bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of GM-CSF in dendritic cell development in vivo (HEM3P.284)
Authors: Bhattacharya Palash , Haddad Christine , Alharshawi Khaled , Prabhakar Bellur
Published: January 2023

We have earlier demonstrated that CD11b+CD11c+ dendritic cells (DCs) derived ex vivo from bone marrow precursors with GM-CSF (GM-BMDCs) can selectively expand natural Foxp3+ regulatory T-cells (Tregs) in a contact dependent manner through OX40L/Jagged-1 co-signaling. However, an ‘in vivo’ counterpart of these GM-BMDCs is not known. In contrast BMDCs derived ex vivo using FLT3L (FL-BMDCs) are considered to be phenotypical and functional equivalents of physiological steady-state DCs. In addition, mice lacking GM-CSF or its receptor do not show a severe defect in DC development while FLt3L deficiency severely affects the physiological development of all types of DCs. To determine the role of GM-CSF in DC-development in vivo, we treated Flt3L deficient mice with GM-CSF and found no evidence of CD11c+ DC development. In contrast, we saw near complete reconstitution of DCs in these mice with Flt3L treatment. Surprisingly, we could successfully generate CD11c+ GM-BMDCs from bone marrow precursors isolated from these Flt3L deficient mice. Interestingly, these GM-BMDCs also express surface markers associated with precursor cells, macrophages and granulocytes. To address these various contradictions, we propose that G-BMDCs generated ex vivo represent a myeloid precursor population rather than fully differentiated DCs. These ‘precursors’ require other cytokines for their terminal differentiation; thus, Flt3L is critical for their differentiation into steady-state DCs.
An automated data analysis strategy to identify and classify hematologic neoplasms (HEM3P.287)
Authors: Tang Mengxiang , Zhu Jason , Zhang Wenchang , Crespo Oliver
Published: January 2023

Cell immunophenotyping using flow cytometry has historically been a powerful tool in screening and diagnosing hematologic neoplasms. The capability of flow cytometers to measure multiple markers simultaneously allows detecting and quantifying normal and aberrant immune cell populations in blood samples. However, analysis of multidimensional flow cytometry data remains cumbersome and limited to serial analysis of bivariate plots. The slow and visually-dependent method relies on an expert’s expertise to identify and gate immune cell populations based on the expression and intensity of targeted protein marker staining. This conventional gating method is subjective, time consuming, and hardly takes advantage of all the dimensionalities of the data. An automated data analysis strategy to identify abnormal populations and classify samples in a gating-free manner has been developed. This automated analysis contains algorithms to extract features from high dimensional data, merge features and create profile for each sample, and classify samples based on established profiles. This generalized analysis strategy could be applied to analyze multidimensional data. An application on hematologic neoplasm classification is presented here. More than 100 samples, including health and patient samples, stained with a same eight color antibody cocktail were acquired using a BD FACSCanto II, and was used to show the accuracy of the automated analysis method is higher than 90%.
ERK signals that promote γδ T cell development require ERK interaction with DEF domain-containing targets (HEM3P.276)
Authors: Wiest David , Lee Sang-Yun , Coffey Francis , Fahl Shawn , Peri Suraj , Hedrick Steven , Fehling Hans , Zuniga-Pflucker Juan-Carlos , Kappes Dietmar
Published: January 2023

Differential induction of ERK signals has been implicated in numerous fate decisions; however, the molecular basis by which gradations in ERK signaling specify alternate fates remains poorly understood. We report here that divergence of the αβ and γδ T cell fates is dependent upon differences in the extent of T cell receptor (TCR) induced activation of ERK signaling. Adoption of the γδ fate is linked to greater amplitude and duration of ERK activation, but ERK activation does not promote γδ development by phosphorylation of substrates like Rsk. Instead, ERK promotes adoption of the γδ fate by physically interacting with DEF domain containing targets through its DEF binding pocket (DBP). The DEF domain-containing targets include immediate early genes (IEG) such as the transcription factor early growth response gene 1 (Egr1). Egr1 protein is normally unstable, but is stabilized by DEF-DBP mediated interaction with ERK. Thus, ERK signals promote γδ development by stabilizing IEG proteins, including transcription factors, thereby enabling them to transactivate targets not possible in the absence of this increase in stability.
Neutrophil extracellular traps as a source of high density lipoprotein oxidation in lupus-related cardiovascular disease. (HUM1P.312)
Authors: Smith Carolyne , Vivekanandan-Giri Anuradha , Pennathur Subramaniam , Kaplan Mariana
Published: January 2023

Lupus patients are fifty times more likely to experience a cardiovascular event, such as stroke or myocardial infarction, compared to age- and sex-matched controls. This is not explained by traditional “Framingham” risk factors, such as obesity and smoking, but possibly by the presence of “proinflammatory”, oxidized high density lipoprotein (HDL). Enzymatic sources of HDL oxidation include myeloperoxidase (MPO) and nitric oxide synthase (NOS), two enzymes which our group has found highly present in neutrophil extracellular traps (NETs). NET structures, composed of chromatin and antimicrobial peptides, are produced by granulocytes under pathogen stimulation or spontaneously in lupus patients. It was therefore predicted that NETs, highly active in lupus patients, can transform HDL into an oxidized form which is linked to proinflammatory and proatherosclerotic pathways. We examined the levels of chloro- and nitro-tyrosine oxidation in HDL from lupus patients and control donors via tandem mass spectroscopy. Finally, we digested lupus and control granulocyte NETs and incubated this with unoxidized HDL in the absence or presence of MPO and NOS-specific inhibitors. Lupus patients exhibited higher levels of regiospecific HDL oxidation versus control. Active NOS and MPO from NETs induced HDL oxidation patterns linked with cardiovascular disease (CVD). This represents a completely unexplored cellular source of HDL oxidation for lupus associated CVD.
Kidney resident B cells and plasmacytoid dendritic cells in the urine of systemic lupus erythematosus patients. (HUM1P.313)
Authors: Scott Eric
Published: January 2023

