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Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets—Th1, Th2, Th17, and regulatory T—was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS–IKAROS heterodimers did not bind the canonical AIOLOS–IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner. The zinc-finger transcription factor IKAROS is essential for B cell development. Taniuchi, Morio and colleagues identify a human kindred presenting with B cell immunodeficiency that was caused by a heterozygous missense mutation in IKZF3 encoding the related AIOLOS protein. AIOLOSG159R is a mutant protein that interferes with both wild-type AIOLOS and IKAROS by forming heterodimers that bind to aberrant DNA-binding sites and prevent normal expression of IKAROS-dependent genes.

Among the BTB-ZF transcription factor family, three amino acids in the BTB domain make Thpok unique in repressing cytotoxic lineage-related genes via recruitment of the NuRD chormatin-remodeling complex (see the related Research Article by Gao et al. ).

MicroRNAs (miRNAs), one of small non-coding RNAs, regulate many cell functions through their post-transcriptionally downregulation of target genes. Accumulated studies have revealed that miRNAs are involved in hematopoiesis. In the present study, we investigated effects of miR-669m overexpression on hematopoiesis in mouse in vivo, and found that erythroid differentiation was inhibited by the overexpression. Our bioinformatic analyses showed that candidate targets of miR-669m which are involved in the erythropoiesis inhibition are A-kinase anchoring protein 7 (Akap7) and X-linked Kx blood group (Xk) genes. These two genes were predicted as targets of miR-669m by two different in silico methods and were upregulated in late erythroblasts in a public RNA-seq data, which was confirmed with qPCR. Further, miR-669m suppressed luciferase reporters for 3′ untranslated regions of Akap7 and Xk genes, which supports these genes are direct targets of miR-669m. Physiologically, miR-669m was not expressed in the erythroblast. In conclusion, using miR-669m, we found Akap7 and Xk, which may be involved in erythroid differentiation, implying that manipulating these genes could be a therapeutic way for diseases associated with erythropoiesis dysfunction.

In classical Hodgkin lymphoma (cHL)—characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells—tumor-associated macrophages (TAMs) play a pivotal role in tumor formation. However, the significance of direct contact between HRS cells and TAMs has not been elucidated. HRS cells and TAMs are known to express PD-L1, which leads to PD-1+ CD4+ T cell exhaustion in cHL. Here, we found that PD-L1/L2 expression was elevated in monocytes co-cultured with HRS cells within 1 h, but not in monocytes cultured with supernatants of HRS cells. Immunofluorescence analysis of PD-L1/L2 revealed that their upregulation resulted in membrane transfer called “trogocytosis” from HRS cells to monocytes. PD-L1/L2 upregulation was not observed in monocytes co-cultured with PD-L1/L2-deficient HRS cells, validating the hypothesis that there is a direct transfer of PD-L1/L2 from HRS cells to monocytes. In the patients, both ligands (PD-L1/L2) were upregulated in TAMs in contact with HRS cells, but not in TAMs distant from HRS cells, suggesting that trogocytosis occurs in cHL patients. Taken together, trogocytosis may be one of the mechanisms that induces rapid upregulation of PD-L1/L2 in monocytes to evade antitumor immunity through the suppression of T cells as mediated by MHC antigen presentation.

Genes encoding B lineage–restricted transcription factors are frequently mutated in B-lymphoid leukemias, suggesting a close link between normal and malignant B-cell development. One of these transcription factors is early B-cell factor 1 (EBF1), a protein of critical importance for lineage specification and survival of B-lymphoid progenitors. Here, we report that impaired EBF1 function in mouse B-cell progenitors results in reduced expression of Myc. Ectopic expression of MYC partially rescued B-cell expansion in the absence of EBF1 both in vivo and in vitro. Using chromosome conformation analysis in combination with ATAC-sequencing, chromatin immunoprecipitation–sequencing, and reporter gene assays, six EBF1-responsive enhancer elements were identified within the Myc locus. CRISPR-Cas9–mediated targeting of EBF1-binding sites identified one element of key importance for Myc expression and pro-B cell expansion. These data provide evidence that Myc is a direct target of EBF1. Furthermore, chromatin immunoprecipitation–sequencing analysis revealed that several regulatory elements in the Myc locus are targets of PAX5. However, ectopic expression of PAX5 in EBF1-deficient cells inhibits the cell cycle and reduces Myc expression, suggesting that EBF1 and PAX5 act in an opposing manner to regulate Myc levels. This hypothesis is further substantiated by the finding that Pax5 inactivation reduces requirements for EBF1 in pro–B-cell expansion. The binding of EBF1 and PAX5 to regulatory elements in the human MYC gene in a B-cell acute lymphoblastic leukemia cell line indicates that the EBF1:PAX5:MYC regulatory loop is conserved and may control both normal and malignant B-cell development.

