bluuebunny/crossref_metadata_2025_split huggingface dataset:
The earliest B-1 precursor cells are present in E10.5 embryos more than HSC precursor cells.
Authors: Yoshimoto Momoko , Tarnawskey Stefan , Mishra Akansha , Kobayashi Michihiro , Bernstein Irwin , Hadland Brandon , Yoder Mervin C. 
Published: January 2023

Abstract
The peritoneal B-1 cells are innate-like B cells, secreting natural antibodies without T cell help. Cell transfer experiments suggest that CD5+ B-1a cells are of fetal origin because adult bone marrow stem/progenitors do not repopulate B-1a cells efficiently, while fetal liver cells do. We have previously reported the B-1 cell potential of extra-embryonic yolk sac (YS) and para-aortic region of E9.5 embryos appearing before the emergence of hematopoietic stem cells (HSCs), and that B-1 progenitors develop in the absence of HSCs. These data imply that at least a part of B-1a cells are HSC independent and derived from early YS/embryos. In addition, it has been reported that fetal liver HSCs failed to repopulate B-1a cells after transplantation. This raises a question whether HSCs at any stage of the embryo or post-natal life produce B-1a cells. Our recent report has demonstrated that E11.5 HSC precursors repopulated both B-1a and B-2 cells at a clonal level after co-culturing with Akt-overexpressing endothelial cells.
In order to test whether the first HSC repopulates B-1a cells, we directly transplanted Ter119− VE-cadherin+ c-kit+ “HSC precursor cells” from E10.5 embryos into sublethally irradiated immune-deficient neonatal mice. Our precise analysis of transplantation assays revealed that “HSC precursor cells” contained both neonatal HSCs that repopulated multi-lineage blood cells and B-1 specific precursors that repopulated only peritoneal B-1a, B-1b, and splenic marginal zone B cells. Surprisingly, the frequency of B-1 precursors is more than that of neonatal HSCs. Thus, the earliest transplantable B-1 specific precursor cells are present in E10.5 embryos showing the same surface markers with cells display HSC activity.
The ion channel TRPM7 is required for B cell lymphopoiesis
Authors: Treanor Bebhinn , Buhari Fathima Hifza Mohamed , Zhao Tiantian , Brauer Patrick M. , Burrows Kyle M. , Cao Eric , Moxley-Paquette Vincent , Mortha Arthur , Zúñiga-Pflücker Juan Carlos , Krishnamoorthy Mithunah 
Published: January 2023

Abstract
The ion channel, transient receptor potential subfamily melastatin, member 7 (TRPM7) was first identified as critical for cell viability in the B cell line, DT40, by regulating cellular Mg2+ homeostasis. However, specific deletion of TRPM7 in T cells results in only a minor defect in thymocyte development and nearly normal numbers of peripheral T cells, raising the question of whether TRPM7 is in fact important for B cell development in vivo. Here, we generated a murine model in which TRPM7 is specifically deleted in B cells under control of the Mb1 promotor. We show that Mb1-Cre mediated deletion of TRPM7 results in a complete loss of peripheral B cells and a developmental block at the pro B cell stage in the bone marrow, coincident with the expression of Mb1. Importantly, we demonstrate using the in vitro OP9 stromal cell system of lymphopoiesis that supplementation with high levels of extracellular Mg2+ can partially support B cell development in TRPM7-deficient mice. Thus, our findings identify a critical role for TRPM7 in B cell lymphopoiesis.
ABCB7 is required for B lymphocyte development
Authors: Lehrke Michael Jonathan , Chung Ji Young , Schwab Aaron , Shapiro Michael , Shapiro Virginia Smith 
Published: January 2023

Abstract
ABCB7 is a mitochondrial iron transporter critical for cellular iron homeostasis, as its loss results in mitochondrial iron accumulation. ABCB7 has been demonstrated to be critical for embryogenesis and hematopoiesis, though its role in specific hematopoietic lineages is unexplored. We found that ABCB7 is required for lymphopoiesis, as CD2-iCre ABCB7 cKO mice have a severe block in both T and B lymphocyte development. The block in B cell development occurs at the Fr. C to Fr. C’ transition, where we found reduced expression of intracellular μ and pre-BCR, as well as reduced proliferation. We observed reduced expression of E2A, Ikaros, and Pax5, transcription factors essential for B cell lineage development. Previous literature has shown that deletion of ABCB7 results in mitochondrial iron accumulation. Because this is toxic to cells, we hypothesized that B cells increase heme synthesis in order to detoxify accumulating iron. In support of this, we found that HO-1, a surrogate marker for intracellular heme concentration, was elevated in B cells lacking ABCB7. Interestingly, heme levels regulate Bach2, a transcriptional repressor important for B cell development as it suppresses myeloid program in lymphocytes. Bach2 expression was found to be decreased in ABCB7-deficient B cells. We are currently examining the role of Bach2 and the mechanism responsible for the block in B cell development upon loss of ABCB7.
mTORC1 and mTORC2 differentially regulate the development of NK cells
Authors: Yang Chao , Siebert Jason , Thakar Monica , Malarkannan Subramaniam 
Published: January 2023