Renal involvement remains the principal cause of morbidity and mortality in SLE. B cells play a prominent role in kidney pathology in mouse models of SLE. In addition to producing autoantibodies, B cells produce cytokines and can present antigen to autoreactive T cells. Recently, we identified a B cell population with high expression of CD19 and CD20 and low expression of CD21 that is expanded in some human SLE patients. The presence of an expanded CD19hi B cell population correlates with poor long-term outcomes of end stage renal disease. High expression of CXCR3 by CD19hi B cells, suggests that they are trafficking to sites of inflammation, such as the kidney. To test this possibility we examined concurrent patient urine and plasma samples for the presence of B cells by flow cytometry. An Interferon alpha assay was used to determine whether levels were elevated in blood and urine.We identified B cells in ~1/3 of patient samples that are likely of kidney origin. Patients fell into two non-overlapping groups based on B cell phenotype.In the first group, urine B cells exhibited a B cell phenotype, among which were CD19hi B cells, present at the frequency equal to the peripheral blood.In the second group, the B cells exhibited a plasmablast phenotype and these cells spontaneously secreted IgG. Moreover, IFNα levels in plasma correlate significantly with the frequency of CD19hi B cells.
Bisphenol A (BPA) exacerbates acute myocarditis in female BALB/c by activating mast cells and the inflammasome (HUM1P.302)
Authors: Stafford Katelyn , Greyner Henry , Molina Frank , Bucek Adriana , Fairweather DeLisa
Published: January 2023

Myocarditis is an inflammatory heart disease that is the leading cause of heart failure in young adults. Sex hormones play a vital role in development of myocarditis with testosterone driving disease in males. Whereas, estrogen, via Estrogen Receptor α (ERα) mediates cardioprotection in females. Since myocarditis is influenced by sex hormones, it is highly probable that endocrine disruptors (EDs); which interfere with natural hormones, will play a part in the progression of the disease. The human population is exposed to Bisphenol A (BPA), a known ED that binds the ER, from plastics, such as water bottles and plastic food containers. BPA has been found to act through ERβ in cardiomyoctes to increase cardiac arrhythmias. Thus, BPA could increase myocarditis through deleterious actions of the ERβ rather than beneficial effects via ERα. To our knowledge no one has examined the role of EDs like BPA on myocarditis. We found that clinically relevant doses (25 µg/L and 250 µg/L) of BPA increased acute myocarditis compared to control water. We found BPA significantly decreased ERα, while ERβ was significantly increased. We found that mast cells (cKit) are largely responsible for the increase in inflammation along with CD4 Thelper cells. IL-1β, IFN-γ, TLR4, Caspase-1, Mmp9, and ST2 were all increased with BPA treatment which all lead to increased inflammation and disease. We believe that BPA is influencing the inflammasome and mast cells leading to exacerbation of disease in females.
Studying the association of plasma S100A12 and noninfectious pulmonary complication in infants and young children following cardiopulmonary bypass (HUM1P.305)
Authors: Shu Qiang , Liu Xiwang , Chen QiXing , Chen Chi , Shi Shanshan , Shi Zhuo , Yu Jiangen , Lin Ru , Tan Linhua
Published: January 2023

Objective To examine the kinetics of plasma S100A12 and sRAGE in infants and young children with CPB, and to investigate whether they could predict the occurrence of noninfectious pulmonary complication (NPC) after cardiac surgery. Methods 20 patients were in NPC group, which included pleural effusion, chylothorax, partial lung collapse, pulmonary hypertensive crisis, airway disorders, pneumothorax, pneumomediastinum, and phrenic nerve palsy, and 40 patients in no-NPC group. Plasma S100A12 and sRAGE were measured at 6 perioperative time points. Results S100A12 and sRAGE increased immediately after CPB (P<0.001). SRAGE dropped to lower than baseline level (P<0.05), while S100A12 was still at high level 24h after operation (P<0.001). S100A12 and sRAGE immediately after CPB in NPC group were significantly higher than no-NPC group (P<0.05). S100A12 were still higher in NPC group than no-NPC at 24h after operation (P<0.01). S100A12 immediately after CPB was a predictor for postoperative NPC (OR=1.042, 95% CI: 1.010-1.076, P=0.011). S100A12 immediately after CPB were positively associated with MV time (r=0.47, P<0.001), duration of surgical Intensive Care Unit (r=0.407, P=0.002) and hospital stay (r=0.421, P=0.001). Conclusions Plasma S100A12 and sRAGE were significantly increased immediately after CPB; The elevated S100A12 immediately after CPB served as an early reliable biomarker of the occurrence and the prognosis of NPC after CPB in infants and young children.
Expansion of the IgD class switched B cell population in carriers of the autoimmune disease associated risk haplotype in BLK (HUM2P.332)
Authors: Simpfendorfer Kim , Manjarrez-Orduño Nataly , Armstead Brandon , Simeone Alyssa , Diamond Betty , Gregersen Peter
Published: January 2023

