bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dissecting a highly immunogenic antigen presentation process triggered by a natural pathogenic antigen: listeriolysin O (APP3P.104)
Authors: Valderrama Adriana , Unanue Emil , Carrero Javier 
Published: January 2023

Abstract
Listeriolysin O (LLO) is the major immunogen and virulence factor secreted by L. monocytogenes. Peptides processed from the LLO are presented at low nanomolar concentration to CD4 T cells. The mechanism for the efficiency of this antigen presentation process remains unknown and may provide novel insight into antigen processing and presentation. We tested LLO mutants with impaired molecular properties, such as membrane binding, cytolytic activity, and catabolism. All LLO variants triggered T cell responses at low nanomolar concentrations. Because LLO presentation was independent of any known activity intrinsic to LLO, we investigated cellular mechanisms that may increase its antigenicity. All LLO mutants were taken up into cells at high amounts in large undefined vesicles. Based on this, we hypothesized that bulk protein uptake of LLO could cause a novel cellular stress mechanism that would explain its immunogenicity. Endoplasmic reticulum stress (ERS) is a cellular response that deals with large amounts of unfolded protein. We investigated if the entry of LLO into APC triggered the ERS. Treatment with nanomolar concentrations of LLO caused an increase in Chop and Gadd34 expression, as well as other canonical markers of the ERS. However, it did not induce Xbp1 splicing or the ER-chaperone response. In conclusion, LLO may be activating the Perk pathway leading to an ERS in APC. The biological and antigen presentation outcomes of this response remain under investigation.
Statins induce microautophagy in RAW 264.7 cell line. (APP3P.107)
Authors: Sahu Ranjit , Singh Satwinder , Davidson Anne 
Published: January 2023

Abstract
Autophagy is process by which cells recycle cellular components to overcome starvation. This process is constitutive in antigen presenting cells such as dendritic cells and macrophages. Among the various autophagy pathways, microautophagy (Mi) has a unique significance as it is implicated in the biogenesis of exosomes which are relevant to the design of therapeutics. Statins have been shown to up regulate macroautophagy and alleviate the effect of LPS apart from inhibiting cholesterol synthesis. However their role in microautophagy has not been reported. In the present study we investigated the effect of statins on microautophagy using the RAW264.7 cell line as a model system. Treatment with statins resulted in an up regulation of the genes for MA as well as Mi as determined by qPCR. Further studies showed that the levels of proteins TSG101 and VPS4 were increased during statin treatment. Pretreatment with statins led to a lowering of LAMP2A levels and decreased LAMP2A on the late endosomes, mimicking a LAMP2A knock down. Pretreatment with statins not only lowered the transcription of iNOS and TNF-alpha genes but also prevented the effect of LPS by decreasing initial levels of LAMP2A and increasing levels of TSG101. We propose that Mi can be a surrogate for Chaperone mediated autophagy (CMA) under conditions where the components of CMA are rendered non-functional due to disruption in normal cellular metabolism.
Trogocytosis efficiency is dependent upon TCR affinity for MHC: Peptide ligands (APP4P.115)
Authors: Wetzel Scott , Kreitinger Joanna , Crane Naomi , Hayes Timmon 
Published: January 2023

Abstract
Antigen recognition by CD4+ T cells leads to large-scale spatial and temporal molecular redistributions, forming the immunological synapse. We have previously shown that upon dissociation from APC, T cells capture large membrane fragments from the antigen presenting cell containing both MHC:peptide ligands and costimulatory molecules directly from the immunological synapse in a process termed trogocytosis. The transferred molecules are retained on the surface of the T cell and sustain intracellular signaling and are associated with the selective survival of the trogocytosis positive cells after removal of the antigen presenting cells. To further characterize trogocytosis, we have examined the impact of TCR affinity for cognate MHC:peptide ligands on the efficiency of trogocytosis. Using moth cytochrome C 88-103 and the altered peptide ligands MCC K99A and MCC-A, we show here that the extent of trogocytosis by primary murine CD4+ T cells correlates with the affinity of the TCR for the MHC:peptide ligands. Preliminary studies also show that sustained intracellular signaling and selective survival of the trogocytosis+ cells varies based upon the TCR affinity for the specific MHC:peptide ligands captured by the T cell.
Requirement for PD-1 in the induction and maintenance of tolerance differs depending on tolerogenic stimulus (BA8P.118)
Authors: Pauken Kristen , Martinov Tijana , Heffernan James , Spanier Justin , Sahli Nathanael , Jenkins Marc , Vezys Vaiva , Fife Brian 
Published: January 2023

