Kurzfassung: Background: RBC transfusion can induce alloimmune responses that lead to hemolysis. However, only a subset of transfused patients become alloimmunized, and there is heightened interest in identifying factors that promote or inhibit alloimmunization. The frequency of alloimmunization has recently been shown to be increased in patients with inflammatory disease. In mouse models, inflammation induced by the viral RNA mimetic, polyinosinic:polycytidylic acid (poly(I:C)), profoundly enhances alloimmune antibody responses. Poly(I:C) induces production of inflammatory cytokines, including Type 1 interferon (IFNαβ) and monocyte chemoattractant protein 1 (MCP-1), which recruits CCR2 expressing monocyte-derived dendritic cells (MoDCs) from bone marrow. We hypothesize that IFNαβ promotes inflammation-induced RBC alloimmunization by regulating MoDC-mediated consumption of transfused allogeneic RBCs. Methods: To investigate the role of inflammation in alloimmunization to a human RBC antigen, we utilized a novel mouse model that expresses high levels of the human KEL glycoprotein specifically on RBCs (KELhi). Poly(I:C) was administered to wildtype (WT), CCR2-/-, and IFNAR1-/- mice, which lack the IFNαβ receptor, at varying time points relative to transfusion of leukoreduced KELhi RBCs. The alloimmune response was assessed by measuring KEL-specific IgG via flow cytometric crossmatch. For dendritic cell activation and RBC consumption experiments, RBCs labeled with fluorescent DiO were transfused following poly(I:C) injection. DiO-containing phagocytes, including Mo-DCs (CD11c+ CD11bhi Ly6C+ F4/80+ MHCIIlo), and activation markers were detected by flow cytometry 6hrs after transfusion. Results: WT Mice treated with poly(I:C) 3 hrs prior to RBC transfusion generated anti-KEL glycoprotein alloantibodies. However, alloantibodies were not detectable when poly(I:C) was withheld or administered at days -7, -1, +1, +4, or +7 in relation to RBC transfusion, indicating that inflammation may promote antigen consumption. Poly(I:C) treatment 3 hrs prior to transfusion of DiO-labeled KELhi RBCs led to elevated activation marker expression and marked erythrophagocytosis by MoDCs in the spleen and peripheral blood. MoDC recruitment and RBC consumption were respectively mediated by CCR2 and IFNAR1-dependent mechanisms. Additionally, compared to WT controls, alloimmunization in the presence of poly(I:C) was significantly reduced in CCR2-/- mice and completely abrogated in IFNAR1-/- mice (Figure 1). Conclusions: Although inflammatory stimuli, including poly(I:C), have been shown to enhance alloimmunization, mechanisms underlying these results have been poorly understood. Here, we demonstrate an important role for MoDC erythrophagocytosis and a critical role for IFNαβ signalingin inflammation associated alloimmunization to a T-dependent human antigen expressed on murine RBCs. Although it is unclear whether these findings will apply to other RBC antigens in mice and humans, MCP-1 and IFNαβ are produced in inflammatory diseases associated with alloimmunization. Thus, identifying risk factors for inflammatory cytokine production in transfusion recipients may allow for personalized transfusion protocols for at risk patients. Figure 1 Monocyte-derived dendritic cells and IFNαβ signaling promote alloimmunization to KELhi RBCs. a) WT mice were treated with or without 100µg poly(I:C) 3 hours prior to transfusion with 75µL leukoreduced DiO+ KELhi RBCs. Plots show CD11bhi MoDCs in peripheral blood (Ter119-TCRb-CD19-CD11c+Ly6C+F480+MHClo). Numbers on plots indicated percent of gated cells. b) Cumulative data of a. c) Anti-KEL specific IgG in serum of WT mice following transfusion of KELhi RBCs in the presence or absence of poly(I:C). d) Anti-KEL IgG in serum of indicated mice pretreated with poly(I:C) and transfused with KELhi RBCs. Anti-KEL IgG antibodies in serum were measured by flow cytometric cross-match, completed by incubating serum from transfused mice with KELhi RBCs, and subsequently staining for RBC bound IgG. The adjusted MFI was calculated by subtracting the reactivity of serum with syngeneic WT RBCs from the reactivity of serum with KELhi RBCs. Data show the peak antibody response. Open circles indicate data from individual mice. Figure 