bluuebunny/crossref_metadata_2025 huggingface dataset:
HMGB1 increases IL-8 secretion under hypoxic condition in human nasal epithelia (INC1P.358)
Authors: Min Hyun Jin , Yoon Joo-Heon , Kim Chang-Hoon
Published: January 2023

Objectives High Mobility Group Box 1 (HMGB1) is a nuclear protein. It has been found that HMGB1 can be secreted into extracellular area via various mechanisms, and extracellular HMGB1 have a potential to mediate proinflammatory signaling. The aim of the present study was to investigate the role of HMGB1 in upper airway epithelial cells under hypoxic condition. Materials and Methods We cultured normal human nasal epithelial cells (NHNE). Cells were incubated in hypoxic condition and evaluated whether HMGB1 was secreted into extracellular area. Next, we harvested human nasal mucosa samples and nasal lavage fluids which were supposed to be hypoxic and non-hypoxic condition. We compared the expression of HMGB1 in human nasal mucosa samples by immunohistochemistry and the amount of HMGB1 in lavage fluids using ELISA assay. Results We found that hypoxia induced translocation of HMGB1 into extracellular area in NHNE cells and it was ROS dependent. We identified that secreted HMGB1 protein was participating in up-regulation of IL-8. In addition, much more cytoplasmic HMGB1 was identified in hypoxic conditioned human sino-nasal mucosa compared to control. The amount of HMGB1 in nasal lavage fluids was also increased in hypoxic conditioned patients compared to control Conlcusion: These findings suggest that HMGB1 plays a role in the pathogenesis of hypoxia-mediated inflammation in upper airway epithelium.
CD6 is a novel positive regulator of natural antibody producing B-1a cells (INC1P.401)
Authors: Enyindah-Asonye Gospel , Li Yan , Singer Nora , Gupta Neetu , Fung John , Lin Feng
Published: January 2023

Although CD6 has recently been identified as a risk gene for multiple sclerosis, its biological function remains unclear, partially due to the lack of CD6 gene engineered animals. It is established that CD6 is present on all T cells in both humans and mice; however, it is believed that mouse B cells do not express CD6. B-1a B cells are the primary source of natural antibodies (Nabs), which provide the first line of defense against infection and facilitate the clearance of oxidized lipoproteins that contribute to the pathogenesis of atherosclerosis. By studying CD6 knockout (KO) and WT mice, we found that CD6 is basally expressed selectively on splenic B-1a B cells. CD6 KO mice exhibit smaller B1a B cell population in the spleen concomitant with lower titers of Nabs in the serum than those in WT mice. Interestingly, B-1a-cell-specified progenitors also express CD6 and the frequencies of these progenitors are significantly reduced in the CD6 KO mice. B cell receptor (BCR) signaling is critical for B1a B cell development and T-independent immunoglobulin response. CD6 KO B1a B cells displayed impaired BCR-induced JNK activation and elicited impaired T-independent antibody responses to NP-Ficoll. These findings suggest a positive role for CD6 in regulating B1a B cell homeostasis and immune response.
West Nile Virus infected human dendritic cells induce natural killer T cells to produce anti-inflammatory cytokines (INC1P.406)
Authors: Kovats Susan , turner sean , Simmons Amie , Alberola-Ila Jose
Published: January 2023

West Nile Virus (WNV) infection is an emerging mosquito-borne zoonosis in the U.S. WNV initially replicates in keratinocytes and skin resident dendritic cells (DCs). Infected DCs in the skin or cutaneous lymph nodes are likely to interact with Invariant Natural Killer T cells (iNKTs), an innate effector cell type important for control of pathogen infections. Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections, thus controlling viral load and regulating adaptive immunity. iNKTs can be activated indirectly by inflammatory cytokines or stimulated by lipid antigens presented by CD1d. To assess the DC-iNKT interaction upon WNV infection, we infected human monocyte-derived DCs with WNV and studied their innate immune responses and their ability to activate isolated blood iNKTs. Infected DCs synthesized viral Envelope mRNA and protein, and upregulated costimulatory molecules but showed decreased expression of CD1d. Infected DCs secreted type I interferon, but not IL-12, IL-23, IL-18 or IL-10. Unexpectedly, the WNV-infected DCs stimulated human iNKTs to upregulate CD69 and produce IL-10, but not proinflammatory cytokines. In contrast, incubation of DCs with the TLR3 ligand polyIC did not lead to iNKT IL-10 production, suggesting that DC activation by viral RNA alone does not trigger this iNKT response. In sum, WNV-infected human DCs producing type I interferon induce iNKTs to secrete IL-10 but not proinflammatory cytokines.
Peptidoglycan recognition protein 3-regulated microbiome maintains healthy levels of Treg cells and protects mice from colitis (INC1P.346)
Authors: Gupta Dipika , Jing Xuefang , Dziarski Roman
Published: January 2023

Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with inflammatory bowel disease in humans. In this study we examined the role of T cells in the increased sensitivity of Pglyrp3-/- mice to dextran sodium sulfate (DSS)-induced colitis. We demonstrate that untreated Pglyrp3-/- mice had significantly lower levels of anti-inflammatory Treg cells (CD4+Foxp3+) and significantly higher levels of pro-inflammatory Th17 (CD4+IL17+) cells in the colon than WT mice. Specifically, Helios- but not Helios+ Treg cells were significantly lower in the colon of Pglyrp3-/- mice than WT mice, which suggests that induced Treg cells were decreased in Pglyrp3-/- mice. WT germ-free mice colonized with Pglyrp3-/- stools had lower levels of Treg cells and higher levels of Th17 cells than WT germ-free mice colonized with WT stools. The lower number of Treg cells and higher number of Th17 cells correlated with the increased sensitivity to colitis in the germ-free mice. We further demonstrate that increasing the numbers of Treg cells in the colon with vitamin A or neutralizing IL17 with antibody protected Pglyrp3-/- mice from increased sensitivity to DSS-induced colitis. Thus our data indicate that Pglyrp3-regulated microbiome promotes Treg cells, inhibits Th17 cells and protects from DSS-induced colitis.
Antigen-specific MAIT cell subsets involved in TB immunity (INC2P.420)
Authors: Sakala Isaac , Kjer-Nielsen Lars , Eickhoff Christopher , Wang Xiaoli , Blazevic Azra , Liu Ligong , Fairlie David , Rossjohn Jamie , McCluskey James , Fremont Daved , Hansen Ted , Hoft Daniel
Published: January 2023

Mucosal associated invariant T (MAIT) cells have a semi-invariant TCRVα chain, and depend on riboflavin metabolites (ribityllumazines, RLAg) produced by commensal flora and presented by the nonpolymorphic class Ib molecule MR1 for optimal development. We used MR1/RLAg tetramers and TCR Vα19 transgenic mice to study MR1-dependency, subset heterogeneity and protective effector functions important for tuberculosis (TB) immunity. We found that Vβ6/8+NK1.1+ MAIT cell subsets maximally expand peripherally in mice expressing MR1. In addition, we show that MR1/RLAg tetramer+ MAIT cells expressing CD4, CD8 or neither contain populations variably co-expressing Vβ6/8 and/or NK1.1, indicating that MAIT cells are more heterogeneous than previously thought. Furthermore, NK1.1 (NKR-P1C) is minimally expressed on mature Vβ6/8+ tetramer+ MAIT cells in the thymus but progressively increases with peripheral activation. Importantly, after virulent mycobacterial pulmonary infection, heterogeneous subsets of tetramer+ MAIT cells (CD4-CD8->CD4-CD8+>CD4+CD8-) co-expressing Vβ6/8 and NK1.1 and the homing molecules α4β1 and CXCR3, were recruited into the lungs and afforded maximal early protection in MR1-sufficient mice. Surprisingly, MAIT cells developing in the absence of MR1 were partially protective. Overall, we demonstrate diversity of MAIT cells, evidence that NK1.1 is an activation rather than developmental MAIT cell marker, and also that MAIT cells are important for TB protective immunity.
Restoration of electric footshock-induced immunosuppression in mice by Modified Aloe Polysaccharide (INC2P.411)
Authors: Lee Youngjoo , Kim Jiyeon , Lee Sungwon , Kwon Junghak , Kong Hyunseok , Song Youngcheon , Lee Chong-Kil , Kim Kyungjae
Published: January 2023

A mutual relationship between stress and immune system has long been studied. To investigate the immunomodulatory effects of MAP (modified aloe polysaccharide) on stressed model, we induced electric foot shock on mice for 17days. Oral administration of MAP was found to prevent the atrophy of body weight and immune apparatus weights. The stress-induced disturbances in cellularity were also restored by MAP administration in both thymus and spleen. In addition, MAP treatment improved lymphocytes proliferation induced by several antigens. The model antigen OVA-peptide was designed to examine the restoration effects of MAP in terms of specific immunity. The oral administration of MAP considerably improved the proliferation of OVA-specific CD4+T cells. We also found that MAP treated mice generated more potent ova specific cytotoxic T cells compared to MAP untreated group. In a sense of humoral immunity, OVA specific IgG production level was increased by the oral administration of MAP. According to the analysis of stress-related hormones including dopamine, serotonin and corticosterone, the oral administration of MAP tends to reduce the changes in hormone levels between the stressed and unstressed groups. Lastly, elevated plus-maze (EPM) test showed the anxiolytic effects of MAP in stressed mice. These results consistently demonstrate that oral administration of MAP could increase host defense toward chronic stress induced by electric foot shock.
Identification of polarized macrophage signatures (INM1P.439)
Authors: Guerau-De-Arellano Mireia , Jablonski Kyle , Amici Stephanie , Popovich Phillip
Published: January 2023

Pro-inflammatory (M1) macrophages are implicated in inflammatory diseases while pro-regenerative (M2) macrophages may be beneficial in Central Nervous System disorders. To modulate these phenotypes for therapeutic purposes, we need further understanding of the signaling pathways that define and distinguish M1 versus M2 macrophages. Here, we investigated the signature, i.e., the patterns of gene up/down-regulation, associated with mouse M1 and M2 polarized macrophages through cDNA microarray. The M1 macrophage signature was formed by 629 up-regulated and 732 down-regulated genes while M2 macrophage up-regulated 388 genes and down-regulated 425 genes. Additional analyses revealed that differentiation into M1 or M2 macrophages shared 81 up-regulated and 125 down-regulated genes which we consider to be linked to macrophage activation. In contrast, we identified 548 genes unique to M1 and 307 genes unique to M2 macrophages, including canonical markers previously used to distinguish phenotypes. While some of these genes have been previously identified, novel candidate M1/M2 markers were identified and validated by Real-Time PCR analysis of independent datasets. Our data reveals that macrophage differentiation into opposing M1/M2 phenotypes is formed by a combination of common and distinct genes expression patterns and provides novel genes as candidate differential markers and therapeutic targets.
Natural cytotoxicity receptor NKp44 and NK - astrocyte interactions in HIV-associated neurocognitive disorders (INM1P.443)
Authors: Bowen Kelly , Borgmann Kathleen , Ghorpade Anuja , Mathew Porunelloor
Published: January 2023

