bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endogenous naive CD8 T cell precursor frequency and infection type control CD62L gene expression (85.6)
Authors: Obar Joshua , Khanna Kamal M. , Pham Quynh-Mai , Lefrancois Leo 
Published: January 2023

Abstract
CD62L is essential for homing of lymphocytes to lymph nodes. Moreover, CD62L expression identifies distinct lineages of memory CD8 T cells. However, the factors regulating memory cell lineage differentiation are ill defined. Our data show that CD62L gene regulation is controlled by the input number of transferred antigen-specific CD8 T cells. To enable analysis of endogenous antigen-specific CD8 T cells, we have now utilized a method to enrich naïve antigen-specific CD8 T cells using MHC class I tetramers in conjunction with magnetic bead separation. Of three specificities tested thus far, naïve precursor frequencies ranged from ~100–1000 cells/mouse. Interestingly, following infection with recombinant vesicular stomatitis viruses, the naïve precursor frequency inversely correlated with the time required for the resulting memory population to shift to CD62L+ cells. Remarkably, in the case of ova-specific CD8 T cells, the shift toward CD62L+ ova-specific memory cells occurred substantially faster following infection with Listeria monocytogenes expressing ovalbumin than after VSV-ova infection. This system will allow us to examine parameters of the endogenous CD8 T cell response to infection from initial activation to memory development.
This work was supported by NIH grants AI41576 and DK45260 (LL) and a Damon Runyon Foundation fellowship (KMK).
Memory B Cells Assort by Phenotype, BCR Repertoire & Genotype (86.1)
Authors: McHeyzer-Williams Louise , O’Connor Adam P , McHeyzer-Williams Michael G 
Published: January 2023

Abstract
Effective vaccination requires the generation of high-affinity B cell memory. After immunization with T-dependent antigens, multiple cellular subsets of antigen-specific memory B cells develop. Specifically, following the germinal center (GC) reaction, three B cell subsets emerge: two non-secreting distinct subsets of memory B cells are apparent (6B2hi post-GC and 6B2lo pre-plasma memory cells) as well as secreting CD138pos plasma cells. All three B cell compartments express isotype switch, affinity matured antigen-binding antibody. In the current study, we focus on the IgG1+ memory B cell response to antigen recall in vivo to deepen the molecular and genetic analysis of the two memory subsets and their short-lived plasma cell progeny. Single cell RT-PCR repertoire analysis indicates a majority of VH186.2 variable region gene usage among all three NP-specific memory responders with distinguisable groups of clonotypes based on junctional and somatic variations in the BCR. Notably, BCR associated with the highest affinity for antigen preferentially assort into the 6B2lo pre-plasma memory compartment. Based on gene expression profile and Q-PCR analysis the two memory B cell subsets express unique developmental programs that control subsequent cell fate and impact memory B cell function upon antigen recall.
The role of c-Rel in EBV-mediated B cell survival (86.10)
Authors: Valentin Anibal Jose , Lu Kristina T. , Covey Lori R. 
Published: January 2023

Abstract
LMP1 is an Epstein-Barr virus encoded protein that is expressed on the surface of latently infected cells and is known to promote cell survival through activation of the NFκB pathway. Previously our lab developed an immortalized B cell line from a patient with non-X-linked HIGM in which LMP1 is conditionally expressed under the control of a tetracycline-inducible promoter (pt1-LCLtet cells). Analysis of these cells revealed an increased level of apoptosis even in the continuous presence of LMP1 stimulation. In addition, analysis of NFκB/Rel subunits showed a markedly lower expression of c-Rel (c-Rel75) and the presence of a novel 25 kD form (c-Rel25) in pt1but not control-LCLtetcells. Western analysis of immunoprecipitated c-Rel revealed several higher mw forms in pt1- vs. control-LCLtet cells. To investigate the phosphorylation status of c-Rel75 in the pt1-LCLtet cells, in-vivo labeling was carried out and immunoprecipitation of extracts with anti-c-Rel antibodies revealed that c-Rel75 is hyperphosphorylated in these cells. Therefore, we hypothesize that hyperphosphorylation of c-Rel75 targets a post-translational processing event that results in both the reduced level of c-Rel75 and the appearance of the 25 kD form observed in pt1-LCLtet cells. Importantly, the low abundance of c-Rel75 appears to be closely linked to ongoing apoptosis which is not inhibited by LMP1 signaling.
(Supported by AI37081)
The Role of STAT-3 and LIF in regulating IgM secretion in B-1 cells: CD25+ B-1 cells and their characteristics (86.12)
Authors: Tumang Joseph R , Holodick Nichol , Kaku Hiroaki , Rothstein Thomas L 
Published: January 2023

