bluuebunny/crossref_metadata_2025_split huggingface dataset:
B cells licensed by DCs are primary antigen presenting cells for DNA plasmid upon passive uptake
Authors: Rastogi Ichwaku , McNeel Douglas G 
Published: January 2023

Abstract
Dendritic cells are considered the primary cells involved in professional antigen presentation. However, our group and others have shown that B cells are similarly capable of presenting antigens leading to activation and proliferation of antigen-specific T cells. In this study, we aimed to understand the mechanism of presentation of antigen, encoded by plasmid DNA when delivered to APCs via passive uptake. We used plasmid DNA encoding ovalbumin, splenic B cells from Bl6 mice, DCs from Flt3L treated Bl6 mice, and CD8 T cells from OT1 mice that are restricted for the SIINFEKL-specific T-cell receptor. We have previously reported that only B cells are capable of transcribing the encoded antigen, whereas DCs and macrophages degrade the plasmid DNA upon passive uptake. In our current study we show that B cells also translate the encoded antigen, but can present the antigen to CD8 T cells only in the presence of DCs. We utilized a trans-well assay to show that physical contact between B cells and DCs is required for successful antigen presentation and that live DCs are essential, as replacing them with DC lysate or DC supernatant or both was not adequate. We also show that the presence of DCs promotes B cell survival, mostly through CD40-CD40L signaling by using CD40 KO mice and that B cells are primary antigen presenting cells in this co-culture by using MHC I KO mice. We also show that exposure of B cells to CD40 or CD40L, which is one of the identified interactions between B cells and DCs, was not sufficient for B cells to present antigen in the absence of DCs. In summary, our data suggests that B cells and DCs communicate through a currently unknown cell-cell interaction that licenses B cells for antigen presentation after passive uptake of plasmid DNA.
Repertoire size and predictability of influenza A virus matrix protein (M158–66)-specific CD8+ TCR
Authors: Li Jiangyuan , Cifello Jeffrey , Lu Jian , Weng Nan-Ping 
Published: January 2023

Abstract
Influenza A virus (IAV) is a major human pathogen that causes seasonal infections worldwide. IAV-specific CD8+ T-cell response provides an essential part of immune protection against IAV infections. In HLA-A2+ humans, the dominant epitope on IAV is matrix protein M1 residues 58–66 (GIL GFVFTL, referred to as GIL). Here, we investigated the TCR repertoire size, predictability, and signaling strength of GIL-specific CD8+ T cells healthy adults. We analyzed over 40,000 GIL-tetramer positive CD8+ T cells by scRNAseq with TCR sequences and identified over 10,000 unique abTCRs sequences. Next, we developed a machine learning algorithm (an ensemble random forest models) to further predict the probability of GIL-binding TCR (ranging from 0.1 to 1 with 1 being highest binding confidence) using reported GIL-TCRs. To confirm the predicting accuracy, we expressed 72 TCR with different RF predicting score and showed that TCR with a RF score greater than 0.8 showed 100% GIL-binding (30 different TCRs). Using RF model and filtering, we identified over 4,000 unique GIL-specific TCRs with less than 800 were previously reported. Overall, our study revealed TCR repertoire size of GIL-specific CD8+ TCRs and indicated the features of GIL-binding TCRs are identified by our ML model to accurately predict this TCR specificity, which paces way to investigate frequency and quality of antigen specific TCR repertoire size and function in the general T cell population.
Reverse Immunology: An approach to identify Mtb antigens recognized by T cells
Authors: Jaiswal Swati , Williams Travis , Gideon Hannah P , Hughes Travis K , Tu Ang Andy , Nyquist Sarah K , Shalek Alex N , Fortune Sara M , Flynn JoAnne L , Behar Samuel 
Published: January 2023

