bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel inhibitory peptide of Toll-like receptor signaling limits LPS-induced production of inflammatory mediators and enhances survival in mice (89.12)
Authors: Hefeneider Steven H , McCoy Sharon L , Billiar Timothy R , Tsung Allan
Published: January 2023

Sepsis resulting from gram-negative bacterial infections is characterized by an excessive inflammatory immune response initiated by exposure of the host innate immune system to either bacteria or bacterial products, primarily LPS. Engagement of the TLR4 receptor on immune cells by LPS induces production of inflammatory mediators leading to tissue damage. We recently identified a peptide, termed P13, which was previously shown to be a potent inhibitor of in vitro TLR-signaling. In this study, we demonstrate that use of this novel peptide significantly reduces the in vitro production of inflammatory mediators seen after exposure of hepatocytes/NPC co-cultures and endothelial cells to LPS. In addition, in vivo treatment of mice with this peptide was effective at inhibiting LPS-induced production of inflammatory mediators and significantly limited liver damage. Peptide treatment significantly increased survival of LPS/D-galactosamine treated mice and mice treated with high dose LPS. These results demonstrate the therapeutic potential of peptide P13 to limit an LPS-induced inflammatory response and enhance survival in models of inflammation.

This study was supported by NIH SBIR 1R43AI065000-01A1
Novel mutations in CRAC channel protein Orai1 cause immunodeficiency associated with non-immunological disease (89.23)
Authors: Feske Stefan , Osmers Ute , Kirschner Janbernd , Roether Jens , Picard Capucine , Ehl Stephan , Plogmann Katrin , Fischer Alain , Rao Anjana
Published: January 2023

Orai1 has recently been identified as a pore subunit of the Ca2+ release activated Ca2+ (CRAC) channel essential for the function of T lymphocytes, mast cells and other cells types. A point mutation in Orai1 abrogates CRAC channel function and causes Severe Combined Immunodeficiency (SCID) in human infants. We here report that the immunodeficiency is accompanied by hypohidrotic ectodermal dysplasia (HED) and congenital myopathy, characterized by absent sweat production, dysplastic dental enamel, non-progressive muscle weakness and histologically a predominance of type I and atrophic type II mucle fibers. A major defect in the activation of NF-kB, known to cause HED accompanied by immune deficiency in patients with mutations in IkBα or IKKγ, was ruled out, although a partial, Ca2+ dependent impairment of NF-kB activation downstream of TCR crosslinking was observed. An novel nonsense mutation in Orai1 was observed in an unrelated SCID patient causing a premature truncation of Orai1 protein. The resulting lack of Ca2+ influx and CRAC channel activity could be rescued by ectopic expression of Orai1 in the patient’s cells. The immunodeficiency in this patient is accompanied by fatal progressive growth retardation and encephalopathy, but HED and myopathy are absent. These clinical phenotypes indicate that Orai1 may not only be necessary for regulating Ca2+ influx in the immune system but also several other tissues.
Chemokine receptor signaling in mast cells (89.27)
Authors: Aye Cho Cho , Ono Santa
Published: January 2023

Mast cells play an important role in IgE-associated allergic disorders and immune response to parasites.

In a former study, we demonstrated that chemokine CCL3 (MIP-1α) acted as a co-stimulator for FcεRI-mediated activation on mast cells. To advance our understanding on this finding, we studied gene profiles in Rat Basophilic Leukaemia mast cells after co-activation of CCR1 (a receptor of CCL3) and FcεRI using the Affymetrix GeneChip Arrays. Emp1 (Epithelial Membrane protein 1), RT1-S3 (non-classical type 1 MHC gene), and Slc35e4 (solute carrier family 35, member E4) were found to be the top possible synergy-related genes. Validations of this data are being carried out using real time PCR, western-blotting and siRNA technologies.

Further to our in vivo findings of the inhibition of the immediate hypersensitivity reactions in the conjunctiva and the attenuation of mast cell degranulation in MIP-1α deficient mice, we would like to check if MIP-1α and CCR1 play a critical role to produce these effects. We reconstituted the bone marrow-derived mice mast cells (BMMC) from naïve mice into mast cell-deficient mice and confirmed that the reconstituted BMMC home to their eye. The next step is to examine whether CCR1 and CCL3 are critical for mast cell activation and allergic conjunctivitis-like symptoms following induction of murine allergic conjunctivitis using CCR1- and CCL3- deficient mice.
Modeling GRB2 and SOS1-mediated Oligomerization of LAT: The Role of Valence Switching (89.30)
Authors: Faeder James R , Nag Ambarish , Goldstein Byron
Published: January 2023

