bluuebunny/crossref_metadata_2025_split huggingface dataset:
CD40L acts as an allosteric activator of integrins for signal transduction independent of inside-out signaling
Authors: Shimoda Michiko , Takada Yoko , Maverakis Emanual , Felding Brunhilde , Takada Yoshikazu 
Published: February 2023

Abstract
CD40L plays a major role in immune response and is a target for inflammatory disease therapy. Besides CD40, CD40L binds to several integrins but their role in signaling is unclear. We showed that integrins αvβ3 and α5β1 bind to the CD40L trimeric interface through classical ligand binding site of integrins (site 1). Several CD40L mutants from HIGM1 (hyper-IgM syndrome type 1) patients were clustered in trimeric interface and defective in integrin binding, and in NF-κB and B cell activation, but still bound CD40 and acted as antagonists of CD40L signaling. Thus, integrins play a critical role in CD40L signaling. Previous studies showed that CX3CL1 and sPLA2-IIA can activate integrins by binding to a recently identified allosteric site (site 2) of integrins. Using cell-free assays, we found that soluble (s)-CD40L activated soluble integrins, α4β1, αvβ3 and α5β1, and induced their binding to known specific ligands independent of inside-out signaling. Also, sCD40L activated cell-surface integrins in CHO cells lacking CD40. Finally, sCD40L bound to a cyclic peptide derived from site 2. These findings indicate that CD40L acts as an allosteric activator of integrins by binding to site 2. Docking simulation predicted that the integrin site 2-binding site of CD40L is located outside of CD40L trimer. Importantly, four HIGM1 mutations are clustered in the predicted site 2-binding site of CD40L. The K143T and G144E mutants were the most defective in integrin activation, indicating that integrin binding occurs around these residues of the predicted site 2-binding site. We propose that allosteric integrin activation by CD40L plays a role in CD40L signaling and that defective integrin site 2-binding may be causal for defective CD40L signaling in HIGM1.
Lin28b promotes innate functions in CD8+ T cells through rapid chromatin remodeling
Authors: Watson Neva B , Patel Ravi K , Oyesola Oyebola O , Laniewski Nathan , Smith Norah L , Mon Kristel Yee , Tabilas Cybelle , Daly Ciaran , West Jessica D , Luyen Vu Tien , Maymi Viviana I , Nzingha Kito , Wesnak Samantha P , Scheible Kristin M , Wojno Elia D Tait , Grimson Andrew W , Rudd Brian D 
Published: February 2023

Abstract
Lin28b serves as a master regulator of fetal lymphopoiesis and promotes the development of more innate-like lymphocytes in early life. However, it is still unclear how Lin28b alters the function of CD8+ T cells and protects the host against infection. In this report we examined how Lin28b transcriptionally and epigenetically programs neonatal CD8+ T cells for innate immune defense. We found that murine neonatal CD8+ T cells possess innate-like transcriptomes, are more responsive to innate cytokines in the absence of antigen and produce a broader spectrum of cytokines compared to their adult counterparts. This unique program of bystander activation in early life corresponded with enhanced innate-like protection against irrelevant strains of bacterial, helminth and virus infections. To understand how this diversity of immune function is mediated, we used flow cytometry and scRNAseq and discovered that the neonatal CD8+ T cell pool is composed of many subsets of cells expressing distinct cytokines. ATACseq analysis revealed that neonatal CD8+ T cells are programmed to display innate-like properties and produce a diverse array of cytokines, which may be due to their ability to undergo extensive chromatin remodeling upon cytokine stimulation. These unique properties may be driven by high Lin28b expression in fetal progenitor cells, as adult cells expressing Lin28b initiated a program of bystander activation and immune protection that was analogous to neonates. Collectively, our data suggests that Lin28b enables neonatal CD8+ T cells to undergo a more diverse program of bystander activation, allowing them to deploy a bet-hedging immune strategy and protect the host against a rapidly changing pathogenic environment.
Modular approach to generating long-lived effector/memory (Teff/Tem) T cells to prolong malaria immunity
Authors: Gbedande Komi , Ibitokou Samad , Brown Michael G. , Degli-Esposti Mariapia A , Stephens Robin 
Published: February 2023

