Murine Bone Marrow Transplantation Model Research Articles

Investigating the effects of radiation, T cell depletion, and bone marrow transplantation on murine gut microbiota.

Microbiome research has gained much attention in recent years as the importance of gut microbiota in regulating host health becomes increasingly evident. However, the impact of radiation on the microbiota in the murine bone marrow transplantation model is still poorly understood. In this paper, we present key findings from our study on how radiation, followed by bone marrow transplantation with or without T cell depletion, impacts the microbiota in the ileum and caecum. Our findings show that radiation has different effects on the microbiota of the two intestinal regions, with the caecum showing increased interindividual variation, suggesting an impaired ability of the host to regulate microbial symbionts, consistent with the Anna Karenina principle. Additionally, we observed changes in the ileum composition, including an increase in bacterial taxa that are important modulators of host health, such as Akkermansia and Faecalibaculum. In contrast, radiation in the caecum was associated with an increased abundance of several common commensal taxa in the gut, including Lachnospiraceae and Bacteroides. Finally, we found that high doses of radiation had more substantial effects on the caecal microbiota of the T-cell-depleted group than that of the non-T-cell-depleted group. Overall, our results contribute to a better understanding of the complex relationship between radiation and the gut microbiota in the context of bone marrow transplantation and highlight the importance of considering different intestinal regions when studying microbiome responses to environmental stressors.

Signaling Pathway Mutations Cooperate with the PICALM/MLLT10 Fusion in a Knock-in AML Mouse Model

The PICALM/MLLT10 (or CALM/AF10; C/A) is a rare but recurring fusion gene detected in patients with T-cell acute lymphoblastic leukemia, malignant lymphoma and acute myeloid leukemia (AML). Our lab has previously established a C/A knock-in mouse model, where the fusion gene preceded by a loxP-flanked transcriptional stop cassette was inserted into the Rosa26 locus (the R26LSLCA strain). To achieve tissue-specific expression of C/A, R26LSLCA mice were crossed with three different Cre-inducer lines, namely the Vav-Cre, MB1-Cre and CD19-Cre. In the Vav-Cre strain, Cre recombinase is expressed in the entire hematopoietic system, while in the MB1-Cre and CD19-Cre lines, Cre is expressed in the B-cell compartment. Using this model, Dutta et al. showed that C/A leads to leukemia only when expressed in all hematopoietic cells, including hematopoietic stem cells and does not lead to induction of leukemia when expressed in B-cells alone ( Dutta et al., Leukemia 2016). To further characterise leukemias driven by the C/A fusion, we established new R26LSLCA and Vav-Cre crosses. Similar to what was found previously, the Vav-Cre/R26LSLCA mice developed leukemia with a long median latency of 1 year (366.5 days; N=24) and complete penetrance. On post-mortem analysis, the Vav-Cre/R26LSLCA mice were found to have splenomegaly and leucocytosis. Expression of the fusion gene was confirmed in these mice by RT-PCR on the cDNA from bone marrow (BM) cells. Immunophenotyping analysis of the BM cells and splenocytes of these mice revealed a predominantly myeloid phenotype. To assess the effect of expression of C/A on the transcriptome, we performed RNA-seq analysis using BM cells from 17 leukemic mice and 12 healthy controls. In accordance with reports on C/A patient samples, the Hoxa genes Hoxa7 and Hoxa9 as well as the Hox-cofactor Meis1 were significantly upregulated in our C/A expressing mice. In addition, genes involved in B-cell development, such as Cd19, Pax5 and Ikzf3, and Gata1, which is essential for the development of erythrocytes, were downregulated. To prove that the Vav-Cre/R26LSLCA mice were indeed leukemic and had the potential to propagate the disease, we serially transplanted BM cells from 4 Vav-Cre/R26LSLCA mice into congenic recipients. The secondary (N=20) and tertiary (N=19) transplanted mice developed leukemia with a significantly shorter median latency of 21 and 17 days, respectively. Our group and others have shown that a single genetic lesion is in most cases not sufficient to cause leukemia. Thus, to determine if additional somatic mutations were acquired by the C/A knock-in mice, we performed whole exome sequencing on the DNA from BM cells of the 4 knock-in mice, some of their daughter secondary and tertiary leukemias as well as the corresponding germline DNA samples (N=19). We identified between 2-32 mutations per leukemia exome, including mutations in genes and/or members of gene families that have been implicated in human leukemias. Mutations were detected in the transcription factors Sfpi1, Zeb1, and Sox9; Aff2, a member of the Af4/Fmr2 gene family; and Sf3a2, a component of the RNA-splicing machinery, among others. In addition, in two C/A sister secondary leukemias and the corresponding tertiary leukemias, we detected a missense mutation at amino acid 507 (G507A) of the Ptpn11 gene, which is homologous to G507 of human PTPN11, a known hotspot mutation site in AML and juvenile myelomonocytic leukemia. Interestingly, Dutta et al. also reported a G507 mutation (G507V) in Ptpn11 in a C/A knock-in mouse. PTPN11 regulates the RAS/MAPK signaling pathway. This finding is reminiscent of what we have previously reported in a C/A driven murine bone marrow transplantation model, where we detected somatic mutations in several AML-associated signal transduction genes, including Flt3, Cbl and Kras ( Desai et al., Leukemia 2020). Taken together, our results strongly suggest that mutations in genes involved in signaling cascades, particularly those in the RAS/MAPK pathway, cooperate with the C/A fusion and lead to complete malignant transformation. We aim to functionally validate these findings by establishing mouse models that harbor the C/A fusion as well as mutations in Ptpn11 and other signal transduction genes. These murine models will be immensely valuable for gaining a deeper insight into PICALM/MLLT10-mediated leukemogenesis, studying cooperating mutations and for testing new targeted therapies.

Glycolysis-Driven IFN-γ Expression in T-Cell Via Histone Lactylation Promotes Acute Graft-Versus-Host Disease

