bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of IL-10 during a streptococcus pneumoniae infection prevents excessive inflammation in lungs (MPF1P.778)
Authors: Peñaloza Hernan , Nieto Pamela , Salazar Francisco , Kalergis Alexis , Bueno Susan
Published: January 2023

Streptococcus pneumoniae (S. pneumoniae) is a Gram positive bacterium able to colonize and translocate the respiratory epithelium causing local and systemic diseases. It is a major cause of death worldwide, and despite the efforts made to develop strategies to prevent pneumococcal diseases, none of them are completely efficient. For this reason, it is necessary to fully understand the immune response against S. pneumoniae, and to develop new strategies that improve the host resistance to infection. One of the key component in the immune response is the anti-inflammatory cytokine IL-10 that modulates the inflammatory response against several pathogens, avoiding the excessive damage produced by the host immune system. In this study we evaluated the importance of IL-10 in the modulation of the innate immune response using a model of pneumococcal pneumonia in wild type (wt) and IL-10-/- mice. Our results show that wt mice overexpress IL-10 after infection, while in the absence of this cytokine, IL10-/- mice show excessive lung inflammation, higher neutrophil recruitment but normal monocytes. As a result, IL-10-/- mice have reduced bacterial load in lungs and deeper organs as compared with Wt mice. Our results support the notion that the production of anti-inflammatory cytokines, such as IL-10, during S. pneumoniae infection is required to avoid excessive inflammation of lungs and improve survival, despite the bacterial clearance is less efficient in the presence of this cytokine.
The fraction of T. cruzi-antigen specific T cells in chronic chagasic patients detected by CD154 and membrane TNFα expression (MPF2P.802)
Authors: Ripoll Juan , Giraldo Nicolás , Bolaños Natalia , Cuellar Adriana , Roa Nubia , Rosas Fernando , Velasco Victor , Cucunubá Zulma , Puerta Concepción , González John
Published: January 2023

Background: Chagas disease is a chronic parasitic infection whose pathogenesis is related to parasite persistence and a dysfunctional cellular immune response. There is no method to determine the pool of antigen-specific T cells in Trypanosoma cruzi-infected patients. Thus, the goal of this work was to determine the fraction of antigen-specific T cells by measuring the expression of CD154 and membrane TNFα on the bulk of parasite-stimulated PBMCs. Methodology: Twenty-one chagasic patients (CP), 11 healthy controls (HC) and 5 non-chagasic individuals with chronic heart disease (NCC) were analyzed. PBMCs were incubated in the presence of anti-CD28, anti-CD49d, anti-TNFα and TACE inhibitor as follows: non stimulus, T. cruzi-lysate or SEB. Later, the samples were stained for CD3, CD4, CD8 and CD154 and analyzed by flow cytometry. Results: Symptomatic (3.07%, SD ±1.98) and asymptomatic CP (1.24 ±0.59) displayed higher frequencies of CD4+/CD154+ cells after incubation with T. cruzi-lysate in comparison with NCC (0.084 ±0.03) and HC (0.11 ±0.08) donors P<0.0001. Likewise, the frequency of CD8+ cells expressing membrane TNFα with parasite stimulus was higher in symptomatic (3.76% ±1.43) and asymptomatic patients (2.22 ±0.53) compared with NCC (1.28 ±0.45) and HC (0.57 ±0.41) donors P<0.0001. Conclusions: These results suggest that membrane TNFα and CD154 after stimulation with T. cruzi-lysate could be useful to determine the fraction of parasite-specific T cells.
Role of IL-17 in intestinal ischemia-reperfusion injury. (MUC2P.829)
Authors: Geha Mayya , Bosse Robin , Tsokos Maria , Tsokos George
Published: January 2023

Background: Ischemia-reperfusion injury (IRI) to the small intestine following clamping of the superior mesenteric artery (SMA) results in an intense local inflammatory response characterized by neutrophil infiltration and villous damage. IL-17A is a cytokine expressed by a variety of cells in response to inflammatory cytokines that are released following tissue injury and/or inflammation. IL-17A induces epithelial cells to secrete neutrophil chemoattractants, such as CXCL2. The cytokine IL-23, which can be produced by epithelial cells, plays an important role in IL-17A production. Objective: To test the hypothesis that IL-17A is critical for intestinal damage following IRI. Materials and Methods: IRI was induced in mice by SMA clamping. IL-17A deficient (Il17a-/-) and IL-23R deficient (Il23r-/-) mice. Intestinal injury was evaluated by Chiu scores of jejunal histological sections. Results: IRI was significantly less in both Il17a-/- mice and Il23r/- mice than in WT controls. We also found that pretreatment of WT mice with neutralizing antibody to IL-17A or IL-23 resulted in significant decrease in IRI compared to mice treated with isotype control antibody. Conclusions: These results indicate that IL-17A plays a critical role in IRI. They also suggest that IL-23 contributes to IRI, possibly by promoting IL-17A production.
Targeting sirtuin-1 alleviates experimental autoimmune colitis by induction of Foxp3+ T-regulatory cells (MUC2P.818)
Authors: Akimova Tatiana , Xiao Haiyan , Jiao Jing , Bhatti Tricia , Liu Yujie , Wang Liqing , Hancock Wayne , Beier Ulf
Published: January 2023

