bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8+ T cells from elite controllers recognize and kill HIV abortively infected CD4+ T cells (VIR6P.1168)
Authors: Monel Blandine , Boucau Julie , McKeon Annmarie , LeGall Sylvie , Walker Bruce
Published: January 2023

Understanding the underlying immunological mechanisms of spontaneous HIV control by HIV+ elite controllers (EC) remains an important step in the development of efficient HIV vaccines and functional cures. To understand control mechanisms triggered by CD8+ T cell we established a system in non-activated peripheral blood mononuclear cells (PBMC) using infectious HIV (NL4.3 IRES GFP) carrying the fusion protein Vpr-Beta lactamase (to assess viral entry) and a staining strategy to study the CD8+ T cell response by flow cytometry. First we showed that non-activated CD4+ T cells are permissive for HIV entry but this leads mostly to abortive infection (b-lac+ GFP-). CD8+ T cells from EC were able to recognize these HIV+ cells, became activated (IFNg+, MIP1b+) and underwent degranulation (CD107a+). This activation was initiated by the presentation of HIV peptides from incoming viruses and HLA-I blockade almost abolished this response. We confirmed by mass spectrometry HIV peptides processed in the cytoplasm of CD4+ cells shortly after HIV entry. These results suggest that the ability of CD8+ T cells to form immunological synapses allows them to be activated by low levels of HIV antigens from incoming particles at the surface of early-infected CD4+ T cells. Killing of abortively HIV-infected cells early after HIV entry might avoid inflammation due to pyroptosis and therefore contribute to HIV control.
Antigen processing constraints on CD4+ T cell epitope dominance: an approach to improved epitope prediction (APP5P.113)
Authors: Landry Samuel , Charles Tysheena , Mettu Ramgopal
Published: January 2023

We present a novel method for CD4+ epitope prediction based on modeling antigen processing constraints. Given an antigen with known three-dimensional structure, our algorithm combines four types of data to construct a profile of conformational stability that allows us to identify regions that are most accessible to proteolysis. Using this profile, we construct a profile of epitope-likelihood based on the pattern of unstable-to-stable transitions. We validate our method using 23 datasets of experimentally measured CD4+ T-cell responses of mice bearing IAb or HLA-DR4 alleles as well as of human subjects. Overall, we show that an antigen processing-based approach has at least as much, and sometimes more, predictive power for epitope prediction than existing, sequence-based methods that rely on MHC class II binding affinity estimates.
CD4 T Cell reactivity to both native and citrullinated-MOG epitopes identified in HLA-DR4 mouse model Of demyelinating disease (BA11P.137)
Authors: Kersh Anna
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and demyelination in the central nervous system (CNS). HLA-DR4 is an MHC class II gene associated with increased risk for MS. Humanized HLA-DR4-transgenic mice were used to probe T cell specificity and affinity during demyelinating disease progression in response to myelin oligodendrocyte glycoprotein (MOG). Using the micropipette adhesion frequency assay to obtain sensitive, physiologically relevant measurements, only 26% of CNS-infiltrating CD4 T cells are reactive to MOG in symptomatic HLA-DR4-expressing mice. Moreover T cells probed for specificity to a modified MOG peptide containing a citrulline residue in position 101 instead of arginine revealed T cells reactive to MOG alone, citrulline-modified MOG, or to both. Of importance, the citrullinated-MOG peptide does not induce paralytic disease on its own. We believe that these results are the first demonstration of T cell reactivity to citrullinated-MOG epitopes in the context of HLA-DR4 presentation to T cells. We suggest that T cell cross-reactivity to native and citrullinated epitopes of MOG provides a mechanism of disease progression and escape from immune tolerance. Further, reactivity to the citrullinated neoepitope has great implications for use as a potential biomarker of disease therapeutic efficacy since citrullination is the result of a deimination reaction that occurs during prolonged inflammation.
T cell intrinsic deletion of the adenomatous polyposis coli gene causes homeostatic proliferation and inflammatory bowel disease (BA11P.142)
Authors: Wong Chun-Shu , Chen Chong , Wu Qi , Liu Yang , Zheng Pan
Published: January 2023

Accumulating data demonstrate a strong association between lymphopenia and autoimmune diseases. However, to our knowledge, none of the known single gene defects that cause lymphopenia are sufficient to cause autoimmune diseases. In addition, the intracellular programing involved in lymphopenia-driven homeostatic proliferation (HP) remains to be elucidated. Using a conditional knockout of adenomatous polyposis coli (Apc) under the control of CD4-cre, we have previously reported that T-cell lineage-specific deletion of the Apc gene causes severe lymphopenia, due to naive T cell death, in part through activation of the cMyc gene. Here, we show that Apc deletion causes both T cell activation, lymphopenia-driven HP. Using this mouse model, we studied the association between lymphopenia-driven HP, and autoimmune diseases. We showed that APC deficiency and the resulting activation of Wnt signaling in T cells causes a fatal inflammatory bowel disease with Crohn’s disease-like pathology. Moreover, using T cells with targeted mutation of beta-catenin, we found that lymphopenia-drivinen HP depends on activation of beta-catenin in T cells, thus providing a missing link between Wnt signaling and homeostatic proliferation. Development of autoimmune diseases requires lymphopenia. This data provide the first genetic evidence that a T-cell intrinsic single-gene defect is sufficient to cause lymphopenia, homeostatic proliferation and autoimmune diseases.
CD4+CD40+ T cells (Th40) drive a rapid and severe form of experimental autoimmune encephalomyelitis (BA11P.123)
Authors: Vaitaitis Gisela , Waid Dan , Wagner David
Published: January 2023

