pro-B Cells Research Articles

Landscape of immune infiltration in entorhinal cortex of patients with Alzheimer's disease.

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases and manifests as progressive memory loss and cognitive dysfunction. Neuroinflammation plays an important role in the development of Alzheimer’s disease and anti-inflammatory drugs reduce the risk of the disease. However, the immune microenvironment in the brains of patients with Alzheimer’s disease remains unclear, and the mechanisms by which anti-inflammatory drugs improve Alzheimer’s disease have not been clearly elucidated. This study aimed to provide an overview of the immune cell composition in the entorhinal cortex of patients with Alzheimer’s disease based on the transcriptomes and signature genes of different immune cells and to explore potential therapeutic targets based on the relevance of drug targets. Transcriptomics data from the entorhinal cortex tissue, derived from GSE118553, were used to support our study. We compared the immune-related differentially expressed genes (irDEGs) between patients and controls by using the limma R package. The difference in immune cell composition between patients and controls was detected via the xCell algorithm based on the marker genes in immune cells. The correlation between marker genes and immune cells and the interaction between genes and drug targets were evaluated to explore potential therapeutic target genes and drugs. There were 81 irDEGs between patients and controls that participated in several immune-related pathways. xCell analysis showed that most lymphocyte scores decreased in Alzheimer’s disease, including CD4+ Tc, CD4+ Te, Th1, natural killer (NK), natural killer T (NKT), pro-B cells, eosinophils, and regulatory T cells, except for Th2 cells. In contrast, most myeloid cell scores increased in patients, except in dendritic cells. They included basophils, mast cells, plasma cells, and macrophages. Correlation analysis suggested that 37 genes were associated with these cells involved in innate immunity, of which eight genes were drug targets. Taken together, these results delineate the profile of the immune components of the entorhinal cortex in Alzheimer’s diseases, providing a new perspective on the development and treatment of Alzheimer’s disease.

Abstract A26: SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice

Abstract Mantle cell lymphoma (MCL) is a highly aggressive B cell lymphoma that accounts for 6% of all non-Hodgkin lymphomas and is characterized by abnormal proliferation of mature antigen- naive B lymphocytes. Despite recent advances in patient risk stratification, MCL remains incurable due to frequent relapses and resistance to therapy. SOX11 is a key transcription factor in the pathogenesis of MCL and is highly expressed in conventional MCL, but it is not expressed in the normal B cells or in the indolent leukemic nonnodal MCL subtype. Although SOX11 was identified as crucial oncogene that supports maintenance of MCL, its role in the initiation of MCL remains largely unknown. Here, we developed a novel mouse model for condition overexpression of SOX11 and a eGFP reporter and found that elevated levels of SOX11 in B-cells are sufficient to drive MCL-like lymphomas in mice. Moreover, SOX11 expression synergized with loss of p53 and overexpression of Ccnd2 to form aggressive MCL-like CD19+CD5+CD23−IgM+IgD+ lymphomas, with a median survival of 314 (p53Mb1 ; n=11), 165 (SOX11/p53Mb1 ; n=19), and 134 (SOX11/Ccnd2/p53Mb1 ; n=15) days. These SOX11-driven lymphomas show transcriptional, immunophenotypic and functional similarities with both murine B1a cells and MCL patients. SOX11 overexpression skews B-cell development towards the B1a and marginal zone B-cell lineage and it block B2-cell development at the Pro-B and Pre-B cell stage, which coincides with onset eGFP that is a measure of expression of Cre and SOX11. In contrast to cyclin D2-driven MCL-like model, SOX11 is inducing a pre-lymphoma stage, in which there is a progressive accumulation of B1a cells in peripheral blood, peritoneal cavity, spleen and bone-marrow. The pre-lymphoma B1a cells have a BCR repertoire which was biased towards binding of self-antigens, such as phosphatidylcholine, and have increased BCR signaling activity. To prove that embryonic-derived B1a cells were the cell-of-origin of SOX11-driven MCL-like lymphoma, we transplanted CD19+ B-cells from SOX11/p53Mb1 fetal livers into secondary recipients and found that these embryonic cells could give rise to MCL-like lymphomas. Next, we wanted to determine how SOX11 is inducing the pre-lymphoma phase, either by expanding existing B1a cells, having a developmental bias towards B1a cells, or by the ability to reprogram B2-cells towards a B1a cell fate. Therefore, we crossed SOX11/p53 mice with a tamoxifen-inducible CreERT2 line and transplanted different sorted B1 and B2 subsets after a short tamoxifen induction ex vivo in immunocompromised mice. We found that SOX11 OE can redirect mature B2 cells to form B1a/MCL lymphomas. Overall, SOX11 is a key B1a lineage factor in mice and its aberrant expression is leading to the formation of MCL-like lymphomas in mice. Citation Format: Tim Pieters, Steven Goossens, Pieter Van Vlierberghe. SOX11 drives B1a lineage commitment and B1a/MCL-like lymphoma formation in mice [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A26.

