mice receiving FL cells but accelerated leukemogenesis in recipients of BM cells. Deletion of Cdkn1a (p21), an Id1 target gene, can rescue the effect of Id1 loss in both models, suggesting that Cdkn1a is a critical target of Id1 in leukemogenesis. It has been suggested that the FL transplant model mimics human fetal-origin (infant) MLL fusion protein (FP)-driven leukemia, whereas the BM transplantation model resembles postnatal MLL leukemia; in fact, the analysis of clinical samples from patients with MLL-FP+ leukemia showed that Id1 expression is elevated in the former and reduced in the latter type of MLL-FP+ AML. Our findings suggest that Id1 could be a potential therapeutic target for infant MLL-AF9–driven leukemia.

High expression of heme oxygenase-1 in target organs may attenuate acute graft-versus-host disease through regulation of immune balance of TH17/Treg

The high incidence of acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Grades III and IV aGVHD are the leading causes of death in allo-HSCT recipients. Heme oxygenase-1(HO-1) has anti-inflammatory and immune-regulatory functions. In this study, we evaluated the none GVHD and grade I–IV patients samples which were collected at the first re-examination after successful allo-HSCT, we found that expressions of HO-1 mRNA in the bone marrow and peripheral blood mononuclear cells of allo-HSCT recipients who had subsequent non-GVHD and grade I aGVHD were significantly higher than those in patients with Grade III–IV aGVHD. We then demonstrated that enhanced expression of HO-1 in target organs by infusing HO-1-gene-modified Mesenchymal stem cells (MSCs) alleviated the clinical and histopathological severity of aGVHD in experimental mice. Flow cytometry revealed a higher expression of Treg cells and a lower expression of TH17 cells in splenic and lymph node tissues of mice with enhanced HO-1 expression, as compared to that in the aGVHD mice. This was further substantiated by lower expression levels of ROR-Υt and IL-17A mRNA, and higher levels of Foxp3 mRNA in the splenic tissue of mice with enhanced HO-1 expression. Our results indicate that high expression of HO-1 may reduce the severity of aGVHD by regulation of the TH17/Treg balance.

AKT2 Promotes Bone Marrow Cell-Mediated Aortic Protection in Mice

Insufficient aortic protection and repair may contribute to the development of aortic aneurysms and dissections (AAD). However, mechanisms of aortic protection and repair are poorly understood. We have shown that the multifunctional kinase AKT2 plays an important role in protecting the aortic wall. Here, we examined whether AKT2 protects against AAD by promoting bone marrow cell (BMC)-mediated aortic protection. Irradiated wild-type mice received green fluorescent protein-expressing BMCs from wild-type mice or Akt2(-/-) mice, followed by challenge with angiotensin II (1000 ng/kg/min) infusion for 4 weeks. We compared BMC recruitment, aortic destruction, and AAD development between groups. The direct effects ofwild-type and Akt2(-/-) BMCs on smooth muscle cell survival were examined in coculture experiments. After angiotensin II infusion, no (0 of 14) wild-type BMC recipients had AAD; in contrast, 64% (9of 14) of Akt2(-/-) BMC recipients had AAD (p= 0.002) with severe aortic destruction. Compared with aortas from challenged wild-type BMC recipients, aortas from challenged Akt2(-/-) BMC recipients showed significantly less BMC recruitment, NG2 (neuron-glial antigen 2) progenitor activation, and FSP1 (fibroblast-specific protein 1) fibroblast activation. In addition, aortas from challenged Akt2(-/-) BMC recipients showed increased apoptosis and inflammation. In coculture experiments, wild-type but not Akt2(-/-) BMCs prevented smooth muscle cells from undergoing oxidative stress-induced apoptosis. After aortic challenge, BMCs are recruited to the aortic wall and provide protection by activating progenitors and fibroblasts and by promoting aortic cell survival. Our findings indicate that AKT2 is involved in these processes and that defects in this pathway may promote progressive degeneration during AAD development.

The reversed paradigm of chimerism induction: Donor conditioning with recipient-derived bone marrow cells as a novel approach for tolerance induction in vascularized composite allotransplantation.

