bluuebunny/crossref_metadata_2025_split huggingface dataset:
T cell receptor V beta usage in HLA-DQ6αβ8αβ mice displaying spontaneous polyarthritis (137.31)
Authors: Sidhu Maninder , Bradley David S. 
Published: January 2023

Abstract
Spontaneous polyarthritis (SP), an autoimmune disease that develops spontaneously in HLA-DQ6 αβ8aαβ transgenic (tg) mice, is characterized by lymphocyte infiltration in joints leading to chronic polyarthritis in the extremities similar to human rheumatoid arthritis. We examine here the CD4 T cell receptor (TCR) expression in the DQ6/8 tg mice during SP. CD4 T cells were isolated from the lymph nodes of arthritic and non-arthritic DQ6/8 tg mice at various time points, phenotyped via flow cytometry with V beta usage characterized in the CD4+CD25+ T cell population. Preferential usage of V β8.2 was increased from 9% in 2 month naïve mice to 15% in 4 month old and 20% by 8mo old arthritic mice. V β17 expressing T cells also increased from 0.02% in 2 month naïve mice to 2.8% in 4 month old arthritic mice. This is consistent with other studies that have indicated an association of Vβ8.2 TCR in the Experimental Autoimmune Encephalomyelitis and collagen-induced arthritis models. These results support our hypothesis that activated CD4 T cells expressing Vβ8.2 TCR are the predominantly pathogenic; autoreactive T cells involved in SP. Selective depletion of these T cells using anti-Vβ8.2 antibodies will confirm their involvement. Application of such strategies of immune intervention can be helpful in humans to control or reduce the disease progression in the stages of disease initiation. The study was supported by NIH-NIAMS AR45657
Synergistic role of diacylglycerol kinases α and ζ in T cell development and self-tolerance (137.37)
Authors: Zhong Xiaoping , Wan Chi-Keung , Guo Rishu 
Published: January 2023

Abstract
Signal for the T cell receptor (TCR) plays critical roles in T cell development and function. TCR signaling can induce positive or negative selection of thymocytes, induces activation or anergy of mature T cells, and induce conventional T cell activation or regulatory T cells to suppress immune function. The mechanisms that modulate TCR signaling to direct these distinct outcomes have been poorly understood. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA) through phosphorylation. In mammals, ten DGK isoforms have been identified. Using both a gain of function and a loss of function approaches, we have found that DGK ( and ( are important regulators for T cells. Enhanced DGK activity inhibits TCR signaling and T cell maturation. Deficiency of either DGK( or ( causes T cells hyperresponsive to TCR stimulation and resistant to anergy induction. Loss of both DGK( and ( in mice results in impairment of positive selection, spontaneous T cell activation, impairment of regulatory T cell function, and autoimmunity. We further demonstrate that DGKs function both as signal terminator by inhibiting DAG-mediated signaling and as signaling initiator by generating PA.
Innate immune CD11b+Gr-1+ cells, myeloid- derived suppressor cells, influence the adaptive immune response during demyelinating disease (137.6)
Authors: Olson Julie K , Bowen Jenna L 
Published: January 2023

Abstract
Innate immune CD11b+Gr-1+ cells are a heterogeneous population of cells which include neutrophils and myeloid- derived suppressor cells (MDSC). CD11b+Gr-1+ cells that express Ly6C are MDSC which can directly suppress T cell responses. Theiler's murine encephalomyelitis virus (TMEV) is a mouse model of multiple sclerosis in which mice develop a chronic progressive demyelinating disease associated with a myelin- specific CD4+ T cell response. We have previously shown that the innate immune response directly affects the initiation and progression of demyelinating disease. Thus, we wanted to determine the role of CD11b+Gr-1+ cells in the innate immune response during TMEV- induced demyelinating disease. TMEV- infected mice had a large number of infiltrating CD11b+Gr-1+ cells composed of Ly6Chigh cells in the central nervous system (CNS) during the innate immune response. Depletion of Gr-1+ cells in TMEV-infected mice resulted in increased expression of IFNγ, IL-12, and IL-17 in the CNS during the innate immune response and altered development of demyelinating disease. Most importantly, CD11b+Gr-1+ cells suppressed the activation of CD4+ and CD8+ T cells in TMEV- infected mice. These results suggest that innate immune CD11b+Gr-1+ cells, specifically the MDSC, influence the adaptive immune response and modulate the development and progression of demyelinating disease.
This work was supported by the National Multiple Sclerosis Society Grant RG3625.
NK cell development from a novel progenitor found in the murine lung (138.10)
Authors: Halim Timotheus You Fu , Takei Fumio 
Published: January 2023

