bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic regulation of TCR activation as a potential mechanism for CD28 co-stimulation
Authors: Raychaudhuri Kumarkrishna , Rangu Rohita , Ma Alison , Samelson Lawrence E 
Published: August 2023

Abstract
T cell receptor (TCR) engagement initiates a chain of proximal TCR signaling events including phosphorylation of TCRζ, recruitment of upstream kinases, followed by recruitment of signaling and adapter proteins. These events occur within seconds of TCR engagement and are important determinants of the T cell response. Yet, we lack a precise understanding of the spatiotemporal relationships between these molecular events due to the limitations of conventional light microscopy. We used stimulatory antibodies and ligands against the TCR to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Formation of these signaling subunits was then studied using TIRF and TIRF-SIM to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events and subsequent effects on the immunoresponse.
Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in the MCs, as revealed by a decreased kinetic lag between detection of TCRζ and the recruitment of ZAP70. We also observed that CD28 co-stimulation markedly changed Lck dynamics in T cells, resulting in Lck enrichment within MCs and a concomitant increase in co-localization of Lck with ZAP70 and TCRζ. CD28 co-stimulation led to spatial segregation between activated and inhibited species of Lck, raising the possibility of formation of localized Lck activation sites within MCs. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs, thereby decreasing the energy barrier for a rate-limiting kinetic step in TCR proximal signaling.
Establishing novel and unique technology to uncover how Activation-Induced Deaminase specifically targets immunoglobulin regions
Authors: Ruiz Stormy Elizabeth , Heltzel Justin M H , Maul Robert W , Gearhart Patricia J 
Published: August 2023

Abstract
Activation-induced deaminase (AID) is the critical enzyme required for somatic hypermutation (SHM) and class switch recombination (CSR). Upon exposure to antigen, B cells become activated and express AID, initiating the process of affinity maturation. While we understand how AID is targeted to the switch region to initiate CSR, relatively little is known about why AID is targeted to the variable region to initiate SHM. The difficulty lies in the requirement to study SHM in vivo in a small population of germinal center B cells. To address this, we developed a knock-in mouse model called JH1, which utilizes a VDJ containing endogenous gene segments in the Variable (V) gene locus where upstream and downstream sequence isintact and intervening DNA is deleted. This unique model’s V region represents what is observed in endogenous B cells generated from the bone marrow. I have used and validated CUT&RUN on our low cell inputs, expanding possibilities for probing critical targets in precise populations of interest. Our lab and others have shown that SPT5 (a subunit of the RNA Polymerase II modulator, DSIF) is required for AID targeting and thus SHM. Unlike a typical gene, promoter-proximal pausing occurs up to 2kb downstream of the V promoter and is necessary for AID activity. We hypothesize distinctive regulation of RNA Polymerase II activity is what allows AID targeting and SHM. I am currently performing analysis of my generated CUT&RUN datasets for well-known transcriptional regulatory proteins to determine if there is a unique signature for SHM. Understanding how AID is targeted to the V region will help us to understand the elusive, yet indispensable process of SHM, which may be useful to fine-tune humoral responses via vaccines and other treatments.
This work was supported entirely through the Intramural Research Program at the National Institutes of Health (NIH), National Institute on Aging (NIA).
Mechanisms of TRAF3 regulation of TLR signaling in B lymphocytes
Authors: Ybarra Tiffany K , Bishop Gail A 
Published: August 2023

Abstract
Toll-like receptors (TLRs) are pattern recognition receptors that bridge the innate and adaptive immune system and are critical for host defense. TLR signaling to B cells is important in normal immunity, but also implicated in autoimmune disease and B cell lymphomas (BCL). Most studies focus on the role of TLRs in myeloid cell populations. However, B cells express most TLRs and are highly responsive to TLR ligands, yet TLR-mediated B cell pathways are understudied. Previous studies from our lab show that TRAF3 inhibits B cell TLR functions, including cytokine production, Ig isotype switching, and antibody production in response to ligands for TLRs 3, 4, 7/8 and 9. A key knowledge gap is identifying the molecular mechanisms by which TRAF3 mediates this regulation. Our working hypothesis is that B cell-TRAF3 regulates the recruitment and/or post-translational modification of specific TLR complex signaling proteins. Our recent data show that B cell-TRAF3 forms a heterocomplex with TRAF6, associating with the tyrosine kinase Syk. In the absence of TRAF3, TRAF6 shows a greater association with several TLR signaling proteins, including MyD88, IRAK1 and Syk. This suggests that TRAF3 may inhibit TRAF6 access to the TLR signaling complex, and thus early TLR signaling. In addition, our work introduces a role for Syk in TLR signaling in B cells. In the absence of TRAF3, activation of Syk is enhanced in response to ligands for TLRs 4 and 7. Inhibition of Syk activation also revealed downstream effects on TLR-mediated NFκB activation. Further studies aim at providing new insights into molecular mechanisms contributing to the regulation of B cell-TRAF3 in TLR signaling.
VA Merit Review I01 BX001702 NIH P50 CA97274 RO1 AI5107312000000113259002001 NIH T32 Training Grant AI07485 RO1 AI1762656
SLP-76 interaction with PLC-γ1 fine-tunes TCR signal strength for appropriate thymocyte selection, T cell homeostasis, and Ag-specific T cell response
Authors: Stassenko Elizabeth , Wada Junya , Lee Mariah , Li Wenmei , Balagopalan Lakshmi , Evans Christine , Hagenau Herbert , Awasthi Parirokh , Chari Raj , Yamane Hidehiro , Samelson Lawrence E 
Published: August 2023