Hypoxia and hypoxia-inducible factor signals regulate the development, metabolism, and function of B cells.

Hypoxia is a common hallmark of healthy tissues in physiological states or chronically inflamed tissues in pathological states. Mammalian cells sense and adapt to hypoxia mainly through hypoxia-inducible factor (HIF) signaling. Many studies have shown that hypoxia and HIF signaling play an important regulatory role in development and function of innate immune cells and T cells, but their role in B cell biology is still controversial. B cells experience a complex life cycle (including hematopoietic stem cells, pro-B cells, pre-B cells, immature B cells, mature naïve B cells, activated B cells, plasma cells, and memory B cells), and the partial pressure of oxygen (PO2) in the corresponding developmental niche of stage-specific B cells is highly dynamic, which suggests that hypoxia and HIF signaling may play an indispensable role in B cell biology. Based on the fact that hypoxia niches exist in the B cell life cycle, this review focuses on recent discoveries about how hypoxia and HIF signaling regulate the development, metabolism, and function of B cells, to facilitate a deep understanding of the role of hypoxia in B cell-mediated adaptive immunity and to provide novel strategies for vaccine adjuvant research and the treatment of immunity-related or infectious diseases.

Tnpo3 enables EBF1 function in conditions of antagonistic Notch signaling.

Transcription factor EBF1 (early B cell factor 1) acts as a key regulator of B cell specification. The transcriptional network in which EBF1 operates has been extensively studied; however, the regulation of EBF1 function remains poorly defined. By mass spectrometric analysis of proteins associated with endogenous EBF1 in pro-B cells, we identified the nuclear import receptor Transportin-3 (Tnpo3) and found that it interacts with the immunoglobulin-like fold domain of EBF1. We delineated glutamic acid 271 of EBF1 as a critical residue for the association with Tnpo3. EBF1E271A showed normal nuclear localization; however, it had an impaired B cell programming ability in conditions of Notch signaling, as determined by retroviral transduction of Ebf1 -/- progenitors. By RNA-seq analysis of EBF1E271A-expressing progenitors, we found an up-regulation of T lineage determinants and down-regulation of early B genes, although similar chromatin binding of EBF1E271A and EBF1wt was detected in pro-B cells expressing activated Notch1. B lineage-specific inactivation of Tnpo3 in mice resulted in a block of early B cell differentiation, accompanied by a down-regulation of B lineage genes and up-regulation of T and NK lineage genes. Taken together, our observations suggest that Tnpo3 ensures B cell programming by EBF1 in nonpermissive conditions.

