bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell types and cell signals important for normal thymic medullary epithelial cell development (HEM7P.231)
Authors: Jeon Hyein , Williams Joy , Hodes Richard
Published: January 2023

Thymic crosstalk between medullary thymic epithelial cells (mTEC) and mature SP thymocytes is critical for tolerance in developing T cells as well as for mTEC development. Several TNF family receptor-ligand pairs, including RANK-RANKL, LTαβ-LTβR and CD40L-CD40, have been shown to be important in thymic crosstalk, in part via RelB mediated non-canonical NFκB pathway. Recently, we identified CD28-CD80/86 as an additional pathway important for mTEC development. CD80/86, LTβR, CD40, RANK, as well as MHCII (also shown to be important for mTEC development) are expressed by both mTEC and by bone marrow (BM) derived cells in the thymus. Thus, it is important to ask which cell types need to express these molecules in order to support mTEC development. To address the question, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. Interestingly, for CD40 and CD80/86, we find that expression on either non-BM or BM-derived cells is sufficient to promote mTEC development. For LTβR, and RANK, we find that expression on radiation-resistant non-BM derived cells is required. The ability of MHCII expression on specific cell populations to drive mTEC development is currently being examined. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other BM derived cell types as well.
MicroRNA-17-92 regulates Tbx1 expression in third pharyngeal pouch endoderm (HEM7P.232)
Authors: Reeh Kaitlin , Bain Virginia , Lorenz Julia , Bai Yan , Martin James , Manley Nancy , Richie Ellen
Published: January 2023

The thymus and parathyroids originate from bilateral 3rd pharyngeal pouch (pp) endoderm. At E11.5, thymus- and parathyroid-fated domains express Foxn1 and Gcm2 respectively, transcription factors that regulate differentiation but do not specify organ fate. Tbx1, which encodes a T-box transcription factor required for segmentation of the pharyngeal endoderm is initially widely expressed in the 3rd pp, but by E10.5 is excluded from the ventral, thymus-fated domain. We recently reported that Tbx1 is a negative regulator of thymus development (Reeh et al. Development 141:2950, 2014). Ectopic expression of Tbx1 in the thymus-fated domain of the 3rd pp suppresses Foxn1 and inhibits thymic epithelial cell proliferation and differentiation. MicroRNAs (miRs) are important regulators of gene expression. We find that members of the miR-17-92 cluster are expressed in the ventral domain of the 3rd pp in wildtype embryos. Moreover, miR-17-92 downregulates Tbx1 in cardiac progenitor cells to promote their differentiation (Wang et al. Developmental Cell 19:903, 2010). Therefore, we proposed that miR-17-92 regulates Tbx1 expression in 3rd pp endoderm. In support of this hypothesis, we find that deletion of miR-17-92 enhances TBX1 and reduces FOXN1 in 3rd pp endoderm. The data support a model in which miR-17-92 plays an essential role in thymus development by regulating Tbx1 expression in the 3rd pp.
Total body sub-lethal irradiation accelerates thymic senescence by reducing lymphoid progenitors in the thymus (HEM7P.236)
Authors: Xiao Shiyan , zhang wen , Manley Nancy
Published: January 2023

Total Body Irradiation (TBI) dramatically damages the BM and thymus organs, however the long-term effects of irradiation and aging on both BM cells and thymocytes are still unclear. In this study, we described that although the kinetics of thymocyte restoration was varied with the sex, dose and exposure age, a long-term and dose dependent reduction of DN1a,b progenitors and a transient over-response peak of thymocytes around 5-6 weeks were shown unanimously in all groups after irradiation, which led to the recovery of thymus maintained in a lower level of total thymocyte number through life. Such events were primarily due to the reduction of functional LSK progenitors in BM rather than the changes to the thymic environment. The damage of irradiation in BM LSK cells was dose dependent and 0.5 Gy was able to result in a significant long-term reduction of LSK progenitors. Responding to the reduction of thymocytes, both DN3 and DN4 subsets played a critical role on the compensation of the reduction of thymocytes with a periodical profile after irradiation, but the recovery potential was dramatically reduced at the age of 12 months. This study provided the evidence that the capability of LSK cells for lymphocyte production in BM is limited and can be dramatically damaged after a single dose of relatively low TBI, which consequently accelerates premature thymic aging and senescence. Our results may also be useful for the evaluation and therapeutic intervention of human TBI events.
LAG-3 modulation of CD4 T cell mediated killing of Mycobacterium tuberculosis infected macrophages (HUM6P.252)
Authors: Phillips Bonnie , Kaushal Deepak
Published: January 2023

Lymphocyte activation gene-3 (LAG-3) is an immunomodulatory protein that decreases Th1-type immune responses in T cells. We believe that this protein is essential during the host immune response to Mycobacterium tuberculosis (Mtb) infections, where the response is dampened to prevent immunopathogenesis within the lungs. Consequently, overproduction of LAG-3 can be detrimental through a greatly reduced response, causing unhampered bacterial replication and activation of tuberculosis (TB) disease. We have demonstrated that LAG-3 expression is significantly induced in the lungs of nonhuman primates (NHPs) with active TB and is directly correlated to bacterial load and level of infection. LAG-3 is primarily expressed in T cells recruited to the lung. To mechanistically assess the effect of LAG-3 production by T cells on the progression of Mtb infection in macrophages, we have developed a co-culture system where differentiated blood monocytes are infected with Mtb, and supplemented with CD4 T cells harvested from blood and lung of NHPs previously infected with Mtb. Utilizing this model, we are silencing the expression of LAG-3 with siRNA to explore the effect it will have on the immune response to Mtb. We hypothesize that the lack of LAG-3 will result in greater bacterial killing, T cell activation, and enhanced production of Th1-type cytokines. This study will allow us to study the exact role of LAG-3 in the modulation of the immune response to Mtb infection within the granuloma.
Chronic alcohol exposure increases viral expression levels in tissue reservoirs of SIV-infected macaques. (HUM6P.243)
Authors: Robichaux Spencer , Nelson Steve , Bagby Gregory , Amedee Angela
Published: January 2023

