bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expansion of the IgD class switched B cell population in carriers of the autoimmune disease associated risk haplotype in BLK (HUM2P.332)
Authors: Simpfendorfer Kim , Manjarrez-Orduño Nataly , Armstead Brandon , Simeone Alyssa , Diamond Betty , Gregersen Peter 
Published: January 2023

Abstract
Polymorphisms in BLK are associated with numerous autoimmune diseases including rheumatoid arthritis and lupus. BLK (B lymphocyte kinase) is a tyrosine kinase involved in signaling through the B cell receptor (BCR). The disease risk haplotype is known to be associated with reduced expression of BLK transcript and the most significant association of reduced BLK transcript is with a candidate causal SNP rs922483. We have previously shown that expression of BLK is reduced in cord blood B cells from carriers of rs922483 risk alleles (HMG 2012). B cell development is perturbed in blk transgenic and knockout mice, and B cells from these mice are hyper-responsive to BCR stimulation. To determine if reduced BLK expression affects the function of primary human B cells we measured CD86 expression in cells stimulated by anti-IgM. As in mice, cord blood B cells from risk allele carriers are hyper-responsive to BCR stimulation. We also assessed if BLK risk variants convey any variation on the composition of circulating B cell populations of healthy adults. We observed that subjects with BLK risk variants have an expansion in the proportion and number of IgD class-switched B cells, and an increase in the total number of class-switched memory B cells. These results suggest that BLK risk haplotypes confer autoimmune disease susceptibility by reducing a negative regulatory effect of BLK, making B cells more likely to receive help from CD4 T cells and to undergo class switch recombination.
Mast cell-derived IL-33 orchestrates antigen-driven tissue inflammation by promoting a unique inflammatory basophil response (HYP3P.352)
Authors: Hsu Chia-Lin , Berdnikovs Sergejs , Bryce Paul 
Published: January 2023

Abstract
While IL-33 is commonly described as an epithelial derived cytokine, we previously showed that IgE activation of mast cells induces IL-33 expression. In vivo, IgE-primed mast cells serve as peripheral sensors for antigen (Ag) and drive an early edema followed by an antigen-driven tissue inflammation (ADTI). Histamine drives the edematous response but the mechanisms controlling local ADTI are unclear. Here, we sought to examine the roles of IL-33 and its receptor, ST2 in this process. Using a passive cutaneous anaphylaxis model, we show that mast cell-derived IL-33 is both sufficient and necessary for the ADTI response, which is dominated by CD45+Gr-1high neutrophils. ST2-expressing basophils are essential for ADTI and we show that IL-33 serves to activate them, rather than cause migration. Using microarray analysis to examine responses to either IgE/Ag or IL-33 activation, we show that IL-33 promotes an atypical basophil response characterized by neutrophilic chemokines. Interestingly, we find that histamine is necessary for both phases but, while H1R or H2R are required for edema, ADTI is dependent on basophil expression of H4R. In summary, our findings define a mechanism through which mast cells regulate local tissue responses to Ag. We propose a model whereby mast cell histamine release initially attracts basophils via H4R. Basophils are then activated by mast cell-derived IL-33, via ST2, to produce an atypical profile of cytokines and chemokines that recruit neutrophils.
Constitutive β-catenin signaling dysregulates DC differentiation resulting in inability to control microbial infection (INM2P.418)
Authors: Cohen Sara , Schneider Anne , Denkers Eric 
Published: January 2023

Abstract
The protozoan Toxoplasma gondii is the second most common cause of foodborne illness-related deaths in the US and can be fatal to infants and immunocompromised patients. Dendritic cells (DC) are required for immunity to T. gondii, and recently, DC Wnt/β-catenin signaling has been suggested to control DC tolerance in the intestine. However, the role of this signaling axis in peripheral DC development and function is unknown. Using transgenic mice expressing DC-specific constitutively active β-catenin (DCEx3-/-), we surprisingly found that the splenic CD8α DC and the developmentally related peripheral CD103 DC compartment were significantly expanded relative to corresponding populations in wild-type (WT) animals. Splenic CD8α DC are a source of IL-12, and infection of DCEx3-/- mice with T. gondii resulted in rapid lethality associated with proinflammatory cytokine overproduction. Transcription factor Irf8 drives CD8α and CD103 DC differentiation, and flow cytometric analysis revealed strong upregulation of Irf8 in CD8α DC upon β-catenin stabilization. Confirming this, WT CD8α+Irf8+ DC displayed higher β-catenin levels relative to splenic CD8- DC. Together, our data show that β-catenin targets Irf8 during DC development, resulting in expansion of CD8α DC in the spleen and CD103 DC in the periphery. This is the first study to implicate β-catenin in DC subset differentiation and provides novel insight into the complex transcriptional network of DC development.
Differential effects of TLR-2 ligands on CD4+ CD25+ Foxp3+ regulatory cells depending on inflammatory cytokine milieu (IRC4P.489)
Authors: Pandiyan Pushpa , Zheng Lixin , Bhaskaran Natarajan , Weinberg Aaron 
Published: January 2023

