Bone Marrow Recipients Research Articles

Caffeic acid phenethyl ester promotes haematopoietic stem/progenitor cell homing and engraftment

BackgroundSeveral studies have suggested that caffeic acid phenethyl ester (CAPE) can induce the expression of hypoxia inducible factor-1α (HIF-1α) protein. We determined whether CAPE has a novel function in improving the homing and engraftment of haematopoietic stem/progenitor cells (HSPCs) by regulating HIF-1α gene expression in the bone marrow (BM) niche.MethodsFor survival experiments, lethally irradiated C57BL/6 mice were injected with a low number of BM mononuclear cells (MNCs) and CAPE according to the indicated schedule. Homing efficiency analysis was conducted using flow cytometry and colony-forming unit (CFU) assays. The influence of intraperitoneal injection of CAPE on short-term and long-term engraftment of HSPCs was evaluated using competitive and non-competitive mouse transplantation models. To investigate the mechanism by which CAPE enhanced HSPC homing, we performed these experiments including Q-PCR, western blot, immunohistochemistry and CFU assays after in-vivo HIF-1α activity blockade.ResultsCAPE injection significantly increased the survival rate of recipient mice after lethal irradiation and transplantation of a low number of BM MNCs. Using HSPC homing assays, we found that CAPE notably increased donor HSPC homing to recipient BM. The subsequent short-term and long-term engraftment of transplanted HSPCs was also improved by the optimal schedule of CAPE administration. Mechanistically, we found that CAPE upregulated the expression of HIF-1α, vascular endothelial growth factor-A (VEGF-A) and stromal cell-derived factor 1α (SDF-1α). The HIF-1α inhibitor PX-478 blocked CAPE-enhanced HSPC homing, which supported the idea that HIF-1α is a key target of CAPE.ConclusionsOur results showed that CAPE administration facilitated HSPC homing and engraftment, and this effect was primarily dependent on HIF-1α activation and upregulation of SDF-1α and VEGF-A expression in the BM niche.

Distinct NG2 proteoglycan-dependent roles of resident microglia and bone marrow-derived macrophages during myelin damage and repair.

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow.

PD-1 and CTLA-4 up regulation on donor T cells is insufficient to prevent GvHD in allo-HSCT recipients.

The expression of checkpoint blockade molecules PD-1, PD-L1, CTLA-4, and foxp3+CD25+CD4+ T cells (Tregs) regulate donor T cell activation and graft-vs-host disease (GvHD) in allogeneic hematopoietic stem cell transplant (allo-HSCT). Detailed kinetics of PD-1-, CTLA-4-, and PD-L1 expression on donor and host cells in GvHD target organs have not been well studied. Using an established GvHD model of allo-HSCT (B6 → CB6F1), we noted transient increases of PD-1- and CTLA-4-expressing donor CD4+ and CD8+ T cells on day 10 post transplant in spleens of allo-HSCT recipients compared with syngeneic HSCT (syn-HSCT) recipients. In contrast, expression of PD-1- and CTLA-4 on donor T cells was persistently increased in bone marrow (BM) of allo-HSCT recipients compared with syn-HSCT recipients. Similar differential patterns of donor T cell immune response were observed in a minor histocompatibility (miHA) mismatched transplant model of GvHD. Despite higher PD-1 and CTLA-4 expression in BM, numbers of foxp3+ T cells and Tregs were much lower in allo-HSCT recipients compared with syn-HSCT recipients. PD-L1-expressing host cells were markedly decreased concomitant with elimination of residual host hematopoietic elements in spleens of allo-HSCT recipients. Allo-HSCT recipients lacking PD-L1 rapidly developed increased serum inflammatory cytokines and lethal acute GvHD compared with wild-type (WT) B6 allo-HSCT recipients. These data suggest that increased expression of checkpoint blockade molecules PD-1 and CTLA-4 on donor T cells is not sufficient to prevent GvHD, and that cooperation between checkpoint blockade signaling by host cells and donor Tregs is necessary to limit GvHD in allo-HSCT recipients.

