bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endothelial-expressed leukocyte-specific protein 1 regulates intravascular and extravascular directionality of chemotaxing neutrophils (P5094)
Authors: Qadri Syed , Xu Najia , Hossain Mokarram , Liu Lixin 
Published: January 2023

Abstract
The endothelial-expressed Ca2+- and F-actin-binding leukocyte-specific protein 1 (LSP1) was shown to play a pivotal role in the regulation of endothelial transmigratory cup formation, microvascular permeability increases, and transendothelial migration of leukocytes. Due to the complexity of leukocyte-endothelial interactions, the cell-specific role of LSP1 is incompletely understood. To elucidate the orchestration of neutrophil dynamics by endothelial LSP1 in vivo, intravital microscopy and time-lapsed video analysis were performed in the microvasculature of cremaster muscle in LSP1-deficient chimeric mice. Absence of LSP1 (Lsp1−/−) in non-hematopoietic tissue resulted in significantly impaired chemotactic directionality (chemotaxis index) but not the neutrophil migration velocity during CXCL2-elicited intraluminal crawling and extravascular migration as compared to the responses in wild type (Lsp1+/+) endothelial cells. The velocity of migration of Lsp1−/− neutrophils in extravascular tissue was significantly lower in both Lsp1+/+ and Lsp1−/− chimeric mice as compared to that of Lsp1+/+ neutrophils in these mice. Analysis of integrin expression of transmigrated neutrophils from CXCL2-induced peritonitis in chimeric mice revealed blunted expression of α6 and β1 on neutrophils only in mice with Lsp1−/− endothelial cells. In conclusion, non-hematopoietic endothelial LSP1 is a novel regulator of neutrophil directionality during neutrophil recrutment.
Signaling via neurokinin-1 receptor stimulates mast cell pro-inflammatory and Th2 biasing functions (P6008)
Authors: Sumpter Tina , Ho Chin , Tkacheva Olga , Shufesky William , Morelli Adrian , Larregina Adriana 
Published: January 2023

Abstract
Pro-inflammatory mast cells (MC) are crucial for initiation and perpetuation of anaphylaxis and atopic diseases. TNFα secretion by activated MC is critical for MC-mediated inflammation. Efficient MC activation depends on exogenous stimuli triggering endogenous mediators, such as pro-inflammatory neuropeptides. Surprisingly, studies linking neuromediators to MC are lacking. Here we analyzed the role of the neuropeptides, substance-P (SP) and hemokinin-1 (HK-1), in the regulation of IgE-mediated MC inflammation. In vitro, we show that MC express the shared receptor for SP and HK-1, the neurokinin-1 receptor (NK1R). FcϵRI ligation of bone marrow derived MC (BMMC) from WT mice increased HK-1 and NK1R mRNA and protein levels but had no effect on SP. NK1R-/- BMMC had significantly decreased degranulation and TNFα release suggesting that autocrine HK-1 promoted MC inflammatory functions. Mechanistic analysis demonstrated that NK1R-/- BMMC had impaired Akt-dependent NFκB activation. In vivo, NK1R-/- mice showed significantly decreased IgE-mediated cutaneous and systemic anaphylaxis and reduced development of chronic asthma, marked by reduced pulmonary eosinophilia, reduced Th2 skewing and low levels of TNFα in bronchial fluid. We conclude that MC utilize a positive feedback loop whereby autocrine HK-1 induces NK1R signaling that, in turn, potentiates TNFα secretion in the course of IgE-mediated anaphylactic and atopic disorders.
Functional expression of the novel C5a receptor C5L2 in human mast cells (P6014)
Authors: Pundir Priyanka , Kulka Marianna 
Published: January 2023

