bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting of antigens to dendritic cells via lentiviral vector induces tolerance of pre-activated T cells and protects mice from autoimmunity (P5215)
Authors: de Andrade Pereira Bruna , Ackermann Mathias , Dresch Christiane , Fraefel Cornel
Published: January 2023

Autoimmune disease is characterized by a breakdown of tolerance and activation of auto-reactive T cells. Current treatment is based on palliative drugs that attenuate or delay disease symptoms. The aim of this project was to develop a new strategy to induce permanent antigen-specific tolerance for treating autoimmune disease. The strategy includes the ex vivo modification of autologous hematopoietic stem cells (HSC) with lentiviral vectors that express myelin oligodendrocyte glycoprotein (MOG), an auto-antigen involved in experimental autoimmune encephalomyelitis (EAE). The MOG-transgene is under the control of a dendritic cell (DC)-specific promoter. After re-infusion, the modified HSC will give rise to MOG-expressing DCs. We have previously demonstrated that 100% of mice that received HSC transduced with MOG-lentiviral vectors were protected. Protection correlated with the depletion of MOG-specific T cells and generation of functional Foxp3+ regulatory T cells. Moreover, we showed that the tolerance induced was antigen-specific while the immune system was not generally suppressed. In the present work, we demonstrate that mice which have received HSC transduced with MOG-lentiviral vectors were also fully protected from EAE after adoptive transfer of pre-activated MOG-specific T cells. This result highlights the applicability of this strategy in a therapeutic model by reverting the pathogenic phenotype of effector MOG-specific T cells.
Proteasome inhibition with bortezomib depletes plasma cells and autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients (P5146)
Authors: Gomez Alejandro , Willcox Nick , Vrolix Kathleen , Hummel Jonas , Duimel Hans , Verheyen Fons , Molenaar Peter , Buurman Wim , Martinez-Martinez Pilar , De Baets Marc , Losen Mario
Published: January 2023

In the autoimmune disease myasthenia gravis (MG), autoantibodies against the muscle AChR are mainly produced by both short- and long-lived plasma cells, which are resistant to standard immunosuppressive drugs (i.e. glucocorticoids). A novel therapy to eliminate plasma cells is the proteasome inhibitor bortezomib, which is used to treat patients with multiple myeloma (MM, a plasma cell malignancy). Previously, we demonstrated that bortezomib also reduced autoantibody titers in an animal model of MG (Gomez, A. M. J. Immunol. 2011). The thymus of MG patients is frequently enriched in germinal centers and contains plasma cells that produce autoantibodies in vitro, even after irradiation (which depletes B and T lymphocytes). We studied the in vitro effects of bortezomib in cultured thymus cells from MG patients undergoing therapeutic thymectomy. Treatment with a single dose of bortezomib blocked the production of these pathogenic autoantibodies, reduced the total IgG levels and eliminated plasma cells. Ultrastructural signs of apoptosis were detected in plasma cells as early as 8 h after addition of bortezomib; at 24 h, no plasma cells could be detected. Moreover, we demonstrated that the minimum concentration of bortezomib that eliminates plasma cells in vitro is 60-fold lower than the peak concentration found in MM patients treated with bortezomib, suggesting that low doses might be effective for eliminating plasma cells in patients with antibody-mediated autoimmune diseases.
Treatment with non-depleting T cell antibodies increases TGFβ levels in the diabetic pancreas and results in enhanced β cell function. (P5213)
Authors: Martin Aaron , Yi Zhouan , Wang Bo , Tisch Roland
Published: January 2023

A recent report from our laboratory detailed the reversal of recent-onset autoimmune diabetes in the NOD mouse following treatments with antibodies against CD4 and CD8. Treatment of mice with these particular antibodies does not deplete T cells, but transiently alters their function, culminating in long-lasting immune tolerance and a restoration of normal glycemic control. Consistent with observations made by other research groups, induction of diabetes remission was dependent on TGFβ, supported by two lines of evidence: 1 - co-administration of anti-TGFβ with anti-T cell antibodies resulted in a failure of remission induction, and 2 - elevated levels of TGFβ RNA and protein can be detected in the pancreata of remission mice. Within one week of antibody treatment, T cells are purged from the pancreas and the draining lymph node in a process that was found to be independent of TGFβ, suggesting that effects of elevated TGFβ are not limited to T cell function. Furthermore, treatment of recent-onset diabetic mice with a reduced dose of antibody resulted in a purging of T cells from the pancreas and draining lymph node, but not a restoration of glycemic control. Normal glycemic control could be achieved at this dose when exogenous TGFβ was administered. Together, these data indicate that TGFβ may directly affect the insulin-producing pancreatic β cells. Recent observations support the notion that TGFβ enhances insulin production and/or induces β cell proliferation.
Immunological studies on promising and selective anti-arthritic effects of Euphorbia hirta (P5155)
Authors: Ahmad Sheikh , Albakheet Saleh
Published: January 2023

Euphorbia hirta L. (Euphorbiaceae) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, wounds, antipyretic, anti-inflammatory activities etc. In the present study we investigated the anti-arthritic effects of fresh leaves of E. hirta ethanol extract, that was found to inhibit the production of inflammatory mediators and cytokines of adjuvant arthritis in mice. The production of the Th1 and Th2 cytokine was estimated by flowcytometric assay by using cytometric bead array (CBA) analysis in adjuvant induced arthritis in mice treated with E. hirta at graded oral doses. CBA uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate five cytokines in a 50 µl sample volume. Oral administration of E. hirta at doses of 25-200 mg/kg showed significant inhibition of Th1 (IL-2, IFN-γ & TNF-α) and up regulation of Th2 (IL-4 and IL-5) cytokines. Also, E. hirta significantly down regulated LPS induced production of nitric oxide production in peritoneal macrophages. These results suggest that E. hirta exhibit an improvement in adjuvant induced arthritis through down regulation of activated macrophages and T lymphocytes functions such unique effects of E. hirta shown on adjuvant arthritis mice model may be advantageous to the long term treatment of clinical rheumatoid arthritis.
Effects of valproic acid and dexamethazone in acute kidney ischemia-reperfusion injury model (P2216)
Authors: Speir Ryan , Andrews Jared , DeHart Mary , Gelnett Mary , Brand Timothy , Salgar Shashikumar
Published: January 2023