P078 Conundrums surrounding organ transplant in a bone marrow stem cell transplant recipient

Bone marrow (BM) donors and their recipients need not have a matching blood type. Eventually, the BM recipient will become the blood type of the BM donor. This scenario can become quite a conundrum if the BM recipient becomes a patient in need of an organ transplant. In order for a patient to receive a donor organ, the patient and donor’s blood type and HLA typing must be compatible. Blood type was determined using gel test cards. HLA typing was determined by using sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP), and sequence based typing (SBT) technologies. Patient #1, originally typed as an A2, had 1 bone marrow donor and 2 cord blood transplants. One of the cord blood transplants successfully engrafted. The engrafted cord blood was from a type O donor. Patient #1 is now typing as type O. Patient #2 was originally typed as A2 and received a bone marrow transplant from a type B donor. Patient #2 is now front-typing as a B and back-typing as an AB. The patient now has an HLA and ABO identical kidney match (his father who is a type B). Previously, the patient and his father were ABO incompatible. The ABO and HLA results on both patient #1 and patient #2 indicate that the BM transplants have engrafted. Results also indicate that the ABO and HLA now match that of the donor and differs from the recipient’s original ABO and HLA type. Due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. Both patients will be entered into the UNet system according to their “new” ABO and HLA types, as UNOS regulations require patients to be listed as per the results of two separate ABO typing tests. The patients’ antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial.

OR46 A novel cellular crossmatch to detect donor HLA specific immunity

Aim To develop a cellular crossmatch method (CXM) for evaluating donor HLA specific cellular immunity in HLA mismatched recipients. Methods HLA Typing: HLA types of all samples were obtained by high resolution typing. CXM: Irradiated donor cells were used as the donor HLA specific stimulant to co-incubate with recipient (responder) cells and Th1 (IFNγ & IL-2) or Th2 (IL-4, 5, 10) cytokine capture beads for overnight in fixed cell:bead ratio. The cells were lysed and the captured cytokines were detected by corresponding fluorescent 2nd step antibodies. The percentage of positive beads (Flowspot %) were calculated which proportionately correlated with numbers of donor specific responding cells. The MFI level correlated with the cytokine secretion capability of responding cells. Results A total 147 paired bone marrow donor and recipient samples were tested by CXM. Compared to the HLA identical group, IL-2 but not IFNγ was significantly increased in HLA-class I (CI) (P Conclusions Unlike mixed lymphocyte reaction (MLR) which detects DNA synthesis by radioisotope, the newly developed CXM is able to detect donor HLA specific immunity in responder within 24 h using standard flow cytometry. CXM detects earlier T cell activation (e.g. cytokine secretions) in a uni-or bi-directional multiplex detecting system. The method of CXM is simple, sensitive, and donor HLA specific. It potentially has wide application in both the research and clinical fields for predicting GVH/GVL in BM transplantation, cellular rejection in solid organ transplantation, and in other applications where cell–cell responses are of interest. Download : Download high-res image (207KB) Download : Download full-size image

P2y12 Receptor Promotes Pressure Overload-Induced Cardiac Remodeling via Platelet-Driven Inflammation in Mice.

Inflammation plays a critical role in adverse cardiac remodeling and heart failure. The P2y12 receptor is one of the predominant activating receptors for platelets, thus initiating inflammatory responses under various diseases. In this study, we investigated the functional significance of P2y12-mediated platelet activation in pressure overload-induced cardiac remodeling. Notably, P2y12 knockout (P2y12-/-) mice exhibited suppressed transverse aortic constriction-induced changes in cardiac hypertrophy, collagen synthesis, inflammatory cell recruitment, and cardiac dysfunction. Activated platelets and platelet-leukocyte aggregates were markedly downregulated in P2y12 knockout mice compared with wild-type counterparts after transverse aortic constriction. Moreover, bone marrow chimera experiments revealed that wild-type recipients of P2y12 knockout bone marrow markedly improved cardiac function and attenuated cardiac remodeling, reversed by wild-type platelets reinjection. Platelet depletion and P-selectin inhibition mimicked these protective effects by limiting the interaction between activated platelets and leukocytes. Furthermore, activated wild-type platelets directly induced cardiomyocyte hypertrophy and collagen synthesis via α-granule exocytosis, vanished in P2y12 knockout platelets or those administered anti-NSF (N-ethlymalimide-sensitive factor) antibodies. The results suggest that P2y12-mediated platelet activation promotes cardiac remodeling by triggering a series of inflammatory changes and interacting with leukocytes and endotheliocytes.