Abstract
C5a regulates immune responses by chemoattracting and activating immune cells, i.e. mast cells (MC). Its postulated that C5a regulates MC function by activating a G protein-coupled receptor (GPCR), however it can bind to C5aR, classical GPCR, or C5L2, not G protein coupled, and thought to activate distinct signaling. The presence and role of C5L2 in MC remain unknown. We hypothesized that human MC express functional C5L2. C5aR and C5L2 expression was analyzed by qPCR and flow cytometry. Degranulation, cytokine/chemokine production, adhesion, and migration were measured using standard assays. Lentivirus short hairpin (sh)RNA was utilized to create a stable C5L2-deficient (LAD2-C5L2ko) and a control MC cell line (LAD2-cntr). LAD2 expressed cell-surface C5L2 but not C5aR. C5a stimulated LAD2 degranulation (25%), TNF (22±1.3pg/ml), GM-CSF (15±0.4pg/ml), MCP1 (53±3.6pg/ml) and IP-10 (32±4.3pg/ml) production, adherence to fibronectin (466±92%), and chemotaxis (148.93±0.94%). Pre-treatment with pertussis toxin, wortmanin, U-73122, and Ro-31-8220 inhibited adhesion. Exposure to C5a reduced cell-surface C5L2 expression by ~45%. LAD2-C5L2ko showed decreased expression of GM-CSF and MIP-1β compared to LAD2-cntr. LAD2-C5L2ko showed reduced adhesion and chemotaxis compared to LAD2-cntr. Therefore, we demonstrated that C5a activates LAD2 through C5L2 which is sensitive to G protein signaling inhibitors and provide the first evidence of the crucial role of C5L2 in regulating MC functions.
The role of germinal centers in T cell receptor revision (P1421)
Authors: Higdon Lauren , Fink Pamela 
Published: January 2023

Abstract
Vβ5 transgenic CD4 T cells are tolerized to the endogenous superantigen Mtv-8 by either deletion or TCR revision. Revision occurs when a peripheral CD4 T cell downregulates Vβ5 and undergoes RAG-mediated recombination and surface expression of an endogenous TCR. Given that revision occurs in germinal centers, and that follicular helper T cells, or Tfh, in Vβ5 Tg mice increase in an Mtv-8-dependent manner, we are studying whether revising cells have a Tfh phenotype and whether germinal center interactions are required for revision. Revising cells have a transcription factor and surface phenotype resembling that of Tfh, with low Blimp-1 and elevated Bcl-6, CXCR5, PD-1, IL-21 receptor, and OX-40 expression. The frequency of post-revision T cells increases in mice heterozygous for Blimp-1, a transcriptional repressor of Tfh formation, but revision intermediates are unaffected. The adaptor molecule SAP is required for prolonged B and T cell interactions in the germinal center, and SAP null mice have reduced numbers of post-revision T cells, but no reduction in revision intermediates. These results suggest that revising T cells share some traits with Tfh and that Tfh formation and prolonged B-T cell interactions are required for the completion, but not initiation, of TCR revision. Ongoing studies will analyze revision in the absence of Bcl-6, a transcription factor required for Tfh formation, and TNFR1, a receptor required for the formation of germinal center structures.
SHP-2 is dispensable for effector CD8+ T cell and iNKT cell development (P1429)
Authors: Miah S. M. Shahjahan , Salter Alexander , Reilly Emma , Yang Wentian , Brossay Laurent 
Published: January 2023

Abstract
Src homology region 2 domain-containing phosphatase-2 (SHP-2) is ubiquitously expressed and is required for gastrulation, hematopoiesis, and embryonic development. Constitutive deletion of SHP-2 in mice is embryonically lethal. During development and maturation, certain subsets of T cells express a variety of inhibitory receptors known to associate with SHP-2. These subsets include iNKT cells, Ly49+CD8+ T cells with a memory phenotype, and exhausted T cells during chronic infection. We hypothesized that conditional SHP-2 deletion would have major effects on T cell development by altering the thresholds for positive and negative selection. Surprisingly, and in contrast to a previous report, we found that SHP-2-deficient CD8+ T cells and iNKT cells develop normally in the thymus and exhibit proper maturation in the periphery. Therefore, SHP-2 is dispensable for development and differentiation of both effector CD8+ T cells and iNKT cells.
Bacteriophage adhered to mucus provide a novel mucosal immune system (P3166)
Authors: Barr Jeremy , Auro Rita , Furlan Mike , Whiteson Katrine , Talago Natasha , Paul Lauren , Erb Marcella , Pogliano Joe , Stotland Aleksandr , Wolkowicz Roland , Cutting Andrew , Doran Kelly , Salamon Peter , Youle Merry , Rohwer Forest 
Published: January 2023