NK cells play important role in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. NK cells were shown to be recruited to the CNS following several pathological conditions. NK cells could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. Natural cytotoxicity receptors (NCRs) play critical role in the cytolytic function of NK cells. Here we have studied the expression and function of natural cytotoxicity receptor NKp44 upon NK-astrocyte interactions in the presence or absence of an HIV peptide (3S peptide) shown to induce NK cell killing of CD4+ T cells during HIV infection. Using a fusion protein consisting of the extracellular domain of NKp44 and human IgG Fc, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study showed that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection and impacts astrocyte role in HAND.
Contribution of adipocytes in the regulation of metabolic inflammation: the role of osteopontin (INM1P.429)
Authors: Lyroni Konstantina , Ieronymaki Eleftheria , Patsalos Andreas , Tsatsanis Christos
Published: January 2023

Adipose tissue is not only involved in energy storage but also functions as an immuno-regulatory organ that participates in the establishment of obesity-related chronic low-grade inflammation. In this work we questioned whether adipocytes themselves could acquire a pro- or anti- inflammatory phenotype and determine potential signaling molecules affecting this process. The results showed that MEF-derived adipocytes were activated by LPS or IL-4. Si-RNA library screening allowed the identification of signaling molecules regulating iNOS and Arg1 expression and showed that PPARγ STAT3 and Akt2 were central regulators. In vivo, we found adipocytes from Akt2-/- mice or from HFD-induced obese mice had increased expression of iNOS and Arg1 and the pro-inflammatory cytokine osteopontin (OPN). Surprisingly, genetic deletion of OPN resulted in increased responsiveness of adipocytes to LPS as determined by elevated expression of iNOS and NO production. Despite the increased NO production Arg1 and IL-10 were also higher in OPN-/- adipocytes suggesting that OPN affects additional signaling events that suppress adipocyte activation. Further analysis determined the signaling components affected by OPN that regulate inflammatory factors in adipocytes. Our findings suggest that adipocytes obtain pro- or anti-inflammatory properties, which are affected by obesity. OPN partly regulates the anti-inflammatory properties of adipocytes by initiating anti- inflammatory cascades.
Myeloid-derived suppressor cells contribute to maintaining allogeneic pregnancies (IRC4P.461)
Authors: Sinha Pratima , Carter Darryl , Ostrand-Rosenberg Suzanne
Published: January 2023

In pregnant women the fetus is an allograft and successful human pregnancies require maternal tolerance to fetal alloantigens. Myeloid-derived suppressor cells (MDSC) are potent inducers of T cell tolerance in tumor-bearing hosts. MDSC levels also increase during normal pregnancy; however, whether they mediate maternal tolerance is unknown. To investigate if MDSC are involved, female BALB/c (H-2d) mice were caged overnight with allogeneic C57BL/6 (H-2b) males. The following morning, females with vaginal plugs indicative of copulation, were started on an MDSC depletion regimen. MDSC-depleted mice produced no offspring, while 46% of control-treated mice had healthy litters. One of the mechanisms MDSC use to inhibit anti-tumor immunity is to down-regulate CD62L (L-selectin) on T cells and prevent T cell entry into lymph nodes and subsequent activation. To determine if a similar mechanism is operative during pregnancy, mice were treated with granulocyte colony stimulating factor (G-CSF), an inducer of MDSC. G-CSF-treated, but not control-treated, mice contained elevated levels of MDSC, and elevated levels of naïve, circulating T cells with reduced expression of CD62L. MDSC-depletion of G-CSF-treated mice restored CD62L expression. These results suggest that MDSC promote maternal-fetal tolerance, and that tolerance during successful pregnancies may be due to the failure of T cells to enter lymph nodes where they could become activated to fetal alloantigens.
NSAIDs inhibit NFkβ activation to prevent inflammation (IRC4P.602)
Authors: Johnson Annie , Myers Michael
Published: January 2023

Non-steroidal anti-inflammatory drugs (NSAIDs) control inflammation through reductions in Prostaglandin E2 (PGE2) levels. Recent work by this laboratory identified NSAID-induced changes in inflammatory gene expression that appear to be independent of changes in PGE2; however, the molecular mechanism is unknown. Our goal is to determine whether NSAIDs affect gene expression via inhibition of NFkβ. Knowledge of NSAID gene control will provide a mechanism for screening novel drugs claiming anti-inflammatory properties. RAW 264.7 cells transfected with a plasmid under NFkβ control to express Secreted Alkaline Phosphatase (SEAP) were pre-treated with NSAID concentrations ranging from 10-3 to 10-7 M and stimulated with 1μg/mL LPS for 24 hours. NSAIDs from the 5 major classes inhibited SEAP release at 10-3 M, while acetylsalicylic acid (ASA) and acetaminophen did not. HEK293 cells transfected with the same plasmid were pre-treated with flunixin, diclofenac, and ASA at concentrations from 10-3 to 10-7 M and stimulated with 10ng/mL TNFα for 24 hours. Diclofenac and flunixin decreased SEAP release at 10-3 M, whereas ASA failed to inhibit SEAP release. These results demonstrate that NSAIDs prevent SEAP release via NFkβ inhibition, suggesting a new target for novel NSAIDs. Experiments to confirm NSAID inhibition of NFkβ activation by measuring the release of p65 are underway. The results of this research may also provide a screening tool for novel NSAIDs.
Dendritic cells regulate human antibody responses to glycan antigens (IRC4P.449)
Authors: Mendoza Daniel , Xu Yi , Rodriguez-Barradas Maria , Lewis Dorothy , Orson Frank , Tweardy David , Corry David
Published: January 2023