Introduction: Renal ischemia-reperfusion (IR) causes acute kidney injury (AKI) with high mortality and morbidity. The objective of this study was to ameliorate kidney IR injury and identify novel biomarkers for AKI. Methods: Left renal ischemia was induced in Wister rats by clamping the renal artery for 45 minutes, followed by reperfusion and right nephrectomy. Thirty minutes prior to ischemia, rats (n=8/group) received Valproic Acid (150 mg/kg; VPA), Dexamethasone (3 mg/kg; Dex) or Vehicle (Saline) IP. Animals were sacrificed at 3h, 24h or 120h IR and blood, urine and kidney were collected. Results: At 3h IR, urine albumin was lower (P>0.05) in VPA (0.84±0.62) and Dex (1.04±0.73) compared to Vehicle (1.47±1.02 mg/ml) group; and Lipocalin-2 was lower (P<0.006) in Dex (0.42±0.15) and higher (P<0.01) in VPA (1.21±0.32) compared to Vehicle (0.77±0.31 µg/ml) group. Osteopontin was higher (P<0.05) at 24h IR in VPA and Dex . TIM-1 did not differ. Serum creatinine (mg/dL) at 24 h IR in VPA (2.7±1.8) and Dex (2.3±1.2) was reduced (P<0.05) compared to Vehicle (3.8±0.5). Histopathology at 3h IR demonstrated reduced (P<0.04) ischemic changes in the renal cortex in VPA (27± 32 %) compared to Vehicle (56±27%) group. Tubular necrosis in the outer medulla was lower (P<0.02) in Dex and VPA (43-45%) compared to Vehicle (70%) at 24h IR. BCL-2 as determined by RT-PCR did not differ. Conclusion: The VPA administration prior to surgery appears to offer renal protection from IR injury.
Bim regulates allogeneic immune responses and transplant arteriosclerosis through opposing effects on T cell activation and death. (P2218)
Authors: von Rossum Anna , Enns Winnie , Shi Yu , Choy Jonathan
Published: January 2023

T cell responses towards allograft arteries are responsible for the development of transplant arteriosclerosis (TA), a vascular condition characterized by intimal thickening of graft arteries. Bim is a pro-apoptotic Bcl-2 protein known to down-regulate immune responses by inducing effector T cell death, but its role in regulating allogeneic T cell responses is poorly understood. We compared allo-activation and death of T cells from Bim+/+, Bim+/- and Bim-/- mice as well as the development of TA in these animals. Unexpectedly, Bim was required for alloantigen-induced proliferation of both CD4 and CD8 T cells, as well as IL-2 production. Partial reduction in Bim expression was sufficient to attenuate T cell activation while complete elimination was required to prevent T cell death driven by cytokine deprivation in vitro. When TA was examined in aortic interposition grafts, intimal thickening was significantly reduced in Bim+/- but not Bim-/- recipients. There was significantly less CD4 T cell accumulation in the intima of arteries from Bim+/- as compared to Bim+/+ recipients, but this effect was not observed in Bim-/- mice. Further, T cell proliferation was significantly reduced in both Bim+/- and Bim-/- mice in response to allograft arteries but T cell death was attenuated only in Bim-/- animals. Taken together, our findings show that Bim controls both T cell activation and death in TA, and that these processes are differentially susceptible to reductions in Bim expression.
ERAAP associates with MHC I molecules for generation of optimal MHC I peptide repertoire. (APP3P.100)
Authors: Yang Soo Jung , Shastri Nilabh
Published: January 2023

Endoplasmic reticulum (ER) aminopeptidase associated with antigen processing (ERAAP) plays a critical role in the generation of the peptide repertoire presented by MHC I by trimming precursor peptides to optimal length. How ERAAP carries out this function and whether ERAAP activity is influenced by MHC I or other molecules in the ER is unknown. Here, we show that ERAAP physically associates with MHC I during peptide trimming events in the ER. ERAAP or its truncated domains II/III interacted with empty MHC I in the ER, but this interaction was independent of the peptide-loading complex. Most importantly, overexpression of domain II/III interrupted ERAAP function in wild-type cells resulting in altered-peptide presentation by MHC I. Thus, ERAAP function can be regulated by its association with MHC I and perhaps other key ER molecules.
Intradermal immunization with polyguanine conjugated antigens enables targeted and sustained delivery of protein antigens to dendritic cells in vivo. (APP3P.111)
Authors: Ciccotelli Jo Erika , Toussaint Helene , Erdos Geza , Carey Cara , Watkins Simon , Falo Louis
Published: January 2023