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter–enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.

A high-precision hypernuclear experiment has been commissioned at the Mainz Microtron (MAMI) to determine the hypertriton Λ binding energy via decay-pion spectroscopy. The method has been successfully pioneered with 4ΛH studies in the last decade. The experiment makes use of a novel high luminosity lithium target with a length of 45mm while being only 0.75mm thick to keep momentum smearing of the decay pions low. The target-to-beam alignment as well as the observation of the deposited heat is achieved with a newly developed thermal imaging system. Together with a precise beam energy determination via the undulator light interference method a recalibration of the magnetic spectrometers will be done to obtain a statistical and systematic error of about 20 keV. The experiment started in the summer of 2022 and initial optimization studies for luminosity and data quality are presented.

In the K1.8 beam-line at Hadron Experimental Facility of J-PARC, a new magnetic spectrometer S-2S is being installed. S-2S was designed to achieve a high momentum resolution of Δp/p = 6 × 10−4 in FWHM. Several strangeness-physics programs which require the high resolution will be realized by S-2S. The present article introduces J-PARC E70 (missing-mass spectroscopy of Ξ12Be) and E94 (missing-mass spectroscopy of Λ7Li, Λ10B, and Λ12C) experiments.

Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Kruppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated Zfp521 genes (Zfp521(-/-)) possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521s role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies. Funding Agencies|National Institutes of Health [R01AI081878, R01AI098417, R21AI115696, R01CA117907, K01DK098315]; Wendy Siegel Fund for Leukemia and Cancer Research; Mary Miller and Charlotte Fonfara-Larose Leukemia and Down Syndrome Research Fund; NIH Institutional National Service Award [2T32AI074491]; NIH [F31HL138754]; Victor W. Bolie and Earleen D. Bolie Graduate Scholarship Fund; Swedish Cancer Foundation; Swedish Medical Research Council

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1-4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein-Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1-4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

Even though leukemia is considered to be confined to one specific hematopoietic cell type, cases of acute leukemia of ambiguous lineage and patients relapsing in phenotypically altered disease suggest that a malignant state may be transferred between lineages. Because B-cell leukemia is associated with mutations in transcription factors of importance for stable preservation of lineage identity, we here investigated the potential lineage plasticity of leukemic cells. We report that primary pro-B leukemia cells from mice carrying heterozygous mutations in either or both the Pax5 and Ebf1 genes, commonly mutated in human leukemia, can be converted into T lineage leukemia cells. Even though the conversion process involved global changes in gene expression and lineage-restricted epigenetic reconfiguration, the malignant phenotype of the cells was preserved, enabling them to expand as T lineage leukemia cells in vivo. Furthermore, while the transformed pro-B cells displayed plasticity toward myeloid lineages, the converted cells failed to cause myeloid leukemia after transplantation. These data provide evidence that a malignant phenotype can be transferred between hematopoietic lineages. This has important implications for modern cancer medicine because lineage targeted treatment of leukemia patients can be predicted to provoke the emergence of phenotypically altered subclones, causing clinical relapse. Funding Agencies|Swedish Cancer Society; Swedish Childhood Cancer Foundation; Swedish Research Council; Knut and Alice Wallenbergs Foundation; Linkoping University