Abstract
Natural killer (NK) cells are innate lymphoid cells that play essential roles in both innate and adaptive immunity. Kinase mTOR is critical for NK cell development; however, the unique roles of mTORC1 or mTORC2 in regulating this process remain unknown. Ncr1iCre-mediated conditional deletion of Rptor or Rictor (genes encoding defining component of mTORC1 or mTORC2, respectively) results in altered homeostatic NK cellularity and impaired development at distinct maturation stages. The transition from CD27+CD11b− to CD27+CD11b+ stage is impaired in Rptor cKO mice, while, the terminal maturation from CD27+CD11b+ to CD27−CD11b+ stage is compromised in Rictor cKO mice. Mechanistically, Raptor deficiency renders large alteration of the gene expression profile including transcription factors governing early NK cell development. Comparatively, loss of Rictor causes a restricted transcriptome changes. The reduced expression of T-bet correlates with the terminal maturation defects and results from impaired mTORC2-AktS473-FoxO1 signaling. To further explore the development of murine NK cells and the effects of Raptor or Rictor deficiency, single cell RNA sequencing analyses were performed. The Lin−NK1.1+ cells from BM of adult WT mice are unbiasedly classified into six different populations including CD3+ cells, ILC1, immature NK cells, two intermediate mature NK cells, and terminal mature NK cells. Most Raptor-deficient NK cells form new cluster that is not seen in either WT or Rictor cKO mice. Rictor deficiency affects the terminal maturation of NK cells and potentially the development of ILC1. Collectively, our results reveal the divergent roles of mTORC1 and mTORC2 in NK cell development.
Characterizing hematopoietic precursors of Langerhans Cell Histiocytosis (LCH) using a humanized mouse model
Authors: Mancusi Rebecca , Bigenwald Camille , Merad Miriam 
Published: January 2023

Abstract
Langerhans Cell Histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by the accumulation of immature dendritic cells in inflammatory lesions. LCH pathogenesis is not fully understood, however recent findings demonstrate that misguided differentiation programs of dendritic cell precursors drive LCH. Importantly, expression of the somatic mutation BRAFV600E in these precursor dendritic cells indicates a “high-risk” LCH diagnosis. Previous studies have utilized mouse models to further investigate the role BRAFV600E in LCH pathogenesis. However, the lack of a human immune compartment in mice prevents a comprehensive analysis on the function of human dendritic cells in LCH. This study aims to study dendritic cell commitment, maturation, and function using a humanized mouse model of LCH. We describe a series of experiments in which we reconstitute GFP+ BRAFV600E+ transduced human hematopoietic precursors in irradiated, immunodeficient mice. Additionally, transduction and transplantation protocols were optimized to improve novel strategies in the development of a humanized mouse model of LCH. Initial results suggest successful human engraftment in vivo based on flow cytometry analysis of GFP expression in bone marrow 7 and 14 days post-transplantation. We expect to observe an LCH-like phenotype in these mice with continued examination. Our study ultimately develops Mount Sinai’s first humanized mouse models of LCH and enables efficient long-term research on the role of BRAFV600E in the human immune compartment of LCH.
Altered TGFβ signaling in non-hematopoietic cells leads to eosinophilic esophagitis.
Authors: Laky Karen , Kinard Jessica , Guerrerio Anthony , Frischmeyer-Guerrerio Pamela 
Published: January 2023