Background Cell metabolism is a highly regulated process essential for sustaining life and driving evolution. Cellular metabolites and metabolic flux intricately govern post-translational modifications of proteins. In the context of acute graft-versus-host disease (aGVHD), T cells undergo enhanced glycolysis, resulting in increased lactate production. Consequently, lactate may act as a substrate for lysine lactylation (Kla), influencing gene expression by modulating histone biology. This study aims to assess T cell glucose metabolism during aGVHD, investigate the role of histone lactylation, and explore its impact on T cell functionality. Methods We conducted a comparative analysis of histone lactylation between aGVHD patients and individuals undergoing allo hematopoietic stem cell transplantation (control). Subsequently, we established a murine bone marrow transplant model and isolated T cells for analysis. Glucose metabolism status was assessed using extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) assays, along with the 2-NBDG glucose uptake assay. To comprehensively investigate histone lactylation in T cells, we employed the cleavage under targets and tagmentation (CUT&Tag) technique to explore its regulatory effects on gene expression. To investigate the impact of lactatylation on aGVHD pathology, we constructed an aGVHD model by applying T cells with a specific deletion of LDHA. Results By analyzing the serum lactate at the onset of aGVHD, we found that patients with grade III-IV aGVHD had significantly higher lactate levels compared to those with grade I-II aGVHD ( Figure 1A). In light of this finding, we further examined the histone lactylation levels in T cells from aGVHD patients. The results demonstrated an enhanced histone lactylation in these T cells compared to controls ( Figure 1B). In the aGVHD mice, the ECAR of T cells exhibited a significant increase ( Figure 1C). Intriguingly, the OCR were also significantly higher in the aGVHD mice compared to the bone marrow T cell-depleted (BM-TCD) mice ( Figure 1D). Additionally, the 2-NBDG revealed a significantly higher fluorescence intensity in aGVHD mice ( Figure 1E). Through analysis of T cell RNA-seq data, we discovered upregulation of most glycolytic enzymes in aGVHD mice similar to human data ( Figure 1F). Notably, LDHA, glucose transporter 1 ( Slc2a1), and monocarboxylate transporter 4 ( Slc16a3) were highly upregulated ( Figure 1G). The upregulation of LDHA in T cells from aGVHD mice prompted us to investigate the presence of histone lactylation. The results revealed that histone in T cells from aGVHD mice exhibited a higher level of lactylation ( Figure 1H). Because lactylation was particularly prominent at the H4K12 site, we conducted CUT&Tag experiments. We found promoters of key regulatory genes of the Th1 subset, such as Tbx21 and Ifng, along with several genes associated with IFN-γ production, including Stim2, Crtam, Il18rap, Fasl, and Phf11a, Cxcr3 were all lactylated ( Figure 1I). Specifically, the Call Peak analysis confirmed the elevated H4K12la levels on the promoters Tbx21 and Ifng ( Figure 1J). The expression of IFN-γ and CXCR3 in CD4 + and CD8 + T cells of aGVHD mice was significantly higher than in BM-TCD mice ( Figure 1K). To confirm the role of histone lactylation in IFN-γ generation, we intervened with T cells using the p300 lactylation writer inhibitor (A485). The results demonstrated a significant reduction in histone lactylation upon A485 treatment, which was effectively restored by the addition of neutral lactate sodium (Nala) ( Figure 1L). Notably, A485 treatment led to a decrease in IFN-γ + T cells, while Nala administration resulted in a substantial recovery of IFN-γ expression ( Figure 1M). Additionally, the infusion of Cd4creLdhafl/fl T cells into recipient mice improved the survival of aGVHD mice ( Figure 1N). Conclusion This study represents the first global investigation of T cell histone lactylation in aGVHD. Importantly, we have uncovered a novel link between histone lactylation and the regulation of IFN-γ-related gene expression in the context of aGVHD. Considering the heightened glycolysis and the pivotal role of IFN-γ in aGVHD pathology, it is plausible to postulate that T cell-produced lactate induces histone lactylation, thereby promoting the transcription of IFN-γ-related genes and contributing to the development of aGVHD.

Intestinal Epithelial-Derived IL-34 Regulates Macrophage Polarization in an Apolipoprotein E Dependent Manner and Mitigates the Severity of Graft Versus Host Disease

Acute graft versus host disease (GVHD) of the gastrointestinal (GI) tract is a major complication of allogeneic bone marrow transplantation. While donor T cells are the proximate trigger of GVHD, innate cells also contribute to disease pathogenesis, although the role of these cell populations is not as well defined. Macrophages, for example, are known contributors in the pathophysiology of chronic GVHD, but their role in acute GVHD is not well delineated. Fundamentally, macrophages are dependent upon exposure to m-CSF and IL-34 which serve as critical trophic factors necessary for their development through binding to the CSF-1R. IL-34 is a more recently discovered cytokine produced by macrophages, but whose role in GVHD pathogenesis is unknown. To examine this question, we employed a murine bone marrow transplantation model (B10.BR [H-2k]®B6[H-2b]) and observed that that there was no difference in GVHD-induced lethality when recipients were reconstituted with marrow grafts from wild type (WT) or IL-34 -/- donors. In contrast, recipient IL-34 -/- animals had significantly worse survival compared to WT mice indicating that host IL-34 expression protected animals from lethal GVHD. We observed no difference in the absolute number of donor macrophages in the colons of WT versus IL-34 -/- mice; however, IL-34 -/- recipient mice had a significant increase in the number of inflammatory Ly6C + macrophages, and a corresponding decrease in Ly6C - cells. Single cell RNA sequence (scRNAseq) analysis of donor macrophages from the colons of WT and IL-34 -/- recipients revealed that macrophages from WT animals had increased expression of anti-inflammatory genes (i.e., CD83, ApoE, Selenop, Lyz1 and Mrc1), whereas macrophages from IL-34 -/- recipients had increased expression of pro-inflammatory genes (i.e., Ifitm1, S100A8, S100A9 and GOs2), indicating that absence of host IL-34 skewed donor macrophages towards an inflammatory phenotype. Since the CSF-1R is not expressed on T cells, we sought to determine the effect of IL-34/CSF-1R signaling on proinflammatory T cells in GVHD target organs. scRNAseq analysis of colonic CD4+ T cells revealed that Csf2 (GM-CSF) was the most differentially expressed gene in IL-34 -/- CD4+ T cells. Functionally, this was confirmed by the observation of a significantly increased number of CD4 + GM-CSF+ T cells in the colons of these mice. Notably, there was no difference in proinflammatory CD8 + T cells, nor any effect on T cell phenotype in the liver or lung, demonstrating that the mitigating effect of host IL-34 expression was confined to the GI tract. To determine the cellular source of IL-34 production, we employed IL-34 LacZ reporter mice and showed that β-Gal co-localized with the epithelial marker, E-cadherin, indicating IL-34 is produced primarily by intestinal epithelial cells. Additional studies using chimeric mice in which IL-34 was restricted to either the hematopoietic or non-hematopoietic compartmentrevealed that absence of IL-34 in the non-hematopoietic compartment phenocopied findings observed with global IL-34 -/- animals, confirming that intestinal epithelial cells were the primary source of this cytokine. The administration of exogenous IL-34 prolonged survival, abrogated pathological damage in the colon and resulted in a corresponding decrease in pro-inflammatory macrophages and T cells, demonstrating this pathway could be pharmacologically targeted. Re-analysis of the scRNAseq data on donor macrophages from the colons of WT and IL-34 -/- mice revealed that apolipoprotein E ( Apoe) was the most differentially expressed gene in macrophages from WT GVHD animals. To confirm a functional role for ApoE, mice were transplanted with T cell depleted (TCD) BM from either WT or ApoE -/- mice and treated with IL-34. These studies revealed that the protective effect conferred by IL-34 was abrogated in animals that received TCD ApoE -/- BM grafts, indicating that ApoE expression in donor macrophages was necessary for agonist driven IL-34-mediated regulation of GVHD. Finally, we confirmed these studies have translational relevance by demonstrating IL-34 and Apoe have increased expression in intestinal epithelial cells and macrophages, respectively, in the colons of humans with GVHD. Thus, these results identify IL-34 as a novel intestinal epithelial cell-derived cytokine that regulates macrophage polarization and mitigates GVHD in an ApoE-dependent manner.