The CD40-CD154 dyad is critically involved in autoimmune diseases including Multiple Sclerosis (MS) and Type 1 Diabetes (T1D). CD4+CD40+ T cells, Th40, are necessary and sufficient to establish T1D in the mouse model. In the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS, CD4 T cells are known to play a dominant role in disease development, however, the contribution of Th40 cells has not been determined. Here we reveal that the EAE induction regimen, specifically the Freund’s adjuvant (CFA), causes a significant expansion of Th40 cells, which in turn play a major role in EAE development. While expansion of Th40 cells is not dependent on specific antigen, the precipitation of central nervous system disease symptoms is MOG-antigen dependent, shaping a different TCR repertoire. We show that surface expression of CD40 on T cells is associated with a more rapid and severe disease course. Th40 cells are present in lesions in the brains and spinal cords of EAE mice and their cytokine profile is skewed toward IFNγ. In EAE facilitated by CD4+CD40- T cells, where disease develops with significantly slower kinetics and lesser severity, the cytokine profile is skewed more towards IL-17. Primary Th40 cells can adoptively transfer EAE, without prior in-vitro expansion, requiring only the injection of CFA in the recipient mice. These data demonstrate the importance of T cell CD40 and its relation to differential expression of IFNγ and IL-17 in EAE disease induction.
Dual roles of GM-CSF in modulating NK-cell migratory properties (CAM4P.147)
Authors: Andalur Nandagopal Saravanan , Upreti Deepak , Santos Susy , Chyi Su Ruey , Ball Blake , Lin Francis , Kung Sam
Published: January 2023

Background: Natural Killer (NK) cells play a key role in innate immunity against viral, microbial infections and transformed cells and their migration for effector function to peripheral tissues or inflamed lymph nodes are tightly regulated. Of interest, production of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by cancer cells is correlated to host immune suppression and tumor metastasis, suggesting an immune evasion property of GM-CSF. Here we examined role(s) of recombinant GM-CSF in the regulation of NK-cell migratory properties in vitro. Methods: Previously published “Y” shape microfluidic platform was used to study the roles of GM-CSF gradient on NK-cell migrations. IL-2 activated human primary NK cells were used in the migration studies. Results: Our microfluidic-based migration study demonstrated a novel role of GM-CSF in regulating repulsive NK-cell migration under the stable GM-CSF gradient (at 20 ng/ml), followed by subsequent arrest in cell migration. Blocking of GM-CSF-Rα abolished the repulsive migratory behavior but not the arrest. In contrast, lower concentrations of GM-CSF induced hyper-polarization, immediate arrest of NK cells, and little/or no NK-cell migrations. Circularity measurement in controls and above experiments confirmed statistically the correlation between hyperpolarization and migration arrest. Future analyses will elucidate the mechanisms underlying the dual roles of GM-CSF in the regulation of NK-cell migratory properties.
The HMGB1-MD-2 axis as a novel therapeutic target in inflammatory diseases (CCR5P.210)
Authors: Yang Huan , Wang Haichao , Chavan Sangeeta , Billiar Timothy , Yousef Al-Abed , Tracey Kevin
Published: January 2023

Innate immune cells express receptors for pathogen-associated molecular patterns (PAMP e.g. LPS) as well as damage-associated molecular patterns (DAMP e.g. HMGB1), and orchestrate inflammatory responses to both infection and sterile injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modulation that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguishes between HMGB1 isoforms. Here we demonstrate that the TLR4 adaptor molecule, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide HMGB1 isoform, to the exclusion of other isoforms. Screening of HMGB1 peptide libraries identified a tetramer (FSSE, designated as P5779) as a specific MD-2-antagonist preventing MD-2/HMGB1 interaction and subsequent TLR4 signaling mediating cytokine and chemokine release. Moreover, P5779 does not interfere with LPS-induced cytokine production, thereby preserving PAMP-mediated TLR4-MD2 responses. P5779 conferred protection in animal models of hepatic ischemia/reperfusion injury, chemical toxicity and sepsis. Taken together, these findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms and suggest strategies to attenuate DAMP-mediated inflammatory responses while preserving anti-microbial immune responsiveness.
Glucosylceramide induced complement activation triggers inflammation in Gaucher disease (CCR5P.212)
Authors: Pandey Manoj , Tinch Stuart , Inskeep Venette , Zhang Wujuan , Setchell Kenneth , Köhl Jörg , Grabowski Gregory
Published: January 2023

Gaucher disease (GD) happens due to mutations in GBA1, which causes a deficiency of acid β-glucosidase critical for degradation of glucosylceramide (GC) into glucose and ceramide. Macrophages (Mɸs) were mainly having this defect and associated with eminent expression of cytokines, chemokines, and recruitment of activated immune cells in GD. The mechanism by which GC triggers such inflammation is still wanted. Excess of GC storage in dendritic and T cells has been reported to be the reason for their activation and production of inflammatory cytokines in Gba1 mouse model (D409V/null; 9V/null). Here, we have identified elevated expression of C5a and C5aR on 9V/null mice APCs. We therefore, hypothesized that GC induced C5a elicits immune inflammation in GD. To confirm this, conduritol B epoxide (CBE), induced chemical model of GD was developed in WT and C5aR-/- mice. We found that C5aR-/- mice were protected from CBE-induced GD development, which was associated with prolonged survival, marked reduction of GC, DCs and T cells positivity for CD40, CD80, CD86, CD40L, CD69, cytokines-chemokines secretion, immune cells recruitment, and decreased loss of red blood cells (RBCs) and platelets. Pharmacological blocking of C5aR in 9V/null mice showed marked reduction in several of these GD manifestations. Our data suggest that GC induced C5a contributes in disease development and targeting C5a signaling may have therapeutic potential for GD.
Determining the effect of BMP signaling on thymus fate specification and differentiation of the third pharyngeal pouch in the mouse. (HEM7P.235)
Authors: Frisbie Trent , O'Neil John , Gordon Julie , Vokes Steven , Richie Ellen , Manley Nancy
Published: January 2023