Abstract
Mucosal surfaces serve as a primary entry point for multiple pathogens and are therefore principal sites of immune defense. Here we demonstrate through in vitro and in silico studies that increased phage adherence to the host mucosal layer, provides a novel immune defense mechanism. We show that compared to the surrounding environment, phage-to-bacteria ratios were increased on all mucosal surfaces sampled ranging from cnidarians to humans. This increased phage abundance protects the underlying epithelium from bacterial infection. Enrichment of phage on mucus occurs via interactions between host mucin glycoproteins and phage immunoglobulin-like protein domains exposed on phage capsids. Metagenomic analysis found these immunoglobulin-like proteins present in many environments, particularly those adjacent to mucosal surfaces. Preliminary glycan microarrays and 2D gel electrophoresis show that phage adherence can rapidly adapt to hosts mucus glycan profiles, and in response, the host may regulate its mucus glycosylation to select for a beneficial phage community. This adaptation between phage and host provide a mechanism for the manipulation and selection of the mucosal microbiota. Based on these observations, we present the Bacteriophage Adherence to Mucus (BAM) model describing a phage-derived mucosal immunity with potential applicability to all mucosal surfaces, thus opening a novel field of immunological study.
Amphiregulin is essential for UVB mediated immunosuppression. (P6231)
Authors: Meulenbroeks Chantal , van Weelden Huib , Rutten Victor , Willemse Ton , Sijts Alice , Zaiss Dietmar 
Published: January 2023

Abstract
UVB exposure of the skin is associated with amelioration of inflammatory diseases, which has been shown to be mediated by activated Tregs. Since we have recently shown that Tregs, for optimal functioning, are dependent on signals induced by the EGF-like growth factor AREG, we tested whether UVB-mediated immunosuppression is dependent on AREG. We irradiated C57BL/6 wt and AREG-/- mice with UVB light and sensitized them with DNFB. Mice then were challenged and ear swelling was measured. Our results show that UVB light irradiation suppressed ear swelling in wt but not in AREG-/- mice. To determine at what point AREG expression would be essential, we transferred lymphocytes isolated from lymph nodes of irradiated and sensitized wt mice into naïve wt or AREG-/- mice, that then were sensitized with DNFB and challenged. In contrast to wt recipients, AREG-/- mice lacked suppression upon challenge. Similar results were found when lymphocytes, derived from UVB-irradiated CD4cre x EGF-Rflox/flox or wt mice, were transferred into wt mice. Mice that had received lymphocytes from irradiated and sensitized wt mice were fully able to suppress challenge induced ear swelling, while those that had received lymphocytes derived from UVB-irradiated CD4cre x EGF-Rflox/flox showed a significantly decreased ability to suppress swelling. Our data shows that AREG at the site of inflammation is essential for UVB irradiation-induced and Treg mediated immunosuppression.
The investigation of the role of Acanthamoeba as an allergen in allergic airway inflammation. (P6218)
Authors: Park Mi Kyung , Cho Min Kyoung , Kang Shin Ae , Park Hye Kyung , Yu Hak Sun 
Published: January 2023

Abstract
Asthma patient rapidly have increase recently, but some of these patients do not react to known allergens in skin prick tests, which suggest the presence of unknown environmental allergens. Although Acanthamoeba are abundant in the environment (it also have been often found in human airway cavities) and possess strong proteases enough to elicit allergic airway inflammation, no study has been conducted to determine whether Acanthamoeba can elicit allergic reactions. We repeatedly inoculated Acanthamoeba trophozoites or excretory-secretory (ES) proteins intranasally into mice, and evaluated symptoms and airway immune responses. We found that Acanthamoeba trophozoites or ES proteins reproducibly elicited immune responses resembling allergic airway inflammation. After boiling, which removed protease activity, treatment no longer elevate Th2 related chemokine gene expression in mouse lung epithelial cells, allergic airway inflammation symptoms and Th2 immune responses were remarkably reduced by de-activating proteases. These responses might be not fully elicited through protease activated receptor 2. Furthermore, asthma patients were found to have higher Acanthamoeba specific IgE titers than normal controls. This study suggests that it might be possible to cause allergic airway inflammation by Acanthamoeba (or their products).
Characterization of a weakly expressed KIR2DL1 allele supports a positive role for the KIR distal promoter in proximal KIR promoter activation. (P1357)
Authors: Anderson Stephen , Li Hongchuan , Huehn Andrew , Cooley Sarah , Miller Jeffrey 
Published: January 2023