Abstract
Immunity to Plasmodium infection or vaccination is known to decay. In mouse models, both B and T cells are required for immunity; however, decay of protection correlates with loss of malaria-specific T cells, not antibody. Whole parasite vaccines, such as the infection and drug cure protocol, protect. However, sterile protection from P. chabaudi or vaccination last less than 200 days. Therefore, we have reverse-engineered a vaccine strategy that first induces a long-lived protective antibody response using live parasite vaccination, and then boosts Teff and Tem response to prolong protection. Cytomegalovirus (CMV), is a known chronic vaccine vector that induces T cell mediated immunity and shows promise against SIV, tuberculosis and liver-stage malaria. Therefore, we used chronic vaccination with MCMV to test our hypothesis that promoting longer generation of Teff in mice would prolong protection by a live vaccine. MCMV encoding P. chabaudi MSP-1 epitope B5 (MCMV-B5) was used to follow the malaria- specific T cell response. Upon infection of mice with MCMV-B5, both adoptively transferred B5 TCR Tg T cells and MCMV-specific CD4 T cells proliferated and were maintained over 2.5 months. Coinciding with this protection, the MCMV vector worked to prolong the protection generated by live malaria vaccine. In investigating the mechanisms of protection, we found that IFN-γ induced by MCMV prolongs the protection, potentially through promotion of IL-12 producing and antigen presenting cell function. Our findings suggest the methods and the mechanisms to effectively prolong immunity to Plasmodium infection based on CMV mechanism to redress the problem of short-lived protection to malaria infection and vaccines.
TRAF3 in T cells restrains negative regulators of LAT to promote TCR/CD28 signaling
Authors: Arkee Tina , Hostager Bruce , Houtman Jon , Bishop Gail A 
Published: February 2023

Abstract
The adaptor protein TRAF3 plays an important role in T cell biology. T cell conditional TRAF3−/− mice, despite normal T cell numbers, have poor responses to immunization and infection, and T cells from these mice have defective TCR/CD28 signaling. TRAF3 associates with, and enhances signaling by, the TCR/CD28 complex upon engagement of both TCR and CD28. The molecular mechanism by which TRAF3 interacts with and impacts TCR/CD28-mediated complexes and function remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, the TCR/CD28 complex. Our work revealed that TRAF3 recruitment did not require direct binding of TRAF3 to CD28; rather, TRAF3 associates with the CD28-binding protein Linker of activated T cells (LAT). The TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 enhanced TCR/CD28 signaling via inhibition of the LAT-associated negative regulatory protein Dok1, which is phosphorylated and inactivated by the tyrosine kinase Breast tumor kinase (Brk/PTK6). In TRAF3−/− T cells, there was increased plasma membrane localization of Protein tyrosine phosphatase 1B (PTP1B), which dephosphorylates Brk at an activating tyrosine residue, resulting in altered Brk activation. Consistent with these findings, inhibiting Brk activity in WT T cells inhibited basal and TCR/CD28-mediated Src family kinase activation, while inhibiting PTP1B activity in TRAF3−/− T cells rescued such activation. These results reveal a new mechanism for the enhancement of TCR/CD28-mediated signaling by TRAF3. We are currently investigating the role of TRAF3 in T cell metabolic reprogramming, which requires effective TCR/CD28 signaling.
LSECtin (CLEC4G) inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling
Authors: Wang Jinghua , Manick Brian , Renelt Mark , Gerassenkov Tatsiana , Bi Ming , Kalabokis Vassilios , Person Anthony , Wu Guoping 
Published: February 2023