Abstract
The inhibitory receptor Programed Death (PD)-1 interacting with PD-L1 is important for tolerance since blocking this pathway accelerates autoimmunity. However, the mechanisms by which PD-1/PD-L1 contribute to tolerance remain unclear. In this study we tracked endogenous insulin-specific CD4 T cells in mice that are susceptible (NOD) or resistant (B6.g7) to Type 1 Diabetes (T1D). Despite the presence of insulin-reactive CD4 T cells in both strains, anti-PD-L1 precipitated T1D in NOD but not B6.g7 mice. Accelerated T1D in NOD mice correlated with increased numbers of insulin-specific CD4 T cells in the spleen, pancreatic LN, and pancreas. The number of cells increased in the pancreatic LN of B6.g7, but not the spleen or pancreas, suggesting that loss of PD-1 signaling was insufficient to promote trafficking to the pancreas in these mice. Unexpectedly, the breakdown of self tolerance in NOD mice did not rely on PD-1 signals as anergic cells did not change with PD-1 blockade. Rather, PD-1 was more important for regulating effector T cells. This contrasts data using antigen-coupled ECDI-fixed cells to induce anergy in potently activated effector cells during T1D in NOD, where PD-1/PD-L1 ligation is required for both the induction and maintenance of tolerance. This work reveals the role PD-1 signaling has for different contexts of tolerance, and has important therapeutic implications for use of PD-1 inhibitors in patients.
Disruption of tolerance by ER stress in type 1 diabetes (BA8P.128)
Authors: Marre Meghan , Profozich Jennifer , Coneybeer Jorge , Geng Xuehui , Bertera Suzanne , Trucco Massimo , Piganelli Jon 
Published: January 2023

Abstract
Type 1 diabetes (T1D) is an autoimmune disease, largely mediated by autoreactive T cells, in which islet β cells are destroyed. The events that break peripheral tolerance in individuals genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D, such as viral infection, chemicals, reactive oxygen species, and dynamic glucose sensing, result in endoplasmic reticulum (ER) stress. ER stress increases the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells and worsen T1D. Indeed, chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased IFNγ secretion from diabetogenic BDC2.5 T cells (28-1500 fold). Chelation of stress-induced calcium (Ca2+) flux reduced this response (27-64%), supporting a role for Ca2+-dependent enzymes such as tissue transglutaminase 2 (Tgase). Tgase activity is higher (18 fold) in β cells under stress, and reduced Tgase expression decreased the BDC2.5 response to β cells under stress (58%). Finally, nonantigenic insulinomas were exposed to normal physiological conditions in vivo. At harvest, these cells had increased ER stress (300 fold), increased Tgase activity (44 fold), and elicited higher BDC2.5 IFNγ responses (22 fold) compared to cultured cells. Thus, β cell ER stress causes formation of neo-antigens that activate autoreactive T cells.
Activation of autoreactive T cells by endogenous microflora induces spontaneous autoimmunity in the immunologically privileged retina (BA8P.130)
Authors: Zarate-Blades Carlos , Horai Reiko , Chen Jun , Silver Phyllis , Dillenburg-Pilla Patricia , Yamane Hidehiro , Chan Chi-Chao , Honda Kenya , Caspi Rachel 
Published: January 2023