Abstract
T cells plays important role in controlling the infection. Previous research findings have suggested that many immunodominant antigens are not similarly immunogenic. We attempt to discover novel antigen that can be developed as a potential vaccine candidate. Low dose Mycobacterium tuberculosis (Mtb) infection in Cynomolgus macaque results in human like granuloma formation. It is comprised of T cells, B cells, NK cells, and several other cells. Upon FDG-PET scan it has been shown that some of these granulomas are permissive or progressive in nature and some are restrictive or controlling.
Our aim is to discover Mtb antigens that can be recognized by the T cells present in the granuloma. To investigate our hypothesis, we have infected Cynomolgus macaque with low dose of Mtb. T cells were isolated from the low and high burden granuloma. They were subjected to scRNA sequencing. Using bioinformatic tools and algorithms we have identified high value TCRs from controlling granulomas. The TCRs were reconstructed and transfected in TCR deficient Jurkat cells with NFAT promoter and luciferase reporter gene. The Mtb ORFeome library was cloned and expressed in E. coli. For antigen preparation, IPTG induced recombinant E. coli were killed by Peracetic acid or heat-killed and presented to the high value TCRs expressed in the reporter Jurkat cells by autologous BLCLs. The recognition was measured using luciferase activity.
The selected unique TCRs show remarkable luciferase activity in response to PMA and Ionomycin stimulation. We believe that the antigen recognized by these TCRs will be equally immunogenic and can be developed as the new target for the vaccine against tuberculosis.
Supported by BAA-NIAID-NIHAI201700104
H-2Kb and H-2Db class I molecules on cerebral endothelium differentially modulate CD8 T cells dynamics and pathological outcomes in experimental cerebral malaria
Authors: Fain Cori E , Zheng Jiaying , Jin Fang , Ayasoufi Katayoun , Chen Maggie , Dropik Abigail , Khadka Roman , Tritz Zachariah Peter , Hansen Michael , Johnson Aaron J 
Published: January 2023

Abstract
Immune mechanisms of neuropathology in human cerebral malaria are not fully understood. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we sought to define the contribution of cerebral endothelial cell (CEC) MHC class I antigen presentation, to the pathogenesis and lethality of ECM. CD8 T cell interactions with class I molecules on CECs has been put forward as a putative mechanism for ECM. However, the importance of this interaction has not been confirmed molecularly in vivo. We therefore hypothesized that conditional ablation of antigen presentation by H-2Kb and H-2Db class I molecules specifically on brain endothelium would result in controlled modulation of activation and entry of parasite-specific CD8 T cells, attenuating pathology. To test this, we crossed endothelium-specific CDH5-Cre mice with our novel H-2Kb and H-2Db LoxP mice to create endothelium-specific cKO of either or both class I molecules. 2-photon imaging revealed that each discreet class I molecule influenced differential T cell dynamics at the neurovasculature during lethal stage of disease. Furthermore, CD8 T cell activation and infiltration of the brain was distinctly altered, per class I interaction, and vascular permeability at the blood-brain barrier was attenuated, resulting in improved survival of H-2Kb and H-2Db endothelial cKO mice. Together these data demonstrate that parasite-specific CD8 T cells undergo Ag-dependent engagement with H-2Kb or H-2Db class I molecules on CECs in order to coordinate activation, entry, and induction of neuropathology. These studies indicate that modulation of CEC class I restricted antigen presentation may hold therapeutic benefit in cases of severe malaria.
Supported by Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship F31 1F31NS116924-01. NIH R01 5R01NS103212-05.
mTORC2 contributes to murine lupus associated immunopathology
Authors: Zeng Hu , Zhou Xian , Li Yanfeng , Davidson Anne 
Published: January 2023