The third pharyngeal pouch is an endodermally derived epithelium that differentiates into thymus and parathyroid cells. Foxn1 (transcription factor) is a thymus-specific marker, which is required for thymic epithelial cell (TEC) proliferation and differentiation. Gcm2 is a parathyroid-specific marker, which is essential for parathyroid survival and differentiation. Loss of function genetic analysis of BMP4 showed that BMP4 is required for organ morphogenesis but not initial thymus fate; however, repression of BMP signaling by transgenic expression of ectopic Noggin inhibitor suppressed Foxn1expression. Based on these data, we hypothesize that BMP signaling is necessary for normal thymus fate specification during initial organogenesis with BMP signaling promoting thymus fate while inhibiting parathyroid fate. We are testing this hypothesis using inducible Gremlin transgenic mice in a tissue-specific Cre-Lox induction system, as well as gain and loss of function experiments in embryo cultures. Current data suggest delayed expression of Foxn1 when Gremlin is ectopically expressed. Explant cultures with dorsomorphin, a BMP inhibiting drug, showed a reduction of both Gcm2 and Foxn1. Also, cultured embryos with a BMP4 bead implant showed a reduction of Gcm2. Surprisingly, these data suggest inhibition of BMP signaling is suppressing Gcm2, while supporting the hypothesis that BMP signaling is necessary for initial thymus fate specification.
Prenatal and postnatal exposure to concentrated ambient particulate matter alters the developing immune system of mice (HEM8P.240)
Authors: Tijerina Pamella , Blum Jason , Hoffman Carol , Park Sung Hyun , Grunig Gabriele , Chen Lung-Chi , Zelikoff Judith
Published: January 2023

Prenatal exposure to certain air pollutants have been shown to cause immunological dysfunction in offspring, however previous experimental studies have yet to evaluate the vulnerability of the immune system throughout the pre- and early postnatal period. Therefore, to more accurately reflect human exposure, mice were exposed to concentrated fine-sized ambient particulate matter (PM2.5) both pre- and postnatally and parameters of immune status were assessed in offspring. Timed-pregnant dams were exposed 6hr/d throughout gestation (GD 0-17) to filtered air or PM2.5. At 5 wks-of-age, blood and spleen were collected from a subset of male and female offspring, and serum samples were analyzed for cytokine levels. Spleens were weighed and splenocytes isolated to assess changes in immune cell profiles by flow cytometry. Of the endpoints analyzed, only female offspring were significantly affected with pre- and postnatal PM2.5 exposure resulting in an 11% reduction of normalized splenic weight and decreased maturation status of splenic B cells (MHCII and CD45R expression) compared to their control counterparts. These sex-specific alterations demonstrate gaps in previous developmental studies that focused only on PM2.5-induced immune effects on male offspring. Together, these results suggest that particulate air pollution negatively impacts the developing immune system, proposing critical consequences for offspring exposed during the pre- and early postnatal period.
A role for Hox5 genes in T cell function during chronic allergic inflammation (HYP7P.268)
Authors: Ptaschinski Catherine , Hrycaj Steven , Wellik Deneen , Lukacs Nicholas
Published: January 2023

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. T helper 2 (Th2) cells contribute to the development of disease through release of the cytokines interleukin (IL)-4, IL-5 and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. While many factors are involved in the development of allergic asthma, the molecular mechanisms behind the disease remain largely unknown. As the three members of the Hox5 gene family (Hoxa5, Hoxb5 and Hoxc5) have been shown to play a role in lung development, we asked whether these genes also play a role in the adult lung in response to disease. We found that Hox5-deficient mice show exacerbated pathology compared to wild-type controls in a chronic allergen model, with an increased Th2 response as well as changes in lung tissue pathology. Examination of T cells from naïve wild-type or Hox5-deficient mice in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression and production of IL-4, IL-5 and IL-13 in Hox5-deficient mice compared to wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of immune cell function and Th2-induced pathology during allergic disease in the lung.
Regulation of allergic inflammation by murine endothelial cell transglutaminase 2 (HYP7P.260)
Authors: Soveg Frank , Abdala-Valencia Hiam , Campbell Jackson , Cook-Mills Joan
Published: January 2023

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that is involved in inflammatory processes. In allergic inflammation, previous research indicates that TG2 is upregulated in human asthma and in the lung endothelium of OVA-challenged mice. We hypothesized that endothelial cell TG2 is required for allergic inflammation. To test this hypothesis, we generated mice in which TG2 was specifically deleted in the endothelium using the Cre-Lox recombinase system. To induce allergic inflammation, mice were sensitized and challenged with OVA. Compared to wild type mice challenged with OVA, OVA-challenged TG2 knockout mice exhibited a significant reduction in lung airway resistance, the number of eosinophils in the BAL, and the number of eosinophils in the airway spaces. Endothelial deletion of TG2 did not affect chemokines and cytokines that regulate allergic inflammation. Taken together, these findings indicate that endothelial cell TG2 is required for allergic inflammation. This suggests that TG2 regulates the recruitment of eosinophils into OVA-challenged lungs. Future studies will focus on identifying mechanisms by which TG2 regulates signals for leukocyte recruitment during allergic lung inflammation.
Tim-3 promotes homeostasis of ulcerative colitis by inhibiting M1 macrophage polarization (INC6P.308)
Authors: Han Gencheng
Published: January 2023