Reconstitution of multifunctional CD56lowCD16low natural killer cell subset in children with acute leukemia given α/β T cell-depleted HLA-haploidentical haematopoietic stem cell transplantation

ABSTRACTWe recently described the CD56lowCD16low subset of Natural Killer (NK) cells that both mediate cytotoxic activity and produce IFNγ, being more abundant in bone marrow (BM) than in peripheral blood (PB) of pediatric normal subjects. Given the multifunctional properties of this subset, we examined its development and functional recovery in a cohort of children undergoing α/β T-cell depleted HLA-haploidentical haematopoietic stem cell transplantation (HSCT). The results obtained indicate that CD56lowCD16low NK cells are present in both PB and BM already at one month post-HSCT, with an increased frequency in BM of graft recipients as compared with normal subjects. During the first 6 months after HSCT, no difference in CD56lowCD16low NK cells distribution between PB and BM was observed. In comparison to normal subjects, CD56lowCD16low NK cells from transplanted patients show lower expression levels of CD25 and CD127 and higher levels of CD122, and accordingly, produce higher amounts of IFNγ after stimulation with IL-12 plus IL-15. The recovery of NK-cell cytotoxicity after HSCT was strictly restricted to CD56lowCD16low NK cells, and their ability to degranulate against K562 target cells or autologous leukemic blasts was completely restored only one year after HSCT.Based on the phenotypic and functional ability of reconstituted CD56lowCD16low NK cells, we suggest that they play an important role in host defense against leukemia relapse and infections after HSCT, and represent an ideal candidate for approaches of adoptive immunotherapy.

Clinical characteristics and outcome of human herpesvirus-6 encephalitis after allogeneic hematopoietic stem cell transplantation

In this retrospective analysis using the Transplant Registry Unified Management Program, we identified 145 patients with human herpesvirus (HHV)-6 encephalitis among 6593 recipients. The cumulative incidences of HHV-6 encephalitis at 100 days after transplantation in all patients, recipients of bone marrow or PBSCs and recipients of cord blood were 2.3%, 1.6% and 5.0%, respectively. Risk factors identified in multivariate analysis were male sex, type of transplanted cells (relative risk in cord blood transplantation, 11.09, P<0.001; relative risk in transplantation from HLA-mismatched unrelated donor, 9.48, P<0.001; vs transplantation from HLA-matched related donor) and GvHD prophylaxis by calcineurin inhibitor alone. At 100 days after transplantation, the overall survival rate was 58.3% and 80.5% among patients with and without HHV-6 encephalitis, respectively (P<0.001). Neuropsychological sequelae remained in 57% of 121 evaluated patients. With both foscarnet and ganciclovir, full-dose therapy (foscarnet ⩾180 mg/kg, ganciclovir ⩾10 mg/kg) was associated with better response rate (foscarnet, 93% vs 74%, P=0.044; ganciclovir, 84% vs 58%, P=0.047). HHV-6 encephalitis is not rare not only in cord blood transplant recipients but also in recipients of HLA-mismatched unrelated donors. In this study, development of HHV-6 encephalitis was associated with a poor survival rate, and neurological sequelae remained in many patients.