Abstract
Eosinophilic esophagitis (EoE) is a chronic immune-mediated disease characterized by esophageal inflammation and dysfunction. Among the top ten single nucleotide polymorphisms (SNPs) that are associated with EoE susceptibility, approximately one third are in genes that encode proteins involved in transforming growth factor beta (TGFβ) signaling. Patients and mice heterozygous for a mutation in the kinase domain of TGFβR1 (TGFBR1M318R+/−) exhibit cardiovascular, skeletal, pulmonary, and immune manifestations of Loeys-Dietz Syndrome (LDS). Immune manifestations of LDS include elevated serum IgE against food antigens and an increased likelihood of developing EoE that is characterized by esophageal dilation, food impaction, basal cell hyperplasia, and an inflammatory infiltrate composed of eosinophils, mast cells, T cells, type 2 innate lymphoid cells (ILC2), and antigen presenting cells. How altered TGFβ signaling leads to EoE is unknown. We made bone marrow chimeras to determine which cells are responsible for the development of EOE. We found that altered TGFβ signaling in radio-resistant non-hematopoietic cells was both necessary and sufficient to cause EoE. Moreover, the development of EoE was not predicated on altered TGFβ signaling in the hematopoietic effector cells. Using RNASeq and immuno-histochemistry analyses we show that altered TGFβ signaling in esophageal epithelial cells results in the expression of cytokines, chemokines, and adhesion molecules that facilitate the recruitment, activation, and accumulation of eosinophils, mast cells, T cells, and ILC2. Targeting these inflammatory mediators in vivo should have therapeutic potential for patients suffering with EoE.
Transcription factor signal transducer and activator of transcription 3 (STAT-3) serves as a negative regulator controlling IgE class switching
Authors: Dascani Paul Lorenzo , Ding Chuanlin , Kong Xiangyu , Yan Jun 
Published: January 2023

Abstract
A mutation in the Signal Transducer and Activator of Transcription 3 (STAT-3) has been linked to incidence of Autosomal Dominant Hyper Immunoglobulin E Syndrome (AD-HIES), a disease characterized by elevated serum IgE antibody and susceptibility to respiratory and skin infection. However, how this genetic mutation leads to the phenotype has not been fully understood. In this study we investigated the specific role of STAT-3 in the germinal center (GC) B cells and plasma cells for IgE class switching. Through the use of CD2-Cre or CD19-Cre driven STAT-3 conditional knockout (cKO) mice in a Th2-type immunization model, we aimed to determine the specific cell subset(s) and signaling mechanism by which STAT-3 mediated class switching is regulated. We demonstrated in vivo and in vitro that CD2-Cre driven STAT-3 cKO mice showed elevated IgE and decreased IgG1 in the serum, and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased while IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of antibody-producing plasma cells. Subsequent experiments using a CD19-Cre B-cell specific cKO mouse established this effect to be B-cell intrinsic. Mice with STAT-3 deficiency in B cells have also demonstrated an increase in lung inflammation when used in an asthma-like disease model. Our data also revealed other transcription factors, including Bcl-6 and Aicda, function as downstream signals of STAT-3 regulation. This model suggests a negative role for STAT-3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a therapeutic target for treatment of AD-HIES and other immune disorders.
Contribution of the glycosyltransferase FucT-VII to allergic skin inflammation
Authors: Khan Ibrahim Mohammmed , Ulrich Benjamin Joseph , Kansas Geoffrey S. , Kaplan Mark H. 
Published: January 2023

Abstract
The development of E and P selectin ligands on the surface of immune cells requires α1,3 fucosyltransferase FucT-VII, an enzyme encoded by the Fut7 gene. T cell activation and cytokine environments regulate glycosyltransferase expression, eventual glycan formation and cell migration. The T helper 2 (Th2) subset are mediators of allergic inflammation, but the requirement for FucT-VII-dependent migration in allergic inflammation is less clear. In this study we determined if Fut7 expression was required for allergic inflammation in the constitutively active STAT6VT model in which mice are predisposed to a spontaneous inflammatory phenotype similar to that of atopic dermatitis. STAT6VT mice were crossed with Fut7−/− animals and compared for the development of disease and inflammation. Decreased incidence of disease was observed in Fut7−/− STAT6VT mice, compared to STAT6VT mice. Interestingly, if disease did occur in Fut7−/− animals, the inflammation was not attenuated and in some cases was more severe. One factor that impacted disease development was related to offspring who inherited the STAT6VT allele from their maternal parent. The dams would have experienced inflammation throughout pregnancy and might have promoted a mechanism of migration independent of Fut7 gene expression. Together, these data indicate that Fut7 promotes, but is not strictly required for, allergic skin inflammation.
Forward genetic screen of allergy reveals genetic basis for allergic disease
Authors: SoRelle Jeffrey , Chen Zhe , Wang Jianhui , Tang Miao , Li Xiaohong , Beutler Bruce 
Published: January 2023