Induced Foxp3+ T-regulatory cells (iTreg) are essential to gastrointestinal immune homeostasis and play an important role in preventing autoimmune colitis. We have previously shown that inhibiting sirtuin-1 (Sirt1) can augment Treg function and hypothesized that targeting of Sirt1 might enhance iTreg development and thereby represent a potential therapy for inflammatory bowel disease (IBD). We adoptively transferred CD4+CD25-Foxp3- T effector (TE) cells from wild-type (C57BL/6) or fl-Sirt1/CD4cre mice into B6/Rag1-/- mice and monitored the mice until they lost 10-15% of their weight. Adoptive transfer of TE cells lacking Sirt1 to B6/Rag1-/- mice resulted in increased iTreg formation compared to mice receiving wild-type TE cells (2.3 ±1.6%, vs. 6.2 ±3%, in wild type and fl-Sirt1/CD4cre TE recipients, respectively, p<0.001). This correlated with attenuated colitis and reduced weight loss in fl-Sirt1/CD4cre vs. wild type TE recipients (1.04 ±1.4% vs. 13.97 ±2.2%, respectively, p<0.001). In a second model of IBD, we used pharmacologic Sirt1 targeting of mice receiving multiple cycles of dextran sodium sulfate (DSS) in their drinking water, alternated with fresh water. Likewise, wild-type mice receiving cyclic DSS and a Sirt1 inhibitor, EX-527, had reduced weight loss (5.8 ±5.9% vs. 13.2 ±6.9%, p=0.03) and increased iTreg formation compared to controls. Sirt1 appears a promising target for pharmacologic therapy of IBD as a result of promoting iTreg development.
Gender differences in Crohn's disease are mediated by differential effects of estrogen on T cell function (MUC2P.825)
Authors: Goodman Wendy , Garg Rekha , Davis Calvin , Katz Jeffrey , Cominelli Fabio , Pizarro Theresa
Published: January 2023

Inflammatory bowel disease (IBD) is one of several autoinflammatory diseases for which a female gender bias has been reported. We hypothesized that hormonal signaling may alter T cell function in IBD, since chronic activation of pathogenic T cells drive intestinal inflammation and symptoms of female patients often change during times of altered hormone signaling, such as puberty and pregnancy. We studied the function of regulatory T cells in experimental murine models of ileitis (SAMP1/YitFc) and colitis (CD45RB transfer model), as well as in human IBD patients. In the CD45RB colitis model, CD45RBlow cells (enriched for Treg cells) from WT female mice were less effective at dampening colitis compared to their male counterparts (p<0.001, n=6/group). Consistent with this finding, female SAMP (SAMP-F) mice developed accelerated, worsened ileitis compared to SAMP-M beginning at 6 weeks of age (p<0.01, n=6/group), and displayed reductions in Foxp3 expression and Treg function in vitro. SAMP-F Tregs were non-responsive to estrogen-mediated increases in Foxp3 expression, suggesting that hormone signaling may influence Treg function in ileitis. Lastly, female IBD patients displayed a reduction in Foxp3 expression by FACS compared to males (p≤0.05, n=3/group); this was in contrast to Foxp3 expression in healthy female controls, which exceeded that of healthy males (p<0.01, n=5/group). Together, these data suggest that hormone signaling may directly influence Treg function in IBD.
pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires (TECH1P.863)
Authors: Vander Heiden Jason , Yaari Gur , Uduman Mohamed , Stern Joel , O'Connor Kevin , Halfer David , Vigneault Francois , Kleinstein Steven
Published: January 2023

Summary: Driven by dramatic technological improvements, large-scale characterization of lymphocyte receptor repertoires via high-throughput sequencing is now feasible. Although promising, the high germline and somatic diversity, especially of B-cell immunoglobulin repertoires, presents challenges for analysis requiring the development of specialized computational pipelines. We developed the REpertoire Sequencing TOolkit (pRESTO) for processing reads from high-throughput lymphocyte receptor studies. pRESTO processes raw sequences to produce error-corrected, sorted and annotated sequence sets, along with a wealth of metrics at each step. The toolkit supports multiplexed primer pools, single- or paired-end reads, and emerging technologies that employ single-molecule barcodes. pRESTO has been tested on data generated from Roche and Illumina platforms. It has a built in capacity to parallelize the work between available processors, and is able to efficiently process millions of sequences generated by typical high-throughput projects. As part of ongoing collaborations, pRESTO has been applied to multiple B-cell repertoire studies, employing both Roche 454 and Illumina MiSeq sequencing technologies. Availability: pRESTO is freely available for academic use. The software package and detailed tutorials may be downloaded from http://clip.med.yale.edu/pRESTO.
Live cell gene expression detection in monocytes and macrophages through flow cytometry (TECH1P.837)
Authors: Weldon Don , Williams Yuko , Liu Haizhen , Ko Alex , Koong Victor
Published: January 2023

Detecting gene expression in immune cells has traditionally been limited to technologies that utilize a reporter construct for a gene of interest or within lysed or fixed cell populations as is the case for RT-PCR. Here we show the ability to detect gene expression in live monocytes and macrophages which allows for more physiologically relevant information based on the cell’s response to given stimuli. Determining which genes were up or down regulated in those cells provides insight into complex gene regulatory networks and immune cell function. Here we present a novel RNA expression detection technology capable of detecting specific mRNA and miRNA in live, intact cells. This technology allows for detection of target RNA, with the ability to perform downstream assays such as Fluorescence-Activated Cell Sorting (FACS) to enrich for a specific subpopulation of live cells based solely on their RNA expression profile. Following FACS sorting based on RNA detection the cells remain viable and fully functional for use in downstream assays allowing researchers the ability to further characterize or determine functionality of the enriched population. Within a population of cells there is inherent homogeneity which can skew results when only looking at the population level as opposed to understanding the single cell resolution of gene expression. Looking at RNA levels in a more dynamic manner proves to be much more informative than looking at duplicate samples over time.
Role of inflammation in preventing the induction of transplantation tolerance (TRAN1P.876)
Authors: Jangalwe Sonal , Cuthbert Amy , Greiner Dale , Brehm Michael
Published: January 2023

