bluuebunny/crossref_metadata_2025_split huggingface dataset:
Niche-specific regulation of hematopoietic stem and progenitors by NR4A1
Authors: Mumau Melanie , Emerson Stephen , Punt Jennifer 
Published: January 2023

Abstract
Hematopoietic stem and progenitor cells (HSPCs) have the potential to generate all cells in the blood. Although the differentiation of HSPCs depends on their interactions with the niche, the exact mechanisms that control lineage specification remain unclear. Adult HSPC development predominates in the bone marrow, but also occurs at extramedullary sites including the spleen and there favors myelopoiesis. To explore how the spleen regulates myeloid differentiation, we transplanted bone marrow-derived, myeloid-biased HSPC subsets, including multipotent progenitor populations MPP2s and MPP3s, and monitored their development. We discovered that MPP2s, not MPP3s, contributed ~2-fold more to the total cell number in the spleen than in the bone marrow. At this same time point, ~25% of the Lin−Sca1+cKit+ population in the spleen, but not the bone marrow, were MPP2-derived. These data indicate that for specific HSPC populations, the splenic environment promotes their differentiation and self-renewal. Since we recently defined a role for the nuclear receptor, NR4A1, in regulating myeloid progenitor differentiation, we wondered if NR4A1 also affected HSPCs. Using NR4A1GFP mice, we discovered that HSPCs expressed higher levels of NR4A1 in the spleen compared to the bone marrow. Almost all NR4A1+ HSPCs in the spleen co-expressed VLA-4, a known HSPC niche retention factor. The few NR4A1+ HSPCs (~10%) in the bone marrow also expressed VLA-4 suggesting that NR4A1 affects the location of HSPCs either within the local niche or via mobilization to distant sites like the spleen. Together, our data highlight the importance of the splenic microenvironment in HSPC maintenance, differentiation, and localization, in part by the influence of NR4A1.
Full Length RAG2 Expression Enhances the DNA Damage Response in Pre-B Cells
Authors: Byrum Jennifer , Rodgers William A. , Rodgers Karla 
Published: January 2023

Abstract
V(D)J recombination of lymphocyte antigen receptor genes occurs via formation of DNA double strand breaks (DSBs) through activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. It is not known how developing immune cells respond to DNA damage in a manner that achieves optimal balance between continued cell development with genomic integrity. Studies from our lab show full length RAG2 exports from the nucleus and enriches at the centrosome within minutes following DNA damage in pre-B cells, exhibiting a response of RAG2 upon DNA damage. The exact mechanism by which RAG2 relocalization occurs is not known, however, mass spectrometry results identified three centrosomal proteins that interact with RAG2 strictly in the presence of DNA DSBs. To determine how RAG2 may affect cell viability upon genotoxic stress, we examined various cell responses versus time following irradiation of cells expressing either full length or mutants of RAG2. Our results indicate that RAG2 plays an active role in bolstering DNA damage responses to genotoxic stresses. Specifically, cells expressing full length RAG2, versus mutant forms, show an increase in the ability to cull damaged cells from the cell population, with evidence of more robust cell division several days after cells were subjected to genotoxic insult. Overall, we propose that while RAG2 functions to cleave DNA during V(D)J recombination, it also performs an alternate function to reinforce the cell’s response to DNA damage, thus facilitating continued development of immune cells to help generate a robust immune system.
Maturation of cord blood natural killer (NK) cells differs among neonates
Authors: Prince Amanda , Watkin Levi , Davis Valerie , Orange Jordan , Aagaard Kjersti , Mace Emily 
Published: January 2023

Abstract
The source, timing of colonization, and stability of the neonatal microbiome is largely unknown. We and other groups recently demonstrated that infant colonization may begin in utero, challenging the notion that neonates are born sterile. However, neonates are particularly vulnerable to viral infection partially due to dampened responses from NK cells and delayed maturation of adaptive immunity. Given the degree of variability in neonatal vulnerability to infection, we hypothesized that intrauterine exposure to microbes enables a greater diversity of NK cells in the neonates, relative to the adult, to allow for immune tolerance. Here, we performed multi-parametric flow cytometry analyses to dissect the normal range and expression of human NK cell developmental markers from umbilical cord (UC) blood (n=37). NK cells were identified as CD45+ CD3− CD56+ using FlowJo X and Cytobank. UC blood had a lower frequency of mature NK cells than adult peripheral blood. Furthermore, examination of NK cell maturation via spanning analysis of density events (SPADE) highlighted inter-individual diversity of neonatal NK cell subsets. Diversity of NK cells subsets between neonates and adults was significantly different with higher diversity in neonates (Simpson’s Diversity Index, p = 0.008). Our findings suggest diversity in stages of NK cell development between individual neonates that may reflect early immune exposure. Given ours and others recent work demonstrating that the intrauterine environment is not sterile, we speculate that microbial exposures during gestation may be a driving difference in the diversification NK cell populations in human infants and hence enable immune tolerance at the risk of neonatal viral vulnerability.
RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the RAG2 PHD region.
Authors: Rodgers William A. , Byrum Jennifer , Rodgers Karla 
Published: January 2023