Polymorphisms in BLK are associated with numerous autoimmune diseases including rheumatoid arthritis and lupus. BLK (B lymphocyte kinase) is a tyrosine kinase involved in signaling through the B cell receptor (BCR). The disease risk haplotype is known to be associated with reduced expression of BLK transcript and the most significant association of reduced BLK transcript is with a candidate causal SNP rs922483. We have previously shown that expression of BLK is reduced in cord blood B cells from carriers of rs922483 risk alleles (HMG 2012). B cell development is perturbed in blk transgenic and knockout mice, and B cells from these mice are hyper-responsive to BCR stimulation. To determine if reduced BLK expression affects the function of primary human B cells we measured CD86 expression in cells stimulated by anti-IgM. As in mice, cord blood B cells from risk allele carriers are hyper-responsive to BCR stimulation. We also assessed if BLK risk variants convey any variation on the composition of circulating B cell populations of healthy adults. We observed that subjects with BLK risk variants have an expansion in the proportion and number of IgD class-switched B cells, and an increase in the total number of class-switched memory B cells. These results suggest that BLK risk haplotypes confer autoimmune disease susceptibility by reducing a negative regulatory effect of BLK, making B cells more likely to receive help from CD4 T cells and to undergo class switch recombination.
Mast cell-derived IL-33 orchestrates antigen-driven tissue inflammation by promoting a unique inflammatory basophil response (HYP3P.352)
Authors: Hsu Chia-Lin , Berdnikovs Sergejs , Bryce Paul
Published: January 2023

While IL-33 is commonly described as an epithelial derived cytokine, we previously showed that IgE activation of mast cells induces IL-33 expression. In vivo, IgE-primed mast cells serve as peripheral sensors for antigen (Ag) and drive an early edema followed by an antigen-driven tissue inflammation (ADTI). Histamine drives the edematous response but the mechanisms controlling local ADTI are unclear. Here, we sought to examine the roles of IL-33 and its receptor, ST2 in this process. Using a passive cutaneous anaphylaxis model, we show that mast cell-derived IL-33 is both sufficient and necessary for the ADTI response, which is dominated by CD45+Gr-1high neutrophils. ST2-expressing basophils are essential for ADTI and we show that IL-33 serves to activate them, rather than cause migration. Using microarray analysis to examine responses to either IgE/Ag or IL-33 activation, we show that IL-33 promotes an atypical basophil response characterized by neutrophilic chemokines. Interestingly, we find that histamine is necessary for both phases but, while H1R or H2R are required for edema, ADTI is dependent on basophil expression of H4R. In summary, our findings define a mechanism through which mast cells regulate local tissue responses to Ag. We propose a model whereby mast cell histamine release initially attracts basophils via H4R. Basophils are then activated by mast cell-derived IL-33, via ST2, to produce an atypical profile of cytokines and chemokines that recruit neutrophils.
Constitutive β-catenin signaling dysregulates DC differentiation resulting in inability to control microbial infection (INM2P.418)
Authors: Cohen Sara , Schneider Anne , Denkers Eric
Published: January 2023

The protozoan Toxoplasma gondii is the second most common cause of foodborne illness-related deaths in the US and can be fatal to infants and immunocompromised patients. Dendritic cells (DC) are required for immunity to T. gondii, and recently, DC Wnt/β-catenin signaling has been suggested to control DC tolerance in the intestine. However, the role of this signaling axis in peripheral DC development and function is unknown. Using transgenic mice expressing DC-specific constitutively active β-catenin (DCEx3-/-), we surprisingly found that the splenic CD8α DC and the developmentally related peripheral CD103 DC compartment were significantly expanded relative to corresponding populations in wild-type (WT) animals. Splenic CD8α DC are a source of IL-12, and infection of DCEx3-/- mice with T. gondii resulted in rapid lethality associated with proinflammatory cytokine overproduction. Transcription factor Irf8 drives CD8α and CD103 DC differentiation, and flow cytometric analysis revealed strong upregulation of Irf8 in CD8α DC upon β-catenin stabilization. Confirming this, WT CD8α+Irf8+ DC displayed higher β-catenin levels relative to splenic CD8- DC. Together, our data show that β-catenin targets Irf8 during DC development, resulting in expansion of CD8α DC in the spleen and CD103 DC in the periphery. This is the first study to implicate β-catenin in DC subset differentiation and provides novel insight into the complex transcriptional network of DC development.
Differential effects of TLR-2 ligands on CD4+ CD25+ Foxp3+ regulatory cells depending on inflammatory cytokine milieu (IRC4P.489)
Authors: Pandiyan Pushpa , Zheng Lixin , Bhaskaran Natarajan , Weinberg Aaron
Published: January 2023

The responsiveness of CD4+CD25+Foxp3+ thymic Tregs (tTregs) towards microbial agents that serve as toll-like receptor (TLR) ligands is unclear. This study investigates the effects of TLR ligands on Tregs. We show that TLR-2 ligands promote IL-2 dependent survival and proliferation of tTregs both in the presence or absence of TCR signals and inflammatory cytokines. The proliferation is directly dependent on TLR-2 expression on Tregs showing that TLR-2 ligands may be involved in Treg proliferation in vivo. Consistent with this, Tlr-2-/- mice harbor fewer Tregs in colon and mesenteric lymph nodes in vivo. However, in the presence of IL-6 and TCR signals, Tregs also acquire effector functions by producing IL-17A. However, they retain their suppressive properties and do not lose Foxp3 expression. Their effector function is dependent on TLR signaling by antigen presenting cells, IL-6 and IL-23 in the milieu, but not on direct TLR-2 signaling by Tregs. IL-17A production in Tregs is also relevant in vivo, as CD4+Foxp3+Tregs are capable of producing IL-17A without losing their suppressive properties during oropharyngeal candidiasis and inflammatory bowel disease. These data show that CD4+Foxp3+ tTregs are a stable lineage of cells, and that TLR-2 ligands promote their proliferation without inducing plasticity. Moreover the data show for the first time, the direct and indirect effects of TLR-2 ligands that differentially regulate tTreg proliferation and effector functions.
Isolation of highly purified mouse CD4+CD25+FoxP3+ T regulatory cells in less than 45 minutes (IRC4P.476)
Authors: Kokaji Andy , Tonelli Luciana , McQueen Karina , Thomas Terry , Eaves Allen
Published: January 2023

