bluuebunny/crossref_metadata_2025 huggingface dataset:
NFIL3 suppresses Rorγt transcription and links intestinal Th17 cell differentiation to the circadian clock network (P1223)
Authors: Yu Xiaofei , Rollins Darcy , Kashiwada Masaki , Rothman Paul , Takahashi Joseph , Hooper Lora
Published: January 2023

Environmental factors can profoundly impact the development and function of the mucosal immune system. The microbiota is one such factor that is known to regulate intestinal interleukin-17 (IL-17) producing CD4+ T helper (Th17) cells, but little is known about other environmental cues that influence their development. Here, we show that the transcription factor NFIL3 suppresses Th17 development by directly binding to the Rorγt promoter and repressing its activity. NFIL3 is linked to the circadian clock network through the transcription factor REV-ERBα, and accordingly we observed altered intestinal Th17 cell frequencies in mice that lacked REV-ERBα or were subjected to disturbed light cycles. Together, our findings reveal a new transcriptional regulatory network that regulates intestinal Th17 cell differentiation and unveil a molecular link between the circadian clock circuitry and Th17 cell development.
A transcription factor network in Th9 cells (P1229)
Authors: Jabeen Rukhsana , Goswami Ritobrata , Awe Olufolakemi , Kaplan Mark
Published: January 2023

Th9 cells are the most recent addition to the spectrum of T helper cell subsets that differentiate in the presence of TGF-β and IL-4. Th9 cells promote allergic inflammation, anti-tumor immunity, and may contribute to the regulation of auto inflammatory disease. Whether Th9 cells express a characteristic transcriptional signature has not been determined. To define the identity of Th9 cells, we performed a microarray analysis and identified 629 genes enriched in Th9 cells compared to Th2 and Treg cells. Of these genes, we chose a panel of 14 genes based on potential relatedness to Th9 function, and confirmed Th9-enriched expression in a larger panel of T helper subsets using quantitative PCR. The Th9 enriched genes encoded transcription factors including Erg, Maf, Ahr and Crem, and surface receptors including Il17rb, Ccr4 and Ccr8. Using mice deficient for the transcription factors that promote Th9 development including STAT6, IRF4 and PU.1, we distinguished the impact of each factor on this subset of genes. Thus our data indicates that Th9 cells have an identifiable transcriptional signature, and we have defined a transcription factor network that regulates Th9 gene expression.
Distinct dendritic cell subsets dictate the fate decision between effector and memory CD8+ T cell differentiation (P6172)
Authors: Kim Taeg , Gorski Stacey , Hahn Steven , Braciale Thomas
Published: January 2023

An effective host response to a microbial pathogen requires both the generation of effector T cells as well as formation of durable T cell memory. Dendritic cells (DCs) play a central role in initiating the adaptive T cell response to microbial infection, and multiple distinct DC subsets have the potential to take up and present microbial antigen. However, the contribution of different DC subsets to the generation of effector and memory CD8+ T cells in vivo following infection and the DC dependent mechanisms to account for this dichotomy remain incompletely understood. Here we show that the two major migratory respiratory DC (RDC) subsets responding in the lung draining lymph nodes to influenza virus infection, CD103+ and CD11bhi RDC, activate naïve virus-specific CD8+ T cells along distinctly different differentiation pathways. CD103+ RDC in vivo stimulate the generation of activated CD8+ T cells with features characteristic of CD8+ T effectors that is activated T cells which efficiently leave the lymph nodes and home to the infected respiratory tract. In contrast, stimulation of naïve virus-specific CD8+ T cells by CD11bhi RDC give rise to CD8+ T effectors which are preferentially retained within the draining lymph nodes during the acute phase of infection, and which exhibits properties of central memory CD8+ cells. DC-dependent cellular and molecular basis for refining this dichotomy for in vivo CD8+ T cell differentiation are further delineated.
Kruppel-like factor 6 and inducible nitric oxide synthase regulates apoptosis during respiratory syncytial virus infection (P6198)
Authors: Segovia Jesus , Mgbemena Victoria , Chang Te-Hung , Bose Santanu
Published: January 2023

Human respiratory syncytial virus is a highly pathogenic lung-tropic virus that causes severe respiratory diseases such as pneumonia and bronchiolitis in infants, children and the elderly. Various innate immune mediators including nitric oxide are produced in response to respiratory syncytial virus infection and play a critical role in host defense. Generation of nitric oxide during respiratory syncytial virus infection is dependent on inducible nitric oxide synthase expression. Although nitric oxide plays a crucial role in combating pathogen invasion and spread, it can also lead to an exaggerated inflammatory response and tissue damage. Nitric oxide-mediated apoptosis is responsible for exacerbated disease pathology during respiratory syncytial virus infection, however the mechanism is unknown. In the current study we have identified kruppel-like factor 6 as a critical transcription factor required for inducible nitric oxide synthase gene expression during respiratory syncytial virus infection. Furthermore, we have also uncovered the role of inducible nitric oxide synthase as a critical host factor regulating apoptosis during respiratory syncytial virus infection.
Interferon lambda confers antiviral protection on the epithelium (P6186)
Authors: Mahlakoiv Tanel , Staeheli Peter
Published: January 2023

