bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intestinal immunity in the experimental model of Taenia solium taeniosis (91.12)
Authors: Flisser Ana , Avila Guillermina , Aguilar Laura , Velasco Sofia , Garcia Francisco 
Published: January 2023

Abstract
Taenia solium can grow in normal hamsters, mature worms are obtained. Expulsion starts at 3 weeks and parasites may remain up to 2 months. An inflammatory reaction in the intestinal mucosa, surrounding the scolex of the worm, is produced; it comprised lymphocytes, plasma cells, eosinophils and goblet cells. Presently we are searching for mRNA of Th1 and Th2 interleukins by in situ hybridization in intestinal biopsies. For this purpose hamsters were infected per os with 8 T. solium cysticerci, necropsies were performed at different days post-infection. One cm biopsies of the small intestine were taken from the area surrounding the scolex of all tapeworms found, fixed in PBS-paraformaldehyde and processed by histological techniques; 3μm sections were obtained and adhered to electrocharged slides. Antisense probes of DNA for the detection of mRNA were constructed according to published literature of IFNγ, IL-4, IL-5 and IL-13. In situ hybridization was performed with digoxigenin labeled oligonucleotides; alpha tubuline was used as a positive control and human papiloma virus (p16-2R) as a negative control. Immunological detection was obtained with antibodies against digoxigenin coupled to alkaline phosphatase or peroxidase. Kinetics of each cytokine was defined by counting the number of positive cells per 100 enterocytes. IFNγ was the first cytokine detected, followed by IL-13, since day 2 and 4 respectively. Then, IL-4 and IL-5 became apparent at the end of week one and continued positive for the following weeks. These data point to a shift from Th1 to Th2 cytokines, since no positive reaction for IFNγ was observed from week 2 onwards.
INF-Gamma-Inducible Lysosomal Thiol Reductase affects the Expression and Function of Manganese Superoxide Dismutase in Mouse Fibroblasts (91.3)
Authors: Bogunovic Branka M , Maric Maja 
Published: January 2023

Abstract
INF-Gamma-Inducible Lysosomal Thiol Reductase (GILT) is a unique member of thiol reductase family with optimal enzymatic activity at low pH. It is located in the endosomal compartment of the cell where the antigen processing takes place. GILT facilitates protein unfolding, which makes protein more accessible to processing by proteases present in endosomes/lysosmes and consequent presentation of resulting peptides by MHCII. In addition, it has been shown that GILT has an APC-unrelated role in the regulation of T cell activation. In this study, we demonstrate that GILT affects the expression and function of manganese superoxide dismutase (SOD2) in mouse fibroblast cell line. In comparison to the wild-type, GILT-deficient mouse ear fibroblasts had significantly lower SOD2 expression level and function. In addition, GILT-deficient mouse fibroblasts have shown increased proliferation. These data raise the possibility that there may be a direct and/or indirect effect of lysosomal protein GILT on mitochondrial SOD2 expression level, and function.
This work was supported by Scientist Development Grant, American Heart Association 0535032N
Talin-Deficient NK Cells Reveal a Critical Role for Talin in LFA-1-Mediated Actin Cytoskeletal Rearrangement (91.4)
Authors: Mace Emily Margaret , Takei Fumio 
Published: January 2023

Abstract
The purpose of this study is to elucidate the role that the cytoskeletal adaptor protein talin1 plays in the regulation of LFA-1 in natural killer (NK) cells. It has been proposed that LFA-1 not only mediates adhesion of NK cells to target cells, but may be an important activating receptor for NK cell cytotoxicity. Talin is known to act as a link between integrins and the actin cytoskeleton and to regulate ligand-binding function of LFA-1. To study the role of talin, we generated NK cells in vitro from talin1−/− ES cells and tested the effects of talin deficiency on LFA-1-mediated functions of these NK cells. Our results showed that talin is required for both adhesion of NK cells to ICAM-1 and for LFA-1-mediated activation signal leading to the re-organization of the actin cytoskeleton. Nevertheless, talin-deficient NK cells still killed some target cells in an LFA-1-independent manner, suggesting an alternative pathway of NK cytotoxicity independent of LFA-1 and talin. Thus we have shown that talin plays a critical role in LFA-1 mediated actin cytoskeletal reorganization in NK cells.
Differential regulation of cyclooxygenase (COX) isoforms in alveolar (AM) and peritoneal macrophages (PM) from heat-killed Mycobacterium bovis BCG treated mice (91.5)
Authors: Shinohara Tsutomu , Yamashita Makiko , Nishiyama Akihito , Tsuji Shoutaro , Henriksen Ruth Ann , Myrvik Quentin N 
Published: January 2023