Abstract
The responsiveness of CD4+CD25+Foxp3+ thymic Tregs (tTregs) towards microbial agents that serve as toll-like receptor (TLR) ligands is unclear. This study investigates the effects of TLR ligands on Tregs. We show that TLR-2 ligands promote IL-2 dependent survival and proliferation of tTregs both in the presence or absence of TCR signals and inflammatory cytokines. The proliferation is directly dependent on TLR-2 expression on Tregs showing that TLR-2 ligands may be involved in Treg proliferation in vivo. Consistent with this, Tlr-2-/- mice harbor fewer Tregs in colon and mesenteric lymph nodes in vivo. However, in the presence of IL-6 and TCR signals, Tregs also acquire effector functions by producing IL-17A. However, they retain their suppressive properties and do not lose Foxp3 expression. Their effector function is dependent on TLR signaling by antigen presenting cells, IL-6 and IL-23 in the milieu, but not on direct TLR-2 signaling by Tregs. IL-17A production in Tregs is also relevant in vivo, as CD4+Foxp3+Tregs are capable of producing IL-17A without losing their suppressive properties during oropharyngeal candidiasis and inflammatory bowel disease. These data show that CD4+Foxp3+ tTregs are a stable lineage of cells, and that TLR-2 ligands promote their proliferation without inducing plasticity. Moreover the data show for the first time, the direct and indirect effects of TLR-2 ligands that differentially regulate tTreg proliferation and effector functions.
Isolation of highly purified mouse CD4+CD25+FoxP3+ T regulatory cells in less than 45 minutes (IRC4P.476)
Authors: Kokaji Andy , Tonelli Luciana , McQueen Karina , Thomas Terry , Eaves Allen 
Published: January 2023

Abstract
Regulatory T cells (Tregs), a subset of lymphocytes, play a key role in maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. They can be broadly classified into natural or adaptive (induced) Tregs. Natural Tregs (CD4+CD25+FoxP3+), develop in the thymus and emigrate to the periphery to help maintain immune homeostasis. Adaptive Tregs can be induced from CD25-negative naïve CD4+ T cells in the thymus and acquire CD25 (IL-2R alpha) and FoxP3 expression in the periphery after adequate antigenic stimulation. They are typically induced by chronic allergic inflammation and disease processes, such as autoimmunity. The isolation of highly purified Tregs is essential for advancing research in this field. To date this has been achieved by lengthy protocols or flow-based cell sorting. We have developed a rapid column-free one-step immunomagnetic cell separation method (EasySep™) to isolate Tregs (natural and induced) from mouse splenocytes in less than 45 minutes. CD25-PE-labelled cells are bound to magnetic particles using antibody complexes and separated using an EasySep™ magnet. The procedure can be automated using RoboSep™. Starting from 2.2±0.4% Tregs, purities of 84±3% (mean±SD, n=20) CD4+CD25+FoxP3+ cells can be achieved. This kit provides a new tool to study the mechanisms by which Tregs exert their influence, which has broad implications for the development of cell-based therapies for autoimmune disease and cancer.
Mechanisms of effector T cell suppression by CD4+Foxp3+ Treg in inflamed peripheral tissue (IRC4P.482)
Authors: Camirand Geoffrey , Rothstein David 
Published: January 2023

