bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting T follicular helper cell development in vivo using CRISPR
Authors: Huang Bonnie , Phelan James D. , Webster Dan E. , Shaffer Arthur L. , Schwartzberg Pamela L. 
Published: January 2023

Abstract
T follicular helper (Tfh) cells are specialized CD4 helper T cells that signal B cells to produce high affinity antibodies and provide long-term humoral immunity. Identifying genes that govern Tfh differentiation and function could provide targets for improving vaccines and therapies for autoimmunity. However, Tfh differentiation is only partially understood, and not fully recapitulated in vitro. To discover novel Tfh-regulating genes, we developed a CRISPR-based functional genetic system to mutate genes in primary mouse T cells, and measured the effects on Tfh cells in vivo.
Transgenic Cas9-expressing mouse CD4 T cells were transduced in vitro with an optimized retroviral vector encoding an sgRNA and GFP. With a guide targeting Tcf7 (encoding a Tfh-essential transcription factor Tcf1), &gt;90% cells could be transduced; up to &gt;80% of GFP+ cells were Tcf1neg by flow and mutational analyses. To evaluate the functional consequence of CRISPR editing, we adoptively transferred virus-specific CD4 T cells that were transduced with either a control or Tcf7 sgRNA construct into wild-type hosts. After viral challenge, control T cells differentiated into both Tfh and non-Tfh cells, while very few Tcf7-edited T cells became Tfh cells. Similarly, guides against the Tfh master transcription factor Bcl6 abrogated Tfh differentiation in vivo. Next, we constructed sgRNA libraries of up to 400 guides, targeting up to 80 genes, including ones implicated in primary immunodeficiencies and druggable targets. NGS analysis of T cells post-viral challenge revealed depletion of multiple guides targeting known Tfh-required genes in the Tfh vs non-Tfh populations, validating this approach. We are currently following up on potential hits from these screens.
The importance of the NFAT5/TonEBP-mediated osmotic stress response in B cells
Authors: Rokvic Ljiljana , Titze Jens , Schuh Wolfgang , Jäck Hans-Martin 
Published: January 2023

Abstract

Introduction
Hyperosmotic stress is well investigated within renal borders, many non-renal tissues are facing fluctuations of osmolality as part of normal, physiological behavior. For example, compared to blood, osmolality of lymphoid tissues, like spleen and thymus, is substantially hypertonic. Therefore, the ability of immune cells to respond and adapt to osmotic stress is crucial for normal immune responses. The osmotic stress response is driven by nuclear factor of activated T cells (NFAT5)/tonicity enhancer binding protein (TonEBP), the only known mammalian osmosensitive transcription factor. It has been shown that mice with a heterozygous deletion of NFAT5 display impaired adaptive immune responses. To specifically analyze the functions of NFAT5 in B lymphocytes, we generated conditional B cell specific knock-out mice.

Objectives
Using a conditional B cell-specific NFAT5 knock-out mouse model, we want to assess the importance of NFAT5 for B cell development, B cell activation and antibody production.

Results
NFAT5-deficient mice fed with high salt diet (HSD; 4% NaCl in food and 0.9% NaCl in water) and immunized with TNP-KLH showed impaired IgM and IgG responses compared to wildtype controls. Accordingly, frequencies and numbers of PC and GC B cells were reduced in immunized NFAT5-deficient mice. In support of these findings, we found lower cell numbers and diminished viability of LPS-stimulated NFAT5-deficient B cells cultured in hypertonic medium (+40mM NaCl).

Conclusion
NFAT5 in B lymphocytes controls antibody responses by controlling B cell activation and survival. Gene expression profiling will now elucidate transcriptional programs and signaling pathways involved in NFAT5-controlled B cell responses.
LVS vaccination allows BALB/c mice to survive virulent F. tularensis pulmonary challenge but does not induce sterilizing immunity
Authors: Soucy Alicia , Metzger Dennis W 
Published: January 2023