Regulatory T cells (Tregs), a subset of lymphocytes, play a key role in maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. They can be broadly classified into natural or adaptive (induced) Tregs. Natural Tregs (CD4+CD25+FoxP3+), develop in the thymus and emigrate to the periphery to help maintain immune homeostasis. Adaptive Tregs can be induced from CD25-negative naïve CD4+ T cells in the thymus and acquire CD25 (IL-2R alpha) and FoxP3 expression in the periphery after adequate antigenic stimulation. They are typically induced by chronic allergic inflammation and disease processes, such as autoimmunity. The isolation of highly purified Tregs is essential for advancing research in this field. To date this has been achieved by lengthy protocols or flow-based cell sorting. We have developed a rapid column-free one-step immunomagnetic cell separation method (EasySep™) to isolate Tregs (natural and induced) from mouse splenocytes in less than 45 minutes. CD25-PE-labelled cells are bound to magnetic particles using antibody complexes and separated using an EasySep™ magnet. The procedure can be automated using RoboSep™. Starting from 2.2±0.4% Tregs, purities of 84±3% (mean±SD, n=20) CD4+CD25+FoxP3+ cells can be achieved. This kit provides a new tool to study the mechanisms by which Tregs exert their influence, which has broad implications for the development of cell-based therapies for autoimmune disease and cancer.
Mechanisms of effector T cell suppression by CD4+Foxp3+ Treg in inflamed peripheral tissue (IRC4P.482)
Authors: Camirand Geoffrey , Rothstein David
Published: January 2023

In secondary lymphoid organs (SLO), CD4+Foxp3+ regulatory T cells (Treg) control immunity by preventing prolonged T cell-DC interactions. Whether Treg suppressor function in inflamed non-lymphoid tissues differs from SLO remains unclear. Thus, we used 2-photon intravital microscopy to dissect how Treg prevent allograft rejection by effector T cells (Teff) in transplanted islets. To restrain the immune reaction to the transplant site, we adoptively transferred Teff, Treg, or both (2-3x106 each) to B6 mice lacking SLO (splenectomized LTβR-/- mice) 3-4 days after transplantation of BALB/c islets. Teff alone induced acute allograft rejection. However, the co-transfer of Teff + Treg led to long-term allograft survival in ~50% of recipients. Analysis of adoptively transferred CFSE-labeled Teff recovered from the graft on day 3 and 5 revealed that Treg did not affect either Teff migration or proliferation at that site (compared to Teff alone), despite the presence of Treg in significant numbers. Intravital microscopy of transplanted islets on day 2 showed that Treg were found in the peri-islet region, where they made brief (<1 min) contacts with DCs. Interestingly, Treg restrained the motility parameters of Teff in the peri-islet region, and significantly reduced Teff intra-islet infiltration, compared to Teff alone. These results indicate that Treg can function directly in inflamed non-lymphoid tissues, but that their mechanisms of action may differ from those used in SLO.
SLAM-associated protein modulates CD8 T cell responses primed by antigen-presenting B cells (IRM4P.503)
Authors: Priatel John , Huang Yu-Hsuan , Tsai Kevin , Harder Kenneth , Tan Rusung
Published: January 2023

Mutations in the SH2D1A gene, that encodes SLAM-associated protein (SAP), cause X-linked lymphoproliferative disease (XLP), a rare congenital immunodeficiency defined by exquisite sensitivity to Epstein-Barr virus (EBV). The inability of boys with XLP to control EBV infection results in fulminant, often fatal, infectious mononucleosis, massive expansions of EBV-infected B cells and malignant B cell lymphomas. However, the precise mechanism of how SAP loss contributes to susceptibility to EBV remains unclear. Given the unique B cell tropism of EBV and the purported role of SAP in regulating lymphocyte/lymphocyte interactions, we hypothesized that SAP expression within CD8 T cells is critical for T cells to mount responses towards antigen-expressing B cells. Wild type and Sh2d1a-/- CD8 T cells responded strongly when stimulated in vitro with cognate antigen-coated whole splenocytes. However, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells in vitro or challenged with antigen-expressing B cell lymphoma in vivo. These results demonstrate that SAP is required for the priming of CD8 T cells when B cells act as the primary APC. Together, our findings suggest that the susceptibility of XLP patients to EBV infection may at least in part be a consequence of an inability of their naïve CD8 T cells to differentiate into effectors upon encountering virally-infected B cells.
Pattern associated modelling for discovery of novel protein motifs in the macrophage scavenger receptors (IRM5P.703)
Authors: Han SeongJun , Bharadwaj Prashant , Novakowski Kyle , Lee Annie , Wong Andrew , Bowdish Dawn
Published: January 2023

