bluuebunny/crossref_metadata_2025_split huggingface dataset:
A direct approach for measuring total TCRαβ diversity (P3354)
Authors: Schoettle Louis , Wei Xixi , Blattman Joseph 
Published: January 2023

Abstract
Two cardinal features of adaptive immunity are diversity and specificity: the immune system must be able to recognize virtually any pathogen while maintaining enough cells specific for each pathogen in order to mount effective responses. T cells accomplish this by expressing clonotypic αβ heterodimeric T-cell receptors (TCR) that recognize pathogen epitopes presented by major histocompatibility (MHC) proteins. Although the diversity of TCR chains have been independently measured, quantitating diversity due to pairing of a and b chains has been problematic; single cell sequencing is too costly while molecular approaches have not worked. We have developed a novel approach for directly measure diversity resulting from TCR αβ pairing employing DNA origami nanostructures to physically link mRNA encoding both TCRα and β chains, coupled with Ion Torrent deep sequencing to obtain CDR3 sequence information for both chains from the same cell. We have designed and generated such DNA origami structures for ligation of TCR mRNA, transfected with high efficiency naive primary T cells with these origami structures, and re-isolated origami with bound TCR mRNA. Thus, we are now able to directly estimate the total diversity of the T cell receptor repertoire (including TCRαβ pairing diversity) and can use this approach to assess how decreased TCR diversity (such as occurs during aging or immune reconstitution) affects the ability to mount effective responses to pathogens.
Analyzing cytotoxicity kinetics with a novel automated high-throughput real-time killing assay (P3366)
Authors: Qu Bin , Schwarz Eva , Bhat Shruthi , Lis Annette , Zhou Xiao , Lyrman Hélène , Backes Christian , Stephan Cora , Bufe Bernd , Zufall Frank , Hoth Markus , Kummerow Carsten 
Published: January 2023

Abstract
Cytotoxicity is an essential parameter to examine killer cell activity, to screen hits for new drugs, and to proof safety evaluation. Cytotoxicity assays are routinely used in basic research, clinical studies, and industrial applications. However, the most commonly applied standard end-point cytotoxicity assays largely suffer from the lack of kinetic information, which is indispensable to assess killing competence. Here we report a quantitative and highly sensitive calcein-based real-time high-throughput cytotoxicity assay on a single cell level and for population use. By determining fluorescence loss as a function of the fraction of lysed cells, this assay measures the kinetics of cytotoxicity over a broad dynamic range. The high time resolution of the assay also provides kinetic information with the potential to reveal neglected/immeasurable properties in the process of cytotoxicity of both purified human cytotoxic T lymphocytes and natural killer cells. To test its clinical potential, we have also applied this assay to screen the cytotoxic potential of (easy to prepare) peripheral blood mononuclear cells in healthy blood donors. In summary, this assay should substantially increase the screening efficiency for clinical and industrial purposes and offer quantitative kinetic data with a high time resolution.
Visualizing the physiological self-reactivity of epidermal γδ TCR (P3280)
Authors: Zal Tomasz , Chodaczek Grzegorz , Zal M. Anna 
Published: January 2023

Abstract
Dendritic epidermal T cells (DETCs) remain activated in vivo at steady state through their monomorphic Vgamma5-Vdelta1 TCR, forming phosphotyrosine-rich aggregates located on projections, called PALPs (Chodaczek et al. 2012). This recent discovery suggests the existence of beneficial self-reactivity on the part of the evolutionarily-preserved γδ TCR. Located at barrier-forming squamous keratinocyte junctions, and remarkably long-lived, the PALPs display multiple hallmarks of true immunological synapses, including TCR clustering and CD3-zeta and ZAP70 tyrosine phosphorylation. Using two independent super-resolution microscopy techniques: the structured illumination microscopy (SIM) and stimulated emission depletion (STED), we show that PALPs consist of several tens of peripherally distributed, tyrosine-phosphorylated TCR microclusters. Individual microclusters are uniformly sized at 150-160 nm and are associated with distinct signaling intermediates, suggesting that they represent supra-molecular signaling assemblies of a defined composition. Intravital fluorescence recovery after photobleaching and high speed time-lapse imaging showed that small intracellular cargo granules trafficked bi-directionally within T cell protrusions between large stationary granules and the PALPs. These studies reveal the PALPs as dynamically active TCR-activatory immunological synapses, and suggest that this novel form of physiological TCR self-reactivity mediates homeostatic secretory functions.
Over-expression of CD200 protects mice from dextran sodium sulfate induced colitis (P3286)
Authors: Chen Zhiqi , Yu Kai , Gorczynski Reginald 
Published: January 2023