Abstract
Autoimmune uveitis (AU) has an incidence similar to multiple sclerosis, it develops spontaneously and is a major cause of blindness. We hypothesized that AU could be triggered by endogenous microbiota in view of anecdotal evidence linking AU to systemic infections. We tested this in R161H mice, a new model of AU developed in our lab. R161H mice express a transgenic T cell receptor (TCR) specific to retinal protein IRBP and develop spontaneous uveitis starting at 4 weeks of age. R161H mice treated with an antibiotic cocktail, or reared under germ free conditions, had a significant delay in disease onset and intensity compared to controls, as well as fewer IL-17-secreting T cells in the gut LP. Notably, conventionally housed R161H mice consistently displayed 2-5x more Th17 cells in their LP compared to WT littermates, even when crossed onto an IRBP-deficient background. Compared to polyclonal T cells, R161H TCR transgenic T cells were more readily activated by extracts of gut contents in vitro and adopted the Th17 phenotype in the gut with higher frequency in vivo. Experiments utilizing Nur77 reporter mice and pZap70, revealed clear evidence that IRBP-specific T cells receive an activation signal through their TCR in the gut. These data not only have implications for etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microflora may be a more common trigger of autoimmune diseases than is currently appreciated
Temporal regeneration of aire-expressing medullary epithelial cells and its impacts on reestablishing central immune tolerance (BA8P.122)
Authors: Tajima Asako , Coppola Antonina , Grupillo Maria , Gualtierotti Giulio , Piccirillo Ann , Geng Xuehui , Rudert William , Trucco Massimo , Fan Yong 
Published: January 2023

Abstract
The essential role of tissue specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs) in mediating central tolerance has been well-established. We have previously shown that mice with mTEC-specific insulin deletion develop autoimmune diabetes within 3 weeks postnatal. To further understand the mechanisms of mTEC-mediated self-tolerance, we generated the Aire-YFP mice by crossing R26R-YFP reporters with Aire-Cre transgenic mice. Three subsets of mTECs were isolated from these mice: the YFPLOWCD80LOWClass IILOW pre-Aire cells; the YFPHICD80HIClass IIHI Aire+ cells; and the YFPHICD80INTClass IIINT post-Aire cells. Diverse TSA transcripts were detected in Aire+ mTECs, but not, or more restricted in pre- and post-Aire subsets, respectively. To investigate the temporal differentiation of mTECs, we generated the Aire-Cre:R26R-DTR (Aire-DTR) mice, which marked both Aire+ and post-Aire mTECs with diphtheria toxin (DT) receptors. Consecutive administration of DT resulted in ablation of both subsets, whereas pre-Aire mTECs remained intact. Upon DT withdrawal, transcripts of Aire and TSAs remained absent at day 3, but became detectable at day 5. Interestingly, no autoimmunity was observed in Aire-DTR mice treated weekly with a single dose of DT, suggesting that the newly generated Aire+ mTECs could reestablish self-tolerance in a timely manner. Our data provide new insights into the temporal regeneration of mTECs and its importance in maintaining central tolerance.
Priming of myelin-reactive CD4+ T cells in the absence of CD11c+ dendritic cells (BA8P.123)
Authors: Waldner Hanspeter , Cheung Julie , Wexler Aaron 
Published: January 2023

Abstract
Experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, is predominantly mediated by pro-inflammatory CD4+ T cell responses to myelin antigens, including myelin proteolipid protein (PLP). Dendritic cells (DCs) are considered critical for inducing T cell responses against infectious agents, but their role in priming of myelin-specific CD4+ T cells is still unclear. To investigate their importance in promoting PLP-specific CD4+ T cell responses, we have established novel SJL mice that express the simian diphtheria toxin receptor (DTR) on CD11c+ DCs, allowing ablation of DCs following diphtheria toxin administration (CD11c-DTR SJL mice). PLP-pulsed spleen cells from DC-depleted mice promoted significantly diminished proliferation and pro-inflammatory cytokine responses of CD4+ T cells from PLP TCR transgenic mice in vitro compared to DC-sufficient splenocytes. However, CFSE-labeled PLP-specific CD4+ T cells transferred into DC-depleted mice showed undiminished proliferation in response to PLP/CFA immunization, suggesting that non-DCs were capable of myelin-specific T cell priming. We determined the importance of DCs in EAE development by transferring PLP TCR transgenic CD4+ T cells into DC-depleted RAG2-deficient mice lacking B cells. Interestingly, PLP-specific CD4+ T cells mediated severe EAE even in the persistent absence of DCs, suggesting that differentiation of encephalitogenic CD4+ T cells required neither B cells nor CD11c+ DCs.
A novel function of RGS10 in effector T lymphocytes to augment mouse EAE (BA8P.126)
Authors: Lee Jae-Kyung , Chung Jaegwon , Kannarkat George , Graham Kareem , Tansey Malu 
Published: January 2023

