bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilization of Myristica fragrans Houtt. reduces serum sialic acid in type II diabetes mellitus (P3103)
Authors: Mollik Md. Ariful Haque
Published: January 2023

Scientists are searching for new leads from the natural sources to combat different diseases. The studies were undertaken to discover new drugs from the natural sources. The beneficial effects of Myristica fragrans Houtt. are not limited to hypoglycemia only, but it also ameliorates diet induced obesity, insulin resistance, and exhibit cardioprotective effects. The studies attempt to investigate the effect of Myristica fragrans Houtt. fruit juice on a newly investigated risk factor, sialic acid in type II diabetes mellitus. A total of 48 type II diabetes mellitus patients, divided into group A (n=24), and group B (n=24) were investigated during the studies. The patients of group A were following Myristica fragrans Houtt. fruit juice treatment along with diet control, whereas the patients of group B were on diet control only. Serum sialic acid decreased in group A from 66.21±2.30 mg/dl to 63.59±2.10 mg/dl (<0.11) but, increased in group B from 66.55±1.70 mg/dl to 68.24±2.50 mg/dl (<0.12), compared to baseline. Post-treatment between group comparison revealed a significant difference (<0.05). The beneficial effects on fasting plasma glucose and glycohemoglobin were also greater in group A compared to group B as was the case with blood lipids, weight, and blood pressure. The studies provide another mechanism for the cardioprotective effect of Myristica fragrans Houtt. and further strengthen its value in the management of type II diabetes mellitus.
Obesity associated inflammation as a risk factor to cancer development: clinical implications (P3113)
Authors: Nehete Pramod , Hanley Patrick , Nehete Bharti , Buchl Stephanie
Published: January 2023

During the last two decades, the percentage of obese adults and children has been steadily increasing. In addition to increasing the risk of coronary heart disease, stroke, hypertension, and diabetes, obesity increases the risk of a variety of cancers such as breast (postmenopausal), endometrial, colon, kidney, esophagus, gallbladder, and pancreas. This elevated risk of cancer appears to be highly correlated with a low-grade systemic inflammation due to the obesity itself. Chimpanzees are our closest genetic relative and share a multitude of human diseases including obesity and cancer. We investigated the inflammatory mediators involved with obesity in a diet controlled animal model to elucidate possible explanations for obesity related cancers. We observed that the obese group has significantly higher plasma levels of pro-inflammatory cytokines (e.g. IL-6, interferon- γ and tumor necrosis factor-α) and also higher amount of adipocytokines (e.g. Leptin, Glucagoan, GLP-1, Amylin (total), C-peptide and Insulin). We further expect that the serum from the obese group to present a unique signature profile that stimulates healthy PBMC’s to induce specific pro-inflammatory cytokines and gene-transcriptional factors. These findings will support that this bioassay may not only have utility as an early mechanistically informative inflammatory biomarker for obesity and cancer but may also prove useful in monitoring changes in the inflammatory state during therapeutic interventions.
c-kit signaling pathways regulate EAE susceptibility in male SJL mice (P3119)
Authors: Short Abigail , Walker-Caulfield Margaret , Brown Melissa
Published: January 2023

Females are more susceptible to autoimmune diseases, including multiple sclerosis (MS), a demyelinating disease of the central nervous system. While genetic, hormonal, and immune differences have been implicated, the mechanism for this female bias remains unclear. The animal model of MS, experimental autoimmune encephalomyelitis (EAE), recapitulates many of the features of the human disease; including the significant sex dimorphism. We previously reported that wild type (WT) female SJL mice exhibit more severe disease than their c-kit mutant counterparts and that this phenotype is mast cell dependent. In contrast, male SJL-Kit-W/W-v mice develop significantly more severe EAE than their WT littermates. This c-kit dependent difference in male disease corresponds to a more robust peripheral CD4+ T cell response at day 10 post disease induction. Male SJL-Kit-W/W-v mice demonstrate a higher percentage and number of encephalitogenic GM-CSF producing PLP(139-151)-specific CD4+ T cells when compared to their WT controls. These data indicate that c-kit signaling exerts its protective effects in the periphery by regulating the magnitude and quality of the autoreactive T cell response. This effect is male specific given that the peripheral T cell GM-CSF responses in WT and Kit-W/W-v females are equivalent. Further studies are ongoing to define the cell type that is the target of c-kit signals and the mechanism by which c-kit exerts its disparate effects in male and female SJL mice.
Neuroprotective effects of bee venom phospholipase 2 by suppression of neuroinflammatory responses in mouse model of Parkinson’s disease: role of CD4+CD25+Foxp3+ regulatory T cells (P3125)
Authors: Chung Eun Sook , Bae Hyunsu
Published: January 2023