Background. Glycans mediate host-pathogen interactions, cancer and autoimmunity. Glycan antigens directly activate B cells without T cell help to produce antibodies. The role of human dendritic cells (DC) in this process is unclear. We hypothesize that DC regulate human antibody responses to glycan antigens. We use pneumococcal polysaccharides (PS) as glycan antigens because PS are crucial determinants of bacterial virulence and are targeted by existing vaccines. Methods. We incubated PBMC of healthy adults with various PS to identify responding B cell subsets by flow cytometry. We incubated isolated B cells with PS with and without autologous monocyte-derived DC to measure IgG/IgM production by ELISA on day 6. We measured IL-8 in DC exposed to PS by flow cytometry, and uptake of fluorochrome-labeled PS by DC by confocal microscopy. Results. Plasmablasts expand in response to PS. B cells produced IgG to PS only in the presence of DC (B cells alone: 1; B cells+DC: 59 ug/ml, p=0.01). IgG production to PS required direct DC-B cell contact (transwell plates: 3, regular plates: 35 ug/ml, p=0.03). Results for IgM were similar. Percent of DC producing IL-8 to PS: media: 17%, PS: 24%, p<0.02); PS was internalized by DC within 5 min and 16h later was at the DC-B cell interface. Conclusion. Human DC regulate antibody responses to PS. We have identified and essential role for DC in immune responses to glycan antigens with important implications for vaccine design and disease processes.
Effect of glycyrrhizin on the intratracheal infection of Klebsiella pneumoniae in NOD/scid IL-2Rγnull mice inoculated with alcoholic patient white blood cells (IRC4P.609)
Authors: Ito Ichiaki , Asai Akira , Ohama Hideko , Tsuchimoto Yusuke , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Humanized mice created with alcoholic patient white blood cells (patient chimeras) are extremely susceptible to infection with K. pneumoniae. M2Mϕ in patient WBC are responsible for impairing the host antibacterial response of the chimeras. Since glycyrrhizin is known as an inhibitor of M2Mϕ activities, in this study, the effect of glycyrrhizin on K. pneumoniae infection in these chimeras was investigated. Patient chimeras (γ-irradiated-NOD/scid IL-2Rγnull mice inoculated 5 x 106 cells/mouse of alcoholic patient WBC) were infected intratracheally with 100 CFU/mouse of K. pneumoniae, and treated with 10 mg/kg of glycyrrhizin (48 hours before and 2 h after infection, Minophargen Pharmaceutical Co., Ltd., Tokyo, Japan). In the results, severe bacterial growth was demonstrated in the liver (2.7 x 107 CFU/g organ), kidney (2.9 x 107 CFU/g organ), and spleen (1.4 x 109 CFU/g organ) of patient chimeras 24 h after the infection. Contrarily, only 0.15 - 6 x 104 CFU/g organ of the pathogen were detected in these organs of patient chimeras treated with glycyrrhizin. Similarly, bacterial growth was minimal in the organs of healthy chimeras (γ-irradiated-NOD/scid IL-2Rγnull mice inoculated with healthy donor WBC). These results suggest that K. pneumoniae infections in alcoholics may be controllable by glycyrrhizin, an inhibitor of M2Mϕ activities.
Supraphysiological myeloid cell activation during mycobacterial immune reconstitution inflammatory syndrome (IRC4P.456)
Authors: McBerry Cortez , Sakai Shunsuke , Kauffman Keith , Barber Daniel
Published: January 2023

Anti-retroviral therapy (ART) of HIV-infected individuals usually leads to clinical improvement, but some individuals develop a worsening of symptoms following ART, referred to as Immune Reconstitution Inflammatory Syndrome (IRIS). CD4 T cells are thought to be a primary mediator of this paradoxical disease and recent work in patients has correlated activated monocytes with the development of IRIS. Here we use a mouse model of Mycobacterium avium associated IRIS to examine myeloid cell responses in IRIS. We find that CD4 T cell reconstitution of chronically infected TCRaKO mice leads to large increases in neutrophils and Ly6C+ CCR2+ monocytes. Prior to CD4 T cell transfer, myeloid cells in chronically infected TCRaKO mice expressed less iNOS but higher TNF compared to WT mice. Within ~12 days after CD4 T cell reconstitution, multiple myeloid cell subsets dramatically upregulated iNOS and TNF reaching levels far above what is ever observed in WT mice at any time during infection. Transfer of IFNgKO CD4 T cells into chronically infected TCRaKO mice failed to drive myeloid cell expansion or increased function, and mice displayed greatly reduced signs of illness. These data indicate that progression of a chronic mycobacterial infection in the absence of CD4 T cell help predisposes myeloid cells to over respond to T cell stimulation when CD4 T cells are later introduced, indicating myeloid cell hyper responsiveness may underly the immunopathogenesis of IRIS.
Phenotypic models of T cell activation (IRC5P.627)
Authors: Lever Melissa , Maini Philip , van der Merwe P. , Dushek Omer
Published: January 2023

T cell activation is a crucial checkpoint in adaptive immunity, and this activation depends on the binding parameters between the T cell receptor (TCR) and peptide-MHC (pMHC) complexes as well as the dose at which the pMHC is presented. Despite extensive experimental studies, it still remains controversial as to how these parameters govern the resultant activation. We present the development of a model that captures the phenotype of T cell activation. This model is influenced by existing models in the literature as well as a quantitative dataset we have generated that probes T cell activation over wide range of pMHC binding parameters and doses. The dataset was generated by stimulating jurkats and primary cells transduced with a therapeutic high affinity T cell receptor with a panel of plate bound pMHC that are mutations of the HLA-A2 NYESO pMHC. These ligands have a 105-fold range in binding time for the TCR. Activation was quantified by measuring IL8 (jurkats) and IFN-ϒ (primary). We have observed from the data that there is an optimal ligand binding time that gives the most potent activation. Of further interest is the observation of an inhibition in T cell activation at high doses of pMHC presentation. Our analysis indicates that a kinetic proofreading with limited signalling mechanism combined with a negative signalling motif can produce this observed phenotype of optimal ligand binding time and optimal ligand dose.
Interleukin-33 is a mediator of murine familial hemophagocytic lymphohistiocytosis (IRC5P.629)
Authors: Rood Julia , Chu Niansheng , Kreiger Portia , Rao Sheila , Stelekati Erietta , Wherry E. , Behrens Edward
Published: January 2023