Abstract
The development of systemic lupus erythematosus (SLE) is associated with overactivation of the type I interferon (IFN) pathway, lymphopenia, and increased follicular helper T (Tfh) cell differentiation. However, the cellular and molecular mechanisms of action of type I IFN in SLE remains incompletely understood. Here we show that type I IFN activates the mechanistic target of rapamycin complex 2 (mTORC2) in T cells to promote T cell lymphopenia. mTORC2 also promotes Tfh differentiation and disrupts Treg homeostasis. Inactivation of mTORC2 greatly ameliorated the immunopathology in a lupus-prone mouse model, associated with reduced Tfh differentiation, normalization of Treg homeostasis and reduced T cell glucose metabolism. These data indicate that mTORC2 acts downstream of type I IFN and costimulatory receptor ICOS, to promote T cell lymphopenia and Tfh differentiation in murine lupus development, suggesting that inhibition of mTORC2 could limit lupus disease progression.
Supported by Lupus Research Alliance Mayo Foundation for Medical Education and Research R01AR077518
Enrichment of Epstein Barr Virus in patients with Multiple Sclerosis
Authors: Kim Ellie , Forney Carmy , Viel Kenyatta , O’Brien Madison , Nelson Catrina , Gecaine Paul , Zabeti Aram , Weirauch Matthew , Kottyan Leah 
Published: January 2023

Abstract
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system affecting more than 2 million people worldwide and has been linked with Epstein-Barr Virus (EBV) infection. Previous studies by the Kottyan and Weirauch labs show that the EBNA2 protein of both EBV type 1 and 2 binds unique MS risk loci potentially causing changes at the transcriptional level contributing to MS disease progress. We hypothesize that type 1 and type 2 EBV will be detectable in the MS patient samples, and we aim to quantify EBV enrichment and identify EBV type in MS patient-derived blood samples. Analysis by qPCR showed that 30% of the control population (n=10) and 66% of the MS patient population (n=41) had detectable levels of EBV whereas the MS patients had a significantly higher frequency of EBV presence (X2 test p=0.0389). Of the samples that tested positive for EBV, 66% showed presence of type 1 EBV while 33% were unknown without a significant difference between MS patients and control group (X2 test p&gt;0.9999). Due to issues with the limit of detection, droplet digital PCR was assessed for higher sensitivity compared to qPCR, but was found to have a comparable limit of detection to qPCR. To overcome the challenge of detecting low copy number viruses, we look toward targeted DNA capture and next generation sequencing to improve the sensitivity of EBV detection and specificity of EBV typing. Understanding the distribution of EBV types among MS patients would elucidate the precise role of EBV in MS development, potentially leading to improved treatment and prevention methods.
Supported by grants from NIH grants R01 HG010730, R01 NS099068, R01 GM055479, and U01 AI130830 to MTW; R01 DK107502, R01 AI148276, U19 AI070235, U01 HG011172, and P30 AR070549 to LCK; R01 AR073228, R01 AI024717, and CCHMC ARC Award 53632 to MTW and LCK.
Cyanobacteria Geitlerinema sp. lipopolysaccharide induces neutrophil-dependent lung inflammation
Authors: Del Toro Rosalinda Monroy , Incrocci Ryan , Williams Philip , Mayer Alejandro M S , Swartzendruber Julie A , Swanson-Mungerson Michelle 
Published: January 2023

Abstract
Cyanobacterial blooms found in freshwater systems are a source of environmental toxins that impact the immune system. Geitlerinema sp., a strain of cyanobacteria, produces toxins including lipopolysaccharide (LPS) which induces fever and potential respiratory disease. One mechanism of respiratory disease and inflammation is through the inhalation of LPS that is associated with neutrophil infiltration of the lungs. Based on our previous data that Geitlerinema sp. induces pro-inflammatory cytokines, we hypothesized that inhalation of Geitlerinema sp. LPS would induce lung inflammation in vivo. To address this hypothesis, c57Bl/6 mice were intranasally exposed to Geitlerinema sp. LPS before lungs and BAL fluid were assessed for inflammation and inflammatory markers 24 hours later. Our data show that inhalation of Geitlerinema sp. LPS induced lung inflammation by recruiting neutrophils into the airways. The data also demonstrate that Geitlerinema sp. LPS induces proinflammatory cytokines and chemokines: IL-6, IL-8, IFN-g, RANTES and TNF-a in the BAL fluid. Additionally, in vitro data using an airway epithelial cell line show that Geitlerinema sp. LPS induced production of pro-inflammatory cytokines is dependent on the NF-κB pathway. These data help to better understand the mechanism of Geitlerinema sp. LPS airway-induced inflammation and implicates the importance of the toxin in promoting human disease near freshwater ecosystems where cyanobacteria bloom.
A new β2 integrin activation reporter mouse reveals localized intra- and extra-vascular neutrophil integrin activation in vivo
Authors: Wen Lai , Marki Alex , Wang Zhihao , Orecchioni Marco , Makings Jeffrey , Kim Kenneth , Kiosses William B , Mikulski Zbigniew , Ley Klaus 
Published: January 2023