One of the most frequently mutated proteins in human B-lineage leukemia is the transcription factor PAX5. These mutations often result in partial rather than complete loss of function of the transcription factor. While the functional dose of PAX5 has a clear connection to human malignancy, there is limited evidence for that heterozygote loss of PAX5 have a dramatic effect on the development and function of B-cell progenitors. One possible explanation comes from the finding that PAX5 mutated B-ALL often display complex karyotypes and additional mutations. Thus, PAX5 might be one component of a larger transcription factor network targeted in B-ALL. To investigate the functional network associated with PAX5 we used BioID technology to isolate proteins associated with this transcription factor in the living cell. This identified 239 proteins out of which several could be found mutated in human B-ALL. Most prominently we identified the commonly mutated IKZF1 and RUNX1, involved in the formation of ETV6-AML1 fusion protein, among the interaction partners. ChIP- as well as PLAC-seq analysis supported the idea that these factors share a multitude of target genes in human B-ALL cells. Gene expression analysis of mouse models and primary human leukemia suggested that reduced function of PAX5 increased the ability of an oncogenic form of IKZF1 or ETV6-AML to modulate gene expression. Our data reveals that PAX5 belong to a regulatory network frequently targeted by multiple mutations in B-ALL shedding light on the molecular interplay in leukemia cells., Author summary The use of modern high throughput DNA-sequencing has dramatically increased our ability to identify genetic alterations associated with cancer. However, while the mutations per se are rather easily identified, our understanding of how these mutations impact cellular functions and drive malignant transformation is more limited. We have explored the function of the transcription factor PAX5, commonly mutated in human B-lymphocyte leukemia, to identify a regulatory network of transcription factors often targeted in human disease. Hence, we propose that malignant conversion of B-lymphocyte progenitors involves multiple targeting of a central transcription factor network aggravating the impact of the individual mutations. These data increase our understanding for how individual mutations collaborate to drive the formation of B—lineage leukemia.

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit + Sca1 + Lineage − cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor κB ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self-antigens (self-Ags). The transport of self-Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7−/− (Ccr7-KO) mice do not show skin auto-immune diseases. To resolve this inconsistency, we examined Ccr7-KO epidermis- or dermis-hyperpigmented transgenic (Tg) mice, in which the transport of skin self-Ags is traceable by melanin granules (MGs). Under CCR7-deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7-KO-dermis-pigmented Tg mice confirmed the presence of CCR7-independent transport from the dermis. Compared with WT-dermis-pigmented Tg mice, the amount of transported melanin and number of MG-laden CD11c+ cells were both approximately 40% of the WT levels, while the number of MG-laden CD205+ or CD207+ cells decreased to about 10% in skin regional LNs of Ccr7-KO-dermis-pigmented Tg mice. Cell sorting highlighted the involvement of CD11c+ cells in the CCR7-independent transport. Here, we show that CCR7-independent transport of skin self-Ags occurs in the dermis. This system might contribute to the continuous transport of self-Ags, and maintain peripheral tolerance.

While murine B- and T-lymphopoiesis require overlapping molecules, they occur in separate organs: the bone marrow (BM) and the thymus, respectively. The BM microenvironment is incapable of supporting T-lymphopoiesis because of insufficient interactions of Notch1 with delta-like ligand (Dll). Notch1/Dll interactions also play a role in the suppression of B-lymphopoiesis in the thymus. However, it is still unclear whether the Notch1/Dll interaction alone explains why the thymus does not support B-lymphopoiesis. In this study, we compared the precursor population colonizing the thymus with that in the BM by culturing them on stromal cells expressing abundant Dll1. We demonstrated that Flt3(+) Il7r(+) B220(+) Cd19(+) BM cells gave rise to B cells under this condition. We defined them as resistant to Dll1. In the thymus, Dll1-resistant cells were undetectable. This suggested that the absence of Dll1-resistant cells might explain the absence of B-lymphopoiesis in the thymus.

Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1). Surprisingly, the adhesion was a consequence of direct cell–cell interaction between MCs and Dll1-expressing stromal cells rather than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)–Dll1 interaction by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch receptor–ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of MCs to inflammatory sites.

Summary Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self-antigens (self-Ags). The transport of self-Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7(-/-) (Ccr7-KO) mice do not show skin auto-immune diseases. To resolve this inconsistency, we examined Ccr7-KO epidermis- or dermis-hyperpigmented transgenic (Tg) mice, in which the transport of skin self-Ags is traceable by melanin granules (MGs). Under CCR7-deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7-KO-dermis-pigmented Tg mice confirmed the presence of CCR7-independent transport from the dermis. Compared with WT-dermis-pigmented Tg mice, the amount of transported melanin and number of MG-laden CD11c(+) cells were both approximately 40% of the WT levels, while the number of MG-laden CD205(+) or CD207(+) cells decreased to about 10% in skin regional LNs of Ccr7-KO-dermis-pigmented Tg mice. Cell sorting highlighted the involvement of CD11c(+) cells in the CCR7-independent transport. Here, we show that CCR7-independent transport of skin self-Ags occurs in the dermis. This system might contribute to the continuous transport of self-Ags, and maintain peripheral tolerance.