Effective vaccine design depends on the ability to target specific antigens to antigen presenting cells (APCs), including dendritic cells (DCs). Conjugation of proteins antigens to polyguanine (Poly(dG)) molecules is a novel immunization approach capable of transforming soluble antigens into aggregated particulates with exposed scavenger receptor (SR) ligands. We have previously shown that Poly(dG) conjugation to protein antigen results in increased antigen-specific helper and cytotoxic T cell responses, memory T cell induction, and antibody titers. Here, we specifically investigate mechanisms of Poly(dG)-conjugated antigen delivery and internalization. Compared to soluble OVA, Poly(dG)-OVA is rapidly and more efficiently internalized by bone marrow derived DCs in vitro, and this internalization is inhibited by scavenger receptor blockade. Importantly, in a mouse model intradermally injected Poly(dG)-OVA results in antigen persistence in the skin for up to 7 days. This is accompanied by increased antigen uptake by skin resident DCs and persistent migration of antigen loaded DCs to the draining lymph nodes. DCs exposed to Poly(dG)-OVA had increased expression of CCR7 and secretion of MCP-1, TNF-α, and IL-6. These results suggest that coupling Poly(dG) to protein antigens enables efficient DC targeting through SRs, prolonged delivery of antigens in vivo, and activation of innate immunity. This approach may be used to design more efficient antiviral and antitumor vaccines.
RBP4 activates antigen-presenting cells leading to adipose tissue inflammation and systemic insulin resistance (APP4P.116)
Authors: Vieira Pedro , Yore Mark , Dwyer Peter , Syed Ismail , Aryal Pratik , Kahn Barbara
Published: January 2023

A major cause of Type 2 diabetes is impaired insulin action which is a critical risk factor for cardiovascular disease and early mortality. Insulin resistance in obesity is highly associated with adipose tissue (AT) inflammation. RBP4, a retinol-transporter, is elevated in insulin resistance and contributes to increased diabetes risk. We aimed to determine the mechanisms for RBP4-induced insulin resistance. Here we show that RBP4 elevation causes AT inflammation by activating innate immunity which elicits an adaptive immune-response. RBP4-overexpressing mice (RBP4-Ox) are insulin-resistant and glucose-intolerant and have increased AT macrophage and CD4 T-cell infiltration. In RBP4-Ox, AT CD206+ macrophages express pro-inflammatory markers and activate CD4 T-cells while maintaining alternatively-activated macrophage markers. These effects result from direct activation of AT antigen-presenting cells (APCs) by RBP4 through a JNK-dependent pathway. Transfer of RBP4-activated APCs into normal mice is sufficient to induce AT inflammation, insulin resistance and glucose intolerance. Thus, RBP4 causes insulin resistance, at least in part, by activating AT APCs which induce CD4 T-cell Th1 polarization and AT inflammation.
Pulmonary dendritic cells require cis-activation to cross-prime T cells (APP4P.117)
Authors: Jakubzick Claudia , Desch A. Nicole , Gibbings Sophie , Clambey Eric , Janssen William , Slanskey Jill , Kedl Ross , Henson Peter
Published: January 2023

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident migratory DC fall into two types, respectively expressing the integrin markers, CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103+ DC as the major cross-presenting DC able to induce CTL. Here we proposed that DC function might also be determined by the nature of the pathogen associated molecular pattern (PAMP) that stimulates it and to which it is able to respond. Thus, in the presence of a TLR3 (CD103+) or a TLR7 (CD11b+) agonist respectively we demonstrated in vivo that both DC subtypes did exhibit the capacity to orchestrate a CTL response. This selectivity did not extend to antigen cross-presentation for T cell proliferation but was required for induction of cytotoxicity and protection in a model of tumor metastasis.
Role of Integrins in mediating Th17 Pathogenesis (CAM3P.203)
Authors: McGeachy Mandy
Published: January 2023

IL-23 plays a key role in autoimmune pathology, as evidenced by the resistance of IL-23 deficient animals to many models of autoimmune disease; susceptibility SNPs in the IL-23 receptor have also been identified in human disease including inflammatory bowel disease and psoriasis. We have previously demonstrated that IL-23 drives effector differentiation and proliferation of primary and memory Th17 cells in vivo. We have now identified a novel integrin that is expressed by Th17 cells in an IL-23-dependent manner. Inflammatory Th17 cells (generated in vitro in the presence of IL-23, IL-6 and IL-1) express high levels of this integrin, while ‘regulatory’ Th17 cells do not, as TGFβ suppresses expression in a dose-dependent manner. Integrin blockade rendered mice resistant to experimental autoimmune encephalomyelitis (EAE), and knockout mice were also resistant to EAE. T cells specifically require integrin expression for EAE induction. While effector functions such as IL-17 are not impaired, integrin knockout cells have reduced migration out of LN and into CNS. Together these data highlight a novel Th17-specific integrin that is regulated by IL-23, and may promote effector Th17 functions and/or proliferation.
CD4+ T cell-derived IL-10 is independent of IL-27 signaling in a recall response. (CCR3P.220)
Authors: Perona-Wright Georgia , Fonseca Nicolette , Redpath Stephen
Published: January 2023

Surviving influenza infection requires a careful balance of pro-inflammatory signals that promote viral clearance and anti-inflammatory signals that prevent immunopathology. IL-10 is a potent immunosuppressive cytokine that is essential to this balance. Evidence suggests that in CD4+ T cells, IL-10 expression is critically dependent on IL-27 signaling. Here we show in vitro and in vivo that CD4+ cells downregulate gp130, the signal transduction component of the IL-27 receptor, upon activation. Gp130 expression remains low throughout CD4+ T cell contraction and memory, and renders the T cells non-responsive to IL-27 stimulation. Despite this, during secondary activation effector CD4+ T cells express IL-10 at a level equivalent to their primary effector counterparts. Cells genetically deficient for il27ra also fail to express IL-10 during primary activation but are equivalent to wildtype cells during a secondary response. Together our data highlight an IL-27 independent mechanism of IL-10 regulation that is unique to secondary CD4+ T cell activation, and plays a decisive role in the balance between effective immunity and immunopathology during influenza-induced respiratory disease.
Premature thymic involution impairs let7 up-regulation in the transition from fetal to adult hematopoietic stem cells in the thymus (HEM4P.236)
Authors: Xiao Shiyun , Zhang Wen , Manley Nancy
Published: January 2023