Abstract We previously found that Tim-3 is involved in the physiopathology of inflammatory bowel diseases (IBD). However, the underlying mechanisms remain to be determined. Here we further demonstrated that Tim-3 contributes to the homeostasis of ulcerative colitis (UC), one type of IBD, by suppressing the polarization of pathogenic M1 macrophages. Downregulation or blockade of Tim-3 in UC is associated with biased pro-inflammatory macrophage (M1) response. Adoptive transfer of macrophages silenced of Tim-3 worsened colitis and enhanced inflammation. On the contrary, transgenic expression of Tim-3 attenuated DSS-induced colitis by down-regulating M1 macrophage response. Co-culture macrophages overexpressed of Tim-3 with intestinal lymphocytes decreased pro-inflammatory response. Tim-3 controls the activity of intestinal macrophages partially dependent on TLR-4 signaling, as in TLR-4 knockout mice blockade of Tim-3 pathway resulted in significantly attenuated colitis and decreased M1 macrophage response. Finally we demonstrated that Tim-3 signaling inhibited the phosphorylation of IRF3, a transcriptional factor regulating macrophages polarization. A better understanding of this pathway may shed new light on the pathogenesis of UC and help to find new therapeutic strategy.
LPS-induced p21 is required for M2 polarization of endotoxin- tolerant macrophages and controls the accumulation of inhibitory p50/p50 NF-κB (INC6P.317)
Authors: Rackov Gorjana , Martínez-A Carlos , Balomenos Dimitrios
Published: January 2023

Gram-negative bacterial infection can lead to systemic inflammation and sepsis — a complex pathology that is the third leading cause of death in the U.S. A critical mechanism in lowering inflammation and septic shock is endotoxin tolerance, a state of hyporesponsiveness to secondary lipopolysaccharide (LPS) challenge. p21 controls macrophage activation independently of its cell cycle inhibitor activity. Here, we investigated M1 to M2 macrophage polarization during endotoxin tolerance, which is regulated by NF-κB composition switch from p65/p50 to p50/p50. p21 deficiency led to delayed formation of inhibitory p50/p50 NF-κB homodimers that control IFN-β promoter, a key cytokine which orchestrates M1 responses. Consequently, lack of p21 enhanced IFN-β production and IFN-β-dependent STAT1 phosphorylation, resulting in upregulation of iNOS and M1 chemokines. Importantly, p21-deficient macrophages had impaired ability to acquire M2-like LPS-tolerant profile, showing reduced induction of M2 genes such as arginase I. The biological significance of our work was reflected in an in vivo model of endotoxin tolerance, where p21-deficient mice failed to develop tolerance and showed high M1-dependent responses and death. Overall, we identified p21 as a key molecule that regulates M2 polarization and endotoxin tolerance, through NF-κB composition switch from p65/p50 to p50/p50. Thus, analysis of the role of p21 in macrophages may be of therapeutic value for optimizing M1/M2 ratio in disease.
TLR stimulation of type II NKT cells requires cell to cell contact with DCs with involvement of CD40-CD40L and IL-12p40 (INC6P.318)
Authors: Tripathi Prabhanshu , Sedimbi Saikiran , Karlsson Mikael , Yrlid Ulf , Kadri Nadir , Cardell Susanna
Published: January 2023

Natural killer T (NKT) cells are CD1d restricted innate like lymphocytes recognizing lipid antigens and mediating immune regulatory functions. NKT cells are divided into two subsets based on the type of T cell receptor (TCR) expressed. Type 1 or invariant NKT cells uses an invariant TCRα-chain (Vα14-Jα18 in mice/Vα24-Jα18 in humans) and type II or diverse NKT cells are all non Vα14 expressing NKT cells. Most of the knowledge about NKT cells have been derived from type I NKT cells, while type II NKT cells are less well explored. Here, we investigated the signals driving Toll-like receptor (TLR) ligand activation of type II NKT cells. We use the 24αβ mouse model expressing a transgenic Vα3.2 and Vβ9 type II NKT cell TCR on the C57/BL6 background. Using in vitro co-culture experiments with 24αβ type II NKT cells and FLT3L derived bone marrow dendritic cells (BMDCs) and TLR ligands, we have analyzed the factors required for type II NKT cell activation, applying BMDCs from different knock out mice. TLR stimulated DCs potently activated type II NKT cells to secrete IFN-γ in a CD1d and type I IFN independent manner. In contrast, an absolute requirement of IL12p40 and partial dependence on CD40, IL15 and IL18 for optimal IFN-γ induction was observed. Thus, our data suggest that TLR ligand - BMDC mediated activation of type II NKT cells requires cell - cell contact that includes CD40 - CD40L ligation, IL15, IL18 and IL-12, but not CD1d - TCR interaction.
Interleukin 4 induces a unique NK cell population in vivo and in vitro (INC6P.325)
Authors: Tsuyoshi Kiniwa , Yutaka Enomoto , Ai Omi , Atsushi Miyajima
Published: January 2023

