bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tapasin-related protein TAPBPR interacts directly with peptide-free MHC-I/β2-microgolbulin complexes (APP3P.101)
Authors: Morozov Giora , Zhao Huaying , Mage Michael , Boyd Lisa , Schuck Peter , Natarajan Kannan , Margulies David 
Published: January 2023

Abstract
The molecular mechanism by which MHC-I molecules are loaded with peptides is crucial to fundamental aspects of antigen presentation relating to self-tolerance and CD8 T cell activation. To gain insight into the mechanism of MHC-I peptide loading we have studied human TAPBPR (TAP binding protein-related) protein, which is 22% identical to tapasin in amino acid sequence, widely expressed and IFN-γ inducible, but is not part of the peptide-loading complex (PLC). To understand the interaction of TAPBPR with MHC-I, we produced recombinant soluble TAPBPR and evaluated its interactions with MHC-I/β2m complexes in vitro. Using recombinant MHC-I molecules refolded with photolabile peptides, we show, by gel-shift analysis, size exclusion chromatography, and analytical ultracentrifugation that TAPBPR binds MHC-I/β2m after photolysis of the bound peptide, in a 1:1 molar ratio. MHC-I molecules loaded with low-affinity peptides also bind TAPBPR. The TAPBPR/MHC interaction is reversed by exposure to high-affinity peptides known to bind the MHC-I molecule, indicating a role of TAPBPR in stabilizing a peptide-receptive form of the MHC-I/β2m complex. Because of the predicted structural similarity of TAPBPR to tapasin, this system not only provides insight into the molecular details of TAPBPR/MHC-I binding, but may also elucidate tapasin interactions in the PLC that have previously eluded experimental interrogation.
LAMP-2C regulation of MHC class II antigen presentation (APP3P.105)
Authors: Pérez Liliana , Deffit Sarah , Blum Janice 
Published: January 2023

Abstract
In autophagy cells divert cytoplasmic components and organelles to lysosomes for degradation impacting innate and adaptive immunity. A selective form of autophagy, known as chaperone-mediated autophagy (CMA), is implicated in adaptive immunity. CMA relies on the lysosome-associated membrane protein (LAMP)-2A to translocate proteins from the cytoplasm into lysosomes. Alternative splicing of the LAMP-2 gene generates 3 highly conserved isoforms LAMP-2A, LAMP-2B and LAMP-2C. The function and regulation of these genes is not well understood. Here we identified a novel role for LAMP-2C in the regulation of CMA. Studies were undertaken here to understand the role of LAMP-2C in MHC class II presentation of cytoplasmic antigens. Ectopic expression of LAMP-2C did not alter the maturation or the enzymatic activity of lysosomal enzymes. Furthermore, LAMP-2C ectopic expression failed to perturb other degradation pathways involved in antigen presentation like macroautophagy and proteasome degradation. Thus, LAMP-2C appears to have a selective regulatory role with respect to the presentation of cytoplasmic proteins. Future studies are underway to determine mechanistically how LAMP-2C controls antigen presentation. This work was supported by NIH 5T32AI060519 and AI079065.
Protein deprivation alters trafficking of the B cell receptor and endogenous antigens in B cells (APP3P.106)
Authors: Deffit Sarah , Blum Janice 
Published: January 2023

Abstract
Protein malnutrition in children has been linked to increased instance of infection and higher fatality likely due to limited T and B cell-mediated host immunity. These findings suggest protein malnourishment may alter antigen presentation and the cognate interaction between T and B cells. Studies here show serum deprivation induced macroauophagy and MHC class II presentation of select antigens which utilize this pathway in B cells. Further studies established serum deprivation inhibited MHC class II presentation of cytoplasmic autoantigen, glutamic acid decarboxylase and the presentation of epitopes derived from the B cell receptor. Serum deprivation did not alter the expression MHC class II proteins but did upregulate proteasome and lysosomal protease activity. Interestingly, B cell receptor accumulation on the cell surface was detected likely due to a reduction in endocytosis. An altered subcellular distribution of glutamic acid decarboxylase protein was observed, suggesting changes in the trafficking of this antigen. Studies are currently underway to examine how protein deprivation regulates antigen trafficking and presentation. This work was supported by the T32DK007519 and NIH RO1AI079065.
Sec61 plays an essential role in facilitating antigen cross-presentation to induce immune glomerular injury: a novel ‘self-organized criticality theory’ explaining cause of SLE (BA8P.129)
Authors: Tsumiyama Ken , Shiozawa Shunichi 
Published: January 2023