Abstract
Association of RAG2 with the epigenetic marker H3K4me3 through a plant homeodomain (PHD) in the RAG2 non core domain is important for relieving auto inhibition of the RAG recombinase activity for V(D)J recombination. However, little is known regarding the properties of RAG2 that regulate its PHD-dependent interactions with H3K4me3. Accordingly, we measured the localization and dynamics of an N-terminal GFP fusion protein of full length RAG2 (GFP-FL); a T490A mutant of full length RAG2 (GFP-T490A) that does not undergo a regulatory phosphorylation proximal to the PHD region of RAG2; and GFP-labeled core domain of RAG2 (GFP-Core). The proteins were expressed in RAG2−/− pro-B cells, and the H3K4me3 was labeled using monoclonal antibody. Cross-correlation analysis showed that GFP-FL and GFP-Core exhibited similar colocalization with H3K4me3, which was significantly less than colocalization of GFP-T490A with H3K4me3. Blocking H3K4 demethylation using 2,4-pyridinedicarboxylic acid (PDA) caused GFP-FL/H3K4me3 colocalization to approach that of GFP-T490A and H3K4me3, whereas GFP-Core/H3K4me3 colocalization was unchanged. Super resolution imaging showed that RAG2 interactions with H3K4me3 were restricted to interfaces with H3K4me3-enriched puncta, and that GFP-T490A exhibited greater overlap with the H3K4me3-enriched puncta than GFP-FL. Finally, measurement of fluorescence recovery after photobleaching demonstrated that GFP-T490A had a lower mobile fraction and rate of diffusion than GFP-FL in conditions where RAG2 interactions with H3K4me3 were enhanced. These data suggest that RAG2 interactions with H3K4me3 are regulated by Thr490 proximal to the PHD region, such as by a phosphoryation/dephosphorylation cycle.
Ikaros regulates epigenetic and transcriptional programs in progenitor B cell leukemia
Authors: Schjerven Hilde , Ayongaba Etapong F. , Rodriguez Princess , Frietze Seth 
Published: January 2023

Abstract
Hematopoietic cell development is tightly controlled by a network of transcriptional and epigenetic regulators, many of which are mutated or have altered expression in hematological malignancies. Ikaros is a transcription factor that is critical for the proper development of several hematopoietic lineages, and essential for the B-cell lineage. It is recognized as a critical tumor suppressor in precursor B-cell lineage acute lymphoblastic leukemia (pre-B ALL), and is emerging to play roles also in other hematopoietic malignancies. In pre-B ALL, Ikaros mutations are particularly prevalent in the Ph+ (BCR-ABL1+) and ‘Ph-like’ subgroups of leukemia, and Ikaros mutations correlate with poor prognosis. To study the mechanisms of Ikaros tumor suppressor function, we developed a mouse model and a human model system with selective perturbation of Ikaros in Ph+ pre-B ALL cells. This has revealed conserved deregulated genes and pathways, and underscored the role of Ikaros in regulating progenitor-restricted gene programs. Furthermore, our recent studies have highlighted the role of Ikaros in regulation of chromatin structure, and revealed a novel role in epigenetic regulation. It is challenging to therapeutically target mutations in a tumor suppressor factor. It is therefore important to understand the mechanism of action and downstream targets to elucidate targetable vulnerabilities of the Ikaros-mutant leukemic cells. Investigating the tumor suppressor role of Ikaros in developing B cells also sheds light on its role in normal B cell development. We are using our newly established models of Ikaros-mutated pre-B ALL to study the underlying molecular mechanisms and downstream targets, and will present our results to-date at the meeting.
The Differential Role of GM-CSF and Flt3L on Myelopoiesis of Lineage Negative Bone Marrow Cells
Authors: Oh Sehwa , Choi Eun Young 
Published: January 2023