Abstract
B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we identify a novel subset of B-1 cells that express the high affinity IL-2 receptor (IL-2Ra/CD25) and preferentially have the activated form of the signal transducer and activator of transcription-3 (STAT3). We show that upon inhibition of pSTAT3, Ig secretion is elevated preferentially in the CD25+ subset. Consistent with the induction of Ig secretion, we detected increases in the expression of key regulators of the plasmacytic program, B lymphocyte inducer of maturation program 1 (BLIMP-1) and X-box binding protein 1 (XBP-1). In contrast to the CD25- subset of B-1, we also found that the CD25+ subset differentially expresses a number of genes including the STAT3 target SOCS3, the cytokine receptor GP130 and the leukemia inhibitory factor receptor (LIF-R). We demonstrate that treatment of B-1 cells with LIF induces Ig secretion preferentially in the CD25+ subset. Together, these data identify STAT3 and LIF as possible regulators of Ig secretion in B-1 cells. Furthermore, these results implicate a role for cytokines in regulating B-1 Ig secretion.
Differential regulation of NF-κB survival signaling in transitional and mature Fo B cells (86.13)
Authors: Castro Iris , Hoek Kristen L. , Shinners Nicholas P. , Khan Wasif N. 
Published: January 2023

Abstract
The earliest transitional (T1) B cells are significantly more sensitive to cell death than the late transitional (T2) and mature follicular (Fo) B cells and aberrant survival of T1 B cells can lead to autoimmune disease. While it is known that survival of splenic B cells is predominantly regulated by B cell antigen receptor (BCR) and B cell activation factor receptor (BAFF-R), the mechanisms by which these receptors mediate survival is less clear. We have investigated the developmental stage-specific regulation of anti-apoptotic genes and the transcription factor NF-κB, which is a major modulator of these genes. We found that in vivo, T1 B cells express the highest levels of Mcl-1 and Bcl-xL, whereas mature Fo B cells predominantly express Bcl-2. Consistent with T1 cell sensitivity to cell death following BCR crosslinking, T1 B cells only weakly induced the expression of anti-apoptotic genes (Mcl-1 and Bcl-xL) relative to robust induction in T2 and mature Fo B cells following BCR stimulation. BCR-induced expression of NF-κB (NF-κB1, RelA, cRel, NF-κB2 and RelB) coincides with that of anti-apoptotic genes; lowest in T1 and highest in mature Fo B cells. These findings suggest that T2 and mature Fo B cell survival may be linked to sustained expression of NF-κB and anti-apoptotic genes.
This study is supported by NIH RO1 A1060729
Gene expression in Blimp-1-transduced fibroblasts (86.16)
Authors: Buldak Gerald L , Frank Matthew , Jackowski Suzanne , Brewer Joseph W. 
Published: January 2023

Abstract
B-lymphocyte-induced maturation protein-1 (Blimp-1) is an important regulator in the development of several immune cell lineages, as well as embryogenesis, where it is expressed in all three primordial germ layers. The best understanding of mechanistic regulation by Blimp-1 comes from B cells, in which it is a master regulator of terminal differentiation into professional secretory plasma cells (PC). Blimp-1 expression results in the induction of another master regulator called X-box-binding protein 1, or XBP-1. Both genes are critical for PC differentiation; it is difficult, however, to identify genes regulated by Blimp-1 independently of XBP-1. In other cell types, knowledge is limited to either the requirement, or temporal and spatial localization, of Blimp-1 expression.
To better understand the genes and pathways regulated by Blimp-1, we have developed a reductionist system to investigate its effects in NIH-3T3 fibroblasts. Blimp-1-expressing fibroblasts are viable to at least 48 hours, exhibit morphological changes, and altered gene expression profiles, without inducing XBP-1. Therefore, this system can be used to analyze XBP-1-independent events downstream of Blimp
J.W.B. is supported by NIH-GM61970, S.J by NIH-GM45737, and G.L.B by NIH-T32 AI007508.
SOCS3 regulates B lymphopoiesis at the immature B cell stage (86.4)
Authors: Le Yi , Zhu Bing-Mei , Hennighausen Lothar , Silberstein Leslie E 
Published: January 2023