To test a new approach of donor conditioning with recipient bone marrow cells (BMC) to induce tolerance in vascularized composite allograft (VCA) transplantation. Lewis rats' (recipients) BMC were stained with PKH-26. The ACI rats (donors) were conditioned with 80×106 Lewis BMC, 24 or 72 hours before VCA (groin flap) transplantation. Forty-eight VCA were performed between ACI donors and Lewis recipients. In groups I and II, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received 7-day anti-αβ-TCR/cyclosporine-A post-transplantation. In groups III and IV, donors were preconditioned (24 and 72 hours before transplantation, respectively), and recipients received no systemic immunosuppression. In group V, recipients received 7-day anti-αβ-TCR/cyclosporine-A post-transplantation. In group VI, recipients received no systemic immunosuppression. Assessment included evaluation of transplant viability and induction of donor-specific chimerism via flow cytometry, immunofluorescence, and PCR. Groups III, IV, and VI rejected allografts, at an average of 14 ± 5.2, 10 ± 2.7, and 8 ± 0.7 days. In groups I, II, and V, the mean survival was 80 ± 18.2 (p = 0.0002), 64 ± 27.4 (p = 0.001), and 30 ± 4.7 (p = 0.02) days. In groups I and II, donor-specific chimerism in the blood decreased from 8.8 ± 3.4% and 8.6 ± 3.4% on day 7 to 3.7 ± 1.32% (p = 0.02) and 4.7 ± 2.7% when the flaps manifested grade 3 rejection. The presence of PKH-26+ Lewis BMC was confirmed in the donor's blood, bone marrow, lymphoid organs, and liver (preconditioned at 24 and 72 hours). Donor preconditioning is a novel approach modifying recipient's responsiveness to donor allograft and prolonging the allograft survival under short-term immunosuppression. © 2015 Wiley Periodicals, Inc. Microsurgery 36:676-683, 2016.

Patient Derived Xenotransplantation Model of Atypical Chronic Myeloid Leukemia (aCML)

Background:Atypical chronic myeloid leukemia (aCML) is a rare disorder classified as one of the MPN/MDS overlap syndromes. aCML usually presents like CML but lacks the pathognomonic BCR-ABL fusion found in CML. Most patients progress to acute myeloid leukemia (AML) with a median time to AML of 11.2 months and have a median overall survival of only 12.4 months (Wang et al. Blood 2014). Recurrently mutated genes found in aCML patients include SETBP1 , CSF3R, NRAS, EZH2, ASXL1, ETNK1, and U2AF1.The pathogenesis of aCML is poorly understood and neither specific nor effective treatments besides hematopoietic stem cell transplantation are available. We therefore aimed at developing a patient derived xenotransplantation model that allows serial transplantation and expansion of human leukemic cells and evaluation of novel treatments and drugs in vivo.Patient and Methods:Bone marrow cells were harvested from a patient diagnosed with atypical CML based on persistent leukocytosis, immature circulating myeloid precursors (16% metamyelocytes, 8% myelocytes, 9% blasts), marked dysgranulopoiesis, minimal monocytosis and basophilia, hypercellular bone marrow with high myeloid/erythroid ratio and 6% myeloid blasts, dysplasia in megakaryocytes and erythroid progenitors, and absence of BCR-ABL and mutated JAK2. The patient had moderate anemia and normal platelet counts and cytogenetic analysis showed a normal karyotype.Eight hundred thousand bone marrow cells were injected intravenously in NOD/SCID IL-2 receptor γ (NSG) deficient mice. We monitored these mice for human cell engraftment by regular eye bleeds every 4 weeks. Bone marrow and spleen cells from engrafted mice were retransplanted in secondary and tertiary mice of the NSG strain transgenic for SCF, IL3 and GM-CSF (NSGS). Patient cells were analyzed for mutations in fifty four genes by next generation sequencing and mutations were confirmed by Sanger sequencing in primary patient cells and cells from tertiary mice.Results: Human CD45+ cells from the aCML patient showed increasing engraftment over time in the NSG mouse reaching 16% in peripheral blood and 35% in spleen at 26 weeks after transplantation. In secondary (n=2) and tertiary (n=4) mice we used NSGS recipient mice and observed considerably accelerated engraftment kinetics leading to 19, 21 and 73% human cells in peripheral blood, spleen and bone marrow, respectively, between 12 and 15 weeks after transplantation. The myeloid marker CD33 was expressed in 86% of human bone marrow cells, while lymphoid markers CD3 and CD19 were absent. The stem and progenitor phenotype CD34+CD38- was found in 11% of human cells. The progenitor marker CD123 was expressed in 42% of cells, while the myeloid marker CD14 was expressed in 6% of cells. Hemoglobin levels and platelet counts were considerably lower in secondary and tertiary recipients of aCML cells compared to control animals. Spleens were enlarged at time of sacrifice with an average spleen weight of 150 mg. Morphological evaluation of bone marrow cells in tertiary recipients revealed a characteristic picture for aCML with 39% neutrophils, 8% blasts and 53% myeloid progenitors and monocytes. Molecular analysis identified mutations in ASXL1, RUNX1 and EZH2 with variant allele frequencies of 49, 48 and 46 percent, respectively that were confirmed in human cells from tertiary recipient mice. Thus, we show that primary aCML cells can be expanded and serially transplanted in immunodeficient mice and suggest clonal stability of this model.Conclusion:We provide the first patient derived xenotransplantation model for atypical CML, which preserves the phenotypic and molecular characteristics of the primary disease and allows serial transplantation and expansion of aCML cells. This model will serve to better understand the pathogenesis of aCML and to test urgently needed novel treatment approaches. DisclosuresNo relevant conflicts of interest to declare.