Costimulation blockade targeting the CD40-CD154 pathway is an effective approach to induce tolerance to allografts in murine and non-human primate models. However inflammation generated during infection and exposure to pathogen associated molecular patterns abrogates costimulation blockade induced tolerance. Here we have characterized the alloreactive CD8 T cell response during costimulation blockade in the absence or presence of inflammation. Costimulation blockade consisting of donor splenocytes (DST)/anti-CD154 antibody (MR1) stimulated proliferation of alloreactive CD8 T cells but resulted in deletion of the cells by apoptosis. Exposure to LPS during blockade prevented this deletion, but surprisingly reduced proliferation kinetics of the alloreactive CD8 T cells. These data suggest that LPS increases the frequency of alloreactive CD8 T cells by providing survival signals during cell division. RNA-seq analyses revealed differential expression of genes involved in apoptosis (like Fasl, Perp, Birc5, E2F transcription factors), cell cycle (Ki-67, Bub1, Nusap1), CD8 T cell differentiation and function (granzyme B, T-bet, Eomes) and type I interferon induced genes (Ifi44, Ifit1, Usp18) between the treatment groups. Such phenotypic differences will facilitate identification of pathways that can be targeted to prevent the abrogation of transplantation tolerance in situations of inflammation.
Notch modulates human NK cell differentiation through regulation of inhibitory killer immunoglobulin-like receptor (KIR) expression and acquisition of effector function (TUM2P.890)
Authors: Felices Martin , Ankarlo Dave , Lenvik Todd , Miller Jeffrey
Published: January 2023

The Notch signaling pathway plays a substantial role on human NK cell development, however the role of Notch on KIR upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood derived NK cells or sorted KIR- peripheral blood NK cells were cultured with IL-15 for 7 days in the presence or absence of gamma-secretase inhibitor, known to inhibit Notch signaling. Inhibition of Notch signaling significantly decreased KIR expression. Conversely, co-culturing the same cells with OP9 cells bearing Notch ligands enhanced KIR expression. Overexpression of the active portion of Notch on cord blood derived NK cells after 28 days of culture resulted in a 2-fold increase in KIR expression indicating that Notch signaling plays a direct, cell intrinsic, role in KIR regulation. By knocking down delta-like 1 (DLL1) on NK cells and co-culturing them with OP9 cells expressing Notch ligands we show that DLL1 mediated cis-inhibition controls KIR expression. Notch signaling also enhances CD16 upregulation that precedes KIR expression. More important than receptor expression is function. We found that Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even on post-transplant samples where NK cell function is inherently defective. Given these attributes of Notch signaling, we propose Notch agonists can enhance NK cell maturation and tumor killing in a post-transplant setting.
Inhibition of tumor growth by targeting the MDA5-IPS-1 pathway requires coordinated induction of cancer cell death and immune activation (TUM2P.891)
Authors: Yu Xiaofei , Fisher Paul , Wang Xiang-Yang
Published: January 2023

Melanoma differentiation-associated antigen 5 (MDA5), a cytosolic innate pattern recognition receptor for sensing double-stranded RNA, functions as a first line of defense against viral infection. Here we report that ectopic expression of MDA5, mediated by a replication incompetent adenovirus (Ad.Mda5), results in prostate cancer cell death and concomitant innate immune activation, e.g., production of type I interferons (IFNs). IFN-β promoter stimulator (IPS)-1, a downstream adaptor protein, is critical for MDA5-stimulated cancer cell apoptosis and type I IFN response. While MDA5-regulated IFN regulatory factor 3-IFN-β signaling cascade remains intact in nontransformed normal cells, these cells are less sensitive to MDA5-induced apoptosis compared with cancer cells. Intriguingly, intratumoral delivery of Ad.Mda5 preferentially remodels the tumor environment toward Th1 polarization, indicated by marked elevation of cytokine IFN-β, IL-12, and IFN-gamma. As a result, in situ overexpression of MDA5 leads to strong suppression of established mouse prostate tumors, which is associated with activation of natural killer (NK) cells and cytotoxic CD8+ T lymphocytes. Genetic ablation or antibody neutralization reveals that antitumor effect of MDA5 therapy depends on IPS1, IFN-β, and IL-12. Therefore, the intrinsic capabilities of ‘danger’ sensing MDA-5 in simultaneously promoting cancer cell death and immune augmentation may be exploited to develop novel cancer therapeutics.
Multifunctional CD26hi Th17 cells eradicate large human tumors (TUM2P.902)
Authors: Nelson Michelle , Bailey Stefanie , Huff Logan , Kundimi Sreenath , Paulos Chrystal
Published: January 2023

Human CD4+ T cells differentiate into multiple effector subsets, but their distinct roles in anti-tumor immunity remain elusive. CD4+ T cell subsets enriched from bulk human CD4+ T cells [Th1 (CXCR3+), Th2 (CCR4+), and Th17 (CCR6+ or CD26hi)] were stimulated with anti-CD3 beads bearing agonists to either CD28 or ICOS and were then engineered with a chimeric antigen receptor that recognizes human mesothelioma. In vitro, ICOS costimulation proved superior to CD28 for augmenting the function of human Th1, Th2, CCR6+ Th17 and CD26hi Th17 cells, as indicated by elevated production of IFN-γ, IL-4 and IL-17A, respectively. Moreover, CD26hi Th17 cells possessed strikingly enhanced polyfunctionality compared to Th1, Th2 or CCR6+ Th17 cells, as demonstrated by their heightened capacity to co-secrete IL-17A, IFN-γ, IL-22, IL-2, and TNF-α simultaneously. Compared to other enriched T cell subsets, a greater percentage of CD26hi Th17 cells exhibit an effector memory phenotype. In vivo, CD26hi Th17 cells more efficiently reconstituted immunodeficient hosts and persisted long-term. Furthermore, CD26hi Th17 cells possessed a superior ability to kill large human tumors (>150mm2) when infused into mice compared to Th1, Th2 or CCR6+ Th17 cells. These results suggest that the generation of multifunctional, long-lived human Th17 populations could be instrumental to the design of novel, effective T cell-based cancer therapies.
Isocitrate dehydrogenase mutant glial cell resist natural killer cell cytotoxicity (TUM2P.906)
Authors: Zhang Xiaoran , Chang Yigang , Brancho Michael , Kohanbash Gary , Leo Madeline , Okada Hideho , Lotze Michael , Amankulor Nduka
Published: January 2023