Type III interferon (IFN-λ) belongs functionally to the interferon family, although it is structurally related to IL-10 family of cytokines. We have used a reporter mouse lacking type I IFN receptor to identify IFN-λ-responsive cells in vivo by staining tissue sections with antibodies that recognize the IFN-induced Mx1 protein. We found that epithelial cells lining the respiratory, gastrointestinal and reproductive tracts are responding most strongly to IFN-λ stimulation. Additionally, IFN-λ-induced Mx1 was detectable in epithelial cells of the skin, placenta, fetal membranes, ovaries, mammory glands, cornea and conjunctiva. In vitro experiments on various epithelial cell lines not only confirmed this finding, but also demonstrated the potent antiviral activity of exogenous IFN-λ against a diverse set of viruses. Using knockout mice lacking either functional type I or type III IFN receptors, we demonstrated that IFN-λ is redundant to type I IFN functions in the lung upon infection with various respiratory viruses, whereas a predominant protection against rotavirus infection of the small intestinal epithelium was mediated by IFN-λ. Interestingly, mice lacking functional receptors for IFN-λ showed higher susceptibility not only to rotavirus, but also to another enteric virus, the reovirus. Taken together, a picture emerges showing that IFN-λ is a potent antiviral cytokine mediating protection against viral pathogens which infect the epithelium.
Transgenic expression of survivin compensates the OX40-deficiency in driving Th2 development and allergic inflammation (P1184)
Authors: Song Jim , Lei Dylan , Haque Rizwanul
Published: January 2023

The inhibitor of apoptosis family molecule survivin has been suggested to be a crucial intermediate in signaling pathways leading to T cell development, proliferation, and expansion. However, the importance of survivin to T cell-driven inflammatory responses has not been demonstrated. Here, we show that survivin transgenic mice exhibit increased antigen-driven Th2 lung inflammation, and that constitutive expression of survivin reverses defective lung inflammation in the absence of OX40 costimulation. We found that while OX40-deficient mice are compromised in generating Th2 cells, airway eosinophilia, and IgE responses, survivin transgenic and OX40-deficient mice generate normal Th2 responses and exhibit strong lung inflammation. These data suggest OX40 costimulation crucially engages survivin during antigen-mediated Th2 responses, and promote the notion that targeting survivin could be therapeutically useful for controlling allergic responses or lung inflammation.
Signaling through the P2X7R for ATP induces human cutaneous innate and adaptive Th17 responses. (P4110)
Authors: Mathers Alicia , Killeen Meaghan , Ferris Laura , Kupetsky Erine , Falo, Jr. Louis
Published: January 2023

Human cutaneous DCs have the ability to prime and bias Th17 lymphocytes. However, the factors that stimulate cutaneous DCs to induce Th17 responses are not well known. We hypothesize that alarmins, such as ATP, play a pivotal role in the induction and maintenance of cutaneous immune responses by stimulating DC maturation, chemotaxis, and secretion of IL-1β and IL-6, Th17 biasing cytokines. Utilizing an ex vivo human cutaneous model, we have determined that signaling purinergic receptors, predominantly P2X7R, via an ATP analog initiates innate proinflammatory inflammation, DC17 differentiation, and the subsequent induction of Th17 biased immunity. Moreover, our results suggest a potential role for P2X7R signaling in the initiation of psoriasis pathogenesis, a Th17 dependent autoimmune disease. In support of this, we observed the increased presence of P2X7R in non-lesional and lesional psoriatic skin compared to normal healthy tissues. Interestingly, there was also a P2X7R variant highly expressed in lesional psoriatic skin compared to non-lesional psoriatic and normal healthy skin. In conclusion, we have established that purinergic signaling in the skin induces innate inflammation leading to the differentiation of human Th17 responses, which have implications in the pathogenesis and potential treatment of psoriasis.
Proatherogenic conditions promote autoimmune Th17 responses in vivo (P4136)
Authors: Chung Yeonseok , Lim Hoyong , Kim Young Uk , Sun Hua , Hanabuchi Shino , Teng Babie
Published: January 2023

Although patients with atherosclerosis have a higher incidence of systemic autoimmune diseases, the relationship between proatherogenic factors and autoimmune T cell responses is poorly understood. Mice lacking both LDL receptor and apolipoprotein B mRNA editing enzyme (Ldlr-/-Apobec1-/-; LDb) are hyperlipidemic and prone to atherosclerosis. Here, we show that LDb mice exhibit increased IL-17 in circulation as well as in the aortic sinus area, which was attributed to a preferential enhancement of Th17 cells in the secondary lymphoid organs. In addition, the environment within LDb mice was substantially favorable for the Th17 polarization of auto-reactive CD4+ T cells during homeostatic proliferation. In vitro, the addition of oxidized LDL, but not native LDL, promoted dendritic cell-mediated Th17 polarization by triggering IL-6 and IL-1 in a MyD88-dependent fashion. Furthermore, myelin oligodendrocyte glycoprotein (MOG)-reactive CD4+ T cells expanded in the presence of oxidized LDL expressed increased levels of Th17 signature genes, and induced more profound experimental autoimmune encephalitomyelitis (EAE) when transferred into naïve mice. Our findings demonstrate that proatherogenic factors induce the polarization and functional maturation of autoimmune Th17 cells, which may be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases.
Human CCR6+CXCR3+ “Th1/17” cells are polyfunctional and can develop from Th17 cells under the influence of IL-1β and IL-12 (P4122)
Authors: Duhen Thomas , Campbell Daniel
Published: January 2023