Abstract
In secondary lymphoid organs (SLO), CD4+Foxp3+ regulatory T cells (Treg) control immunity by preventing prolonged T cell-DC interactions. Whether Treg suppressor function in inflamed non-lymphoid tissues differs from SLO remains unclear. Thus, we used 2-photon intravital microscopy to dissect how Treg prevent allograft rejection by effector T cells (Teff) in transplanted islets. To restrain the immune reaction to the transplant site, we adoptively transferred Teff, Treg, or both (2-3x106 each) to B6 mice lacking SLO (splenectomized LTβR-/- mice) 3-4 days after transplantation of BALB/c islets. Teff alone induced acute allograft rejection. However, the co-transfer of Teff + Treg led to long-term allograft survival in ~50% of recipients. Analysis of adoptively transferred CFSE-labeled Teff recovered from the graft on day 3 and 5 revealed that Treg did not affect either Teff migration or proliferation at that site (compared to Teff alone), despite the presence of Treg in significant numbers. Intravital microscopy of transplanted islets on day 2 showed that Treg were found in the peri-islet region, where they made brief (&lt;1 min) contacts with DCs. Interestingly, Treg restrained the motility parameters of Teff in the peri-islet region, and significantly reduced Teff intra-islet infiltration, compared to Teff alone. These results indicate that Treg can function directly in inflamed non-lymphoid tissues, but that their mechanisms of action may differ from those used in SLO.
SLAM-associated protein modulates CD8 T cell responses primed by antigen-presenting B cells (IRM4P.503)
Authors: Priatel John , Huang Yu-Hsuan , Tsai Kevin , Harder Kenneth , Tan Rusung 
Published: January 2023

Abstract
Mutations in the SH2D1A gene, that encodes SLAM-associated protein (SAP), cause X-linked lymphoproliferative disease (XLP), a rare congenital immunodeficiency defined by exquisite sensitivity to Epstein-Barr virus (EBV). The inability of boys with XLP to control EBV infection results in fulminant, often fatal, infectious mononucleosis, massive expansions of EBV-infected B cells and malignant B cell lymphomas. However, the precise mechanism of how SAP loss contributes to susceptibility to EBV remains unclear. Given the unique B cell tropism of EBV and the purported role of SAP in regulating lymphocyte/lymphocyte interactions, we hypothesized that SAP expression within CD8 T cells is critical for T cells to mount responses towards antigen-expressing B cells. Wild type and Sh2d1a-/- CD8 T cells responded strongly when stimulated in vitro with cognate antigen-coated whole splenocytes. However, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells in vitro or challenged with antigen-expressing B cell lymphoma in vivo. These results demonstrate that SAP is required for the priming of CD8 T cells when B cells act as the primary APC. Together, our findings suggest that the susceptibility of XLP patients to EBV infection may at least in part be a consequence of an inability of their naïve CD8 T cells to differentiate into effectors upon encountering virally-infected B cells.
Pattern associated modelling for discovery of novel protein motifs in the macrophage scavenger receptors (IRM5P.703)
Authors: Han SeongJun , Bharadwaj Prashant , Novakowski Kyle , Lee Annie , Wong Andrew , Bowdish Dawn 
Published: January 2023

Abstract
Class A scavenger receptors, including scavenger receptor A and macrophage receptor with collagenous structure, are surface proteins that bind modified endogenous ligands and bacterial components. As these receptors play a key role in innate immunity, they are associated with a range of infectious diseases such as pneumonia. Thus, understanding the physical properties of these receptors will provide insight for the development of novel therapeutics. Macrophage receptor with collagenous structure is amongst the least well-characterized members of this family and is expressed in macrophages to mediate pathogen recognition. Our investigation addresses the development of an efficient and highly accurate bioinformatics technique through combinatorial usage of Aligned Pattern Clustering, and Multiple Sequence Alignment Pattern Retrieval Program for novel motif discovery in the macrophage scavenger receptor. By utilizing scavenger receptor A and its validated motifs as a model, we successfully validated the feasibility of this bioinformatics technique with a minimum sensitivity of 54% and positive predictive value of 60%. As a subsequent validation of the newly discovered motifs, the top sub-sequences of macrophage receptor with collagenous structure, selected by both programs, were analyzed for their biological functionality. Here, we propose that RGRAE, VFCRMLG, EDAGVE and WGTICDD motifs in the scavenger receptor cysteine-rich domain play an important role in pathogen recognition.
Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex (IRM6P.719)
Authors: Lam Eric , Pareek Tej , Letterio John 
Published: January 2023