Abstract
There is no licensed vaccine for protection against virulent strains of Francisella tularensis, an intracellular pathogen that replicates within lung macrophages and causes lethal acute infection in mice at an intranasal challenge dose of ~10 CFU. Immunization with the attenuated F. tularensis LVS strain provides some level of protection against virulent F. tularensis SchuS4 infection; however, the basis for this induced resistance remains unknown. We found that LVS vaccination allowed complete survival of BALB/c mice (but not C57Bl/6 mice) from lethal SchuS4 pulmonary challenge for 30 days, and 50% survival for at least 100 days, yet the surviving mice contained large numbers of SchuS4 organisms in their lungs and spleens (~104 CFU/organ). Early after challenge, resistant BALB/c mice produced greater amounts of Th17-associated cytokines compared to susceptible C57Bl/6 mice. However, IL-17 and IL-22 did not appear to be important for restricting chronic infection. On the other hand, depletion of either CD4+ or CD8+ cells three weeks after SchuS4 challenge caused resistant BALB/c mice to succumb to infection within one week. Similarly, treatment of vaccinated BALB/c mice with anti-IFNγ monoclonal antibodies three weeks after SchuS4 led to fatal acute tularemia. We conclude that LVS vaccination of BALB/c mice induces an immune response that allows the animals to constrain and tolerate a chronic SchuS4 infection. Long-term resistance is dependent on CD4+ cells, CD8+ cells, and IFNγ.
CD4+ T cells prevent abacavir hypersensitivity reactions in HLA-B*57:01 transgenic mice
Authors: Cardone Marco , Garcia Karla , Yano Masahide , Mattson Elliot , Juengst Leslie , Roderiquez Gregory , Tilahun Mulualem E , Boyd Lisa F , Natarajan Kannan , Puig Montserrat , Margulies David H , Norcross Michael A 
Published: January 2023

Abstract
Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be associated with the development of abacavir (ABC) hypersensitivity syndrome (AHS) and with the induction of liver injury by the β-lactam antibiotic flucloxacillin. The nucleoside analog ABC, an inhibitor of the HIV reverse transcriptase, can induce severe multi-organ toxicity in &gt;50% of HLA-B*57:01+ patients with HIV infection. In a previous study we showed that ABC induced binding to the HLA-B*57:01 of altered self-peptides containing predominantly isoleucine or leucine residues at the carboxyl terminus. Recognition of these self-peptides drives in vitro activation of cytotoxic CD8+ T cells. However, the early immune events/danger signals required to overcome the immune tolerance that otherwise suppress ADR are still unknown. In order to study HLA-linked drug hypersensitivity in vivo and to understand better drug immune tolerance, HLA-B transgenic mice were generated. Here, we show that ABC activates HLA-B*57:01 transgenic CD8+ T cells in vitro via CD8 and HLA. A systemic, but partial, early response to the drug is also observed in vivo, however, ABC fails to promote skin hypersensitivity in immunocompetent transgenic mice. Instead, depletion of CD4+ T cells breaks immune tolerance to ABC and promotes ADR in HLA-B*57:01 transgenic mice.
Supported by the Intramural Research Program of the NIAID, NIH, and CDER, FDA.
Functions of the ‘alarmin’ cytokine IL-25 in house dust mites-induced allergic asthma-like chronic pulmonary inflammation
Authors: Etienne Estefania , Wang Hongshan , Tang Wanhu , Ha Hye-Lin , Siebenlist Ulrich 
Published: January 2023

Abstract
Allergic inflammatory lung disease is a complex immune response orchestrated by many cytokines, chemokines and other factors signaling danger. One of these is the alarmin IL-25, an epithelial cell-derived cytokine secreted at mucosal sites in response to various allergens. Although IL-25 is a member of the IL-17 cytokine family and it is the only member to promote type-2 responses. IL-25 activities overlap with those of IL-33 and TSLP, two other cytokines secreted by mucosal epithelial cells; all three activate innate lymphoid cells type 2 (ILC2s).
We set out to elucidate unique/significant roles of IL-25 in the chronic asthma-like lung inflammation model induced by repeated exposures to house dust mites. If unique/significant contributions could be identified in this physiologically relevant model, IL-25 could be a potential therapeutic target in allergic asthma. To carry out these studies we generated mice with global or conditional deletion of IL-17RB and CIKS (a.k.a. Act-1, Traf3ip2). Surprisingly we found that blocking IL-25 signaling in lung CD11c+ cells reduced expression of IL-9 and IL-13, cytokines that are critical in asthma and that derive primarily from Th9 and Th2 cells, respectively, during the chronic stages. IL-25 signaling in CD11c+ cells also contributed significantly to mast cell accumulation and ultimately lung remodeling. We will present investigations of potential mechanisms underlying the effects of IL-25 signaling in CD11c+ lung cells. Our data demonstrate that IL-25 signaling in CD11c+ cells makes physiologically important contributions in the chronic HDM-induced model of allergic asthma, findings that should encourage future studies of this cytokine as a therapeutic target.
Cognate CD40-CD40L interaction maintains thymic B cells in absence of TCR-MHC interaction between thymic T and B cells
Authors: Alkhaleel Farrah , Fujihara Chiharu , Rivera Hiraldo Luis E. , Williams Joy A. , Hodes Richard J. 
Published: January 2023