Abstract
The liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), also known as CLEC4G, belongs to the calcium-dependent lectin family, acts as a co-inhibitor of human T cell immunity, and mediates tumor immune escape in melanoma. However, the influence of LSECtin on T cell activation remains controversial. In this study, we demonstrate that the LSECtin protein inhibits human T cell activation and proliferation through the butyrophilin family receptor, BTN3A1. LSECtin markedly suppressed human T cell proliferation in the presence of T cell receptor signaling. Furthermore, LSECtin significantly reduced IFN-γ, IL-2, IL-17 and TNF-a cytokine and IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and TARC/CCL17 chemokine production in activated human T cells. Interestingly, a functional ELISA binding assay revealed that LSECtin bound to BTN3A1, LAG-3 and CD44, but very weakly bound to BTN3A3. Anti-BTN3A1 neutralizing antibody attenuated the binding of LSECtin to BTN3A1. BTN3A1 is expressed on both resting and activated human T cells, and pretreatment with an anti-BTN3A1 antibody partially attenuated the ability of LSECtin to inhibit IL-2 secretion from human T cells, suggesting that BTN3A1 acts as a receptor for LSECtin to deliver a negative signal to T cells. Thus, we show that LSECtin/BTN3A1 represents a novel negative regulatory pathway on T cells and may serve as a unique immune checkpoint for developing new immunotherapy strategies for the treatment of immune-related diseases, including cancers.
Single Cell Accessibility Profiling of Murine DN3 Thymocytes
Authors: Collins Patrick , Lee Bella , Stevens Patrick , Nganga Vincent K , Rincon Mercedes , Bassing Craig , Oltz Eugene M 
Published: February 2023

Abstract
Heterogeneity is critical for immune system health, else our defenses are easily evaded by pathogens. Likewise, epigenetic diversity likely governs lineage decisions during development. Some enhancers appear to open prior to the transcription of proximal genes, which we hypothesized would create gradients of epigenetic activity in developmentally intermediate, but otherwise uniform, cells. To evaluate regulatory diversity in a fixed, well defined population, we performed single cell accessibility measurements on Rag-deficient double negative 3 (DN3) thymocytes. Computationally defined models predicted epigenetic stratification within this homogenous population. Sequence-based imputations further separated the DN3 cells via enrichment for lineage-defining transcription factor motifs, suggesting the epigenetic spectra reflects early versus late DN3 stages. As antigen receptor (AgR) loci recombine within DN3 cells, we also examined if single cell accessibility of AgR promoters and recombination signal sequences could predict pre-selection T Cell Receptor Beta (TCRb) repertoire. Compared to algorithms that use bulk measurements of chromatin composition or DNA sequence, single cell accessibility had slightly improved accuracy in predicting Vb segment choice. Collectively, our results illustrate the application, and perhaps complexities, of interpreting single cell accessibility data, and identify possible regulatory dynamics within an otherwise homogenous DN3 population.
Proteomic analysis of microglia during LPS-induced acute neuroinflammation.
Authors: Cartier Anna , Moyron-Quiroz Juan , Huang Tse Shun , Tam Miguel 
Published: February 2023

Abstract
Dysregulation of microglia function is associated with neuroinflammation. A major limitation in understanding microglial contribution to cellular processes and their role in disease has been the lack of tools to specifically distinguish these cells from other myeloid cells. We generated and validated a rat monoclonal antibody against a selective microglia marker P2RY12. We utilized LPS administration as a model for neuroinflammation to assess alterations in P2RY12 expression, a homeostatic protein. Mice were injected with either PBS, low or high dosages of LPS daily for 4 days after which microglia were harvested from the brain. Isolated cells were co-stained with CD11b, CX3CR1, and P2RY12 as general markers for microglia. Flow cytometric quantification using live cells showed a significant increase in CD11b+ expression in both low and high dose LPS treated mice compared to the control untreated group. Low and high dose LPS injections led to a significant reduction in P2RY12 and CX3CR1 expression in these cells. These data are consistent with a decrease in P2RY12 expression under inflammatory conditions. We assessed microglial proteomic profile using BioLegend’s proprietary technology, TotalSeq™, which allows simultaneous analysis of both the transcriptome and proteome at the single-cell level. We utilized a panel of 200 antibody-oligonucleotide conjugates, including P2RY12, to analyze differentially expressed proteins under normal and LPS-induced inflammation in mice. These studies help elucidate the diversity and physiology of microglia, and potentially aid in the discovery of novel markers in response to neuroinflammation.
Human-type artificial lymphoid tissues (organoids) induce antigen-specific immune responses upon antigen-stimulation
Authors: Watanabe Takeshi 
Published: February 2023