The postnatal thymus is a highly efficient microenvironment for promoting T cell specification and differentiation. However, hematopoietic stem cells (HSCs) possess age-dependent differences in differentiation potential into T or B cells, with fetal-type HSCs having a higher potential for producing B cells. The switch between fetal- and adult-type HSCs occurs in the perinatal period, associated with a transition from Lin28b to Let7 expression. We found that in Foxn1lacZ/lacZ mutant mice, which undergo premature thymic involution beginning at 1-2 weeks postnatally, the ETP and DN1a/b subsets were immediately and progressively reduced, while thymic B cells showed a transient increase peaking at around 3-4 weeks. Heterochronic chimeras showed that the capacity to produce B cells in mutant thymus was dependent on the age of HSCs, rather than the age of thymus. We further demonstrate that in thymic progenitor B cells, while Lin28b down regulation occurred normally, Let7g up regulation was impaired, repressing differentiation and promoting proliferation of progenitor B cells in Foxn1lacZ/lacZ thymus. Changes in the thymic microenvironment included progressive down regulation of CCL25, accelerated reduction of DL4 and a transient increase in IL7. Taken together, these data show that the switch from fetal-type to adult-type HSCs within thymus is controlled by thymic microenvironment at multiple levels, including lymphoid progenitor entry, fate specification, and differentiation.
Ribosomal protein L22 (Rpl22) controls the development of αβ lineage T cells by regulating ER stress pathways (HEM4P.239)
Authors: Solanki Patel Nehal , Stadanlick Jason , Cecile-Duc Ann , Rhodes Michele , Wiest David
Published: January 2023

While mature αβ and γδ T cells arise from a common CD4-CD8- thymocyte progenitor, the pathways controlling separation of these lineages remain poorly understood. We have identified the ribosomal protein Rpl22, a novel effector that plays a crucial role in αβ T cell development, but is dispensable for γδ T cell development. Indeed, Rpl22-deficiency selectively blocks the development of αβ T cells at the β-selection checkpoint in a p53-dependent manner. Here, we investigate the basis for p53 induction caused by Rpl22-deficiency and its restriction to αβ T cell progenitors. We found that Rpl22 can directly bind and suppress the translation of p53 mRNA, and that the absence of Rpl22 induces p53 through translational de-repression. Rpl22 appears to specifically regulate p53 in αβ T cell progenitors by controlling the activity of signaling processes that induce p53, such as endoplasmic reticulum (ER) stress. αβ T cell development is dependent on the activation of ER stress responses, which are known to induce p53. Pharmacologic exacerbation of ER stress blocks development of αβ, but not γδ T cells. Moreover, Rpl22-inactivation exacerbates signals from all three known ER stress sensors (PERK, Ire1α, and ATF6). Finally, inhibiting the ER stress response mediated by PERK partially restores development of Rpl22-deficient αβ progenitors. Together, our data suggest that Rpl22 regulates αβ T cell development by controlling the physiologic ER stress upon which their development depends.
Characterization of the NOTCH1-driven miRNA network that controls normal human T-cell development. (HEM4P.246)
Authors: Taghon Tom , Van der Meulen Joni , Durinck Kaat , Van de Walle Inge , Mestdagh Pieter , Pipelers Peter , De Preter Katleen , Vandesompele Jo , Thas Olivier , Rondou Pieter , Van Vlierberghe Pieter , Frank Speleman
Published: January 2023

We have recently shown that miRNAs can cooperate with NOTCH1 activation in malignant T-cell lymphoblasts (Mavrakis et al. Nature Genetics 2011), but little information is available regarding NOTCH1-regulated miRNAs that mediate human T-cell development under normal physiological circumstances in the thymus. Therefore, we first screened for miRNAs that positively correlate with NOTCH1, HES1 and DTX1 expression during normal human T-cell development. To select for miRNAs that are directly NOTCH1 regulated, NOTCH1 expressing CD34+ thymic T-cell progenitor cells from two healthy donors were cultured in vitro in either the absence (OP9-GFP) or presence of four different NOTCH1 ligands (DL1, DL4, J1, J2) and differential miRNA and mRNA expression patterns were monitored. Next, we validated the candidate NOTCH1 driven miRNAs by implementing publically available NOTCH1 ChIP-sequencing data (Wang et al. PNAS 2011). This showed binding of ICN1 to the promotor regions of some putative target miRNAs. To identify key mRNA targets of these NOTCH1-regulated miRNAs, we used an integrated genomic analysis in which we screened for genes with strongly anti-correlated expression patterns to NOTCH1 and its downstream miRNAs that were additionally predicted targets of these NOTCH1-driven miRNAs. Currently, we are validating these miRNA/mRNA interactions and functionally analyzing the roles of these miRNAs and their targets on human T-cell development in the OP9-DL1 system.
The presence of LAG-3 in nonhuman primates undergoing an active Mycobacterium tuberculosis infection (HEM4P.252)
Authors: Phillips Bonnie , Kaushal Deepak
Published: January 2023