Abstract
Objective: Our novel ‘self-organized criticality theory’ which may explain the cause of SLE shows that immune glomerular injury is caused by CTL generated via antigen cross-presentation. Here we show essential role of Sec61 in antigen cross-presentation and immune glomerular injury. Methods: Bone marrow-derived dendritic cell (BMDC) was cultured with fluorescent-labeled OVA. Early endosome antigen 1 (EEA1), calnexin and Sec61 were detected using immunofluorescent staining. Sec61 was immunoprecipitated from OVA-pulsed BMDC lysate, followed by immunoblotting of OVA. Mice were repeatedly immunized with OVA to induce SLE. Splenic DC (spDC) was cultured with fluorescent-labeled OVA to examine localization of engulfed antigen. Further, exotoxin A was co-immunized with OVA to inhibit Sec61. Results: In BMDC, OVA was co-localized with an endosomal marker EEA1, and subsequently separated from EEA1. OVA was co-localized with Sec61, but not the ER marker calnexin. The result was similar when spDC was used. Engulfed OVA was co-precipitated with Sec61, and the amount of Sec61 in endosome was increased after repeated immunization with OVA. Instead, inhibition of Sec61 did not induce CTL nor renal injury, suggesting that OVA interacts with Sec61 hence exported from endosome to cytoplasm via Sec61. Conclusion: Increase of Sec61 in endosome and the export of antigen from endosome to cytoplasm via Sec61 are important for antigen cross-presentation and as the cause of immune glomerular injury.
Self-tolerance in the polyclonal CD4+ T cell repertoire (BA8P.132)
Authors: Malhotra Deepali , Linehan Jon , Jenkins Marc 
Published: January 2023

Abstract
Autoimmunity affects millions of people throughout the world. CD4+ T cells express T cell receptors (TCRs) that recognize peptides loaded on major histocompatibility complex II molecules (p:MHCII). Recognition of self-p:MHCII ligands may promote autoimmunity, necessitating the existence of tolerance mechanisms that regulate self-reactive CD4+ T cells. Study of TCR transgenic cells that recognize model self-antigens has identified several potential mechanisms of tolerance including clonal deletion, regulatory T cell induction, and anergy. However, TCR transgenic systems are unable to determine how each mechanism contributes to tolerance within a polyclonal repertoire comprised of a diverse set of TCRs. Thus, we have produced p:MHCII tetramers to track and study how tolerance is maintained in a polyclonal pool of self-reactive CD4+ T cells. We find that 50% of self-reactive CD4+ T cells in this polyclonal population are deleted, and following priming the remaining non-deleted self-reactive CD4+ T cells exhibit diminished proliferation and altered differentiation patterns. Ongoing work is focused on understanding if and how these tolerance pathways differentially regulate individual CD4+ T cell clones in the polyclonal repertoire and whether TCR affinity is the guiding factor for this decision.
Real time visualization of the differential mTEC and DC contributions to T cell central tolerance (BA8P.119)
Authors: Jones Jessica , Rey-Villamizar Nicolas , Sasiponganan Chayanit , Selden Hilary , Roysam Badrinath , Ehrlich Lauren 
Published: January 2023

Abstract
T cell central tolerance for peripherally expressed tissues is established by thymocyte:stromal cell interactions that occur within the thymic medulla. However, the roles of the medullary thymic epithelial cell (mTEC) and dendritic cell (DC) subsets in mediating negative selection against tissue-restricted antigens are unclear. While genetic models provide evidence regarding the selecting potential of either stromal cell subset in the absence of the other, these models interfere with the normal thymocyte:stromal cell crosstalk necessary to establish a properly differentiated thymic microenvironment; this leaves open the relative contribution of either stromal cell type in mediating negative selection when both subsets are intact. Here, using two-photon fluorescence microscopy, we observe thymocyte:stromal cell interactions at the onset of TCR signaling as they occur in real time in the thymic medulla. We have quantitated CD8+ and CD4+ thymocyte contacts with either Aire+ mTECs or DCs in the presence of membrane-bound or secreted soluble forms of antigen, and find that both thymocyte MHC-restriction and subcellular localization of the antigen modulate the contributions of mTECs and DCs to negative selection. These experiments provide insight into the cellular mechanisms of central tolerance that deter autoimmunity, as they occur in a physiological environment.
Generation and characterization of immune-complex specific T cells (BA8P.121)
Authors: Giles Josephine , Pinilla Clemencia , Shlomchik Mark 
Published: January 2023

