bluuebunny/crossref_metadata_2025_split huggingface dataset:
Overexpression of total STAT1 during lymphopenia-induced proliferation leads to decrease CD4 T cell survival
Authors: Catalfamo Marta , Cecile Le Saout , Luckey Megan A , Villarino Alejandro , Smith Mindy , Myers Timothy G. , Hasley Rebecca B. , Park Jung-Hyun , O’Shea JJ , Lane H. Clifford 
Published: January 2023

Abstract
To preserve the size and diversity of the T cell repertoire throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 signals through a heterodimer receptor composed of the IL-7Rα and the γc chains. Binding of IL-7 to its receptor leads to activation of STAT5 and induction of target genes implicated in survival and proliferation signals. IL-7/IL-7R signaling can also activate STAT1 and STAT3 but the function of IL-7-dependent activation of these transcription factors is largely unknown. Under lymphopenic conditions, IL-7 modulates the expression levels of total STAT1 (t-STAT1) leading to an IL-7-dependent STAT1 signaling in addition to STAT5. We generated a transgenic mouse that overexpress STAT1 in CD4 and CD8 T cells to understand the contribution of IL-7-dependent STAT1 activation on T cell homeostasis in the setting of lymphopenia. We found that under lymphopenia-induced proliferation T cells from the STAT1 transgenic mouse upregulates t-STAT1 leading to diminish CD4 T cell survival. This mechanism could be a physiological mechanism to regulate the expansion of the CD4 T cell pool under lymphopenic conditions.
Glycogen metabolism supports effector function and energy homeostasis of dendritic cells
Authors: Thwe Phyu M. , Beauchamp Saritha , Amiel Eyal 
Published: January 2023

Abstract
As professional antigen presenting cells of the immune system, dendritic cells (DCs) serve as a bridge between innate and adaptive immune responses. Activation of DCs by a stimulus through toll-like receptors (TLRs) is coupled with an increase in metabolic demand, which is fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides metabolites and energy required for DC effector function, and inhibition of glycolysis impairs the survival and effector function of these cells. The prevailing model in the field proposes that TLR-driven glycolysis is sustained primarily by an increase in glucose uptake. While non-immune cells store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen metabolism in DCs has not been reported. Our data indicate that glycogen metabolism supports the activation of DCs, particularly before these cells increase the surface expression of glucose transporter-1 (GLUT1). We show that DCs express the enzymes essential for glycogen metabolism, that the glycogen metabolism is regulated upon TLR stimulation, and that inhibition of the glycogen metabolism in DCs impairs the expression of key maturation-associated molecules in these cells. Additionally, our data suggest that glycogen-derived glucose carbons feed into the tricarboxylic acid (TCA) cycle to support early maturation of DCs. These data indicate that the glycogen metabolism in DCs plays an important role in energy homeostasis in these cells to support their effector function. Understanding the role and regulatory mechanisms of glycogen metabolism in DCs has important implications for the future development of enhanced cellular based immunotherapy.
Downregulation of human IL4 and IL13 expression by CRISPR/Cas9-mediated deletion of DNase I hypersensitive site HS11/12 from a transgenic human Th2 cytokine locus
Authors: Pivniouk Vadim I , Rosenbaum David , Pivniouk Oksana , Vercelli Donata 
Published: January 2023

Abstract
Production of the Th2 cytokines IL4 and IL13 is frequently dysregulated in allergy and asthma. Multiple genetic elements regulating expression of murine Th2 cytokines have been characterized by gene targeting. To study the regulation of human Th2 cytokines, we created BAC transgenic mice (BAC5TG) carrying a 160 kb fragment of the human Th2 locus encompassing RAD50, IL13 and IL4. This transgene includes all the regulatory elements required for physiological expression of mouse Th2 cytokines.
BAC5TG mice appear normal, are born at the expected Mendelian ratio, and demonstrate tissue-specific and transgene copy number-dependent expression of human Th2 cytokines. DNA methylation profiles across the transgenic human Th2 locus are highly comparable if not identical to those observed in human cells.
A DNase I hypersensitive site (HS) in the human IL13-IL4 intergenic region, previously identified in our lab and designated HS11/12, colocalizes with an area of extensive and profound CpG hypomethylation both in human and mouse transgenic T cells. However, unlike most Th2 regulatory elements identified to date, HS11/12 is not conserved between humans and mice. Using the CRISPR/Cas9 approach, we deleted HS11/12 from the human Th2 transgene in BAC5TG mice. Notably, production of both human IL13 and IL4 by in vitro differentiated Th2 cells from BAC5DHS11/12 TG mice was reduced by ~50%.
Our results suggest that HS11/12 represents a novel cis-regulatory element required for optimal production of the human Th2 cytokines IL4 and IL13. The application of CRISPR/Cas9 technology to BAC transgenic mice may provide a powerful tool to study human gene regulation.
Activation of Interferon Regulatory Factor 3 (IRF3) is Required in Both T cells and APCs for Optimal Expression of IFN-γ, IL-17, and Granzyme B during T cell responses.
Authors: Petro Thomas M , Guinn Zachary , Lampe Anna 
Published: January 2023