Abstract
We had previously reported that the immunologically active artificial lymphoid tissues (aLTs) could be stably generated in mice by implantation of lymphoid stromal cell-embedded biocompatible scaffold (collagen sponge) first in 2004 (Suematsu and Watanabe) as well as later by applying the chemokine/lymphokine-trapped gel-embedded scaffold in 2016 (Kobayashi and Watanabe). The generation of the functional human-type aLTs might be beneficial in future for clinical application as the immunotherapy against cancers as well as the normalization of diseases caused by malfunction of human immune system. It may be applied for the evaluation of vaccines. We first established the human stromal cell lines suitable for the formation of the human aLTs. Then, spheroids composed of the human stromal cells were prepared. The mixtures of stromal cell spheroids and human peripheral blood lymphocytes (PBMCs) were transplanted into renal subcapsular spaces of the immunodeficient NOG-dKO mice. After 2–3 weeks, human type aLTs (hPBMC-aLTs) were stably formed. The human type aLTs are composed of the clusters of human B and T cells including scattered human DC cells. The human type aLTs were immunized with the inactivated varicella zoster virus (VZV) or with NP-conjugated ovalbumin (NP-OVA) in adjuvant. Anti-VZV or anti-NP-hapten specific antibody-producing cells were strongly induced in the human-aLTs. IFNg was produced from human T cells in aLTs upon the immunization. These data indicate that the immunologically functional human aLTs could be successfully generated. We are currently investigating anti-cancer activity of the human aLTs as well as attempting to construct them in vitro system.
Immunophenotypic and functional characterization of circulating cytotoxic T lymphocytes and natural killer cells using the BD FACSymphony™ A1 Cell Analyzer
Authors: Baracho Gisele Vanessa , Kara Nihan , Rigaud Stephanie , Widmann Stephanie , Tyznik Aaron 
Published: February 2023

Abstract
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells have become attractive therapeutic targets for cancer immunotherapy because of their efficient mechanisms for killing cancerous cells. Both effector CTLs and mature NK cells are endowed with secretory granules containing pre-formed cytolytic proteins that, once released at the immunological synapse, initiate a cascade of reactions leading to the apoptosis of the target cell. CTLs and NK cells may also promote cell killing through death-receptor-mediated and cytokine-induced cell death mechanisms. Here we show a 16-color flow cytometry panel allowing clear identification of CTLs, NK cells and related cell subsets in whole blood. We also describe an optimized intracellular staining protocol for analysis of granules content, including granzyme B, granzyme K and perforin. Optimal resolution of cell populations was achieved by employing a selection of BD Horizon Brilliant™ Reagents and BD Horizon™ Red 718 Reagent, a new small molecule fluorochrome option for the red laser. Cell fixation and permeabilization were carried out directly in whole blood using the BD Phosflow™ Reagents, adding convenience to the workflow and practical analysis of cell frequencies. The data also showed the expression of selected activation and inhibitory surface receptors. These results combined with the analysis of intracellular proteins contribute to characterization of the functional status of cytotoxic cells.
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Proteomic stabilization of Cynomolgus Macaque blood with PROT-1 for flow cytometric analysis of activated innate and adaptive immune cells after extended storage
Authors: Williams Jamie , Genell Caroline A , Nuti Sandra , Delamere Patricia A , Maier Curtis C , Marshall Nikki B 
Published: February 2023

Abstract
Flow cytometry is a widely used, and rapidly evolving, immunophenotyping application; however, limited advancements around sample stability and preservation continue to strain scientists in resource-limited settings. As flow cytometric samples are labile, data acquisition is generally performed within 24 hours, with traditional fixation methods, to minimize impact to the quality of the analysis. Here, we describe the development of an optimized proteomic stabilization method and 13-color immunophenotyping panel that allows for long-term storage of Cynomolgus Macaque whole blood and retrospective phenotyping of resting and activated populations of innate and adaptive immune cells including T cells, NK cells, B cells, monocytes and dendritic cells. Taken together, these methods highlight a key advancement around flow cytometry sample stability and preservation, allowing for greater resource flexibility without compromising quality of the analysis.
Sertoli cell induction of Tregs: a critical component of Sertoli cell immune-regulation in transplantation
Authors: Hibler Taylor , Kaur Gurvinder , Dufour Jannette 
Published: February 2023