Class A scavenger receptors, including scavenger receptor A and macrophage receptor with collagenous structure, are surface proteins that bind modified endogenous ligands and bacterial components. As these receptors play a key role in innate immunity, they are associated with a range of infectious diseases such as pneumonia. Thus, understanding the physical properties of these receptors will provide insight for the development of novel therapeutics. Macrophage receptor with collagenous structure is amongst the least well-characterized members of this family and is expressed in macrophages to mediate pathogen recognition. Our investigation addresses the development of an efficient and highly accurate bioinformatics technique through combinatorial usage of Aligned Pattern Clustering, and Multiple Sequence Alignment Pattern Retrieval Program for novel motif discovery in the macrophage scavenger receptor. By utilizing scavenger receptor A and its validated motifs as a model, we successfully validated the feasibility of this bioinformatics technique with a minimum sensitivity of 54% and positive predictive value of 60%. As a subsequent validation of the newly discovered motifs, the top sub-sequences of macrophage receptor with collagenous structure, selected by both programs, were analyzed for their biological functionality. Here, we propose that RGRAE, VFCRMLG, EDAGVE and WGTICDD motifs in the scavenger receptor cysteine-rich domain play an important role in pathogen recognition.
Cdk5 controls IL-2 gene expression via repression of the mSin3a-HDAC complex (IRM6P.719)
Authors: Lam Eric , Pareek Tej , Letterio John
Published: January 2023

Cyclin-dependent kinase 5 (Cdk5) is a unique member of a family of serine/threonine cyclin-dependent protein kinases. We previously demonstrated disruption of Cdk5 gene expression in mice impairs T-cell function and ameliorates T-cell-mediated neuroinflammation. Here, we show Cdk5 modulates gene expression during T-cell activation by impairing the repression of gene transcription by histone deacetylase 1 (HDAC1). This effect is mediated through a direct phosphorylation of the mSin3a protein by Cdk5, which prevents the accumulation of the mSin3a protein and the formation of a functional mSin3a/HDAC1 complex during T cell activation. Disruption of Cdk5 activity in T-cells enhances HDAC activity and binding of the HDAC1/mSin3a complex to the IL-2 promoter, ultimately leading to suppression of IL-2 expression. These data point to essential roles for Cdk5 in regulating gene expression in T-cells and transcriptional regulation by the co-repressor mSin3a.
Lymphocyte activation gene 3 (LAG-3) regulates activation-induced CD4+ T cell metabolic transition (IRM6P.725)
Authors: Previte Dana , Delmastro-Greenwood Meghan , Piganelli Jon
Published: January 2023

LAG-3 negatively regulates CD4+ T cell proliferation and effector function by competing with CD4 for MHC class II binding. Others have demonstrated that loss of LAG-3 results in CD4+ T cell hyperresponsiveness and accelerated autoimmunity, further supporting LAG-3’s regulatory capabilities. In order for optimal CD4+ T cell activation, naïve T cells must transition their metabolic program from oxidative phosphorylation (OXPHOS) to aerobic glycolysis (AG), a faster process that drives macromolecule production. We hypothesized that LAG-3 regulates the metabolic transition of CD4+ T cells from OXPHOS to AG, and in turn, loss of LAG-3 expression results in a faster transition to AG, promoting T cell hyperproliferation. At baseline, B6.LAG-3-/- splenocytes exhibited enhanced spare respiratory capacity as compared to WT controls, which correlated with increased mitochondrial mass and expression of the mitochondrial biogenesis transcription factor TFAM (2-fold), and mitochondrial complexes I-IV. Upon suboptimal Con A stimulation, LAG-3-/- splenocytes demonstrated greater upregulation of glycolysis-associated genes by 24 hrs (1.5 - 3 fold). These results correlated with increased Myc expression and mTOR signaling, both of which are crucial for driving AG. Taken together, these data indicate that LAG-3 regulates CD4+ T cell activation by controlling metabolic programing since the loss of LAG-3 results in a more metabolically primed phenotype.
CK2α protein levels and CK2 kinase activity are induced upon CD4+ T cell activation and are essential for Th17 cell differentiation (LYM3P.736)
Authors: Gibson Sara , Qin Hongwei , Liu Yudong , Rowse Amber , Benveniste Etty (Tika)
Published: January 2023

CK2 is a highly conserved serine/threonine kinase that is ubiquitously expressed in all cell types and essential for cell viability. The kinase activity of CK2 promotes a number of pro-survival and pro-inflammatory signaling pathways including PI3K/Akt, NF-κB, and JAK/STAT. These pathways are also essential for CD4+ T cell activation and polarization into effector phenotypes. Here we demonstrate that protein levels of the catalytic subunit of CK2, CK2α, increase dramatically upon in vitro activation of murine CD4+ T cells isolated from the spleen. Furthermore, in in vitro polarizing conditions, CK2α protein levels correlate with expression of Th1 and Th17 lineage specific transcription factors T-bet and RORγt, respectively. CK2 kinase activity also increases upon activation, as measured by phosphorylation of the CK2 specific site S129 on Akt. Using the small molecule inhibitor CX-4945, we demonstrate that CK2 kinase activity is essential for Th17 cell polarization. Our data suggest that CK2 could be targeted in Th17 cell driven autoimmune disorders such as multiple sclerosis and colitis.
T cell intrinsic SerpinB1 and a cathepsin protease serve as yin and yang to control Th17 differentiation (LYM3P.732)
Authors: Hou Lifei , Cooley Jessica , Remold-O’Donnell Eileen
Published: January 2023