Abstract
β2 integrins (LFA-1, Mac-1, CD11c-CD18, and CD11d-CD18) are leukocyte-specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). Human β2 integrin activation can be detected by reporter antibodies including mAb24 and KIM127. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA-1, Mac-1 and CD11c-CD18 in all major leukocyte populations. Using a CXCL1-induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high-dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo.
This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L.) and a Postdoctoral Fellowship (19POST34450228 to L.W.) from the American Heart Association, USA.
Isoflurane targets BLT1 to attenuate lung injury
Authors: Yuki Koichi , Koutsogiannaki Sophia 
Published: January 2023

Abstract
Sepsis-associated lung injury is one of major comorbidities in sepsis, largely responsible for its mortality. Lack of specific therapeutic for lung injury is an urgent issue to be addressed. We found that volatile anesthetic isoflurane attenuated lung injury associated with experimental polymicrobial abdominal sepsis. This was associated with a reduction in the number of recruited neutrophils in the lung and bronchoalveolar space. Furthermore, isoflurane attenuated neutrophil chemotaxis induced by major chemoattractant leukotriene B4 (LTB4), not by C5a. Although LTB4 level did not differ by isoflurane exposure, isoflurane was shown to interact with LTB4’s neutrophil receptor BLT1 in silico. The interaction site was predicted to overlap with BLT1 antagonist BIIL260 binding site, suggesting isoflurane as a BLT1 antagonist. Five biotinylated compounds with a range of spacer lengths were developed and subjected to calcium mobilization test using CHO-BLT1 cells to determine their property as BLT1 antagonist. Two compounds showed adequate inhibition and we performed the competition assay for BLT1 between the compound and isoflurane. Isoflurane significantly competed with the BIIL260 biotinylated compound, indicating that isofluane bound to the BIIL260 binding site on BLT1. Isoflurane is considered as an alternative sedative in Europe and Canada, and this result could support the potential use of isoflurane as an ICU sedative in lung injury setting.
Supported by GM118277
Inflammation depletes humoral immunity by limiting plasma cell access to the bone marrow survival niche
Authors: Aaron Tonya , Benet Zachary , Fooksman David 
Published: January 2023

Abstract
The survival of plasma cells (PCs) is directly correlated to the longevity of prophylactic titers against pathogens. The bone marrow contains hematopoietic and non-hematopoietic cells that contribute to the maintenance of PCs. Studies have shown that pathogens contain a variety of mechanisms that result in the depletion of bone marrow PCs and antibody titers. Most, if not all, forms of infection induce inflammation, however, the role of inflammation in bone marrow PC depletion is unclear. Here, we show that acute Tumor Necrosis Factor Alpha (TNFa) treatment can deplete pre-existing antibody titers in a variety of models. We also found that recombinant TNFa treatment limits PC retention in the bone marrow via adoptive transfer experiments. To determine if PC retention in the bone marrow was mediated in a cell-intrinsic or − extrinsic manner, we generated bone marrow chimeras with wild-type and TNFa receptor knockout mice. Results showed that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells regulates PC retention in the bone marrow, indicating that this mechanism is cell-extrinsic. Another cell-extrinsic mechanism that we identified is TNFa regulation of syndecan-1, a cell surface proteoglycan found to regulate PC survival and motility. These findings demonstrate that inflammation limits PC access to the bone marrow survival niche, ultimately leading to the reduction of antibody titers and long-term humoral protection.
Supported by grants from the NIH (R01 HLI141491) and the Eric Heyer M.D. Ph.D Translational Research Grant
XIST is a source of TLR7 ligands underlying the sex bias in Systemic Lupus Erythematosus
Authors: Crawford Jonathan D , Wang Hong , Talbot C Conover , Curran Ashley M , Goldman Daniel W , Petri Michelle , Antiochos Brendan , Darrah Erika 
Published: January 2023