Robust patient-derived xenografts of MDS/MPN overlap syndromes capture the unique characteristics of CMML and JMML

AbstractChronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) are myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap disorders characterized by monocytosis, myelodysplasia, and a characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Currently, there are no available disease-modifying therapies for CMML, nor are there preclinical models that fully recapitulate the unique features of CMML. Through use of immunocompromised mice with transgenic expression of human GM-CSF, interleukin-3, and stem cell factor in a NOD/SCID-IL2Rγnull background (NSGS mice), we demonstrate remarkable engraftment of CMML and JMML providing the first examples of serially transplantable and genetically accurate models of CMML. Xenotransplantation of CD34+ cells (n = 8 patients) or unfractionated bone marrow (BM) or peripheral blood mononuclear cells (n = 10) resulted in robust engraftment of CMML in BM, spleen, liver, and lung of recipients (n = 82 total mice). Engrafted cells were myeloid-restricted and matched the immunophenotype, morphology, and genetic mutations of the corresponding patient. Similar levels of engraftment were seen upon serial transplantation of human CD34+ cells in secondary NSGS recipients (2/5 patients, 6/11 mice), demonstrating the durability of CMML grafts and functionally validating CD34+ cells as harboring the disease-initiating compartment in vivo. Successful engraftments of JMML primary samples were also achieved in all NSGS recipients (n = 4 patients, n = 12 mice). Engraftment of CMML and JMML resulted in overt phenotypic abnormalities and lethality in recipients, which facilitated evaluation of the JAK2/FLT3 inhibitor pacritinib in vivo. These data reveal that NSGS mice support the development of CMML and JMML disease-initiating and mature leukemic cells in vivo, allowing creation of genetically accurate preclinical models of these disorders.

Abstract 1126: Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5

Abstract Human cytomegalovirus (HCMV), also referred to as human herpesvirus-5 (HHV-5), can cause serious and even fatal disease in immunocompromised individuals and newborns, namely individuals with AIDS, solid organ transplant recipients, chemotherapy patients and recipients of bone marrow and stem cell transplants. HCMV is a ubiquitous virus found throughout all geographic regions and socioeconomic groups, infecting greater than 50% of adults in industrialized countries and as many as 100% in developing countries. Although current therapeutics improves clinical outcomes, they are limited by toxicity, intravenous infusion and the development of resistance by the virus. Thus, there is a pressing need to develop novel therapeutics to prevent HCMV infections with concomitant organ toxicity. Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. We have identified a compound Retro94, which potently inhibits production of infectious HCMV virions in cells by operating against a host cell process, rather than directly targeting the virus. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs (99-99.99%). Here we discuss in vitro, in vivo, stability, and binding study of Retro94. Overall, our findings have identified Retro94, as novel agent that affects key cellular trafficking factors important for supporting HCMV infection. Citation Format: Dhimant H. Desai, Linda Cruz, Nicholas T. Streck, Trisha D. Desai, Aron Lukacher, Shantu G. Amin, Nicholas J. Buchkovich. Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2017-1126

Regulatory T Cells Promote Natural Killer Cell Education in Mixed Chimeras.

Therapeutic administration of regulatory T cells (Tregs) leads to engraftment of conventional doses of allogeneic bone marrow (BM) in nonirradiated recipient mice conditioned with costimulation blockade and mammalian target of rapamycin inhibition. The mode of action responsible for this Treg effect is poorly understood but may encompass the control of costimulation blockade-resistant natural killer (NK) cells. We show that transient NK cell depletion at the time of BM transplantation led to BM engraftment and persistent chimerism without Treg transfer but failed to induce skin graft tolerance. In contrast, the permanent absence of anti-donor NK reactivity in mice grafted with F1 BM was associated with both chimerism and tolerance comparable to Treg therapy, implying that NK cell tolerization is a critical mechanism of Treg therapy. Indeed, NK cells of Treg-treated BM recipients reshaped their receptor repertoire in the presence of donor MHC in a manner suggesting attenuated donor reactivity. These results indicate that adoptively transferred Tregs prevent BM rejection, at least in part, by suppressing NK cells and promote tolerance by regulating the appearance of NK cells expressing activating receptors to donor class I MHC.