1. Monocyte-derived dendritic cells and IFNαβ signaling promote alloimmunization to KELhi RBCs. a) WT mice were treated with or without 100µg poly(I:C) 3 hours prior to transfusion with 75µL leukoreduced DiO+ KELhi RBCs. Plots show CD11bhi MoDCs in peripheral blood (Ter119-TCRb-CD19-CD11c+Ly6C+F480+MHClo). Numbers on plots indicated percent of gated cells. b) Cumulative data of a. c) Anti-KEL specific IgG in serum of WT mice following transfusion of KELhi RBCs in the presence or absence of poly(I:C). d) Anti-KEL IgG in serum of indicated mice pretreated with poly(I:C) and transfused with KELhi RBCs. Anti-KEL IgG antibodies in serum were measured by flow cytometric cross-match, completed by incubating serum from transfused mice with KELhi RBCs, and subsequently staining for RBC bound IgG. The adjusted MFI was calculated by subtracting the reactivity of serum with syngeneic WT RBCs from the reactivity of serum with KELhi RBCs. Data show the peak antibody response. Open circles indicate data from individual mice. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: Abstract 250 The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme. Mediator acts as the end-point integrator of a variety of activators and intracellular signaling, and conveys these signals to the general transcription machinery. Among circa 25 subunits, MED1/TRAP220 subunit is crucial for many biological events through the interaction with distinct activators such as nuclear receptors (NRs) and GATA family activators. In hematopoiesis, MED1 plays an important role for optimal NR-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we analyzed the role of MED1 in the niche. We used mouse embryonic fibroblasts (MEFs) as an in vitro niche model, since MEFs have a nuance of the osteoblastic precursor and support long-term culture-initiating cells (LTC-ICs). When syngenic normal bone marrow (BM) cells were cocultured with mitomycin C-treated Med1−/− or Med1+/+ MEFs (in p53−/− background), the number of live cells (cell counts, DNA contents and MTT assays) and DNA synthesis (BrdU incorporation) was significantly suppressed on Med1−/− MEFs compared with the control during the two-week period. However, they recovered to the control level when cocultured on Med1−/− MEFs into which MED1 was stably introduced (Rev-Med1−/− MEFs). Furthermore, when LTC-ICs were assayed in complete methylcellulose media (Methocult M3434, StemCell Technologies) after the 8-week coculture, the number of LTC-ICs was attenuated for BM cells cocultured on Med1−/− MEFs compared to the control, but recovered to the control level when cocultured on Rev-Med1−/− MEFs. In order to identify the direct target(s) of MED1 that was responsible for these phenotypes, a microarray analysis of mRNA comparing Med1+/+ and Med1−/− MEFs was performed, and disclosed approximately 15 genes whose expressions were profoundly attenuated in Med1−/− MEFs. Among molecules encoded by these genes we focused on osteopontin (OPN) because solely OPN was known to have a role in niche to support hematopoietic stem/precursor cells (HSPCs). Angiopoietin-1 and Jagged-1 expressions were comparable. The expression of Opn mRNA was distinctly downregulated in Med1−/− MEFs but recovered to the control level in Rev-Med1−/− MEFs. The Opn expression in Med1+/+ MEFs was also similar to those in MC3T3-E1 mouse osteoblastic, and OP-9 BM stromal, cells. Western blot analysis of Med1+/+ MEFs using the polyclonal antibody that recognized the N-terminus of mouse OPN disclosed the abundant expression of the full-length form of OPN (ca. 70kDa), which was much less in Med1−/− MEFs. In contrast, the N-terminal cleaved form of OPN was barely visible in both MEFs. Next a chromatin immunoprecipitation (ChIP) assay was performed to know if Mediator complex was actually recruited to the Opn promoter. As expected, when the sheared chromatin was immunopurified with the antibody against MED10 subunit, Mediator was proved to be recruited to the Opn promoter more abundantly in Med1+/+ than Med1−/− MEFs, and the recruitment in Rev−Med1−/− MEFs recovered to the level of the former. Transient transfection and luciferase reporter assays disclosed that the basal level transcription as well as vitamin D receptor (VDR)- and Runx2-mediated transcriptional activation