Abstract
There is ample evidence that, in systemic autoimmune diseases such as systemic lupus erythematosus, T cells contribute to disease by both providing help for B cells and directly attacking target tissues. However, there is relatively little information on the nature and specificity of such autoreactive T cells. Moreover, there are few if any systems in which they can be identified and studied in vivo. To this end, we have generated a series of autoreactive T cell hybridomas using rheumatoid factor (RF) B cells fed with immune complexes (ICs). We are using this cognate autoreactive T and B cell system to investigate two major questions. 1) What are the T cells specific for within the immune complex? We have used a positional scanning strategy using synthetic combinatorial libraries. Using peptide reactivity patterns to search proteome databases, we identified several candidate peptides, some of which have been synthesized and validated, leading to putative implication of several DNA and RNA interacting proteins. 2) How do autoreactive T cells function in vivo? We have generated retrogenic mice expressing the autoreactive TCR cloned from one of the hybridomas. These cells are positively selected into the CD4 compartment but do not become nTregs. These mice will be used to characterize the autoreactive T cells in vivo, including their interactions with cognate RF B cells as well as their potential to promote disease.
Polyfunctionality of naive T cells is enhanced in older age (CCR3P.223)
Authors: Van Eps Puja , Banks Richard , Aung Htin , Betts Michael , Canaday David 
Published: January 2023

Abstract
Reduced number of naïve T cells along with an accumulation of differentiated cell types in aging has been described but little is known about the polyfunctionality of T cells. In this study we compared the individual cytokine and polyfunctional profiles of T cell subsets, including newly described stem cell like memory T cells (TSCM) in response to stimulation with submaximal concentration of non-specific mitogen in older (median age 80, n=23) versus younger (median age 27; n=23) adults using multicolor flow cytometry. Secretion of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 were measured. Polyfunctionality was determined using FlowJo and SPICE (NIAID). Aging was associated with a marked decline in CD8+ naïve cells (11% vs. 47 %; p&lt;0.0001) and an expansion in memory cell types including central memory (p&lt;0.05), effector memory and effectors (p&lt;0.001 for both) compared to younger subjects. There was also a decline in CD4+ naïve cells with age (33% vs. 45%; p=0.02). There were no differences in frequencies or cytokine profiles of TSCM between groups. CD8+ naïve cells in the older group had increased secretion of all cytokines measured compared to the younger subjects and exhibited greater polyfunctionality (p=0.03). CD4+ naïve cells among the older group also had greater polyfunctionality with a TNF-α and IL-2 dominance (p=0.001). The decline in naïve T cells in aging, rather than their polyfunctional potential maybe contributing to immunosenescence.
Initial IL7R signaling regulate the induction of effector CD8+ T cells and subsequent anti-tumor immune response in lymphopenic host (CCR3P.208)
Authors: Suzuki Toshihiro , Kishimoto Hidehiro , Abe Ryo 
Published: January 2023

Abstract
Induction of lymphopenia and adoptive transfer of T cells are followed by lymphopenia-induced proliferation (LIP), and generated a anti-tumor immune response. However, the distinct role of redundant IL7 in lymphopenic host and subsequent LIP of T cells for anti-tumor immune response has been unclear, since multiple mechanisms are involved in the anti-tumor immune response under lymphopenic conditions. To clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited by IL7R blockade with anti-IL7Ra mAb at various stages, and the anti-tumor effect was assessed. When LIP was occurred, the growth of transplanted LLC expressing model antigen gp33, was markedly regressed. Using IL7R blockade with mAb at various stages, we found that IL7R signaling at the start of LIP is crucial for the anti-tumor immune response. Although some reports focused on the benefit of IL7 for the improvement of effector T cell function, later IL7R blockade did not arrest the expansion of CTLs, and anti-tumor effect was occurred. IFN-g production also depend on IL7R signaling during induction phase, but not later phase, and, the expression of co-inhibitory molecules, PD-1, on CTLs was unchanged in IL7R blockade hosts. In conclusion, our results demonstrated that initial IL7R signaling and subsequent LIP regulated the expansion of IL7Ra+ effector precursors mainly, but that the effector function of CTL precursors and migration into tumor did not require IL7R signaling.
Notch signaling regulates T follicular helper cell function and maintenance during allergic immunity (CCR3P.209)
Authors: DellAringa Mark , Reinhardt Richard 
Published: January 2023