Introduction: Mutations in IDH 1 and 2 are common in gliomas. Methylation-related epigenetic repression of gene expression is a feature of IDH mutant (IDHmut) tumors. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut tumors, and correlate this with decreased susceptibility to natural killer (NK) cell-mediated cytolysis in vitro Methods: Microarray: We compared IDHmut and IDHwt gene expression and NKG2DL mean methylation values in glioma patients using TCGA data. RT-PCR: NKG2DL expression was assayed using primers for NKG2D ligands. Cytotoxicity: Astrocytes were cultured with activated NK cells. Cytotoxicity was measured by flow cytometry. Results: mRNA expression values for NKG2DLs ULBP1 and ULBP3 were significantly reduced in IDHmut gliomas. Conversely, methylation values were significantly increased. NKG2DL hierarchical clustering of gene expression of shows genotype-based expression clusters. IDHmt astrocytes expressed a two to eight-fold reduction in the relative gene expression of NKG2DLs compared with IDHwt astrocytes. Cytotoxicity is significantly lower in IDHmut astrocytes/NK cell co-cultures compared with IDHmut/NK or U87/NK co-cultures. Conclusions: Our findings demonstrate evidence for the transcriptional repression of NKG2DLs in IDHmut gliomas in silico and in vitro. Reduced NKG2D levels in IDHmut cells correlates with impaired NK-mediated cytotoxicity and suggests a role for NK cell resistance in IDH mutant tumors.
Clonally expanded T cells from skin biopsies of patients with systemic sclerosis recognize DNA topoisomerase I and cytomegalovirus antigens (HUM3P.258)
Authors: Zacharakis Nikolaos , Oleszak Emilia , Gaughan John , Nanajian Anthony , Myers Allen , Tsygankov Alexander , Platsoucas Chris
Published: January 2023

Systemic sclerosis (SSc) is a chronic autoimmune disease, characterized by immune system dysregulation, uncontrolled deposition of collagen and microvascular damage in the skin and several internal organs. T cells have a key role in the initiation and propagation of SSc. Our laboratory has previously identified the presence of high proportions of identical α- and β-chains TCR transcripts in skin biopsies from patients with SSc of recent onset, demonstrating clonal expansion of T cells in response to one or more antigens. In order to identify these antigens, pairs of those in vivo clonally expanded α- and β-chain TCR transcripts from two patients with SSc (SSc-21 and SSc-22) were expressed in mutant TCR negative T cells, J.RT3-T3.5, generating a total of 52 T cell lines, including 10 controls. An intracellular Ca2+ mobilization assay was employed to examine whether these transduced Jurkat T cell lines recognize putative SSc antigens presented by autologous EBV-transformed B cell lines. The self-antigen, DNA topoisomerase I and CMV and parvovirus antigens were tested. Statistically significant intracellular Ca2+ mobilization was observed in response to 3 DNA topoisomerase I and 2 CMV peptides by 5 T cell lines expressing clonally expanded α- and β-chain TCR transcripts from patient SSc-21 and SSc-22. These results demonstrate that clonally expanded T cells recognizing DNA topoisomerase I or CMV antigens are present in skin biopsies of recent onset of patients with SSc.
Broad macrophage infiltration leads to a proinflammatory state in streptozotocin-induced diabetic mice (HUM3P.261)
Authors: Niu Shuo , Bian Zhen , Tremblay Alexandra
Published: January 2023

Our studies demonstrate significantly enhanced neutrophil (PMN) recruitment during zymosan-induced peritonitis in mice with strepotozocin (STZ)-induced diabetes. Cross-leukocyte adoptive transfer further reveals that, while the PMN response is potentiated under diabetes, the existence of a proinflammatory state in the peritoneum of diabetic mice, which is reflected by more than two-fold increases in peritoneal macrophages and significant elevations of IL-6, IL-1β, TNF-α, and the PMN-driven chemokine CXCL1 (KC/IL-8) upon zymosan challenge, plays a dominant role in exaggerating the peritonitis. Insulin treatment not only alleviates hyperglycemia, but also reduces macrophage infiltration in the peritoneum and eliminates the enhanced PMN recruitment during the peritonitis, confirming that such proinflammatory state is associated with hyperglycemia but not STZ. Strikingly, dissections of various tissues from diabetic mice reveal increased macrophages infiltration in additional essential organs including the liver and kidneys. Exposing diabetic mice to a sub-effective dose of adriamycin, a potential renal damage drug, leads to acute nephritis and proteinuria toward which insulin treatment precludes. To address how diabetes induces broad macrophage infiltration, we analyzed peripheral leukocytes and observed a significant increase in the circulating proinflammatory F4/80+Gr-1+ monocytes, which infiltrate tissues and trigger exaggerated responses upon inflammatory challenges.
Cardiometabolic abnormalities associate with an inflammatory cytokine profile in breast adipose tissue and plasma of obese African American women (HUM3P.264)
Authors: Denis Gerald , Strissel Katherine
Published: January 2023