Ligand-induced receptor aggregation is a well-known mechanism for the initiation of intracellular signals, but recent evidence also indicates that oligomerization of distal signaling molecules may be required for signal propagation. Formation of signaling complexes containing oligomers of the transmembrane adaptor protein LAT has recently been identified as a critical step in TCR-mediated signaling [Houtman et al., Nat Struct Mol Biol, 13, 798 (2006)]. Cross-linking of LAT arises from the formation of a 2:1 complex between GRB2 and SOS1, which bridges two LAT molecules through the interaction of an SH2 domain on each GRB2 with a phosphotyrosine of each LAT. We have developed a mathematical model to describe this oligomerization and determined the distribution of LAT oligomers and the kinetics of oligomer formation, which we compare with recent experimental results. The valence of LAT for binding GRB2, i.e., the number of LAT phosphotyrosines that can bind the GRB2 SH2 domain, ranges from 0 to 3 and is a critical factor in determining both the nature and extent of aggregation. A dramatic rise in oligomerization occurs between valence 2 and 3, a phenomenon we call valence switching because the valence is controlled by the extent of phosphorylation of specific LAT tyrosines.

Supported by grant R37 GM35556 from the National Institutes of Health and the Department of Energy through contract DE-AC52-06NA25396.
Signaling pathways involved in foreign antigen expression by a recombinant vaccinia virus (89.38)
Authors: Hu Ningjie , Shikuma Cecilia , Shiramizu Bruce , Koo Raymond , Yewdell Jonathan W
Published: January 2023

BACKGROUND: Vaccinia virus (VV) was used as a live vaccine to eradicate smallpox and has in recent years received considerable attention for the development of live vaccine vectors due to its broad host range and ability to express a variety of foreign antigens, which can induce humoral and cellular immunity against virus infections. However, little is known about the cellular signaling pathways involved in the foreign antigen expression by a recombinant VV.

METHODS: Using a recombinant EGFP-VV, we studied four cellular signaling pathways, PI3K, p38 MAPK, JNK, and ERK, by assessing EGFP production in infected human peripheral blood monocytes and monocyte-derived dendritic cells (MDDCs).

RESULTS: EGFP expression was inhibited significantly in both monocytes and MDDCs infected with EGFP-VV by blocking the PI3K, p38 MAKP, and JNK pathways by specific inhibitors LY294002, SB203580, and SP600125, respectively. PI3K was the most critical pathway involved in antigen expression by VV-infected cells, whereby LY294002 (25 uM) exhibited 90.1% decrease of EGFP expression compared to non-inhibitor-treated control (DMSO). Whereas, at the same concentration (25 uM), SB203580 and SP600125 inhibited only 69.3% and 30.4% of EGFP expression, respectively. Inhibition of the ERK pathway by PD098059 did not alter EGFP expression.

CONCLUSIONS: VV is able to infect APCs and efficiently express inserted gene(s) through cellular signaling pathways of PI3K, p38 MAPK, and JNK, but not ERK.

ACKNOWLEDGMENTS: This work was supported by University of Washington CFAR (Award AI 27757), Hawaii Community Foundation (Award 20061466), UH INBRE, and HACTU grant from NIAID (A134853).
Regulation of CD1d expression by vitamin D results in fewer hyporesponsive iNKT cells in the vitamin D receptor knockout mouse. (89.7)
Authors: Yu Sanhong , Cantorna Margherita T
Published: January 2023

CD1d-reactive natural killer T (NKT) cells with an invariant T cell receptor Vα14 rearrangement (i) are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. Vitamin D and the vitamin D receptor (VDR) are important regulators of T cell function, yet their role in iNKT cells remains unknown. Expression of the vitamin D receptor (VDR) is required for normal development of iNKT cells. Mice lacking the VDR had significantly fewer iNKT cells and the remaining iNKT cells were hyporesponsive. iNKT cell development is blocked at a late stage in iNKT cell development, the CD44high NK1.1− stage. Bone marrow transplantation showed that VDR knockout (KO) hematopoetic cells fail to repopulate iNKT cells in the wild type thymus. The block was not due to fewer iNKT precursors in the VDR KO mice but instead CD1d expression was diminished in the VDR KO thymus. The decreased CD1d results in fewer, hyporesponsive iNKT cells in the VDR KO mouse. The data suggest that vitamin D through the vitamin D receptor is critical for proper iNKT cell development and function.
The Immunomodulatory Effects of Ascaris suum Pseudocoelomic Fluid in Th1-Mediated Systems (90.10)
Authors: Hodges Marcus Gomez , Smith Allen , Urban Joseph , Keane-Myers Andrea
Published: January 2023