Abstract
NK cells in the lung are phenotypically different from those in the spleen or bone marrow (BM). We have identified NK cell progenitors (NKPs) in normal mouse lung. Lung NKPs are lineage- NK1.1- CD122+ B220+/- and are phenotypically similar to conventional BM NKPs. Cells with the same phenotype are also found in peripheral blood. Lung NKPs cultured on OP9 stromal cells with cytokines expand over 2×104 fold and mature into NK1.1+CD3- NK cells. The frequencies of NKPs among the lineage-NK1.1-CD122+B220+ population in the lung and lineage-NK1.1-CD122+B220- cells in the BM are 0.16 and 0.14, respectively. However, NK cells generated in vitro from lung NKPs and BM NKPs significantly differ in the expression of the Ly49 family of NK cell receptors. Most NK cells develop in vitro from lung NKPs express multiple Ly49s whereas most of those develop from BM NKPs express CD94/NKG2 but not Ly49. Lung NKPs develop into CD3-NK1.1+ NK cells upon adoptive transplant into NOD/SCID IL2Rγ-/- hosts. Thus, we have identified NKPs in the lung. They likely develop in the BM and migrate to the lung through peripheral blood. This supports the emerging hypothesis that NK cell development includes final maturation stages outside of the BM, where the local microenvironment influences development. The contribution of lung NKPs to the lung NK cell population in vivo is currently being investigated.
KPNA1 is a putative substrate of the RAG1 ubiquitin ligase (138.11)
Authors: Jones Jessica M , Simkus Carrie , Bhattacharyya Anamika 
Published: January 2023

Abstract
The RAG1 V(D)J recombinase encompasses DNA binding/cleavage and ubiquitin ligase activities. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1 in a reaction supported by the ubiquitin conjugating (E2) enzyme UbcH2/Rad6 or UbcH5a. KPNA1 is the first putative substrate identified for the RAG1 ubiquitin ligase. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. RAG1 did not promote rapid ubiquitin chain extension following mono-ubiquitylation of substrate, regardless of the E2 used. Substitutions of amino acids surrounding the third, non-canonical Zn coordination site of the RAG1 RING domain abrogated functional interaction with E2 enzymes, and this was significantly correlated with reduction in the ability of full length RAG1 to support recombination of extra-chromosomal substrates. These data suggest that RAG1-dependent mono-ubiquitylation of a substrate, possibly KPNA1, is required for optimal levels of V(D)J recombination.
Polychromatic immunophenotyping and probability binning algorithm reveal distinct disease clusters in patients with Common Variable Immunodeficiency (138.12)
Authors: Kalina Tomas , Stuchly Jan , Janda Ales , Ruzickova Sarka , Litzman Jiri , Sediva Anna , Vlkova Marcela 
Published: January 2023

Abstract
Common Variable Immunodeficiency (CVID) is a disease where only small fraction of cases has known molecular defect. Classification of CVID patients is based on B-cell phenotype and count.
Using polychromatic flow cytometry we set out to analyze B-cell phenotype in a single six-color tube in a cohort of 49 patients and 50 controls. The aim of the study was to find an expert free approach for phenotype analysis that would cover all differences in the 6-color space and form groups of patients with similar phenotype. We used "probability binning" algorithm for a creation of 1024 bins (eg. six-color gates) that were optimally covering the cells' distribution in the whole B-cell compartment. We have created a matrix file with numerical representation of all cells in all bins. Next, we analyzed this matrix in MeV software, which was developed originally for the microarray data analysis. "Self-Organizing Tree Analysis" created a tree of related individuals. First branching split the whole cohort into control-like phenotype branch (28% of CVID patients, 5 clusters) and CVID branch (94% of CVID patients in 6 clusters). Independent sample from the same patient 3-13 month later was clustered to either the same (11 pts), neighbor (4 pts) or further away (2 pts), thus confirming the reproducibility of this approach.
The "signature" bins for each cluster were characterized.
Grant support MSM0021620813 and IGA NR/9198-3
Role of Ikaros in mast cell development and differentiation (138.21)
Authors: Rao Kavitha N , Gregory Gregory D , Winandy Susan , Brown Melissa A 
Published: January 2023