Abstract
Success of most current vaccines relies on the production of high-affinity antibodies. The generation of high-affinity antibody producing B cells occurs within the germinal center, and CD4+ T follicular helper cells (Tfh) play an indispensable role during the maturation of these B cells. A critical step in this process is the secretion of interleukin-4 (IL-4) by Tfh cells. IL-4 signaling in germinal center B cells is required for affinity maturation of IgG1. IL-4 production by Tfh cells appears to occur independently from the classical Th2 differentiation pathway. Currently, the mechanisms regulating IL-4 production by Tfh cells are unknown. One intriguing possibility for non-canonical IL-4 production in Tfh cells is the Notch pathway. Notch signaling is required for the differentiation of Tfh cells, and has been shown to modulate IL-4 production. Here, we provide mechanistic evidence for Notch signaling in the production of IL-4 by Tfh cells. Inhibition of Notch signaling after germinal center formation resulted in a significant decrease in the percentage and number of Tfh cells actively producing IL-4. Furthermore, inhibition of Notch signaling led to decreased expression of the chemokine receptor CXCR5 in IL-4 producing CD4+ T cells, which corresponded to a repositioning of IL-4-competent cells outside of the germinal center. These findings suggest a model in which Notch signaling is not only important in Tfh differentiation, but also in Tfh function and maintenance.
Biallelic T cell receptor rearrangement enhances the efficiency of thymocyte development and positive selection. (HEM4P.234)
Authors: Schuldt Nathan , Auger Jennifer , Hogquist Kristin , Binstadt Bryce 
Published: January 2023

Abstract
Biallelic rearrangement of TCR loci has been proposed to increase the likelihood of productive gene rearrangement, thereby promoting thymocyte survival. When allelic exclusion is incomplete, this process also results in dual TCR-expressing T cells, which may increase repertoire diversity, but may also increase autoreactive potential. We investigated the effects of biallelic TCR gene rearrangement by comparing wildtype (wt) T cells to those from mice hemizygous for the genes encoding TCR α, TCR β, or both. Using bone marrow chimera competition assays we found that Tcrb hemizygous thymocytes were impaired in the DN1 to DN2 transition relative to wt cells. In addition, Tcrb hemizygous mice exhibited a higher frequency of T cells committed to the γδ lineage. Bone marrow chimera competition assays revealed a dramatic increase in the ratio of wt thymocytes to Tcra hemizygous thymocytes between the pre-selection and post-selection DP compartments, demonstrating that biallelic Tcra rearrangement increases the rate of positive selection. Despite their impairments in thymocyte development, mice hemizygous for both Tcra and Tcrb generated polyclonal T cell repertoires similar to wt mice as assessed by numbers of antigen-specific naïve T cells, responses to multiple self and foreign antigens, and TCRβ sequence diversity. Thus, biallelic rearrangement of Tcra and Tcrb loci enhances the efficiency of thymocyte development, but is not necessary for normal TCR repertoire diversification.
Signaling through FcRγ receptors on dendritic cells drives IL-33 dependent Th2-type responses (HYP6P.262)
Authors: Tjota Melissa , Blaine Kelly , Barrett Nora , Sperling Anne 
Published: January 2023