Abstract
Macrophage COX-2, a rate-limiting enzyme for the increased synthesis of PGE2, a mediator of inflammatory and immune responses against infections, is induced by intracellular bacteria. However, little is known about the regulation of expression of COX-2 or COX-1 by different tissue macrophages in response to bacteria in vivo. In this study, AM and PM were isolated from C57Bl/6 mice 24 hr following intranasal or intraperitoneal administration of BCG, 0.5 mg or 1 mg, respectively. As comparison controls, normal AM and PM were treated in vitro with 20 μg/ml BCG. Expression of COX-1 and COX-2 was determined by confocal microscopy and western blot. We found that in vivo BCG induced COX-2 expression in local AM or PM. The constitutive expression of COX-1 in PM was down-regulated by in vivo BCG. For AM, COX-1 levels, which were minimal in untreated cells, were slightly increased by in vivo BCG. Following in vivo BCG, neither COX-1 nor COX-2 in PM or AM was localized in the nuclear envelope (NE), indicating catalytically inactive forms. Treatment with BCG in vitro induced catalytically active COX-2 expression in AM and PM, but there was no effect on COX-1 expression or activity. Thus, following in vivo treatment with BCG, the profile of COX-1 expression by local macrophages is tissue dependent. However, the formation of catalytically inactive COX-2/COX-1 in response to in vivo BCG is tissue-independent. The formation of macrophages with catalytically inactive COX isoforms and diminished PGE2 production may result in enhanced immune responses against intracellular infections. (NIH RO1 HL71711, DOD DAMD 17-03-1-0004)
Loss of function in effector T cells is a consequence of residence in the normal lung environment (92.3)
Authors: Arimilli Subhashini , Miller Martha Alexander 
Published: January 2023

Abstract
It is well accepted that the lung environment is immunosuppressive. This is in part related to the presence of immunosuppressive mediators even in the absence of ongoing infection. Recently a number of studies have reported the loss of function of CD8+ effector cells that enter the lung following viral infection. The impaired function of these cells was proposed to be the result of infection induced changes in the lung. However, our recent findings suggest an alternative model. Our data provide evidence that the normal lung environment is sufficient to promote loss of function in effector cells. Transfer of an established CTL line into naïve BALB/c mice resulted in a significant loss of function in lung resident cells within 48 hours. At the time of transfer cells were 95% functional; however 48 hours post-transfer only 25% of cells produced IFN&#947; in response to peptide stimulation. Similar findings were obtained following transfer of effector cells generated by short-term culture of Listeria p60-specific TCR transgenic splenocytes or by in vivo Listeria infection. Importantly loss of function was restricted to lung parenchymal cells as airway or spleen resident cells retained function at levels similar to pre-transfer cultures. These findings suggest a new paradigm for the immunoregulation of effector cells in the lung. Understanding the mechanism responsible for the loss of function may lead to therapeutics that can prevent this outcome in cases where an increased number of effectors is desired or promote it in cases where pathogenic effector cells are present.
Automated quantitation of events with protein localized to the T:APC synapse using the ImageStream imaging flow cytometer (92.4)
Authors: George Thaddeus , Burbach Brandon J , Shimizu Yoji , Harmon Ian , Peterson Erik J , Hall Brian E , Morrissey Philip J 
Published: January 2023

Abstract
Initiation of antigen-specific adative immune responses requires interaction between a T cell and an antigen presenting cell (APC) that presents a peptide/MHC complex recognized by the T cell receptor. Maintenance of strong contact between the T:APC conjugate and subsequent activation of the T cell involve recruitment towards the conjugate interface (or ‘immune synapse’) of adaptor proteins that regulate cytoskeletal reorganization and T cell gene transcription. Evaluation of molecular recruitment to the immune synapse is traditionally performed using microscopy. However, because manual image acquistion and quantitative image analysis are time consuming processes, analyses of protein recruitment to the immune synapse have remained largely qualitative. Here we describe a method for quantifying events with protein localized to the immune synapse using the ImageStream imaging flow cytometer, which automatically collects large numbers of images per data set and provides quantitative image analysis tools. Lymph node T cells from OVA-specific TCR-transgenic DO11.10 mice were incubated with OVA-pulsed splenocytes to induce T:APC conjugates. Immunofluorescently-stained samples were acquired on the ImageStream and analyzed to 1) specifically distinguish T:APC conjugates from singlets and other doublets using a combination of photometric and morphometric features, and 2) quantify the number of T:APC conjugates with specifc protein recruited to the immune synapse using am intensity-weighted distance metric.
The effect of Bisphenol A on the gap junction-mediated intercellular communication among cultured mouse macrophage cells (92.5)
Authors: Rhee Seung Keun 
Published: January 2023