Familial hemophagocytic lymphohistiocytosis (FHL) is a life-threatening disorder caused by uncontrolled, systemic immune activation. The murine model, in which lymphocytic choriomeningitis virus (LCMV) infection of perforin knockout (PKO) mice triggers FHL, demonstrates that the disease is driven by an excess of interferon-γ (IFNγ)-producing LCMV-specific CD8+ T cells. While this overactive T cell response is thought to arise from excess antigen stimulation through the TCR, data from our lab suggest that non-TCR, MyD88-dependent signaling pathways may be equally important. We show that interleukin-33 (IL-33), a cytokine released by damaged tissue, plays a critical role in driving FHL. LCMV-infected PKO mice receiving IL-33 receptor-blocking antibody (α-IL-33R) show markedly improved survival compared to isotype-treated controls (p=0.0005) and reduced severity of multiple disease parameters, including serum IFNγ (p=0.0005). α-IL-33R-treated mice have fewer gp33-specific CD8+ T cells (p=0.0044) and total effector CD8+ T cells (p<0.0001). IL-33R blockade also reduces the frequency and IFNγ MFI of IFNγ+ gp33-specific CD8+ T cells (p=0.0004 and p=0.0016, respectively). We observe similar effects on LCMV-specific CD4+ T cells, as well. These data demonstrate that disruption of IL-33 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ, leading to improved morbidity and mortality, and suggest blockade of this pathway as a viable treatment strategy for FHL.
Effect of 1,25(OH)2D3 on in vitro differentiation of human naïve and CD4+ T cells to Th17 and Treg subsets (IRC5P.630)
Authors: Fawaz Lama , Mrad May , Sayegh Souraya , Kazan Jalal , akika reem , Khoury Samia
Published: January 2023

The balance between Th17 and Tregs influences the pathogenesis of autoimmune diseases. The active form of vitamin D, 1α,25-dihydroxyvitamin D3, was shown to exert anti-inflammatory effect. The aim of this study is to investigate the effect of vitamin D on the differentiation of naïve and total CD4+ T cells into pathogenic Th17 subset and regulatory T cells ex-vivo in healthy controls, and to characterize their phenotype and its modulation by vitamin D. Purified naïve and total CD4+ T cells were activated in Th17 and Treg polarizing conditions in the presence or absence of 1,25(OH)2D3 for 6 days. Cells were processed for multiparameter staining and flow cytometry, and cell culture supernatants were reserved for cytokine quantitation by cytometric bead array for IL-17A, IL-10, IL-6, TNF-α, IL-2, IFN-γ, and IL-4. In Th17 cell cultures, overall expression of CCR6 and the percentage of CCR6+IL-17A+CD4+ T cells is decreased with Vitamin D. Interestingly, IL-23R and IL-23R+IL-17A+ cells, as well as IL-17+IFN-γ+ cells associated with pathogenic Th17 cells are reduced, while Foxp3+ cells are increased. In the presence of vitamin D, Treg polarized CD4+ T cells upregulated CD25 and CTLA4 expression but not Foxp3. Foxp3 expression was reduced in both CD25hi and CD25lo cells. Cytokine analysis by CBA and Treg suppression assays are underway. Results obtained thus far point to an effect for Vitamin D in reducing pathogenic Th17 cells, and modulating the induction of regulatory T cells.
CD73 protects Treg and ILC in the control of Th cell mediated colitis (IRC5P.632)
Authors: Drygiannakis Ioannis , Kurtz Courtney , Klann Jane , Farrow Norma , Thai Rosarlin , Gomez Bobbie , Wilson Jeffrey , Borowitz Molly , Bekiaris Vasileios , Ware Carl , Ernst Peter
Published: January 2023

CD73 is a membrane-bound enzyme catalyzing the final step of the conversion of extracellular ATP to adenosine, which then binds to its receptors and signals via cAMP and non-cAMP-mediated pathways. While CD45RBlow helper T cells from wild-type and CD73 KO mice were both highly enriched in the FoxP3+ regulatory T cell transcription factor, cells from CD73 KO mice produced more IFN-γ and IL-17A and could not inhibit effector T cell proliferation and function in vitro. In vivo CD73 expression by both regulatory (CD45RBlow) and effector (CD45RBhigh) T cells was required for Treg to prevent effector T cell-induced colitis following adoptive transfer into immunodeficient Rag1 KO recipients. The cytokine profile in the intestine was skewed compared to the protective combination of wild-type CD45RBlow and wild-type CD45RBhigh. Donor CD45RBlow recovered from colitic mice had reduced FoxP3 and CD45RBhigh phenotype shifted from Th17 to Th1. If recipients lacked both Rag1 and CD73, wild-type CD45RBlow Th cells did not protect from T cell-induced colitis. CD45RBlow Th cells lost FoxP3, acquired Th1 and Th17 transcription factors, depleted recipient’s innate lymphoid cells and caused disease. Prior adoptive transfer of innate lymphoid cells protected these recipients from disease and maintained FoxP3 expression on cells of the CD45RBlow pool. Thus, CD73 on cells of both adaptive and innate immunity is required for effective regulatory T cell function in intestinal homeostasis.
Fibroblastic reticular cell subsets derived from human lymph node resembles bone marrow stromal cells and interfere with the expression of homing markers in autogous and allogeneic lymphocytes (IRC5P.624)
Authors: Marti Luciana , Alvarenga Heliene , Macedo Antonio , Andrade Maria Claudina , Cury Adriano , Salvalaggio Paolo , Pasqualin Denise , Bromberg Silvio , Rizzo Luiz , Hamaerschlak Nelson
Published: January 2023