Abstract
We previously demonstrated that maintenance of the thymic B cell population requires cross-talk between thymic B cells and T cells, analogous to the cross-talk that had been demonstrated between thymic epithelial cells and developing T cells. Maintenance of thymic B cells specifically requires interaction between CD40 ligand (CD40L) and cell autonomous CD40 on thymic B cells. Co-stimulatory interactions such as those mediated by CD40 commonly exert their effects in concert with pMHC-specific T cell receptor (TCR) recognition. We have therefore analyzed the requirement for MHC-TCR interaction for thymic B cell maintenance. Analysis of mixed bone marrow (BM) chimeras constructed from MHCI/MHCII double knockout (DKO) and WT donor BM revealed that there is no requirement for expression of MHCI or MHCII on thymic B cells for their maintenance. Thus, the thymic B cell population is regulated by cognate CD40-CD40L interaction in the absence of direct pMHC-TCR interaction with T cells. Experiments are currently in progress to determine whether CD40-dependent thymic B cells are sustained by direct interaction with T cells expressing membrane-bound CD40L, or by soluble CD40L in the absence of direct T cell-B cell interaction.
N-glycan branching inhibits pro-inflammatory B cell activity by controlling TLR4 and BCR signaling.
Authors: Mortales Christie-Lynn , Demetriou Michael 
Published: January 2023

Abstract
N-glycosylation is a critical regulator of T cell function. The branching and number of N-glycans on cell surface glycoproteins drive binding to galectins, forming a galectin-glycoprotein lattice that controls T cell growth, differentiation and development by regulating receptor clustering, distribution and endocytosis. N-glycan branching is ~1.5–2 fold higher in B cells compared to T cells, yet effects on B cell function are unknown. Here we report that branching regulates pro-inflammatory signaling via Toll Like Receptor – 4 (TLR4) and the B cell receptor (BCR). Mechanisms controlling pro-inflammatory (cell surface) versus anti-inflammatory (endosomal) TLR4 signaling in B cells is poorly understood. Loss of branching in B cells de-couples LPS induced proliferative from pro-inflammatory responses by inhibiting TLR4 endocytosis. Similar de-coupling is observed with BCR signaling, where loss of branching inhibits BCR driven proliferation and Lyn/Syk/PLCγ2/Ca2+ signaling via reduced CD19 surface expression while concurrently driving pro-inflammatory effector functions and PI3K/Akt/mTOR activation. Together, these data reveal N-glycan branching as a critical negative regulator of TLR4 and BCR triggered pro-inflammatory B cell responses.
Ezh2 is required for the division-specific epigenetic programming of plasma cell differentiation
Authors: Scharer Christopher , Guo Muyao , Haines Robert , Kania Anna , Boss Jeremy M 
Published: January 2023

Abstract
Following activation of naïve B cells, epigenetic remodeling is required to facilitate the induction of the plasma cell gene expression program. Ezh2 is an epigenetic repressor, catalyzing the trimetylated state of histone H3 lysine 27 (H3K27me3), and is essential for B cell development and the maintenance of a germinal center reaction. Recently, Ezh2 has been shown to directly interact with the master transcriptional regulator of plasma cell fate Blimp1. To investigate a wider role in regulating other aspects of B cell fate, a tamoxifen-inducible deletion of Ezh2 was developed, allowing deletion during distinct phases of B cell differentiation. Ezh2 was not required for mature B cell homeostasis, however; in response to differentiation signals, a cell-intrinsic reduction in antibody secreting plasma cells was observed. Additionally, Ezh2 deficient plasma cells secreted fewer antibodies, indicating a failure to fully induce the plasma cell fate. To determine the molecular processes regulated by Ezh2, chromatin accessibility and the transcriptome were profiled by ATAC-seq and RNA-seq, respectively. Ezh2 deficient plasma cells demonstrated an up regulation of inflammatory related genes and B cell lineage transcription factors. Chromatin accessibility data indicated a failure to repress B cell enhancers and promoters. Additionally, Blimp1 target genes displayed increased expression in Ezh2 deficient plasma cells providing a genetic link to the interaction of these two repressors. Together, these data indicate that Ezh2 facilitates the epigenetic repression of the B cell program and is required for full induction of plasma cell fate and function.
POLD3 Plays an Essential Role in Somatic Hypermutation
Authors: WU WENJUN , XIAO ZHIHUI , Poltoratsky Vladimir 
Published: January 2023