Abstract
CD200:CD200R interactions have been shown to lead to potent immunosuppression, resulting in enhanced allograft survival; reduced collagen-induced arthritis; and increased growth of leukemic, lymphoma cells and breast cancer cells. We have investigated the effect of "knockout"of CD200R, or over-expression of CD200 in transgenic mice (CD200tg), on susceptibility to dextran sodium sulfate (DSS) induced colitis in C57BL/6 female mice. Control animals given DSS developed weight loss (~15%) and diarrhoea over 7 days, before slow recovery. These effects were more pronounced in CD200RKO mice, with ~40% mortality at day 7. In contrast CD200tg mice showed attenuation of all symptoms. Immunohistological analysis and protein expression array confirmed severe inflammation in CD200RKO and controls, with loss of villi, inflitration by macrophages, neutrophils and CD3+ cells, and high expression levels of IL-1β and TNF-α in the intestine. In CD200tg mice intestinal tissue showed enhanced inflitration by Foxp3+Tregs, with increased expression of IL-10. Taken together these data suggest for the first time that CD200:CD200R interactions play a role in regulating inflammatory colitis in mice, which may open new avenues for treatment of inflammatory diseases in man.
TRAF6 in DCs controls intestinal immune tolerance (P3292)
Authors: Han Daehee , Walsh Matthew , Cejas Pedro , Surh Charles , Choi Yongwon 
Published: January 2023

Abstract
The intracellular signaling mediator TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). We now report that DC-specific deletion of TRAF6 (TRAF6ΔDC) results, quite unexpectedly, in loss of mucosal tolerance, characterized by spontaneous rise in lamina propria Th2 cells, and development of eosinophilic enteritis and fibrosis of the small intestine. Loss of TRAF6ΔDC mucosal tolerance requires the presence of commensal microbiota in a manner that appears independent of the MyD88 pathway. Further, TRAF6ΔDC mice exhibit decreased regulatory T cell (Treg) numbers in the small intestine, and diminished induction of iTregs in response to model antigen. Evidence suggests this defect may be associated with diminished DC-expressed IL-2. Finally, we demonstrate that aberrant Th2-associated responses in TRAF6ΔDC mice can be mitigated via restoration of Treg activity. Collectively, our findings reveal a novel role for TRAF6 in directing DC maintenance of intestinal immune tolerance through balanced induction of Tregs versus Th2 immunity.
Skin-resident T cells limit UV damage through sensing the danger signal ATP (P3246)
Authors: MacLeod Amanda , Havran Wendy 
Published: January 2023

Abstract
Exposure to ultraviolet radiation (UVR) is a major risk factor for skin cancers and skin inflammation. However, molecular and cellular mechanisms to minimize UV damage in the skin are incompletely understood. Based on their sentinel function, we hypothesized that resident T cells in the skin, murine dendritic epidermal T cells (DETC) and human skin-resident T cells, sense UVR-induced danger signals such as extracellular adenosine triphosphate (ATP) to initiate protection and repair. Supporting this hypothesis, we find that epidermal keratinocytes release ATP upon UVR to activate DETC and human skin-resident T cells in a direct and IL-1β-mediated manner and this response could be inhibited by ATPases. Using mice deficient in DETC, we show a previously unrecognized role for DETC in repairing keratinocyte DNA damage upon UVR. Functionally, ATP enhanced TCR-mediated DETC proliferation and IL-17A production. IL-17A induced molecules relevant to DNA repair and apoptosis in keratinocytes, including TNF related weak inducer of apoptosis (TWEAK) and Growth arrest and DNA damage gene (GADD45), providing a possible link to the observed UV protective mechanisms exerted by DETC. Ongoing studies quantifying UVR-induced DNA damage and apoptosis in wildtype, TCRδ-/-, and IL-17A-/- skin cells and in human skin cultures will provide novel information on skin-resident T cell function, and may identify new targets to treat and prevent sunburn and skin cancer in humans.
Fucosylation is a hallmark of inflammatory macrophages and a novel therapeutic target in rheumatoid arthritis (P5137)
Authors: Li Jun , Hsu Hui-Chen , Yang PingAr , Wu Qi , Spalding David , Mountz John 
Published: January 2023