Th1 T cells are essential in the immune response against Mycobacterium tuberculosis (Mtb) infections. Lymphocyte activation gene-3 (LAG-3) regulates this response through the diminishment of effector T cells and the impairment of monocyte differentiation. We hypothesize that LAG-3 up-regulation within Mtb infected nonhuman primates (NHPs) will suppress the Th1 portion of the host immune response resulting in conditions that favor the pathogen and create active tuberculosis (TB) within the host. Here we are studying LAG-3 expression in rhesus macaques infected with aerosolized Mtb. We have chosen the macaque model due to their similarities to humans in the spectrum of TB disease, as well as the architecture of Mtb induced lung granulomas. Previous studies have implied that LAG-3 may have a role in the activation of Mtb infections. There is increasing evidence that up-regulation of LAG-3 occurs in the lungs of animals experiencing TB activation. Further investigation of the lung tissue of these infected animals with fluorescent immunohistochemistry visualized with confocal microscopy has illustrated LAG-3 to be largely present within the outer periphery of the lung. Cells expressing LAG-3 have shown to include T cells and NK cells, with large numbers of these cells appearing in close vicinity to certain populations of antigen presenting cells. These results show that in animals undergoing TB activation, LAG-3 has potential to play an immunomodulatory role in this process.
Type-2 innate lymphoid cells facilitate antigen-induced CD4+ TH2 cell immune response that exacerbates chronic allergic asthma (HYP7P.291)
Authors: Liu Bo , Lee Jee-Boong , Chen Chun-Yu , Burwinkel Karen , Wang Yui-Hsi
Published: January 2023

Type-2 innate lymphoid cells (ILC2s) and acquired CD4+TH2 and TH17 cells were shown to contribute to the pathogenesis of allergic asthma; however, their relative roles in mounting antigen recall response that exacerbate chronic allergic asthma remain elusive. Here we reported that repeated OVA re-challenge induced significant increases of eosinophil, not neutrophil accumulation and serum OVA specific IgE and IgG1 in rested mice that previously developed allergic asthma. Subsequent analysis revealed that these rested mice with allergic asthma preferentially mounted elevated OVA specific CD4+TH2 cell response against intranasal OVA re-challenge, while the frequency of lung OVA specific TH17 cells or ILC2s remained constant. While ILC2s and TH17 cells developed normally, STAT6 KO mice that lacked TH2 cell compartment or mice ablated of CD4+ T cells failed to mount allergic recall response to OVA re-challenge. Notably, we observed that cocultured ILC2s could greatly enhance TH2 cytokine production by OVA specific TH2 cells after OVA stimulation ex vivo. Indeed, mice co-transferred with ILC2s and OVA specific TH2 cells exhibited significantly increased inflammatory cell infiltration and goblet cell hyperplasia, compared to those transferred with ILC2s or TH2 cells alone. Collectively, our results suggest that antigen re-exposures preferentially induce lung resident TH2 cells to mount antigen recall immune response that can be enhanced by ILC2s to exacerbate allergic inflammation
Suppression of established IgE-mediated food allergy with anti-FcεRIα mAb (HYP7P.310)
Authors: Krishnamurthy Durga , Khodoun Marat , Jensen-Jarolim Erika , Finkelman Fred
Published: January 2023

Food allergy, in mouse models, is mediated by mast cells, FcεRI and IgE. We recently demonstrated that rapid desensitization with a hamster mAb to mouse FcεRIα (MAR-1) safely suppresses IgE-mediated anaphylaxis (JACI, 131:1555-64, 2013). Consequently, we evaluated whether the same approach could suppress established food allergy in BALB/c mice that had been sensitized intraperitoneally with ovalbumin/alum or egg white/alum and challenged repeatedly by oral gavage with ovalbumin or egg white until they had developed shock and diarrhea. Treatment of these mice with 50 µg of MAR-1 every 4 days suppressed both the induction of hypothermia by oral gavage with ovalbumin or egg white and mast cell MMCP1 secretion by 85-90% and decreased the incidence of diarrhea by 70-85%. Although MAR-1 suppression of food allergy was eventually limited by mouse development of IgG antibodies to hamster IgG, this could be prevented by injecting mice with anti-CD4 mAb (GK1.5) at the time of the initial MAR-1 injection. Although GK1.5, by itself, had little effect on established food allergy, the combination of MAR-1 plus GK1.5 completely suppressed the hypothermia, diarrheal, MMCP1, IL-4 and IL-13 responses to oral gavage with ovalbumin or egg white. These observations suggest that rapid desensitization, followed by continuing treatment with a non-immunogenic (humanized) anti-FcεRIα mAb might effectively suppress IgE-mediated food allergy in people with this disorder.
FADD and caspase-8 mediate priming and activation of the canonical and non-canonical Nlrp3 inflammasomes (INC5P.321)
Authors: Gurung Prajwal , Kanneganti Thirumala-Devi
Published: January 2023

The Nlrp3 inflammasomes are essential for protection against several infections and autoimmune diseases. Despite the plethora of research focused on understanding the roles of NLRP3 in various disease settings, the mechanisms controlling its activation remain enigmatic. Herein, we show through genetic studies that FADD or caspase-8 is required for both transcriptional priming and posttranslational activation of the canonical and non-canonical Nlrp3 inflammasomes. FADD and caspase-8 were required for caspase-1 activation during canonical Nlrp3 inflammasome activation by both soluble (LPS+ATP) and particulate (Silica) ligands. We showed that FADD and caspase-8 also regulate Nlrp3 expression. Furthermore, both FADD and caspase-8 were critical for caspase-11 and caspase-1 activation during non-canonical Nlrp3 inflammasome activation by C. rodentium and E. coli. These requirements of FADD and caspase-8 for caspase-1 activation were specific to the Nlrp3 inflammasome since the activation of Nlrc4 inflammasome by S. typhimurium was not hampered by deletion of FADD or caspase-8. In vivo LPS challenge or C. rodentium infection of FADD or caspase-8-deficient mice resulted in impaired IL-1β production compared to their littermate controls. Thus, our results demonstrate that FADD and caspase-8 are critical mediators of the canonical and non-canonical Nlrp3 inflammasomes.
Impact of obesity on intraepithelial lymphocyte homeostasis (INC6P.350)
Authors: Park Christa , Costanzo Anne , Cheung Kitty , Jameson Julie
Published: January 2023