Abstract
The transcription factor Ikaros has been implicated in the regulation of hematopoiesis at the level of T, B, NK and dendritic cells. In this study we examine the role of Ikaros in the development and differentiation of mast cells, using mice lacking Ikaros (Iknull). In vitro derived bone marrow mast cells (BMMCs) from Iknull mice expressed decreased levels of mRNA encoding transcription factors required for optimal mast cell development (MITF, GATA1, GATA2 and STAT5a), and mast cell-specific proteases (chymase, tryptase and carboxypeptidase). Upon activation through FcεRI crosslinking, Iknull mast cells revealed a hyper-activation phenotype as assessed by increased degranulation, cytokine secretion and expression of co-stimulatory molecules including OX40 ligand. Furthermore, Iknull BMMCs expressed reduced levels of several cell surface adhesion molecules and integrins including CD49d (integrin alpha 4), which is required for homing of mast cell precursors to the intestine. Consistent with this observation, in vivo examination of Iknull mice for presence of mast cells in various tissues revealed a striking deficiency of mast cells in the peritoneal cavity, and limiting dilution analysis demonstrated severely reduced number of mast cell precursors in the intestine. Taken together, our data suggest that Ikaros plays an important role in mast cell development, differentiation and activation.
This research has been supported by JDRF-3-2007-617 and NIH RO1 CA047992.
The transcription factor E47 controls the cell cycle quiescence and development of multipotent hematopoietic progenitors (138.3)
Authors: Yang Qi , Kardava Lela , Leger Anthony St , Martincic Kathleen , Varnum-Finney Barbara , Bernstein Irwin D , Milcarek Christine , Borghesi Lisa 
Published: January 2023

Abstract
It is of great interest to understand the mechanisms controlling the development and maintenance of multi-potential bone marrow progenitors, the key hematopoietic compartment that seeds the entire immune system. Using E47 knockout (KO) mice, we found that the transcription factor E47 plays a critical role in the cell cycle regulation and development of the multipotent hematopoietic subsets. Multipotent LSKs (Lineage-Sca-1+c-Kit+) from E47 KO mice had significantly increased BrdU incorporation, decreased proportion of progenitors in G0, and hypersensitivity to a mitotoxin 5-Fluorouracil, suggesting loss of cell cycle quiescence. Total LSKs contain both self-renewing hematopoietic stem cells (HSCs) and non-renewing multipotential progenitors (MPPs). Using three independent phenotypic definitions, we found that E47 KO mice have numerically unperturbed HSCs but a 2-4 fold reduction in the MPPs. Moreover, the residual MPPs in E47 KO mice fail to fully up-regulate flk2 and lack V(D)J rearrangement, suggesting compromised lymphoid differentiation potential. Finally, both loss-of-function and gain-of-function experiments identified a key stem cell regulator, p21, as E47 target in the multipotent LSKs. Taken together, our data define an important role of the transcription factor E47 in controlling the development, differentiation and cell cycle regulation of multipotent hematopoietic progenitors.
Vasorin Expression During Mouse B cell Development (138.31)
Authors: Franco Daniel A. , Reardon Christopher L. 
Published: January 2023

Abstract
The stages of B cell development are delineated by stage-specific timing of gene and cell surface marker expression coinciding with exposure to cytokines and growth factors. Transforming growth factor beta (TGF-β) has been previously shown to affect lymphopoesis, as well as more mature processes, such as B-cell isotype switching. Receptors for TGF-β are diverse and widely distributed but display some tissue specificity. Here we describe expression of a Type-I TGF-β membrane receptor, vasorin, previously reported to be almost exclusively expressed by vascular tissue. Using RT-PCR analysis, the message for vasorin was identified in mouse pre-B cell lines prior to BCR gene rearrangement. Thus, vasorin expression may be closely linked to early lymphocyte development at the pro and pre-B stages. To verify that this finding was not an artifact of the cell lines, we looked for vasorin message in primary cell suspensions of spleen, thymus and liver from neonatal mice. Vasorin gene expression is readily detected in the primary repositories of B cell development, the spleen and liver, but not in primary thymocytes. These data suggest that receptor binding of TGF-β may be more vital to B-cell than to T-cell development. Vasorin expression by progenitor B cells may ensure measured proliferation via TGF-β receptor signaling.
VH Gene Usage and Restricted B cell Development (138.4)
Authors: Robbins Gregory R. , Knight Katherine L. 
Published: January 2023