Impairment of myeloid-derived suppressor cell expansion and enhancement of dendritic cell differentiation of MyD88-deficient bone marrow cells in graft-versus-host disease

Abstract Myeloid differentiation 88 (MyD88) is an adaptor molecule in toll-like receptor-associated signaling. To examine the effect of MyD88-deficiency in myeloid cell differentiation under inflammatory condition, we transplanted bone marrow (BM) cells isolated from MyD88-deficient C57BL/6 (B6) mice together with B6 T cells into MHC-matched allogeneic BALB.B strain mice. This transplantation induced more serious graft-versus-host disease in the BALB.B BM recipients, compared with the GVHD induced in the control BALB.B mice which received wild type B6 BM and T cells. The aggravation of GVHD was associated with impaired expansion of CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) from the MyD88-deficient BM cells during the GVHD development. Instead of MDSC expansion, myeloid cells from MyD88-deficient BM cells showed enhanced apoptosis and dendritic cell (DC) differentiation, which resulted in enhancement of activation of allo-reactive B6 T cells. These results provide information helpful for the understanding of the role of MyD88 in myeloid cell differentiation and development of strategy to reduce the GVHD severity.

Haemolysis, pure red cell aplasia and red cell antibody formation associated with major and bidirectional ABO incompatible haematopoietic stem cell transplantation.

Acute and delayed haemolysis, alloimmunisation and pure red cell aplasia (PRCA) are potential complications after ABO incompatible haematopoietic stem cell transplantation (HSCT). The aims of this study were to investigate acute and delayed red blood cell (RBC) antibody-associated complications, including haemolysis, PRCA and alloimmunisation in major and bidirectional ABO incompatible HSCT. We retrospectively examined the transplant courses of 36 recipients of bone marrow or peripheral blood stem cells from ABO incompatible donors and evaluated the current practice of performing plasmapheresis in patients with higher isoagglutinin titres. We investigated the role of ABO incompatibility in haematopoietic recovery, transfusion requirements, alloimmunisation and PRCA. Laboratory signs of acute haemolysis were noted in five (14%) patients, one (3%) of whom had clinically overt haemolysis. Patients with haemolysis had IgM titres ≥1:8 and received >16 mL of RBC in the HSCT. In patients with higher titres, plasmapheresis performed prior to the transplant prevented acute haemolysis. Delayed haemolysis was not recorded in the follow up. Haematopoietic recovery and transfusion requirements did not differ notably between patients with and without haemolysis. De novo RBC antibodies were detected in two (5.5%) patients after HSCT, and PRCA was noted in one (3%) patient. Carried out with adequate graft processing, plasmapheresis and blood component support, haemolysis is not a common complication after HSCT. Our results confirm that the occurrence of haemolysis depends on larger RBC volumes and higher isoagglutinin titres. Despite the reduction of patients' isoagglutinin titres by plasmapheresis, we still noted a critical combination for the development of laboratory signs of haemolysis (IgM titre ≥1:8 and RBC volume >16 mL). De novo immunisation to RBC antigens and PRCA are rare events following ABO incompatible HSCT.

Short-course rapamycin treatment enables engraftment of immunogenic gene-engineered bone marrow under low-dose irradiation to permit long-term immunological tolerance

BackgroundApplication of genetically modified hematopoietic stem cells is increasingly mooted as a clinically relevant approach to protein replacement therapy, immune tolerance induction or conditions where both outcomes may be helpful. Hematopoietic stem and progenitor cell (HSPC)-mediated gene therapy often requires highly toxic pretransfer recipient conditioning to provide a ‘niche’ so that transferred HSPCs can engraft effectively and to prevent immune rejection of neoantigen-expressing engineered HSPCs. For widespread clinical application, reducing conditioning toxicity is an important requirement, but reduced conditioning can render neoantigen-expressing bone marrow (BM) and HSC susceptible to immune rejection if immunity is retained.MethodsBM or HSPC-expressing OVA ubiquitously (actin.OVA) or targeted to MHC II+ cells was transferred using low-dose (300 cGy) total body irradiation. Recipients were administered rapamycin, cyclosporine or vehicle for 3 weeks commencing at BM transfer. Engraftment was determined using CD45 congenic donors and recipients. Induction of T-cell tolerance was tested by immunising recipients and analysing in-vivo cytotoxic T-lymphocyte (CTL) activity. The effect of rapamycin on transient effector function during tolerance induction was tested using an established model of tolerance induction where antigen is targeted to dendritic cells.ResultsImmune rejection of neoantigen-expressing BM and HSPCs after low-dose irradiation was prevented by a short course of rapamycin, but not cyclosporine, treatment. Whereas transient T-cell tolerance developed in recipients of OVA-expressing BM administered vehicle, only when engraftment of neoantigen-expressing BM was facilitated with rapamycin treatment did stable, long-lasting T-cell tolerance develop. Rapamycin inhibited transient effector function development during tolerance induction and inhibited development of CTL activity in recipients of OVA-expressing BM.ConclusionsRapamycin acts to suppress acquisition of transient T-cell effector function during peripheral tolerance induction elicited by HSPC-encoded antigen. By facilitating engraftment, short-course rapamycin permits development of long-term stable T-cell tolerance.