‘Crown-like structures’ (CLS) of CD68+ macrophages in human subcutaneous, visceral and breast adipose depots associate with cardiometabolic and cancer risks. Rather than surgical fat biopsy, which is difficult for vulnerable humans, informative blood-based assays are preferred for medical decision making and risk assessment. HYPOTHESIS. Breast CLS and adipocyte parameters, as measures of metabolic health, meaningfully associate with pro-inflammatory imbalances of specific cytokines and adipokines in plasma of obese women. RESULTS. We obtained breast histological samples and matched plasma of 20 obese, unhealthy African American women in the Komen Tissue Bank and the Black Women’s Health Study, and compared to 20 age-, sex- and race-matched subjects who were obese but healthy; and to a matched cohort who were lean and healthy. We measured 41 cytokines and adipokines in plasma, and determined CLS and adipocyte size (a measure of metabolic health) in breast fat. We used clinical determinations of cardiometabolic status as independent variables and evaluated patterns of cytokine expression and tissue immunometabolism as dependent variables. We found that obese and healthy women clearly resolve from obese and unhealthy as well as from lean and healthy, and that a limited set of cytokines and adipokines are sufficient to define risk cross-sectionally. IMPACT. Immunometabolic profile of patient plasma may offer a robust tool to assist patient-provider discussion of risk.
IL-7Ralow memory CD8+ T cells are clonally anergic cells and defect in aerobic glycolysis pathway (HUM4P.274)
Authors: Sim Ji Hyun , Kim Jin-Hee , Seol Min A , Lee Sun-Kyung , Cho Bon-A , Ko Youngho , Oh Keunhee , Lee Dong-Sup , Kim Joon , Park Ae , Kang Insoo , Choi Kyungho , Kim Hang-Rae
Published: January 2023

We have found two subsets of CD8+ T cells expressing IL-7Rahigh and IL-7Ralow with different cell survival responses to IL-7 in peripheral blood of human. Treating IL-7Rαlow memory CD8+ T cells with anti-CD3/CD28 Abs did not produce IL-2, but comparably expressed IFN-γ and TNF-a. But the mechanism for this is unknown. These cells have reduced calcium flux and subsequent inhibited nuclear translocation of NF-AT1 upon TCR stimulation as well as PMA/ionomycin treatment, suggesting that IL-7Rαlow memory CD8+ T cells also have defected in distal-TCR signaling. Importantly, this anergic status was reversed by exogenous IL-2 stimulation, clearly distinct from adaptive tolerance. When TCR stimulation on IL-7Rαlow memory CD8+ T cells exhibited markedly decreased GATA3 and c-myc expression and increased NUAK1, which are key regulators in aerobic glycolysis, compared to those of IL-7Rαhigh memory CD8+ T cells. The expression of GATA3 and c-myc in IL-7Rαlow memory CD8+ T cells were restored after IL-2 stimulation. In conclusion, we firstly show that specific subset of human CD8+ T cells, IL-7Rαlow memory CD8+ T cells, are T-cell clonally anergy in vivo. We also demonstrate that GATA3-c-myc-NUAK1 system may control the maintenance of clonally anergic IL-7Rαlow memory CD8+ T cells in vivo and provides insight into human immunology. Acknowledgement: This research was supported by National Research Foundation of Korea (2014M3A7B4052194) and by the SNUH Research Fund (34-2013-0206)
Statin treatment suppresses IgE-mediated mast cell activation in vitro and in vivo (HYP4P.316)
Authors: Barnstein Brian , Kolawole Elizabeth , Oskeritzian Carole , Ryan John
Published: January 2023

Statin drugs are HMG-CoA reductase inhibitors used in the treatment of hypercholesterolemia and cardiovascular disease. They have also been shown to suppress the immune response. Given the critical role that mast cells play in allergic and inflammatory diseases, we assessed the effect of statin drugs on mast cell function. We demonstrate that Fluvastatin suppresses IgE-mediated degranulation and production of pro-inflammatory cytokines and chemokines, without altering IL-10 secretion. These effects were reversed by the addition of mevalonic acid, and mimicked by geranyl geranyl tranferase blockade, suggesting that Fluvastatin inhibits lipid modifications to small G proteins downstream of HMG CoA reductase. In vivo studies further showed that Fluvastatin suppressed IgE-mediated anaphylaxis. Interestingly, Fluvastatin-mediated suppression was completely absent in 129/SvJ mast cells, which showed a 2-fold induction of HMG CoA reductase upon statin treatment. Human mast cells from eight donors showed a range of responses to Fluvastatin, indicating that genetic background may influence statin-mediated suppression. These data demonstrate the possible use of statins, a widely-prescribed group of drugs, as a possible therapy for mast cell-associated disorders.
Transcriptional profiling of constitutive mast cells reveals a unique gene expression signature and highlights their heterogeneity across tissues (HYP4P.303)
Authors: Dwyer Daniel , Barrett Nora , Austen K.
Published: January 2023