Abstract
Signaling through the preBCR is an important checkpoint in B cell development in which the capacity for μ-chain to pair with surrogate light chain (SLC) is tested. Mutant ali/ali rabbits that lack the predominantly rearranged gene, VH1, use VH4 in most VDJ gene rearrangements. Surprisingly, the periphery of these rabbits is initially dominated by VHn-utilizing B cells, not VH4-utilizing B cells. We hypothesize that development of VH4-utilizing B cells is blocked in the bone marrow at the preB cell stage due to weak interaction of the μ-chain with SLC. The IgH CDR3 is important for formation of, and for signaling through the preBCR. Previous crystal structure analysis demonstrated that IgH CDR3 interacts with both components of the SLC, and raises the possibility that VH4-utilizing μ-chains require CDR3 of a particular size and/or amino acid sequence for SLC pairing. We compared IgH CDR3 sequences of VH4- and VHn-utilizing B cells and although we found no obvious differences in amino acid sequences, we did find that the CDR3 of VH4-utilizing B cells was significantly shorter than those found associated with VHn VDJ genes. We hypothesize that long IgH CDR3s encoded by VH4-utilizing VDJ genes prevent pairing with SLC. We are using in vitro experiments to test if pairing of VH4-derived μ-chains with SLC is restricted by CDR3 length. These studies will further identify the structural parameters required for preBCR formation.
IgE Signaling Selectively Suppresses FceRI Beta Chain Expression (139.2)
Authors: Ryan John J. , Barnstein Brian , Brenzovich Jennifer 
Published: January 2023

Abstract
Mast cells are critical players in allergic disease mediated by IgE and FcεRI interactions. In mice, FcεRI functions as a tetramer, composed of an IgE binding alpha subunit, two ITAM bearing gamma subunits, and a signal enhancing/amplifying beta subunit. Little is known about beta chain regulation. We activated mast cells with IgE + antigen and determined the resulting effects on beta chain mRNA, protein levels and surface expression. IgE crosslinkage inhibited beta chain mRNA expression, with a nadir at 5 hours post-stimulation. This was matched by reduced expression of the beta chain, with no change in the alpha or gamma subunits. A truncated form of beta, termed beta-t, may suppress IgE receptor expression. We found that IgE-mediated signaling also inhibited beta-t expression, and that beta-t was slower to return to baseline levels than conventional beta. We were able to classify the beta chain mRNA regulation pathway as primarily involving Fyn, Syk, PI3K, and NFκB. Monomeric IgE is known to greatly increase surface FceRI expression. Interestingly, we found that IgE crosslinkage returned membrane FceRI to basal levels in cells previously incubated with monomeric IgE. Furthermore, the presence of antigen during culture with IgE prevented FceRI upregulation. These data suggest that FceRI signaling not only activates mast cells but may also convey a homeostatic signal returning FceRI expression to a reduced basal level, perhaps to suppress continued signaling. Loss of this suppressive effect could be art of atopic disease etiology. Supported by NIH grants 1R01 AI59638 and U19A1077435.
The peptidyl-prolyl isomerase Pin1 suppresses Bax activation and mitochondrial targeting in human eosinophils (139.7)
Authors: Shen Zhong-Jian , Malter James S 
Published: January 2023

Abstract
Pulmonary eosinophilic inflammation is a defining feature of allergic asthma. Within a few days of allergen challenge, airway eosinophils (Eos) increase by ~20-fold in both human and animals. Genetic ablation of Eos markedly attenuates submucosal matrix deposition, airway smooth muscle hyperplasia as well as airway hyperresponsiveness, suggesting a critical role of these cells in asthmatic lung pathology. Normally short-lived (~3 days) peripheral blood Eos acquire prolonged survival (&gt;7 days) in the lung by contacting with anti-apoptotic cytokines GM-CSF and IL-5, which increase after allergen exposure. How these cytokines suppress Eos apoptosis remains largely unknown. Here we show that Pin1 mediates GM-CSF/IL-5 signaling, inhibits activation of proapoptotic Bax and its mitochondrial disruption. Both cytokines considerably increased Pin1 isomerase activity while induced Erk1/2-mediated phosphorylation of Bax at Pin1 recognition sites, allowing Bax interaction with Pin1. Pin1 blockade disrupted this interaction, resulting in Bax N-terminal activation, mitochondrial translocation and downstream caspase 9 activation even in the presence of survival cytokines. Our study uncovers previously unknown mechanism by which Pin1 isomerization regulates Bax activation. Pin1 may be an attractive therapeutic target for the asthma and other eosinophilic diseases.
Negative Regulation of Human Th2 cells by Type I Interferon (140.15)
Authors: Huber Jonathan P , Chowdhury Fatema Z , Gill Michelle A , Gruchalla Rebecca S , Farrar J David 
Published: January 2023