Introduction: Canonical notion demonstrates that Cell fate is determined by transcriptional factor. Accordingly, the lineage specific transcriptional factors have been investigated for various kinds of cells. Especially, in the hematopoietic system, the extensive research for lineage specific transcriptional factors had elucidated the transcriptional factors which regulated lineage commitment. B cell commitment and development requires the activities of multiple transcription factors, including the early B cell factor (EBF), PAX5, and E2A. These transcription factors regulate B cell development in a stage specific manner. The hematopoietic progenitor cells which are deficient for any of them, cannot commit B cells. Among them EBF1 is presumed to be more potent. Rescue early pro B cell to induce the expression of several key proteins including RAG that enable gene rearrangement to occur by opening of IgH locus. We found that the B cell developmental arrest caused by EBF1 deficiency can be rescued by a single non coding RNA. These B cells which are deficient EBF1 but showed the expression of CD19, B cell lineage specific surface marker and VDJ recombination, molecular markers of B cell commitment in vitro B cell differentiation system cocultured with Tst4 cells, stromal cell lines. We further investigated the quality and differentiation potential of these B lineage commitment cells in the in vivo mouse model and elucidated the mechanism of this phenomenon. Material and methods: We collected EBF1−/− fetal liver hematopoietic progenitor (Lin−) cells and cultured them on TSt-4 stromal cells after infecting with non coding RNA and control vector in IMDM medium containing cytokines and then injected it into NOG mice. Collect bone marrow (BM), thymus and spleen from those mice. Then comprehensive Gene-Expression analysis, real time PCR for VDJ recombination analysis was performed and checked surface marker by flowcytometry. Result: We analyzed BM and spleen of non coding RNA infected EBF1 KO cells injected mice and found the expression of CD19 in BM as well as in spleen and upregulated of B220 also, comparing with control vector expressed cells. Furthermore, surface IgM expression of CD19 positive cells in the spleen is upregulated compared with the cells in the BM (Figure 1). Several target genes of the non-coding RNAs were identified by use of cDNA array analysis and luciferase reportor assay. Among them, several genes were involved in TGF beta pathway. As TGF beta family and the pathway, has been reported a critical factor which is negatively regulating B lymphopoiesis (Figure 2). We hypothesized that TGF beta family genes such as Tgfβr3, Acvr2a, are responsible for B cell differentiation for which EBF1 is dispensable .We cultured EBF KO cells for 14 days with and without TGF beta 1,2,3 antibody and Activin A antibody on TST4 cells. We found that increase mean intensity (MFI) of B220 into antibodies positive cocultured cells (Figure 3) to suggest, the suppression of TGF beta pathway is partially responsible for B cell differentiation under EBF1 deficiency. Conclusion: Canonical notion of cell fate determination of B cells defines that EBF1 is an indispensible factor for B lymphopoiesis. However, from our previous and present study it is proved that without EBF1 B cell development can progress to pro B to pre B cell and Immature B cell stage and “VDJ recombination” occur in the absence of EBF. Furthermore, in vivo mouse model, EBF1 deficient hematopoietic progenitor cells differentiated into IgM positive cells. Therefore we can conclude that EBF is dispensable of VDJ recombination, opening of IgH locus, binding of RAG protein and B cell differentiation to the mature stage. One of the mechanisms is possibly due to the stimuli from microenvironment, such as TGF beta family and pathway. Furthermore, the detail mechanism of IgH locus opening, epigenetic changes and chromatin remodeling around the IgH locus in the absence of EBF is under investigation. Disclosures Chanda: Japan Society for the Promotion of Science(JSPS): Research Funding.