According to the Center of Disease Control and Prevention, more than one-third of the U.S. adult population is obese. Obesity is caused by overnutrition and accumulation of excess body fat leading to a state of chronic inflammation that impacts the function of epithelial tissues such as the intestine. The intestine consists of villi that increase the overall surface area to digest food and absorb nutrients. The epithelial layer of the villi contains immune cells such as intraepithelial intestinal lymphocytes that are important in maintaining epithelial homeostasis and repairing damaged intestinal tissue. Mice were placed on a high fat diet to define the mechanism by which obesity impacts intestinal epithelia and barrier function. Our data show that obese mice exhibit an increase in the severity of DSS-induced colitis. Using immunofluorescent microscopy we observe an increase in intestinal villi length with a simultaneous reduction in IEL number. The lamina proprial macrophages become skewed toward M1 CD11b+ CD11c+ proinflammatory type macrophages. Short-term diet reduces the severity of colitis for obese mice suggesting that dieting improves intestinal homeostasis in obesity. These data show the detrimental effects of obesity in the small intestine and future studies should focus on finding therapeutic treatments to fully restore IEL number and function to promote optimal intestinal homeostasis and barrier function.
Ectodomain shedding of the NKG2D ligand MULT1 regulates sensitivity to NK cells (INC6P.400)
Authors: Deng Weiwen , Wang Lin , Nice Tim , Lau Stephanie , Zhang Lily , Iannello Alexandre , Raulet David
Published: January 2023

NKG2D is one of the most important activating receptors expressed by NK cells in terms of tumor cell recognition. NKG2D binds to different ligands, and triggers cytolysis and cytokine release by NK cells. Certain human NKG2D ligands can be shed and are thought to inhibit NK-mediated surveillance of cancer. Here we showed that a mouse NKG2D ligand, MULT1, is cleaved efficiently from cells, and accumulates in the serum of tumors and inflammatory diseases. Cleaved MULT1 binds to NKG2D with high affinity (~10 nM). Remarkably, an engineered form of the shed MULT1, when secreted by tumor cells lacking NKG2D ligands, causes tumor rejection, rather than inhibiting it. Rejection is mediated by NK cells and in some cases T cells. Provision of recombinant MULT1 along with B16 melanoma cells resulted in induction of NK function ex vivo and tumor rejection in vivo. We propose a model in which expression of NKG2D ligands by non-tumor cells, persistently stimulate NK cells, resulting in desensitization of the NK cells; interruption of the interaction by soluble MULT1 restores NK cell functionality, causing tumor elimination. Consistent with this model, Rag2-/- mice which also lack NKG2D show higher NK activity than Rag2-/- mice, resulting in better B16 tumor rejection. Our study represents the first instance of NKG2D ligand shedding in the mouse, and provides the first evidence that such ligands are immunostimulatory, reversing the conventional understanding of soluble NKG2D ligands.
Antiviral activity of human OASL is mediated by mimicking ubiquitin chains to activate RIG-I pathway (INM6P.421)
Authors: Zhu Jianzhong , Zhang Yugeng , Cuevas Rolando , Forero Adriana , Dhar Jayeeta , Ibsen Mikkel , Schmid-Burgk Jonathan , Schmidt Tobias , Ganapathiraju Madhavi , Fujita Takashi , Hartmann Rune , Chen Zhijian , Barik Sailen , Hornung Veit , Coyne Carolyn , Sarkar Saumendra
Published: January 2023

Virus infection is sensed in the cytoplasm by RIG-I, which requires RNA and poly-ubiquitin binding to induce type I interferon (IFN), and activate cellular innate immunity. We show that unlike mouse, the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduces RIG-I signaling and enhances virus replication in human cells. Conversely, OASL expression suppresses replication of a number of viruses in a RIG-I-dependent manner and enhances RIG-I-mediated IFN induction. OASL interacts and colocalizes with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhances RIG-I signaling. Importantly, inactive RIG-I mutants defective in ubiquitination and polyubiquitin binding still bind to OASL, and their signaling activity is rescued by OASL. Our findings show a novel mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.
Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock (INM6P.406)
Authors: Miao Edward , Hagar Jon , Powell Daniel , Aachoui Youssef , Ernst Robert
Published: January 2023

Inflammatory caspases, such as caspase-1 and -11, mediate innate immune detection of pathogens. Caspase-11 induces pyroptosis, a form of programmed cell death, and specifically defends against bacterial pathogens that invade the cytosol. During endotoxemia, however, excessive caspase-11 activation causes shock. We report that contamination of the cytoplasm by lipopolysaccharide (LPS) is the signal that triggers caspase-11 activation in mice. Specifically, caspase-11 responds to penta- and hexa-acylated lipid A, whereas tetra-acylated lipid A is not detected, providing a mechanism of evasion for cytosol-invasive Francisella. Priming the caspase-11 pathway in vivo resulted in extreme sensitivity to subsequent LPS challenge in both wild type and Tlr4-deficient mice, whereas caspase 11-deficient mice were relatively resistant. Together, our data reveal a new pathway for detecting cytoplasmic LPS.
Leukotriene B4 dictates sterile inflammation in type 1 diabetes (INM7P.424)
Authors: Serezani Henrique , Filgueiras Luciano , Brandt Stephanie , Wang Sue , Morris David , Wang Zhuo , Mirmira Mirmira , Evans-Molina Carmella , Jancar Sonia
Published: January 2023