Abstract
CXCL12-mediated FAK phosporylation and sustained pro-adhesive responses occur uniquely in progenitor bone marrow (BM) B cells, but not in mature peripheral B cells. Since CXCR4 receptor expression is similar at all B cell stages, we hypothesized that progenitor and mature B cells possess different mechanisms to control FAK phosphorylation. Here we demonstrate that Suppressor of Cytokine Signaling 3 (SOCS3) regulates CXCL12-induced FAK phosphorylation through the ubiquitin-proteosome pathway. CXCL12 triggers increased FAK ubiquitination in peripheral B cells and spleen B cells, but not in BM B cells. In accordance with this finding, SOCS3 expression is relatively low in progenitor BM B cells and high in peripheral blood and spleen B cells. Over-expression of SOCS3 results in decreased CXCL12-induced FAK phosphorylation and impaired adhesion to VCAM1. Next, we examined B lineage cells from MMTV-Cre SOCS3fl/flmice in which SOCS3 protein expression was reduced greater than 95%. SOCS3 deficient splenic B cells have increased and prolonged FAK phosphorylation and enhanced sustained adhesion to VCAM-1, similar to progenitor B cells. MMTV-Cre SOCS3fl/flmice have a two-fold increase in the absolute number of IgM+,IgD−, immature B cells in BM compared to wild type mice (n=8, p&lt;0.01). IL-7 dependent pre-B cell proliferation was unaffected by SOCS3 deficiency. However, withdrawal of IL-7 induced the generation of immature B cells that survived longer in culture compared with wild type cells. Taken together, our results suggest that SOCS3 may be an important negative regulator of immature B cells by affecting their survival and pro-adhesive responses in the bone marrow microenvironment.
THE HEAVY METAL MERCURY MODULATES p-ERK1/2 IN ACTIVATED B-CELLS (86.7)
Authors: Laiosa Michael , Rosenspire Allen , McCabe Michael 
Published: January 2023

Abstract
The heavy metal mercury (Hg) is known to have immunomodulatory properties affecting lymphocyte signal transduction, death receptor signaling and autoimmunity. We tested the hypothesis that Hg exposure modulates B-cell activation in response to BCR stimulation. To test this hypothesis; WEHI-231 immature B-cells, were stimulated with Goat-anti-IgM in the presence or absence of 5 μM HgCl2. We found a significant reduction in p-ERK1/2 in the presence of Hg by Western blot. However, it is unclear whether the reduction of BCR dependent pERK is a consequence of Hg modulating the amount of p-ERK1/2 in each cell, or whether Hg is reducing the number of responding cells in the population. Single cell proteomics were employed to address whether pERK1/2 is modulated on a per-cell-basis using a ERK phosphospecific fluorochrome labeled antibody. We found nearly 80% of the WEHI cells, possess pERK1/2 by 5 minutes after stimulation. Interestingly, pre-incubation with HgCl2 causes a 25% decrease in the percentage of responding cells after IgM stimulation. Moreover, the MFI of pERK1/2, is reduced by more than 30% following exposure to HgCl2. Additional studies in primary mouse B-cells have found Hg also modulates phosphorylation of the proximal BCR kinase Syk. Taken together these data suggest Hg modulates BCR signal strength which may have profound implications for establishment of self-tolerance within the B-cell repertoire.
FcγRIIB1 regulates plasma cell apoptosis through a c-Abl-dependent pathway (86.8)
Authors: Tzeng Shiang-Jong , Bolland Silvia , Bonvini Ezio , Pierce Susan 
Published: January 2023

Abstract
In B cells the FcγRIIB1 blocks BCR signaling when both receptors are coengaged by binding to immune complexes. Inhibition is mediated by the recruitment of SHIP to the FcγRIIB1’s ITIM motif that has been phosphorylated by Lyn. In contrast, FcγRIIB1 regulates B cell apoptosis when clustering without BCR engagement. We show that the FcγRIIB1 triggers apoptosis by a mechanism that is distinct from the mechanism by which the FcγRIIB1 inhibits BCR signaling. Following clustering the FcγRIIB1 is tyrosine phosphorylated in an ITIM- and SHIP-independent fashion. The phosphorylation of the FcγRIIB1 and the apoptosis mediated by FcγRIIB1 are abolished in c-Abl and Arg double knockout cells. We then determined that long lived plasma cells that express FcγRIIB1 but not BCR and memory B cells when stimulated by a BCR-independent, TLR-dependent pathway are likely the targets of FcγRIIB1-mediated apoptosis. In both naïve and immunized mice, FcγRIIB1 clustering on bone marrow CD138+ plasma cells supported by TLR stimuli, e.g. CpG, resulted in a decrease in the number of Ig-secreting cells and an increase in the number of cells undergoing apoptosis. In human CD27+ memory B cells we found that FcγRIIB1 clustering blocked the CpG-induced differentiation to plasma cells. Taken together, our findings provide strong evidence that FcγRIIB1 serves as a peripheral checkpoint to maintain tolerance by regulating both apoptosis and differentiation of plasma cells.
Differential Regulation of SOCS Gene Expression by IL-27 in Astrocytes and CD4+ T Cells (87.11)
Authors: Qin Hongwei , Cong Yingzi , Roberts kevin L. , Baker Brandi J. , Elson Charles O. , Benveniste Etty N. 
Published: January 2023