Abstract
TCR signal strength regulates T cell fate at various stages of thymocyte development and the differentiation of peripheral T cells into distinct T cell subsets. We previously showed that TCR engagement forms a heterotetrametric complex composed of LAT, Gads, SLP-76, PLC-γ1 and that this complex is essential to activate PLC-γ1 to further transduce the TCR signal. The interaction of the SLP-76 proline-rich region (PRR) with the PLC-γ1 SH3 domain is relatively weak, and these two domains are highly conserved amongst many mammalian species. Therefore, we hypothesized that the SLP-76 PRR sequence with low affinity for PLC-γ1 might be evolutionarily favored to prevent excess TCR signals. To test this hypothesis, we developed mice carrying a conditional T cell-specific knock-in mutation in the SLP-76 PRR with a 2.5-fold increased affinity for PLC-γ1 by CRISPR/Cas9-mediated gene editing and Cre/LoxP system (cKI). Notably, cKI increased the expression of active caspase-3 in signaled SP4 and SP8 thymocytes and decreased the numbers of mature medullary thymocytes, suggestive of enhanced negative selection. Under steady-state conditions, cKI enhanced the generation of CD8+ central memory T cells, implying an increased sensitivity to self-Ag/MHC I in the periphery. Moreover, cKI rendered CD8+ T cells more sensitive to OVA altered peptide ligands with a weaker affinity for OT-I TCR. Finally, cKI enhanced priming of OT-I CD8+ T cells that had not undergone massive cell divisions for IFNγ-producing effectors in vivo, implying an increased TCR signal strength in weakly activated CD8+ T cells by cognate Ag. Our data indicate that the SLP-76 PRR/PLC-γ1 SH3 interaction plays a critical role in fine-tuning TCR signal strength to avoid excess signals via TCR.
NCI/NIH Intramural Support
Tunable B cell response through allosteric modulation of Bruton’s tyrosine kinase
Authors: Reboldi Andrea , Berthelette Alyssa , Kolli Nilima , Amatya Neha , Andreotti Amy , Berg Leslie J , Der Sandy , Kang Joonsoo 
Published: August 2023

Abstract
The B cell receptor (BCR) is essential for both the initial burst of B cell proliferation in response to antigens, and for the differentiation of plasma cells and memory B cells during germinal center reactions. However, the BCR’s precise role in germinal center selection has been difficult to assess: mechanistic investigations into its modulation during the decision of B cell fate suffer from experimental limitations due to the fundamental role of the BCR in B cell biology.
Bruton’s tyrosine kinase (BTK), a member of the TEC family, is a key component of BCR signaling. Using a computational screening method based on small molecule docking outside of the BTK active site, we have identified putative allosteric modulators of BTK. In vitro primary B cell Nur77 upregulation, calcium flux, proliferation and plasma cell differentiation confirmed that these molecular probes alter B cell activation strength, kinetics, and fate only when the BCR is engaged. In vivo immunization using an antigen model reveled that BTK agonism strongly favors B cell differentiation into switched plasma cells. BTK agonism, either at the time of the immunization or at the peak of germinal center formation, enhanced humoral immunity, proving for the first time that transient, augmented BCR signaling during germinal center reaction has a direct impact on B cell differentiation ability.
In addition to being powerful tools for basic B cell biology investigation, allosteric agonists of BTK could be developed into antigen-licensed B cell adjuvants for vaccines in at risk populations, as well as into cancer immunotherapy drugs which hyperactivate B cells in tumor microenvironments during checkpoint inhibitor blockade therapies.
A cellular platform for the evaluation of the effect of O-glycan modification on B cell receptor signaling
Authors: Angata Takashi , Hsiao Po-Long , Tsai Ho-Yang , Huang Wan-Chen , Lin Kuo-I 
Published: August 2023