The main functions of human bone marrow stromal cell are related to bone marrow homeostasis, even though immune functions already have been described to these cells. A similar cell type named fibroblastic reticular cell was described in animal models secondary lymphoid organs, which the main function was to favor and regulate the immune responses. Therefore, we have obtained informed consent from patients that undergone to organ removal surgery and requested a lymph node donation (Ethics committee approval number - 06592712.4.0000.0071). After treating the lymph node tissue, we were able to isolate stromal cells from several lymph nodes. These cells were cultured for 4 passages, than characterized; we observed that there were two subsets of stromal cells in lymph nodes, one that express gp38 (40%) and meet the already described fibroblastic reticular cell phenotype and another gp38 negative (60%). Either cell subset expressed CD29, CD44, CD73, CD90 or CD105 and do not expressed CD35, CD45, CD19, HLA-DR, CD14, CD34, CD31 or CD106. Both subsets were able to differentiate in adipocytes, chondrocytes and osteoblast, resembling human mesenchymal stem cell bone marrow derived. Regarding their immune function, we observed that either subset was able to interfere with proliferation and CCR6, CXCR4 expression of stimulated allogeneic and autologous T cells.
LPS inhibits ILC2 mediated type 2 immunity in the lung (IRC5P.625)
Authors: Poon Grace , Takei Fumio
Published: January 2023

The hygiene hypothesis states that the lack of early childhood exposure to infectious agents, which promotes type 1 immunity, results in increased incidents of allergy and asthma, which are predominantly type 2 immune responses. Recently identified group 2 innate lymphoid cells (ILC2s) are essential for the development of protease allergen induced type 2 lung inflammation. To better understand how type 2 immunity is regulated, we tested the effect of LPS-induced type 1 inflammation on ILC2s and type 2 lung inflammation. Intranasal LPS pretreatment of mice suppressed both papain- and IL-33-induced ILC2 activation and eosinophilic lung inflammation. Specifically, the number of activated ILC2s that express IL-5 and IL-13 was decreased with LPS treatment. The LPS effect on ILC2s was dose and time dependent. The in vivo findings were reproduced in vitro. Pretreatment with LPS inhibited the production of type 2 cytokines IL-5 and IL-13 by ILC2s cultured in the presence of IL-33 and TSLP. These results indicate that LPS triggers an ILC2 inhibitory signal, which leads to the suppression of ILC2 activation and type 2 inflammation in the lung upon allergen exposure.
Spontaneous aggregation and novel signalosome organization of BENTA-associated gain-of-function CARD11 mutants in lymphocytes. (IRM5P.641)
Authors: Stinson Jeffrey
Published: January 2023

We recently described BENTA (B cell Expansion with NF-kB and T cell Anergy), a novel lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte-specific scaffolding protein CARD11. Normally, antigen receptor (AgR) stimulation induces CARD11 phosphorylation to evoke an active conformation that nucleates a signalosome incorporating BCL10, MALT1, and other proteins required for IkB kinase (IKK) activation and NF-κB nuclear translocation. However, ectopic expression of CARD11 mutants triggers constitutive NF-kB activity in B and T cell lines without AgR stimulation. Here we show that these CARD11 mutants form large, cytosolic protein aggregates with a novel substructure, dubbed mCADS (Mutant CARD11 Dependent Shells). Endogenous mCADS are also detected in B cell lymphomas harboring similar CARD11 mutations. In contrast, WT CARD11 cannot form mCADS -/+ AgR stimulation. Only MALT1 and phospho-IKK are stably colocalized with mCADS, indicative of active, sustained signaling. Surprisingly, mCADS formation requires interaction with BCL10 but not NEMO (IKKg). Our results suggest that gain-of-function CARD11 mutants spontaneously assemble into an active, highly-ordered signalosome that is structurally and functionally distinct from WT CARD11, regardless of AgR ligation. The unique structure and composition of mCADS may represent a novel therapeutic target for treating BENTA or lymphoma without disrupting normal AgR-induced NF-kB activation.
The nucleolus contains immune-related RNAs during infection. (IRM5P.644)
Authors: LEE TAEYUN , Lee Sungwook , Park Boyoun
Published: January 2023

The nucleolus is a subnuclear structure that is involved in ribosomal RNA (rRNA) synthesis and ribosome assembly, and it is also the location of viral replication or cellular stress sensing. However, the function of the nucleolus in the immune response is still unclear. Here, we report that the nucleoli are fused in both macrophages and dendritic cells during lipopolysaccharide (LPS) or CpG-DNA stimulation, which activates the Toll-like receptor 4 (TLR4) or Toll-like receptor 9 (TLR9) signaling pathway. Our results from RNA-sequencing analysis reveal that the nucleolus contains diverse RNA transcripts in response to LPS. Furthermore, gene ontology term analysis shows that these RNA species are highly correlated with immune-related functions. More interestingly, intron-containing RNA transcripts are existed in the nucleolus. Our results provide new insight into another mechanism of nucleolus in the immune system.
Regulation of CD4+ T cell activation and effector function by the histone acetyltransferase Myst1 (IRM6P.662)
Authors: Carson William , Mertz David , Cavassani Karen , Mukherjee Sumanta , Harissa Batoul , Kroetz Danielle , Schaller Matthew , Dou Yali , Kunkel Steven
Published: January 2023