Immune regulatory effects of microRNA9-3

MicroRNAs are known to regulate cell proliferation, differentiation, and apoptosis. However, the immunological mechanism and role of microRNA9-3 (miR9-3) are unknown. This study used CRISPR/cas9 technology to knock out miR9-3 to modulate its expression level. FACS results showed that the absolute number of total B cells declined in miR9-3-deficiency in the spleen (Sp), bone marrow (BM), and lymph node (LN) to different levels compared to the wild-type. Also, the absolute numbers of Fo, T1, and T2 cells decreased both in Sp and LN. The absolute numbers of total T cells in Sp and LN declined sharply; CD4+ and CD8+ T cells showed a dramatic decrease in Sp, LN, and Th (thymus) of the miR9-3− group. In BM, the cells number of immature B cells, pro-pre-B cells, pro-B cells, and pre-B cells reduced to different levels, while mature B cells were comparable to wild-type. These data illustrated that miR9-3-deficiency impaired the development of B cells in BM. Also, the development of T cells was severely impaired. In Th, the numbers of DN and DP cells were remarkably reduced in the miR9-3 mutant mice. Also, the numbers of DN-1, DN-3, and DN-4 cells decreased. The absolute number of cells in the hematopoietic stem cell (HSC) system such as LT-HSC (long-term HSC), ST-HSC (short-term HSC), MPP (multipotent progenitor), GMP (granulocyte-macrophage progenitor), CMP (common myeloid progenitors), MEP (megakaryocyte-erythroid progenitor), and CLP (common lymphoid progenitor) all were decreased in miR9-3 deficient mice. These results showed that miR9-3 deficiency initiated the damage to the entire hematopoietic system. Moreover, the absolute number of myeloid cells in both Sp and BM decreased in mutant mice. The cells number of NK cells showed a sharp reduction in Sp whereas the change was not significant in BM. The above results suggest that miR9-3 participates in the immune regulation of B cells, T cells, and the HSC system, highlighting its regulatory roles.

P337: LONG TERM INFECTION-DRIVES CHANGES IN THE BONE MARROW MICRO-ENVIRONMENT OF PAX5+/- MICE PREDISPOSED TO B CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Pax5 heterozygosity (Pax5+/-) in mice mimics germline or somatic Pax5 dysregulation (resulting in reduced Pax5 levels) observed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). While a link between general non-specific infectious exposure and BCP-ALL has been demonstrated in the Pax5+/- mouse model, the effects of a specifically tractable infection are poorly understood. In particular, little is known about potential interactions of pre-leukemic early progenitor B cell populations and immune cells present in the bone marrow microenvironment (BME) during an infection. Aims: We aimed to characterize the short-term and long-term effects of a viral infection in the BME of the Pax5+/- mouse model employing the Lymphocytic choriomeningitis virus (LCMV). LCMV is a non-cytopathic virus that has been extensively utilized to investigate virus-induced immunopathology, effector responses and immune tolerance. Methods:Pax5+/- mice backcrossed to the C57BL/6J background (N10) were infected with LCMV. Using flow cytometry, ELISA and plaque assay, innate and late adaptive immune responses in infected Pax5+/- and WT (Pax5+/+) were assessed. Pre-leukemic early progenitor B cell populations, antigen-specific CD8+ T cells and regulatory T cells (Treg) were evaluated in the BME. This was complemented by short-term ex vivo assays where B cells were stimulated with differing immune stimuli. Results: When we investigated immune responses in a short-term ex vivo culture system using the bone marrow of Pax5-/+ and WT mice, we found differences in the induction of the interleukin 7 receptor (IL-7r) on immature Pro-B cells as well as the production of cytokines (IL-6 and TNF-▫▫) in response to the toll-like receptor (TLR7) agonist R848 and the lymphocytic choriomeningitis virus (LCMV) Docile strain. When we infected Pax5-/+ and WT mice with LCMV-Docile, we found that innate (early interferon production) and adaptive immune responses (tetramer positive CD8+ T cells and early viral titers) were not intrinsically impaired in Pax5-/+ mice. However, at day 90 post-infection, LCMV-specific T cells were present within the BME and tetramer specific CD8+ T cells raised against the LCMV nucleocapsid protein (np 396) were increased in the BM of Pax5-/+ infected mice (Figure 1A). Furthermore, when we evaluated Major Histocompatibility Complex Class II (MHC-II) expression on different B cell subsets in the BME of infected and uninfected mice, we found that there was a significant upregulation in all immature B-cell subsets (Pro-B, PreBI-II) in response to infection in the WT but not in the Pax5-/+ infected mice. Pax5-/+ mice were susceptible to LCMV infection resulting in shorter long-term survival compared to infected WT mice (Figure 1B). Image:Summary/Conclusion: This study demonstrates that the BME environment as well as survival of pre-leukemic Pax5-/+ mice is differently affected by LCMV infection supporting the observations that immune dysregulation could contribute to the emergence of the leukemic clone1. 1Supported by the Carreras foundation project no. DJCLS07R/2019