Recent studies found that adoptive transfer of helminth-primed dendritic cells impairs host protection to the enteric pathogen, Citrobacter rodentium. In the present study, we used Ascaris suum pseudocoelomic fluid (PCF) in lieu of infection, and demonstrated that pre-exposure of bone-marrow derived dendritic cells to PCF, prior to LPS stimulation, suppressed activation marker expression and production of IL-12. Similar to LPS, heat-inactivated and extract preparations of C. rodentium induced dendritic cell activation. This activation was suppressed in cells previously exposed to PCF, resulting in decreased expression of CD40, CD86, and MHC II. These findings suggest that PCF may modify Th1 responses in the host. In preliminary studies, BALB/c mice were sensitized with PCF (100 μg), followed by oral inoculation with C. rodentium (2.5 x 108 CFU). Bacterial colonization and colonic hyperplasia was assessed at days 3 and 7 post-infection. Mice receiving two sensitizations prior to inoculation had increased C. rodentium colonization in the cecum and colon by day 7 post-infection, compared to those receiving no pre-treatment. We are currently repeating this experiment using a higher concentration of PCF (200 μg) to enhance differences in bacterial colonization. This model may help determine if PCF alters the course of Th1-mediated immunoregulation in allergic inflammatory responses.
Synergistic effect of TNF-α and ceramide on the production of IL-8 in A549 cells and A549 cells transfected with human ceramide kinase (CERK). (90.11)
Authors: Hinkovska-Galcheva Vania , Van Way Susan M , Cornell Timothy , Sun Lei , Gueorguieva Iana , Zlatarov Nikolay
Published: January 2023

Many inducers of the stress response result in ceramide (Cer) accumulation as a result of activation of either sphingolyelinases (SMases) or the de novo pathway. Recently, there is strong evidence for a role of sphingolipids in inflammatory responses such as leukocyte recruitment. The attraction of leukocytes to tissues is essential for inflammation and the host response to infection. Following stimulation by TNF-?, the production of IL-8 increased over time in a concentration dependent manner. Simultaneously, Cer levels increased gradually from 7.56 % during 15 minute stimulation to 35% for 16 hour stimulation. Using Fumonisin B1, an inhibitor of de novo synthesis of Cer, which inhibits Cer synthesis by 25%, we observed 17% decrease of IL-8 expression. When the inhibitor for neutral (SMase) GW4869 was used on A549 cells, there was a significant reduction of Cer as well as IL-8. Subsequently, two serine-threonine phosphatases (PPas), protein phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) have been shown to be Cer responsive in vitro and in vivo. These results suggest that TNF-? stimulates the production of IL-8 by a mechanism that involves the sphingomyelin-Cer system. Cer may be important in increasing the production of IL-8 in A 549 cells. Since Cer can be phosphorylated by CERK to C1P, we predict that CERK expression may affect the balance between these sphingolipids and influence the Cer dependent PPas.
Cyclic patterns of respiratory burst activity in zebrafish (Danio rerio) (90.12)
Authors: Hannum Lynn , Kaplan Jessica
Published: January 2023

The cyclic expression of clock genes creates daily variations in physiology, including immune system components. To investigate circadian patterns of immune function in zebrafish, we have focused on respiratory burst activity in phagocytic cells from kidney, the principal lymphoid organ in fish. Respiratory burst responses were assayed at four hour intervals using 2’,7’-dichlorofluorescein diacetate (H2DCFDA) to indicate reactive oxygen species (ROS) levels. We initially stimulated cells with phorbol 12-myristate 13-acetate (PMA). While responses to PMA did not vary throughout the day, the levels of ROS in control cells varied significantly peaking prior to dawn, followed by a low several hours later. Next, we analyzed responses to stimulation with zymosan A. The relative increase in ROS production after exposure to zymosan varied daily, being highest daily at 2 AM, with a low at 10 PM. Interestingly, the kinetics of the response to zymosan varied throughout 24-hour periods as well; it took significantly longer to reach the peak production of ROS when cells were stimulated with zymosan at 6 AM than at 10 PM, which had the shortest time to peak response. Our data highlight the potential of zebrafish as a model of circadian control of immune function in vertebrates.