In humans, memory CD4+ T cells co-expressing the chemokine receptors CCR6 and CXCR3, and producing both IL-17 and IFN-γ, have been proposed to be highly pathogenic in several autoimmune disorders. However, their developmental pathways, relationship with “classic” Th17 and Th1 cells and physiological role in normal immune responses are not well understood. Here, we examined CCR6+CXCR3+ T cells from healthy individuals, and found that ex vivo those cells expressed higher levels of IL12Rβ2 and IL23R compared to Th17 and Th1 cells and displayed an enhanced response to IL-12 and IL-23. Interestingly, Th17 cells cultured in the presence of IL-1β upregulated IL-12Rβ2, and IL-1β together with IL-12 induced IL-17+ IFN-γ+ cells and strongly increased the expression of T-bet, IL-12Rβ2 and IL-23R without inhibiting RORγt. CCR6+CXCR3+ T cells shared antigen specificities with Th17 and Th1 cells, and T cells specific for extracellular bacteria such as S. pneumoniae, E. coli and L. rhamnosus were highly enriched in this subset. Furthermore, a significant proportion of CCR6+CXCR3+ T cells expressed the gut-homing molecule α4β7. Thus, CCR6+CXCR3+ CD4+ T cells are a functionally distinct population of T cells with broad antigen specificity and migratory potential that can develop from “classic” Th17 cells under the influence of IL-1β and IL-12. Further characterization of these cells will reveal new insights into CD4+ T cell differentiation and function in infection and autoimmunity.
Glucose metabolism in CD4+ T cell subsets modulates inflammatory disease and autoimmunity (P4126)
Authors: Gerriets Valerie , Macintyre Andrew , Nichols Amanda , Rathmell Jeff
Published: January 2023

Upon stimulation, CD4+ T cells differentiate into effector (Teff) or regulatory (Treg) T cells, the balance of which is crucial to prevent inflammation or autoimmunity. One approach to treat autoimmune disease is to modulate T cell differentiation to favor Treg over Teff cells. We examined the metabolite and metabolic gene expression profiles of the subsets and found that they have distinct metabolic requirements and programs. Teff have a high expression of the glucose transporter GLUT1 as well as glucose metabolism genes and a high rate of glycolysis. In contrast, Treg instead have high expression of mitochondrial metabolism genes and low GLUT1 levels. The effect of metabolic differences to modulate the Teff:Treg balance can be tested directly via genetic modulation of GLUT1. In vitro, T cells from GLUT1 transgenic mice are more inflammatory than wildtype littermates. Alternatively, T cells from GLUT1 knockout mice are unable to increase glucose metabolism upon activation and have fewer Th1, Th2 and Th17 cells. Despite less total CD4+ T cells, these mice have normal natural and inducible Treg numbers. In a mouse model of inflammatory bowel disease (IBD), deletion of GLUT1 in T cells inhibits IBD disease progression. The mice have less CD4+ T cells in the mesenteric lymph nodes and decreased inflammatory cytokine production than wildtype control T cells. Thus, the modulation of glucose metabolism may provide an alternative treatment for inflammatory and autoimmune disease.
TCR repertoire analysis of La/SS-B autoantigen-responsive peripheral blood CD4+ T cells using single-cell RT-PCR: a proof of principle case study (P3249)
Authors: Joachims Michelle , Pan Zijian , Moore Jacen , James Judith , Scofield Robert , Sivils Kathy , Thompson Linda , Farris A.
Published: January 2023

TCR repertoire analysis in a variety of biological settings has largely consisted of TCR spectratyping of V-beta expression or V-specific antibody staining. Here, we combine in vitro stimulation of CFSE-labeled antigen specific T cells with a single cell nested multiplex RT-PCR to efficiently capture the unique T cell repertoire of autoantigen La/SS-B specific CD4+ T cells in a systemic lupus erythematosus and Sjögren’s syndrome patient with precipitating levels of anti-La and Ro antibodies. CFSE-labeled peripheral blood cells were cultured with either purified La protein or a mixture of human La peptides, and CD4+CFSElow cells were single sorted into PCR plates. TCR alpha and beta chain cDNAs were amplified and sequenced. The efficiency of paired TCR chain recovery was 60-65%, and was similar between the populations analyzed. While the magnitude of proliferative responses to the two types of La antigen-specific stimuli were similar, the responding T-cells displayed little overlap in TCR sequence. The sequences derived from the peptide pool-stimulated CD4+ cells were pauci-clonal, with 2 clones dominating more than half the response (58/93 cells). Conversely, the purified protein-responsive CD4+ TCRs were distributed among multiple clonotypes. Our approach maps the T cell repertoire of human antigen responsive cells and may have great utility for elucidating the properties of a variety of antigen-specific T cells in diverse clinical settings.
T cell evaluation in cytotoxicity studies (P3271)
Authors: Gillis Katherine , Clor Julie , Khan Asima , Tyagarajan Kamala
Published: January 2023

A comprehensive mechanistic understanding of cytotoxic compounds action requires knowledge of their impact on different immune sub-populations such as CD4 and CD8 T cells. In this study, we explored the mechanism of action of Gambogic acid, a natural product with potent anti-tumor activity and Anisomycin, an antibiotic that inhibits protein synthesis on peripheral blood mononuclear cells (PBMCs) using assays that combine CD4 and CD8 T cell identification with cell health parameters followed by microcapilllary cytometry. Our studies demonstrate that gambogic acid results in significant apoptosis in both CD4 and CD8 T cells. Further, mitochondrial depolarization plays a significant role in gambogic acid induced cytotoxicity of both CD4 and CD8 T cells with a greater percentage of cells demonstrating loss of mitochondrial membrane potential at lower concentration compared to other apoptosis markers. Anisomycin also results in significant mitochondrial membrane potential loss in T cells. Significant Caspase activation was observed in PBMC’s on treatment with anisomycin; with Caspase 3/7, Caspase 8 and Caspase 9 activity being observed in CD4 and CD8 T cells. Data on dose and time based responses in the sequence of cell health impacts will be further presented for these compounds on PBMC. Evaluating mechanistic impact of compounds on T lymphocytes can provide a more enriched understanding of complete activity of compounds that induce cytotoxicity.
Immune activating effects of stimulation of TLR agonists and cytokines on keratinocytes from a patient with a CARD14 mediated pustular psoriasis (P6283)
Authors: Chen Yongqing , Liu Yin , Huang Yan , Yee Carole , McBride Alison , Morasso Maria , Bowcock Anne , Lowes Michelle , Goldbach-Mansky Raphaela
Published: January 2023