Abstract
The importance of N-glycosylation, a ubiquitous post-translational modification (PTM) of cell-surface and secreted proteins in vertebrates, in the regulation of B cell receptor (BCR) signaling has been well documented. On the other hand, the importance of O-glycosylation, another type of ubiquitous PTM, in B cell function is getting recognized only recently. A simplified system to study the effects of O-glycan modifications in BCR signaling would facilitate the efforts to understand the mechanism of B cell regulation by O-glycans. To this end, we reconstituted BCR components and CD45 with orthogonal self-labeling protein tags in the mouse myeloma J558L cell line, which is deficient in the components of BCR and several co-receptors. The enzymes that modify O-glycans were further introduced, and the cells were subjected to imaging-based quantitative analysis of the distance between BCR and CD45, as well as the evaluation of BCR signaling by Western blotting. Gene expression profiles among the cells were also compared by RNA sequencing. Our study revealed that core 2 branching of O-glycans has minimal effect on the distance between BCR and CD45, whereas the addition of an α2–8-linked sialic acid by ST8Sia-VI reduces the distance between them, facilitating the downstream BCR signaling. Our system may be adapted for the functional evaluation of other PTMs in BCR signaling.
Supported by a grant from the Ministry of Science and Technology (MOST)/National Science and Technology Council (NSTC) of Taiwan (MOST 109-2311-B-001-009-MY3)
QRICH1 is a negative regulator of T cell activation via the CARD11 signalosome
Authors: Carter Nicole M , Pomerantz Joel L 
Published: August 2023

Abstract
Controlled lymphocyte activation is crucial for defending against pathogens while preventing diseases such as autoimmunity and lymphoma. In T lymphocytes, the scaffold protein and signaling hub CARD11 mediates NF-κB, JNK, and mTOR pathway activation downstream of T cell receptor (TCR) engagement. We recently identified QRICH1 as a previously unrecognized CARD11 interactor recruited to the CARD11 complex downstream of TCR engagement and discovered that QRICH1 protein levels are upregulated in wild-type activated mouse primary CD8+ T cells. Overexpression of QRICH1 in 293T and Jurkat T cells dose-dependently inhibited NF-κB activation by wild-type CARD11 as well as by constitutively active and oncogenic CARD11 mutants. QRICH1-deficient Jurkat T cells had increased NF-κB activation and IL-2 secretion downstream of TCR stimulation. To study the in vivo role of QRICH1 in immunity, we generated a novel QRICH1 conditional KO mouse model. Compared to control mice, QRICH1 T cell KO mice had decreased splenic CD4+ and CD8+ T cells, despite no differences in thymic CD4+, CD8+, or CD4+CD8+ T cell populations. The CD8+ T cell population in QRICH1 T cell KO mice was skewed towards an effector memory phenotype. QRICH1-deficient primary CD8+ T cells had increased proliferation and production of pro-inflammatory cytokines IFN-γ and TNF-α in response to ex vivo stimulation with anti-CD3/CD28, and QRICH1-deficient OT-1 CD8+ T cells also had increased cytokine production in response to antigen-specific stimulation with Ova peptide. Our findings reveal that QRICH1 negatively regulates T cell activation by modulating CARD11’s signaling output.
This work was supported by NIH grants R01AI148143, F31CA254167, and T32GM007445.
Understanding the involvement of sphingosine kinase-1 and two-pore channels in calcium signaling in T cells
Authors: Soares Giselle Amanda Borges E , George Emma H , Hamzah Ali H , Walseth Timothy F , Slama James T , Wall Katherine Ann 
Published: August 2023