Abstract
Cyclin-dependent kinase 5 (Cdk5) is a unique member of a family of serine/threonine cyclin-dependent protein kinases. We previously demonstrated disruption of Cdk5 gene expression in mice impairs T-cell function and ameliorates T-cell-mediated neuroinflammation. Here, we show Cdk5 modulates gene expression during T-cell activation by impairing the repression of gene transcription by histone deacetylase 1 (HDAC1). This effect is mediated through a direct phosphorylation of the mSin3a protein by Cdk5, which prevents the accumulation of the mSin3a protein and the formation of a functional mSin3a/HDAC1 complex during T cell activation. Disruption of Cdk5 activity in T-cells enhances HDAC activity and binding of the HDAC1/mSin3a complex to the IL-2 promoter, ultimately leading to suppression of IL-2 expression. These data point to essential roles for Cdk5 in regulating gene expression in T-cells and transcriptional regulation by the co-repressor mSin3a.
Lymphocyte activation gene 3 (LAG-3) regulates activation-induced CD4+ T cell metabolic transition (IRM6P.725)
Authors: Previte Dana , Delmastro-Greenwood Meghan , Piganelli Jon 
Published: January 2023

Abstract
LAG-3 negatively regulates CD4+ T cell proliferation and effector function by competing with CD4 for MHC class II binding. Others have demonstrated that loss of LAG-3 results in CD4+ T cell hyperresponsiveness and accelerated autoimmunity, further supporting LAG-3’s regulatory capabilities. In order for optimal CD4+ T cell activation, naïve T cells must transition their metabolic program from oxidative phosphorylation (OXPHOS) to aerobic glycolysis (AG), a faster process that drives macromolecule production. We hypothesized that LAG-3 regulates the metabolic transition of CD4+ T cells from OXPHOS to AG, and in turn, loss of LAG-3 expression results in a faster transition to AG, promoting T cell hyperproliferation. At baseline, B6.LAG-3-/- splenocytes exhibited enhanced spare respiratory capacity as compared to WT controls, which correlated with increased mitochondrial mass and expression of the mitochondrial biogenesis transcription factor TFAM (2-fold), and mitochondrial complexes I-IV. Upon suboptimal Con A stimulation, LAG-3-/- splenocytes demonstrated greater upregulation of glycolysis-associated genes by 24 hrs (1.5 - 3 fold). These results correlated with increased Myc expression and mTOR signaling, both of which are crucial for driving AG. Taken together, these data indicate that LAG-3 regulates CD4+ T cell activation by controlling metabolic programing since the loss of LAG-3 results in a more metabolically primed phenotype.
CK2α protein levels and CK2 kinase activity are induced upon CD4+ T cell activation and are essential for Th17 cell differentiation (LYM3P.736)
Authors: Gibson Sara , Qin Hongwei , Liu Yudong , Rowse Amber , Benveniste Etty (Tika) 
Published: January 2023

Abstract
CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in all cell types and essential for cell viability. The kinase activity of CK2 promotes a number of pro-survival and pro-inflammatory signaling pathways including PI3K/Akt, NF-κB, and JAK/STAT. These pathways are also essential for CD4+ T cell activation and polarization into effector phenotypes. Here we demonstrate that protein levels of the catalytic subunit of CK2, CK2α, increase dramatically upon in vitro activation of murine CD4+ T cells isolated from the spleen. Furthermore, in in vitro polarizing conditions, CK2α protein levels correlate with expression of Th1 and Th17 lineage specific transcription factors T-bet and RORγt, respectively. CK2 kinase activity also increases upon activation, as measured by phosphorylation of the CK2 specific site S129 on Akt. Using the small molecule inhibitor CX-4945, we demonstrate that CK2 kinase activity is essential for Th17 cell polarization. Our data suggest that CK2 could be targeted in Th17 cell driven autoimmune disorders such as multiple sclerosis and colitis.
T cell intrinsic SerpinB1 and a cathepsin protease serve as yin and yang to control Th17 differentiation (LYM3P.732)
Authors: Hou Lifei , Cooley Jessica , Remold-O’Donnell Eileen 
Published: January 2023