This work was supported by NIH Grant Number P20 RR-016463 from the INBRE Program of the National Center for Research Resources.
Estrogen augments the number and activity of CD11c+CD49b+NK1.1int cells in vivo (90.4)
Authors: Siracusa Mark , Overstreet Mike , Scott Alan L , Klein Sabra L
Published: January 2023

Estrogens may increase innate immune responses, which may augment subsequent adaptive responses and lead to female-biased resistance to infection. Both dendritic cells (DCs) and natural killer (NK) cells are key players in innate immunity; thus, the goal of the present study was to test the hypothesis that the functioning of DCs and NK cells is affected by exposure to 17β-estradiol (E2) via estrogen receptors. To test whether E2 alters the number, phenotype, or function of mature cells, CD11c+ DCs and NK cells were isolated from the spleens of female C57BL/6 mice that were intact, ovariectomized, or ovariectomized with E2 replaced exogenously. Neither removal nor replacement of E2 affected the number of CD11c+CD11b+ cells in the spleen. E2 did not affect the expression of MHCII, B220, CD8α, CD40, or CD86 on monocytes. The presence of E2 increased the number of CD11c+CD49b+NK1.1int cells, but reduced numbers of CD11c+CD49b+NK1.1hi cells, which also were B220+, CD49b+, and Gr1-, a phenotype reported for interferon-producing killer DCs (IKDCs). CD11c+CD49b+NK1.1int cells expressed MHC II, CD40, CD11b, B220, and CD8α. E2 did not affect the lytic activity of CD11c+ cells. CD11c+ cells produced more IFNγ, but neither IL-12 nor IFNα, in the presence of E2. In vitro, E2 facilitated differentiation of bone marrow precursor cells into functional CD11c+ DCs and increased the expression of CD11b, MHCII, CD40, and CD86, but not NK1.1 or CD49b. These data provide in vivo evidence that E2 differentially affects distinct populations and functions of CD11c+ cells.

Support: NIH grant AI054995 and JHMRI pilot award
Atractylodes japonica enhances CD4+ T-cell activities and modulates Th1/Th2 differentiation (90.8)
Authors: Lee hee kyung , Bae Hyunsu , park namhee
Published: January 2023

Atractylodes japonica (AJ) is a common used herbal medicine in Asian countries such as Korea, China and Japan. The present study was designated to evaluate the direct effects of AJ on helper T cell activities and on Th1/Th2 lineage development in vitro. The results demonstrated that AJ had no mitogenic effects on unstimulated CD4+ T cells, but augmented CD4+ T-cell proliferation upon activation with anti-CD3/anti-CD28 antibodies in a dose-dependent manner. AJ treatment significantly increased CD4+T cell population and the IFN-γ expression was significantly enhanced, while IL-4 expression was significantly decreased. In addition, in vitro Th1/Th2 polarization experiments revealed that AJ enhanced IFN-γ secretion in Th1 cells, but reduced the IL-4 in Th2 cells in dose-dependent manner.

Therefore, these results suggest that AJ treatment could be a desirable alternative therapy for the prevention or correction of Th2 dominant pathological disorders, such as allergy and asthma.
TCR- independent induction of IL-17 production in TH17 Cells (91.11)
Authors: Lee Yun Kyung , Weaver Casey T.
Published: January 2023

Effector CD4 T cells have been classically divided into two lineages, T-helper type 1 (TH1) and TH2. TH1 cells develop in response to intracellular viruses and bacteria and secrete interferon-γ (IFNγ) while TH2 cells produce interleukin (IL)-4 in response to extracellular pathogens. Recent studies have identified a third distinct lineage of effector CD4 T cells called as TH17 that characteristically produce IL-17. The development of TH17 cells depends on TGFβ and IL-6. The published reports establish a direct link between IL-23 and the TH17 cell subset in mediating inflammatory pathology in certain autoimmune diseases although IL-23 is not required for TH17 commitment. We observed that IL-23 acts synergistically with IL-1 or IL-18 to induce IL-17 cytokine production in TH17 cells independent of T cell receptor (TCR) stimulation, analogous to the induction of IFNγ by IL-12 and IL-18 in TH1 cells. Our results suggest that IL-1 or IL-18 signaling might be preceded by IL-23 signaling through the IL-23 receptor (IL-23R), which is upregulated in TH17 by TGFβ. The TH17 effector function is augmented by these non-antigen-specific responses subsequent to their previous antigen-induced differentiation. Our findings help to delineate the role of IL-23 in TH17 cell biology and identify potential targets for prevention of TH17-mediated immunopathology.