Epithelial-specific Toll-like Receptor (TLR)5 Activation Mediates Barrier Dysfunction in Experimental Ileitis.

A large body of evidence supports a central role of TLR5 and its natural ligand, flagellin, in Crohn's disease (CD), with the precise mechanism(s) still unresolved. We investigated the role of flagellin/TLR5 in SAMP1/YitFc (SAMP) mice, a spontaneous model of Crohn's disease-like ileitis. Ileal Tlr5 and serum antiflagellin IgG antibodies were increased in SAMP before the onset of inflammation and during established disease; these trends were abrogated in the absence of colonizing commensal bacteria. Irradiated SAMP receiving either wild-type (AKR) or SAMP bone marrow (BM) developed severe ileitis and displayed increased ileal Tlr5 compared with AKR recipients of either SAMP or AKR bone marrow, neither of which conferred ileitis, suggesting that elevated TLR5 in native SAMP is derived primarily from a nonhematopoietic (e.g., epithelial) source. Indeed, ileal epithelial TLR5 in preinflamed SAMP was increased compared with age-matched AKR and germ-free SAMP. TLR5-specific ex vivo activation of SAMP ileal tissues decreased epithelial barrier resistance, indicative of increased permeability, and was accompanied by altered expression of the tight junction proteins, claudin-3, occludin, and zonula occludens-1. Our results provide evidence that aberrant, elevated TLR5 expression is present in the ileal epithelium of SAMP mice, is augmented in the presence of the gut microbiome, and that TLR5 activation in response to bacterial flagellin results in a deficiency to maintain appropriate epithelial barrier integrity. Together, these findings represent a potential mechanistic pathway leading to the exacerbation and perpetuation of chronic gut inflammation in experimental ileitis and possibly, in patients with Crohn's disease.

Young Bone-Marrow Sca-1+ Stem Cells Rejuvenate the Aged Heart and Improve Function after Injury through PDGFR\u03b2-Akt pathway

Bone marrow (BM) reconstitution with young BM cells in aged recipients restores the functionality of cardiac resident BM-derived progenitors. This study investigated the cell type primarily responsible for this effect. We reconstituted old mice with BM cells from young or old mice and found that the number of stem cell antigen 1 (Sca-1) cells homing to the heart was significantly greater in young than old chimeras. We then reconstituted old mice with young BM Sca-1+ or Sca-1− cells. We found that Sca-1 cells repopulated the recipient BM and homed to the heart. The number of BM-derived cells in the aged myocardium co-expressing PDGFRβ was 3 times greater in Sca-1+ than Sca-1− chimeric mice. Sca-1+ chimeras had more active cell proliferation in the infarcted heart and improved ventricular function after MI. The improved regeneration involved activation of the PDGFRβ/Akt/p27Kip1 pathway. Sca-1+ stem cells rejuvenated cardiac tissue in aged mice. Restoration of the Sca-1+ subset of stem cells by BM reconstitution improved cardiac tissue regeneration after injury in aged mice.

Mesenchymal stem cell transplantation in tight-skin mice identifies miR-151-5p as a therapeutic target for systemic sclerosis.