Abstract
Inflammatory macrophages (MΦ) play an important role in rheumatoid arthritis (RA). Fucosylation is a regulator of inflammation and we have observed a highly positive correlation between Fucosyltransferases (Fut)1 or Fut3 and Tnfα (p=0.0001) in RA synovial tissues. In sorted cells from human RA synovial fluid, Fut1 and Fut3 was highly expressed in M1 inflammatory MΦ (CD68+CD80+), but not in M2 MΦ (CD68+CD80-), synovial fibroblasts, Th1, Th17, and other T cells (p&lt;0.01, n=5). Fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ derived by GM-CSF from mouse bone marrow or human PBMC monocytes, strongly indicating the indispensable role of fucosylation in M1 MΦ differentiation. Additionally, phalloidin staining showed 2-D-gal disrupted MΦ actin, suggesting a potential effect of fucosylation in antigen processing and presentation. Indeed, 2-D-gal treatment of fully differentiated M1 MΦ for 2 days dramatically reduced their uptake, processing and presentation of GFP-Eα (from I-Edα) and FITC-CII antigens in a dose dependent manner (p&lt;0.01). In vivo, 2-D-gal dramatically blocked the bovine CII-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p&lt;0.01) with reduced inflammatory MΦ in draining LN (1.3±0.3% vs 0.5±0.1%, p&lt;0.05), decreased TNF-α (130 vs 39 pg/ml, p&lt;0.05), and anti-CII in the serum. Our study indicated that M1 MΦ differentiation and function is orchestrated by fucosylation, which is a novel therapeutic target in RA.
Kv1.3 deletion induces Foxo1 expression independently of Foxp3 and biases Th cells toward an immunoregulatory phenotype (P5127)
Authors: Gocke Anne , Grishkan Inna , Calabresi Peter 
Published: January 2023

Abstract
Myelin-reactive Th cells in patients with multiple sclerosis (MS) express Kv1.3, a voltage-gated K+ channel essential for Th cell activation and function. Recently, we have demonstrated that Kv1.3 deletion prevents experimental autoimmune encephalomyelitis, a murine model of MS, by inducing a regulatory Th cell phenotype independently of Foxp3. However, the mechanism by which Th cells with regulatory properties develop in the absence of Kv1.3 remains unclear. Foxo1 has been shown to function in induction of tolerance and in Treg cell lineage commitment. Foxo1 nuclear exclusion is induced in effector Th cells by downstream T cell receptor (TCR) signaling. Herein, we demonstrate an increase in nuclear Foxo1, with concomitant reduction in T-bet, in Kv1.3 KO Th cells. Surprisingly, Kv1.3 deletion does not impair TCR-induced intracellular Ca2+ flux or the activation of the Ca2+-sensitive transcription factor NF-AT. Instead, the absence of Kv1.3 leads to p56lck relocalization from TCR to the IL-2R complex, thereby contributing to the increase in Foxo1 and the differentiation of Treg cells in the absence of changes in Foxp3 expression. Herein, we demonstrate that regulatory Th cells are generated in the absence of Kv1.3 as a consequence of disrupted TCR signaling during formation of the immunological synapse, rather than impaired Ca2+ flux, resulting in upregulation of Foxo1. These data highlight Foxo1 as a novel target for induction of Treg cells in autoimmune diseases such as MS.
MyD88 plays a role in deciding between apoptotic and necroptotic cell death after UV irradiation (P1238)
Authors: Harberts Erin , Fishelevich Rita , Liu Juan , Atamas Sergei , Gaspari Anthony 
Published: January 2023

Abstract
Ultraviolet irradiation (UV) induced cellular damage is classically associated with apoptosis and known to result in systemic immunosuppression. How the decision to undergo apoptosis is made following UV is not fully understood. We hypothesize that a central mediator of TLR signaling, MyD88, determines cell fate after UV. Survival after UV of immortalized bone marrow-derived macrophages (BMM) from MyD88 germline-deficient mice (MyD88-/-) was significantly higher and apoptotic morphology reduced, vs. wild-type (WT) BMM. UV-induced DNA laddering in peritoneal macrophages (PM) and epidermis of MyD88-/- animals, vs. WT, was decreased. In MyD88-/- PM, decreased cleavage of pro-necroptotic protein RIP1, elevated mRNA levels of RIP1 and RIP3, and significantly increased TNF-α release suggests that necroptosis rather than apoptosis has been initiated. In vivo studies after UV showed significantly less TUNEL-positive cells and more profound inflammation in MyD88-/- skin sections. MyD88-/- mice are also found to be resistant to UV-induced immunosuppression. Considering that MyD88 participates in many TLR pathways, TLR2-/-, TLR4-/-, and WT BMM were compared for evidence of UV-induced apoptosis. Only TLR4-/- BMM had a similar phenotype to MyD88-/-, suggesting that the TLR4-MyD88 axis importantly contributes to cell fate decision. Our study describes a new cellular consequence of MyD88 signaling after UV, and may provide rationale for therapies to mitigate UV-induced immunosuppression.
Differential RNA expression of TLR pathway signaling pathways in macrophages from chickens stimulated with Interferon γ in disease resistant B2 haplotypes versus susceptible B19 haplotypes. (P1216)
Authors: Drechsler Yvonne , Keeler Calvin , Carr Thomas , Griggs Lisa , Collisson Ellen 
Published: January 2023