Introduction: EBV positive diffuse large B-cell lymphoma of the elderly (EBV positive DLBCL, elderly) has been newly categorized in 2008 WHO classification. The incidence is higher in Asian countries than Western countries. The prognosis of DLBCL has been improved by the introduction of rituximab, while EBV positive DLBCL remains unknown. Recently we reported that EBV positive DLBCL of the elderly did have quit inferior prognosis than EBV negative DLBCL. The mechanism lying under the inferiority has yet been elucidated. We hypothesize that tumor microenvironment plays a role in the mechanism, as EBV related lymphoma are usually accompanied with massive infiltration of non-tumor cells. We have previously found that tumor secreted small RNAs were selectively taken by macrophages and dramatically change the character into tumor supporting phenotype in the EBV positive lymphoma. Accordingly, we focus the interaction between tumor cells and macrophages in EBV positive lymphoma microenvironment. Methods: We investigated the number of CD163-positive macrophage (CD163+M2) in the DLBCL specimen with and without EBV positivity. The effect of EBV positivity in the tumor cells on the macrophages infiltrated in the tumor were studied by use of the coculture system using human monocytic cell line (THP-1) and human Burkitt lymphoma cells lines (Akata) which has two subclones such as EBV positive and negative. PMA-induced macrophages from THP1 cells were cocultured with EBV positive or negative Akata, then the expression of the several cytokines such as TNF-a, IL-10, CXCL10, and VEGF were measured by real-time PCR. Finally, we tried to clarify the impact of the macrophages on tumor formation in vivo by using xeno-transplantation model. Hematopoietic humanized NOG mice were infected with EBV to induce EBV related lymphoproliferative disease (LPD). After the appearance of symptoms of the LPD such as body weight loss, mice were treated with Clodronate to deplete macrophages. Result: The multivariate analysis for DLBCL patients demonstrated the statistically significant association between both high scores of CD163+M2 macrophages (CD163-positive cell > 20%) and EBV positivity, and poor prognosis (p = 0.0084, 0.0020, respectively.), which implies that EBV affected the quantity of CD163+M2 macrophages in the tumor microenvironment. Co-culture of THP1 with EBV positive lymphoma cells significantly upregulated CXCL10 and VEGF, when compared with EBV negative cells. (p = 0.0073, 0.0161, respectively.). (Figure 1) Most surprisingly, EBER positive B cells almost completely disappeared by macrophage depletion by Clodronate treatment (Figure 2). These results suggest that the macrophages in the EBV positive tumor microenvironment are crucial for survival of EBV+ tumor cells. Conclusion: EBV status of the lymphoma cells affected on TAMs in the way such as up-regulation of CXCR10 and VGEF, angiogenetic factors which are presumed to support tumor survival. The in vivo depletion of macrophages by Clodronate also demonstrated that they were indispensable for EBV positive tumor cell survival. Taken together, the interaction of EBV positive tumor cells and tumor associated macrophages is of crucial importance in the biology and formation of EBV positive lymphoma. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Introduction Cell fate and lineage specification have been thought to be largely regulated at the level of transcription. However, regulation of differentiation at the transcriptional level alone does not appear to explain all hematopoietic cell fate decisions, suggesting the presence of other as-yet-unknown mechanisms. EBF1 is critical transcription factors in B-lymphopoiesis. Ectopic EBF1 expression promotes B cell development in multipotent progenitor cell (MPP). EBF1is deleted and downregulated in different B cell malignancy like some ALLs and Hodgkin lymphoma. MicroRNAs are noncoding RNAs that are vital to many cell functions and that post-transcriptionally repress target mRNAs. Each miRNA is thought to have several target mRNAs, and computational predictions suggest that more than a third of all human genes are targets of miRNAs. However, in cell fate determination, it has been thought to act as only a fine tuner. Recently we reported that miR-126 drives MLL-AF4 ALL cells, exhibiting both myloid and B cell surface markers, towards B cell without upregulating of EBF1, E2A, and PAX5, which are critical transcriptional factors in B cell lineage commitment. Moreover, miR-126 could induce partial B lymphopoiesis in EBF1 deficient hematopoietic cells (HPCs) (Okuyama.K et al. PNAS). In the present study, in order to challenge the canonical notion that cell fate is governed solely by transcriptional factors, we investigated whether miRNA have full potentiality to induce B cell development in EBF1 deficiency. Material and methods We introduce several different miRNA in EBF1-/-hematopoietic progenitor (Lin-) cells and cultured them on TSt-4 stromal cells in IMDM medium containing stem cell factor, IL-7, and Flt3 ligand (10 ng/mL each). Then comprehensive Gene-Expression analysis, genomic PCR for VDJ recombination analysis and flowcytometric analysis for B-cell lineage markers were performed. Result Among the miRNA we analyzed, miR-195 induced expression of CD19, an established B lineage marker, and upregulated of B220 , B cell associated marker, comparing with control vector expressed EBF1 KO HPCs (Figure 1a). Then we investigated the molecular marker of B committed cells,“ VDJ recombination”. In miR-195 transduced EBF1KO cells, V to DJ recombination were completed, while the control was not (Figure 1b). Based on the results obtained from surface and molecular markers, miR-195 transduced EBF1KO cells were identified as B committed cells suggesting that miR-195 can rescue B lymphopoiesis in EBF1 deficiency. Finally our microarray data shows that overexpression of miR-195 in EBF1 KO cells upregulates B cell lineage commitment genes such as Pax5, Bcl11a, VpreB1, Cd79a, lamda5 and E2A which are downsteam targets of EBF1 and downregulates the myeloid lineage specific genes which are also antagonized by EBF1 in B cell lineage specification (Figure 2a). Next in order to clarify its mechanism, we studied the target genes of miR-195. Tgfβr3, Smad7, Acvr2a, Lats, HGF, and Pbx3 were identified as the targets of miR-195 by luciferase assay in 293T cells (Figure 2b). TGF beta family and the pathway, has been reported to suppress B lymphopoiesis. We hypothesized that TGF beta family genes such as Tgfβr3, Smad7, and Acvr2a, are responsible for transcription factor independent B cell differentiation by miR-195. Further analysis is now under investigation. Conclusion MiR-195 induces CD19 expression, completion of VDJ recombination and upregulation of B cell related genes in EBF1-/-HPCs while neither PAX5 nor E2A are able to upregualte B cell related genes or induce CD19 expression in the absence of EBF1. It suggests that miR-195 is more potent than E2A and Pax5 in this system and acts more than a fine tuner in B cell lineage specification. In the canonical notion, cell fate is determined solely by transcriptional factors. Our study challenges this notion, proposing that miRNA might have potential to be an alternative of transcriptional factors in some condition (Figure 2c). As we all know, impaired differentiation by deregulation of transcriptional factors is one of “hit” to leukemia development. Accordingly miRNA can potentially rescue this deregulation to become promising therapeutic target. Disclosures: Ando: Alexion: Research Funding.