Abstract
Recently we saw a link between activation of Interferon Regulatory Factor (IRF)-3 and expression of IFN-γ, as well as Granzyme B (GrB) from T cells during viral infections in C57Bl/6 (B6) and IRF3KO mice. To investigate this further, we stimulated splenic mononuclear cells (SMC) from B6 and IRF3KO mice in vitro with anti-CD3±anti-CD28± poly I:C, a TLR3/IRF3 agonist. We show during responses with anti-CD3/anti-CD28 that IRF3 is activated within 24 h in B6 SMC and expression of IFN-γ, IL-17 and GrB is impaired in IRF3KO SMC. Adding poly I:C with anti-CD3/anti-CD28 accelerated IRF3 activation and significantly increased IFN-γ, but not IL-17 or GrB, from IRF3KO SMC. Interestingly, Foxp3 expression and percentage of Foxp3+ T cells were significantly lower in unstimulated IRF3KO SMC compared with B6 SMC. To determine the contribution of IRF3 activation in T cells vs APCs, B6 and IRF3KO bone marrow dendritic cells (DCs; 3×104) were mixed in various combinations with WT or IRF3KO purified T cells (3×105), as well as anti-CD3±anti-CD28± poly I:C. After 48 h, IFN-γ, IL-17, and GrB expression was greatest in cultures where both B6 DCs and B6 T cells were mixed. IFN-γ, IL-17 and GrB decreased when either IRF3KO DCs or IRF3KO T cells were part of the mixture. Least production of IFN-γ, IL-17, and GrB occurred when DCs and T cells were from IRF3KO mice. These results suggest that IRF3 in both APCs and T cells is required to achieve optimum production of IFN-γ, IL-17, and GrB during T cell responses. Moreover, the data unexpectedly show that IRF3 is activated during T cell immune responses without a TLR3 agonist. The factor(s) responsible for IRF3 activation during APC/T cell responses is unclear but appears to be a soluble factor whose expression is stimulated through CD28.
The LFA-1 signal for cell migration influences Notch and TGF-β pathways with functional consequences for T-cells
Authors: Verma Navin Kumar , Fazil Mobashar Hussain Urf Turabe , Theng Ong Seow , Chalasani Madhavi Latha S. , Praseetha P , Kumar Sunil , Panda Aditya K , Freeley Michael , Smith Sinead , Boehm Bernhard Otto , Kelleher Dermot 
Published: January 2023

Abstract
The integrin LFA-1 plays a key role in T-cell motility, which is critical for host defense and is also main driver of autoimmune diseases. However, whether and how LFA-1 signalling can modulate T-cell effector functions remain to be understood. We performed gene expression analysis of LFA-1-stimulated human T-cells and identified genomic signatures associated with Notch and TGF-β pathways, both involved in T-cell differentiation. The activation of Notch pathway in motile T-cells was confirmed by the nuclear translocation of cleaved Notch intracellular domain and up-regulation of target genes Hey1 and Hes1. LFA-1 stimulation of naïve peripheral blood mononuclear cells was found to promote T-cell Th1 polarization through a GSK3β signalling-dependent Notch pathway. We further detected that LFA-1 signalling up-regulated Stat3 and/or JNK activation-dependent expression of Smad7, Smurf2 and Ski causing T-cell TGF-β unresponsiveness. LFA-1-stimulated naïve T-cells were refractory to TGF-β-mediated induction of Foxp3+ iTreg or RORγt+ Th17 differentiation. Of note, peripheral or splenic T-cells isolated from patients with rheumatoid arthritis, type1 diabetes or thyroiditis showed constitutively high expression of Tbet. Pretreatment of cells with blocking anti-LFA-1 antibody, specific inhibitors or siRNA against identified molecules restored LFA-1-mediated modulation of T-cell functional phenotypes. Thus, LFA-1-mediated signalling is crucial for T-cell immunoregulation concurrent with T-cell motility, involving both Notch and TGF-β pathways. These findings have implications for normal immunologic functions and also have therapeutic relevance for inflammatory diseases.
Function of protein kinase CK2 in CD4+ T cell polarization and EAE
Authors: Gibson Sara , Qin Hongwei , Liu Yudong , Rowse Amber , Benveniste Etty 
Published: January 2023