Abstract
During the inflammatory response, multiple cytokines drive myelopoiesis to expand innate cells. Of those cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and FMS-like tyrosine kinase 3 ligand (Flt3L) are known cytokines secreted during the immune response and drive myeloid and dendritic cell (DC) development. Though GM-CSF and Flt3L have been used for bone marrow differentiation culture, their specific role in myeloid and DC differentiation derived from lineage negative hematopoietic cells has not been studied. To compare the kinetics of myeloid and DC development under GM-CSF and Flt3L culture, we sorted and cultured lineage negative bone marrow (Lin− BM) cells from B6 mice in vitro using GM-CSF- or Flt3L-containing media. We found that Flt3L had the potential to produce both cDC1 (XCR1+) and cDC2 (CD11b+) DCs from the Lin− BM cells, whereas GM-CSF lacked the potential to produce XCR1+ cells. Flt3L culture preferentially differentiated Lin− BM cells into immature (MHCIIlo CD11cint) and mature (MHCII+ CD11chi) DCs, and monocytic Ly6C+ myeloid cells. However, GM-CSF culture condition influenced differentiation towards immature and mature cDC2s, and Ly6C+ monocytes and Ly6Ghi granulocytes. Flt3L and GM-CSF conditions were found to preferentially differentiate not only dendritic cell subsets but also myeloids. This study demonstrates the different roles of GM-CSF and Flt3L on myeloid and DC differentiation during in vitro Lin− BM culture.
HSC-requirement for macrophage regeneration is tissue-specific
Authors: Ghosn Eliver , Kuipers Hedwich , Waters Jeffrey , Sosa Jasmine , Phillips Megan , Herzenberg Leonore A. 
Published: January 2023

Abstract
The current paradigm posits that a single long-term hematopoietic stem cell (LT-HSC) is capable of regenerating all components of the immune system. However, recent studies have overturned this paradigm by showing that tissue-resident macrophages (in brain, skin, and liver) develop from fetal progenitors in the yolk sac that emerge separate from, and prior to, the LT-HSC.
Recently, we have identified novel subsets of tissue-resident macrophages in the mouse peritoneum, namely Small and Large Peritoneal Macrophages (SPM and LPM). Here, using Runx1 lineage-tracing assays, we show that, similar to brain microglia, both SPM and LPM emerge at around embryonic day 8 (E8), prior to, and independent of, the conventional LT-HSC. However, after E11, the fetal liver LT-HSC can be called upon to regenerate both SPM and LPM, but not microglia. Single-cell high-dimensional phenotypic and gene expression assays reveal that LT-HSC-derived SPM and LPM are phenotypically and functionally comparable to their E8-derived yolk sac counterpart. After birth, and throughout adulthood, both peritoneum and liver, but not the brain, call upon LT-HSCs in the bone marrow to continuously regenerate their fetal-derived tissue-resident macrophages.
These results suggest that the requirement for LT-HSCs to regenerate macrophages is tissue-specific. In rodents, both peritoneum and liver, but not the brain, might be exposed to injuries and other exogenous insults that could require constant regeneration of their tissue-resident macrophages. Collectively, these findings add a new layer to the complex developmental landscape of the myeloid lineage and challenge the current notion that tissue-resident macrophages develop independently of the LT-HSCs.
Matrix protein Tenascin-C expands and reversibly blocks maturation of eosinophil progenitors
Authors: Doan Ton C. , Jeong Brian M. , Coden Mackenzie , Abdala-Valencia Hiam , Berdnikovs Sergejs 
Published: January 2023