Abstract
Calcium (Ca2+) signaling is an essential requirement for cell activation, proliferation, differentiation, homeostasis, etc. In the tumor microenvironment, however, an upregulation of the Ca2+ signaling pathway has been associated with cancer cell proliferation and malignancy, which further contributes to immune response suppression. Although nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ signaling secondary messenger known, the NAADP-Ca2+ signaling pathway remains elusive and controversial. Studies have so far revealed the interaction of NAADP with accessory proteins such as Jupiter microtubule-associated homolog 2 (JPT2) and Lsm12 along with the poorly characterized ‘two-pore channels’ (TPC1 and TPC2) located in the acidic endo-lysosomal compartments of cells.
In 2018, a screening campaign revealed the ability of PF-543 to inhibit NAADP-mediated Ca2+ signals in sea urchin egg homogenates and in human U2OS cells. As PF-543 is marketed as a potent sphingosine kinase 1 (SphK-1) inhibitor and is believed to possess TPC inhibitory function, the mechanism behind PF-543’s ability to inhibit NAADP-mediated Ca2+ signals (through inhibition of SphK-1 or the TPCs, or both) needed investigation. Therefore, we tested structural analogs of PF-543 and evaluated their ability to inhibit both SpK-1 and Ca2+ signals in DO.11.10 cells, a T cell hybridoma that possesses the T cell receptor (TCR). Thus, in addition to identifying novel targets for the development of Ca2+ signaling modulators and anticancer therapeutics, our results also reveal the contributions of SphK-1 and the TPCs in the NAADP-mediated Ca2+ signaling pathway in T cells.
NIH R15GM131329
Single Cell Sequencing Suggests Mucosal Origins of T Cells in HLA-B27-associated Inflammation
Authors: Paley Michael A , Hassman Lynn , Yang Xinbo , Linskey Nicole , Esaulova Ekaterina , Paley Grace L , Ruzycki Philip , Laurent Jennifer , Feigl-Lenzen Lacey , Artyomov Max , Garcia K Chris , Yokoyama Wayne M 
Published: August 2023

Abstract
HLA-B27 was one of the first Major Histocompatibility Complex alleles associated with an autoimmune disease, i.e., Ankylosing Spondylitis (AS) and acute anterior uveitis (B27AU), which cause joint and eye inflammation, respectively. As HLA-B27 presents antigens to CD8 T cells, an antigen-specific CD8 T cell response may be central to disease pathogenesis. Consistent with this hypothesis, our recent work identified clonal expansion of public T cell receptors (TCRs) shared across patients with AS and B27AU and enriched in the joint or eye over the blood. These TCRs recognized both bacterial and self-peptides, suggesting a role for molecular mimicry in multiple organs afflicted by HLA-B27-associated inflammation.
The main bacterial peptide recognized by these public TCRs is derived from yeiH, a gene present in multiple enteric microbiota. It remains unclear, however, whether these T cells have had a prior antigenic encounter within the gastrointestinal tract. We therefore performed single-cell RNA sequencing (scRNAseq) and T cell receptor sequencing (scTCRseq) on immune cells from the eye and blood during B27AU (n=5) and non-B27AU (n=14). In this analysis, we found that the expanded TCRs in B27AU were associated with CD161 expression and enriched for a transcriptional program from intestinal CD8 T cells. Moreover, we were able to identify an additional expanded TCR sequence that also recognized YEIH. Finally, expanded T cell clones contained higher expression of TNF and IFNG, suggestive of a pro-inflammatory transcriptional program.
Collectively, our data suggest that, in HLA-B27-associated inflammation, the early antigen exposure and differentiation of putative pathogenic CD8 T cells may have occurred in enteric organs.
This study received support from NIH 5R01AI103867 (KCG), U19AI057229 (KCG), the Howard Hughes Medical Institute (KCG), the Rheumatology Research Foundation (MAP), Arthritis National Research Foundation (MAP), the Rheumatic Diseases Research Resource-based Center at Washington University (NIH P30-AR073752, MAP, WMY), the Bursky Center for Human Immunology and Immunotherapy Programs (WMY), and the Barnes-Jewish Hospital Foundation (WMY).
T cell subsets in Systemic Sclerosis patients show features of exhaustion and reduced anti-fibrotic activities
Authors: Dooms Hans , Volfson-Sedletsky Victoria , Belkina Anna C , Bujor Andreea 
Published: August 2023

Abstract
Systemic Sclerosis (SSc) or Scleroderma is a chronic autoimmune disease where immune cells contribute to the initiation and/or progression of microvascular damage and fibrosis, leading to devastating tissue malfunction often resulting in high morbidity and mortality. We recently discovered that PBMCs from SSc patients show increased expression of various co-inhibitory receptors, consistent with the presence of a state of chronic immune activation and dysfunction. Here, we sought to identify and functionally characterize dysregulated T cell subsets in SSc, and determine their role in pathogenic fibroblast activation. We used multi-parameter spectral flow cytometry and in vitro T cell/fibroblast co-culture assays to understand the role of T cell dysfunction and exhaustion in fibrosis, using clinical samples from a well-defined cohort of SSc patients. Our immunophenotyping analysis revealed that T cell populations in SSc patients show features of T cell exhaustion, such as enhanced expression of the co-inhibitory receptors PD-1 and TIGIT, and reduced production of the anti-fibrotic cytokine IFN-γ. PD-1+TIGIT+ T cells were found in peripheral blood as well as lung and skin of SSc patients. In addition, we found a significant reduction of immune cell subsets expressing the transcription factor T-bet in the blood of SSc patients, suggesting a reduced presence of immune cells that counteract a pro-fibrotic Type 2 immune response in SSc. In support of this, isolated T cell subsets from SSc patients showed reduced capacity to limit collagen production in T cell/fibroblast co-cultures. Together, our data suggest that T cells with anti-fibrotic activities are driven into a state of exhaustion in SSc, enabling the progression of fibrosis.
Supported by grants from NIH (R21 AR071580) and the National Scleroderma Foundation (Walter & Marie Coyle Research Grant)
Distinct PD-1hi CD4+ T helper cell populations promote CD11c+ T-bet+ B cell expansion in liver and spleen during obesity
Authors: Enslow Benjamin , Vanz Carlo , Dudley Elizabeth , Hägglöf Thomas , Leadbetter Elizabeth A 
Published: August 2023