Chronic alcohol abuse is associated with increased susceptibility to HIV infection and an accelerated disease progression. Using rhesus macaques infected with SIV and exposed to chronic binge alcohol (CBA), our studies have also shown increased susceptibility to mucosal SIV infection. Additionally, higher viral loads and accelerated disease progression were observed in CBA animals, despite the development of comparable humoral and cell-mediated immunity. In our current studies, we sought to evaluate viral replication kinetics by quantifying viral RNA and DNA levels in tissue reservoirs over 10 weeks of SIV followed by treatment with ART for 40 weeks. Viral RNA and DNA burdens in lymph nodes at peak viremia were similar between treatment groups. At viral set point, comparable proviral DNA levels were also found in lymph nodes of both groups, while CBA-treated animals had significantly higher levels of viral RNA (p>0.01). Following ART treatment, similar levels persisted, with comparable proviral DNA burdens found and significantly higher SIV expression levels observed in CBA-animals (p<0.05). Together, these observations show CBA increases viral transcription to increase viremia and accelerate disease progression. Future studies will investigate the immunological mediators affected by CBA and how they influence on viral expression.
Herpes simplex virus type 1 glycoprotein B-specific effector memory CD8+ T cells are associated with asymptomatic ocular herpes disease in both humans and "humanized" HLA transgenic mice (HUM6P.245)
Authors: Benmohamed Lbachir , Khan Arif , Srivastava Ruchi , Wechsler Steven
Published: January 2023

We have dissected the phenotype and the function of HSV-1 glycoprotein B- (gB-) specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found that: (i) healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM, CD45RAlowCCR7lowCD44highCD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM, CD45RAlowCCR7highCD44lowCD62Lhigh); (ii) ASYMP individuals had significantly higher proportions of multi-functional effector CD8+ T cells, which responded mainly to gB342-350 and gB561-569 “ASYMP” epitopes, and simultaneously produced IFN-g, CD107a/b, granzyme B and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly mono-functional and were directed mainly against non-overlapping gB17-25 and gB183-191 “SYMP” epitopes; (iii) immunization of HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine.
Different allergenicity profile of egg white protein extract vs. purified ovalbumin in an adjuvant-free mouse model (HYP7P.267)
Authors: Gangur Venugopal , Ortiz Tina , Parvataneni Sitaram , Gonipeta Babu , He Yingli , Thi San Su , Nolkemper Martha
Published: January 2023

Food allergies are the leading cause of life-threatening allergic reactions with incompletely understood mechanisms. Here we initially evaluated the allergenicity of egg white protein extract (EWPE) in a novel transdermal sensitization model without adjuvants such as alum/cholera toxin/super-antigen that are commonly used in mouse models of egg allergy. Groups of mice received transdermal exposure (TDE) to EWPE (1 mg/exposure/week) for four weeks. Sensitization was measured by specific IgE antibody (SIgE Ab) levels in plasma using ELISA. Systemic anaphylaxis was assessed upon oral challenge (OC) with EWPE using clinical scoring method, and hypothermia reactions. Four TDEs were sufficient to elicit robust SIgE Ab responses. Upon OC (15 mg/mouse), EWPE elicited near-fatal clinical signs of anaphylaxis associated with rapid hypothermia reactions. We then evaluated allergenicity of purified ovalbumin (OVA) using this model. Groups of mice received TDE to OVA at 4 different doses. Robust OVA SpIgE responses were observed upon TDE. Surprisingly, sensitized mice did not exhibit significant clinical symptoms or hypothermia reactions upon OC with a wide dose range. Nevertheless, robust anaphylaxis was evident upon i.p., injection with OVA. These data suggest that mechanisms of oral egg protein-induced reactions in adjuvant-free model likely depends on whether protein extracts or pure protein from the same food source (egg) is used in model development.
TLR7 is required for both plasmacytoid dendritic cell IFN-I production and subsequent exhaustion during chronic viral infection (INC6P.309)
Authors: Macal Monica , Chang Aaron , Zuniga Elina
Published: January 2023

Type I Interferons (IFN-I) are potent cytokines essential for immune surveillance against many pathogens including persistent viruses such as human immunodeficiency virus (HIV) in humans and Lymphocytic Choriomeningitis Virus (LCMV) in mice. Plasmacytoid dendritic cells (pDCs) are specialized to produce IFN-I upon TLR stimulation, but the molecular mechanisms underlying their initial IFN-I production and the subsequent exhaustion that occurs during chronic viral infection remain to be fully elucidated. Using chronic LCMV infection in mice, we demonstrated that pDCs initially sensed and produced abundant IFN-I in a TLR7-dependent manner. Strikingly, cell-intrinsic TLR7 signaling was also essential for the later pDC IFN-I exhaustion observed in chronic LCMV infection and compromised innate responses against secondary opportunistic pathogens. Interestingly, when analyzing pDC bone marrow progenitors, we found that they were reduced in LCMV chronically infected mice, they had compromised developmental potential and only generated exhausted pDCs. Overall, our studies highlight a dual role for TLR7 in contributing to both induction and subsequent exhaustion of innate responses and suggest a pDC progenitor defect underlying IFN-I exhaustion during chronic viral infection.
Lactic acid attenuates LPS-induced cytokine production in mast cells (INC6P.320)
Authors: Caslin Heather , Abebayehu Daniel , Spence Andrew , Abdul Qayum Amina , Ryan John
Published: January 2023