Abstract
SerpinB1, an endogenous inhibitor of neutrophil serine proteases, was recognized to restrict infection-associated inflammation. Recently, we found that loss of SerpinB1 leads to expansion of IL-17A+ γδ and Th17 cells even at steady-state. To date, little is known about how proteases and their inhibitors influence the differentiation of T cell subsets. Here, using differentiation of sorted naïve CD4+ T cells, we show that, under Th17 conditions (IL-6 and TGF-β), deficiency of SerpinB1 significantly increased the IL-17+ population (~ 50% increase over WT) and reciprocally diminished the FoxP3+ population. In contrast, there were no genotype-differences in Th1 differentiation. Although neutrophil elastase is considered the primary target of SerpinB1, elastase-/- mice have IL-17+ γδ and Th17 cell numbers comparable to WT mice. Systematic screening of pure proteases using peptidase assays revealed that SerpinB1 also inhibits a subset of cysteine cathepsin proteases. Treating naïve CD4 cells with the broad cysteine protease inhibitors E64d and FA-fmk decreased differentiation to IL17+ cells and reciprocally increased FoxP3+ cells. Use of specific cathepsin inhibitors including CLIK195 and LHVS further suggests a role for cathepsin-L or a cathepsin-L like protease. Thus, these findings show that, in addition to the known transcriptional regulation paradigm, the balance between Th17 cells and Tregs is tightly regulated by SerpinB1 acting through a cysteine cathepsin.
Signaling through Tsc2-mTOR regulates memory CD8 T cell differentiation. (LYM4P.755)
Authors: Sowell Ryan , Rogozinska Magdalena , Marzo Amanda 
Published: January 2023

Abstract
Inhibition of the mTOR pathway with the pharmacological inhibitor rapamycin, can enhance the formation of virus-specific memory CD8 T cells. While mTOR signals through two separate protein complexes, mTORC1 and mTORC2 the role of each in memory CD8 T cell differentiation remains unclear. The Tsc1/2 protein complex inhibits activation of mTORC1, and genetic deletion or inactivation of Tsc1/Tsc2 leads to hyperactivation of downstream mTORC1 targets. mTOR signaling is increased after T cell activation and presumably down modulated during transition to memory. In order to characterize the role of Tsc1/2-mTOR signaling in the formation of memory CD8 T cells, mice that express a dominant negative form of Tsc2 were first crossed with the OT-I transgenic mice (Tsc2-DN OT-I). Subsequently, wild-type or Tsc2-DN OT-I cells were adoptively transferred into naïve congenic hosts that were then infected with Vesicular Stomatitis Virus expressing ovalbumin. We report that disruption of Tsc2 resulted in an impaired generation and/or maintenance of memory CD8 T cells. However, the number of effector Tsc2-DN OT-Is were increased compared to WT OT-Is. These data suggest that Tsc2 signaling controls the survival of effector CD8 T cells during contraction.
Functional heterogeneity of the CD8 T cell response: implications for fate and memory formation (LYM4P.746)
Authors: Kahan Shannon , Bakshi Rakesh , Luther Rita , Harrington Laurie , Weaver Casey , Zajac Allan 
Published: January 2023

Abstract
Although anti-viral CD8 T cells display differences in their portfolios of effector functions, dissecting this ensemble has been impeded by the lack of effective methods for segregating, tracking, and defining these distinct subsets based upon their cytokine production profiles. To address this we utilized novel IL-2 and IFN-γ double cytokine reporter mice. We demonstrated that within 6 hours of activation naïve CD8 T cells undergo a burst of cytokine production and populations are detectable that produce only IL-2, only IFN-γ, co-produce IL-2 and IFN-γ, or do not produce either cytokine. These patterns of cytokine production are reconfigured by changes in the inflammatory milieu. As effector and memory CD8 T cells develop a shift towards IFN-γ production occurs, with a smaller subset capable of co-producing IL-2 and IFN-γ, and the IL-2 only population typically not detectable. By harnessing the cytokine reporter system to separate viable IL-2+ and IL-2- effector subsets, and conducting a series of adoptive transfer studies, we discovered that IL-2 producing effector CD8 T cells transition into memory populations that rapidly and vigorously proliferative upon secondary challenge. Taken together these studies illustrate that dissecting the functional complexity of CD8 T cells has the potential to pin-point the properties that enable these cells to thrive and form the memory compartment, and reveal possible biomarkers of successful responses.