Abstract
Auto-reactive CD4+ T cells drive many autoimmune diseases including multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). CK2 is a constitutively expressed and highly conserved serine/threonine kinase that promotes a number of pro-survival and pro-inflammatory signaling pathways including PI3K/Akt/mTOR, NF-kB and JAK/STAT. These pathways are essential for CD4+T cell activation and polarization, but little is known about how CK2 functions in T cells. Here we demonstrate that protein levels of the major catalytic subunit of CK2, CK2α, are induced in murine CD4+ T cells upon activation, correlating with an increase in overall kinase activity. In addition, CK2α expression is elevated in CD4+ T cells that are activated and infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Targeting the activity of CK2α using the small molecule inhibitor CX-4945 in vitro significantly inhibited Th17 cell polarization and promoted FOXP3 expression in both Th17 and Treg polarizing conditions with no appreciable effect on the generation of Th1 cells. In vivo treatment with CX-4945 during EAE resulted in a dampened Th17 response in the periphery and a decrease in IL-17A+ and IL-17A+IFN-γ+ CD4+ T cells present in the spinal cord. Continued CX-4945 treatment significantly reduced the severity of EAE clinical symptoms and increased the percentage of FOXP3+ Tregs in the spinal cord during disease resolution. Our data implicate CK2 as a novel regulator of the Th17/Treg cell axis, and suggest that CK2 could be targeted for the treatment of CD4+ T cell-driven autoimmune disorders.
Image Based Assessment of NFkB Nuclear Translocation in Murine Cells with ImageStream Cytometry
Authors: Kong Raymond K , Pugsley Haley , Friend Sherree , Morrissey Phil 
Published: January 2023

Abstract
Nuclear Factor kappa B (NFkB) transcription factor plays a central role in regulating many key processes in mammalian cells, including differentiation, cell growth, inflammation, immunity, and stress responses. The activation of NFkB and its translocation from the cytoplasm to the nucleus has become a focal point for studying cellular function, signal transduction pathways, disease mechanisms and drug discovery efforts. The nuclear translocation of NFkB in human cells has been extensively studied with the ImageStream technology during the last decade. However, similar studies in murine cells has not been done. In this project, NFkB nuclear translocation in a murine cell line is measured using ImageStream cytometry. Images of activated and non-activated cells labeled with anti-NFkB antibody and a nuclear dye are acquired on the ImageStream cytometer. Images analysis is performed using IDEAS image analysis software. A Similarity score is determined for every cell based on a pixel by pixel correlation of the nuclear image to the NFkB image. As a result the mean similarity values in the non-activated and activated cell groups are determined. The increased mean similarity value is correlated with higher level of nuclear translocation. The project result will provide a robust method for measuring the NFkB nuclear translocation in the murine cells using ImageStream technology.
A diverse repertoire of memory CD4 T cells is maintained in the absence of the common gamma chain
Authors: Matson Courtney Alexandra , Johnson Lakesha , Warner Ross , Singh Nevil J 
Published: January 2023