Chromatin remodeling events are central molecular mechanisms that serve to govern CD4+ T cell function through regulation of gene accessibility. Myst1 is a histone acetyltransferase responsible for the acetylation of the protein tail of histone 4 at lysine residue 16, and is thought to participate in the activation of gene transcription. Studies of murine and human CD4+ T cells indicate that Myst1 is upregulated upon T cell receptor stimulus. Deletion of the gene encoding Myst1 resulted in a significant proliferation defect in these cells that is unaffected by the addition of exogenous IL-2. Myst1-deficient T cells also exhibit modulations in cytokine production and lineage commitment following skewing to various effector lineages in vitro. Additionally, signal transduction via STAT5 may play a role in Myst1 expression, as downregulation of STAT5a/b expression via siRNA transfection significantly decreased Myst1 expression levels in T cells. These results suggest an important role for the STAT5-Myst1 axis in governing the activation and effector function of CD4+ T cells.
Characterization of human immune phenotypes using a novel transcriptome analysis approach (IRM6P.656)
Authors: Scheid Adam , Van Keulen Virginia , Felts Sara , Zhang Yuji , Markovic Svetomir , Pease Larry
Published: January 2023

To test the hypothesis that complex immune phenotypes can be defined by stable gene expression profiles we are using a RNAseq-based transcriptome analysis to relate phenotypes of activated peripheral blood CD4+ cells from healthy subjects and stage IV melanoma patients to their freshly-isolated counterparts. We have defined molecular immune response signatures (MIRS) containing genes that consistently differentiate individuals from one another. Two different phenotypic groups are defined using this approach, both of which are mathematically distinct from randomly-selected groups. First, a MIRS generated using healthy donor T cells stimulated with α-CD3/CD28 beads defines a distinct response phenotype and successfully predicts 17 independent healthy subject and melanoma patient responses, demonstrating the predictive capability of the approach. Second, our comparison of melanoma patient signatures with those of healthy subjects revealed a group of healthy individuals with distinct expression signatures that did not cluster with those of any of the melanoma patients. These results indicate that our RNAseq-based transcriptome analysis has the potential to identify phenotypic groups as well as gene sets that have response-predictive capabilities. Our work has both discovery and translational implications, as our approach can be used to investigate complex genetic contributions to immune responses and predict how individual patients will respond to therapeutic interventions.
Differential long noncoding RNA signatures in activated plasmacytoid dendritic cells (IRM6P.659)
Authors: Joslyn Rochelle , Savan Ram
Published: January 2023

Long noncoding RNAs (lncRNAs) represent a novel class of RNA which act through diverse mechanisms to exert control over a wide range of cellular processes. Through control of transcription, post-transcriptional regulation, or cellular signaling events, lncRNAs can function as potent modulators of innate immune responses, however very few lncRNAs have been investigated. We utilized whole exome sequencing of plasmacytoid dendritic cells (pDC) treated with IFN (interferon)-β, TLR7 agonist (toll like receptor, R848), and R848 with type I IFN blockade to identify 910 noncoding transcripts which are differentially regulated by IFN signaling and TLR7 activation. Through dissection of the stimulatory events controlling lncRNA expression, we have identified subsets of type I IFN- and TLR7-induced or suppressed lncRNAs. We hypothesize that many of these transcripts participate in the orchestration of innate and adaptive antiviral immunity through their modulation of pDC function. Dysregulation of these lncRNAs could significantly alter pDC maturation, IFN production, antigen presentation, turnover or localization, events which would be consequential during viral infection or IFN-driven autoimmune disease.
The intrinsic and extrinsic regulation of γδ-T17 cells through the IL-17-IL-17r axis during homeostasis (LYM2P.729)
Authors: Fleming Christopher , Cai Yihua , Xue Feng , Wei Yu-Ling , Jala Venkatakrishna , Worth Christopher , Liu Na , Chien Yueh-Hsiu , Ding Chuanlin , Yan Jun
Published: January 2023

IL-17 producing γδ T cells (γδ-T17) are major players in promoting several autoimmune inflammatory diseases and cancers. However, γδ-T17 homeostatic regulation has not been fully understood. In naïve, adult IL-17r-/- and IL-17A-/-/F-/- mice we observed higher frequencies of total γδ T cells, predominantly γδ-T17, in the lung, spleen, gut, LN and skin but not in the thymus. We hypothesized that the IL-17-IL-17r axis regulates peripheral γδ-T17 homeostasis. Two different regulatory pathways were identified to be involved in this regulation: extrinsically through microbiota-host interaction and intrinsically through IL-17r-IL-7r signaling interaction. The extrinsic pathway was discovered in vitro using CFSE-labeled IL-17r-/- lymphocytes that had endogenous γδ-T17 proliferation dependent upon DCs, particular CD103+ DCs, and IL-1β. Using mice delivered from IL-17r-/- pregnant females treated either with lymphotoxin beta receptor-Ig to remove LNs or antibiotics (ampicillin, vancomycin, neomycin, and metronidazole) to deplete gut microbiota we found significantly decreased frequency of γδ-T17 in peripheral tissues. Also, intrinsically IL-17r-/- γδ-T17 expressed higher levels of RORγt that suppressed BTLA expression, which is a known inhibitor of IL-7-driven γδ-T17 proliferation. Taken together, our findings suggest that the IL-17-IL-17r axis regulates γδ-T17 via both intrinsic and extrinsic pathways.
The role of miRNAs in the homeostasis of peripheral T cells (LYM2P.732)
Authors: Pobezinsky Leonid
Published: January 2023