Elevated lactate levels and attenuated lactate clearance following hospital admission have been associated with increased mortality in sepsis patients. While some effects of lactate on LPS stimulation have been examined in monocytes and macrophages, mast cells have not been examined. Therefore, the purpose of this study was to examine the effect of lactic acid on LPS-induced mast cell activation. Methods: Murine bone marrow derived mast cells were cultured in medium with or without lactic acid for 24 hours. Additionally, sodium lactate and formic acid were examined. Following the pre-incubation period, LPS was added and supernatants were collected after 16 hours for cytokine ELISA analysis. Results: Pre-incubation with lactic acid significantly reduced cytokine (TNF, IL-6) and chemokine (MCP-1, MIP-1a) secretion following LPS stimulation. Sodium lactate pre-incubation did not change cytokine production. However formic acid, which has a similar pKa to lactic acid, also reduced cytokine production compared to the control. Discussion: Lactic acid attenuates LPS-induced mast cell activation, an effect that appears to be dependent upon pH rather than acid chemical structure. Our results suggest that lactic acid may play a suppressive role in mast cell activation during sepsis.
Dectin-1 expressed on pDCs and mDCs displays opposing roles in TH2 polarization (INC6P.321)
Authors: Upchurch Katherine , Joo HyeMee , Zhang Wei , Xue Yaming , Li Dapeng , Li Xiao-Hua , Hori Toshiyuk , Zurawski Gerard , Oh SangKon
Published: January 2023

Dendritic cells (DCs) can induce and control host immune responses. DC subset-dependent functional specialties and their ability to display functional plasticity, which is mainly driven by signals via pattern-recognition receptors (PRRs), identify DCs as immune orchestrators. A PRR, Dectin-1 is expressed on myeloid DCs (mDCs) and is known to play an important role in TH17 responses. Here, we demonstrate that human plasmacytoid DCs (pDCs) express Dectin-1. Interestingly, Dectin-1-activated pDCs promote TH2 induction and activation, whereas Dectin-1-activated mDCs decrease both. This counter-regulation of TH2 by the two DC subsets is mainly due to their distinct abilities to control OX40L expression through different mechanisms. This study provides new insights for the regulation of host immune responses by DCs during microbial infections.
Lactic Acid suppresses IL-33-mediated mast cell inflammatory responses while increasing VEGF production: an Akt- and miR-155-dependent pathway (INM6P.336)
Authors: Abebayehu Daniel , Spence Andrew , Abdul-Qayum Amina , Taruselli Marcela , McLeod Jamie , Ndaw Victor , Baker Bianca , Kolawole Elizabeth , Ryan John
Published: January 2023

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments that also include mast cells and IL-33. The goal of this work is to evaluate how LA modifies IL-33 activation in mast cells. Bone marrow derived mast cells (BMMC) were cultured in the presence of LA and activated with IL-33. BMMC cultured in LA had reduced IL-33-mediated cytokine production. However, LA increased IL-33-mediated VEGF production. Using an MCT1 inhibitor or replacing LA with sodium lactate revealed that LA effects are MCT1 and pH-dependent. Examining IL-33 signaling demonstrated that LA decreased TAK1, JNK, and NFkB phosphorylation, yet increased Akt phosphorylation. An Akt inhibitor blocked VEGF production, but surprisingly enhanced IL-33-mediated cytokine production. Because miR-155 has recently been reported to suppress Akt activation in mast cells, we examined LA effects on miR-155. LA decreased miR-155-5p expression in BMMC. Moreover, BMMC transfected with miR-155-5p were resistant to LA-mediated suppression. Our data demonstrate that lactic acid, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation while increasing angiogenesis.
Modulation of immune responses toward Helicobacter pylori infection by NLRs (INM6P.339)
Authors: Washington Philipson Casandra , Bassaganya-Riera Josep , Viladomiu Monica , Kronsteiner-Dobramysl Barbara , Michalak Pawel , Hontecillas Raquel
Published: January 2023

Helicobacter pylori (HP) colonizes 50% of the world’s population resulting in a decades-long gastric infection. Bacterial interaction with host intracellular environment occurs via injection of bacterial components through a TIVSS or intracellular replication. HP has been recognized for its ability to modulate intracellular NOD-like receptors (NLR). Host responses toward the bacterium can result in asymptomatic, pathogenic or even favorable immunity. Mechanisms underlying the dual role of HP as a commensal versus pathogen are not completely understood. We combined computational modeling, bioinformatics and experimental validation to investigate intracellular host-HP interactions. Global transcriptomic analysis on bone marrow-derived macrophages (BMDM) in a gentamycin protection assay unveiled that intracellular colonization of HP upregulated NOD1, NOD2, NLRP3, NLRC5 and inflammasome components (Caspase-1 and -11) but suppressed regulatory NLRX1 which was inversely correlated to TRAF6, NF-B, proinflammatory cytokines and reactive oxygen species. Loss of NLRX1 facilitates bacterial clearance in BMDM and infected mice. Lastly, we constructed a computational model to shed light on complex immune responses and pathway crosstalk regulated by NLRX1 during infection. In conclusion, NLRX1 is associated with chronic bacterial persistence during H. pylori infection and it may represent an immune evasion mechanism employed by the bacterium to facilitate long-term host colonization.
Cell-type specific control of immune responses mediated by inflammasomes in vivo (INM6P.333)
Authors: Nichols Randilea , von Moltke Jakob , Vance Russell
Published: January 2023