Integrated Genomic Analysis Identifies a Downstream Regulatory Element Oncogenically Activating Hematopoietically Expressed Homeobox ( HHEX) in ETP-ALL

Hematopoietically-expressed Homeobox ( HHEX) is required for the maintenance of hematopoietic stem cells (HSPCs) and common lymphoid progenitor cells. HHEX is also the second most frequent integration site in retroviral insertional mutagenesis screens of leukemias and lymphomas arising in AKXD recombinant inbred mouse strains, implying that it is an important oncogene. Enforced expression of HHEX induces T-cell acute lymphoblastic leukemias (T-ALL) in murine bone marrow transplantation models. In human studies, HHEXmRNA expression is upregulated in human T-ALL studies, especially in Early T-cell Precursor (ETP-) ALL subtypes where it is concordantly expressed with LMO2. The HHEX locus is not rearranged in ETP-ALL where it is upregulated so the mechanism of HHEX activation is not clear. We analyzed T-ALL induced by retroviral mutagenesis and found an intergenic site of frequent integration, 65kb 3‘ of the Hhex coding exons. Integrations clustered within a 3 kb genomic area that is also an open chromatin region (OCR) by ATAC-seq analysis and that induced Hhex upregulation. The syntenic human region was highly enriched for H3K27 acetylation and for occupancy by multiple transcription factors, notably GATA1 and TAL1, per ENCODE. Our prior data had shown that HHEX was a downstream target of the LMO2 complex comprised of GATA1/2, TAL1 or LYL1, and nucleated by the scaffolding protein, LIM domain binding protein 1 (LDB1). We had previously shown by ChIP-PCR that the LMO2 complex was bound to the intron 1 enhancer. Thus, we performed ChIP-exonuclease analysis of LDB1 in the human ETP-ALL model cell line, LOUCY. We confirmed its occupancy at the intron 1 enhancer but we also observed LDB1 occupancy 68kb 3‘ of the HHEX coding exons, in the exact region syntenic to the murine common insertion sites. ChIP-exo analysis allowed us to pinpoint a core element with composite GATA/E box sites that was highly conserved across multiple mammalian species. LDB1 occupancy at intron 1 and at +68kb of the HHEX locus was reminiscent of LDB1's occupancy at the beta globin locus where LDB1's occupancy and homodimerization mediates chromatin looping between composite E box/GATA sites in the locus control region (LCR) and the beta globin proximal promoters. To test whether a similar interaction was occurring at the HHEX locus, we analyzed virtual 4C (chromatin conformation capture) data. We confirmed looping between the +68kb element and the proximal promoter of HHEX in human ETP-ALL primary samples and in human HSPCs. The experiments showed that looping occurs in certain developmental contexts in HSPCs and is recapitulated in ETP-ALL and mediated by the LMO2/LDB1 protein complex. HHEX is a downstream oncogene of the LMO2/LDB1 complex and is activated through this distal +68kb regulatory element. Our studies raise the possibility that dissection of this regulatory element and interference with chromatin looping are potential therapeutic mechanisms that could disrupt oncogene expression.

Peritransplantation Glucocorticoid Haploidentical Stem Cell Transplantation Is a Promising Strategy for AML Patients With High Leukemic Burden: Comparison With Transplantations Using Other Donor Types

Using a murine haploidentical bone marrow transplantation (BMT) model, we recently showed that peritransplantation administration of glucocorticoid (GC) redistributed donor T cells from the gastrointestinal tract to bone marrow, which resulted in a significant reduction of graft-versus-host disease (GVHD) while promoting graft-versus-leukemia effects. Furthermore, in a retrospective clinical study of patients with acute myelogenous leukemia (AML) undergoing transplantation in non-remission, we also showed that haploidentical stem cell transplantation (haplo-SCT) using peritransplantation GC administration led to a significantly lower relapse rate and better overall survival rate compared with haplo-SCT using post-transplantation cyclophosphamide. In the present study, using the same dataset of patients undergoing GC haplo-SCT, we retrospectively compared with patients with AML undergoing transplantation in non-remission using 3 other donor types: matched sibling donor (MSD), matched unrelated donor (MUD), and umbilical cord blood (UCB). For GC haplo-SCT, 44 patients underwent peripheral blood stem cell transplantation in a single center (Hyogo College of Medicine), with the conditioning treatment consisting of fludarabine, melphalan, anti-thymocyte globulin (2.5 mg/kg), and TBI 3 Gy. Methylprednisolone was given from the start of conditioning treatment, and the GVHD prophylaxis consisted of tacrolimus and methylprednisolone (1 mg/kg). The transplantation outcomes were compared with data of 1889 patients undergoing MSD-SCT (n = 449), MUD-BMT (n = 493), or UCB transplantation (UCBT) (n = 947) in non-CR, which were extracted from the Transplant Registry Unified Management Program data, the largest data registry in Japan. For donor engraftment, significantly faster neutrophil and platelet engraftment was achieved with GC haplo-SCT compared with allo-SCT using the 3 other donor types. Neutrophil engraftment was achieved at a median of 10 days for GC haplo-SCT, and 20 days for MSD-, MUD-, and UCB-transplants. Platelet engraftment was achieved at a median of 19.5 days for GC haplo-SCT, 42 days for MSD-SCT and MUD-BMT, and 43 days for UCBT, respectively. The incidence of grade II-IV acute GVHD was lower after allo-SCTs using MSD (hazard ratio [HR] = 0.465, P = .003), MUD (HR = 0.524, P = .010), and UCB (HR = 0.647, P = .067) compared with GC haplo-SCT. There was no significant difference in the incidence of chronic GVHD between GC haplo-SCT and allo-SCT using the other 3 donor types. Regarding relapse, GC haplo-SCT was associated with a significantly lower risk compared with MSD-SCT (P < .001) or MUD-BMT (P = .004). GC haplo-SCT tended to have a lower risk compared with UCBT (P = .063). Especially, all the 43 evaluable GC haplo-SCT recipients achieved CR after transplantation, whereas 23.9%, 22.8%, and 27.0% of patients who underwent MSD-SCT, MUD-BMT, and UCBT could not achieve CR after transplantation, respectively. Regarding non-relapse mortality, GC haplo-SCT was associated with a significantly higher risk compared with MUD-BMT (P = .014), and tended to have a higher risk compared with MSD-SCT (P = .061). There was no significant difference between GC haplo-SCT and UCBT (P = .600). Allo-SCTs using MSD (HR = 2.548, P < .001), MUD (HR = 2.134, P = .005), and UCB (HR = 2.376, P = .001) lead to significantly higher overall mortality compared with GC haplo-SCT; the adjusted overall survival at 3 years was 19.8% for MSD, 26.1% for MUD, 28.0% for UCB, and 65.1% for GC haplo. Thus GC haplo-SCT is a promising treatment option for patients with AML with a high leukemic burden.