Abstract
Although much is known about how mammals respond to allergens, it is still unclear how allergies are acquired. Allergies arise from environmental triggers, but the genetic causes are not clearly understood. We undertook a forward genetic approach to discover genes with non-redundant function in allergic sensitivity. Our screen measured antigen-specific IgE antibody response to a model cysteine protease allergen, papain, in N-ethyl-N-nitrosourea (ENU) mutagenized mice. Males heterozygous for ENU-induced mutations (G1) were bred to wild-type females, and their daughters (G2) were backcrossed to the G1 to produce homozygous mutations in G3 mice, which were screened for IgE responses to papain. Whole exome sequencing of the G1 detects single nucleotide mutations, which are genotyped in all G3 progeny. This method allows for real-time mapping, where phenotype (papain-specific IgE) and genotype are statistically compared using dominant, additive, and recessive models of inheritance to determine linkage. 14,694 G3 mice in 636 pedigrees were screened with estimated genomic saturation of 7.6% of all genes damaged or destroyed in 3 homozygous mice. Mutations with altered allergen-specific IgE segregated into two pathways: class switch recombination (Increased IgE: Itch, Itk. Decreased IgE: Jak3, Stat6, and Ikbkb) and B cell receptor signaling (increased IgE: Blk, Plcg2, Rasgrp3, CD19, and Pik3r1). Interestingly, many BCR mutations were dominant or semi-dominant and did not cause significant immunodeficiency. This first forward genetic screen of allergic disease unveiled several genes with unknown roles in allergy and provides a potential genetic explanation for some patients who present with allergy without immunodeficiency.
Type 2 innate lymphoid cells contribute to a mixed-type allergic response following topical exposure to the quaternary ammonium compound didecyldimethylammonium chloride.
Authors: Shane Hillary L. , Lukomska Ewa , Anderson Stacey E. 
Published: January 2023

Abstract
Didecyldimethylammonium chloride (DDAC) is an antimicrobial dialkyl-quaternary ammonium compound used in numerous industrial and commercial products. Clinical and animal data suggest that DDAC exposure elicits multiple types of hypersensitivity reactions. To examine the immunological mechanism behind this mixed type response and the potential involvement of ILCs, we assessed early immune responses in the skin following dermal DDAC exposure (0.25% and 0.5%) in a BALB/c murine model. DDAC exposure resulted in a rapid and dramatic increase in the Th2-skewing and ILC2 activating cytokine TSLP in the skin. Correspondingly, dermal ILC2s were activated 24 hours after DDAC exposure, resulting in increased levels of CD25, ICOS and KLRG1, and decreased CD127 on a per cell basis throughout a 7 day period of exposure. Following ILC2 activation, the Th2 cytokine IL-4 was found to be elevated compared to control mice in total ear protein lysate in the 0.5% DDAC treated mice. To determine a functional role for ILC2s in DDAC induced sensitization we used Rag2−/− mice, which lack mature T and B cells. ILC2s from Rag2−/− were similarly activated by DDAC and importantly, produced significant levels of IL-4 (0.5%DDAC) and IL-5 (0.25% and 0.5%) in the skin. These data indicate that ILC2s contribute to Th2 immune responses early after DDAC exposure. ILC2s have been previously implicated allergic responses, but to our knowledge have not been thoroughly investigated in chemical sensitization. These results indicate that skin resident ILC2s become activated and produce TH2 cytokines following exposure to DDAC, providing a possible mechanism for the development of a mixed-type allergic response commonly observed with chemical sensitizers.
The B cell IL-10 receptor suppresses antibody production.
Authors: Dooley Abigail , Quintana Montserrat , Cheung Michael , Sun Li Lauren , Gupta Neetu 
Published: January 2023