of Opn was specifically attenuated in Med1−/− MEFs, and that the basal transcription and ligand-dependent activation were dependent on the N-terminal domain (amino acids 1 to 602) and the two NR-recognition motifs of MED1, respectively. If OPN was the direct target of MED1, OPN might be responsible for, and the addition or depletion of OPN might alter, the BM cell growth and LTC-ICs support. Indeed, the addition of recombinant full-length OPN to Med1−/− MEFs restored, and the addition of the anti-OPN (N-terminus) polyclonal antibody to Med1+/+ MEFs attenuated, both the growth of cocultured BM cells during the two-week coculture and the numbers of LTC-ICs after the long-term coculture. Finally, to exclude the possibility that these phenotypes might have been restricted to MEFs, OP-9 cells, which had widely-accepted niche function, were used for similar experiments. Indeed, the addition of the anti-OPN antibody to OP-9 cells attenuated both the growth of cocultured BM cells and the numbers of LTC-ICs. Taken together, these data suggest that MED1 in niche cells, through upregulating VDR- and Runx2-mediated transcription on the Opn promoter, plays an important role in HSPCs support. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Introduction The prognosis of classic Hodgkin lymphoma (cHL) in young adults has improved following advances in current therapeutics. However, evidence of elderly patients with cHL has limited due to its rarer. To analyze clinical outcomes and risk factors in elderly patients with advanced-stage cHL who received a doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) regimen, we conducted a nationwide multi-center retrospective study in Japan (UMIN000033264). Methods The key eligibility criteria of the current study were as follows: 1) patients with histologically diagnosed cHL between 2007 and 2016 in each institution; 2) advanced-stage cHL (stage III, IV or IIB with bulky or extranodal lesion); 3) age at diagnosis & gt; 60 years; and 4) had received at least one cycle of an ABVD regimen as initial treatment including a modified ABVD regimen (dacarbazine dose reduced to 250 mg/m 2 at first cycle). Patients with human immunodeficiency virus infection or methotrexate-associated cHL were excluded. The primary endpoint was 5-year overall survival (OS). Secondary endpoints included progression-free survival (PFS) and event-free survival (EFS), the latter defined as the time from a diagnosis of cHL to disease progression or relapse, subsequent systemic chemotherapy for cHL, or death due to any cause in this study. The average relative dose intensity (ARDI) of ABVD was calculated as the sum of the relative dose intensity of each drug divided by four. Results A total of 171 patients from 45 institutions were included in this study. The central pathological review could be performed in 140 cases (82%), in which histological subtypes of cHL were determined as follows: mixed cellularity, 89 (64%); nodular sclerosis, 33 (24%); lymphocyte-deleted, 4 (3%); lymphocyte-rich, 2 (1%); not otherwise specified, 12 (9%). The median age was 71 years (interquartile range [IQR] 65-76). The number of patients in each 10-year age group was 79 (46%) for 61-70 years, 78 (46%) for 71-80 years, and 14 (8%) for & gt; 81 years. The numbers of patients with ECOG performance status (PS) ≥2 and B symptoms were 33 (19%) and 85 (50%), respectively. Bulky mediastinal diseases were found in four patients (2%). The risk factors used for the International Prognostic Score (IPS), including male sex, stage IV, hypoalbuminemia, anemia, leukocytosis, and lymphopenia, were not significantly different among these age groups. The median number of cycles of ABVD was 6 (IQR:5-7), and 66% of patients completed at least 6 cycles. The median ARDI was 77%. The median ARDI in each 10-year age group was 88% for 61-70 years, 71% for 71-80 years, and 54% for & gt; 81 years. With a median follow-up time of 52 months (IQR:31-76), the estimated OS, PFS, and EFS at 5 years were 72%, 59%, and 54%, respectively. Among 131 patients whose Epstein-Barr virus (EBV) status was analyzed using EBV-encoded small RNA1 (EBER1) in situ hybridization, 61 (46%) were positive for EBER1. The OS was not significantly different between EBV-positive and EBV-negative