Isocitrate Dehydrogenase 2 Mutation Drives Bone Marrow Macrophage Dysfunction without a Complete Block in Hematopoietic Differentiation

Myelodysplastic syndromes (MDS) represent a broad category of myeloid malignancies characterized by myeloid skewed hematopoiesis, blood cytopenias and increased risk of transformation to leukemia. While not common, isocitrate dehydrogenase 2 (IDH2) mutations are represented in clonal hematopoiesis and in MDS, are found in patients with higher International Prognosis Scoring System (IPSS) disease scores and confer a higher risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite which disrupts hematopoiesis. However, the mechanisms that induces clonal progression and transformation remains incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutation on mature myeloid cell functions and the bone marrow microenvironment (BMME) remains to be elucidated. Using RAW264.7 cells, a monocyte/macrophage-like murine cell line, we retrovirally infected cells with human wt IDH2 or the R172K IDH2 mutant, a common IDH2 mutation. After confirming a 30-fold increase of 2HG (p<.005) and observing evidence of hypermethylation in the IDH2 mutant RAW cells, we tested several key functions of bone marrow macrophages: ability to engulf apoptotic cells (efferocytosis), erythropoietic support, and cytokine production. After labeled apoptotic neutrophils were fed as efferocytic targets to RAW cells at a 10:1 ratio for 6 hours, we observed a 10% decrease of efferocytosis in IDH2 mutants (p < 0.05). Next, we cocultured RAW cells with primary murine late-stage erythroid progenitors and quantified their capacity to form colonies (CFU-E) in the presence of erythropoietin. Compared to wt, IDH2 mutant RAW cells inhibited CFU-E formation by 25% (p < 0.0075). Cytokine array on cell culture supernatant from mutant RAW cells had elevated proinflammatory chemokines/cytokines (CCL5, CXCL10, TNF-a) and decreased cytokines related to inflammation resolution and efferocytosis (GDF-15, Gas6, Endostatin, CCL2). To test these findings further in vivo we generated a murine bone marrow transplant model with retrovirally infected hematopoietic stem cells (HSC: lineage-, c-Kit+, Sca-1+) containing the human wt or R172K IDH2 mutation with an mCherry reporter and transplanted into CD45.1 congenic mice (10 mutant, 10 wt, 1:1 male:female). Mutant IDH2 HSCs demonstrated better engraftment (85-90%) compared to wt (70-80%) and also had higher levels of mCherry positivity within CD45.2+ cells (80-90% mutant vs 40-60% in wt). Longitudinal blood draws showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes in IDH2 mutants by 16 weeks post-transplant. In contrast to current literature, IDH2 mutant cells did not exhibit a complete block in myeloid cell differentiation, as the mutant clone gave rise to mature monocytes, macrophages, and neutrophils. We also harvested bone marrow at 16 weeks post transplantation. Compared to wt IDH2, the mutant IDH2 bone marrow cells exhibited myeloid skewing hematopoiesis and a 3-fold increase in bone marrow monocytes (CD45+, Ly6G-, Ly6C++, F4/80+, p < 0.00001). In the IDH2 mutant bone marrow cells, myeloid progenitors were also expanded: common myeloid progenitors (CMPs: 4-fold increase p < 0.001, CD45+, lineage-, c-Kit+, Sca-1-, CD34+, CD16/32-), and granulocytic monocytic precursors (GMPs: 3-fold increase p < 0.0001, CD45+, c-Kit+, Sca-1-, CD34+, CD16/32+). We also observed a previously unreported loss of long-term hematopoietic stem cells (LT-HSC: 13.5-fold reduction, p < 0.00001, CD45+, lineage-, c-Kit+, Sca-1+, Flt3-, CD48-, CD150+). When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow derived macrophages fed normal apoptotic neutrophils at a 10:1 ratio for 3 or 24 hours. In summary, we identify defects in efferocytic function and cytokine production in monocytes and macrophages derived from IDH2 mutant hematopoietic stem and progenitor cells. We have previously shown that defects in the efferocytic function of bone marrow macrophages induce increases in inflammatory signals and contribute to skewing of hematopoiesis. Since these macrophage dysfunctions have not previously been attributed to IDH2 single mutation, they represent a potential novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and transformation to leukemia.

Melatonin Improves Ischemia-Induced Circulation Recovery Impairment in Mice with Streptozotocin-Induced Diabetes by Improving the Endothelial Progenitor Cells Functioning.

Patients with diabetes mellitus tend to develop ischemia-related complications and have compromised endothelial progenitor cell (EPC) function. Melatonin protects against ischemic injury, possibly via EPC modulation. We investigated whether melatonin pretreatment could restore EPC function impairment and improve circulation recovery in a diabetic critical limb ischemia mouse model. Under 25 mM high-glucose medium in vitro, EPC proliferation, nitric oxide production, tube formation, and endothelial nitric oxide synthase (eNOS) phosphorylation were significantly suppressed. Hyperglycemia promoted EPC senescence and apoptosis as well as increased reactive oxygen species (ROS) production. Melatonin treatment reversed the harmful effects of hyperglycemia on EPC through adenosine monophosphate–activated protein kinase-related mechanisms to increase eNOS phosphorylation and heme oxygenase-1 expression. In an in-vivo study, after a 4-week surgical induction of hindlimb ischemia, mice with streptozotocin (STZ)-induced diabetes showed significant reductions in new vessel formation, tissue reperfusion, and EPC mobilization in ischemic hindlimbs compared to non-diabetic mice. Mice with STZ-induced diabetes that received melatonin treatment (10 mg/kg/day, intraperitoneal) had significantly improved blood perfusion ratios of ischemic to non-ischemic limb, EPC mobilization, and densities of capillaries. In addition, a murine bone marrow transplantation model to support these findings demonstrated that melatonin stimulated bone marrow-originated EPCs to differentiate into vascular endothelial cells in femoral ligation-induced ischemic muscles. In summary, this study suggests that melatonin treatment augments EPC function along with neovascularization in response to ischemia in diabetic mice. We illustrated the protective effects of melatonin on EPC H2O2 production, senescence, and migration through melatonin receptors and modulating eNOS, AMPK, and HO-1 activities at the cellular level. Thus, melatonin might be used to treat the impairment of EPC mobilization and circulation recuperation in response to ischemic injury caused by chronic hyperglycemia. Additional studies are needed to elucidate the applicability of the results in humans.

Chimerism through the activation of invariant natural killer T cells prolongs graft survival after transplantation of induced pluripotent stem cell-derived allogeneic cardiomyocytes.

The loss of functional cells through immunological rejection after transplantation reduces the efficacy of regenerative therapies for cardiac failure that use allogeneic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Recently, mixed-chimera mice with donor-specific immunotolerance have been established using the RGI-2001 (liposomal formulation of α-galactosyl ceramide) ligand, which activates invariant natural killer T (iNKT) cells. The present study aimed to investigate whether mixed chimerism, established using RGI-2001, prolongs graft survival in allogeneic iPSC-CM transplantation. Mixed-chimera mice were established via combinatorial treatment with RGI-2001 and anti-CD154 antibodies in an irradiated murine bone marrow transplant model. Luciferase-expressing allogeneic iPSC-CMs were transplanted into mixed-chimera and untreated mice, followed by in vivo imaging. RGI-2001 enhanced iNKT cell activation in mice, and mixed chimerism was successfully established. In vivo imaging revealed that while the allografts were completely obliterated within 2 weeks when transplanted to untreated mice, their survivals were not affected in the mixed-chimera mice. Furthermore, numerous CD3+ cells infiltrated allografts in untreated mice, but fewer CD3+ cells were present in mixed-chimera mice. We conclude that mixed-chimera mice established using RGI-2001 showed prolonged graft survival after allogeneic iPSC-CM transplantation. This donor-specific immunotolerance might increase the efficacy of regenerative therapies for heart failure with allogeneic iPSC-CMs.