SerpinB1, an endogenous inhibitor of neutrophil serine proteases, was recognized to restrict infection-associated inflammation. Recently, we found that loss of SerpinB1 leads to expansion of IL-17A+ γδ and Th17 cells even at steady-state. To date, little is known about how proteases and their inhibitors influence the differentiation of T cell subsets. Here, using differentiation of sorted naïve CD4+ T cells, we show that, under Th17 conditions (IL-6 and TGF-β), deficiency of SerpinB1 significantly increased the IL-17+ population (~ 50% increase over WT) and reciprocally diminished the FoxP3+ population. In contrast, there were no genotype-differences in Th1 differentiation. Although neutrophil elastase is considered the primary target of SerpinB1, elastase-/- mice have IL-17+ γδ and Th17 cell numbers comparable to WT mice. Systematic screening of pure proteases using peptidase assays revealed that SerpinB1 also inhibits a subset of cysteine cathepsin proteases. Treating naïve CD4 cells with the broad cysteine protease inhibitors E64d and FA-fmk decreased differentiation to IL17+ cells and reciprocally increased FoxP3+ cells. Use of specific cathepsin inhibitors including CLIK195 and LHVS further suggests a role for cathepsin-L or a cathepsin-L like protease. Thus, these findings show that, in addition to the known transcriptional regulation paradigm, the balance between Th17 cells and Tregs is tightly regulated by SerpinB1 acting through a cysteine cathepsin.
Signaling through Tsc2-mTOR regulates memory CD8 T cell differentiation. (LYM4P.755)
Authors: Sowell Ryan , Rogozinska Magdalena , Marzo Amanda
Published: January 2023

Inhibition of the mTOR pathway with the pharmacological inhibitor rapamycin, can enhance the formation of virus-specific memory CD8 T cells. While mTOR signals through two separate protein complexes, mTORC1 and mTORC2 the role of each in memory CD8 T cell differentiation remains unclear. The Tsc1/2 protein complex inhibits activation of mTORC1, and genetic deletion or inactivation of Tsc1/Tsc2 leads to hyperactivation of downstream mTORC1 targets. mTOR signaling is increased after T cell activation and presumably down modulated during transition to memory. In order to characterize the role of Tsc1/2-mTOR signaling in the formation of memory CD8 T cells, mice that express a dominant negative form of Tsc2 were first crossed with the OT-I transgenic mice (Tsc2-DN OT-I). Subsequently, wild-type or Tsc2-DN OT-I cells were adoptively transferred into naïve congenic hosts that were then infected with Vesicular Stomatitis Virus expressing ovalbumin. We report that disruption of Tsc2 resulted in an impaired generation and/or maintenance of memory CD8 T cells. However, the number of effector Tsc2-DN OT-Is were increased compared to WT OT-Is. These data suggest that Tsc2 signaling controls the survival of effector CD8 T cells during contraction.
Functional heterogeneity of the CD8 T cell response: implications for fate and memory formation (LYM4P.746)
Authors: Kahan Shannon , Bakshi Rakesh , Luther Rita , Harrington Laurie , Weaver Casey , Zajac Allan
Published: January 2023

Although anti-viral CD8 T cells display differences in their portfolios of effector functions, dissecting this ensemble has been impeded by the lack of effective methods for segregating, tracking, and defining these distinct subsets based upon their cytokine production profiles. To address this we utilized novel IL-2 and IFN-γ double cytokine reporter mice. We demonstrated that within 6 hours of activation naïve CD8 T cells undergo a burst of cytokine production and populations are detectable that produce only IL-2, only IFN-γ, co-produce IL-2 and IFN-γ, or do not produce either cytokine. These patterns of cytokine production are reconfigured by changes in the inflammatory milieu. As effector and memory CD8 T cells develop a shift towards IFN-γ production occurs, with a smaller subset capable of co-producing IL-2 and IFN-γ, and the IL-2 only population typically not detectable. By harnessing the cytokine reporter system to separate viable IL-2+ and IL-2- effector subsets, and conducting a series of adoptive transfer studies, we discovered that IL-2 producing effector CD8 T cells transition into memory populations that rapidly and vigorously proliferative upon secondary challenge. Taken together these studies illustrate that dissecting the functional complexity of CD8 T cells has the potential to pin-point the properties that enable these cells to thrive and form the memory compartment, and reveal possible biomarkers of successful responses.
Environmental cues during infection determine effector CD8 T cell differentiation (LYM4P.748)
Authors: Plumlee Courtney , Lefrançois Leo , Khanna Kamal
Published: January 2023

Naive antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells and short-lived effector cells. In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an immune response is not well understood. Here we show that EECs were pre-programmed to differentiate based on early signals received from distinct infectious environments, but surprisingly, when these same EECs were transferred into infected hosts, the EECs could be diverted from their original fate by a disparate inflammatory environment. After observing plasticity at the population level, we also examined the antiviral and antibacterial responses of single CD8 T cells from the polyclonal repertoire. The progeny of naive clonal CD8 T cells displayed unique profiles of differentiation based on extrinsic pathogen-induced environmental cues, with some clones demonstrating extreme bias toward a single developmental pathway. These results, taken together, demonstrate that stochastic and instructive events differentially contribute to shaping the CD8 T cell response and provide insight into the underlying forces that drive effector differentiation and protective memory formation.
Resistance of rhesus monkeys to homotypic challenge after Chlamydia trachoma tis genital tract infection (MPF2P.816)
Authors: Qu Yanyan , O'Connell Catherine , F. Tarantal Alice , Andrews Charles , O'Connor Shelby , Vallejo Abbe , Darville Toni
Published: January 2023