Caspase recruitment domain-containing protein 14 mutations are the first monogenic defect causing psoriasis and pityriasisi rubra pilaris (PRP), linking psoriasis and PRP into one disease spectrum. Interestingly the disease responds well to the treatment with anti-p40 antibody (Ustekinumab) blocking IL-12 and IL-23. The mutant protein is mainly expressed in keratinocytes, leading us to explore whether keratinocyte dysfunction triggers the inflammatory phenotype through the recruitment of hematopoietic cells to the skin. CAR14 mutant keratinocytes produced high level of IL-8 to Toll-like receptor 3, 5, 9, IL-1a/b, IL-36, and TNF stimulation than control cells. IL-17A synergizes with Poly IC, Flagellin, and TNF, but not with IL-12/23, suggesting that Ustekinumab may not directly block keratinocytes but indirectly through inhibit IL-17A produced from likely hematopoietic cells. Using siRNA knock down assay of CMB (CARD-MALT1-BCL10) complex, we show that IL-17A induced IL-8 production is MALT1-dependent; thus raising the question what is the role of CARD14 in IL-17 mediated signal. Our data suggests that while IL-12/23 does not activate keratinocytes, IL-17A is a potent activator and even synergistically with other cytokine stimulant from hematopoietic cells during skin inflammation.
Deubiquitinase A20 mediates feedback inhibition of IL-17 receptor signal transduction (P6267)
Authors: Garg Abhishek , Ahmed Mushtaq , Vallejo Abbe , Ma Averil , Gaffen Sarah
Published: January 2023

IL-17A (IL-17) is a proinflammatory cytokine that mediates host defense against extracellular pathogens but also contributes to the pathogenesis of various autoimmune diseases, such as rheumatoid arthritis (RA), multiple sclerosis (MS) and psoriasis. Despite some homology with the toll-like receptors, IL-17 receptor subunits (IL-17RA and IL-17RC) do not recruit adaptors such as MyD88, but instead associate with an E3 ubiquitin ligase Act1. Upon recruitment, Act1 ubiquitinates Traf6, resulting in the activation of the NF-κB and MAPK pathways. Details of the molecular events involved in negative regulation of IL-17 signaling still remain poorly understood, but are important clinically in understanding IL-17 mediated autoimmune pathology. Since both Act1 and Traf6 have ubiquitin ligase activity, deubiquitination could be an effective strategy of inhibiting IL-17 signaling. We focused on the enzyme A20 (Tnfaip3), as mutations in its gene locus are associated with many autoimmune diseases. Indeed, we found that absence of A20 results in increased IL-17-dependent proinflammatory gene expression and is associated with increased NF-κB activation. Unexpectedly, A20 associates with IL-17RA at its distal C-terminal domain, a domain that has been previously demonstrated to play a role in mediating negative regulation of IL-17 signaling. In summary, regulation of IL-17 signaling by A20 might provide an interesting mechanistic approach for inhibiting this pathway.
The adaptor protein CIKS/Act1 is essential for imiquimod-induced psoriasis. (P6269)
Authors: Ha Hye-Lin , Siebenlist Ulrich
Published: January 2023

Psoriasis is a skin disease characterized by abnormal keratinocyte (KC) proliferation and differentiation and by an influx of inflammatory immune cells. The IL-23/IL-17 axis is thought to play an important role in the pathogenesis of psoriasis. Imiquimod (IMQ) can induce/exacerbate psoriasis in patients and it induces a psoriasis-like condition in mice. In addition to IL-17A/F, IL-17C has also recently been implicated in IMQ-induced psoriasis. To determine by what mechanisms IL-17 cytokines critically contribute to psoriasis, we investigated a mouse model in which signaling by all IL-17 cytokines is abrogated due to the loss of the obligate adaptor protein CIKS/Act1. Importantly, the gene for CIKS has also been identified as a strong susceptibility locus in psoriasis. Here we show that IMQ induced many of the typical phenotypes of human psoriasis in wild-type mice, but largely failed to do so in CIKS-deficient mice. In addition to promoting the influx of inflammatory cells, IL-17 signaling via CIKS contributed to increased proliferation of KCs and inhibited their terminal differentiation. Mice lacking CIKS in KCs only were protected from some, but not all of the pathologic phenotypes associated with psoriasis in this model. These data demonstrate that CIKS is essential for IMQ-induced psoriasis by mediating the signaling of IL-17 cytokines in several distinct cell types. Thus our findings identify CIKS as a promising target for therapeutic intervention in this disease.
Follistatin-like protein 1 regulates IL-17 signalling by influencing the expression of IL-17 receptor A. (P6271)
Authors: Campfield Brian , Hirsch Raphael , Kolls Jay
Published: January 2023