Abstract 6187: ICP-723 is a potent pan-TRK Inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations of NTRK fusions

Abstract Background: Somatic oncogenic fusions of NTRK with other genes, such as ETV6, LMNA, TPM3, PAN3, etc. occur in a wide range of adult and pediatric tumors. Clinical application of 1st generation TRK inhibitors, i.e., larotrectinib and entrectinib, for the treatment of NTRK fusion-positive cancer patients has achieved high response rates regardless of tumor types. However, prolonged treatment often leads to acquired drug resistance attributed to various mutations in TRK kinase domains. Here we report the development of a 2nd generation pan-TRK inhibitor, ICP-723, potently inhibits activities of wild-type and some mutant forms of TRK. Results: ICP-723 effectively inhibits kinase activities of TRKA, TRKB, TRKC with IC50 values < 1 nM. In the KINOMEscan, at a concentration of 100 nM, ICP-723 competitively binds to TRKA/B/C with 100% of inhibition rate, indicating high selectivity for pan-TRK. To evaluate the anti-proliferative activity of ICP-723 in TRK-driven tumor cells, KM12 colorectal cancer cell line bearing TPM3-NTRK1 fusion, as well as a panel of 19 Ba/F3 pro-B murine cell lines over-expressing wild-type or mutant forms of NTRK fusions are tested. ICP-723 demonstrates robust in vitro efficacy in all wild-type TRK-driven tumors. It also overcomes solvent front mutations (e.g., TRKA G595R, TRKC G623R/E) often developed following 1st generation TRK inhibitor treatment. Amongst other TRK mutations, ICP-723 exerts reduced activity in gatekeeper mutations (e.g., TRKA F589L, TRKB F633L, TRKC F617L) and some xDFG mutations (e.g., TRKA G667C/S, TRKB G709C); but retains activity in other xDFG mutations (e.g., TRKA G667A, TRKC G696A/C) and unclassified mutations (e.g., TRKA V573M, TRKA A608D, TRKB V689M). In vivo efficacy studies further demonstrate its robust anti-tumor effects in xenograft models. Treatment with 1 mg/kg of ICP-723 results in 89.5% TGI of KM12 tumors, similar to the efficacies of larotrectinib or selitrectinib at 30 mg/kg dose levels. Treatment with 1 or 3 mg/kg of ICP-723 leads to 100% survival of mice bearing Ba/F3 LMNA-NTRK1 G595R tumors, confirms the effectiveness of ICP-723 against solvent front mutant TRKA. The in vivo efficacy of ICP-723 is also well accompanied by pharmacodynamic modulation of TRK phosphorylation, where ICP-723 exposure levels correlate with the inhibition of TRK phosphorylation. The pharmacokinetic parameters of ICP-723 are overall favorable, with high oral bioavailability. Preclinical safety evaluations also exhibit acceptable drug tolerance in SD rats and Beagle dogs. Conclusions: ICP-723 is a novel, 2nd generation pan-TRK inhibitor with broad-spectrum anti-tumor activities against wild-type and various acquired drug-resistant mutations of NTRK gene fusions. ICP-723 is now in phase I clinical trial in China and United States. Citation Format: Ruixia Liang, Chao Zhou, Yingrui Han, Prince Lu, Charles Ying Wang, Zuopeng Wang, Richard Liu, Norman Kong, Jason Bing Zhang, Jasmine Jisong Cui, Davy Xuesong Ouyang. ICP-723 is a potent pan-TRK Inhibitor with robust anti-tumor activities against wild-type and acquired drug-resistant mutations of NTRK fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6187.

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors.

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.

ABCB7 is required for B cell development, proliferation, and class switch recombination

Abstract Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination. Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.