Abstract
A broad range of immune-based therapeutic drugs have been available for treatment of various autoimmune diseases but many have had limited success. Therefore, it is critical to identify additional cellular targets that can regulate the pathogenic response of immune cells. We hypothesize that GPCR modulator RGS proteins are important modulators of the immune responses involved in the development of multiple sclerosis (MS). Our hypothesis is based on reports that various SNPs in RGS proteins is highly correlated with the diagnosis of MS. Especially, an in-depth search of the GEO profiles database also revealed higher levels of RGS10 transcripts in MS patients’ PBMCs. Here, we showed RGS10-null mice displayed significantly milder clinical symptoms of EAE with reduced incidence and delayed disease onset. We observed that there were less numbers of CD45+ cells and CD4+ T cells in MOG35-55-immunized RGS10-null mice in the CNS. RGS10-null LN T cells were less proliferatative and produce less IFN-γ and IL-17 cytokines in response to MOG. Our data suggest a critical role for RGS10 in modulating disease by attenuating lymphocyte infiltration and/or peripheral immune responses during EAE. This is the first study ever conducted to elucidate the function of RGS10 in effector lymphocytes in the context of EAE. The identification of RGS10 as a central regulator of the inflammatory processes will open a possibility for developing more specific targeted therapy for the treatment of MS.
Cutaneous B-1 B cells can secrete IL-10 and require integrin α4β1 (VLA-4) for migration into skin (CAM3P.201)
Authors: Debes Gudrun , Wilson R. , Lauer Uta , Hamann Alf , Geherin Skye 
Published: January 2023

Abstract
B1 B cells are innate-like B cells that provide key effector functions during infection and inflammation. Despite their importance, the target organs, function and trafficking of this B cell subset are largely unknown. Here we show that, unexpectedly, B cells, including B1(-like) cells with the potential to secrete IL-10, were part of the normal cutaneous immune system of mice and humans. In the mouse, we found steady-state migration of peritoneal B1 B cells into skin. In addition, B1, but not follicular B2, B cells efficiently migrated into inflamed skin by virtue of α4β1 integrin (VLA-4) expression. Importantly, B1 B cells expressed high affinity VLA-4 at steady state, indicating that B1 cells were poised to migrate into inflamed sites. Our data suggest that IL-10+ innate-like B cells are recruited into skin inflammation to dampen cutaneous inflammation, and we propose that dysregulation of the recruitment or function of B1(-like) B cells contributes to inflammatory skin diseases, such as psoriasis.
TNFR2 expression by CD4 effector T cells is required to induce full-fledged experimental colitis (CCR3P.204)
Authors: Chen Xin , Xiao Haitao , Howard O.M.Zack , Oppenheim Joost 
Published: January 2023

Abstract
There is now compelling evidence that TNFR2 is constitutively expressed on Tregs and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of CD4+Foxp3+ regulatory T cells (Tregs). However, we also showed that the expression of TNFR2 could be up-regulated on CD4+Foxp3- effector T cells (Teffs) upon TCR stimulation, and TNFR2-expressing Teff cells were more activated and more resistant to Treg-mediated inhibition. In order to further define the role of TNFR2 in the proliferative expansion of pathogenic CD4 T cells in a setting of autoimmune disorder, we transferred naïve CD4 cells from WT mice and TNFR2-/- mice into Rag1-/- recipients. The results showed that TNFR2-deficient Teff cells failed to induce full-fledged colitis unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to produce proinflammatory Th1 cytokines on a per cell basis. In vitro, TNF blockade inhibited the up-regulation of TNFR2 expression, as well as proliferative responses and cytokine production, by TCR-stimulated naïve CD4 cells. This effect of TNF blockade can be replicated by anti-TNFR2 Ab, but not by anti-TNFR1 Ab. Therefore, TNFR2 is critical for the proliferative expansion of pathogenic CD4 T cells, and thus may be a therapeutic target in the treatment of CD4 T cell-mediated autoimmune inflammation, although at a cost of attenuation of Treg activity.
IFN-α induces soluble IL-15 complexes via ADAM17 mediated cleavage (CCR3P.206)
Authors: Anthony Scott , Howard Megan , Stonier Spencer , Dai Zhimin , Khong Hiep , Overwijk Willem , Schluns Kimberly 
Published: January 2023