Constitutive mast cells (MC) are long-lived sentinel cells residing in peripheral connective tissue near the microvasculature and nerve endings. Similar to basophils (Ba), MC express the high-affinity IgE receptor FcεR1α, and are frequently implicated in the pathobiology of allergic diseases. Although protease expression in murine MC varies between tissues, the full extent of their diversity across compartments is not known, and the study of MCs is hindered by their rarity and location. In collaboration with the Immunological Genome Project, we developed techniques for isolating MC from a wide variety of murine tissues for microarray analysis. Hierarchical clustering and principal component analysis of MC versus Ba and other immune cells indicate that Ba are transcriptionally more similar to other circulating granulocytes (eosinophils, and neutrophils) than to MC. Furthermore, granulocytes and other tissue resident myeloid cells (macrophages and dendritic cells) are closer to each other and to lymphoid cells than either group is to constitutive MC. Further analysis revealed a core MC transcriptional signature not seen in other immune cell populations, but MC heterogeneity across tissue compartments was similar to that seen with dendritic cells, indicating that their diversity goes far beyond protease expression. Our results provide novel insights into this unique cell type and lay the groundwork for future studies on the activation and function of MC in peripheral tissue.
Developing an antibody targeting CεmX of mIgE for the treatment of IgE-mediated diseases (HYP4P.305)
Authors: Chan Yueh-Hsuan , Lee Yu-Tzu , Chou Hung-Wen , Wu Pheidias , Chen Jiun-Bo , Li Chau-Hong , Cheng Tien-Tien , Chen Nien-Yi , Chang Tse-Wen , Lue Ko-Haung
Published: January 2023

CεmX (also referred to as M1’) is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ε heavy chain of membrane-bound IgE (mIgE) on human B lymphocytes. Antibodies that target CεmX are potentially useful in controlling IgE production for treating allergic and other IgE-mediated diseases. Herein we report that an anti-CεmX mAb, 4B12, was shown to be effective in reducing allergen-specific IgE and IL-5 production upon the challenge of the allergen in an asthma model employing CεmX gene knocked-in mice that express mIgE containing human CεmX domain on B cells. 4B12 could also alleviate airway hyper-responsiveness (AHR), allergen-induced eosinophil infiltration, and lung inflammation in those mice. Furthermore, we demonstrated that a humanized 4B12 mAb (referred to as FB825) could inhibit the production of human IgE in mice that had been reconstituted with human peripheral blood mononuclear cells. Based on the abilities of 4B12 to bind to mIgE and to lyse mIgE-expressing B cells by apoptosis, ADCC, and other cytolytic mechanisms and on the above results on animal models, a phase-I human clinical trial of FB825 to investigate its IgE-related biological effects is being performed.
Unexpected pro-inflammatory effects of IL-10 on mouse mast cells (HYP4P.306)
Authors: Abdul Qayum Amina , Ryan John
Published: January 2023

Mast cells play critical roles in allergic and inflammatory diseases as effector and immunoregulatory cells. A well understood mast cell activation pathway is antigen-mediated crosslinkage of IgE bound to its high affinity receptor (FcεRI). Cytokines produced during the immune response may alter mast cell function. We have shown that, contrary to its better-known anti-inflammatory role, IL-10 induces pro-inflammatory cytokines in peritoneal and bone marrow derived mast cells stimulated with IgE+antigen. The increase in pro-inflammatory cytokines and chemokines (IL-6, MCP-1, MIP-1a, and IL-13) is seen both at the mRNA and protein level. Consistent with previously published data, IL-10 suppresses TNF production. Using an in vivo IgE-induced passive systemic anaphylaxis model, we show that IL-10 pre-treatment causes a more severe anaphylactic response and increased serum IL-6 and MIP-1a, suggesting that IL-10-mediated enhancement can occur in vivo. These data support a more nuanced view of IL-10 as a key immunoregulatory cytokine capable of curbing and promoting different inflammatory mediators in hypersensitivity reactions.
Identification of a novel T. cruzi B cell superantigen (INC4P.340)
Authors: Brown Eric
Published: January 2023

Catalytic antibodies represent an innate defense mechanism that allows a subset of antibodies to bind and then hydrolyze their targets, making these antibodies up 1000-fold more efficient than conventional immunoglobulins. Pathogens have evolved B-cell superantigens (BC-SAgs) as an immunevasion strategy designed to neutralize catalytic antibody activity. Exposure to BC-SAgs such as the Staphylococcus aureus extracellular fibrinogen binding protein (Efb) and the HIV gp120 glycoprotein decreased catalytic activity to these antigens, respectively. We describe for the first time the identification and characterization of a Trypanosoma cruzi BC-SAg (Tc24). Immunization with Tc24 in the presence of different adjuvants significantly decreased the ability of purified IgM to hydrolyze Tc24 compared hydrolysis rates observed for IgM purified from unimmunized controls. Infection with T. cruzi resulted in a similar decrease in catalytic activity. In both immunization and infection models, the dose (of either Tc24 or the number of T. cruzi used to infect mice) affected Tc24 hydrolysis rates. Analysis of IgMs purified from infected humans also demonstrated reduced hydrolysis of Tc24 compared to hydrolysis rates observed for IgM purified from uninfected individuals. These data suggest that Tc24 is a BC-SAg that may impact the host's defenses early in the infection process.
Macrophages modulate bacterial persistence and gastric pathology during Helicobacter pylori infection (INC4P.346)
Authors: Viladomiu Monica , Bassaganya-Riera Josep , Kronsteiner-Dobramysl Barbara , Washington Philipson Casandra , Michalak Pawel , Eden Kristin , Hontecillas Raquel
Published: January 2023

Based on preliminary data suggesting that macrophages are critical regulators of Helicobacter pylori colonization and gastric pathology, we investigated how macrophage phenotypes shape the outcome of infection and persistence. The loss of PPARγ in the myeloid compartment favors a 5 to 10-fold decrease in bacterial loads at the expense of more severe gastric lesions. PPARγ-deficient macrophages present altered control of transcription factors such as NFκB, which results in a pro-inflammatory phenotype. Stomach lamina propria analyses at weeks 0 to 4 post-infection revealed increased levels of CD11b+ F4/80hi CD64+ CR3CR1+ macrophages in WT mice, while PPARγKO mice failed to expand and maintain such population. Macrophage depletion using clodronate liposomes resulted in a significant reduction of gastric H. pylori in WT mice, thus abrogating the differences in bacterial loads observed between WT and PPARγKO mice. In vitro co-culture of H. pylori with bone marrow derived macrophages showed that either PPARγ loss or pharmacological blockade enhances bactericidal activity, which is associated to significant differential expression of chil1, etv5, iigp1, ptger4, sqle, osm, hspa2 and rptoros levels as revealed by global transcriptome analyses. Hence, macrophages facilitate H. pylori infection by 1) serving as bacterial reservoirs and allowing intracellular replication, and 2) favoring a gastric regulatory response that favors persistence.
Novel scaffolding proteins and mechanisms mediating RIG-I signaling complex assembly (INM2P.351)
Authors: Krishnan Manoj , Pulloor Niyas , bist pradeep
Published: January 2023