patients (75% and 76% at 5 years, respectively). Univariate analysis for OS revealed that age, PS ≥ 2, hypoalbuminemia, anemia, lymphopenia, and the presence of B symptoms were significantly associated with short OS. In multivariate analysis, age (hazard ratio [HR] 1.568 per 10-year increase, 95% confidence interval [CI] 1.020-2.411) was only an independent risk factor for OS, whereas lymphopenia (HR 1.967, 95% CI 1.196-3.235) was an independent risk factor for EFS. Higher ARDI was significantly associated with improved OS and EFS. Bleomycin-induced lung toxicity (BLT), observed in 41 (24%) patients, was not associated with OS. In this study, we found that 55 patients died: 23 within two years after diagnosis mainly due to cHL progression (n = 10, 43%) and treatment-related toxicity (infection [n = 4, 17%] and BLT [n = 4, 17%] ). The remaining 32 patients died more than two years after diagnosis mainly due to second primary malignancies (n = 14, 44%), including solid tumors (n = 6), other types of lymphoma (n = 4), and myelodysplastic syndrome/acute myeloid leukemias (n = 4). Conclusions The HORIZON study showed relatively good prognosis for elderly patients with advanced-stage cHL who received an ABVD regimen (estimated five-year OS rates was 72%). Age and lymphopenia were an independent risk factors for OS and EFS, respectively. The development of novel therapies is warranted to improve the outcomes of such patients. Disclosures Makita: SymBio: Honoraria; Novartis: Honoraria; Eisai: Honoraria; Daiichi-Sankyo: Consultancy; CSL Behring: Honoraria; Chugai: Honoraria; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Kusumoto: Kyowa Kirin: Honoraria; Chugai: Honoraria, Research Funding; Daiichi Sankyo: Research Funding. Tsujimura: Takeda: Honoraria; Chugai: Honoraria; Kyowa Kirin: Honoraria; Eisai: Honoraria; Janssen: Honoraria. Takayama: Chugai: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding. Kuroda: Otsuka Pharmaceutical: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Abbvie: Consultancy, Honoraria; Ono Pharmaceutical: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Sysmex: Research Funding; Pfizer: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Shionogi: Research Funding; Asahi Kasei: Research Funding; Taiho Pharmaceutical: Research Funding; Fujimoto Pharmaceutical: Current Employment, Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria, Research Funding; Dainippon Sumitomo Pharma: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Bristol-MyersSquibb: Consultancy, Honoraria, Research Funding; Janssen Pharmaceutical K.K: Consultancy. Shimada: BMS: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Janssen: Honoraria; Chugai: Consultancy, Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; AstraZeneca: Honoraria; Otsuka: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; SymBio: Honoraria. Okamoto: Chugai: Research Funding; Kyowa Kirin: Research Funding; Ono: Research Funding; Taiho: Research Funding; Takeda: Research Funding. Asano: Takeda: Honoraria. Maruyama: Janssen: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Novartis: Research Funding; MSD: Honoraria, Research Funding; Otsuka: Research Funding; Ono: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas Pharma,: Research Funding; Celgene: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Amgen: Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Honoraria; Kyowa Kirin: Honoraria; Zenyaku: Honoraria; AstraZeneca: Honoraria; Nippon: Honoraria; SymBio: Honoraria. Yamaguchi: Chugai: Honoraria, Research Funding; Genmab: Research Funding; MSD: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Takeda: Honoraria; Ono: Honoraria; Celgene: Honoraria; Otsuka: Honoraria; Sumitomo Dainippon: Honoraria; Eisai: Honoraria; AstraZeneca: Consultancy. Nagai: AbbVie: Research Funding; AstraZeneca: Honoraria, Research Funding; Bayer: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Chordia Therapeutics: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Mundipharma: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; Novartis: Honoraria; Ono Pharmaceutical: Honoraria; Sanofi: Honoraria; Sumitomo Dainippon Pharma: Honoraria; SymBio Pharmaceuticals: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Zenyaku Kogyo: Research Funding.