Upon pathogen recognition, certain cytosolic nucleotide-binding domain, leucine-rich repeat containing proteins (NLRs) form multi-protein complexes called inflammasomes, which activate CASPASE-1. CASPASE-1 initiates inflammation by processing IL-1beta and -18 into their active forms, and by inducing a rapid, lytic cell death called pyroptosis. The NLRC4 inflammasome responds to the cytosolic presence of specific bacterial proteins, such as flagellin, via NAIPs. A fundamental question is whether and how activation of innate immune responses triggers pathogen-specific adaptive immune responses. We seek to test the hypothesis that cell-type-specific activation of the inflammasome is sufficient to induce inflammation and adaptive immune responses in vivo. To test our hypothesis, we are using a novel ‘knock-in’ mouse strain. This mouse expresses a fusion protein, which selectively activates the NLRC4 inflammasome. Preliminary data indicate extensive NLRC4-dependent inflammatory disease in mice expressing the fusion protein in myeloid-derived cells.
Disabled-2 negatively regulates lipopolysaccharide-stimulated Toll-like receptor 4 signaling and inflammation response in murine macrophage (INM7P.349)
Authors: HUNG WEI-SHAN , Chou Chein-Shen , Tseng Ching-Ping
Published: January 2023

Toll-like receptor 4 (TLR4)-mediated host response to lipopolysaccharide (LPS) stimulation plays a pivotal role in host defense against from Gram-negative bacterial infection. Upon ligand binding, TLR4/MD2 complexes are internalized to form endocytic signalsomes and extend LPS response. However, the underlying mechanism of LPS-stimulated TLR4 internalization and signaling has not yet been completely understood. Here we delineated whether the endocytic adaptor protein Disabled-2 (Dab2) that is highly expressed in macrophage plays a role in TLR4 signaling and trafficking. In vivo analysis of the myeloid lineage-restricted Dab2 knockout (Dab2-/-) mice revealed that the mice were more susceptible to LPS challenge leading to an overproduction of the chemokine RANTES in serum and poor survival. The functional role of Dab2 in TLR4 signaling was investigated further using RAW264.7 macrophage with Dab2 knockdown. We found that attenuation of Dab2 expression increased the basal level of cell surface TLR4/MD2 complexes and accelerated the rate of LPS-induced down-regulation of these complexes from the cell surface. Consequently, TLR4 endocytic pathway-dependent IRF-3 activation was augmented with an increased expression for the subsets of inflammatory cytokine and IFN-inducible genes. These data indicate that Dab2 functions as a negative regulator of TLR4 endocytic pathway and signaling, supporting a novel role of Dab2 in the inflammatory response of macrophage to endotoxin.
Analysis of genes associated with pulmonary mucosal defense against influenza A virus infection (INM8P.402)
Authors: Samarasinghe Amali , Lin Yanyan
Published: January 2023

Influenza A virus infects affect a large population worldwide every year. Antigenic drifts and shifts, and re-assortments in influenza A viruses contribute to reduced host protection. Early host immune defenses may also vary based on the virus strain. A murine model system is one of the best in vivo models to study the host immune defense against Influenza A virus. We hypothesized that host mucosal defenses are initiated in the lung mucosa by expression of antimicrobial peptides. We infected C57BL/6 female mice with 1000 TCID50 A/CA/04/2009 (pH1N1) or A/PR/08/1934 (PR8) strains of influenza A virus and quantified changes in cell recruitment and antimicrobial peptide gene expression at 0, 1, 3, 9, 24, 48, and 120 hours. Both viruses efficiently replicated in mice with peak viral production at 48 hours. However, PR8 caused more damage to the host, marked by increased recruitment of neutrophils, lymphocytes and macrophages and viral burden compared to pH1N1. Genes associated with antimicrobial peptides (RELM, MBD4, CRAMP, PLUNC) were upregulated starting at 3 hours post infection. These genes were more highly expressed in response to PR8 virus. Our data suggest that the proteins encoded by these genes may initiate the immune response against influenza A virus infections and that the host mucosal response varies depending on the strain of virus.
Identification of mouse cytomegalovirus immunoevasins that modulate NK cell recognition via the NKR-P1B:Clr-b axis (INM8P.403)
Authors: Carlyle James , Samaniego Jackeline , Suzuki-Sampaio Isabella , Lau Timothy , Rahim Mir Munir , Popovic Branka , Krmpotic Astrid , Jonjic Stipan , Makrigiannis Andrew , Allan David , Aguilar Oscar
Published: January 2023

Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing pathological target cells through germline-encoded receptor-ligand interactions. Mouse cytomegalovirus (MCMV) is a beta-herpesvirus that has co-evolved with its murine host, and as such, a significant portion of its genome encodes immunoevasins that directly target NK cell receptor-ligand interactions. Here, we identify two putative immunoevasins that modulate host-pathogen interactions via the inhibitory NKR-P1B:Clr-b recognition system. First, we identify an m145 family member that actively prevents MCMV infection-mediated Clr-b downregulation. Ectopic expression of this m145 family member in mouse fibroblasts increases Clr-b expression at the cell surface. In contrast, infection with an m145-family deletion mutant shows more substantial Clr-b loss at the cell surface compared to wild-type MCMV infection. Importantly, enhanced Clr-b loss upon infection with the deletion mutant could be reversed by complementation experiments involving overexpression of the m145 family immunoevasin. Secondly, we have identified an immunoevasin that directly interacts with the NKR-P1B inhibitory receptor, suggestive of decoy ligand function. This interaction is MCMV strain-dependent and host NKR-P1B allele-specific, suggesting that polymorphisms have evolved at both the pathogen and host levels that affect NK cell recognition of infected target cells.
The ameliorative effects of JAK1 inhibition by hypnotic bromvalerylurea in CLP induced rats sepsis model. (INM8P.362)
Authors: Kikuchi Satoshi , Kawasaki Shun , abe Naoki , Watanabe Yuji , Aibiki Mayuki
Published: January 2023

Presently, there are no approved drugs to treat sepsis, despite clinical trials of many agents that have distinct targets. Therefore a novel effective treatment should be developed. Objectives: We recently observed marked immunosuppressive effects of an old hypnotic drug, bromvalerylurea (BU), on lipopolysaccharide (LPS)-activated macrophages. The objective was to elucidate molecular mechanisms underlying the anti-inflammatory effects of BU and to determine whether BU ameliorated cecum ligation and puncture (CLP)-induced sepsis. Interventions: LPS-activated macrophages were incubated in vitro with BU. After CLP, some rats were subjected to subcutaneous injection with BU. Methods: Immunoblotting, quantitative real-time RT-PCR, immunohistochemical staining and, protein-knock down with siRNA. Results: BU suppressed a variety of pro- and anti-inflammatory mediators. LPS induced a two-step program of pro-inflammatory activation of macrophages; LPS first induced nuclear translocation of NF-κB, and then caused activation of Janus kinase 1/Signal transducer and activator 1 (JAK1/STAT1) that lead to interferon regulatory factor 1 expression as a consequence of NF-κB translocation. BU suppressed JAK1 activity. BU prevented the onset of MOF and increased survival in the septic rats. Conclusions: The JAK1/STAT1-dependent pro-inflammatory pathway could represent a promising novel drug target for treating sepsis. BU is worth reevaluating as a novel agent for sepsis and related disorders.
Ligand binding and signaling by IFI16 (INM8P.400)
Authors: Xiao Tsan , Jin Tengchuan
Published: January 2023

IFI16 plays important roles in inflammatory immune responses to viral infections as well as in autoimmune disorders such as lupus. It has been implicated in interferon induction through a STING-mediated pathway, as well as in inflammasome activation during recognition of viral DNA in the nucleus. Despite recent progress that highlighted the mechanisms of dsDNA binding by the IFI16 HINb domain, there are major gaps in our understanding of the ligand-induced structural changes, the polymerization of IFI16 mediated by dsDNA and its PYD domain, its interaction with downstream signaling partners, the regulation of IFI16 by host and microbe-derived molecules, and the cell-type specific immune responses mediated by IFI16. We have characterized the key structural determinants in both the recognition of dsDNA by IFI16 and the potential downstream signaling events. We will outline the implications of our findings and discuss future directions in clarifying the structure and function of IFI16 in infectious diseases and autoimmune disorders.
Analysis of Tpl2-deficient mice reveals functional redundancies in the regulation of IFNγ production by NK cells during MCMV infection (INM8P.401)
Authors: Watford Wendy , Rowley Sean , Tarrio Margarite , Lee Seung-Hwan , Hardwick Kara , Boissel Laurent , Kirkland Rebecca , Tsichlis Philip , Biron Christine
Published: January 2023

The serine-threonine kinase, Tpl2, promotes innate inflammatory responses and is considered as a therapeutic target for autoimmunity. We have demonstrated that Tpl2 is required for efficient IFNγ secretion by CD4+ T cells and Th1 effector functions, but little is known about how Tpl2 regulates IFNγ production by other cells, including natural killer (NK) cells. Herein, we addressed the contribution of Tpl2 to the development and function of NK cells. NK cell development and cytotoxic activity occurred independently of Tpl2. In contrast, IL-12-induced IFNγ secretion was nearly abrogated in Tpl2-/- NK cells but could be restored by concomitant IL-18 stimulation. Impaired IFNγ secretion was not attributed to differences in IL-12R expression or Stat4-dependent signals. Furthermore, only a modest reduction in IL-12-induced IFNγ mRNA expression was observed, suggesting that Tpl2 regulates IFNγ production primarily via translational control. Consistent with in vitro findings, Tpl2-/- mice mounted a diminished NK-derived systemic IFNγ response to murine cytomegalovirus (MCMV) infection, which could be compensated for in ex vivo restimulation assays by the presence of MCMV infection-induced IL-18 secretion. These data demonstrate the importance of Tpl2 in IL-12-induced NK cell IFNγ secretion. Furthermore, the synergism and functional redundancy of the IL-18 pathway with the IL-12-Tpl2 pathway underscores the criticality of ensuring appropriate NK-derived IFNγ to anti-viral immunity.
Depletion of gut microbiota protects the kidney against ischemia/reperfusion injury (IRC10P.409)
Authors: Emal Diba , stroo Ingrid , Teske Gwendolin , Claessen Nike , Butter Loes , florquin sandrine , Leemans Jacklien , Dessing Mark
Published: January 2023