Abstract
MHC has been associated with immunity in several species and studies have demonstrated association of MHC B haplotype in chickens and resistance to a variety of viral pathogens including Marek’s disease virus, avian leucosis virus, Newcastle disease virus and Rous sarcoma virus. Previous studies in our laboratory have shown that chicks with the homozygous B2 MHC haplotypes are more resistant to respiratory illness following avian coronavirus infection, than homozygous B19 chicks. Further studies demonstrated more vigorous responses of B2 macrophages to Poly I:C and IFNγ stimulation than responses of B19 derived macrophages. Macrophages play a critical role in the early innate response and mobilization of the adaptive immune responses. Next Generation Sequencing of IFNγ stimulated macrophages show differential expression of genes of the toll-like receptor signaling pathway. Macrophages were stimulated for 0, 1, 2, 4, 8, 16, 24 and 48 hours. At most time points more genes were stimulated in macrophages of the disease resistant B2 haplotype and quantification of RNA expression (fold-increase) indicates earlier and more robust stimulation of several genes.
Fli-1 transcription factor is involved in cytokine production in spleen cells upon Toll-like receptor stimulation (P1218)
Authors: Brandon Danielle , Williams Sarah , Zhang Xian 
Published: January 2023

Abstract
Friend leukemia integration-1, a member of the Ets family of transcription factors, is highly expressed in splenocytes. Dysregulation of Fli-1 occurs in Systemic Lupus Erythematosus patients and lupus prone mice. Systemic Lupus Erythematosus is an autoimmune disease utilizing the innate and adaptive immune response. Reduction of Fli-1 expression in NZM 2410 and MRL/lpr lupus prone murine models resulted in decreased severity of disease. Studies of Toll-like receptor 7 and 8 reveal various implications in autoimmunity. Inflammatory cytokine IL-6 plays a pivotal role in the pathogenesis of SLE. The role of Fli-1 in Toll-like receptor signaling pathways in splenocytes is unknown. In this study, we generated Fli-1 heterozygous knockout (Fli-1+/-) B6 mice that expressed decreased levels of Fli-1 and investigated the impact of Fli-1 on splenocyte development and cytokine production. Spleen cells were isolated from Fli-1+/- B6 mice and wild-type controls. Examination of cell populations in the spleen revealed unaffected populations of B, T, Natural Killers and Dendritic cells between Fli1+/- mice and wild-type littermates. Spleen cells isolated from Fli-1+/- mice produced increased levels of IL-6 compared to wild-type mice upon stimulation with Toll-like receptor 7 ligand R848. Our results suggest that Fli-1 is involved in IL-6 cytokine production in spleen cells.
An alternative role for FoxP3 as an effector T cell regulator controlled through CD40 (P1027)
Authors: Wagner David , Carter Jessica 
Published: January 2023

Abstract
The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly not. CD4+ cells expressing the CD40 receptor defined as Th40 cells are highly diabetogenic in NOD mice and BDC2.5.TCR.Tg mice have a high number of these effector T cells. Given the importance of CD40 for pathogenic T cells, BDC2.5.CD40-/- mice were created where Tregs, CD4+CD25hiFoxP3+, develop normally and pathogenic effector cells are severely ablated. Tregs in both wild type, BDC2.5.wt and CD40 knockout , BDC2.5.CD40-/-, mice express membrane TGFbeta and are fully functional; however Th40 cells are less responsive to Treg control than CD4+CD40- cells. Blocking membrane TGFbeta significantly ablates suppression of CD40- effector T cells with little impact on Th40 cells. Th40 cells from diabetic BDC2.5.wt mice rapidly induces diabetes in NOD.scid recipients but cells from pre-diabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, those effector T cells are FoxP3+; but at diabetes onset Th40 cells become FoxP3-. CD28 engagement induces FoxP3 but CD40 induces a smaller FoxP3 not yet reported in murine T cells. This poses a new consideration for the function of FoxP3, not as a molecule defining Tregs, but rather a molecule controlling effector T cell function. The smaller form of FoxP3 therefore is associated with effector cell pathogenicity.
CD4+ T regulatory cells mitogenic signaling is activated by IL-1 and IL-6 produced by serum amyloid A-stimulated monocytes (P1037)
Authors: Macaubas Claudia , Nguyen Khoa , Truong Phi , Wang Nan , Hou Tieying , Yoon Taejin , Mellins Elizabeth 
Published: January 2023