Background: IL-17 is a critical proinflammatory cytokine in Collagen Induced Arthritis (CIA) and lung host defense. Follistatin-like protein-1 (FSTL-1), a proinflammatory glycoprotein mediates the development of CIA. FSTL-1 mediates IL-17-dependent stimulation of IL-6 by an unknown mechanism. Methods: ST2 cells with shRNA for FSTL-1 (shFSTL-1) and control (shCntrl) sequences were stimulated with 8ng/mL IL-17 and 2ng/mL TNFα. Transcript abundance was assessed by qRT-PCR. Protein secretion was assessed by Luminex. IL-17 receptor surface expression was assessed by FACS analysis. Results: Compared to shCntrl cells, shFSTL-1 cells had 1)decreased transcripts of IL-6 (275±3 vs. 43±3), G-CSF (1802±7 vs. 1325±93), IL-17RA (0.798±0.02 vs. 0.504±0.03) and IL-17RC (1.28±0.01vs. 0.72±0.1) relative to unstimulated cells, 2)decreased protein secretion IL-6 (25142 ± 229pg/ml vs. 2542 ± 37), G-CSF (388 ± 32 pg/ml vs. 27.8 ± 3.9 ) secretion, and 3)decreased IL-17RA surface expression when unstimulated (86.6% vs. 44.8%) or stimulated with ligand (14.1% vs. 5.2%) despite similar IL-17RC surface expression when unstimulated (93.6% vs. 91.3%) or stimulated (59.2% vs. 49.5%). Conclusions: FSTL-1 mediates IL-17 stimulated IL-6 and G-CSF production in ST2 cells. FSTL-1 influences transcript abundance of IL-17RA and IL-17RC, but shFSTL-1 attenuates surface expression of IL-17RA alone, suggesting that FSTL-1 regulates IL-17 signalling by influencing the cell surface expression of IL-17RA.
Overexpression of IK cytokine ameliorates the development of murine autoimmune arthritis (P5148)
Authors: Park Hye-Lim , Cho Mi-La , Min Jun-Ki , Nam Jae-Hwan
Published: January 2023

Rheumatoid arthritis (RA) is a systemic autoinflammatory disorder characterized by synovial hyperplasia and infiltration of inflammatory cells, as well as by the progressive destruction of cartilage and bone. Self-antigen presentation through aberrant major histocompatibility complex (MHC) class II expressing B cells produces autoantibodies, leading to development of more erosive RA. IK cytokine functions as an inhibitor of interferon γ (IFNγ)-induced MHC class II on B cells. Although its cellular and molecular mechanisms have remained unclear, IK cytokine has previously been linked to autoimmune diseases such as lupus nephritis and alopecia areata. Here, we investigated the role of IK cytokine in the pathogenesis of murine autoimmune arthritis. We generated immunodeficient arthritis mice (tIK-Tg-IL-1RaKO) by cross-breeding transgenic mice expressing truncated IK (tIK-Tg) with knockout mice lacking the IL-1 receptor antagonist (IL-1Ra-KO). In this regard, the Type II collagen (CII) immunized tIK-Tg-IL-1RaKO mice showed mild type arthritis and lower incidence than CII immunized IL-1RaKO mice. tIK-Tg-IL-1RaKO mice also showed decreased proinflammatory cytokine expression and immune cell expansion. Taken together, these results suggest that overexpression of IK cytokine may suppress the development of autoimmune arthritis by modulating inflammatory response in the case of autoimmune arthritis. Key words: IK cytokine, MHC class II, Rheumatoid arthritis
Blockade of IL-21 promotes the induction of T regulatory cells in healthy controls and IBD patients (P5158)
Authors: Wu Rebecca , Cox Jennifer , Blumberg Hal , Levin Steven , Lundsgaard Dorthe
Published: January 2023

T regulatory cells (Treg) play a critical role in maintaining immune homeostasis and tolerance to self-antigen. A subset of Treg induced by activation in the presence of TGF-β (induced or iTreg) has been shown to play a role in regulating immune responses and preventing autoimmune disease (AID). IL-21 is a pleiotropic cytokine involved in both innate and adaptive immune responses. Recent data in mouse models has shown that modulation of IL-21 function can influence Treg development and function, but the effect of IL-21 on human Treg is unknown. Therefore, we investigated the role of IL-21 in development of iTreg from T cells from healthy control (HC) and Crohn’s disease (CD) patients. Induced Treg were generated from CD4+CD25- T cells by activation in the presence of IL-2 and TGFβ. Exogenous IL-21 or blocking anti-IL-21 monoclonal antibody was also included on day 0 and cells were stained for intracellular IL-21 and FoxP3 expression on day 7. There was a significant reduction in the number of Foxp3+ T cells (p<0.03) in the presence of exogenous IL-21 and a significant increase when anti-IL-21 antibody was included (p<0.02). These effects on iTreg development were seen in both HC and CD patients. These data suggest targeting IL-21 with neutralizing antibodies can increase iTreg development and hence has promising potential as a novel therapeutic approach for the treatment of AID such as CD.
Redox modulation of the processing and presentation of myelin-associated antigens in experimental autoimmune encephalomyelitis. (P5184)
Authors: Allan Euan , Tailor Pankaj , Yates Robin
Published: January 2023

NOX2 is well known for its microbicidal activity within phagolysosomes of phagocytes. Our research group has recently found that NOX2 activity also impacts two key antigen processing chemistries within the phagosome of macrophages and dendritic cells. NOX2 activation inhibits disulfide reduction and alters proteolysis through the oxidative inactivation of cysteine cathepsins B, S, and L. Both disulfide reduction and proteolysis are vital for antigen and invariant chain processing, and thus are required for efficient antigen processing/presentation. Our objective is to determine if NOX2 influences the progression of EAE, which requires processing of endogenous myelin, by modulating antigen processing and changing the immuno-dominance of MOG peptides. We examined the susceptibility of WT and NOX2-deficient mice to EAE. Varying MOG components were used to induce EAE in WT and NOX2-deficient mice. NOX2-deficient mice were found to be partially clinically protected from MOG35-55 induced EAE, and less so from recombinant MOG induced EAE. Antigen presentation efficiency of MOG35-55 by NOX2-deficient macrophages to 2D2 T cells was reduced compared to WT macrophages when the antigenic source was recombinant MOG, but not when the macrophages were pulsed with MOG35-55. These results suggest that NOX2 may have a role in the modified processing of MOG proteins, which may be partially responsible for the differential susceptibility of NOX2-deficient mice to EAE.
Anti-IFN-gamma autoantibodies in adults with disseminated NTM infection (P4547)
Authors: Lin Chia-Hao , Chi Chih-Yu , Chu Chen-Chung , Liu Jing-Pei , Ku Chung-Lung
Published: January 2023