Kurzfassung: Hematopoietic stem cell (HSC) self-renewal and lineage output are orchestrated by multiple regulatory layers, including RNA modifications. N6-methyladenosine (m6A) is an abundant modification found in RNAs which affects the translation and stability of modified transcripts. The effects of m6A are determined by m6A writers (install m6A), erasers (remove m6A) and readers (recognize m6A). In embryonic stem cells, deletion of the m6A writer METTL3 enforces a naïve pluripotent state. This raises the question of whether m6A RNA methylation analogously regulates stem cell self-renewal and differentiation in somatic stem cells such as HSCs. Using a Vav-Cre/Mettl3 (VCM3) hematopoietic-specific knockout mouse model, we show that loss of the RNA m6A writer METTL3 in fetal HSCs results in hematopoietic failure and perinatal lethality. At E14.5 (FL) hematopoiesis loss of Mettl3/m6A results in hematopoietic failure with expansion of Lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) that are defective in the production of progenitors and mature blood cells, as evidenced by failure to rescue lethally irradiated congenic recipient mice in transplant experiments. The relative defect I hematopoiesis was further demonstrated by competitive transplant experiments, in which transplanted KO FL were consistently out-competed by WT FL. Interestingly, BrdU/7AAD labeling reveals a significant proliferative defect with reduced BrdU uptake in VCM3 KO FL cells, and specifically in Lin-c-Kit+Sca-1- (LK) progenitor cells. RNA-seq analysis of FL LSK cells reveals that loss of m6A results in upregulation of multiple 2'-5'-oligoadenylate synthetase (OAS) family genes. Interestingly, the majority of OAS family genes are not m6A modified in several m6A sequencing data sets. We therefore hypothesized that the OAS genes might be regulated at the transcriptional level. We performed cleavage under targets and release using nuclease (CUT & RUN) analysis and found that OAS family genes are transcriptionally activated, as evidenced by significant increase in H3K4 trimethylation (H3K4me3) at their respective promoter regions. The OAS family genes are activated by the presence of double stranded RNA (dsRNA), which can arise either endogenously or as a pathogen-associated trigger of the innate immune system in the context of viral infection. The dsRNA response includes three major response mechanisms. First, OAS genes facilitate RNase L dimerization, which mediates cleavage of cellular tRNA and rRNA, resulting in translation and proliferation arrest. Second, activation of the PKR/eIF2a pathway also results in translation arrest. Lastly, activation of the MDA-5/RIG-I/MAVS response induces interferon signaling, which further limits cellular proliferation. Staining of VCM3 WT & KO FL cells with the dsRNA-specific J2 antibody directly demonstrated significant accumulation of dsRNA in KO FL cells. We further demonstrated activation of the OAS/RNaseL axis in KO cells by quantification of tRNA-His-36 cleavage, which acts as a sensitive marker of RNase L activity. Phosphorylation of both PKR and eIF2a in KO cells demonstrated enhanced activity of this pathway. Lastly, we found a significant upregulation of interferon pathway genes in KO cells, and demonstrated that CRISPR mediated knockout of Mavs in VCM3 KO FL cells diminishes the activity of interferon response genes and rescues the Mettl3 KO phenotype in colony forming unit (CFU) assays. These results suggest that in the absence of m6A, endogenous dsRNA formation is enhanced, resulting in an associated inflammatory response which partially accounts for the hematopoietic defect observed in Mettl3 KO mice. In conclusion, our study suggests a novel, protective role for the m6A RNA modification in preventing endogenous dsRNA formation and aberrant activation of a detrimental innate immune response during mammalian hematopoietic development. Disclosures Fan: IsoPlexis: Other: served on the scientific discovery board, Patents & Royalties; Singleron Biotechnologies: Other: served on the Scientific Advisory Board; BioTechne: Other: served on the Scientific Advisory Board. Flavell:Rheos Biomedicines: Equity Ownership; GSK: Consultancy; Artizan Biosciences: Equity Ownership; Troy: Equity Ownership; SMOC: Equity Ownership; Zai labs: Consultancy.

Kurzfassung: Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.