The lifelong presence of mature T cells in the organism is vital for immune responses. Despite being extensively studied, the molecular mechanisms governing T cell homeostasis are still not fully understood. Here we investigated how microRNAs, acting through post-transcriptional regulation, contribute to the maintenance of mature naïve T cells in the periphery. We found that T cell-specific Dicer ablation caused lymphopenia, due to survival defects in mature T cells. Using miRNA knockout mice, we identified specific miRNAs which, when deleted, resulted in T cell death, and overexpressing these miRNAs in the T cells of transgenic mice provided complete protection. Finally, we characterized the downstream molecular mechanisms responsible for miRNA-induced suppression of cell death. Altogether our findings represent a novel miRNA-mediated mechanism that controls homeostasis of peripheral naïve T cells in vivo.
GATA-3 regulates the development of ILC3 subsets at multiple stages (LYM2P.721)
Authors: Zhong Chao , Hu Gangqing , Zhao Keji
Published: January 2023

Innate lymphoid cells (ILCs) are emerging lymphocytes that maintain the homeostasis between the host and commensals, and play essential roles during secondary lymphoid organogenesis and innate immune responses. Mature ILCs, similar to their adaptive counterparts of CD4 T cells, can be divided into three major subsets based on their signature cytokine production and selective expression of master transcriptional regulators, T-bet, GATA-3 and RORγt. However, how the development and functions of these subsets are precisely regulated, especially by transcription factors including GATA-3 that are expressed at various levels, is still elusive. We have previously reported that in addition to its critical role in the development and maintenance of ILC2s, GATA-3 is also required for the development of other IL-7Rα+ ILCs including ILC3s. Here we show that GATA-3 is continuously expressed by all ILC3 subsets and has crucial regulatory functions at multiple stages during the development of ILC3 subsets. Deletion of Gata3 gene after RORγt expression by Rorc-Cre resulted in a significant reduction of ILC3s, especially for the NKR+ ILC3 subset. Genome wide analysis of gene expression in the wild type and Gata3-deficient ILC3s revealed that the expression of Il7r and a number of genes associated with the functions of ILC3s was dramatically reduced in the absence of GATA-3. Thus, our results shed light on the regulatory function of GATA-3 at multiple developmental stages of ILC3 subsets.
Defining the mechanisms and cues that influence cytotoxic T cell homeostasis during malnourishment (LYM2P.727)
Authors: Gubbels Bupp Melanie , Scott Samantha , Murphy Sarah , Edwards Whitney , Litvin Seth
Published: January 2023

T cell homeostasis is clearly disrupted by malnutrition, but the precise cues and mechanisms through which it occurs are unknown. Short-term malnutrition reduces the size of the peripheral CD8+ T population in mice. Perhaps paradoxically, malnourished CD8+ T cells are less sensitive to death-by-neglect and express higher levels of CD127, a component of the IL-7 receptor. We aimed to identify whether vitamin and mineral deficiency or reduced caloric intake triggers these changes and to better understand the molecular mechanisms through which such changes occur. While vitamin and mineral deficiency is not sufficient to reduce total CD8+ T cell numbers or trigger CD127 up-regulation, it is sufficient to reduce CD8+ T cell sensitivity to death-by-neglect. Mechanistically, we have determined that the reduced sensitivity to death-by-neglect observed in malnourished CD8+ T cells is Foxo1-dependent. Given that CD127 is a transcriptional target of Foxo1, additional studies were conducted to determine if CD127 is up-regulated or if CD127low CD8+ T cells are simply out-competed in a malnourished environment. Our results suggest that during malnourishment, Foxo1 induces the up-regulation of CD127 on CD8+ T cells, which may relate to the cells’ reduced sensitivity to growth factor withdrawal. While different mechanisms may be triggered by calorie versus vitamin and mineral deficiency, both deficiencies reduce CD8+ T cell sensitivity to death-by-neglect.
Decreasing initial stimulation increases generation of early effector cells that survive the contraction phase in malaria infection (MPF2P.754)
Authors: Stephens Robin , Opata Michael , Carpio Victor , Ibitokou Samad , Dillon Brian , Obiero Joshua
Published: January 2023

CD4 T cells orchestrate immunity against blood-stage malaria. However, a major challenge in designing malaria vaccines is understanding generation of memory from effector T cells (Teff) in chronic infection. We use a transgenic mouse model with T cells specific for the Merozoite Surface Protein (MSP)-1 of Plasmodium chabaudi to show that activated T cells generate three Teff (CD127-) subsets representing progressive activation. The earliest observed subset (CD62LhiCD27+) divides first, and maintains memory gene expression. An intermediate (CD62LloCD27+) effector subset has more multi-cytokine producing T cells and a terminal phenotype, including expression of PD-1 and Fas. Fully activated (CD62LloCD27-) late effector cells contain fewer cytokine+ and more AnnexinVhi apoptotic cells. Using an adoptive transfer system, we show the ability of CD62Lhi Teff to generate the terminal Teff subsets but also to survive the contraction phase better than the CD62Llo subsets. Furthermore, we show that progression towards terminal differentiation increases with duration of infection, but is inhibited by IL-2, which promotes expansion but not progression. Therefore, we have defined markers of progressive activation of CD4 Teff at the peak of malaria infection, and shown that CD127-CD62Lhi Teff survive the contraction phase. Therefore, antigen and cytokine levels during CD4 T cell expansion influence the proportion of activated cells that can survive T cell contraction in malaria infection.