Abstract
Suppressor Of Cytokine Signaling (SOCS) proteins are critical feedback inhibitors of the JAK/STAT signaling pathway. In addition, several studies have demonstrated that SOCS-1 and SOCS-3 have neuroprotective effects. Recently, several reports have shown that the cytokine IL-27 functions as an inhibitory regulator, and represses the development of TH17 cells associated with autoimmune brain inflammation. Stimulation with IL-27 results in the activation of members of the STAT family, predominantly STAT-1 and STAT-3. Astrocytes, the major glial cell in the CNS, have a number of important physiological properties related to CNS homeostasis, and have also been shown to produce IL-27. In this study, we describe that IL-27 induces SOCS-3 mRNA expression in primary astrocytes, but not in the macrophage cell line RAW264.7, the microglial cell line BV2 and primary microglia. IL-27 induces STAT-1α and STAT-3 activation as assessed by tyrosine phosphorylation in astrocytes, which is correlated with the induction of SOCS-3 gene expression. There was no induction of SOCS-1 mRNA after IL-27 treatment in primary astrocytes. Furthermore, we demonstrate that IL-27 induces STAT-1α and STAT-3 activation, and both SOCS-1 and SOCS-3 mRNA expression in naïve CD4+ T cells, in a time-dependent manner. These results indicate that IL-27 differentially induces SOCS-1 and SOCS-3 expression in primary astrocytes and naïve CD4+ T cells by activation of STAT-1α and STAT-3 in a cell-type specific manner. We propose that SOCS-1 and SOCS-3 may participate in IL-27-mediated suppression of TH17 cell production and autoimmune brain inflammation.
Disruption of Rxra Gene in Thymocytes and T Lymphocytes Modestly Alters Lymphocyte Frequencies, Proliferation, Survival and Th1/Th2 Balance (87.12)
Authors: Stephensen Charles Bolt , Borowsky Alexander D. , Lloyd Kevin C. Kent 
Published: January 2023

Abstract
Retinoid X receptor (RXR) agonists, including the vitamin A metabolite 9-cis retinoic acid, decrease T lymphocyte apoptosis and promote Th2 development ex vivo. To examine the in vivo role of RXR-alpha in T lymphocytes we disrupted the Rxra gene using Lck-targeted, cre-loxP-mediated excision of Rxra exon 4. Successful disruption was seen in thymocytes and T lymphocytes. Mice were healthy and the thymus, spleen and lymph nodes appeared normal. However, knockout mice had a lower percentage of double-positive (CD4+CD8+) and a higher percentage of double-negative thymocytes than wild type mice. The percentages of splenic T and B lymphocytes were lower in knockout mice. Ex vivo proliferation was decreased and apoptosis increased in T lymphocytes from knockout mice. Memory CD4+ T lymphocytes from knockout mice produced more IFN-γ and IL-2 and less IL-5 and IL-10 than memory cells from wild type mice, indicating a Th1 bias in vivo. However, Rxra disruption did not similarly bias ex vivo differentiation of naïve CD4+ T lymphocytes, nor did Rxra disruption alter the serum IgG1/IgG2a response to immunization. In summary, disruption of Rxra altered T and B lymphocyte percentages, produced a modest Th1 bias in vivo, and altered T lymphocyte proliferation and apoptosis ex vivo.
This work was supported by NIH grant # R01 AI 50863, USDA CRIS Project # 5306-51530 and USDA cooperative agreement # 585306-0-233.
The Tec kinase Itk regulates conventional versus non-conventional CD8 T cell development (87.14)
Authors: Horai Reiko , Handon Robin , Broussard Christine , Mueller Kristen , Venegas Ana M. , Fowlkes B.J. , Schwartzberg Pamela L. 
Published: January 2023