This work is supported by the National Institutes of Health (C.T.W.).
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

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

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

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

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

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 IFNγ 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

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

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

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

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

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

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

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

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

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.
Reactive Nitrogen species process a microbial carbohydrate presented by the MHCII Pathway (93.20)
Authors: Duan Jinyou , Avi Fikri Y , Kasper Dennis L
Published: January 2023

The class II major histocompatibility complex (MHCII) pathway in antigen-presenting cells (APCs) is the conduit by which extracellular proteins are processed by proteases, resulting in foreign peptides being presented to T cells. After depolymerization to a markedly decreased molecular size, zwitterionic polysaccharides (ZPSs) are also presented by this pathway. However, nothing is known about the chemistry of carbohydrate processing apart from its dependence on the presence of inducible nitric oxide synthase (iNOS).

By examining the ability of known nitric oxide-derived species, peroxynitrite, nitroxyl, and nitric oxide itself, to degrade polysaccharide A (PSA), a ZPS from Bacteroides fragilis, we determined that deamination was the predominant mechanism of PSA processing in APCs. Structural characterization of the nitric oxide-derived product PSA-NO indicated that the depolymerization didn¡t disturb PSA¡s repeat units with exception of the deamination cleavage of 4-amino groups of 3-linked 2-acetamido-4-amino-2,4,6-trideoxy galactopyranosyl (AATp) residues.

PSA-NO presented by APCs induced CD4+ T cell proliferation that was blocked by inhibitors of endosomal processing and by an antibody to HLA-DR. A novel mechanism (deaminative reaction) differentiates carbohydrate from protein processing during the oxidative burst.
Partial MHC Class II molecules preferentially bind to B cells (93.22)
Authors: Link Jason M , Meza-Romero Roberto , Afentoulis Michael , Agotsch Marisa , LaTocha Dorian , Burrows Gregory , Vandenbark Arthur A
Published: January 2023

Recombinant, two-domain MHC Class II proteins (α1β1MHCII) that lack sequence from the membrane proximal α2 and β2 domains of MHC Class II adhere preferentially to B cells in vivo and ex vivo; whereas, full length (four-domain) constructs do not bind. Because α1β1MHCII molecules that contain only native MHC Class II sequence have preferential affinity for B cells, we hypothesize that a natural, cell-free form of MHC Class II (similar to recombinant α1β1MHCII) exists and has affinity for a receptor expressed by B cells. Evidence that endogenous, natural MHC Class II molecules transfer to B cells and that α1β1MHCII has inter- and intra-species affinity for B cells suggest that this is a natural and evolutionarily conserved property. And evidence that α1β1MHCII can stimulate CD4+ T cells suggests that there is an immune function for partial, cell-free MHC Class II. Transfer of immunoreactive, cell-free α1β1MHCII/Ag complexes to B cells suggests a here-to-fore unrecognized pathway for T cell activation.
HLA-DQ dimer formation pattern and its possible contribution to the sleep disorder narcolepsy (93.23)
Authors: Miyadera Hiroko , Tokunaga Katsushi
Published: January 2023

Allelic polymorphisms of HLA class II are associated to the susceptibility/resistance to various diseases. In particular, the susceptibility to sleep disorder narcolepsy is strongly linked to DQB1*0602. In addition, some other DQB1 alleles also affect the susceptibility to the disease when present in addition to DQB1*0602. In order to understand how these alleles contribute to the susceptibility/resistance to narcolepsy, we have analyzed the abilities of these and other DQB1 alleles to form dimer with various DQA1 alleles. The residues which may contribute to the association of DQA1 and DQB1 were also identified and will be presented.
Nonsense Mediated Decay associated “Pioneer round of translation” peptides as source for MHC I mediated antigen presentation (93.3)
Authors: Schmidt Andreas , Shastri Nilabh
Published: January 2023

Nonsense mediated decay (NMD) plays a critical role in the mRNA quality control mechanism protecting cells from aberrant mRNAs containing premature termination codons (PTC). Recognition of these PTCs requires a “pioneer round of translation” prior to the bulk translation of mRNAs for cellular protein synthesis, therefore producing a pool of immediate early peptides.

Effectiveness of the immune surveillance system is insured by the timely and complete MHC I mediated presentation of a pool of precisely cleaved peptides representing all cellular proteins. So far sources of antigenic peptides are known to include regular degraded proteins, defective ribosomal products (Drips) and cryptic peptides starting with a non-AUG start codon. Here the contribution of peptides originating from the pioneer translation to the pool of peptides presented by MHC I molecules is described. In Hela cells RNAi mediated depletion of the essential NMD factor hUpf1 as well as the pioneer translation initiation complex factors CBP80 and PABP2 reveal impaired MHC I antigen presentation. In sharp contrast, targeted interference with conventional translation without effecting pioneer translation permits antigen presentation. Taken together these findings put products of the pioneer translation into an important position as novel resource for antigenic peptides and reveal a new relationship between NMD and immunosurveillance.