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection for which a vaccine is needed. Here, 2 groups of 5 female rhesus monkeys were infected with either plasmid-cured attenuated CTD153 or wildtype virulent C. trachomatis D/UW-3/Cx, and then challenged with D/UW-3/Cx. Cervical infection was monitored, blood and cervical secretions were collected at intervals for T cell and antibody assays and genital tissues were harvested at wk 12 for histopathology. Results showed 3/5 monkeys infected with D/UW-3/Cx or CTD153 were resistant to challenge. However, 2/5 in each group were susceptible to challenge and 2 monkeys primarily infected with CTD153 exhibited prolonged challenge infections. No chronic genital tract pathology was observed. Serum anti-elementary body (EB) IgG was detected in all; mucosal anti-EB IgG/IgA were detected only in the 2 with prolonged infection. Bioassays showed enhanced proliferation in CD4 and CD8 central (TCM) and effector (TEM) memory compartments, and IFNγ production in response to EB at wk 4, that declined to baseline by wk 12. In vivo expansion of CD4 TCM, CD8 TEM, and NKG2A cells at wk 4 coincided with infection resolution in 8/10. T cell proliferative responses to EB were highest in monkeys with prolonged infection. Three reinfection-resistant monkeys shared Mamu-DRB1*03 and DRB1*10. Resistance to chlamydial reinfection is likely linked to MHC II and driven by chlamydial antigens minimally expressed by inactivated EBs.
Biomarkers of pulmonary tuberculosis identified in multiplexed proteomic assay (SOMAscan) of human serum (MPF2P.808)
Authors: Ochsner Urs , Green Louis , De Groote Mary , Sterling David , Ostroff Rachel , Janjic Nebojsa
Published: January 2023

Background: A rapid, accurate, and inexpensive tuberculosis (TB) diagnostic test in the developing world would allow earlier treatment and reduce transmission. Methods: We used slow off-rate modified aptamers (SOMAmers) in a highly multiplexed proteomic assay (SOMAscan) to measure 1129 human proteins and 16 M. tuberculosis proteins in serum samples collected in S. Africa, Vietnam and Peru, provided by the Foundation for Innovative New Diagnostics. Results: Among the top host serum biomarkers distinguishing TB from non-TB were factors involved in cell-matrix interactions such tissue remodeling and fibrosis (Kallistatin, TSP4, gelsolin, CDON) which were lower in TB compared to non-TB, and factors of innate immunity, acute phase reactants, and inflammation (LBP, ITI heavy chain H4, NPS-PLA2, IP-10), which were higher in TB, regardless of the HIV status. A 9-marker model performed well in a training set of 173 TB vs. 160 non-TB samples (sensitivity 90% / specificity 85%, AUC=0.94), which was confirmed in a blinded verification set of 132 TB vs. 118 non-TB samples (sensitivity 80% / specificity 84%, AUC=0.88). Conclusions: The discovery of robust, quantitative, non-culture based diagnostic biomarkers of active pulmonary TB has great potential to facilitate the rapid and accurate diagnosis of TB disease.
Epigallocatechin-3-gallate Reduces Mast Cells Activity TNF-α and NFKB in Colitis by Interrupting an Inflammatory Cascade (MUC2P.827)
Authors: Hajj Hussein Inaya , Zgheib Zeina , Zeenny Marie Noel , Chams Sana , Bou Assi Tarek , Chams Nour , Jurjus Abdo
Published: January 2023

There is increasing evidence that (-)-epigallocatechin-3-gallate (EGCG) inhibits carcinogenesis and inflammation among other properties. However, its mechanism is not fully elucidated.To investigate the mechanism of action of EGCG in improving experimental colitis. 35 male Sprague-Dawley rats were randomly divided into 4 groups: Normal control group (n=5), EGCG group (n=9), TNBS group (n=9), and TNBS+EGCG group (n=12). For both, TNBS treated group and EGCG group, 1 mg/Kg EGCG was administered daily by intraperitoneal injection, starting one week before the induction. At days 3, 10 and 17, rats were sacrificed and the descending colon was collected. The score of histological alterations of the colonic mucosa was evaluated. The mast cells were assessed by toluidine blue staining. The mRNA expression of Tumor Necrosis Factor-α (TNF-α), and nuclear factor (NF-KB) was measured and ROS was tested by immunofluorescence. EGCG decreased significantly the erosions and the inflammatory cellular infiltration of the mucosa and submucosa. In addition, the mast cells were markedly reduced in number and degranulation. The increased levels NF-KB and TNF-α after TNBS administration were significantly and concurrently reduced in colonic scrapings after EGCG treatment at all the time points and did ROS. EGCG probably prevented the transcription of inflammatory genes by stabilizing mast cells and reducing its secretory pro-inflammatory factors such as TNF-α.
Role of non-hematopoietic β-arrestin1 in intestinal inflammation (MUC2P.828)
Authors: Lee Taehyung , Lee Eunhee , Obey Kimberly , Michelin Erika , Chung Misoo , Vlachos Elena , Parameswaran Narayanan
Published: January 2023