A Conserved Cis-Regulatory Retinoic Acid Responsive Element Is Essential for Maintenance of Primitive Hematopoietic Stem Cells through Regulation of Hoxb Cluster

Hematopoietic stem cells (HSCs) sustain lifelong production of multiple blood cell types through a finely-tuned balance between stem cell maintenance and activation to prevent bone marrow exhaustion or overgrowth. The highly conserved Hox family of homeodomain containing transcription factors have been identified as key regulators and contributors in both normal hematopoiesis and leukemogenesis. Most previous work has focused on individual Hox genes; however, it remains largely unknown whether and how multiple Hox genes in a cluster are regulated and function in hematopoiesis. We initiated a study to perform systematic, high-throughput transcriptome analysis in the following 17 cell types from the bone marrow (BM) of C57BL/6J mice: 4 hematopoietic stem and progenitor cells (CD49blo long-term (LT)-HSC, CD49bhi intermediate-term (IT)-HSC, short-term (ST)-HSC, and MPP); and 4 committed progenitors (CLP, CMP, GMP and MEP); and 9 mature lineage cells (B cell, T cell, NK cell, dendritic cell, monocyte, macrophage, granulocyte, megakaryocyte and nucleated erythrocyte). Intriguingly, as part of a unique fingerprint observed in the most primitive CD49blo LT-HSCs, we detected expression from the Hoxb cluster. Further analysis on all the four Hox clusters revealed that most of the genes from the Hoxb cluster, and not from the other Hox clusters, were predominantly expressed in the CD49blo LT-HSCs. This suggests that they might function as a cluster to maintain CD49blo LT-HSCs. A previous study has shown that one cis -regulatory retinoic acid responsive element (RARE), is conserved among vertebrate species and regulates multiple Hoxb gene expression in central nervous system development. Thus, we asked whether RARE is essential for maintenance of primitive CD49blo LT-HSCs by regulation of Hoxb cluster. To test this hypothesis, we utilized a RAREΔ knockout mouse model and assayed for HSC numbers in BM. We observed that homozygous deletion of RARE led to 2-fold reduction in both the frequency and absolute number of CD49blo LT-HSCs. Functionally, we first conducted limiting dilution, competitive repopulating unit (CRU) assays by transplanting 2.5×104, 7.5×104 or 2×105 of BM cells from RAREΔ mutants and their control littermates, together with 2×105 recipient BM cells derived from the Ptprc mutant strain, into lethally irradiated recipient mice. Our data showed a 2.5-fold decrease in functional HSCs in RAREΔ HSCs (1/20,326) compared to control (1/50,839). To further evaluate the long-term effect of RARE on HSCs, we performed serial BM transplantation and observed a 12.9-fold reduction of reconstitution ability after secondary transplantation. These data indicate that deletion of RARE compromised HSC long-term reconstitution capacity. Collectively, our work provides evidence showing that RARE is essential for maintenance of the primitive HSCs by regulation of Hoxb cluster genes. DisclosuresNo relevant conflicts of interest to declare.