Systemic sclerosis (SSc), an autoimmune disease, may cause significant osteopenia due to activation of the IL4Rα/mTOR pathway. Mesenchymal stem cell transplantation (MSCT) can ameliorate immune disorders in SSc via inducing immune tolerance. However, it is unknown whether MSCT rescues osteopenia phenotype in SSc. Here we show that MSCT can effectively ameliorate osteopenia in SSc mice by rescuing impaired lineage differentiation of the recipient bone marrow MSCs. Mechanistically, we show that donor MSCs transfer miR-151-5p to the recipient bone marrow MSCs in SSc mice to inhibit IL4Rα expression, thus downregulating mTOR pathway activation to enhance osteogenic differentiation and reduce adipogenic differentiation. Moreover, systemic delivery of miR-151-5p is capable of rescuing osteopenia, impaired bone marrow MSCs, tight skin, and immune disorders in SSc mice, suggesting that miR-151-5p may be a specific target for SSc treatment. Our finding identifies a previously unrecognized role of MSCT in transferring miRNAs to recipient stem cells to ameliorate osteopenia via rescuing a non-coding RNA pathway.

Comparison of Peripheral Blood Stem Cells (PBSC) to Bone Marrow (BM) for T-Replete HLA-Haploidentical Donor Transplantation Using Post-Transplant Cyclophosphamide

T cell-replete haploidentical donor transplants (HAPLO-HCT) using post-transplant cyclophosphamide for control of alloreactivity is now being increasingly utilized. HAPLO-HCTs were originally performed using BM grafts. However, recently, a few single center studies have reported good outcomes using G-CSF mobilized PBSC grafts for HAPLO-HCT. No prospective randomized comparisons of BM to PBSC grafts for HAPLO-HCT have been performed. Therefore, we analyzed outcomes for 687 adults (496 BM, 191 PBSC) who received HAPLO-HCT for hematologic malignancies using post-transplant cyclophosphamide + mycophenolate + calcineurin inhibitor for GVHD prophylaxis between 2009 and 2014 in the United States. The primary outcome was overall survival. The characteristics of recipients of BM and PBSC were similar except BM recipients were older, more likely to have a performance score ≥90, HCT-CI index ≤2, be CMV seronegative, have a lymphoid malignancy and receive reduced-intensity conditioning. Most PBSC transplants occurred between 2012 and 2014. The median follow-up was 35 and 20 months for recipients of BM and PBSC grafts, respectively. Cox regression models were built to study the effect of graft type adjusted for other significant factors on overall mortality, non-relapse mortality, relapse and graft-versus-host disease (GVHD) and outcomes censored at 2-years to accommodate differential follow-up between treatment groups (Table 1). After adjusting for age, CMV serostatus, disease risk index (disease type/disease status for myeloid and lymphoid malignancy and cytogenetic risk for acute leukemia and myelodysplastic syndrome) and transplant conditioning regimen there were no significant differences in risks for overall mortality (HR 1.00, p= 0.98; 2-year overall survival: 54% and 57%) or non-relapse mortality (HR 0.92, p=0.74; 2-year non-relapse mortality: 17% and 16%) after transplantation of BM compared to PBSC, respectively. However, relapse risks were higher after transplantation of BM compared to PBSC (HR 1.49, p=0.009; 2-year relapse: 45% and 28%). Subset analyses explored the effect of graft type separately for myeloablative and reduced intensity conditioning regimen adjusting for age, CMV serostatus and disease risk index. Consistent with the main analysis there were no differences in overall or non-relapse mortality risks and relapse risks were higher with BM compared to PBSC with myeloablative regimens (Table 1). Although this may in part be explained by lower chronic GVHD risks with transplantation of BM grafts, chronic GVHD was not significantly predictive of relapse risk when modeled as a time-dependent covariate (HR= 0.73, p=0.49). Grade II-IV acute GVHD risks (HR 0.45, p<0.001; 22% and 37%), adjusted for conditioning regimen were lower after transplantation of BM compared to PBSC. Chronic GVHD risks adjusted for age and performance score were also lower after transplantation of BM compared to PBSC (HR 0.35, p<0.001; 20% and 41%) but rates of moderate and severe chronic GVHD were not significantly different (28% and 32%). There were no differences in incidence of hematopoietic recovery by graft type. In conclusion, compared to BM grafts HAPLO-HCT with PBSC are associated with similar overall survival and non-relapse mortality risks but lower relapse risks with myeloablative conditioning regimens. Longer follow up is needed to ascertain whether survival differences may occur later. The observed adverse effect of BM grafts on relapse with myeloablative regimens must be studied further in the setting of carefully controlled trials. [Display omitted] DisclosuresCiurea:Cyto-Sen Therapeutics: Equity Ownership; Spectrum Pharmaceuticals: Other: Advisory Board. Hamadani:Takeda: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees. Wingard:Ansun: Consultancy; Gilead: Consultancy; Astellas: Consultancy; Fate Therapeutics: Consultancy; Merck: Consultancy.