Abstract
Type I interferon (IFN-α/β) is a potent anti-viral cytokine that regulates multiple aspects of innate and adaptive responses to viral infections. As some respiratory viral infections have been linked to the etiology and exacerbation of asthma, we wished to determine the effects of IFN-α/β on driving the development of human Th2 cells. In these studies, we found that IFNα/β inhibited the ability of IL-4 to promote Th2 differentiation and significantly de-stablized cytokine expression from fully polarized Th2 cells. Further, this effect was specific to IFNα/β as neither IL-12 nor IFNγ inhibited IL-4-regulated Th2 development or stability. IFNα/β inhibited Th2 development by blocking the induction of GATA-3, and this effect was found to bypass the induction of GATA3 via IL-4-mediated STAT6 activation. Finally, IFNα/β also potently inhibited Th2 cytokine secretion from fully committed CRTH2+ cells isolated directly from peripheral blood. Thus, we have found a novel function of IFNα/β in the inhibition of Th2 responses that may be useful as a therapeutic approach for the treatment of asthma.
Humanization of an anti-CεmX monoclonal antibody 4B12 for potential clinical use in targeting mIgE+B cells (140.19)
Authors: Wu Pheidias Che-Hao , Chen Jiun-Bo , Hung Alfur Fu-Hsin , Hsueh Ying-Chao , Chang Tse Wen 
Published: January 2023

Abstract
The CεmX domain of 52 a.a. residues, located between the CH4 domain and membrane-anchor segment of human membrane-bound ? chain on mIgE+ B cells, is a unique antigenic site for the immunological targeting of those B cells. We have demonstrated that a chimeric form of anti-CεmX monoclonal antibody (mAb), 4B12, can induce apoptosis and ADCC against a human mIgE.Fc+ B cell transfectoma via effector cells from PBMC in vitro (presented by another abstract in this meeting). To enhance the clinical applicability of 4B12, we have humanized it by first grafting its six complementary-determining regions (CDRs) into human Ig VH subgroup IV and V? subgroup II frameworks (FWs). Those residues in the FWs of VH and VL of the parental murine 4B12 that are essential for Fv dimer interaction and CDR conformation were identified by analyzing the binding affinity of a number of designed Ab variants with site-directed mutations, using competitive ELISA and surface plasmon resonance analysis. A humanized 4B12 retaining the affinity of its parent was obtained by adopting all FWs of the selected human V? template without any change and all FWs of the selected VH with only one residue change. The Val at position 71 of the human VH was replaced by Arg at the corresponding position of the murine VH. Based on molecular modeling analysis, it is likely that such a change is probably crucial for maintaining the proper structural packing of Arg71 with Ile29 in the CDRH1 and Gly55 in the CDRH2. The KD of the chimeric and humanized 4B12 are 5.37×10-9 M-1 and 5.76×10-9 M-1, respectively. Affinity enhancement of humanized 4B12 by selective CDR mutations is in progress.
Anti-CmεX monoclonal antibodies potentially applicable for targeting mIgE-expressing B cells (140.20)
Authors: Chen Jiun-Bo , Wu Pheidias C. , Hung Alfur Fu-Hsin , Yu Hui-Ming , Chang Tse Wen 
Published: January 2023

Abstract
On B lymphocytes, membrane-bound IgE (mIgE) is part of BCR and essential for generating isotype-specific IgE response. On mIgE+ B cells in man, the membrane-bound ε chain (mε) exists predominantly in the long form (mεL), containing 52-a.a. CεmX domain between CH4 and the C-terminal membrane-anchoring segment; the conventional form of mε (mεS) exists in minor proportions. CεmX thus provides an attractive site for immunological targeting of mIgE+ cells. Here we have shown that 9 newly prepared CεmX-specific monoclonal antibodies (mAbs), as well as previously reported a20, can bind to mIgE.FcL-expressing CHO cells, while only 4B12 and 26H2 can bind to mIgE.FcL-expressing B cell line Ramos cells. In epitope mapping studies using synthetic peptides representing different parts of CεmX, 4B12 was shown to bind to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CεmX. These results suggest that the C-terminal part of CεmX is sterically hindered for mAb binding by associated components in the BCR. The chimerized mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, could lyse mIgE.FcL-expressing Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 could cause ADCC against mIgE.FcL-expressing Ramos cells in a does-dependent fashion. These results indicate that anti-CεmX mAbs, 4B12 and 26H2, can bind to native mIgE on B cells and are potentially useful for targeting mIgE+ B cells for controlling IgE production.