Adult patients with disseminated nontuberculous mycobacterial (dNTM) infections usually have severe immune system defects, such as AIDS or autoimmune diseases. Recently, several reports have shown that anti-IFN-γ autoantibodies may play an important role in the pathogenicity of dNTM infections. However, a considerable proportion of reported anti-IFN-γ autoantibodies cases either have clinical or laboratory evidence of autoimmune disease. In this study, we identified 19 previously healthy adults who suffered from dNTM infections, and 17 received further immunological function assays. High-titer anti-IFN-γ autoantibodies with activity inhibiting IL-12 production were found in the plasma of all of the patients. In addition to dNTM infection, 35% and 71% of our patients also suffered from salmonellosis and herpes zoster, respectively; both types of infections have rarely been reported in previous cases. This observation suggests that IFN-γ may be crucial in controlling salmonella infection and reactivating latent varicella zoster virus infection in humans. Two particular HLA alleles, DRB1*16:02 DQB1*05:02 (odds ratios of 8.68, 95% CI: 3.47-21.90, P=1.1×10-6, Pc=3.08×10-5 and 7.16, 95% CI: 3.02-17.05, P=1×10-7, Pc=1.4×10-6, respectively), were found in 82% (14/17) of our patients. In conclusion, our data suggest that anti-IFN-γ autoantibodies may play a critical role in the pathogenesis of dNTM infections and are associated with HLA-DRB1*16:02 and DQB1*05:02 among healthy adults.
The role of lymphoid micro-architecture in dendritic cell-mediated antigen capture and initiation of cellular adaptive immunity (P5200)
Authors: Gerner Michael , Kastenmuller Wolfgang , Germain Ronald
Published: January 2023

Dendritic cells (DCs) specialize in antigen (Ag) sampling and presentation and are critical for promoting T cell-mediated immune responses. DCs are composed of multiple subsets with divergent functional capabilities, with lymph node (LN)-resident CD8+ vs. CD11b+ DC preferentially processing captured Ags for MHC-I vs. MHC-II presentation, respectively. Utilizing a novel microscopy method, “Histo-Cytometry,” we have recently found that these subsets reside in differential spatial micro-compartments in murine LN, with preferential but not exclusive localization of CD8+ DCs to the central T cell zone and the CD11b+ DCs to the LN periphery. Here, we show that DC spatial positioning has profound functional effects on DC-mediated Ag capture and downstream MHC processing after protein immunization. Specifically, poor Ag penetration deep inside the LN paracortex led to decreased Ag sampling by the centrally localized CD8+ DCs and absence of CD8+ T cell responses at low levels of immunized Ag, as compared to the more effective Ag capture by peripheral CD11b+ DC and CD4+ T cell priming. Moreover, DC subset regionalization directly influenced the location of early CD8+ vs. CD4+ T cell activation, with centrally localized CD8+ T cell vs. predominantly peripheral CD4+ T cell priming. Collectively, these findings indicate that spatial organization by DC subsets creates micro-anatomical domains with specialized functional capacities affecting the development of cell-mediated immunity.
IL-20 receptor signaling inhibits cutaneous IL-1β and IL-17A production to promote methicillin-resistant Staphylococcus aureus infection (P6350)
Authors: Myles Ian , Fontecella Natalia , Valdez Patricia , Vithayathil Paul , Naik Shruti , Belkaid Yasmine , Ouyang Wenjun , Datta Sandip
Published: January 2023

Staphylococcus aureus causes the majority of human skin and soft tissue infections, and is a major infectious cause of mortality. However, host defense mechanisms against S. aureus are incompletely understood. Interleukin (IL)-19, -20 and -24 signal through type I and type II IL-20 receptors and have been associated with inflammatory skin diseases such as psoriasis and atopic dermatitis. We hypothesized these cytokines may play a role in S. aureus skin infections. We show here that this family of cytokines promotes cutaneous S. aureus infection in mice by downregulating IL-1β- and IL-17A-dependent inflammatory pathways required for control of infection. Our findings identify an immunosuppressive role for these cytokines during infection, and suggest these pathways may contribute to infectious susceptibility.
The ‘atypical’ chemokine receptor CCRL1 aids dendritic cell migration from inflamed skin. (P6352)
Authors: Bryce Steven , Luster Andrew , Graham Gerry , Nibbs Rob
Published: January 2023

Chemokine-directed leukocyte migration is essential for immune and inflammatory responses. The chemokine receptor CCR7, by responding to chemokines CCL19 and CCL21, is critical in regulating the recruitment of dendritic cells (DC), from peripheral tissues to draining lymph nodes (LNs). CCR7 is required for DC entry into lymphatic vessels, and for the passage of these cells across subcapsular sinus of the lymph node. Here, they initiate adaptive immune responses by priming antigen-specific T cells. CCRL1, an unexpected second receptor for CCL19 and CCL21, belongs to the family of atypical chemokine receptors and is able to bind and sequester extracellular chemokine without exhibiting any form of classical chemokine signaling. CCRL1 function in vivo is still unclear. We have generated CCRL1 deficient mice but find no perturbations in DC numbers in the LNs at rest, and DC trafficking from the skin is normal after painting with hapten (FITC). However, if FITC is applied after the induction of cutaneous inflammation, FITC+ DCs fail to reach draining LNs. Using a combination of microscopy and flow cytometry, we show that DCs are unable to enter lymphatic vessels and remain trapped in the skin. These data will be presented in the context of new insights into the identity of CCRL1-expressing cells in the skin and lymph node. Collectively, our data indicate that CCRL1-mediated regulation of CCL19 and/or CCL21 is required to maintain DC departure from the skin during inflammation.
Divergent roles for AdipoR1 and AdipoR2 in mediating revascularization and metabolic dysfunction (P6358)
Authors: Duffen Jennifer , Nakamura Kazuto , Silver Marcy , Zuriaga María , Aprahamian Tamar , Walsh Kenneth
Published: January 2023