Abstract
Dendritic cells (DCs) play a critical role in the development of Th2 responses during atopic asthma, but the mechanism by which allergens drive DCs to promote Th2 responses is still unclear. Previously, we demonstrated that allergen-specific IgG immune complexes (ICs) enhance secondary Th2 responses via an IL-33-dependent mechanism by ligating FcγRIII on antigen presenting cells. To identify if this pathway was common to other Th2 stimuli, we used house dust mite (HDM) extract. While HDM-mediated allergic inflammation was not dependent on FcγRIII, it was reliant on the common signaling component FcRγ as FcRγ-/- mice failed to develop Th2 responses in the lung. Notably, ligation of FcRγ associated receptors, FcγRIII and Dectin-2, on DCs treated with ICs or HDM respectively, upregulated IL-33 in an FcRγ and PI3-kinase dependent manner suggesting a common pathway by which Th2 stimuli can direct the development of DCs that induce Th2 differentiation. Reestablishment of Th2 responses in the FcRγ-/- mice with an adoptive transfer of HDM-sensitized FcRγ+/- DCs indicated that the lack of response in the FcRγ-/- mice was due to a deficient response from the DCs. Interestingly, addition of recombinant IL-33 during HDM sensitization was sufficient to restore airway inflammation in FcRγ-/- mice. Overall, these data identify a mechanism whereby Th2 stimuli signal through FcRγ associated receptors on DCs to upregulate IL-33 production and induce Th2-mediated allergic airway inflammation.
Dissecting mechanisms underlying the pathogenesis of eosinophilic esophagitis (HYP6P.263)
Authors: Tait Wojno Elia , Noti Mario , Kim Brian , Siracusa Mark , Nair Meera , Benitez Alain , Ruymann Kathryn , Muir Amanda , Yearley Jennifer , Menard-Katcher Paul , Kubo Masato , Obata-Ninomiya Kazushige , Karasuyama Hajime , Comeau Michael , de Waal Malefyt Rene , Sleiman Patrick , Hakonarson Hakon , Cianferoni Antonella , Falk Gary , Wang Mei-Lun , Spergel Jonathan , Artis David 
Published: January 2023

Abstract
Eosinophilic esophagitis (EoE) is an allergic disease characterized by esophageal eosinophilia, inflammation, and dysfunction. EoE has become increasingly common, but current management strategies are nonspecific. Thus, there is an urgent need to identify new pathways that could be targeted to treat EoE. Recently, EoE was associated with a gain-of-function polymorphism in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation and peripheral basophilia. However, how TSLP and basophils might contribute to the development of eosinophil responses during EoE remains unknown. Here, we employed a new murine model of EoE-like disease to investigate the role of TSLP and basophils in promoting esophageal eosinophil responses. Development of esophageal eosinophil responses was dependent on TSLP-elicited basophils, and antibody-mediated neutralization of TSLP or depletion of basophils ameliorated established esophageal eosinophilia. In addition, we examined how sort-purified human basophils influence eosinophil responses in vitro. Finally, elevated TSLP levels and exaggerated basophil responses observed in esophageal biopsies from EoE patients correlated with eosinophil responses. Together, these data indicate that TSLP-elicited basophil responses may play a key role in mediating eosinophil responses in EoE, suggesting that the TSLP-basophil axis could represent a new and promising therapeutic target to treat this disease.
Diesel exhaust particles potentiate allergic Th2/Th17 recall responses (HYP7P.301)
Authors: Brandt Eric , LeCras Timothy , Khurana Hershey Gurjit 
Published: January 2023

Abstract
Diesel exhaust particles (DEP) are a major component of traffic related air pollution and have been implicated in asthma exacerbations. In mice intra-tracheally exposed 9 times over the course of 3 weeks to either saline, DEP, house dust mite (HDM) or HDM and DEP, we assessed the rate of DEP clearance from the lung and examined its impacts on HDM recall responses. DEP alone did not induce the classic hallmarks of asthma but promoted a Th17 response resulting in long lasting chronic neutrophilia. DEP was still observed in a majority of alveolar macrophages 6 weeks after exposure. Coexposures to HDM and DEP generated a mixed Th2/Th17 response with a more severe asthma phenotype that completely resolved within 4 weeks. However, upon in vitro restimulation with HDM, Th2/Th17 responses were significantly enhanced in lung and draining lymph node cell cultures of HDM+DEP exposed mice compared to mice exposed to HDM alone. In conclusion, the prolonged presence of DEP in the lungs is associated with chronic neutrophilia. DEP-mediated exacerbation of allergen-induced asthma promotes long-term expansion of effector/memory Th17 and Th2 populations.