Type 1 diabetes (T1D) is characterized by chronic systemic inflammation that could be involved in the enhanced susceptibility to sepsis. The IL-1b/IL-1bR axis drives inflammation in a MyD88-dependent manner, but the molecular mechanisms involved in IL-1b actions in macrophages during T1D are unknown. We hypothesized that leukotriene (LT) B4 promotes IL-1b responsiveness in T1D by enhancing MyD88 expression. Peritoneal macrophages (PMs) from T1D mice expressed higher levels of MyD88 and its transcription factor STAT-1 than PMs from nondiabetic mice. 5-LO expression, LTB4 synthesis and IL-1b levels were also increased in both PM and serum from T1D mice. In vitro and in vivo insulin treatment restored LTB4 and STAT-1/MyD88 levels. Genetic and pharmacologic inhibition of the LTB4/BLT1 axis abolished MyD88 and STAT-1 induction in diabetic mice. ChIP assay showed that AP-1/cJun drove basal STAT-1 levels in an LTB4-dependent manner in T1D. We speculated that high LTB4 levels contribute to enhanced susceptibility to sepsis in T1D. Compared to WT or untreated T1D mice, genetic and pharmacologic inhibition of 5-LO prolonged T1D mice survival after polymicrobial sepsis; associated with decreased blood bacteria and reduced production of proinflammatory cytokines. We conclude that LTB4, by increasing MyD88 and IL-1b levels in a c-Jun/STAT-1 dependent manner, is the driving force involved in inflammation associated with T1D that contributes to high mortality associated to sepsis T1D.
Dual role of Interleukin-37 in the regulation of inflammation triggered by Propionibacterium acnes in human monocytes (INM8P.450)
Authors: Qin Min , Park Andrew , Agak George , Garban Hermes , Kim Jenny
Published: January 2023

IL-37 is an anti-inflammatory cytokine that is highly concentrated in the epidermis. In this study, we sought to investigate the immunomodulatory role of IL-37 in acne pathogenesis. In human monocytes isolated from peripheral blood mononuclear cells, we show that P. acnes upregulates expression of IL-37 in a pathway dependent on Toll-like receptor 2, NLRP3 and caspase-1 signal transduction. Using small interfering RNA to knock down IL-37 gene in monocytes, our results show upregulation of IL-1β, IL-6, and IL-8, and downregulation of IL-10, suggesting anti-inflammatory properties in P. acnes -stimulated monocytes. We demonstrate that IL-37 may have a reversal property when acting extracellularly. Recombinant IL-37 alone and P. acnes both induce IL-1β, IL-6, and IL-8 in a concentration-dependent manner. A greater cytokine response occurs when both IL-37 and P. acnes are used to co-treat monocytes. As we show that IL-37 is released only after cell membrane digest in monocytes but not actively secreted by viable cells, our data suggests a dualistic role of IL-37. While it may be anti-inflammatory intracellularly, IL-37 may act as a pro-inflammatory cytokine when released after cell damage and death. Intracellular IL-37 is a potent inhibitor of P. acnes -induced inflammatory response, yet has a reversal of properties when acting to signal nearby immune cells.
Neutrophils are critical in the pathogenesis of atypical EAE in LysMCre-SOCS3fl/fl mice (IRC5P.471)
Authors: Liu Yudong , Qin Hongwei , Benveniste Etty (Tika)
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS) characterized by demyelination, inflammatory lesions and axonal damage. We previously demonstrated that deletion of Suppressor of Cytokine Signaling 3 (SOCS3) in myeloid cells resulted in atypical EAE with a striking neutrophil infiltration in the brain. In this study, we investigated the role of neutrophils in the pathogenesis of atypical EAE. Kinetics studies show that accumulation of neutrophils in LysMCre-SOCS3fl/fl mice correlate with disease development. Ab-mediated depletion of neutrophils delays the onset of atypical EAE and prolongs survival. Importantly, brain-infiltrating neutrophils display high expression of CXCR2, and produce high levels of CXCL2, CCL2 and nitric oxide (NO). In addition, LysMCre-SOCS3fl/fl recipients develop atypical EAE in Th1- and Th17-adoptive transfer models. Our findings indicate that, in LysMCre-SOCS3fl/fl mice, neutrophils facilitate the disruption of the blood-brain barrier (BBB), and once they gain access to the brain, they play an essential role in the development of atypical EAE by promoting their own recruitment through production of CXCL2, and promoting macrophage recruitment through production of CCL2. More importantly, the activated neutrophils produce high levels of NO, resulting in inhibition of regulatory T-cells, oligodendrocyte damage and axonal degeneration, which leads to atypical EAE in LysMCre-SOCS3fl/fl mice.
Cognate CD40-CD40L interaction is important for maintenance of thymic B cells. (IRM8P.705)
Authors: Fujihara Chiharu , Williams Joy , Jeon Hyein , Hodes Richard
Published: January 2023