Abstract
We have previously identified serum amyloid A (SAA) as a selective mitogen for regulatory T cells (Treg). However, the precise molecular mechanism by which SAA induces Treg proliferation has been unknown. Here we provide evidence that IL-1 and IL-6 are directly involved in the SAA-mediated reversal of Treg anergy. By engaging its several cognate receptors, SAA induces IL-1 and IL-6 secretion by monocytes. This monocyte-derived inflammatory milieu is required for Treg expansion as pharmacologic inhibition and genetic deletion of IL-1 and IL-6 abrogate the ability of SAA to reverse Treg anergy. Furthermore, while both IL-1 and IL-6 are required for ERK1/2 activation, only the former is necessary for AKT signaling in proliferating Treg. Collectively, these results point to a novel mechanism, by which SAA initiates a monocyte-dependent production of inflammatory cytokines to drive mitogenic signaling in Treg.
MHC composition and variation in turkeys of the Americas (P6066)
Authors: Reed Kent , Monson Melissa , Settlage Robert , Mendoza Kristelle , Dalloul Rami 
Published: January 2023

Abstract
Variation in the MHC is associated with disease susceptibility/resistance in avian species of agricultural importance. Variation includes individual sequence differences, but also structural differences (gene rearrangement) and copy number variation. The directionality of structural changes can be determined through comparison of closely related species in a phylogenetic context. The MHC has now been described for multiple galliform species including the chicken and the domestic turkey (Meleagris gallopavo). Analysis of the turkey MHC is limited primarily to broad taxonomic contrasts with chicken. This study applies high-throughput sequencing approaches to investigate MHC structure of turkeys of the Americas. Sequence assemblies of the Eastern wild turkey (M. g. silvestris) and the Ocellated turkey (M. ocellata; native to the Yucatán Peninsula of Mexico, Guatemala and Belize) were constructed from whole genome sequencing efforts. Assemblies were compared with the MHC of the domestic turkey. In addition, an MHC-specific SureSelect capture array was developed for sequencing multiple birds from different genetic backgrounds. Sequence libraries were created for 14 turkeys (domestic, heritage and wild birds), a second Ocellated turkey, and a chicken. Libraries were hybridized to over 12K baits (120 bp) and the resulting pools were sequenced producing an average of 439K paired reads per library. Results of the sequence analysis and variant discovery are presented.
Cranberry products alter immunity after a Salmonella challenge in pigs (P6084)
Authors: Eicher Susan , Stanley Megan , Richert Brian , Rostagno Marcos 
Published: January 2023

Abstract
Cranberry products decrease bacterial adhesion and alter some immune functions. Our objective was to determine if feeding 3 different cranberry products to pigs would alter mesenteric lymph node (MLN) or spleen cell (SPL) immune functions, with or without a Salmonella challenge. Pigs were fed 1) whole cranberry powder (1-2 g/d) in dry feed plus free choice water, 2) cranberry fiber (4%) in dry feed with free choice water, 3) control dry feed with cranberry juice (10%) as the only liquid for 10 d post-weaning and 2 d pre-challenge, or 4) control diet with free choice water. Pigs were sampled (MLN and SPL) at weaning (19 days), 10, 17, and 24 days post-weaning. On d 28 post-weaning, an oral 10^5 CFU Salmonella enterica serovar Typhimurium dose was given, and 24 h and 7 days post-challenge MLN and SPL tissues were collected. CD14 and CD18 expression and ability to phagocytize fluorescently labeled bioparticles was determined by flow cytometry. SPL did not differ before the challenge; phagocytosis by SPL was increased at 24 hours post-challenge by the powder supplement. However, juice decreased phagocytosis by SPL (% and mean fluorescence) and CD14 expression at 14 days post-weaning. In contrast, on d 14 post-challenge, MLN of the powder supplemented pigs expressed more CD14 and CD18. Phagocytosis was not different for any cells on d 14 post-challenge. Cranberry products affected SPL and MLN differentially, but primarily enhanced MLN activation after the challenge.