The cytosolic innate-immune receptor RIG-I is very important for the detection and host response against several RNA-viruses. During viral challenge, RIG-I activates mitochondria-associated adaptor MAVS to recruit several downstream cytosolic signaling proteins, resulting in transcription factor IRF3 activation and Type-I interferons transcription (TI-IFN). However, the mechanisms coupling activation of organelle-anchored MAVS with key downstream cytosolic components is incompletely understood. Through a human genome-wide RNAI screen, we identified several novel regulators of RIG-I signaling. Furthermore, we identified a key novel scaffolding protein (named RIG-I assembly mediator protein, RAMP) that links MAVS with downstream TBK1 activation, IRF3 phosphorylation and TI-IFN transcription during virus infection. RAMP functioned as a scaffolding platform that interacted and assembled a complex containing MAVS, TBK1, NEMO and IRF3. Lack of RAMP expression led to the failure of MAVS to interact with downstream NEMO, TBK1, and IRF3. Recombinant RAMP supported IRF3 activation in a cell free assay. RAMP was essential for antiviral cellular resistance, and infection induced subcellular re-localization of RAMP. Furthermore, our data demonstrated that RAMP and TRAF proteins act in parallel to transduce signals from MAVS to IRF3. This study provided important novel insights into the regulation and assembly of the RIG-I signalosome and TI-IFN response.
Ubiquitination-mediated regulation of RIP2 in NOD2 signaling and MDP tolerance induction (INM2P.354)
Authors: Sukumaran Bindu , bist pradeep , Ng Aylwin , Kim Bae Hoon , Kudukkil Pulloor Niyas , Khameneh Hanif , Hedl Matija , Dikshit Neha , Shenoy Avinash , Balamuralidhar Vanniarajan , Hong Michelle , Neutzner Albert , Chin Keh-Chuang , Kobayashi Koichi , Bertoletti Antonio , Mortellaro Alessandra , Abraham Clara , MacMicking John , Xavier Ramnik
Published: January 2023

The cytoplasmic innate immune receptor NOD2 is required for antibacterial immune response. Optimal regulation of NOD2 is essential for controlling bacterial infections and inflammatory disorders. Recently, ubiquitination has emerged as an important posttranslational modification required to control NOD2-signaling. Yet, the specific ubiquitination mechanisms that negatively regulate NOD2-signalling during both acute and chronic (e.g., MDP-tolerance) stimulations are incompletely understood. Through a human genome-wide-RNA-interference screen, we identified an E3-ubiquitin ligase {termed as NOD2 Signaling Regulator (NSR)} as a novel negative-regulator of NOD2-mediated NF-KB, cytokine, and antibacterial responses. Mechanistically, NSR induced K48-linked ubiquitination of RIP2 (NOD2-adaptor), and promoted RIP2 degradation through the endoplasmic-reticulum-associated-degradation (ERAD) pathway. We identified the endoplasmic reticulum as a new regulatory site for NOD2-signaling, where RIP2 was localized and interacted with NSR and ERAD. In vivo NSR knock-down in mice resulted in increased NOD2-mediated NF-KB activation. NSR-mediated RIP2 degradation was critically required for MDP-tolerance induction both in vivo and in vitro. Thus, this study unravels a key molecular mechanism by which NOD2 pathway is attenuated, and provide new directions to understand the role of NOD2 pathway in both infectious and inflammatory diseases.
NLRP12-deficiency results in TNFα-mediated bone marrow apoptosis during emergency hematopoiesis (INM2P.355)
Authors: Linz Brandon , Neely Crystal , Kartchner Laurel , Malik Julia , Brickey Willie , Ting Jenny , Cairns Bruce , Maile Robert
Published: January 2023

The hematopoietic system can rapidly expand in response to tissue damage. NLRP12, expressed in leukocytes and bone marrow cells, has been implicated in regulation of immune responses but remains controversial if it acts as an inflammasome or a regulator of NF-κB signaling. We tested the hypothesis that NLRP12 regulates immune responses and reconstitution as a direct regulator of NF-κB signaling using an emergency hematopoiesis model. To test this, C57BL/6 wild type and Nlrp12-/- female mice underwent a radiation-thermal combined injury (RCI) consisting of 20% total body surface area burn and 5Gy γ-radiation. In wild type mice, we observed elevated NLRP12 expression in target tissues after RCI compared to controls. Following RCI, Nlrp12-/- mice failed to reconstitute peripheral innate neutrophil and monocyte populations, had increased levels of serum TNFα, and TNFα-mediated apoptosis of bone marrow progenitor cells. These data suggest that NLRP12 is essential for immune reconstitution after combined radiation and burn injuries. We have uncovered a central role for NLRP12 in regulating granulopoiesis following immune ablation and NFκB cytokine- and receptor-expression. Accordingly, we propose that NLRP12 acts as an important regulator of NF-κB signaling during events that trigger hematopoeisis, suggesting a novel role of NLRP12 in immune reconstitution following injury and immune depletion.
Streptolysin O hijacks the membrane repair system to blind natural killer cell (INM3P.405)
Authors: Bhattacharjee Pushpak , Romero Matthew , Keyel Peter
Published: January 2023