Abstract
Mutations affecting the Tec kinases Itk and Rlk impair both positive and negative selection and alter CD4 and CD8 T cell development. We previously demonstrated that mature CD8 SP thymocytes and peripheral CD8 T cells in Itk−/− and Rlk−/−Itk−/− mice do not resemble conventional CD8 T cells. Instead, these cells express memory markers and can be selected on MHC class I expressed on hematopoietic cells, suggesting these cells resemble non-conventional MHC class Ib-selected “innate-type” cells. Indeed, Itk-deficiency increases positive selection on MHC class Ib in the Kb−/−Db−/− background. We further find that either
improving TCR signaling with stronger signals orblocking CD28 costimulation, partially corrects memory phenotypes of Itk−/− CD8 T cells, arguing that different strength or quality of signals may lead to development of this distinct CD8 T cell population.
Our results suggest that Tec kinases differentially regulate development of conventional versus non-conventional lymphocytes. The requirement for signals from hematopoietic cells for selection of these “innate-type” CD8 T cells in Itk−/− mice will be discussed.
CD28:CD80 interactions mediate anitgen independent ring junction formation and signalling in T cells (87.15)
Authors: Thomas V. K , Dustin Michael L. 
Published: January 2023

Abstract
The two-signal model of T cell activation has been focused on what costimulatory signals are necessary to facilitate and enhance T cell response to a specific antigen. However, it is also intriguing to study the ability for costimulatory interactions to induce signaling in the absence of specific antigen recognition. We examine the signaling of CD4+ T cells engaging high densities of CD80 molecules in the presence of ICAM-1 in an antigen-independent T cell response.
Examination of freshly isolated naive mouse T cells interacting with CD80 and ICAM-1 reveals a novel role for CD28:CD80 interactions in antigen independent ring junction formation in which CD80:CD28 interactions were located in the central region. The unengaged T-cell receptor is also localized to this central region. These cells exhibit increased cytoplasmic Ca++ compared to cells interacting with ICAM-1 alone. With as many cells fluxing Calcium as seen in antigen dependent stimulation. Mouse T cells interacting with ICAM-1 and CD80 also show an increased level of phosphorylated tyrosine residues. Although there is signaling in an antigen independent T cell response, it is not sufficient for inducing IL-2 secretion. The higher expression levels of CD80 achieved in the bilayer distinguish these results from earlier studies (Bromley et al, 2001; Markiewicz et al 2005) showing no interaction of mouse CD28 and CD80 in the absence of antigen.
Thus, CD28, as well as NKG2D (Somersalo et al, 2004; Markiewicz, 2005), can stimulation formation of antigen independent ring junctions. These experiments also demonstrate that CD4+ T cells can be induced to form antigen independent adhesion rings under conditions of high CD80 density. The function of these structures is under investigation. NIH AI44931
MUC1 Mucin Expressed no Activated T Cells has a Regulatory Function (87.17)
Authors: Su Ee W , Kane Lawrence P , Finn Olivera J 
Published: January 2023

Abstract
MUC1 (CD227) is a large (&gt;400kDa), heavily glycosylated, type-I transmembrane protein present most notably on the apical surface of ductal epithelia. In epithelial adenocarcinomas MUC1 is overexpressed as a hypoglycosylated protein in a non-polarized fashion. Because of these attributes, MUC1 has been studied foremostly as a tumor-associated antigen. The role of MUC1 in other cell types remained largely unexplored until MUC1 was found to be expressed by activated human T cells from healthy donors. Preliminary studies we have performed on the role of MUC1 in T cells point towards MUC1 as a negative regulator of effector T cell function. Crosslinking of MUC1 limits the proliferative capacity of primary human T cells activated with anti-CD3, as well as the ability of these cells to make IL-2 and upregulate IL-2Rα. Studies using the Jurkat T cell line have yielded intriguing results. When MUC1 is present and crosslinked, Jurkat T cells activated with anti-CD3 do not secrete IL-2. Paradoxically, knockdown of MUC1 in Jurkat T cells with siRNA also renders them unable to produce IL-2 when similarly activated. Because failure to secrete IL-2 and proliferate following TCR ligation are characteristics of T cell hyporesponsiveness, elucidating the exact role of MUC1 in T cell activation in the context of other co-inhibitory or co-activating receptors is important. To do so, we have been employing luciferase reporter assays to determine the effect of MUC1 expression and crosslinking on well-characterized signaling pathways activated during the engagement of TCR/CD3 and CD28. This will add to our current, limited, knowledge on MUC1 signaling in T cells.