Abstract
Somatic hypermutation (SHM) increases the affinity of antibodies to antigen. While the initial steps of SHM, including development of the lesion, its excision and formation of an abasic site, are well established, the repair and mutation events are still under investigation. Multiple DNA repair pathways have been suggested for the SHM.
POLD3, as a part of a scaffold complex, connects error free or error prone DNA polymerases with the processivity factor PCNA and this interaction plays an essential role in mutagenesis and recombination.
In this study we tested the role of POLD3 in SHM. We demonstrated elevated expression of POLD3 in activated mice B cells and accumulation of this protein at DNA damaged sites. We created a Burkett’s lymphoma Raji cell clone (Raji 2C9 clone) expressing endogenous mutant POLD3 lacking C-terminus PCNA interaction site, using the CRISPR system. Functional studies shown that the Raji cells expressing mutant variant demonstrated abnormal cell cycle distribution, hyper-sensitivity to genotoxic agents and compromised SHM, compare to the control cells.
The frequency of mutations in locus encoding immunoglobulin heavy chain variable region was are five times lower in cells expressing mutant POLD3 compare to control cells.
In control cells, mutations accumulated at the RGYW/WRCY “hot spots”, and majority of mutations were C to T or G to A transitions. In cells expressing mutant POLD3 mutations accumulated outside “hot spots” and are represented by transversions. The overall results indicate that POLD3 plays an important role in SHM.
Tetraspanin 33 (TSPAN33) regulates cytoskeleton rearrangements and the Notch signaling pathway in human B cells.
Authors: Maravillas-Montero Jose Luis , Cervantes-Diaz Rodrigo , Acevedo-Ochoa Ernesto , Romero-Ramirez Sandra , Navarro-Hernandez Itze Cecilia , Santos-Argumedo Leopoldo 
Published: January 2023

Abstract
TSPAN33 is the last described member of the tetraspanin protein family; it is highly expressed in malignant B cells and is one of the most controversial tetraspanins on regard of its function in leukocytes. In human B cells, we observed that the expression of TSPAN33 is rapidly induced by IL-4 and anti-CD40 stimulation and its antibody-induced cross-linking reduced the cell proliferation rate. Furthermore, by over-expressing TSPAN33 in Raji B cells, we detected dramatic alterations at different membrane features of these lymphocytes. Our results show that TSPAN33 promotes the formation and extension of microvilli and membrane protrusions generated during cell spreading by altering the cortical actin cytoskeleton and the surface expression of LFA-1, CD29 and CD44. In this way, we also demonstrate that TSPAN33 participates in adhesion events such as immune synapse formation with T cells and extracellular matrix substrate adhesion. TSPAN33 belongs to the TspanC8 tetraspanin subgroup, which is associated with the metalloprotease ADAM10 regulating its traffic and activity in different cell membranes. ADAM10 mediates the proteolytic cleavage of several proteins including the Notch receptors. We observed that TSPAN33 does not just alter the expression ADAM10 but also modifies the expression and activity (measured by proliferation and target genes activation assays) of Notch receptors in B cells. Taken together, all these findings demonstrate that TSPAN33 plays a role in the cytoskeleton dynamics and Notch signaling pathway in B cells, by altering different membrane features which in turn modify the surface ADAM10/Notch interactions, regulating its signaling capacity.
PSGL-1 is a negative regulator of effector, exhausted, and memory T cells
Authors: Tinoco Roberto , Carrette Florent , Barraza Monique L , Otero Dennis C , Bradley Linda M 
Published: January 2023