Abstract
After successfully combating a pathogen, a small number of antigen-experienced T cells are retained in the body to offer better protection against future infections by the same agent. In the case of CD8 T cells, the stable maintenance of these memory phenotype T cells critically requires homeostatic turnover driven by cytokines such as IL-7 and IL-15. Intriguingly, CD4 T cells that are deficient in the shared receptor for IL-2, IL-7 and IL-15 – i.e. the cytokine common gamma chain (γc), can still generate memory phenotype (MP) T cells in vivo. Here we show that such naturally generated γc-deficient CD4 MP T cells have a diverse T-cell receptor repertoire usage, despite a profound deficiency in the naïve CD4 T cell compartment in such mice. Furthermore, adoptive transfer experiments show that γc-deficient CD4 MP T cells can be stably maintained even in a competitive environment (of a γc+ host) where other CD4 MP T cells can utilize IL-2, IL-7 and IL-15. CD4 MP T cells from γc-deficient animals also differentiated to IFN-γ producers. Therefore, unlike CD8 memory T cells, the maintenance of a diverse repertoire of CD4 memory T cells is independent of trophic cytokines such as IL-7 and IL-15.
Fast and easy isolation of CD27-positive human memory B cells using EasySep&[trade] Releasable RapidSpheres&[trade]
Authors: Maeshima Nina M , Jovanovic Vida K , Ewen Catherine L , Clarke Samuel J , Kokaji Andy I , Woodside Steven M , Thomas Terry E , Eaves Allen C 
Published: January 2023

Abstract
Over the course of a lifetime, memory B cells are key to maintaining antibody-mediated protection from viral and bacterial pathogens and they are one outcome of an effective vaccine. Since memory B cells are typically found at frequencies of less than 5% of normal human peripheral blood mononuclear cells (PBMC), research into their function would be aided by a fast and easy isolation method. To meet this need, we have developed an improved EasySep™ kit for the isolation of CD27-positive (CD27+) human memory B cells from fresh PBMC.
First, CD27+ cells are labeled using an antibody cocktail and EasySep™ Releasable RapidSpheres™ and are positively selected using a hand-held EasySep™ magnet. Next, magnetic particles are released from the positively-selected cells, and then non-B cells within the CD27+ fraction are targeted for depletion using a second antibody cocktail and EasySep™ Dextran RapidSpheres™. Following an additional magnetic separation step, the particle-free, CD27+ memory B cells are poured off into a new tube and ready for use. With one additional step, CD27− naive B cells can be isolated from the same starting sample. The entire isolation protocol is completed in under 40 minutes without any centrifugation steps.
Using this method, CD19+CD27+ memory B cells can be isolated to 97±2% purity and 37±14% recovery (average of n=19 ± S.D.). The CD19+CD27− naive B cells can be isolated to 93±5% purity and 28±11% recovery (n=9 ± S.D.). When stimulated with CpG and IL-15, the isolated memory and naive B cells are functional, as assessed by both proliferative responses and the secretion of IgG.
Experimental food-induced anaphylaxis is driven by C5aR1 actvivation on mast cells
Authors: Koehl Joerg , Kordowski Anna , Wu David , Hogan Simon P 
Published: January 2023

Abstract
Food-induced anaphylaxis is a serious allergic reaction mainly caused by antigen cross-linking of IgE-loaded Fce-receptors on mast cells (MCs). Such MC activation leads to the release of pro-inflammatory mediators which results in the manifestation of the disease. Here, we demonstrate that the complement fragment C5a plays an important and until now not recognized role in the development and severity of food allergy. We subjected C5aR1−/− and wild-type (wt) Balb/c mice to oral ovalbumin (OVA)-induced anaphylaxis. We observed that 78% of wt but only 9% of C5aR1−/− mice suffered from diarrhea and hypothermia after the 7th oral OVA challenge. Importantly, peritoneal MCs strongly expressed C5aR1 after the 7th oral OVA treatment. OVA-specific serum IgE and MCPT-1 levels were significantly decreased in C5aR1-deficient as compared with wt mice. To directly examine the impact of the C5a/C5aR1 axis on MC function, we generated bone marrow-derived MCs (BMMCs) and tested their activation and degranulation potency in response to FceR1 cross-linking. Degranulation of C5aR1−/− BMMCs was reduced by 50% and IL-6 production was decreased by 35% as compared with wt cells. Also, FceR1 cross-linking markedly upregulated C5aR1 MC expression. Further, in contrast to wt mice, C5aR1−/− mice were largely protected from histamine-induced temperature drop in vivo. In line with this finding, C5aR targeting protected human endothelial cells from histamine-induced barrier dysfunction and disruption of tight junction proteins in vitro. Our findings identify a critical role of the C5a/C5aR1 axis in IgE-mediated oral antigen-induced anaphylaxis. C5aR1 may serve as a novel therapeutic target to suppress the inflammatory response in food-induced anaphylaxis.
Peptide-deficient forms of HLA-B: Prevalence and Function
Authors: Geng Jie , Altman John , Krishnakumar Sujatha , Raghavan Malini 
Published: January 2023