Abstract
While eosinophil depletion is a sought after strategy in treating allergic disease, little is known about the local tissue factors regulating eosinophils. In disease, eosinophils encounter provisional extracellular matrix (ECM) glycoproteins such as Tenascin-C (TNC), which strongly correlates with eosinophil expansion in tissues. While restricted in healthy adult lungs, TNC is a hematopoietic niche component in bone marrow stroma and is expressed de novo during wound healing or in pathologic epithelial remodeling in asthma. RNA-Seq shows that exposing naïve murine eosinophils to TNC upregulates immaturity markers (CD34, CD117, and Sca-1) and suppresses IL-5Rα. Thus, we hypothesized that TNC represents a factor in the provisional allergic tissue microenvironment that could support in situ eosinophil progenitor expansion. In murine bone marrow-derived cultures, TNC: 1) 3-fold expanded the available Lin−Sca1+ early precursor pool; 2) downregulated IL-5Rα expression on Lin− CD45+ c-kit+ cells during the eosinophil lineage commitment phase; 3) served as a reversible eosinophil maturation block, as TNC withdrawal rescued maturation and increased final eosinophil yield. Moreover, TNC knockout mice lack in situ lung expansion of Lin− c-kit+ CD34+ common myeloid progenitors and Lin− Siglec-F+ Sca-1+ cells in an allergic inflammation model as well as exhibit accelerated lung eosinophil maturation ex vivo. Adding TNC to lung homogenates cultured in IL-5 ex vivo suppressed eosinophil maturation in a manner consistent with TNC block of eosinophil maturation in bone marrow-derived cultures. Together, our results stress the local tissue factor potential to promote in situ expansion and eosinophil persistence in allergic disease.
An adjuvant-free mouse model of anaphylaxis to saline-soluble wheat protein
Authors: Gangur Venugopal , Jin Yining , Ng Perry KW. , Olson Eric 
Published: January 2023

Abstract
Wheat is a major food that can trigger life-threatening systemic anaphylaxis. Mechanism of sensitization to wheat anaphylaxis is not completely understood at present. For example, whether or not skin exposure to saline-soluble wheat protein (SSWP) can cause sensitization is unknown. Here we tested the hypothesis that transdermal exposure (TDE) to SSWP from durum wheat results in IgE response and clinical sensitization for anaphylaxis. Groups of Balb/c mice weaned onto a plant free diet received six TDEs with SSWP (1 mg/mouse/week) or saline over a 6 week period without adjuvant. Blood was analyzed for IgE responses by ELISA, and anaphylaxis and mucosal mast cell protease (mMCP-1) release upon challenge were quantified. Spleen tissue was analyzed for cytokines using a protein microarray system. Results showed significant IgE response, hypothermia shock response and elevation of mMCP-1 upon challenge. Spleen analysis showed significant elevations of IL-4/5/7/10/17B,E,F, and TSLP and significant reductions of IL-1a and IL-6. The following cytokines were not significantly altered: IL-1b/2/9/13/17A/22/23, IFNg and TNFa.
Engineered ovalbumin-expressing regulatory T cells protect from chicken egg white-induced allergy in vivo
Authors: Abdeladhim Maha , Zhang Aihong , Kropp Laura E. , Kim Yong Chan , Mitre Edward E.S. , Scott David W. 
Published: January 2023

Abstract
Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells. Our lab recently developed antigen-specific regulatory T cell therapies as a treatment for adverse responses in hemophilia (Kim et al. BLOOD 125: 1107, 2015; Yoon et al. BLOOD 129: 238–245, 2017). In this report, we created a chimeric antigen receptor (CAR) approach in which we engineered the target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells to induce tolerance in an allergy model. We named this receptor “BAR” for B-cell Antibody Receptor. Murine Tregs, retrovirally transduced with a BAR containing OVA or control cells, were successfully expanded in vitro and tested in a standard murine OVA-alum allergy model with high titered circulating anti-OVA IgE antibodies. We first confirmed that intravenously injected OVA-BAR Tregs did not directly lead to temperature drops indicative of anaphylaxis. Forty eight hours later, mice were challenged intraperitoneally with 200μg of OVA to induce an anaphylactic reaction, and temperature taken immediately again after challenge for 30 minutes. Our results demonstrated that OVA-BAR Tregs protected mice from hypothermia, whereas the control BARs or PBS-treated mice showed temperature drops indicative of anaphylaxis. These results were repeated and confirmed using human Tregs (CD4+CD25hiCD127low) transduced and expanded and injected into mice. These data provide proof of principle that engineered OVA-BAR Tregs may be useful to treat IgE based allergies.
Revisiting the role of B7-CD28 co-stimulation in thymic negative selection: B7-CD28 co-stimulation enforces central tolerance by clonal deletion and Treg generation as independent cellular processes
Authors: Watanabe Masashi , Hodes Richard J. 
Published: January 2023