NK cells are cytolytic and cytokine-producing effector cells that play major roles for clearance of tumors and virus-infected cells. NK cells are known to be activated by IL-12, a Th1-type cytokine, and secrete a large amount of IFN-γ that accelerates Th1-type responses. However, the roles of NK cells in Th2-type responses are largely unknown. As IL-4 acts as an initiator of Th2-type responses, we examined the phenotype of NK cells in mice over-expressing IL-4 by hydrodynamic tail vein injection. IL-4 expression induced a unique NK cell population (NK1.1high/CD11blow/IL-18Ralow), which was different from conventional mature NK cells (NK1.1high/CD11bhigh/IL-18Rahigh). Interestingly, over-expression of IL-13, whose biological activities largely overlap with IL-4, failed to induce this population. Moreover, these IL-4-responsive NK cells produced a large amount of IFN-γ in response to IL-12 and exhibited a high cytotoxicity compared to conventional mature NK cells. We also found that incubation of conventional mature NK cells with IL-15 and IL-4 changed their phenotype to that similar to IL-4-responsive NK cells, i.e., they became IL-18Ralow and secreted a large amount of IFN-γ in response to IL-12, and produced cytotoxic granules without additional stimulation. Collectively these results suggested that IL-4 induces a unique NK cell population with strong NK activity independent of IL-13 and that IL-4 has a specific role in Th2 responses through this IL-4-responsive NK cells.
Extracellular superoxide dismutase promotes immature neutrophil egress from the bone marrow (INC6P.303)
Authors: Witter Alexandra , Break Timothy , Indramohan Mohanalaxmi , Mummert Mark , Berg Rance
Published: January 2023

Extracellular superoxide dismutase (ecSOD) regulates extracellular concentrations of reactive oxygen species (ROS) to protect tissues during infection and inflammation. Using three groups of mice with varying levels of ecSOD activity, we have previously shown that ecSOD activity enhances neutrophil recruitment to the liver, yet inhibits the innate immune response against Listeria monocytogenes (LM) leading to increased host susceptibility. However, it is unclear whether ecSOD activity affects neutrophil recruitment and function in a cell-intrinsic manner or by modulating the extracellular environment. Using adoptive transfer experiments, we observed that ecSOD activity does not affect neutrophil recruitment or function in a cell-intrinsic manner. Additionally, we determined that ecSOD activity protects the extracellular matrix (ECM) and leads to an increase in phenotypically immature neutrophils in the bone marrow and liver. Collectively, our data suggest that ecSOD activity inhibits degradation of the ECM and promotes egress of immature neutrophils out of the bone marrow and into the liver where they provide inadequate protection against LM. These studies highlight the potential therapeutic value of ecSOD inhibitors to enhance immune responses during bacterial infections.
Fluvastatin elicits apoptosis in primary and transformed mast cells (INC6P.305)
Authors: Paez Patrick , Ryan John , Taruselli Marcela , Ndaw Victor
Published: January 2023

Mast cell leukemia is a rare subtype of acute myelogenous leukemia. A majority of mast cell leukemias express the c-Kit mutation, D816V, which yields factor-independent growth. Understanding and treating mast cell hyperplasias and leukemias could be beneficial to both allergic and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) in a dose-and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and Caspase-3/7 activation. Interestingly, BMMC from the C57BL/6 background were responsive to Fluvastatin-mediated death, while those from 129/SvJ mice were completely resistant. Resistance was matched by a 2-fold induction of HMG CoA Reductase after Fluvastain treatment. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 between 5-10µM, and maximum effects after 72 hours. Mitochondrial staining detected membrane disintegration after 48-72 hours, while cells tested positive for active Caspase-9 as early as 24-48 hours. These preliminary data support the theory that statin family drugs may be a new therapeutic approach for suppressing growth of both normal and transformed mast cells, and emphasize the contribution of genetic background to drug responsiveness.
Macrophages are a central hub of innate immunity and inflammatory signaling programs induced by HCV/HIV coinfection. (INM6P.344)
Authors: Aarreberg Lauren , Gale Michael
Published: January 2023

Hepatic inflammation is a hallmark feature of pathogenesis due to HCV/HIV-1 coinfection but the processes that propagate this inflammatory response are not understood. Coinfection of HIV-1 within the HCV-infected liver results more commonly in inflammatory liver disease than monoinfection of either virus, with a quicker progression to end-stage disease. HCV causes significant liver inflammation by triggering inflammasome activation after uptake by hepatic macrophages. Also known as “Kupffer cells”, these CD4+ cells are targets of HIV-1 infection. Phagocytosed HCV particles are potent activators of the NLRP3 inflammasome driving IL1β secretion from macrophages to impart liver inflammation. Here, we evaluated coinfection in human THP1 macrophages as a model of Kupffer cells. We demonstrate that like HCV, HIV-1 activates the inflammasome in these cells upon infection. Coinfection synergizes the inflammatory response, resulting in the production of pro-inflammatory mediators known to drive immune infiltration, tissue damage, and liver fibrogenesis. Furthermore, we have defined the macrophage-secreted cytokine milieu that accompanies monoinfection vs coinfection, revealing how each virus directs an innate immune response on its own and during coinfection. Our studies identify the hepatic macrophage as a hub of inflammatory signaling and reveal crosstalk between virus-responsive inflammatory signaling pathways that underlie hepatic disease and destruction in HCV/HIV-1 coinfection.
Novel role for scavenger receptor B-I in neutrophilic asthma (IRC10P.419)
Authors: Gowdy Kymberly , Madenspacher Jennifer , Thomas Seddon , Nakano Hideki , Cook Donald , Fessler Michael
Published: January 2023