Abstract 3533 microRNAs (miRNAs) control many developmental and physiological processes. However, it is assumed to work as a fine tuner in cell fate determination, which has been shown to be regulated by transcription factors. Here, we challenge this canonical notion. miR-126 is downregulated in MLL-AF4 ALL, biphenotypic leukemia expressing both B cell and myeloid markers, compared with other types of ALLs. CD19 and CD20, B cell differentiation markers, were upregulated when miR-126 is reexpressed in MLL-AF4 ALLs (Figure 1). Interestingly, we found that miR-126, in leukemic cells, induces B cell differentiation (Figure 2a) without changing the expression of E2A, EBF1, or PAX5, (Figure 2b) which are assumed to be critical regulators of B cell development indicating that miR-126 induced B cell differentiation independently of transcriptional factors, such as PAX5, E2A, and EBF1. To test the hypothesis, we tried to rescue block of B cell differentiation in EBF1 knock-out hematopoietic progenitor cells (EBF1 KO HPCs) by exogenous expression of miR-126. As a result, significant upregulation of B cell markers, such as B220, RAG1/2, and CD79a/b was induced by miR-126 in EBF1 KO HPCs which show complete block at pre-pro B cell stage.(Figure 3a) Moreover, miR-126 increased cell proliferation, indicating EBF1 KO HPCs differentiate into large preB cells. Moreover, miR-195, which is upregulated toward pro B cells then downregulated in B cell differentiation, also can partially rescue block of B cell differentiation in EBF1 KO HPCs. (Figure 3b) The block in B lymphopoiesis imposed by the absence of E2A or Pax5 can be overcome by exogenous expression of EBF1. Conversely E2A or Pax5 failed to compensate the effect of EBF1. Thereafter miR-126 and miR-195 potentiates to play more roles than E2A or Pax5 in this system and act as more than a fine tuner in B cell differentiation. Our results elucidate a novel mechanism for the regulation of cell fate independent of transcriptional factor, establish an important role for miRNAs in mammalian lineage specification, and suggest that miRNA could be a new possible therapeutic target for dedifferentiation induced by deregulation of transcriptional factors in ALL, which calls for further studies. Disclosures: No relevant conflicts of interest to declare.