Abstract
Self-Ag-reactive T cells are eliminated from the conventional mature T cell repertoire by clonal deletion and/or fate diversion to the Treg during thymocyte development as a consequence of strong recognition of self-peptide-MHC complex by the TCR, a process termed negative selection. However, the role of the B7-CD28 costimulatory pathway in this process has not been fully elucidated. We analyzed the role of B7-CD28 interaction in thymic negative selection using pMHCII tetramer binding to directly measure the endogenous T cell repertoire specific for distinct self-Ags. Ablation of expression of a self-Ag or of the Aire gene resulted in increase in the number of self-peptide-specific CD4SP thymocytes, consistent with the physiologic process of ongoing negative selection. We found that absence of B7 also resulted in an increase in numbers of conventional CD4SP (non-Treg) thymocytes specific for some but not all self-Ags. Observed increases in conventional CD4SP were far in excess of the concomitant decrease in the number of Treg that occurred in the absence of B7. In addition, the identity of B7-expressing cell populations required for clonal deletion was distinct from the requirements for Treg generation. Thus, the increase of CD4SP cell number specific for self-Ag was largely due to defect in B7 dependent clonal deletion, but not simply conversion of “want-to-be Treg” to conventional CD4SP cells. We propose that 1) thymic clonal deletion occurs through B7-CD28 dependent and independent pathways; 2) B7-CD28 dependent negative selection is mediated by clonal deletion and/or fate diversion to the thymic Treg lineage; and 3) B7-CD28 dependent clonal deletion is a largely independent cellular process from B7-CD28 dependent Treg generation.
Increased antioxidant activity mitigates age-associated dysfunction of thymic stromal cells in aged mice.
Authors: Cepeda Sergio , Xiao Yangming , Hester Allison K. , Venables Thomas , Griffith Ann 
Published: January 2023

Abstract
Age-associated thymic atrophy results in diminished production of new T lymphocytes and a concomitant decrease in responsiveness to new pathogens and vaccines. In addition to loss of thymic size with age, critical stromal functions, including tissue-restricted antigen (TRA) expression, are diminished with age. We previously identified deficiency of the hydrogen peroxide quenching enzyme catalase (CAT) in thymic stromal cells as a cause of thymic atrophy during aging, and established that thymic atrophy can be mitigated by genetic or dietary complementation of catalase antioxidant activity. Ongoing studies indicate that, in addition to increasing thymic cellularity, life-long overexpression of catalase in mitochondria of transgenic mice (mCAT Tg) results in increased TRA expression and decreased numbers of T cells binding self-peptide MHCII tetramers in aged mice. We also find mitigated acquisition of an aged global gene expression signature in aged mCAT Tg mice relative to non-transgenic littermates. Together, these results suggest that complementing catalase activity in thymic stromal cells mitigates age-associated declines in thymic function.
Supported by N.I.H. R01AI121367 and N.I.H. R01AI121367-S
The Role of B7-CD28 and CD40L-CD40 costimulatory signaling in thymic Invariant NKT Cell subset development
Authors: Alkhaleel Farrah , Watanabe Masashi , Hodes Richard J. 
Published: January 2023

Abstract
Invariant Natural Killer T (iNKT) cells are a unique subset of NKT cells that express a highly restricted αβ T cell receptor (TCR) and are selected in the thymus by recognition of Glycolipid/CD1d complex expressed by double-positive thymocytes. We previously demonstrated that thymic iNKT cell development is highly sensitive to B7-CD28 costimulatory interactions, with substantially decreased numbers of thymic iNKT cells in B7 and CD28 knockout mice. Advances in iNKT subset identification have allowed the detailed study of three major iNKT subsets, NKT1, NKT2, and NKT17, defined by characteristic transcription factor expression. We have analyzed the requirement for B7-CD28 and CD40-CD40L costimulatory signaling for development of thymic iNKT subsets. Comparison of BALB/c wild-type, B7 knockout, and CD28 knockout mice showed a substantial decrease in number of all thymic iNKT subsets in B7 and CD28 knockouts, demonstrating a critical role for B7-CD28 signaling in development of all thymic iNKT subsets. In contrast, CD40 knockout and CD40L knockout mice showed a substantial selective decrease in NKT2 cells with less effect on NKT1 and NKT17 subsets. These findings identify distinct requirements for B7-CD28 costimulation in thymic development of all iNKT subsets and for CD40L-CD40 costimulation selectively in NKT2 subset development. Further experiments are currently in progress examining the cellular requirements for B7 and CD40 expression in NKT development using conditional B7 and CD40 knockouts and complementary bone marrow mixed chimera approaches.