279. Efficient and Safe Lentiviral Vector-Mediated Hematopoietic Stem Cell Gene Therapy in MNGIE Mice

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients are deficient in thymidine phosphorylase(TP) resulting in systemic thymidine(Thd) and deoxyuridine(dUrd) accumulation affecting mtDNA replication and causing mitochondrial dysfunction. Common symptoms are gastrointestinal dysmotility, progressive ophthalmoplegia and leukoencephalopathy. Allogenic hematopoietic stem cell (HSC) transplantation has been shown to reduce disease symptoms, but is not well tolerated due to the inherent toxicity of the procedure. Therefore, syngeneic ex vivo lentiviral vector HSC gene therapy overexpressing the native cDNA or the codon optimized (TPco) sequence driven by the phosphoglycerate kinase (PGK) or spleen focus forming virus (SFFV) promoters in Tp−/-Upp−/- double knockout mice, a model for MNGIE disease, was investigated. At 1 month post transplantation after sublethal total body irradiation, very low TP activity was detected in blood of control wild type mice (0.07±0.03nmoles/h/mg), but enzyme activities in PGK treated mice were at least 90-fold higher (PGK-TP = 150±4 and PGK-TPco = 96±4 nmoles/h/mg), and in SFFV recipient mice 400-fold higher (450±5 nmoles/h/mg). Consequently, a significant reduction of plasma and urine Thd and dUrd levels was observed. Long-term follow up (14 months) showed on average 1.2-fold wild type TP activity levels increase in LV-PGK-TP and LV-PGK-TPco and 36-fold in SFFV-TPco treated mice. This was sufficient for sustained reduction of plasma and urine nucleoside levels, which was achieved at 76.5±8.2% donor chimerism levels with low LV vector copy numbers (1.0±1.1VCN/donor cell). The LV integration profile in bone marrow cells of primary recipients was analyzed; LVs displayed the expected tendency to integrate within highly expressed genes and the integration pattern did not differ from that of other SIN-LV vectors in other disease models (primary immune deficiencies and lysosomal enzyme storage disorders). Overall, stem cell gene therapy provided stable TP expression and long-term biochemical correction in MNGIE mice without genotoxicity or apparent phenotoxicity, which will be further evaluated for somatic and neurological phenotype correction and optimized to develop a clinical protocol to treat MNGIE patients.

Ex vivo exposure of bone marrow from chronic kidney disease donor rats to pravastatin limits renal damage in recipient rats with chronic kidney disease.

IntroductionHealthy bone marrow cell (BMC) infusion improves renal function and limits renal injury in a model of chronic kidney disease (CKD) in rats. However, BMCs derived from rats with CKD fail to retain beneficial effects, demonstrating limited therapeutic efficacy. Statins have been reported to improve cellular repair mechanisms.MethodsWe studied whether exposing CKD rat BMCs ex vivo to pravastatin improved their in vivo therapeutic efficacy in CKD and compared this to systemic in vivo treatment. Six weeks after CKD induction, healthy BMCs, healthy pravastatin-pretreated BMCs, CKD BMCs or CKD pravastatin-pretreated BMCs were injected into the renal artery of CKD rats.ResultsAt 6 weeks after BMC injection renal injury was reduced in pravastatin-pretreated CKD BMC recipients vs. CKD BMC recipients. Effective renal plasma flow was lower and filtration fraction was higher in CKD BMC recipients compared to all groups whereas there was no difference between pravastatin-pretreated CKD BMC and healthy BMC recipients. Mean arterial pressure was higher in CKD BMC recipients compared to all other groups. In contrast, 6 weeks of systemic in vivo pravastatin treatment had no effect. In vitro results showed improved migration, decreased apoptosis and lower excretion of pro-inflammatory Chemokine (C-X-C Motif) Ligand 5 in pravastatin-pretreated CKD BMCs.ConclusionsShort ex vivo exposure of CKD BMC to pravastatin improves CKD BMC function and their subsequent therapeutic efficacy in a CKD setting, whereas systemic statin treatment did not provide renal protection.

Pretransplant replacement of donor liver grafts with recipient Kupffer cells attenuates liver graft rejection in rats.

Rejection of liver grafts is a difficult issue that has not been resolved. Preoperative replacement of liver cells in the graft with cells from the intended recipient may attenuate rejection. We investigated whether preoperative transplant of recipient bone marrow cells (BMCs) to the donor replaced liver allograft cells and attenuated rejection. We used a rat model of allogeneic liver transplant (LT) from Dark Agouti (DA) to Lewis (LEW) rats. In BMC group, DA rats received BMC transplants from LacZ-transgenic LEW rats at 1 week before LT. In the control group, DA rats received no preoperative treatment. We evaluated graft damage at 7 days after LT and the survival of the recipient rats. Rats in the BMC group experienced prolonged survival that was abrogated by the administration of gadolinium chloride to donors at 24 h before LT. Serum concentrations of total bilirubin and hyaluronic acid on day 7 were significantly lower in the BMC group, and histopathological analyses revealed that rejection of the liver graft was attenuated. X-gal staining and immunohistostaining of the liver graft revealed that BMCs engrafted in the sinusoidal space differentiated into Kupffer cells. Preoperative transplant of recipient BMCs to LT donors replaced donor Kupffer cells and attenuated post-LT rejection, indicating that this strategy may increase the success of LT.