Abstract
There has been a number of evidence that macrophages establish direct intercellular communication through connexin-forming gap junction channels. In the present study the effect of bisphenol A (BPA), a potent endocrine disruptor, on the gap junction-mediated intercellular communication (GJIC) among cultured mouse macrophage cells (J774 cell-line) was characterized. In J774 cells connexin43 is expressed as a major channel-forming protein. BPA has been described as a xenoestrogen, and detected in human serum and amniotic fluid. The cytotoxicity of BPA toward the cultured cells was evaluated by using both MTT reduction and LDH leakage assay systems. The results showed no appreciable loss in cell viability in the presence of increasing concentrations of BPA (from 0.1 to 3.2 μM) for 1 hr incubation. However, most of cell viability was lost when cells were incubated for 24 hr with the same concentrations of BPA. The BPA acted as an antagonist on GJIC among the cultured macrophage cells, and the phenomenon was dose-dependent and irreversible. According to the data obtained from scrape-loading dye-transfer experiments, three quarters of normal GJIC was reduced by concentration of 0.4 μM BPA for 1 hr incubation. To identify the relevance of this retardation upon BPA treatment, the mRNA and protein levels of Cx43 were assessed with RT-PCR and Western-blotting, respectively. The total protein level of Cx43 was almost constant in a wide range of BPA concentrations, as well as in Cx43 mRNA level. These results suggest that BPA inhibits GJIC through a modulation of the gating of gap junction channels, not through a genomic modulation of Cx43 in macrophage cells.
[This study was supported by Korea Research Foundation Grant 041-D00192]
The role of CTLA-4 in Treg-mediated suppression of T cell LIP (92.6)
Authors: Winstead Colleen , Khoruts Alexander 
Published: January 2023

Abstract
Regulatory T cells are known to express CTLA-4 constitutively, however the role this inhibitory signaling molecule plays in Treg homeostasis and suppression of T cell responses is not well defined. Published reports suggest CTLA-4 expression is associated with Treg production of immunosuppressive cytokines TGF-β and IL-10 and is essential for cell-to-cell-suppression in vitro. Determining what role CTLA-4 expression on Tregs plays in vivo is complicated, however, due to differential expression on regulatory and non-regulatory T cells and development of fatal lymphoproliferative disease in CTLA-4-deficient mice. Recently published reports suggest blockade of CTLA-4 on Tregs abrogates their in vivo function, allowing the generation of effective anti-viral and anti-tumor T cell responses and/or emergence of autoimmune disease. Potential Treg suppressive mechanisms include CTLA-4-to-DC outside-in signaling through B7 and resultant upregulation/activation of tryptophan catabolism and suppression of T cell function/viability. It is thought naturally occurring (thymus-derived) Tregs may function this way to prevent activation and expansion of potentially pathogenic T cell clones in response to infectious stimuli. Our preliminary data suggests outside-in signaling plays a role in suppression of spontaneous T cell LIP by CD4+CD25+Foxp3+ cells (TR). Follow-up experiments will explore this suppressive mechanism in the context of T cell LIP and CD4 T cell LIP-induced autoimmunity.
Diminished FDC activation within germinal centers of CD19-deficient mice (92.8)
Authors: Myers Riley C. , You Yuying , Wang Yue , Carter Robert H. 
Published: January 2023