β-arrestin1 (β-arr1), an intracellular scaffolding protein has been shown to regulate inflammation by modulating Toll-like receptor (TLR) signaling in immune cells. We recently demonstrated that β-arr1 deficiency attenuates experimental colitis in mice. However the cell-type-specific role of β-arr1 in colitis is not known. To test this we generated bone marrow chimeras with β-arr1 deficiency only in non-hematopoietic compartment (non-hβ-arr1KO), and subjected the WT and non-hβ-arr1KO mice to DSS-induced colitis. Interestingly, our results demonstrate that β-arr1 expression in the non-hematopoietic compartment protects mice from early weight loss during colitis. To understand the signaling mechanisms, we knocked down β-arr1 in human intestinal epithelial cells (SW480) and tested the effect of TNF-α on MAPK and NFκB signaling pathways. Even though β-arr1 knockdown did not affect TNF-α-induced NFκB activation, p38 and JNK pathways were enhanced in the knockdown cells. Intriguingly, ERK1/2 activation by TNF-α was significantly inhibited in β-arr1-knockdown cells. Differential modulation of the MAPK signaling by β-arr1 could lead to higher apoptosis of intestinal epithelial cells in non-hβ-arr1 deficient mice. Together, our results demonstrate that β-arr1-dependent signaling in non-hematopoietic cells likely plays a critical role in the pathogenesis of colitis.
Loss of regulatory intestinal dendritic cell subsets during gut inflammation in humans and mice (MUC2P.830)
Authors: Mann Elizabeth , Shi Jie , Wang Dezhi , Zhai Huihong , Jin Shi , Jiang Weiwei , Li Xuhang
Published: January 2023

Background: Dendritic cells (DC) and macrophages (Mφ) mediate intestinal immune tolerance. Recent evidence suggests intestinal CD103+CD11b- DC are involved in generation of regulatory T cells (T-reg), with CD103+CD11b+ DC stimulating Th17 cell differentiation. Information regarding gut DC function during inflammation is scarce. Methods: We compared murine and human gut DC and Mφ in the steady state and during intestinal inflammation, by flow cytometry. Results: Proportions of CD103-CD64+ Mφ were enhanced in the inflamed IL-10 knockout (KO) murine colon compared to WT, alongside a loss of CD103+CD11b- DC. Proportions of CD4+FoxP3+ T-reg were reduced in the inflamed IL-10 KO colon, with enhanced production of IL-17A and TNFα by T cells but a loss of regulatory TGFβ production by CD103+ DC. Proportions of CD103-CD64+ Mφ were also enhanced in the human Crohn’s disease colon compared to non-inflamed control tissue (diverticulitis), alongside a loss of CD103+ DC. CD103 was co-expressed with either CD141 or CD1c on human intestinal DC, corresponding to CD103+CD11b- and CD103+CD11b+ murine DC respectively. Proportions of all CD103+ gut DC subsets were reduced in human Crohn’s disease. Conclusions: Lack of IL-10 may skew DC and Mφ differentiation in the gut, leading to loss of DC able to generate T-reg. Further experiments will determine the ability of intestinal DC and Mφ subsets to stimulate specific T cell responses in vitro and the roles of IL-10 and TGFβ during this process.
The microbiota conditions CX3CR1+ intestinal mononuclear phagocytes to limit intestinal inflammation. (MUC2P.822)
Authors: Diehl Gretchen , Galan Carolina , Longman Randy , Egawa Takeshi , Yang Yi , Littman Dan
Published: January 2023

Regulation of intestinal immune responses requires distinguishing normal beneficial bacteria (the microbiota) from pathogens capable of inducing disease. While it is clear that the intestine and intestinal immune system recognize and respond to the microbiota, it is unclear how this recognition influences subsequent immune responses. One intestinal antigen presenting cell population, characterized by expression of the chemokine receptor CX3CR1, has been described as tissue resident macrophages. In contrast to previous reports, we recently demonstrated that CX3CR1+ cells migrate from the intestine to the mesenteric lymph node, suggesting that they can behave as dendritic cells (DCs). By utilizing both genetically modified mice and antibiotics to manipulate the intestinal microbiota, we demonstrated that the microbiota influences intestinal immunity by modulating CX3CR1+ cells. In the presence of an intact microbiota, CX3CR1+ cells limit the induction of inflammatory immune responses. In contrast, in conditions where the microbiota has been perturbed, CX3CR1+ cells promote the induction of inflammatory immune responses. We further demonstrate that antigen presentation by CX3CR1+ cells is critical for priming T cell responses to intestinal antigens in vivo, confirming CX3CR1+ cells can act as DCs. Together, our findings indicate that CX3CR1+ cells play a central role in integrating signals from the microbiota to maintain intestinal immune homeostasis.
Antimicrobial peptides are induced in the lower bowel after acute Helicobacter hepaticas infection (MUC2P.824)
Authors: Wang Chuanwu , Fox James , Horwitz Bruce
Published: January 2023

Antimicrobial peptides (AMPs) play an essential role in innate immunity to enteric bacterial infection. Previous studies have demonstrated higher antimicrobial peptides RegIIIβ and RegIIIγ expression in the colon of AOM treated 129 RAG-deficient mice 12 weeks post Helicobacter hepaticus (Hh) infection. Blocking of IL-22 interferes with AMP induction. However, whether Hh infection alone acutely induces AMP expression, and whether the ability of Hh to induce AMP expression varies in the different regions of the large bowel has not been evaluated. To examine these issues, several anatomical regions of the lower bowel from 129 Rag2-/- mice were examined at 2 and 6 weeks post Hh infection. Surprisingly, we found that while AMP expression at baseline was markedly lower in the cecum than the colon, AMP expression in the cecum was strongly induced within 2 weeks of Hh infection. This was associated with higher expression of IL-22, and cecal tissue infiltration by neutrophils and inflammatory monocytes. In contrast, Hh infection markedly inhibited IL-25 mRNA expression in both the cecum and colon. Interestingly, AMP expression is not regulated by production of IL-10 within the innate immune system. We hypothesize that Hh mediated induction of AMP may lead to alterations in the cecal microbiome. Further, we propose that this rapid model of Hh-induced AMP expression will be useful for delineating key cellular and molecular pathways that regulate intestinal innate inflammatory pathways.