Abstract
Effective T cell responses depend on adhesion mechanisms that guide T cells to sites of infection and inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to impact T cell migration; however, its role in the differentiation of responding T cells has not been investigated. Since PSGL-1 is highly expressed on naïve, effector, and memory T cells with varying degrees of glycosylation that impact ligand binding, we hypothesized that PSGL-1 may regulate T cell fates. Using mice deficient in PSGL-1 and three murine models we found that PSGL-1 limits the magnitude of the ensuing T cell response. PSGL-1-deficient mice had an increased accumulation of anti-viral CD4+ and CD8+ T cells after both acute and chronic LCMV infection. Virus-specific PSGL-1-deficient T cells had enhanced survival and were not hyper proliferative. Furthermore, PSGL-1-deficient T cells had superior effector function, downregulation of PD-1 and other inhibitory receptors, and enhanced viral control. The enhanced T cell response resulted in significant mortality of PSGL-1-deficient mice infected with chronic LCMV, whereas all PSGL-1-deficient mice survived the acute LCMV infection. After viral clearance, more CD4+ and CD8+ PSGL-1-deficient T cells survived to form memory cells. Interestingly, resting memory T cells from PSGL-1-deficient mice sustained PD-1 downregulation implying that PSGL-1 may regulate inhibitory receptors in memory T cells. We found that PSGL-1-deficient T cells had superior function in a tumor model and were more effective in controlling melanoma tumors. Our findings in three separate models show a previously unrecognized role for PSGL-1 in negatively regulating T cell responses and may have high therapeutic potential.
Antigen presentation profiling reveals T-cell recognition of lymphoma immunoglobulin neoantigens
Authors: Olsson Niclas , Khodadoust Michael , Wagar Lisa , Swaminathan Kavya , Haabeth Ole Audun Werner , Chen Binbin , Rawson Keith , Liu Chih Long , Steiner David , Lund Peder J. , Rao Samhita , Zhang Lichao , Marceau Caleb , Stehr Henning , Newman Aaron M. , Czerwinski Debra K. , Carlton Victoria , Moorhead Martin , Faham Malek , Kohrt Holbrook E. , Carette Jan , Green Michael R. , Davis Mark M. , Levy Ron , Alizadeh Ash A. , Elias Joshua E. 
Published: January 2023

Abstract
Presentation of novel antigenic peptides (neoantigens) that distinguish malignant from normal cells by major histocompatibility complexes (MHC) can serve as potent substrates for specific anti-tumor immune responses. We sought to identify mantle cell lymphoma (MCL) neoantigens by taking an integrated genomic and proteomic strategy that interrogates antigen peptides presented by MHC-class I and class II. Peptides bound to MHC were purified via immunoprecipitation followed by identification using mass spectrometry. Mass spectra were searched against patient-specific proteome databases generated by whole exome sequencing and targeted immunoglobulin gene sequencing. This approach was applied to systematically characterize over 36,000 immunopeptides from 17 patients’ tumor specimens. Interestingly, 52 neoantigenic peptides were derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we identified MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. Furthermore, somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC-II. T-cells specific for Ig-derived neoantigens were found in two patients. Following ex vivo activation and expansion, the T-cells were remarkably able to mediate killing of autologous lymphoma cells. These results demonstrate that combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to MCL implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Fatty Acid Uptake Unexpectedly Supports a Th17 cytokine signature in Type 2 Diabetes (T2D) Inflammation
Authors: Nicholas Dequina , Proctor Elizabeth A. , Jones Albert , Raval Forum , Habib Chloe , Corkey Barbara , Apovian Caroline M. , Lauffenburger Douglas A. , Nikolajczyk Barbara S. 
Published: January 2023

Abstract
Metabolism controls T cell function, but the role of cellular metabolism in regulating our published T2D-associated Th17 cytokine signature is untested. Published work showed Th17 function requires fatty acid (FA) uptake, and suggested Th17s burn fat to support inflammation. We thus hypothesized that chronic exposure of immune cells to FAs, which occurs in T2D patients, drives Th17 inflammation. We used extracellular flux (XF) and cytokine analyses to show that CD3/CD28-stimulated PBMCs from BMI-matched T2D vs. non-T2D subjects were more glycolytic despite overall equal mitochondrial respiration. Consistent with this difference, T cell stimulation in glucose-free media followed by partial least squares analysis of cytokines showed glucose impacted cytokine profiles from T2D and non-T2D cells differently. To challenge the resulting prediction that PBMCs (predominantly T cells) from the two groups used different mechanisms to fuel cytokine responses, we performed XF analysis in the presence of FA +/− glucose. Non-T2D cells utilized FA as a fuel source, but T2D cells were dependent on glucose, despite data showing diabetic vs. non-T2D PBMCs store more lipid in response to elevated exogenous FA. Etomoxir, an inhibitor of FA transport into the mitochondria, caused non-T2D cells to increase glycolysis, but had no effect on T2D cells. These data confirmed that T2D cells exclusively burn glucose, not FA, to generate energy, and that T2D cells cannot be forced to use FA as a fuel. Finally, blockade of FA transport into the mitochondria revealed that FAs were associated with Th17 cytokine production. Together, our results support the unexpected conclusion that FA uptake is uncoupled from FA oxidation to drive the Th17 inflammation in T2D.