B cells are present in the thymus in numbers similar to dendritic cells and epithelial cells, but the functional roles of thymic B cells and the mechanisms that support this population are unclear. Previously, we showed B cells are essential for endogenous superantigen-mediated thymocyte negative selection. In this study, we investigated mechanisms regulating thymic B cells. Thymic B cell frequency and absolute number are markedly lower in TCRa KO mice, indicating the necessity of SP-T cells for maintenance of thymic B cells. Thymic B cell numbers are also reduced in mice expressing a TCR Tg in absence of antigenic ligand, but dramatically increased in mice expressing both TCR and corresponding ligand, demonstrating TCR recognition and thymocyte activation strongly influence thymic B cells. To elucidate molecular interactions that underlie the requirement for activated T cells, we tested the role of CD40L expressed on activated SP-T cells interacting with CD40 to mediate thymic B cell maintenance. In CD40L and CD40 KO, thymic B cell frequency is substantially reduced. In mixed BM-chimeras in which CD40 KO and WT donor-derived cells co-exist, cell autonomous CD40 expression is important for thymic B cell maintenance, whereas mixed BM-chimeras using MHCII KO and WT showed that cell autonomous MHCII expression plays no detectable role. Thus, CD40L-CD40 cognate interaction, but not MHCII-TCR cognate interaction, between T cells and B cells supports the thymic B cell population.
CRACR2A encodes a novel Ca2-binding Rab GTPase important for T cell receptor signalling (IRM10P.748)
Authors: Gwack Yousang , Srikanth Sonal , Kim Kyun-Do , Gao Yuanyuan , Ghosh Shubhamoy
Published: January 2023

Ca2+ signalling is important for T cell stimulation and its impairment leads to immune deficiency. In T cells, Ca2+ entry is mediated by CRAC (Ca2+-release activated Ca2+) channels that consist of the pore component, Orai1 and the endoplasmic reticulum Ca2+ sensor, STIM1. We recently identified CRACR2A (CRAC channel regulator 2A) as an interacting partner of Orai1 and STIM1. Here we investigate the role of a novel isoform of CRACR2A that is predominantly expressed in immune cells. CRACR2A contains multiple functional domains including Ca2+-binding motifs and a C-terminal Rab GTPase domain. Different from small Rab GTPases, CRACR2A utilized a two-step activation mechanism of GTP binding and T cell receptor (TCR) stimulation to mediate its translocation into the immunological synapse. Defect in GTP binding or hydrolysis activity results in loss and gain of function respectively. CRACR2A played a predominant role in Ca2+ and mitogen-activated protein kinase (MAPK) signalling pathways. Furthermore, CRACR2A-deficient T cells showed impairment in Th1 differentiation. In support of these data, mice with T cell-specific deletion of CRACR2A showed a strong resistance to experimental autoimmune encephalomyelitis. Previously, small GTPase has been shown to play an important role in TCR signaling. Our study identifies the unexpected usage of a large Rab GTPase as a molecular switch to regulate TCR signalling pathways.
Structural mechanism of TCR-pMHC catch bond revealed by molecular dynamics, mutagenesis, and single-molecule kinetics analyses (IRM10P.752)
Authors: Liu Baoyu , Chen Wei , Martinez Ryan , Lou Jizhong , Zhu Cheng , Evavold Brian
Published: January 2023

Mechanical force is increasingly recognized as an important axis in regulation of T cell function. Recently, we showed that force elicits agonist-specific TCR-pMHC catch bond (where force counterintuitively prolongs bond lifetime), which provides a biophysical mechanism of force-triggered T cell Ca2+ flux (Liu et al., Cell, in press). Here, we performed steered molecular dynamics (SMD) simulation and single-bond lifetime experiments to investigate the molecular mechanism of TCR-pMHC catch bond. SMD simulation of 2C TCR-agonist dissociation showed that pulling TCR-pMHC bond leads to characteristic rotation of MHCI α1/α2 domain relative to TCR variable region, yielding multiple new atomic interactions (e.g., hydrogen bond) that are absent in crystal structure. Force-regulated bond lifetime measured on naïve 2C T cells validated two major predictions of such catch bond mechanism. (1) Pulling with a geometry that favors the relative domain rotation (by pulling pMHC β chain rather than c-terminal of heavy chain) produced a more pronounced catch bond; (2) a single point mutation on pMHC that abolishes new hydrogen bond formation eliminated catch bond and lowered EC50 (proliferation) by more than three orders of magnitude with only two fold decrease in affinity and a longer zero force lifetime. These data highlight a dynamic perspective of TCR-pMHC structure that reveals novel insights on TCR-pMHC interaction and T cell function.
Regulation of the intracellular localization of the transcription factor Ets1 (IRM10P.754)
Authors: Dalton Justin , Blair Sarah , Garrett-Sinha Lee Ann
Published: January 2023

Ets1 is a DNA binding transcription factor that regulates B and T cell responses. To activate transcription Ets1 must be present in the nucleus of the cell. The major isoform of Ets1 (designated p54) has both a nuclear localization signal (NLS) and a nuclear export signal (NES). We have identified a novel isoform of Ets1 (p68) having an alternate N-terminus with 71 distinct amino acids that replace the first 28 amino acids of p54. Unexpectedly, we found that transfected p68 is localized in the cytoplasm of cells, rather than the nucleus where p54 is found, despite the fact that p68 contains both the NLS and NES sequences of Ets1. This suggests that sequences in the first 28 amino acids of p54 Ets1 regulate its nuclear localization. Contained within this region of p54 Ets1 is a lysine residue (K15) known to undergo sumoylation. We made point mutations in this site (to either alanine or arginine) and showed that this residue is essential for nuclear localization of Ets1. Studies are currently underway to determine whether sumoylation of Ets1 is required for its nuclear localization. Interestingly, there may be a conserved mechanism that regulates nuclear localization of Ets proteins. This interpretation is based on our observation that another Ets family member Elf5 also requires an intact N-terminus to be localized within the nucleus of the cell. Our studies predict that nuclear localization of Ets proteins may be a regulated event and under the control of signaling pathways.