Intra-Bone Marrow Transplantation of Endosteal Bone Marrow Cells Facilitates Allogeneic Hematopoietic and Stromal Cells Engraftment Dependent on Early Expression of CXCL-12.

BackgroundHematopoietic stem cell transplantation (HSCT) has been considered as an effective approach at inducing allogeneic hematopoietic reconstitution and immune tolerance. However, it remains critical to find the optimal HSCT delivery method and robust sources of hematopoietic stem cells (HSCs).Material/MethodsWe introduced a new method by infusing allogeneic endosteal bone marrow cells (BMCs) harvested from long bones endosteum through intra-bone marrow transplantation (IBBMT) into irradiated mice. Recipient mice that were transplanted with central BMCs or through intravenous bone marrow transplantation (IVBMT) were used as controls (n=6 per group). We compared the new method with each control group for allogeneic HSCs homing pattern, peripheral blood chimerism level, skin allograft survival time, and donor stromal cell percentage in recipient BM. AMD3100 was injected to determine whether chemokine stromal cell-derived factor-1 (CXCL-12) was critical for the new method.ResultsMore allogeneic HSCs homed into spleen and bone marrow for the new method as compared to each control group. IBBMT of endosteal BMCs led to a higher peripheral blood chimerism and skin allograft survival. At 18 weeks, donor stromal cell percentage in recipient BMCs was higher for the new method than in each control group. By AMD3100 blockade at day 1, peripheral blood chimerism level and donor stromal cell percentage were significantly reduced as compared to the control group without AMD3100 blockade.ConclusionsOur study suggests that IBBMT of endosteal BMCs is an effective approach for HSCT in inducing allogeneic hematopoietic reconstitution. The advantage is dependent upon the early expression of CXCL-12 after bone marrow transplantation.

Transplant Surgeons can Xenograft Kidneys from untreated Pigs without Rejection

The histocompatibility system exists for defence against viruses. It is responsible for the rejection of allograft. The immune system attempts to counter the explosive speed of viral replication by directing the defensive immune attack by cytotoxic T cells on to histocompatibility antigens on the infected cell’s surface. This enables destruction of the virus factories before the cytotoxic T cells are swamped by the myriad numbers of new virions, a thousand coming from each infected cell every 10 hours. The histocompatibility system mistakes alloantigen on grafts for virus-infected host cells that need swift destruction. For surgical transplantation, Henry Kaplan discovered that immune ablation of the recipient followed by inoculation with donor bone marrow prevents rejection of allogeneic grafts. Sykes has improved Kaplan’s technique by adding recipient bone marrow cells to the donor ones injected for reconstitution of the recipient after immune ablation. Kaplan’s technique, used on untreated pigs, should be the standard procedure for transplantation.

Co-transplantation of Hematopoietic Stem Cells and Cxcr4 Gene-Transduced Mesenchymal Stem Cells Promotes Hematopoiesis.

Mesenchymal stem cells (MSCs) are a promising candidate for cellular therapies. Co-transplantation of MSCs and hematopoietic stem cells (HSCs) promotes successful engraftment and improves hematopoietic recovery. In this study, the effects of co-transplantation of HSCs and mouse bone marrow (BM)-derived MSCs overexpressing CXCR4 (CXCR4-MSC) on CXCR4-MSC homing capacity and the reconstitution potential in lethally irradiated mice were evaluated. Recovery of donor-derived peripheral blood leukocytes and platelets was accelerated when CXCR4-MSCs were co-transplanted with BM cells. The frequency of c-kit(+)Sca(+)Lin(-) HSCs was higher in recipient BM following co-transplantation of CXCR4-MSCs compared with the EGFP-MSC control and the BMT only groups. Surprisingly, the rate of early engraftment of donor-derived BM cells in recipients co-transplanted with CXCR4-MSCs was slightly lower than in the absence of MSCs on day 7. Moreover, co-transplantation of CXCR4-MSCs regulated the balance of T helper cells subsets. Hematopoietic tissue reconstitution was evaluated by histopathological analysis of BM and spleen. Co-transplantation of CXCR4-MSCs was shown to promote the recovery of hematopoietic organs. These findings indicate that co-transplantation of CXCR4-MSCs promotes the early phase of hematopoietic recovery and sustained hematopoiesis.