Abstract
The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR. Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of the three known promoter regions revealed a cluster of 3 single nucleotide polymorphisms (SNPs) in the distal promoter region approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a binding site for the inhibitory ZEB1 transcription factor that overlaps a c-Myc binding site previously implicated in the activation of KIR genes by distal transcription. We therefore propose that the generation of this novel ZEB1-binding site antagonizes the Myc-associated activation of the KIR2DL1 gene by the distal promoter.
Multiplex IP-FCM reveals network-scale protein-protein interaction differences between pro-immunogenic and pro-tolerogenic TCR signaling (P1130)
Authors: Smith Stephen , Neier Steven , Davis Tessa , McCluskey James , Burrows Scott , Sicotte Hughes , Gil-Pages Diana , Pittelkow Mark , Neuhauser Claudia , Schrum Adam 
Published: January 2023

Abstract
T cell receptor signaling involves interactions of various kinases, phosphatases, ubiquitin ligases and adaptor proteins. However, the network-scale protein-protein interactions (PPIs) that mediate signaling, as well as the key differences between pro-immunogenic (agonist) and pro-tolerogenic (antagonist) signals, remain unclear. We have developed a novel method to examine large networks of signaling PPIs, multiplex immunoprecipitation measured by flow cytometry (mIP-FCM). Using multiple classes of Luminex® microspheres, we created a 96-well-formatted matrix array capable of measuring 484 PPIs in a single overnight assay. The array is focused on the most proximal signaling proteins associated with the TCR/CD3 complex. As a model system, we have used Jurkat cells that express the public LC13 TCR, stimulated by antigen presenting cells loaded with either LC13 agonist (FLRGRAYGL) or antagonist (FLRGRFYGL) peptide. Time-course studies have revealed both quantitative and kinetic differences between the two types of signal. We present our latest results mapping the PPI network that mediates TCR signaling. Our results have significantly impacted our understanding of the difference between pro-immunogenic and pro-tolerogenic TCR signals.
Single cell mass cytometry of human peripheral blood reveals an endogenous immune response to surgical trauma (P1144)
Authors: Fragiadakis Gabriela , Gaudilliere Brice , Angst Martin , Nolan Garry 
Published: January 2023

Abstract
An integral component of understanding immune function is measuring how the system responds to perturbation. We hypothesized that studying the immune response to the physiological stress of surgical trauma in vivo would uncover endogenous immune signaling networks critical for the function of the human immune system. Mass cytometry enables the measurement of 45 parameters on single immune cells with antibodies to both surface antigens and intracellular signaling proteins. Upon analyzing peripheral blood from patients at several time points pre- and post-surgery, we discovered that surgical trauma produces distinct population size changes, phenotypic changes, and intracellular signaling responses. Single cell analysis of immune signaling networks identified serial waves of six signaling proteins (STAT1, STAT3, STAT5, P38, S6 and CREB) that coordinated the functional response of the innate and adaptive immune systems. In addition, a subset of myeloid cells expands from 1% to 8% of hematopoietic cells in the 24 hours following surgery in all patients. These cells signal through STAT3, STAT1, CREB, and p38, and are phenotypically similar to previously described myeloid-derived suppressor cells. This systems-level single cell analysis generates a personalized surgical immune response profile of unprecedented resolution. In future work we plan to determine the relationship between these profiles and differences in clinical outcomes.
Transcriptomic and cistromic comparison of the effects of glucocorticoid receptor activation in B-1 and B-2 cells. (P1118)
Authors: Diehl Cody , Spann Nathan , Kaikkonen Minna , Glass Christopher , Witztum Joseph 
Published: January 2023