Abstract
Human leukocyte antigen class I (HLA-I) molecules comprise a highly polymorphic heavy chain complexed to a conserved light chain, b2-microglobulin (b2m), and peptide. When bound to antigenic peptides, HLA-I molecules mediate CD8+ T cell responses against cancers and intracellular pathogens. The transporter associated with antigen processing (TAP) is the major source of cellular peptides for the assembly of HLA-I molecules in the endoplasmic reticulum (ER). Consistent with previous findings of varying stabilities of peptide-deficient forms of HLA-B, we show allotype-dependent variations in the cell surface expression of HLA-B molecules in TAP-deficient and TAP-inhibited cells. HLA-B allotypes that are resistant to inhibition of TAP (RIT-HLA-B) exist on the cell surface in empty or suboptimally loaded forms under TAP-deficiency conditions, even at physiological temperatures. Furthermore peptide-deficient forms of RIT-HLA-B allotypes bind to primary CD8+ T cells via a mode of binding that is CD8-dependent. Together, these findings indicate allotype-dependent variations in the prevalence of peptide-deficient HLA-B molecules and demonstrate previously unrecognized functions for “empty” HLA-I.
Indispensable role of IL-10 in the development of antigen cross-presentation capacity of the cDC1 subset
Authors: Hanakawa Sho , Kitoh Akihiko , Kabashima Kenji 
Published: January 2023

Abstract
The conventional type 1 dendritic cell (cDC1) is a distinct DC subset with specialized functions including cross-presentation of antigens to CD8 T cells and CD103 expression. cDC1s require GM-CSF signal for gaining their signature functions; however, the involvement of other cytokines remains unknown.
Here we found that cDC1s were normal in number but lacked CD103 expression in IL-10-deficient (Il10−/−) mice, suggesting that IL-10 has some effects on cDC1 function in vivo. Thus, next we assessed the CD8 T-cell priming capacity of cDC1s from Il10+/+ and Il10−/− mice by coculture with OT-I CD8+ T cells in the presence of ovalbumin in vitro. In contrast to Il10+/+ cDC1s, Il10−/− cDC1s failed to induce the expansion and IFN-γ production of OT-I cells. In addition, Il10−/− mice had a significantly lower number of memory CD8 T cells in the lymph nodes in the steady state. These results indicate that cDC1s in Il10−/− mice were defective in antigen cross-presentation. We also found that, whereas Flt3-ligand-induced DCs derived from Il10+/+ bone marrow cells upregulated CD103 expression by and CD8 T-cell cross-priming capacity of the cDC1-equivalent population in response to GM-CSF stimulation, Il10−/− bone marrow-derived DCs did not, suggesting that dysfunction of cDC1s in Il10−/− mice is likely due to unresponsiveness to GM-CSF. Finally, we found that mRNA expression of Csf2ra, which encodes GM-CSF receptor α-chain, was completely deficient in both cDC1s in Il10−/− mice and Il10−/− cDC1-equivalent bone marrow-derived DCs. Collectively, our data suggest that IL-10 critically controls the development of antigen cross-presentation capacity of cDC1s under homeostatic conditions, likely through regulating GM-CSF receptor expression.
An allosteric site in the T cell receptor β-chain constant domain plays a critical role in T cell signaling
Authors: Sgourakis Nikolaos , Natarajan Kannan , McShan Andrew , Jiang Jiansheng , Zhao Huaying , Schuck Peter , Boyd Lisa F , Tilahun Mulualem E , Ying Jinfa , Bax Ad , Margulies David H 
Published: January 2023

Abstract
The molecular mechanism through which the interaction of a clonotypic αβ TCR with peptide/MHC (p/MHC) complexes leads to T cell activation is not yet fully understood. Here we exploit a high affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd) by combining crystallographic, NMR, and functional data. In addition to conformational changes in complementarity determining regions (CDR) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β chain dynamics reveals significant chemical shift effects in sites removed from the MHC binding site. In particular, a remodeling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests an allosteric mechanism for TCR signaling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T cell functional assays.