Adiponectin is a well-described anti-inflammatory adipokine with a collectin-like structure. Previous reports have found that its deficiency leads to cardiovascular and metabolic dysfunction whereas its overexpression is protective. Two adiponectin receptors, AdipoR1 and AdipoR2, are poorly characterized and their metabolic functions remain controversial. Here, we subjected AdipoR1- and AdipoR2-deficient mice to hind limb ischemia surgery, a mouse model of peripheral artery disease. Blood flow recovery in AdipoR1-deficient mice was similar to wild-type; however, revascularization in AdipoR2-deficient mice was severely attenuated. Treatment with adiponectin enhanced the recovery of wild-type mice but failed to rescue the impairment observed in AdipoR2-deficient mice. These data show for the first time that AdipoR2 is functionally important in an in vivo model of vascular disease and that its expression is essential for the revascularization actions of adiponectin. Given the connection between cardiovascular and metabolic disease, AdipoR1- and AdipoR2-deficient mice were also evaluated in a model of diet-induced obesity. Strikingly, AdipoR1-deficient mice developed severe metabolic dysfunction compared to wild-type littermates. In contrast, AdipoR2-deficient mice were protected from diet-induced weight gain and metabolic perturbations. Taken together, AdipoR1 and AdipoR2 have divergent roles in mouse models of vascular and metabolic disease.
The contribution of individual TCR signaling components to thymic selection (P4540)
Authors: Brockmeyer Claudia , Herzmark Paul , Robey Ellen
Published: January 2023

In the thymus, T cell progenitors undergo an extensive selection program that shapes the repertoire of T cell receptors to ensure that mature T cells released into the periphery are tolerant to peptides derived from self proteins but able to recognize foreign peptide antigens. The engagement of the T cell receptor on a developing thymocyte triggers the formation of a proximal signaling complex at the plasma membrane and initiates a calcium signal, which together activate multiple downstream signaling cascades crucial for the thymocyte’s survival and maturation. One of the major signaling pathways initiated is the Ras/MAP kinase cascade, which plays an essential role during the positive selection of thymocytes. As thymic positive selection does not occur efficiently in 2-dimensional co-cultures, we are using a thymic slice preparation that provides the 3-dimensional stromal architecture of the thymus. We aim to study the signaling properties of MHC class I-restricted T cell receptor transgenic CD4+CD8+ thymocytes during positive selection either by flow cytometry or by two-photon laser scanning microscopy. We are using pharmacological inhibitors to understand the contribution of individual T cell receptor signaling components to positive selection. We hope to dissect both their longer-term effect on the thymocytes’ development, as well as their interconnection and potential compensatory mechanisms.
Innate and adaptive immunity against oxidative damage cooperate in the pathogenesis of age-related macular degeneration (P3367)
Authors: Cruz-Guilloty Fernando , Saeed Ali , Duffort Stephanie , Ballmick Asha , Tan Yaohong , Salomon Robert , Perez Victor
Published: January 2023

While our knowledge of acute immune responses is extensive, the cooperation of innate and adaptive immunity in chronic inflammatory diseases remains poorly defined. Age-related macular degeneration (AMD) is the leading cause of legal blindness in the developed world. Recently, the immune system has been shown to play a role in AMD. However, the specific roles of the adaptive immune system and different types of macrophages have not been studied in detail. Oxidative stress is a major contributing factor in the development of pathological inflammatory conditions, including atherosclerosis and AMD. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD and mice immunized with CEP-adducted self proteins develop AMD-like pathology. Here, we show CEP-specific T cell activation, pro-inflammatory cytokine production (IFN- γ, IL-17) and infiltration of M1 macrophages in the subretinal space, linking both the adaptive and innate immune systems in the onset of AMD. In vitro, CEP-specific T cells specifically promote M1 macrophage differentiation, while CEP directly induces TLR-2-independent M1 polarization. Moreover, CEP-immunized Ccr2-deficient mice lack macrophage infiltration and retinal lesions, suggesting a deleterious role for macrophages in this model. Our data provide new insights into the pathogenesis and possible treatment of AMD, while providing a more general view of chronic inflammation applicable to other settings.
Successful immunotherapy with interleukin 2 for hypoxia-induced pulmonary hypertension (P3371)
Authors: Bauer Eileen , Zheng Han , Bauer Philip , Lotze Michael
Published: January 2023

The development of therapies to treat pulmonary hypertension (PH) has primarily focused on targeting intrinsic endothelial dysfunction and vasoreactivity. Recent meta-analysis of controlled trials utilizing these therapies, however, shows only modest decreases in mortality. Despite evidence that inflammatory mechanisms complicate/possibly initiate events in PH, no strategies have been developed to target specific endothelial-extrinsic abnormalities of the inflammatory cells involved. We previously reported on the contributory role of the inflammatory damage associated pattern molecule HMGB1 in patients with PH and in a well-established murine model of hypoxia-induced PH. With reports on the dysfunctional behavior of natural killers (NK) cells in PH patients and on its prevention by adoptive transfer of CD4+CD25bright Tregs in a rat model, we decided to target PH using immunotherapy. Treatment with low-dose Interleukin 2 (IL-2,6x104 IU BID) not only attenuated the development of hypoxia-induced PH in mice but more importantly reversed established PH. IL-2 treated mice demonstrated decreased: 1) pulmonary vascular remodeling 2) right ventricular systolic pressures indicative of pulmonary pressures and 3) right heart hypertrophy, all of which are standard assessments of PH, but increased NK and CD4 T-cell number. These findings demonstrate for the first time that PH is an immune-responsive disorder and suggest IL-2 administration as a viable and novel therapeutic strategy.
A single peptide-MHC triggers digital cytokine secretion in both naive and activated CD4+ T cells (P1368)
Authors: Huang Jun , Davis Mark
Published: January 2023