Abstract
Glucocorticoids are widely used clinically to potently modulate the activity of many immune cells. They bind to the glucocorticoid receptor, which functions as a ligand inducible transcription factor capable of either transactivation or trans-repression of target genes. Surprisingly, little is known regarding the molecular mechanisms by which glucocorticoids modulate B cells in general and B-1 cells in particular. We have previously reported that the glucocorticoid receptor is prominently expressed in both mouse B-1 and B-2 cells. Ex-vivo treatment of primary B-1 cells with dexamethasone inhibits proliferation and antibody secretion as well as strongly induces apoptosis. Utilizing RNA- sequencing the effect of glucocorticoid receptor activation on genome-wide mRNA transcript levels was analyzed and compared in quiescent and activated primary B-1 and B-2 cells. Furthermore, the genome-wide binding of glucocorticoid receptor was analyzed in B-1 and B-2 cells using chromatin immunoprecipitation and deep sequencing. While in general the dexamethasone dependent changes in glucocorticoid receptor binding and mRNA levels were similar between B-1 and B-2 cells, reflecting B cell specific effects, there were notable differences as well, possibly reflecting lineage specific differences between B-1 and B-2 cells. Current work is exploring the reasons for these differential responses in B-1 and B-2 cells to glucocorticoid receptor ligation, and the consequences to B cell function.
The adaptor protein HSH2 regulates the terminal differentiation of B cells resulting in qualitative and quantitative differences in the humoral response (P1120)
Authors: Justement Louis , King R. 
Published: January 2023

Abstract
Endogenous expression of the adaptor protein HSH2 is regulated in a dynamic manner during B cell maturation and differentiation. Developing B cells lack detectable HSH2, whereas T1 and T2 B cells in the periphery exhibit increasing levels of expression. Mature follicular B cells exhibit decreased expression of HSH2 compared to T2 cells and expression is further downregulated in germinal center B cells. In contrast, marginal zone B cells and B1a/b B cells exhibit high-level HSH2 expression. Regulation of HSH2 expression plays a critical role in determining the outcome of the humoral immune response as demonstrated using HSH2 transgenic (Tg) and HSH2 hypomorph (HSH2-Lo) mice. Constitutive expression of HSH2 in B cells results in decreased serum Ig titers for all subclasses with the exception of IgA. HSH2-Tg mice immunized with T-dependent or T-independent antigens exhibit a moderate decrease in the production of antigen-specific IgM, whereas class switched isotypes are significantly decreased compared to control mice. In contrast, HSH2-Lo mice exhibit accelerated and enhanced class switching in response to challenge with antigen. Analysis of HSH2 Tg versus HSH2-Lo B cell activation in vitro demonstrated that altered HSH2 expression does not affect class switching, but it does lead to altered expression of the transcriptional regulatory proteins Bach2 and Blimp-1. Thus, HSH2 appears to play an important role in regulating terminal differentiation of B cells.
Skin pathology in Wiskott-Aldrich syndrome mice is caused by hyperactive CD8+ T cells and increased cross-presentation by DCs (P1048)
Authors: Baptista Marisa , Dahlberg Carin , Liedén Agne , Kuo I-Chun , Sunahara Karen , Wallin Robert , Forsell Mattias , Snapper Scott , Eidsmo Liv , Scheynius Annika , Karlsson Mikael , Susanne Nylén , Westerberg Lisa 
Published: January 2023

Abstract
It remains puzzling how immunodeficiency with hypo-responsive cells can be associated with increased immune cell activation to seemingly harmless substances such as allergens. Here, we have investigated skin pathology in the severe primary immunodeficiency Wiskott-Aldrich syndrome (WAS). Wildtype and WAS protein-deficient (WASp KO) mice were challenged with the allergen Der p 2 to induce eczema or infected with Leishmania major to induce robust skin inflammation. Der p 2 challenge of wildtype mice induced eczema with epidermal hyperplasia and egress of Langerhans cells from the skin. WASp KO mice had increased accumulation of mature DCs in the skin and, in contrast to wildtype cells, Der p 2 induced expansion of WASp KO CD8+ T cells that produced IFNγ. Similar to challenge with Der p 2 and unlike wildtype mice, WASp KO mice infected with L. major showed a skewed immune response with increased expansion of IFNγ-producing CD8+ T cells in the draining lymph node. Finally, we show that WASp KO DCs have increased capacity to cross-present exogenous antigen and activate CD8+ T cells. Our results imply that WASp deficiency in skin inflammation causes a breach in tolerance and skews the immune system to DC mediated CD8+ T cell responses at incorrect sites.