An accumulating body of evidence shows that gut microbiota play an important role in health and disease by modulating local and systemic immunity. Peptidoglycan, known to be turned over from the microbiota and translocated into the circulation, might play a central role in this immunomodulation via interaction with its receptors NOD1 and NOD2 present on leukocytes. The importance of the microbiome in several inflammatory diseases is known but its role in kidney disease is limited. To further study this, we performed renal ischemia reperfusion injury (IRI) in Wt and Nod1/2 KO mice with or without depleted gut microbiota using broad-spectrum antibiotics. Our results showed that in Wt mice, depletion of microbiota significantly attenuated renal damage, dysfunction and granulocyte influx after renal IRI. Isolated granulocytes from antibiotic treated Wt mice were less responsive to several TLR/NOD ligands compared to untreated Wt mice while migration and oxidative burst were similar. No evidence was found for bacterial translocation during renal injury, intestinal permeability was present but unaffected by the severity of kidney damage. Nod1/2 KO mice displayed similar renal dysfunction and damage compared to Wt mice following renal IR. In conclusion, we show that depletion of gut microbiota reduced renal IRI in a NOD1/2-independent way.
Two-photon imaging of endogenous regulatory T cell dynamics and suppression of helper T cell priming mediated by CTLA-4 (IRC11P.441)
Authors: Othy Shivashankar , Matheu Melanie , Greenberg Milton , Dong Tobias , Schuijs Martijn , Deswarte Kim , Hammad Hamida , Lambrecht Bart , Parker Ian , Cahalan Michael
Published: January 2023

Despite the major role played by regulatory T cells (Tregs) in immune homeostasis, our knowledge on mechanisms involved is still incomplete. Using two-photon microscopy we characterize the behavior of endogenous Tregs under steady-state conditions, during inflammation, and during an antigen-specific T cell response in lymph nodes. Under steady-state conditions, endogenous Tregs maintain the highest average velocities of any cells in the lymph node and exist as two non-overlapping populations that explore the T-zone and the B cell follicle. In the T-zone, Tregs migrate more rapidly than conventional T cells (Tconv), extend longer processes, and interact with resident dendritic cells (DC) and Tconv. Under inflammatory conditions, Tregs engage migratory DCs near the lymph node capsule, occasionally form long-lived Treg-DC interactions. During antigen specific immune response, Tregs interact with antigen-induced DC:Tconv clusters, while continuing to form contacts with activated Tconv. We investigated the role of CD80/86-CTLA4 interactions during Tconv-Treg cell interactions. Blocking CTLA-4 reduces Treg-Tconv interaction times, increases the volume of DC:Tconv clusters, and enhances proliferation of antigen-specific Tconv in vivo. Furthermore, genetic deletion of CD80/CD86 on Tconv makes them less susceptible to inhibition by Tregs. Our results demonstrate a role for altered cellular choreography of Tregs through CTLA4-based interactions to limit helper T cell priming.
IRF4 regulates the ratio of T-bet to eomesodermin in CD8+ T cells responding to persistent LCMV infection (IRM14P.447)
Authors: Nayar Ribhu , Schutten Elizabeth , Jangalwe Sonal , Kenney Laurie , Daniels Keith , Brehm Michael , Welsh Raymond , Berg Leslie
Published: January 2023

CD8+ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8+ T cell effector functions in previously exhausted CD8+ T cells. TCR stimulation is also important for the differentiation of Eomeshi CD8+ effector T cells from T-bethi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8+ T cells by modulating levels of IRF4. Reduced IRF4 expression results in lower proportions and numbers of T-bet+ Eomes- precursors and poor control of LCMV Clone 13 infection. Altering the ratio of T-bet to Eomes by generating compound heterozygotes of Irf4 and Eomes restores the differentiation of T-bet+ Eomes- precursors and leads to efficient viral control. These data highlight a critical role for IRF4 in regulating CD8+ T cell responses by ensuring a protective ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.
Studying the antagonistic relationship between NKG2D and TGF-β in CD8+ T cells (IRM14P.449)
Authors: Prajapati Kushal , Guevara-Patino Jose
Published: January 2023

One of the major hurdles in cancer vaccine and adoptive T cell transfer therapy (ACT) development is diminished effectiveness and persistence of T lymphocytes due to immuno-suppressive factors. TGF-β is highly prevalent immuno-suppressive factor secreted by tumor which negatively regulates persistence and functionality of CD8+ T cells. Natural killer group 2, member D (NKG2D) is a stimulatory receptor majorly expressed on the surface of NK and CD8+ T cells. Others including our lab have shown that NKG2D activation on CD8+ T cells enhances their function, proliferation and persistence. Accordingly, we hypothesized that NKG2D signaling reverses the inhibitory effects of TGF-β on CD8+ T cells. We found that in vitro NKG2D activation prevented TGF-β mediated inhibition of proliferation and granzyme B expression in human CD8+ T cells. Interestingly, we also found that TGF-β down regulates the surface expression of NKG2D in human and mouse CD8+ T cells. In the presented research we will test two alternative hypotheses to elucidate molecular mechanism underlying NKG2D and TGF-β’s antagonistic effects. NKG2D opposes TGF-β’s suppressive effects in CD8+ T cells by i) repressing levels of transcription factor T-bet ii) increasing levels of inhibitor of TGF-β signaling-regulator of G-protein signaling 3 (RGS3). The findings from this research will form basis to develop a therapeutic strategy to reverse TGF-β’s immunosuppressive effects in vivo that results in better anti-tumor immunity.
Foxo3 controls CD4 T cells differentiation and severity to neuroinflammation (IRM15P.604)
Authors: Stienne Caroline , Michieletto Michael , Carrié Nadège , Saoudi Abdelhadi , Dejean Anne
Published: January 2023