Abstract
Systemic Lupus Erythematosus (SLE) is among the most sex-biased autoimmune diseases identified to date, affecting 9-times more women than men. Recognition of self-RNA by Toll-like receptor 7 (TLR7) is implicated as a central pathogenic process leading to the aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules contributing to this process have not been defined. Given the role of self-RNA and biological sex in SLE pathogenesis, we investigated which sex-biased self-RNAs are potentially responsible for aberrant TLR7 activation in SLE. We used recent revelations about TLR7 sequence specificity and publicly available RNA sequencing data to identify sex-biased sources of self-RNA containing TLR7 ligands. We found X-inactive specific transcript (XIST) to be a particularly rich source of TLR7 ligands that is specifically expressed in women. We then investigated the capacity of XIST to act as a TLR7 ligand in vitro and found that multiple fragments of XIST induce IFNα production in a TLR7-dependent manner more than control RNA of equal length. Furthermore, we found that RNA isolated from XIST-knockout cells have significantly reduced capacity to stimulate TLR7 compared to RNA from wild-type cells. Finally, we used flow cytometry and publicly available RNA sequencing data to investigate the connection between XIST and SLE disease variables. We found that higher XIST expression correlated with SLE disease status, higher SLE disease activity index (SLEDAI) scores, and the interferon signature. Our data suggest that XIST is a source of TLR7 ligands that may underlie the sex bias in SLE.
Supported by grants from National Institute of Dental and Craniofacial Research (NIDCR), NIH (R21 DE028391-02), the Jerome L. Greene Foundation and Scleroderma Research Foundation.
T cells dominate peripheral inflammation in obesity-associated diabetes
Authors: Pugh Gabriella , Fouladvand Sajjad , SantaCruz-Calvo Sara , Agrawal Madhur , Zhang Xiaohua D , Chen Jin , Kern Philip , Nikolajczyk Barbara 
Published: January 2023

Abstract
Many studies have examined obese/insulin-resistant mice to conclude that macrophages dominate pathogenic inflammation in metabolic disease, but few studies have ranked chronic sources of peripheral inflammation in obese people across a natural spectrum of metabolic health. We compared the relative contributions of myeloid and T cells to peripheral inflammation in samples from metabolically healthy and unhealthy obese people to address disappointing outcomes from clinical trials of anti-inflammatories in obesity-associated type 2 diabetes (T2D). We stimulated PBMCs from obese subjects with euglycemia, prediabetes, or T2D with T cell-targeting CD3/CD28 or myeloid-targeting LPS, and measured supernatant cytokines over time. Cytokines increased with time following CD3/CD28, but LPS responses were time-independent. A series of surface marker and intracellular staining assays taking into account these temporal differences in cytokine production unexpectedly showed that T cells contribute more TNFα to peripheral inflammation than do myeloid cells from all subject groups. Bioinformatic modeling combining cytokine outcomes from all subjects, stimuli, and time points, indicated IFNγ generated by CD3/CD28 stimulation was most important for differentiating peripheral inflammation in T2D compared to non-T2D samples. This result was consistent with the previously identified Th1/Th17 profile in T2D. We conclude that human T cells dominate peripheral inflammation in obesity-associated T2D, and thus targeting T cells may be an effective approach for prevention/management of obesity-associated metabolic diseases.
Supported by NIH Training Grants T32 DK007778 and TL1 UL1TR001998, R01DK108056, the Shared Resource Facility of the University of Kentucky Markey Cancer Center P30 CA177558, University of Kentucky College of Medicine, Barnstable Brown Diabetes and Obesity Center, The Center for Clinical and Translational Research (UL1TR000117), and NIH National Center for Advancing Translational Sciences UL1TR001998.