Abstract
Sertoli cells (SC) are immunoregulatory testicular cells that protect and nurture germ cells, which develop after central tolerance is established and are classified as non-self by the immune system. This ability to protect germ cells makes SC a promising alternative to immunosuppressive therapy in transplantation; indeed, they have been co-transplanted with various tissues in animal models with significant success. It has been suggested that regulatory T cells (Tregs) play a role in SC graft survival and protection of germ cells. The goal of this study was to determine if SC promote graft survival through induction of Tregs. To test this, B6 SC were transplanted into BALB/c mice in absence of immunosuppressive drugs. 100% of SC grafts survived to day 20, while 100% of controls (nonimmune privileged cells) rejected by day 20. Analysis of the grafts through immunohistochemistry and flow cytometry revealed that SC grafts had significantly increased Tregs compared to rejecting control grafts. To determine if Tregs were critical for SC survival, B6 SC were transplanted into BALB/c mice depleted of Tregs. 100% of SC grafts without Tregs rejected within 20 days. Excitingly, 57% of SC grafts in Treg depleted mice survived. Flow cytometry revealed that all of the surviving grafts had Tregs, indicating that SC can induce Tregs from naïve T cells and that these Tregs are critical for SC graft survival. The presence of Tregs in surviving pig SC grafts was also shown in a xenogeneic model. Interestingly, the xenograft model showed a significant decrease in T effector cells, indicating that SC are both inducing Tregs and inhibiting effector cells. In summary, SC can survive as both allo- and xenografts without immunosuppression through the activation of Tregs.
Immunosuppression by cyclosporin A via inhibition of TCR-dependent but NFAT-independent functions
Authors: Otsuka Shizuka , Melis Nicolas , Gaida Matthias M , Dutta Debjani , Weigert Roberto , Ashwell Jonathan D 
Published: February 2023

Abstract
Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and graft-versus-host disease (GVHD), for which CNI inhibition of NFAT-dependent gene expression is thought to be the major mechanism. We recently reported that CNIs also inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification. Here we have utilized mice expressing LckS59A, which cannot accept a phosphate at residue 59, to explore the biological significance of CsA-mediated inhibition of TCR signaling. We find that whereas CsA inhibited NFAT activation normally, TCR-signaling activation in T cells expressing LckS59A was resistant to CsA. Importantly, although upregulation of NFAT-dependent effector cytokines such as IL-2 and IFN-g was inhibited, CsA was largely ineffective in suppressing acute GVHD when the allo-aggressive T cells expressed LckS59A. Two important T cell effector pathways were found to be resistant to CsA in LckS59A T cells from acute GVHD mice: upregulation of the cytolytic protein perforin in CD8+ T cells and induction of antigen specific LFA-1/ICAM-mediated T cell:DC (dendritic cell) adhesion as detected by 2-photon imaging. These results demonstrate that therapeutic effective treatment of aGVHD by CsA requires inhibition of TCR-associated calcineurin and proximal signaling.
Blockade of the immune checkpoint B7-H3 alters glucose metabolism in non-small lung cancer cells
Authors: Zhao Qi , Yang Shuo 
Published: February 2023

Abstract
B7-H3 an immune-checkpoint molecule, a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. An anti-B7-H3 antibody omburtamab has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Elicitation of cellular mechanism behind the anti-B7-H3 blockade is important to reveal the immune-independent functions of B7-H3. Warburg effect is one feature of metabolic dysregulation during malignant transformation. To investigate the effect of anti-B7-H3 blockade in the regulation of glucose metabolism in cancer cells, we measured dynamic ECAR and OCR as indicatives of glycolysis and oxidative respiration in NSCLC cells. We demonstrated that anti-B7-H3 antibody blockade switched cell metabolism from glycolysis to oxidative phosphorylation. The alteration of glucose metabolism probably was associated with cellular ROS-mediated pathway. These results clearly showed that, in addition to immune-regulatory function, B7-H3 possessed immune-independent functions in NSCLC. The study is supported by the Science and Technology Development Fund, Macau SAR (FDCT/0015/2918/A1).