Abstract
Generation of high affinity, broadly neutralizing antibodies has remained a holy grail in influenza and malaria. Appropriate B cell responses are essential for producing neutralizing antibodies to combat these infections. Additionally, cytokines and their receptors are known to orchestrate the immune response to infections on several levels. However, the regulation of antibody production by cytokine receptors on B cells is not well understood. The anti-inflammatory cytokine, IL-10 activates the JAK-STAT pathway upon binding to the IL-10 receptor (IL-10R), and inhibits production of the pro-inflammatory cytokines. IL-10 is also thought to regulate antibody production but the specific role of IL-10R expressed on B cells is unclear. We hypothesized that the B cell-expressed IL-10R regulates B cell antibody response to infections. To address this, we generated mice with conditional deletion of IL-10R in the B cell lineage. Using these mice, we first examined B cell antibody production in response to LPS and B cell receptor stimulation. Surprisingly, the loss of IL-10R in B cells increased antibody production. Both IgM and IgG antibody secreting cells were significantly increased when the IL-10R was deleted in B cells. Further, higher IL-10 levels were detected in culture supernatants of IL-10R-deficient B cells stimulated with LPS. Our data suggest that the IL-10R on B cells negatively regulates antibody production. Our ongoing experiments are aimed at addressing the molecular basis of this inhibitory influence of the B cell IL-10R on antibody production. This study has implications in the treatment of seasonal infectious diseases such as influenza and malaria, in which a robust antibody response is essential for protective immunity.
Deletion of the highly conserved functional region of the Cd4 enhancer NCE in RLM11 cells using CRISPR/Cas9 reduces CD4 expression
Authors: Sarafova Sophia D. , Futral Cameron , Brennan Paul , Guzynski Mia 
Published: January 2023

Abstract
The glycoprotein cell surface marker CD4 is a key mediator in the adaptive immune response and T-cell development. Signaled CD4+ CD8+ double positive cells (DP) upregulate CD4 on the cell surface, allowing for a persistent TCR signal in the thymus and differentiating into the CD4+ single positive helper lineage. Previously, we have demonstrated that NCE (novel cis element), located in the first intron of Cd4, acts as an enhancer in RLM11 cells (CD4+, CD8−, Th-Pok− thymoma, arrested at the intermediate stage of development), but not in AKR-1G1 or VL3-3M2 cells (DP thymomas). We have identified a highly conserved region within NCE, labeled core-NCE, which may be vital to the enhancer’s function. To investigate the contribution of core-NCE to the level of CD4 expression, we utilized CRISPR/Cas9 technology to target the region for deletion. Here we describe the construction of plasmids containing guide sequences for targeted deletion of core-NCE by Cas9 cutting and NHEJ repair. We have successfully generated and sequence verified plasmids with three upstream and three downstream guides and introduced them in RLM11 cells. After single cell sorting, we obtained one clone that has the 300bp core-NCE deleted on one Cd4 allele, which resulted in 66% reduction in CD4 expression from that allele. As we removed the core-NCE in cells that have already established CD4 expression, we conclude that the core NCE has an enhancer function at the intermediate/transitional stage of thymocyte development that is separate from the previously described epigenetic function of the same intronic region, making NCE a good candidate for the regulatory element responsible for CD4 upregulation during differentiation into the CD4 lineage.
Thymic selection of innate self-specific T cells: Both Timing and Signal Strength matter
Authors: Verstichel Greet , Thiault Nicolas , Chen Angeline , Cheroutre Hilde 
Published: January 2023

Abstract
Naïve CD4 and CD8 T cells are selected in the thymus in response to low-avidity TCR interactions. Such weak TCR signals are crucial for installing a quiescent program that guides them to the lymph nodes yet allows for immune activation upon encounter of high-affinity ligands in the periphery. In contrast, agonist-selected T cells receive strong self-based TCR signals in the thymus that imprint them to guard the mucosal borders and maintain quiescence at steady state. Although the TCR signal strength is a decisive factor in instructing these different outcomes, the mechanism that generates protective self-specific T cells remains largely enigmatic. Previous work suggests that the self-specific double negative (DN) T cell lineage already diverges early during T cell development. We have explored the role of developmental timing by Cre-mediated deletion of components of the TCR signaling pathway. The Lck proximal promoter drives Cre expression before b-selection and the CD4 promoter drives Cre expression after b-selection but before positive selection. As TCR targets we investigated the lack of Themis expression, essential for the generation of conventional naïve T cells, but dispensable for agonist selection. Vice versa, we analyzed the requirement for Stim1 and Stim2, driving agonist selection, but not conventional selection. Both Lck prox Cre- and CD4 Cre-driven deletion of Themis lead to a drastic decrease in naïve T cells. However, DN T cells are particularly dependent on early strong TCR signals at the Pre-TCR stage, as only Lck prox Cre- but not CD4 Cre-driven deletion of Stim1/2 eliminates this subset. Our results suggest that strong signals at the Pre-TCR checkpoint are crucial for the generation of innate self-specific T cells.