Asthma is a heterogeneous airway disease that has risen significantly in recent decades. While there has been a major research focus on eosinophilic asthma, the neutrophilic, Th17-predominant disease that characterizes severe asthmatics is less understood. Scavenger Receptor B-I (SR-BI) is a receptor mostly studied in the context of cellular uptake of cholesterol. Recently, we reported that SR-BI regulates the pulmonary innate immune response, but the role of SR-BI in asthma is unknown. Therefore, SR-BI+/+ and SR-BI-/- mice were sensitized on days 0 and 7 by oropharyngeal aspiration of ovalbumin (OVA) together with LPS and then challenged on day 14 with an aerosol OVA. Airway inflammation and cytokines were quantified 48hrs after challenge. Bone marrow-derived dendritic cells (BMDCs) from SR-BI+/+ and SR-BI-/- mice were co-cultured in the presence of OVA/LPS with CD4+ T cells from OT-II mice that carry a transgenic TCR specific for the MHC class II-restricted OVA peptide (OT-II Tg). Compared to SR-BI+/+ counterparts, SR-BI-/- mice had increased pulmonary neutrophils, Th17 cells, G-CSF and CXCL5 after OVA challenge, but had equivalent amounts of IL-4, IL-5, and eosinophils. BMDCs from SR-BI-/- mice have elevated levels of CD80 and CD86, and CD4+ OT-II Tg T cells produced more IL-17 after culture with OVA/LPS. SR-BI modulates the lung’s response to environmental adjuvants such as LPS by regulating Th17 polarization, suggesting a novel role for SR-BI in neutrophilic asthma.
IL-2 produced by memory CD4 T cells synergizes with influenza A virus to amplify detrimental lung inflammation (IRC10P.404)
Authors: Strutt Tara , McKinstry Karl , Swain Susan
Published: January 2023

Memory CD4 T cells capable of producing IL-2 in addition to IFNγ are often more effective at combating pathogens than those producing IFNγ alone. The impact of IL-2 on protective memory responses, however, is not well defined. To test if IL-2 contributes to memory CD4 T cell protection, we transferred wild type or IL-2-deficient memory CD4 T cells to unprimed mice and challenged with lethal doses of influenza A virus (IAV). We find comparable expansion of both donor populations and kinetics of viral clearance in both recipients. Strikingly, recipients of IL-2-deficient cells exhibit reduced weight loss, improved lung function, and earlier recovery. The improved outcome in recipients of IL-2-deficient memory cells correlates with significantly tempered inflammatory responses. To more directly assess the pro-inflammatory capacity of IL-2, we administered IL-2: anti-IL-2 antibody complexes targeting delivery to cells expressing distinct IL-2 receptors. IL-2 administration dramatically up-regulates a remarkably broad array of inflammatory mediators systemically and particularly in the lung. Multiple adaptive and innate cell populations contribute. IL-2 administration rapidly impaired lung function in unprimed animals and synergized with IAV infection to cause lethal lung damage. Our findings highlight that IL-2 produced by memory CD4 T cells can contribute to pulmonary inflammation during protective responses and have implications for improving IL-2 based therapies.
TGF-β1-induced regulatory T cells directly suppress B cells through non-cytotoxic manner (IRC11P.423)
Authors: Zheng Song , Liu Ya , Chen Weiqian , Xu Anping , Wang Julie , Olsen Nancy
Published: January 2023

Methods: Naïve CD4+ T cells and B cells were isolated from C57BL/6 mice. Peripherally induced CD4+Foxp3+ regulatory T cells (itregs) was induced with IL-2 and TGF-β. The thymus-derived naturally CD4+Foxp3+ regulatory T cells (ntregs) was obtained from C57BL/6 mice. iTregs and nTregs cells were cultured with B cells for 3-5 days in vitro. The activation marker, proliferation, Annexin-V and 7-AAD expression by B cells was determined by FACS. For in vivo experiment, iTregs and nTregs cells were transferred to endogenous T cell-deleted lupus prone mice. The IgG antibody production was measured by ELISA at the indicated time points. Results: We found that iTregs share similar functional characteristics with nTregs, both iTregs and nTegs can directly suppress the proliferation and production of autoantibodies of B cells. Unlike nTregs, iTregs suppressed B cells through non-cytotoxic manner. Moreover, the TGF-β signal is important for the effects of iTregs on B cells. We further showed that iTregs directly suppress B cells in vitro and in vivo in lupus prone mice. Conclusions: Our data implicate the role and advantage of iTregs in treating B cell-mediated autoimmune diseases.
IRF3 deficiency contributes to impaired memory T cell function in response to influenza infection (IRM14P.453)
Authors: Vogel Alexander , Moore Tyler , Petro Thomas , Brown Deborah
Published: January 2023

The role of IRF3 in initiating the type I interferon response in innate immune cells has been well studied in the context of viral infection. However, it remains unclear what impact IRF3 has in shaping and maintaining adaptive immune responses. To address this, IRF3 deficient (IRF3 KO) mice were infected with influenza A virus (IAV) and T cell responses measured. At day 9 post infection, the frequency of IFN-γ and Granzyme B (GrB) positive T cells were similar between IRF3 KO and wildtype mice. Despite similar primary responses, IRF3 KO mice exhibited significant deficits in recall memory T cell responses to IAV with reduced expression of IFN-γ and GrB. Since IRF3 has been implicated in the expression of cytokines that influence effector and memory T cells responses, cytokine supplementation studies were performed. Addition of IL-12 was the only cytokine tested that was able to rescue the IFN-γ deficiencies in IRF3 KO T cells. Interestingly, peptide specific GrB re-expression could not be restored by any cytokines tested. These results suggest that IRF3 plays a role in the maintenance of antigen specific memory T cell responses and that IRF3 deficiency has a substantial impact on the cytotoxic potential of memory T cells and recall responses. Together, this work provides further insight into the molecular regulation of adaptive immune responses to viral infections and may serve to inform future vaccine strategies that require the development of memory T cells.
Insights into MHC class II regulation of T cell responses (IRM15P.463)
Authors: LeGuern Christian , Germana Sharon
Published: January 2023