Abstract
Type I Interferons (IFN) are potent inducers of IL-15 transcription and cell surface expression. Previous studies have shown that sIL-15 complexes, which act as IL-15 agonists, are generated by TLR stimulation and after non-myeloablative lymphodepletion. Since TLR stimulation induces type I IFNs, we investigated the role of type I IFNs in generating sIL-15 complexes. We report that treatment with a plasmid inducing IFN-α production potently increased serum sIL-15 complexes levels in mice. Production of sIL-15 complexes by bone marrow derived dendritic cells (BMDC) also increased after treatment with IFN-α in an IFNAR-dependent manner. Increases in sIL-15 complexes after total body irradiation coincided with appearance of serum IFN-α and were significantly impaired in the absence of Type I IFN signaling. In addition, we found that IFN-α induced activity of the metalloprotease, ADAM17 in BMDCs, which was critical as induction of sIL-15 complexes by IFN-α was abolished after deleting ADAM17. We also determined whether sIL-15 complexes are generated during a Vesicular stomatitis virus (VSV) infection as VSV induces Type I IFNs. One day after infection, sIL-15 complexes are transiently induced, which was surprisingly unabated in the absence of Type I IFN signaling. Overall, our findings show that Type I IFNs induce production of sIL-15 complexes by activating ADAM17-mediated cleavage, thus identifying a new mechanism regulating sIL-15 complexes during inflammation.
The association of ROS levels in blood cells with age, past radiation exposure, and IL6R gene polymorphisms (CCR3P.211)
Authors: Hayashi Tomonori , Hu Yiqun , Furukawa Kyoji , Ohishi Waka , Geyer Susan , Weng Nan-ping , Hayashi Ikue , Yoshida Kengo , Kajimura Junko , Kyoizumi Seishi , Kusunoki Yoichiro , Nakachi Kei 
Published: January 2023

Abstract
Reactive oxygen species (ROS) play an important role in cell-mediated immune responses, although overproduction and excessive accumulation of ROS may also enhance risks of inflammation-related diseases. To address these concerns, we have developed an assay system to determine ROS (H2O2 and O2.-) levels in blood cells to investigate the effects age and of radiation exposure on ROS levels in the blood cells obtained from 2,789 Japanese atomic-bomb survivors, as well as associations between intracellular ROS levels and immune-/inflammation-related gene polymorphisms. The results showed that H2O2 levels in monocyte and granulocyte fractions increased with age (P&lt;0.001) but not with radiation dose. No effects of age or radiation dose on H2O2 levels were observed for T-cell subsets. On the other hand, O2.- levels in both lymphocyte and granulocyte fractions increased with age and radiation dose (P&lt;0.05). In addition, O2.- levels in T cells, especially in CD8+ T cells, increased with age and radiation dose (P&lt;0.05). Analysis of the association between intracellular ROS levels and gene polymorphisms revealed a significant difference in O2.- levels in T cells by IL6R genotypes. These results suggest that ROS levels in selected types of immune cells are affected by aging, radiation, and genetic factor. Thus, the intracellular ROS levels may serve as a biomarker for age of the cells as well as for estimating the risk of inflammation-related disease development.
β-Catenin induces T-cell transformation by promoting genomic instability (HEM4P.237)
Authors: Emmanuel Akinola , Dose Marei , Keerthivasan Shilpa , Aghajani Katayoun , Gounari Fotini 
Published: January 2023

Abstract
Deregulated activation of β-catenin has been correlated with genomic instability in cancer. During thymocyte development β-catenin activates transcription in partnership with Tcf-1, an essential T-cell specific DNA-binding protein. We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination-activating-gene (RAG) and c-Myc activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG-recombination events and resembled oncogenic translocations in ponies. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. Interestingly, ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including sites near the translocation. Pre-transformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG induced DNA double strand breaks (DSBs). Importantly these cells were able to survive despite DNA damage as activated β-catenin promoted an anti-apoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas and this is associated with compromised DSB repair and increased survival of thymocytes with damaged DNA.