The Foxo3 transcription factor regulates cell cycle progression, survival and DNA repair pathways. In addition to its role as a tumor suppressor gene, studies have established the crucial role of Foxo3 in immune cells, notably in dendritic cells and CD8 T cells in the context of a viral infection. However, the role of Foxo3 in CD4 T cell function is still unknown. Here, we show that Foxo3 expression levels are increased in CD4 T cells after TCR engagement. We also show that Foxo3 deficiency in CD4 T cell leads to decreased secretion of IFN-γ and GM-CSF but not of IL-17. By dissecting the underlying molecular mechanisms, we have identified a new direct target gene of Foxo3 that participates to IFN-γ production and that was previously identified as a susceptibility gene for multiple sclerosis. This led us to hypothesize that Foxo3 could be involved in the susceptibility to central nervous system inflammation. To test this hypothesis, we used the well-established animal model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE) induced by MOG immunization. We show that Foxo3 deficiency is associated with significantly decreased EAE severity and that Foxo3-deficient MOG-specific CD4 T cells fail to differentiate into pathogenic IFN-γ+GM-CSF+cells. Thus, our results show for the first time that Foxo3 plays a crucial role in the pathophysiology of neuroinflammation by directly controlling the expression of genes involved in CD4 T cell differentiation.
Essential role of the Ets transcription factor GABP in control of T cell responses to antigen stimulation (IRM15P.454)
Authors: Luo Chong , Osmanbeyoglu Hatice , Leslie Christina , Li Ming
Published: January 2023

Upon antigen stimulation, naive T cells transition from cellular quiescence into cell cycle progression and initiate functional differentiation. Such a coordinated response is orchestrated by numerous transcriptional programs, among which include Ets protein-dependent regulation. The Ets family of transcription factors, characterized by an evolutionarily conserved DNA-binding domain, is modulated by T cell receptor (TCR) signaling; yet the specific Ets member that is crucial for antigen receptor-mediated response remains elusive. GA-binding protein (GABP), which is composed of GABPα and GABPβ, is the only obligate multimeric complex among Ets factors. T cell-specific ablation of Gabpa gene in mice leads to a profound reduction in peripheral T cells. In response to antigen stimulation in vitro, GABPα-deficient T cells show diminished proliferation, dysregulation of reactive oxygen species and impaired cell survival. In addition, mice lacking GABPα fail to mount an antigen-specific T cell response to Listeria Monocytogenes infection. Transcriptome analysis coupled with chromatin immunoprecipitation sequencing identifies GABPα as a key regulator of the folate-dependent one carbon metabolism as well as cellular balance of redox. Our findings reveal critical functions of GABP-dependent transcriptional program in the control of TCR-stimulated metabolic reprogramming.
COX-1 derived thromboxane A2 plays an essential role in early B cell development via regulation of JAK/STAT5 signaling in mice (LYM7P.619)
Authors: Zhou Jie
Published: January 2023

Cyclooxygenases (COXs) and their prostanoid products play important roles in a diverse range of physiological processes, including in the immune system. Here, we provide evidence that COX-1 is an essential regulator in early stages of B cell development. COX-1-deficient mice displayed systematic reduction in total B cells, which was attributed to the arrest of early B cell development from pro-B to pre-B stage. We further demonstrated that this defect was mediated through down-regulation of the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling and its target genes, including Pax5, in COX-1-/- mice. Mechanistic studies revealed that COX-1 derived thromboxane A2 (TxA2) could regulate JAK3/STAT5 signaling through cAMP-PKA pathway, via binding with its receptor-TP. Administration of TP agonist could rescue the defective B cell development and JAK/STAT5 signaling activity in COX-1-deficient mice. Moreover, administration of low-dose aspirin caused a significant reduction in total B cells in peripheral blood of healthy human volunteers, coincidentally with reduced TxA2 production and downregulation of JAK/STAT5 signaling. Taken together, our results demonstrate that COX-1 derived TxA2 plays a critical role in the stage transition of early B cell development through regulation of JAK/STAT5 signaling, and indicate potential immune-suppressive effect of low-dose aspirin in human. 
LAT-mediated TCR signaling is required for optimal function and clonal expansion but not long-term survival and homeostatic turnover of DETCs (LYM7P.610)
Authors: Zhang Baojun , BOCK CHERYL , Wu Jianxuan , Jiao Yiqun , Chen Benny , Chao Nelson , Zhang Weiguo , Zhuang Yuan
Published: January 2023