Besides their ability to present antigenic peptides and stimulate antigen-specific T lymphocytes, MHC class II (MHCII) molecules have also been implicated in the regulation of ensuing T cell responses. However, the substance of MHCII regulation remains elusive to this day. Defining the variables of this phenomenon appears to be critical for developing rational therapeutic approaches. Using models in which MHCII regulation has been demonstrated, we have revisited the concept by evaluating the impact of MHCII-restricted CD4 regulatory T cells (Tregs) on T cell reactivity to either allogeneic or self-antigens. Studies performed in vitro and in vivo showed that graft host Tregs specific to donor MHCII peptides / host MHCII complexes promoted T cell tolerance of fully allogeneic grafts. These pMHCII complexes - hereafter called TLo - were also involved in Treg-mediated suppression and in initial host Treg activation by graft MHCII. Extending these observations to Treg biology of normal animals, we have also shown that substantial amounts of TLo complexes selectively decorate antigen-presenting cells (APC) from the thymic medulla and participate in the differentiation of suppressive, TLo-specific Treg cells. Together these results suggest that, under steady-state conditions, MHCII genes regulate T cell responses by 1) generating self-TLo complexes on thymic APC to direct thymic Treg development and 2) modulating peripheral T cell activation via TLo-specific Tregs.
Hepatic stellate cells inhibit T cells through active TGFβ1 from a cell-surface-bound latent TGFβ1/GARP complex (IRM15P.609)
Authors: Li Yan , Kim B , Qian Shiguang , Letterio John , Fung John , Lu Lina , Lin Feng
Published: January 2023

Hepatic stellate cells (HSCs) potently inhibit T cells, a process that could help the liver to maintain its immunoprivileged status. HSCs secrete latent TGFβ1, but the detailed mechanisms by which latent TGFβ1 is activated and whether it plays any role in HSC-mediated T-cell suppression remain unclear. Glycoprotein A repetitions predominant (GARP) is a surface marker of activated regulatory T cells (Tregs). GARP binds latent TGFβ1 for its activation, which is critical for Tregs to suppress effector T cells; however, it remains unclear whether GARP is present on HSCs and whether it has any impact on HSC function. We found that TGFβ1+/‑ HSCs, which produce reduced levels of TGFβ1, showed decreased potency in inhibiting T cells. Pharmaceutical inhibition of the TGFβ1 signaling pathway reduced the T-cell-inhibiting activity of HSCs. SMAD3‑/‑ T cells with genetically impaired TGFβ1 signaling were resistant to HSC-mediated suppression. We demonstrated that GARP was constitutively expressed on primary HSCs. Blocking GARP function with antibodies or knocking down GARP expression using shRNAs significantly impaired the potency of HSCs to suppress T cells. These results demonstrate the presence of GARP on HSCs and its critical role in regard to the ability of HSCs to activate latent TGFβ1 and thereby inhibit T cells. Our study reveals a novel mechanism for HSC-mediated immune regulation and potentially for other liver conditions that involve HSC-secreted TGFβ1.
Molecular chaperone gp96/grp94 is critical for immunosuppressive functions of regulatory T cells (IRM15P.458)
Authors: Zhang Yongliang , Wu Bill , Alessandra Metelli , Hong Feng , Ansa-Addo Ephraim , Sun Shaoli , Liu Bei , Li Zihai
Published: January 2023

Regulatory T (Treg) cells are essential for self tolerance and immune homeostasis, which is subjected to regulation by multiple molecular switches. gp96/grp94 is an endoplasmic reticulum (ER) resident chaperone, and it plays an important immune regulatory function by folding Toll-like receptors and integrins. In this study, we demonstrated that gp96 is critically required for immune suppressive function of Treg cells. Disruption of gp96 in mice through Treg-specific gp96 knockout (KO) leads to impairment of suppressive function of Treg cells in vivo and development of fatal inflammatory diseases. Mechanistically, we found that Treg cells showed lineage instability in the absence of gp96, and underwent conversion to IFN-γ producing pathogenic T cells. Moreover, we unveiled that GARP, a cell surface docking receptor for latent membrane-associated TGF-b (mLTGF-b), is the client protein of gp96. The dual loss of GARP and integrins renders gp96 KO Treg cells incapable of expressing mLTGF-b and inefficient in producing active TGF-b. In the B16 tumor bearing mice, transient and inducible depletion of gp96 in Treg cells robustly improved therapeutic efficacy of adoptively transferred gp100-specific CD8 T cells without causing auto-inflammatory diseases. In summary, our study demonstrates that gp96 regulates multiple facets of Treg cell biology, and it suggests a manipulation of gp96 expression in Treg cells as a promising strategy to enhance cancer immunotherapy.
The transcription factor Bcl-3 regulates the development of marginal zone, germinal center and B1 B cells (LYM7P.624)
Authors: Hoevelmeyer Nadine , Wörns Marcus , Reissig Sonja , Adams-Quack Petra , Waisman Ari
Published: January 2023