The pore-forming toxin Streptolysin O (SLO) secreted by Group A Streptococcus binds to cholesterol and oligomerizes on the membrane to form a lytic pore. Immune cells survive pore-formation through a Ca2+ dependent process that sequesters and sheds pores on vesicles. These vesicles are enriched in cholesterol and glycosylphosphatidylinositol (GPI)-anchored proteins, raising the possibility that immune receptors are shed during the repair process. NKG2D, IL-18 receptor and IL-12 receptor promote Interferon-γ (IFNγ) production in natural killer (NK) cells and all associate with cholesterol-rich microdomains or GPI-anchored proteins. Here we found SLO challenge impaired IFNγ secretion from NK cells when stimulated with K562 cells or IL-12 and IL-18 without affecting NK cell viability. We found that the IL-12 Receptor β1 was shed from NK cells on microvesicles along with SLO in a Ca2+ dependent fashion. These data suggest that shedding of activation receptors during membrane repair impairs NK cell function. Taken together, these data describe one way in which bacterial toxins can hijack membrane repair to blind cells to the presence of pathogens.
USP18 as a novel regulator of PD-1 and IFNAR1-mediated immune dysfunction (IRM11P.629)
Authors: Wood Laurence , Mavinkurve Vikram , Muthukumaran Geetha , cabasal marian , dauphinee shauna , Malo Danielle , Paterson Yvonne
Published: January 2023

Immune dysfunction induced by cancer and chronic viral infection contributes to the pathology and progression of each of these diseases. Recent studies have identified a novel player associated with poorly functioning immune cells during chronic viral infection, the ubiquitin-specific protease Usp18. Usp18 is a negative regulator of innate anti-viral responses through direct inhibition of Type I IFN receptor signaling. However, the role of Usp18 in maintaining functional immunity is still unclear. To investigate the role of Usp18 in immunity, we have utilized Usp18-deficient mice and assessed immune function during infection with the Th1 pathogen Listeria monocytogenes (Lm). We find that Usp18 deficiency results in severe susceptibility to acute Lm infection and an inability to develop functional adaptive immunity to Lm. Severe susceptibility to Lm infection in the Usp18-/- mouse is associated with poor inflammatory cytokine responses and blunted splenic T cell function. The immune tolerant state in Usp18-/- mice is partially regulated by the PD-1 inhibitory receptor as blockade partially restores innate responses to Lm. However, blockade of IFNAR1 signaling resulted in significant reversal of this immunosuppressive state and a restoration of T cell function. This study identifies Usp18 and IFNAR1 signaling as critical mediators of immune dysfunction, a finding that may have broad implications in the treatment of cancer and chronic viral infection.
Molecular screening of Mycobacterium vaccae for identification of novel anti-inflammatory compounds (IRM12P.651)
Authors: Smith David , Cantley Alexandra , Clardy Jon , Lowry Christpher
Published: January 2023

Mycobacterium vaccae is a saprophytic, non-pathogenic bacterium. Administration of M. vaccae in activates regulatory T cells (Treg) and attenuates allergic responses. M. vaccae-induced activation of Treg may be initiated through interactions with sentinel immune cells, such as macrophages. We are interested in identifying the particular immunoregulatory molecules produced by M. vaccae that suppress inflammatory signaling in macrophages. Consequently, we developed a reiterative cell-based screen as an unbiased approach to search the M. vaccae chemical landscape for potential anti-inflammatory small molecules. M. vaccae was grown in 7H9 broth as a mixture of both smooth and rough variants.We prepared hexane, ethyl acetate, and aqueous extracts of live M. vaccae cultures; cell-based assays were used to guide successive steps of purification. For cell-based assays, 100 µg/ml of the extracts were incubated with freshly isolated murine peritoneal macrophages, and, after one hour, the cultures were stimulated with 1 µg/ml of lipopolysaccharide (LPS). Interleukin 6 (IL-6) concentrations in the supernatant 12h after stimulation were measured via ELISA as a determinant of inflammatory response. After 5 iterations of screening and evaluation of 80 fractions, we identified 3 fractions from the ethyl acetate extract with potent anti-inflammatory properties. From mass spectrometry, we expect the molecules to be lipid conjugates, and we are in preparation for a structural analysis.
SLAM family receptors are key drivers of CD8 T cell immunity towards B cells (IRM12P.657)
Authors: Priatel John , Huang Yu-Hsuan , Tsai Kevin , Harder Kenneth , Tan Rusung
Published: January 2023

Epstein-Barr virus (EBV) poses serious health consequences for immunocompromised individuals, such as patients undergoing transplant or chemotherapeutic regimens, and is strongly associated with an expanding list of human cancers. To gain insight into immunity against EBV, a rare congenital immunodeficiency defined by exquisite sensitivity to EBV but not other pathogens called X-linked lymphoproliferative disease, a syndrome caused by mutations in the SH2D1A gene that encodes SLAM-associated protein (SAP), has been the subject of intense investigation. The unique B cell tropism of EBV led us to speculate that SAP and SLAM family receptors are especially critical for optimal CD8 T cell immunity against antigen-expressing B cells and B lymphoma cells. Consistent with this hypothesis, 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 or B lymphoma cells in vitro. By contrast, both wild type and Sh2d1a-/- CD8 T cells responded robustly when stimulated with either antigen-coated B cell-depleted splenocytes or antigen-presenting tumor cells lacking SLAM family receptor expression. Collectively, these observations suggest that the recognition of SLAM family receptors on the surface of antigen presenting-B cells and -B lymphoma cells are essential for antigen priming and immunosurveillance by CD8 T cells.