Abstract
Obesity is associated with increased deposition of fat in ectopic tissues, such as within the liver and visceral adipose depots, including the omentum and mesentery. Increased visceral fat is an accompanying feature of the metabolic syndrome and is associated with increased end-organ damage in NAFLD. Emerging evidence implicates intrahepatic B cells in the progression of NAFLD, and increased number of proinflammatory B cells have been found in the steatotic livers of HFD-fed mice. Our preliminary studies demonstrate accumulation of inflammatory T-bet+ CD11c+ B cells in the subcutaneous adipose tissue of obese humans and perigonadal adipose tissue of mice on HFD. We now extend those findings into other sites of ectopic fat deposition to determine the obesity-related changes in T-bet+ B cell frequencies in the liver, omenta, and MAT. Flow cytometry identified increased frequencies of T-bet+ B cells in the spleen and livers, but not visceral adipose, of HFD mice. Furthermore, HFD-associated expansion of T-bet+ B cells in the spleen and liver correlated with a rise of TFH and TPH frequencies, respectively. Future studies will determine the mechanism of tissue-specific T and B cell interactions associated with obesity-induced chronic inflammation.
B.T.E. is supported by the NIH award (K12GM111726). T.H. is supported by a postdoctoral fellowship from the Swedish Research Council. This study was supported by NIH R01 AI32798-01A1 and the Voelcker Fund Young Investigator Awards (E.A.L).
Role of complement activation in cellular activation by systemic lupus erythematosus (SLE) immune complexes (ICs)
Authors: Khandelwal Sanjay , Myoung Sooho S , Lambris John D , Rogers Jennifer L , Pisetsky David S , Arepally Gowthami M 
Published: August 2023

Abstract
Complement (C’) activation is a prominent feature of SLE ICs, but the mechanism by which C’ mediates cellular activation by ICs is not well understood. To investigate this question, we formed SLE ICs in situ in healthy donor (HD) whole blood (WB) by adding SLE plasma (n=1–3;10% v/v) or HD plasma (as a control) along with apoptotic bodies (generated from RAW 264.7 cells) or RNP-Sm antigen (Arotec Diagnostics, New Zealand) with and without C’ inhibitors (CI). For cellular binding studies, WB was incubated for 45 minutes at 37°C and deposition of C3c and human IgG (ICs) on blood cells was examined by flow cytometry. In these studies, we noted that C’ inhibition significantly reduced deposition of C3c and ICs on B cells {% reduction: C3c (97%) and IgG (97%)}, monocytes {% reduction: C3c (40%) and IgG (49%)} and neutrophils {% reduction: C3c (69%) and IgG (70%)}. To investigate cellular activation, WB was incubated for 22 hours and cellular activation was measured by HLA-DR expression on monocytes by flow cytometry and cytokine release (IL-8, IL-6 and TNF) by ELISA. Incubation of WB with SLE ICs, but not ICs made with HD plasma (control ICs), resulted in increased HLA-DR expression on monocytes (MFI control ICs: 1609 V SLE ICs: 11096), which was susceptible to C’ inhibition (MFI SLE ICs with CI: 4975). Similarly, inflammatory cytokine release by SLE ICs was also significantly inhibited with C’ inhibition {% inhibition: IL-8 (75 %), IL-6 (99%) and TNF (99 %)}. Taken together, our results show significant C’-dependent effects of SLE ICs on cellular binding and activation. Additional studies are underway to investigate the prevalence of C’ activating ICs in SLE patient and correlate their presence with clinical disease.
Wendell F. Rosse Complement Research Award, Duke University Medical Center (SK) and Duke Scholars Award, Duke University Medical Center (GMA)