Modeling acute graft-versus-host disease (aGVHD) in murine bone marrow transplantation (BMT) models with MHC disparity.

For more than 50years, hematopoietic stem cell transplantation (HSCT) has been the major curative therapy for hematological malignancies and genetic disorders, but its success is limited by the development of graft-versus-host disease (GVHD). GVHD represents a post-transplantation disorder representing the immune-mediated attack of transplant-derived T cells against recipient tissue finally leading to increased morbidity and mortality of the recipient. GVHD develops if donor and recipient are disparate in major or minor histocompatibility antigens (MHC, miHA). Most of the initial knowledge about the biology of GVHD is derived from murine bone marrow transplantation (BMT) models. Of course, GVHD mouse models do not reflect one to one the human situation, but they contribute significantly to our understanding how conditioning and danger signals activate the immune system, enlighten the role of individual molecules, e.g., cytokines, chemokines, death-inducing ligands, define the function of lymphocytes subpopulations for GVHD development and have significant impact on establishing new treatment and prevention strategies used in clinical HSCT. This chapter describes in detail the procedure of allogeneic BMT and the development of GVHD in two commonly used allogeneic murine BMT models (B6→B6.bm1, B6→B6D2F1) with different MHC disparities, which can be used as a basis for advanced studies of GVHD pathology or the development of new treatment strategies.

Abrogation of a Histone Chaperone Pathway Mitigates Inflammation-Driven AML Progression

Background: Genetic heterogeneity makes clinical interventions challenging for acute myeloid leukemia (AML) patients. Identifying and targeting microenvironment-driven pathways that are active across AML genetic subtypes should allow the development of more broadly effective therapies. Previously, we have shown that AML microenvironment is rich in proinflammatory cytokine interleukin-1β (IL-1β) and significantly promotes the growth of AML progenitors while suppressing healthy progenitors. To elucidate this paradoxical effect, we performed transcriptome (RNA-seq) analysis from IL-1β-stimulated CD34+ AML and normal progenitors and found that ASF1B (anti-silencing function-1B) is one of the most differentially expressed genes. ASF1B is a histone chaperone, which recruits H3-H4 histones onto the replication fork during S-phase. This process is regulated by tousled-like kinase 1 and 2 (TLKs). TLKs and ASF1B are overexpressed in multiple solid tumors and associated with poor prognosis. However, their functional roles in hematopoiesis and inflammation-driven leukemia are unexplored. Here, we reveal a novel molecular mechanism that IL-1β promotes leukemia progression by activating the TLK-ASF1B pathway.Methods and Results: We first confirmed that IL-1β stimulation upregulates ASF1B expression at both mRNA and protein levels in FLT3-ITD, MLL-ENL, and NPM1 positive primary AML samples. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, further suggesting that ASF1B is downstream of IL-1β/p38 signaling. Next, we stably knocked down ASF1B in an AML cell line MOLM-14 using doxycycline-inducible shRNA. ASF1B-depleted AML cells exhibited reduced cell viability, inhibited growth, blocked cell cycle progression, and impaired colony formation ability. We xenografted shASF1B expressing AML cells into NSG mice and induced knockdown in vivo. Flow cytometry analysis of bone marrow cells 3 weeks post-engraftment showed 80 percent reduction in leukemia burden following ASF1B silencing compared to controls.To determine whether upregulation of ASF1B contributes to IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that daily IL-1β exposure accelerated leukemia progression compared to vehicle-treated group (median survival = 64 vs. 85 days, p<0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, p<0.05). Conversely, heterozygous and complete Asf1b deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice. Furthermore, Asf1b deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, p <0.01). Immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Together, these data suggested that targeting of the ASF1B pathway may spare healthy cells. Additionally, we found that TLK2 which regulates ASF1B activation, is also upregulated in AML progenitors compared to healthy cells at the baseline levels and upon IL-1β stimulation. We next knocked down TLKs in human AML cells and observed a similar pattern with growth arrest, higher replication stress and DNA damage response. Finally, we generated Vav-Cre+ Tlk2 mice allowing Tlk2 deletion only in hematopoietic cells and found that Tlk2 deletion prolongs survival of leukemic mice in a dose dependent manner with and without IL-1β stimulation.Conclusions: We demonstrate that increased TLK-ASF1B expression promotes survival of AML cells. We also provide the first in vitro and in vivo evidence that the TLK-ASF1B pathway plays a critical role in potentiating IL-1β-dependent AML growth. Therefore, we establish TLK-ASF1B pathway as a novel route for therapeutic strategy to suppress inflammation-driven growth in various AML genetic subtypes. DisclosuresNo relevant conflicts of interest to declare.

Magnetism-Induced Cell-Targeting Transplantation Improves Early Hematopoietic Reconstitution in a Murine Bone Marrow Transplantation Model