Abstract
Germinal centers (GC) are sites within secondary lymphoid organs where affinity maturation and somatic hypermutation occur, producing high affinity antibody forming cells and memory B cells. We reported previously that mice with mutant CD19 form GC with dark and light zones, although GC B cell differentiation and proliferation are defective in the light zone of these mice. We now show that PNA+ B cells are induced after SRBC immunization and form a dark zone but fail to enter the FDC (light) zone in mice that lack CD19. These mice also lack marginal zone (MZ) B cells, which have been reported to contribute to GC responses through delivery of immune complexes to follicular dendritic cells (FDC), suggesting a possible link between the lack of MZ B cells and the impaired GC reaction. Therefore, we compared GC in the spleen with those in the LN, which does not have a MZ. We used induction of VCAM-1 expression as a marker for FDC activation. Immunization of CD19-deficient mice induced expression of VCAM-1 on LN FDC. In contrast, induction of VCAM-1 expression on FDC in splenic GC was severely reduced in the same mice. These data suggest distinct mechanisms for FDC activation in the LN, which is independent of CD19 on B cells, and in the spleen. In contrast to the LN, immunization fails to activate FDC in splenic GC in CD19-deficient mice, which leads to defective light zone formation. These and other data suggest that the deficiency of MZ B cells and the failure to activate FDC in mice that lack CD19 may be linked: MZ B cells may play a vital role in activation of FDC in splenic follicles that is critical for GC function.
Supported by NIH R01 AI42265.
Dendritic epidermal T cell dysregulation in the cutaneous diabetic environment (92.9)
Authors: Taylor Kristen R , Mills Robyn E , Jameson Julie M 
Published: January 2023

Abstract
Dendritic epidermal T cells (DETC) are specialized epithelial T cells that express a canonical γδ T cell receptor (TCR), which recognizes stressed or damaged keratinocytes. Once activated, DETC release cytokines and growth factors that facilitate cutaneous wound healing. In diabetes, wound healing is delayed and patients often suffer from chronic infections. We hypothesize that DETC are dysregulated in the diabetic environment, contributing to deficiencies in cytokine and growth factor production. Using the leptin receptor deficient mouse model of Type 2 diabetes, we have determined that DETC are present and exhibit their characteristic dendritic morphology. However, DETC in diabetic mice display decreased levels of T cell activation markers including CD69, CD25 and the γδ TCR. Glucose transporter levels are decreased in diabetic DETC, suggesting that the DETC may be insulin resistant. Experiments investigating diabetic keratinocytes demonstrate increased apoptosis and DETC antigen presentation, possibly contributing to DETC dysregulation. Taken together, our studies suggest that the diabetic environment has negative effects on the activation status of DETC, which may render them unable to properly respond to stressed or damaged epithelial cells. Alterations in DETC function may influence the production of growth factors and contribute to immune and repair deficiencies in diabetic patients.
This work is supported by NIH DK073098 and a Department of Immunology Institutional Training Grant 5T32 AI007244-24.
MR1, a novel class Ib molecule, utilizes the endocytic pathway to activate mucosal associated invariant T cells (93.1)
Authors: Huang Shouxiong , Thompson Bruce , Sant Andrea , Hansen Ted H 
Published: January 2023

Abstract
MR1 is a non-MHC linked class Ib molecule that is highly conserved among mammals and its expression is required for the in vivo development of mucosal associated invariant T (MAIT) cells. MAIT cells, with a memory phenotype, express a canonical TCR α chain and preferentially reside in the gut in a manner dependent upon B cells and commensal flora. Although the nature of the ligand for MR1 is unknown, our previous structure-function data strongly suggested MAIT cell activation by MR1 is ligand dependent. Here we show that, unlike other class I molecules, MR1 surface expression and activation of MAIT cells are independent of TAP, tapasin, calreticulin and proteasome function. Alternatively, using fibroblast cells overexpressing MR1 and the class II chaperone Ii, we showed MR1/Ii were physically associated, which significantly enhanced the MAIT cell activation. The association with Ii suggested that MR1 might traffic to endocytic compartments, a hypothesis we tested in a B cell line since B cells express endogenous Ii and are though to be the physiologic APC for MAIT cells. Confocal staining of the B cells displayed colocalization of MR1 and Lamp1, and treatment with lysosome inhibitors was found to inhibit MR1 expression and MAIT cell activation. Thus the endocytic pathway used by MR1 mimics that of class II and certain CD1 isoforms, thereby providing a potential to sample both endocytosed and endogenous antigens.
Viral Sequestration of Antigen Subverts Cross Priming (93.14)
Authors: Tewalt Eric Franklin , Grant Jean M , Granger Erica L , Donohue Keri B , Norbury Chris C 
Published: January 2023