A mechanism of TNK1 activation by C‐terminal gene truncation in human lymphomas

Thirty‐eight‐negative kinase 1 (TNK1) is a poorly characterized non‐receptor tyrosine kinase (NRTK) first isolated from umbilical cord blood. TNK1 differs from most NRTKs in that it possesses a functional ubiquitin association (UBA) domain at its C‐terminus. We previously observed that the TNK1 UBA domain interacts with a variety of poly‐ubiquitin chains, is essential for full TNK1 activation, and facilitates the clustering of active TNK1 into ubiquitin‐rich condensates.1 Paradoxically, a genetic inversion that truncates the TNK1 C‐terminus (including the UBA domain) in human lymphoma hyperactivates the kinase and coverts it into an oncogenic driver. Our current research focuses on how deletion of the UBA, which is important for native TNK1 function, helps activate the kinase. Here we show that immediately adjacent to the UBA domain on TNK1 is an inhibitory 14‐3‐3 binding site, which is also truncated in lymphoma. Furthermore, point mutations that disrupt the 14‐3‐3 binding site are sufficient to convert TNK1 into a hyperactive oncogenic kinase. In addition, we show that although truncation of the UBA perturbs the normal function of TNK1, it also stabilizes TNK1 protein levels. Thus, the lymphoma‐associated truncations in TNK1 activate and stabilize the kinase, resulting in a highly expressed and active TNK1. Finally, we show that truncation of the 14‐3‐3 binding site and UBA domain of TNK1 is sufficient to transform pro‐B cells to growth factor‐independence and grow tumors in vivo. Thus, our data explain how genetic inversions convert TNK1 into an oncogenic driver in human cancers.1. Chan, TY., Egbert, C.M., Maxson, J.E. et al. TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021). https://doi.org/10.1038/s41467‐021‐25622‐3

A Genetic Bottleneck of Mitochondrial DNA During Human Lymphocyte Development.

Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Although the elimination of pathogenic mitochondrial DNA (mtDNA) mutations in somatic cells has been observed, the mechanisms to maintain proper functions despite their mtDNA mutation load are poorly understood. In this study, we analyzed somatic mtDNA mutations in more than 30,000 single human peripheral and bone marrow mononuclear cells. We observed a significant overrepresentation of homoplasmic mtDNA mutations in B, T, and natural killer (NK) lymphocytes. Intriguingly, their overall mutational burden was lower than that in hematopoietic progenitors and myeloid cells. This characteristic mtDNA mutational landscape indicates a genetic bottleneck during lymphoid development, as confirmed with single-cell datasets from multiple platforms and individuals. We further demonstrated that mtDNA replication lags behind cell proliferation in both pro-B and pre-B progenitor cells, thus likely causing the genetic bottleneck by diluting mtDNA copies per cell. Through computational simulations and approximate Bayesian computation (ABC), we recapitulated this lymphocyte-specific mutational landscape and estimated the minimal mtDNA copies as <30 in T, B, and NK lineages. Our integrative analysis revealed a novel process of a lymphoid-specific mtDNA genetic bottleneck, thus illuminating a potential mechanism used by highly metabolically active immune cells to limit their mtDNA mutation load.

Myc-Interacting Zinc Finger Protein 1 (Miz-1) Is Essential to Maintain Homeostasis and Immunocompetence of the B Cell Lineage.

Simple SummaryThe immune system of mice and humans acts against pathogenic threats and intrinsic risks such as cancer. B cells, as antibody-producing cells, provide the ability to specifically target these risks. However, aging leads to a progressive loss of this ability and molecular causes of the gradual loss of immunocompetence remain unknown. Using genetically modified mice, we unravel the transcription factor Miz-1 as a key player of B cell aging during bone marrow lymphopoiesis and peripheral maturation. This enables the investigation of B cell-specific aging mechanisms and how to counteract them for therapeutic approaches to improve immunocompetence in the elderly.Aging of the immune system is described as a progressive loss of the ability to respond to immunologic stimuli and is commonly referred to as immunosenescence. B cell immunosenescence is characterized by a decreased differentiation rate in the bone marrow and accumulation of antigen-experienced and age-associated B cells in secondary lymphoid organs (SLOs). A specific deletion of the POZ-domain of the transcription factor Miz-1 in pro-B cells, which is known to be involved in bone marrow hematopoiesis, leads to premature aging of the B cell lineage. In mice, this causes a severe reduction in bone marrow-derived B cells with a drastic decrease from the pre-B cell stage on. Further, mature, naïve cells in SLOs are reduced at an early age, while post-activation-associated subpopulations increase prematurely. We propose that Miz-1 interferes at several key regulatory checkpoints, critical during B cell aging, and counteracts a premature loss of immunocompetence. This enables the use of our mouse model to gain further insights into mechanisms of B cell aging and it can significantly contribute to understand molecular causes of impaired adaptive immune responses to counteract loss of immunocompetence and restore a functional immune response in the elderly.