We have developed a single molecule imaging technique that uses quantum dot labeled pMHCs to study CD4+ T cell functional sensitivity. We find that both naive and activated T cells can be triggered by a single pMHC to secrete cytokines with a rate of ~1,000 and ~10,000 molecule/min respectively and that additional pMHCs do not increase secretion, thus these cells display a digital response pattern. We also find that a single pMHC induces the formation of a long-lasting TCR microcluster, consistent with a serial engagement mechanism. These data show that scaling up T cell cytokine responses must involve efficient T cell recruitment rather than increasing individual cell cytokine production.
Guardian angels in the graveyard: B1 B cells promote thymic Treg survival (P1386)
Authors: Zhong Xuemei , Alaimo Danielle , Hall Matthew , Lovell John , Vo Hung , Bai Chunyan , Lau Stanley
Published: January 2023

The thymus is the primary site for T cell development. Within the medulla of the thymus, negative selection of immature T cells causes a large number of cells to undergo apoptosis, appropriately deeming this thymic region a “grave yard.” In observing the thymus medulla of BXSB mice, we noted a significant increase of B cells in lupus-symptomatic males as compared to female controls or lupus- asymptomatic males. Composition analysis of these thymic B cells revealed that both B1 and B2 cells are present. Immunohistochemistry analysis suggests possible chemo-attraction for B cells in the medullar of the thymus due to the expression of CXCL13 in lupus male mice, but not control females. In vivo tracking of peritoneal B cells over a two-week time period indicated that some of the thymic B cells originated from the peritoneal cavity. To test the possible influence of B cells on thymic T cell development, we co-cultured peritoneal and spleen B cells with thymocytes in a 3D hanging drop plate. Our results suggest a “guardian angel” effect by B1 B cells, depicting an enhanced central tolerance by thymic-migration and promoting regulatory T cell survival.
Ultra-violet irradiation and riboflavin treatment blocks capping of cellular adhesion molecules and abrogates immunogenicity of antigen presenting cells (P1388)
Authors: Carrillo Ralf , Norris Philip , Jackman Rachael , Marschner Susanne , Goodrich Raymond
Published: January 2023

UV light has known immunomodulatory activity and is used in clinical situations such as for psoriasis. Mirasol treatment, a pathogen reduction technology based on ultra-violet light irradiation in the presence of riboflavin (UV+R), suppresses the ability of treated WBCs to stimulate proliferation of allogeneic T cells in vitro. Prior work showed that surface expression of adhesion molecules was down-regulated after UV+R treatment. We now show that, UV+ R treatment also decreased capping of the intercellular adhesion molecules (ICAMs)-1, -2, -3 and lymphocyte function-associated antigen (LFA)-3. UV+R treated U937 antigen presenting cells (APCs) induce decreased cell-cell conjugation with PBMCs 25% (p = 0.003) and show a 66% (p = 0.0005) reduction in stimulating T cell proliferation in a mixed lymphocyte reaction compared to untreated or gamma-irradiated U937 APCs. Additionally, UV+R treated U937 APCs failed to induce expression of the early T cell activation marker CD69 and also have decreased membrane viscosity when compared to untreated APCs. Diminished surface expression and perturbation of adhesion receptors within the U937 APCs plasma membrane down-regulates T cell stimulation and proliferation in vitro by lowering the surface expression of adhesion receptors needed for cell-cell conjugation and by perturbation of the ability to mobilize and aggregate adhesion receptors to form a cap, a critical component needed for T cell activation.
Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-κB activation (P1370)
Authors: Kang Jung-Ah , Jeong Sang Phil , Park Daeho , Hayden Matthew S , Ghosh Sankar , Park Sung-Gyoo
Published: January 2023

Strong NF-κB activation requires ligation of both the CD28 co-receptor and TCR. PDK1 acts as a scaffold by binding both PKCθ and CARMA1 and is therefore essential for signaling to NF-κB . Here, we demonstrate the importance of PDK1 threonine (Thr)-513 phosphorylation in regulating the intermolecular organization of PDK1 homodimers. Thr-513 is directly involved in heterotypic PDK1 homodimer formation, in which binding is mediated through the pleckstrin homology (PH) and kinase domains. Upon activation, phosphorylated Thr-513 instead mediates homotypic intermolecular binding through the PH domains. Consequently, cell permeable peptides with a Thr-513 to Ile derivative (PTD-PDK1-Thr-513-Ile) bound the kinase domain and while a Thr-513 to Asp peptide (PTD-PDK1-Thr-513-Asp) bound the PH domain. PTD-PDK1-Thr-513-Ile blocked binding between PDK1 and PKCθ, phosphorylation of PKCθ Thr-538, and activation of both NF-κB and AKT. In contrast, PTD-PDK1-Thr-513-Asp selectively inhibited binding between PDK1 and CARMA1 and blocked TCR/CD28 induced NF-κB activation. Therefore, Thr-513 phosphorylation regulates a critical intermolecular switch governing PDK1 homodimer structure and the capacity to interact with downstream signaling pathway components. Given the pleiotropic functions of PDK1, these data may open the door to the development of immunosuppressive therapies that selectively target the PDK1 to NF-κB pathway in T cell activation.