Dendritic Epithelial T Cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout life. DETCs have limited antigenic specificity due to their exclusive usage of a canonical TCR. While the requirement of TCR in DETC development has been clearly demonstrated, the role of TCR in DETC function and lifelong maintenance is still poorly defined. To investigate the role of TCR signaling in DETCs, we used a γδ lineage specific and inducible Cre allele to delete the gene encoding Linker Activation in T cells (LAT), an essential signaling molecule downstream of the TCR. We found that LAT-mediated TCR signals in DETCs were required for proper activation of effector genes and optimal clonal expansion. The function of DETCs in wound healing was also dependent on LAT. Lack of proper TCR signaling prevented DETCs from efficient production of growth factors and cytokines involved in wound healing. Surprisingly, we found that long-term maintenance of DETCs in the skin was not affected by deletion of LAT. These findings demonstrate that LAT-mediated TCR signaling is required for optimal function and clonal expansion of DETCs and implies a LAT-independent mechanism for long-term survival and homeostatic turnover of DETCs in the skin epithelium.
Notch is a signal integrating hub that controls differentiation of effector CD8+ T cells (LYM7P.614)
Authors: Amsen Derk , Backer Ronald , Helbig Christina , Kent Andrew , Dominguez Claudia , Laidlaw Brian , willemsen marcel , van Kampen antoine , Rimmelzwaan Guus , Kaech Susan , Magarian Blander Julie
Published: January 2023

Activated CD8+ T-cells must choose between different effector cell fates. Two major fates are the short lived terminal effector cell (TEC) fate and the memory precursor cell (MPC) fate. Differentiation of immediately protective TECs is proportional to the severity of the infection, suggesting the existence of signals that relay information about the infection to promote the generation of this effector cell type. We here show that the choice between the TEC and MPC fates is governed by inductive signaling via Notch. This cell surface receptor promotes differentiation of TEC and is correspondingly required for clearance of influenza-virus infection. Notch activates a major portion of the TEC-specific gene expression program and suppresses the MPC-program, thus creating maximal separation between the two cell fates. Influenza infection induces ligands for Notch on lung derived migratory dendritic cells in mediastinal lymph nodes. Notch receptors are induced on naïve CD8+ T-cells by inflammatory mediators and IL-2 via mTOR and T-bet dependent pathways. These pathways are subsequently amplified downstream of Notch, thus creating a positive feedback loop to bolster full adoption of the TEC fate. Our results show that Notch functions as a central hub where signals from different sources of information converge and refuel to match effector T-cell differentiation to the demands of the infection.
An epigenetic mechanism of Dll4-facilitated Foxp3+ regulatory T cells differentiation (LYM8P.637)
Authors: Ting Hung-An , Schaller Matthew , Ptaschinski Catherine , de Almeida Nagata Denise , Kunkel Steven , Maillard Ivan , Lukacs Nicholas
Published: January 2023

Notch signaling facilitates inducible Foxp3+ regulatory T cells (iTreg) differentiation. Epigenetic modifications, which include histone modifications, are critical in cell differentiation. Recent genome-wide studies showed that iTreg have increased trimethylation on histone H3 at lysine 4 (H3K4me3) around Foxp3 loci, but the role of Notch signaling and its underlying epigenetic mechanisms remain elusive. Here we report that Delta-like ligand 4 (Dll4) but not Jagged1 can activate Notch signaling, facilitate Foxp3+ iTreg differentiation, and increased H3K4me3 around Foxp3 promoter and conserved noncoding sequences (CNS). We performed epigenetic enzyme array analysis and found one histone methyltransferase—SET and MYND domain containing protein 3 (Smyd3) that is abundant in iTreg. Dll4/Notch signaling amplified and accelerated Smyd3 expression in vitro during iTreg cell differentiation via an RBPJ-mediated transcription activation. Inhibition of Notch signaling decreased Smyd3 expression. When Smyd3 was genetically knocked out in CD4 T cells, the Dll4-enhanced H3K4me3 around the Foxp3 loci and Foxp3+ Tregs were decreased in vitro and in vivo. These results demonstrate that Notch ligand Dll4 can epigenetically regulate Foxp3+ Tregs through Smyd3.
Systems biology approach to determine critical T cell receptor signaling events for the induction of regulatory T cells (LYM8P.639)
Authors: Sheehan Robert , Hawse William , Morel Penelope , Faeder James
Published: January 2023

Antigen stimulation of the T cell receptor (TCR) in a naïve T cell triggers a complex network of signaling pathways that determine whether the cell becomes a tolerogenic T regulatory (Treg) or immunogenic T helper (TH) cell. We previously developed a Boolean model of T cell differentiation that predicted variation of TCR signal duration could be used to optimize the production of Tregs, a strategy that we confirmed in vitro. This model suggested that dynamics of PTEN, a lipid phosphatase that opposes the activation of Akt, might be predictive of cell fate. Subsequent experiments have demonstrated that several mechanisms for regulating PTEN, including posttranslational modification, proteasomal degradation, and transcriptional inhibition, play an important role in T cell fate determination. A more fine-grained computational model of T cell signaling, which considers site-specific phosphorylation of Akt and PTEN, negative regulation of mTORC2 by kinases downstream of Akt, and negative regulation of PTEN through several Akt-dependent and independent mechanisms, recapitulates the dose-dependent dynamics of PTEN and Akt activity. The net effect of these positive and negative feedback interactions is to create a strength- and time-dependent threshold for TCR stimulation to drive the sustained Akt activity required for the differentiation and proliferation of TH cells. The model also shows that sub-threshold TCR signals lead to transient Akt activation leading to a Treg phenotype.