Whole-Transcriptome Profiling and circRNA-miRNA-mRNA Regulatory Networks in B-Cell Development.

The generation and differentiation of B lymphocytes (B cells) is a flexible process with many critical regulatory factors. Previous studies indicated that non-coding RNAs play multiple roles in the development of lymphocytes. However, little has been known about the circular RNA (circRNA) profiles and their competing endogenous RNA (ceRNA) networks in B-cell development and differentiation. Here, four B-cell subsets were purified from single-cell suspensions of mouse bone marrow. Then RNA sequencing (RNA-Seq) was used to display expression profiles of circRNAs, miRNAs and mRNAs during B-cell differentiation. 175, 203, 219 and 207 circRNAs were specifically expressed in pro-B cells, pre-B cells, immature B cells and mature B cells, respectively. The circRNA-associated ceRNA networks constructed in two sequential stages of B-cell differentiation revealed the potential mechanism of circRNAs in these processes. This study is the first to explore circRNA profiles and circRNA-miRNA-mRNA networks in different B-cell developmental stages of mouse bone marrow, which contribute to further research on their mechanism in B-cell development and differentiation.

A novel intramolecular negative regulation of mouse Jak3 activity by tyrosine 820.

Jak3, a member of the Janus kinase family, is essential for the cytokine receptor common gamma chain (γc)-mediated signaling. During activation of Jak3, tyrosine residues are phosphorylated and potentially regulate its kinase activity. We identified a novel tyrosine phosphorylation site within mouse Jak3, Y820, which is conserved in human Jak3, Y824. IL-2-induced tyrosine phosphorylation of Jak3 Y824 in human T cell line HuT78 cells was detected by using a phosphospecific, pY824, antibody. Mutation of mouse Jak3 Y820 to alanine (Y820A) showed increased autophosphorylation of Jak3 and enhanced signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and transcriptional activation. Stably expressed Jak3 Y820A in F7 cells, an IL-2 responsive mouse pro-B cell line Ba/F3, exhibited enhanced IL-2-dependent cell growth. Mechanistic studies demonstrated that interaction between Jak3 and STAT5 increased in Jak3 Y820A compared to wild-type Jak3. These data suggest that Jak3 Y820 plays a role in negative regulation of Jak3-mediated STAT5 signaling cascade upon IL-2-stimulation. We speculate that this occurs through an interaction promoted by the tyrosine phosphorylated Y820 or a conformational change by Y820 mutation with either the STAT directly or with the recruitment of molecules such as phosphatases via a SH2 interaction. Additional studies will focus on these interactions as Jak3 plays a crucial role in disease and health.

An instructive role for Interleukin-7 receptor \u03b1 in the development of human B-cell precursor leukemia

Kinase signaling fuels growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Yet its role in leukemia initiation is unclear and has not been shown in primary human hematopoietic cells. We previously described activating mutations in interleukin-7 receptor alpha (IL7RA) in poor-prognosis “ph-like” BCP-ALL. Here we show that expression of activated mutant IL7RA in human CD34+ hematopoietic stem and progenitor cells induces a preleukemic state in transplanted immunodeficient NOD/LtSz-scid IL2Rγnull mice, characterized by persistence of self-renewing Pro-B cells with non-productive V(D)J gene rearrangements. Preleukemic CD34+CD10highCD19+ cells evolve into BCP-ALL with spontaneously acquired Cyclin Dependent Kinase Inhibitor 2 A (CDKN2A) deletions, as commonly observed in primary human BCP-ALL. CRISPR mediated gene silencing of CDKN2A in primary human CD34+ cells transduced with activated IL7RA results in robust development of BCP-ALLs in-vivo. Thus, we demonstrate that constitutive activation of IL7RA can initiate preleukemia in primary human hematopoietic progenitors and cooperates with CDKN2A silencing in progression into BCP-ALL.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eμ-Myc transgenic mice and significantly improves their survival.