BackgroundEarly hematopoietic reconstitution is essential for improving survival and reducing complications after hematopoietic stem/progenitor cell (HSC/HPC) transplantation (HSCT/HPCT). Increasing HSC/HPC homing to the bone marrow is a potential approach for promoting hematopoietic reconstitution.MethodsWe proposed the transplantation of HSCs/HPCs with a magnetism-induced cell-targeting transplantation (MagIC-TT) strategy. HSCs/HPCs were magnetized with CD45 microbeads. The biological characteristics (morphology, proliferation, viability, and ferroptosis) and target migration ability of these cells were studied in vitro. The hematopoietic reconstitution experiments were constructed in vivo in autologous and allogeneic bone marrow transplantation models with grouping showed as Table 1. The therapeutic effects were assessed by survival, donor chimerism, routine blood examination and histological analysis. We also performed transcriptomic sequencing for further mechanistic studies.ResultsThe biological characteristics was found no significant difference between the MagIC-TT and non-MagIC-TT groups, while migration ability was greatly improved with MagIC-TT (Data not showed). The survival rate was higher in the MagIC-TT group and significantly different in the allogeneic model (P<0.05). Hematopoietic reconstitution of donor chimerism, WBCs, HGB, and PLTs was faster in the MagIC-TT groups (within 22 days) (Figure 1). Confocal observation showed that more donor cells (eGFP +) were retained in the injected femur of the MagIC-TT group than in the femur of the non-MagIC-TT group 7 days after transplantation (P<0.05) (Figure 2). The severity of aGVHD (Assessed by survival, body weight, back arching, fur losting, diarrhea etc.) was reduced in the MagIC-TT groups in the allogeneic model (P<0.05) (Data not showed). Transcriptome sequencing revealed differentially expressed genes (DEGs) involved in localization/locomotion and pathways, cytokine-cytokine receptor interactions and chemokine signaling pathways between the two groups (Figure 3).ConclusionThe MagIC-TT strategy improves HSC/HPC homing, resulting in faster hematopoietic reconstitution in a murine bone marrow transplantation model.Key wordsMagnetism; Hematopoietic Stem Cell Transplantation (HSCT); Cell Homing; Hematopoietic Reconstitution; Ferroptosis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Synergism between IGF2BP1 and ETV6-RUNX1 in the Pathogenesis of ETV6-RUNX1 Positive B-Acute Lymphoblastic Leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common pediatric haematological malignancy with a prevalence of around 85% cases of B cell origin. ETV6-RUNX1 is the most common genetic aberration in childhood B-ALL with a prevalence of around 25% in Western countries and a lower prevalence in Asian countries. ETV6-RUNX1 presence portends a good prognosis in B-ALL. ETV6-RUNX1 translocation has been observed in 1-5% of healthy newborns, indicating that it is not sufficient for leukemogenesis, and the need to accumulate secondary mutations for leukemogenesis.We have previously demonstrated the specific overexpression of Insulin like Growth Factor 2 Binding Protein 1 (IGF2BP1), an oncofetal RNA binding protein, in ETV6-RUNX1 positive B-ALL. Overexpression of the ETV6-RUNX1 fusion protein in 7OZ/3, a murine B-ALL cell line increased endogenous Igf2bp1 levels in a dose dependent manner.Conversely, knockout of IGF2BP1 in Reh-Cas9-GFP expressing B-ALL cell line, a modified version of Reh (cell line positive for ETV6-RUNX1 translocation), using 2 different guide RNAs led to a decrease in the ETV6-RUNX1 transcript expression in the IGF2BP1 KO Reh-Cas9 cells. This was accompanied by a decrease in cell proliferation and increased chemosensitivity to prednisolone.A similar phenotype was observed in Reh-Cas9 cells after knockout of ETV6-RUNX1 fusion transcript hinting towards a synergism between the two proteins in tumour cell survival and chemosensitivity. Interestingly, there appears to be a positive correlation between prednisolone resistance and ETV6-RUNX1 positivity in our patient cohort too, thus supporting the in vitro finding. Previous studies have shown the binding of IGF2BP1 to the ETV6-RUNX1 fusion transcript which was also confirmed by us.To study the in vivo synergistic effect between ETV6-RUNX1 fusion protein and IGF2BP1, we used a murine bone marrow transplant model. The individual genes were overexpressed alone (IGF2BP1/ETV6-RUNX1) or together (IGF2BP1+ETV6-RUNX1) in separate groups of lethally irradiated mice (n=8 per group). In the group where both the genes were overexpressed together, there was clonal expansion and hypercellularity in the bone marrow. This was accompanied by an increase in the number of lineage negative cells (Lin-), progenitors including HSCs, LMPPs and c-kit+ cells which also showed high Ki67 positivity. The marrow hypercellularity led to extramedullary haematopoiesis which was seen as significant splenomegaly.In addition, analysis of the endogenous levels of Igf2bp1 in the bone marrow of the primary murine samples belonging to the ETV6-RUNX1 fusion protein overexpression group resulted in an increase in its levels, thus confirming our in vitro finding suggesting its induction by ETV6-RUNX1 fusion protein.RNA-Seq of IGF2BP1 KO cells identified enrichment of targets of the TNF alpha/NFκB signalling and K-Ras pathways hinting towards their dependency on IGF2BP1. The direct targets of IGF2BP1 were identified by RIP-Seq (RNA-Immunoprecipitation-Seq) analysis in Reh cell line. Numerous genes from the oncogenic signalling pathways including the TNF alpha/ NFκB signalling pathway were identified as IGF2BP1 targets. We validated some overexpressed genes from the TNF alpha/NFκB signalling and PI3K pathway in primary patient samples. We found significant overexpression of some of the genes (IL6ST, NFAT5, CDK6, MDM2, CCND1, NGFR) belonging to these pathways in the ETV6-RUNX1 positive group (n=39) compared to the negative cohort (n=111) as well as MACS sorted CD19+ B-cells (n=5). The decrease in the ETV6-RUNX1 transcript levels after downregulation of IGF2BP1 and overlap between the oncogenic pathways clearly shows an interdependency between the two genes.Overall, our results suggest a potential feedback mechanism between ETV6-RUNX1 fusion protein and IGF2BP1 in the pathogenesis of leukemia development and IGF2BP1 can be utilised as an ideal therapeutic target for this particular subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 531: Disruption of a histone chaperone pathway delays inflammation-driven AML progression

Abstract Background: Inflammation plays a crucial role in normal and malignant hematopoiesis. Patients with acute myeloid leukemia (AML) exhibit aberrant interleukin-1β (IL-1β) signaling which results in AML progression. Previously, we have shown that IL-1β promotes the expansion of leukemic progenitors while suppresses the growth of healthy cells. To understand this paradoxical effect, we applied transcriptome analysis of IL-1β-stimulated AML versus healthy progenitors and revealed differentially expressed genes involved in inflammation, cell cycle, and chromosome organization. We identified that ASF1B (anti-silencing function1B) is one of the highly expressed genes between AML and healthy progenitors upon IL-1β stimulation. ASF1B is a histone chaperone and delivers H3-H4 histone dimers onto DNA during replication and DNA damage response. ASF1B is overexpressed in various solid tumors and associated with poor prognosis. However, the functional roles of ASF1B in hematopoiesis and inflammation-driven leukemia have not been established. Methods and Results: We observed higher ASF1B expression in AML progenitors from various AML genetic subtypes (e.g. FLT3-ITD, NPM1, and MLL-ENL) compared to healthy cells at baseline levels and upon IL-1β stimulation. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, suggesting that ASF1B is downstream of IL-1β/p38 signaling. To determine whether ASF1B mediates IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that IL-1β treatment accelerated AML progression compared to the vehicle-treated group (median survival = 64 vs. 85 days, P&amp;lt;0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, P&amp;lt;0.05). Conversely, both heterozygous and complete ASF1B deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice (median survival = 67 and 68 days vs. 62 days, P&amp;lt;0.01). Additionally, ASF1B deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, P&amp;lt;0.01). Bone marrow and spleen cells harvested from the moribund mice exhibited myeloid phenotypes, indicating that observed survival differences are due to the impact of ASF1B deficiency on leukemic cell growth. Interestingly, immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Conclusion: We demonstrate that the ASF1B pathway is activated in response to microenvironmental cues such as IL-1β in primary AML cells. In vivo evidence using murine AML and genetic models suggests that ASF1B contributes to IL-1β-driven AML progression. Therefore, we provide rationales for using ASF1B as a surrogate marker for AML progression and a safer therapeutic target to inhibit inflammation-driven growth in various AML genetic subtypes. Citation Format: Hsin-Yun Lin, Mona Mohammadhosseini, Marina Villamor, John McClatchy, Sophia Jeng, Andrew Adey, Shannon McWeeney, Travis Stracker, Anupriya Agarwal. Disruption of a histone chaperone pathway delays inflammation-driven AML progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 531.