Abstract
Antigens driven by poxvirus late promoters are produced in much larger quantities than those driven by early promoters both in vitro and in vivo. Despite the abundance of protein produced, antigens driven by late poxvirus promoters typically induce a low or undetectable CD8+ T cell response following immunization in vivo, while those driven by early promoters induce a significant response. We show that antigen driven by late promoters is not expressed in primary dendritic cells, preventing induction of naïve CD8+ T cells via the direct presentation pathway. However, it is puzzling why the cross priming pathway does not compensate for this lack of direct presentation as the relevance of the pathway is based upon its ability to induce CD8+ T cells in the absence of viral infection of DC or upon the expression of viral modulatory molecules that may prevent direct presentation. Our studies demonstrate that when present in similar quantities, late promoter driven antigen is not available for cross priming in vivo. In contrast, cellular antigen from virus-infected cells can access the cross priming pathway under comparable conditions. The mechanism involved does not require the shutoff of host cell protein synthesis but is due to the sequestration of late promoter driven antigen in viral factories inhibiting antigen donation to DC. This observation indicates a novel mechanism of viral evasion, whereby antigen is not available for use in either the direct or cross priming pathways.
Energetic Impact of Individual Peptide Residues on Binding by High-Affinity TCRs: Implications for Antigen Specificity (93.17)
Authors: Bowerman Natalie , Colf Leremy , Bankovich Alexander J. , Garcia K. Christopher , Kranz David M. 
Published: January 2023

Abstract
The mouse CD8+ clone 2C recognizes the syngeneic pMHC ligand SIYR:Kb and the allogeneic pMHC ligand QL9:Ld. High-affinity mutants of the 2C TCR have been generated previously against these ligands by directed evolution using yeast display. In order to generate specific probes for the detection of these pMHC ligands, we have developed a strategy to express high-affinity TCRs that are biotinylated in vivo in E. coli. The biotinylated TCRs and a collection of single amino acid variants of peptides SIYR and QL9 were used to quantitate relative energy contributions of each peptide residue to TCR binding. Generation of a quantitative binding energy map allowed comparison of two structurally very different MHC (Kb versus Ld) that bind to the same TCR. The energetic contributions of different residues from the peptides SIYR and QL9 were distinct. Analysis of QL9:Ld showed that four central positions of peptide QL9 contributed a significant amount of binding free energy to both wild type and high-affinity TCRs. Analysis of SIYR:Kb showed that there are distinct differences, compared to QL9:Ld, in recognition by the same family of mutated TCRs. Using crystal structures of TCR/QL9:Ld and TCR/SIYR:Kb complexes, it was possible to assess whether binding energies were influenced directly through contacts with the TCR, or indirectly through peptide induced conformational changes in the peptide or MHC.
Supported by grants from the NIH.
X-ray Crystallographic Structures of Murine Cytomegalovirus MHC-I-like Molecules Reveal Distinct Modes of Exploitation of the MHC-I Fold (93.18)
Authors: Mans Janet , Tiemessen C. T. , Robinson H. , Balbo A. , Schuck P. , Natarajan Kannan , Margulies D. H. 
Published: January 2023

Abstract
The mouse cytomegalovirus (mCMV), a β-herpesvirus that encodes more than 170 open reading frames (ORFs), serves as a model for infection by the human CMV. Protein homology suggests that at least 10 of the mCMV ORFs might be related in structure to MHC-I molecules. Several of these putative viral MHC-I like molecules (MHC-Iv) have been shown to function as immunoevasins. To establish a structural basis for the function and evolution of MHC-Iv molecules, we have initiated a structural survey, and report here the X-ray crystallographic structures of two mCMV molecules, m144 and m153. m144 crystallized in complex with the light chain, β2-microglobulin (β-2m), and its structure was determined to 1.9 Å resolution by molecular replacement. m153 formed fragile crystals that diffracted to 2.4 Å and its structure was solved by the single anomalous dispersion method. Both molecules reveal readily identifiable α1 and α2 helices and Ig-like α3 domains. Neither molecule has any bound peptide or other small molecule ligand. m144 interacts with β2-m, while m153 lacks a light chain. m153, which purifies as a non-covalently associated dimer, reveals an extended amino terminus stabilized by a unique disulfide bond, and has a lengthy α2 helix that extends into the α3 domain. The m153 dimer is confirmed by sedimentation analysis. Comparison of m144 and m153 structures reveals conservation in the α3 domain, and shows unique features in both α1 and α2. These structures offer insight into the evolution of MHC-Iv molecules and illustrate the versatility of the MHC-I fold.
This research was supported in part by the Intramural Research Program of the NIH, NIAID.