Inhibition of CML Development Following Conditional SIRT1 Deletion in Transgenic BCR-ABL Mice

▪Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mesenchymal Stromal Cells, Instigator or Suppressor for the Development of MLL-AF9 Induced Acute Myeloid Leukemia?

It has been reported that bone marrow (BM) hematopoietic niche is critical for the maintenance of normal hematopoiesis and the development of hematopoietic disorders. However, little is known about how the different BM niche components contribute to the initiation and progression of acute myeloid leukemia (AML). To explore the role of BM stromal cells in the development of AML, we here characterized the BM stromal cells in AML mouse model induced by transplantation of mouse BM cellstransduced with MLL-AF9 oncogene. We subdivided the BM stromal cells (CD45-TER119-) into mesenchymal stem cells (MSCs, CD31-CD44-CD51+SCA1+), mesenchymal progenitor cells (MPCs, CD31-CD44-CD51+SCA1-), and endothelial cells (CD31+) in the mice after development of AML by multicolor fluorescent activated cell sorting (FACS) (Qian et al., Mol Cell Biol, 2013). We found significantly increased frequencies of BM MSCs (p < 0.0001), MPCs (p < 0.0001), and endothelial cells (p < 0.0001) in the AML mice, indicating an AML-induced alteration of BM stromal cell composition. The increased frequency of MSCs in AML mouse BM was confirmed by the increased number of colony foming unit-fibroblasts in the AML mice (p < 0.0001). Furthermore, in vitro multi-lineage differentiation assay revealed increased adipogenic and osteogenic differentiation capacities of the BM MSCs in the AML mice. Consistent with the increased osteogenic differentiation capacity of AML MSCs, Runx2 mRNA expression was increased in freshly sorted AML MSCs compared to that in the control mice. In addition, quantitative real time PCR on freshly sorted BM MPCs showed a dramatic downregulation of the genes important in hematopoietic stem cell regulation, including angiopoietin like 1 (Angptl1), C-X-C motif chemokine (Cxcl12), and kit ligand (Kitl) in the AML mice. The altered gene expression might contribute to the AML progression.To specifically determine the contribution of MSCs to AML development, we analyzed the MSC population expressing Early B cell factor 2 (Ebf2) (Qian et al., Mol Cell Biol, 2013) by establishing the AML in Ebf2-Egfp transgenic recipient mice. We detected an increased frequency of Ebf2+ cells in the recipient BM after development of AML (p = 0.0018). In addition, by using the triple transgenic Tg(Ebf2-Egfp x Ebf2-CreER x Rosa26-loxpStoploxp-Tomato) mouse model, where Ebf2+ MSCs and their progenies can be traced by tomato fluorescent protein after tamoxifen treatment, we found that the MSCs and MPCs generated from the Ebf2+ MSCs were also increased in the AML mouse BM (p < 0.05).To further investigate the functional contribution of Ebf2+ MSCs to the AML progression, we took advantage of the transgenic Tg(Ebf2-CreER x Rosa26-loxpStoploxp-DTA) mouse model allowing specific deletion of the Ebf2-expressing MSCs. Strikingly, we observed an accelerated initiation of AML, illustrated by the shorter survival of the MSC depleted recipients (Figure 1), compared to the non-depleted recipients, suggesting that Ebf2+ MSCs could suppress AML progression.Taken together, our data show that BM stromal cell composition and function are altered in the mice with AML and the BM MSCs may act to suppress the progression of the MLL-AF9 induced AML. These findings provide new evidence for the niche contribution to the development of AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.