Microrna-31 Promotes T-Cell Pathogenicity in Chronic Graft-Versus-Host Disease

MicroRNAs (miRs) are short, non-coding RNAs that repress gene expression at post-transcriptional level. Certain miRs play critical roles on T-cell responses in inflammation and autoimmunity. To understand the biology of miRs in T-cell allo-responses, we measured expression profiles of miRs in donor T cells in allogeneic vs. syngeneic recipients following bone marrow transplantation (BMT). We found miR-31 was dramatically up-regulated in T cells under alloantigen-driven vs. homeostatic proliferation in vivo(Fig. A). MiR-31 was shown to regulate autoimmunity and cancer. However, its roles in regulating T-cell response remain largely undetermined. Recent study shows miR-31 represses peripheral Treg differentiation in EAE development. In contrast, miR-31 inhibits CD8 T-cell function in anti-virus response. We hypothesize that miR-31 is required for CD4 T-cell pathogenicity in chronic graft-versus-host disease (cGVHD), but is dispensable for CD8 T-cell cytolytic function in graft-versus-leukemia (GVL) activity.To study how miR-31 regulates T-cell allo-response, we used mice with miR-31 conditional knock out (KO) in T cells (miR-31fl/fl CD4Cre). No differences were observed in the baseline composition of naïve WT (miR-31fl/fl) and miR-31 KO splenocytes. After being transferred into allogeneic recipients, KO T cells had impaired ability to proliferate, reflected by fewer divided (% CFSElow) cells and reduced donor T-cell expansion in recipient spleens (Fig. B). To further evaluate how miR-31 contributes to T-cell pathogenicity in cGVHD, we used a murine model of BMT (B6 → BALB/c), in which low dose spleonocyes can induce scleroderma manifestation. Significant reduction of clinical scores (Fig. C), including improved skin scores and body weight loss, were found in the recipients of miR-31 KO grafts compared to those with WT grafts. This finding was validated in another haploidentical BMT model of scleroderma (B6 → B6D2F1), indicating miR-31 is required for T-cell to induce cGVHD. Consistently, the pathological damage was dramatically reduced in skin, gut and lung in the recipients of miR-31 KO grafts (Fig. D).Mechanistically, miR-31 deficiency in donor T cells resulted in significantly reduced numbers of CD4 T cells that produce IFNγ, IL-4/5 or IL-17 in recipient spleen and mesenteric lymph nodes. In absence of miR-31, fewer effector and central memory cells were generated from donor CD4 T cells. Further, T cells that are capable of localizing to B-cell follicles (CXCR5+PD-1hi) were significantlyreduced in spleen (Fig. E) and peripheral lymph nodes in recipients of miR-31 KO grafts. Interestingly, among these cells, more foxp3+ follicular T-regulatory (Tfr) but fewer foxp3- follicular T-helper (Tfh) cells were observed in the recipients of KO grafts (Fig. F), indicating miR-31 contributes to the unbalance of Tfr and Tfh cells during cGVHD development. Consistently with the role of miR-31 in regulating Tfr and Tfh cells, we found Blimp1 is the predicted targets of miR-31, which is critically involved in the homeostasis of Tfr and Tfh cells. On donor B-cell compartment, lower levels of CD86 and MHC-II expression were observed in the recipients of miR-31 KO grafts. In addition, germinal center B cells and plasma cells were also reduced in those recipients, indicating miR-31 indirectly regulates B-cell activation and differentiation through controlling T-cell pathogenicity.For translational purpose, we evaluated how miR-31 impacts T cell-mediated GVL effect. Using B6 → BDF1 BMT model, we consistently found attenuated skin manifestation in recipients of KO T cells. Importantly, in the ongoing experiment, miR-31 KO T cells were capable of controlling P815(mastocytoma) growth as efficiently as WT T cells (Fig. G), suggesting miR-31 may be dispensable for T cells to mediating GVL activity after allogeneic BMT.Taken together, the current work reveals that miR-31 plays pleotropic roles in enhancing CD4 T-cell pathogenicity in cGVHD. MiR-31 represents a promising therapeutic target for the control of cGVHD while sparing GVL effects after allogeneic hematopoietic stem cell transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Early Administration of Low-Dose IL-2 Intensify Graft-Versus-Leukemia Effect without Worsening GVHD through Sequential Enhancement of Effector T Cell and CD62L+ Regulatory T Cell Subset

Allogeneic hematopoietic stem cell transplantation (HSCT) is the curative treatment of many hematological malignancies, but the relapse is one of the major causes of treatment failure and mortality. Previous clinical studies have shown that low-dose IL-2 was well tolerated and could lower the relapse rate in human T cell depleted allogeneic BMT. On the other hand, we also demonstrated that low-dose IL-2 could preferentially enhance regulatory T cell (Treg) and improve the symptoms of chronic GVHD (NEJM 2011,Sci Trans Med 2013). In murine bone marrow transplantation (BMT) model showed that Treg can protect from lethal GVHD while preserving graft-versus-leukemia (GVL) effect. Especially, CD62L+ Tregs has a higher capacity in suppressing GVHD than their CD62L- counterpart. Our recent study reported that exogenous low-dose IL-2 induced the expression of PD-1 on Treg preferentially and it was most evident in CD44+CD62L+central-memory Treg (Blood 2016). However, the impact of CD62L+ Treg enhanced by low-dose IL-2 on GVHD and GVL has not been well investigated. In this study, we studied the effect of exogenous IL-2 on the lymphocyte phenotypic profiles including naïve- versus memory-type balance and also examined donor-derived immunity, especially GVHD and GVL, by using leukemia-bearing BMT model. Lethally irradiated B6D2F1 recipients were transplanted spleen cells and BM cells from B6 donors and 5000 IU Teceleukin (recombinant IL-2) was injected once a day for 14 days. The number of CD4+CD25+Foxp3+ Treg, CD4+CD25-Foxp3- conventional T cell (Tcon) and CD8+ T cell was compared between IL2- and vehicle-treated groups. The expressions of CD44, CD62L, CCR4, CCR7, Ki-67, PD-1, CTLA-4, LAG3, GITR, ICOS and TIM-3 in each subset were also examined. Lymphocytes profiling analysis showed that IL-2 treatment in the first 7 days resulted in the dominant expansion of effector T cells without Treg increase (Treg 0.10 vs 0.16,p=0.35, Tcon 2.83 vs 4.20, p=0.01, CD8 T cell 5.52 vs 10.4, p=0.01, million). On the contrary, the extended IL-2 treatment in the next 7 days increased CD62L+ Treg (21.5 vs 48.2 % of total Treg, p<0.001, 0.02 vs 0.07 million, p=0.006) without increasing effector T cells. PD-1 on Treg was very high in BMT-recipients even without receiving IL-2, so further enhancement of PD-1 expression by IL-2 was not observed. However, the expression of other inhibitory molecules including CTLA-4 (MFI, 6.61 vs 9.92, p=<0.001), LAG-3 (1.73 vs 2.17, p=0.04) and ICOS (2.41 vs 3.84, P<0.001) was enhanced by the IL-2 treatment. To elucidate the mechanisms of the increase in CD62L+ Treg, the in vivo violet dye dilution assay was performed. We first sorted out CD62L+ T cells by micro-beads from spleen cells, and then adaptively transferred into recipients. Five days treatment of low-dose IL-2 resulted in the enhancement of the division of CD62L+ Treg (% divided cells; 19.5 vs 29.8 % of Treg, p=0.005, 62.3 vs 108 in number, p=0.002). Interestingly, when CD62L- T cells were transferred, IL-2 treatment also induced the division of the cells and, of note, induced the phenotypic change of CD62L- into CD62L+ Treg (7.63 vs 19.8 % of Treg, p=0.009, 91.3 vs 361, p=0.01). These results suggested that IL-2 have the possibility to enhance the proliferative activity of Treg with the up-regulation of CD62L expression. Lastly, we transplanted lucipherase+P815 leukemia cells into this BMT model and evaluated the clinical impact of low-dose IL-2 on GVL by using in vivo bioluminescence system. To assesse the GVL and GVHD independently, we used two different tumor burden models. In the high tumor burden model, IL-2 injection decreased bioluminescence intensity (BLI) of P815 (BLI at week3, 8.3x10E7 vs 8.1x10E6 photon/second, p<0.001) and significantly extends overall survival. On the other hand, in the low tumor burden model, both treatment groups did not die from leukemia but vehicle-treated control group showed a significant higher clinical GVHD score and died from GVHD. These data demonstrated the clinical benefits on anti-tumor effect of IL-2, which could enhance the GVL activity without the detrimental effect on GVHD. In conclusion, our data indicate multiple effects of IL-2 on post-transplant immunity can coordinate to intensify GVL separately from GVHD on the mechanism of sequential enhancement of effector T cell and CD62L+ Treg in the different phases. These data provide the important information for developing Treg-targeted therapy. DisclosuresMaeda:Toko Pharmaceutical Industries: Other: Investigational drug is provided free of charge.