Different cell imaging methods did not significantly improve immune cell image classification performance.

Developments in high-throughput microscopy have made it possible to collect huge amounts of cell image data that are difficult to analyse manually. Machine learning (e.g., deep learning) is often employed to automate the extraction of information from these data, such as cell counting, cell type classification and image segmentation. However, the effects of different imaging methods on the accuracy of image processing have not been examined systematically. We studied the effects of different imaging methods on the performance of machine learning-based cell type classifiers. We observed lymphoid-primed multipotential progenitor (LMPP) and pro-B cells using three imaging methods: differential interference contrast (DIC), phase contrast (Ph) and bright-field (BF). We examined the classification performance of convolutional neural networks (CNNs) with each of them and their combinations. CNNs achieved an area under the receiver operating characteristic (ROC) curve (AUC) of ~0.9, which was significantly better than when the classifier used only cell size or cell contour shape as input. However, no significant differences were found between imaging methods and focal positions.

HLA-G-ILT2 interaction contributes to suppression of bone marrow B cell proliferation in acquired aplastic anemia.

Acquired aplastic anemia (AA) is an autoimmune disease characterized by hematopoietic stem and progenitor cell destruction in bone marrow. The non-classic human leukocyte class I antigen (HLA-) G interacts with multiple cell subsets, such as T cells and B cells. HLA-G exerts powerful immune suppression by binding with its receptors, immunoglobulin-like transcripts (ILTs). Here, we compared 46 AA patients and 28 healthy controls. Soluble HLA-G levels in bone marrow supernatants from AA patients were higher than controls. The proportion of bone marrow B cells was decreased and the ILT2-expressing cells among CD19+ cells were increased in AA patients. In addition, the percentage of mature B cells among marrow B cells was increased in AA patient, while the percentage of pro-B plus pre-B cells was decreased. More immature B cells and pro-B plus pre-B cells expressed ILT2 in AA patients than in controls, while mature B cells expressing ILT2 did not differ significantly. Functional studies demonstrated that high-level soluble HLA-G inhibited bone marrow B cell proliferation by interacting with ILT2 in AA, and was blocked by anti-HLA-G and anti-ILT2 monoclonal antibodies. Together, these results suggest that the abnormal decrease of pro-B plus pre-B cells in AA patients was related to the enhanced suppression by the excess HLA-G and ILT2 proteins. Therapeutic blockade of the HLA-G-ILT2 interaction may help to normalize bone marrow B cell proliferation.

A Novel Multicolor Flow Cytometry Scoring System for the Diagnosis and Prognostic Stratification of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a class of myeloid neoplasms featuring inefficient maturation and differentiation of hematopoietic cells, blood cytopenia, and a high risk of leukemia onset. The diagnosis of MDS remains a challenging task owing to its complexity, heterogeneity, and the lack of specific characteristics. To look for an easy and inexpensive diagnostic method for MDS, we tried to establish an FCM scoring systems (FCSS) with a combination of antibodies for diagnosis and prognostic stratification of MDS. This FCSS adopted four parameters; i.e., the frequency of myeloblasts in nucleated cells, the ratio between pro-B cells and CD117+ cells, the ratio of CD45 mean fluorescence intensity between lymphocytes and myeloblasts, and the ratio of SSC peak values between mature granulocytes and lymphocytes. We tested the correlation between the total FCSS score with conventional IPSS-R. Additionally, the correlation between the score of each FCSS parameter and IPSS-R was also evaluated. We found that total FCSS score had a positive correlation with IPSS-R, while FCSS parameter 1 and 4 were also correlated with IPSS-R. Furthermore, this FCSS had a sound sensitivity and specificity in the diagnosis of MDS. The FCSS represents a convenient and affordable approach for the diagnosis and prognostic stratification of MDS.

Acetyltransferases GCN5 and PCAF Are Required for B Lymphocyte Maturation in Mice

B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region, and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for the successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacked both GCN5 and PCAF in B cells. Double-deficient mice possessed low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We concluded that both GCN5 and PCAF are required for B-cell development in vivo.