Bcl-3 is an atypical member of the IkB family and functions as a transcriptional suppressor or promoter by associating with the NF-kB members p50/NF-kB1 and p52/NF-kB2 homodimers. The bcl3 gene was first identified at the breaking point of recurring chromosomal translocations in B cell chronic lymphocytic leukemias, and therefore supposed to function as a proto-oncogene via regulation of cell proliferation and apoptosis. To investigate the role of Bcl-3 on B cells we generated mice that overexpress Bcl-3 specifically in B cells and Bcl-3 deficient mice. Interestingly, mice overexpressing Bcl-3 lack marginal zone B and germinal center B cells and exhibit a significant reduction of B-1 B cells. Further, B cells of these mice are impaired in their proliferative capacity, especially upon anti-IgM stimulation. In line with these findings Bcl-3 deficient mice showed increased numbers of marginal zone and germinal center B cells. Our data demonstrate that the transcription cofactor Bcl-3 is important for B cell maturation, especially in the maintenance of marginal zone, germinal center and the B1 B cell compartment probably by inhibiting NF-kB activation.
Insulin-like growth factors are essential for the differentiation of Th17s in EAE (LYM8P.635)
Authors: DiToro Daniel , Frank Stuart , Weaver Casey
Published: January 2023

Insulin-like growth factors (Igfs) are highly conserved proteins similar to pro-insulin in both sequence and structure. They function primarily by binding the Igf1 receptor (Igf1R) and exert pleiotropic effects on many tissues throughout life. These factors were shown to be essential for the normal development and proliferation of B and T lymphocytes, and to positively influence their proliferation and survival following activation. But early attempts to identify potential roles in T cell differentiation and function lead to conflicting results, and the field was largely abandoned. More recent advances in our understanding of T cell biology, however, including the discovery of Th17 and Treg C4s, have permitted a more sophisticated analysis of the role of this system in T cells. Our data indicate that Th17 and Treg CD4s express Igf1R and insulin-like growth factor binding protein 4 (Igfbp4), a critical regulator of Igf activity, at much higher levels than other CD4 T cells, and appear uniquely capable of responding to Igfs. Exogenous Igfs promote Th17 and suppress Treg differentiation in-vitro, yet fail to influence Th1 or Th2 differentiation. Deletion of Igf1R leads to a defect in Stat3 phosphorylation and IL17a production, and enhanced Treg differentiation in-vitro. Mice lacking Igf1R on T cells are substantially protected from Experimental Autoimmune Encephalomyelitis, an effect that can be recapitulated through the in-vivo administration of Igf1R blocking antibodies.
GITR costimulation redirects regulatory T cells into Th9 cells to promote antitumor immunity (LYM8P.643)
Authors: Shi Xiaomin , Xiao Xiang , wu minhao , Fan Yihui , zhang xiaolong , wu chenglin , Ghobrial Rafik , Li Xian
Published: January 2023

GITR is a potent T cell costimulatory molecule with diverse effects on effector and regulatory T cells, and because of this, GITR is of high potential for clinical development including anti-tumor immunity. However, the underlying mechanisms remain unclear. Here, we found that GITR ligation converted iTregs into Th9 cells in vitro. Mechanistically, GITR induced the sustained expression of p50 in activated CD4+ T cells, and p50 then recruited histone deacetylases HDAC1 and Sirt1 to the Foxp3 locus upon binding to the locus; this subsequently led to hypoacetylation, thus resulting in a closed epigenetic configuration of Foxp3 locus. Consistent with this, both p50-/- cells and treatment of HDAC inhibitor rescued iTregs from GITR-mediated suppression. We also found that Foxp3 is a potent repressor of IL-9, mainly through recruiting histone deacetylases to the IL-9 locus, and absence of Foxp3 rendered IL-9 locus accessible. In addition, p50 also promoted the expression of Th9 permissive transcription factors, including PU.1, Batf and Irf4, and all these factors together led to the induction of Th9 cells. Importantly, in a melanoma model in vivo, ligation of GITR using agonistic anti-GITR Ab blocked iTreg differentiation and inhibited tumor progression. In conclusion, our study identifies a new mechanism in which GITR ligation redirects iTregs into Th9 cells to mediate potent anti-tumor immunity. This new finding may have important clinical implications in cancer therapy.
BCL6 orchestrates Tfh cell differentiation via multiple distinct mechanisms (LYM8P.628)
Authors: Crotty Shane , Hatzi Katerina , Nance John Philip , Kroenke Mark , Bothwell Marcella , Haddad Elias , Takemori Toshitada , Melnick Ari
Published: January 2023

Generation of long term humoral immunity is a complex process predominantly dependent on germinal centers and CD4 T cell help to B cells. Follicular helper T cells (Tfh) are the specialized CD4 T cells for B cell help. We and others have now resolved many of the stages of Tfh differentiation, and molecules involved. BCL6 is the defining transcription factor of Tfh cells. However, the functions of BCL6 in Tfh have largely remained unclear. We have defined the BCL6 cistrome in primary human germinal center Tfh cells to assess mechanisms of BCL6 regulation of CD4 T cells, comparing and contrasting BCL6 function in T and B cells. BCL6 primarily acts as a repressor in Tfh cells, and BCL6 binding was associated with control of Tfh cell migration, Tfh differentiation, and repression of alternative cell fates. Interestingly, although some BCL6 bound genes possessed BCL6 DNA binding motifs, more BCL6-bound loci were instead characterized by the presence of DNA motifs for AP1 or STAT. AP1 complexes are key positive downstream mediators of TCR signaling and external stimuli. We show that BCL6 can directly bind AP1, and AP1 and BCL6 co-occupied BCL6 binding sites with AP1 motifs, suggesting that BCL6 subverts AP1 activity. These findings reveal that BCL6 has broad and multifaceted effects on Tfh biology, and provide insight into how this master regulator mediates distinct cell-context dependent phenotypes.