Pretransplant Short-Term Exposure of Donor Graft Cells to ITK Selective Inhibitor Ameliorates Acute Graft-versus-Host Disease by Inhibiting Effector T Cell Differentiation while Sparing Regulatory T Cells.

Graft-versus-host disease (GVHD) remains to be a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). IL-2-inducible T cell kinase (ITK), a TEC cytoplasmic tyrosine kinase, has an essential role in T cell development and receptor signaling. The ITK/Bruton tyrosine kinase inhibitor ibrutinib has been shown to improve chronic GVHD symptoms; however, the effect of ITK selective inhibition on acute GVHD remains unclear. In this study, we evaluated the pharmacological effects of an ITK selective inhibitor (ITKsi) on acute GVHD using murine bone marrow transplantation models. First, we found that CD4+ T cell differentiation toward Th1, Th2, or Th17 was inhibited following ITKsi treatment in a dose-dependent manner while maintaining regulatory T cells in the presence of alloantigens both invitro and invivo. ITKsi preferentially inhibited inflammatory cytokine production and invivo proliferation of alloreactive T cells. We then demonstrated that short-term exposure of donor graft cells to ITKsi significantly delayed the onset of GVHD-associated mortality without compromising the donor cell engraftment and the graft-versus-tumor effect, indicating the potential of ITK selective inhibition in the setting of clinical allogeneic HSCT. These findings suggest that ITK is a potential therapeutic target against GVHD, and the pharmacological ITK inhibitor may serve as a novel strategy for immune regulation after HSCT.

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions.

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

Flt3 ligand expressed on the surface of T-cells contributes to effector function

Abstract Flt3 ligand (FL) is a growth factor that contributes to the homeostasis of the dendritic cell (DC) compartment. FL provides signals for differentiation, development, and survival by binding to the Flt3 receptor tyrosine kinase. Flt3 expression is maintained on DCs through their terminal differentiation. Endogenously, FL is produced by stromal cells and select immune cells, including T-cells and some DC subsets. FL may be expressed as a membrane-bound form (mFL), with no intracellular signaling domain, and may undergo cell-autonomous shedding to generate soluble FL. This is believed to contribute to a localized source of FL for those DC subsets, however T-cells do not express Flt3 and have no FL requirement. As such, it remains unclear why T-cells express mFL on their surface. Interestingly, FL-driven DCs are more steady state-like, secreting less pro-inflammatory cytokines, and can suppress graft-versus-host disease (GvHD). We hypothesized that mFL on T-cells may regulate effector responses through binding to Flt3 on DCs. To test this, we compared GvHD in a murine bone marrow transplant model using WT compared to FL−/− donor cells. Engraftment, T-cell reconstitution, and activation were monitored weekly. We also performed mixed leukocyte reactions (MLR) to measure T-cell proliferation in WT versus FL−/− T-cells. Infusion of FL−/− donor cells resulted in significantly worse GvHD and higher expression of T-cell activation markers CD25 and CD69 in vivo. The absence of mFL also resulted in significantly enhanced alloreactive T-cell proliferation in vitro. These studies indicate that mFL expressed on the surface of T-cells can alter their effector function, which may be a result of the engagement of mFL with Flt3 receptor expressed on DCs.

Impact of bone marrow ATP-binding cassette transporter A1 deficiency on atherogenesis is independent of the presence of the low-density lipoprotein receptor

Background and aimsThere is extensive evidence from bone marrow transplantation studies that hematopoietic ATP binding cassette A1 (Abca1) is atheroprotective in low-density lipoprotein receptor (Ldlr) deficient mice. In contrast, studies using lysosyme M promoter-driven deletion of Abca1 in Ldlr deficient mice failed to show similar effects. It was hypothesized that the discrepancy between these studies might be due to the presence of Ldlr in bone marrow-derived cells in the transplantation model. In this study, we aim to determine the contribution of Ldlr to the atheroprotective effect of hematopoietic Abca1 in the murine bone marrow transplantation model. MethodsWild-type, Ldlr−/−, Abca1−/−, and Abca1−/−Ldlr−/− bone marrow was transplanted into hypercholesterolemic Ldlr−/− mice. ResultsBone marrow Lldr deficiency did not influence the effects of Abca1 on macrophage cholesterol efflux, foam cell formation, monocytosis or plasma cholesterol. Ldlr deficiency did reduce circulating and peritoneal lymphocyte counts, albeit only in animals lacking Abca1 in bone marrow-derived cells. Importantly, the effects of Abca1 deficiency on atherosclerosis susceptibility were unaltered by the presence or absence of Ldlr. Bone marrow Ldlr deficiency did lead to marginally but consistently decreased atherosclerosis, regardless of Abca1 deficiency. Thus, Ldlr expression on bone marrow-derived cells does, to a minimal extent, influence atherosclerotic lesion development, albeit independent of Abca1. ConclusionsThis study provides novel insight into the relative impact of Ldlr and Abca1 in bone marrow-derived cells on macrophage foam cell formation and atherosclerosis development in vivo. We have shown that Ldlr and Abca1 differentially and independently influence atherosclerosis development in a murine bone marrow transplantation model of atherosclerosis.