In the present study, we examined whether bee venom phospholipase 2 (bvPLA2) promotes the survival of dopaminergic (DA) neurons in the MPTP-induced mouse model of Parkinson’s disease (PD). Treatment of bvPLA2 prevented degeneration of DA neurons in the substantia nigra (SN). This nueroprotection of bvPLA2 associated with the suppression of microglia and inhibition of infiltration of CD4+ T cells into the SN. Additionally, CD4+CD25+Foxp3+ Tregs were significantly increased in vivo and in vitro after treatment of bvPLA2. The increased proportion of Tregs by bvPLA2 treatement remained highly suppressive ex vivo. To determine whether this neuroprotective effects of bvPLA2 associated with Tregs, we used transgenic DEREG (depletion of regulatory T cells) mice, which express a diphtheria toxin receptor under control of the Foxp3 locus, allowing selective depletion of Foxp3+ Tregs. Interestingly, bvPLA2 treatment did not prevent MPTP neurotoxicity in mice depleted of Tregs by dt treatment. Therefore, our present studies indicate that the neuro-protective effect of bvPLA2 might be associated with its anti-inflammatory properties resulting from modulation of peripheral immune tolerance by Tregs.
Beryllium activates innate pattern recognition receptors, promotes migration and activation of dendritic cells from the lung to draining lymph nodes and drives development of memory CD4 T cells (P3129)
Authors: McKee Amy , Mack Doug , Fontenot Andrew
Published: January 2023

Beryllium exposure results in chronic beryllium disease, a granulomatous lung disorder characterized by the presence of a beryllium-specific adaptive immune response. The impact of beryllium on the innate immune system is poorly understood. The aim of this study was to determine whether beryllium promotes activation of innate pattern recognition receptors that drive the development of pathological CD4 T cell responses. Similar to aluminum hydroxide, a common vaccine adjuvant, beryllium particles induce IL-1β secretion in an Nlrp3-dependent manner and the release of host DNA during inflammation. Unlike aluminum, beryllium-induced IL-1β secretion requires TLR4. A single beryllium exposure in the lungs of mice promotes migration of DCs to the draining lymph nodes and upregulation of DC costimulatory molecules in a MyD88-dependent manner. Beryllium enhances priming and maintenance of memory CD4 T cells to conventional protein antigen and can generate beryllium-specific CD4 T cell responses in HLA-DP2 transgenic mice in the absence of an additional adjuvant. Together, these data suggest that beryllium activates multiple innate immune receptors and promotes pathological immune responses in susceptible individuals by promoting activation of DCs that drive long-lived CD4 T cell responses. Future work is aimed at understanding which innate receptors are required for beryllium to activate DC in vivo and drive CD4 T cell effector and memory function.
Chronic inflammasome activation promotes the development of spontaneous colitis (P3149)
Authors: Zhang Jinyu , Guo Beichu
Published: January 2023

The inflammatory cytokine IL-1β plays a critical role in the pathogenesis of inflammatory bowel disease. IL-1β production is controlled by inflammasomes, a multiple protein complex that has emerged as a critical player in the pathogenesis of inflammatory and autoimmune diseases. However, the role of inflammasomes in intestinal inflammation remains controversial, while clinical studies show elevated levels of IL-1β protein in intestinal tissues and cells of inflammatory bowel disease patients, several recent studies suggest that inflammasome and IL-1β provide protection against chemically induced colitis. In this study, we demonstrate a critical role of chronic inflammasome activation in the development of colitis. Our data showed that chronic intestinal inflammation was associated with increased enhanced inflammasome activities and IL-1β production in colon tissues. Moreover, ATP or Alum-induced IL-1β processing significantly increased in bone marrow-derived macrophages in IL-10 deficient mice compared with wild type mice. We also found that IL-10 could directly inhibit NLRP3 inflammasome activation and IL-1β processing. Furthermore, our results indicate that inhibition of inflammasome activities with IL-1R antagonist or caspase inhibitor could suppress IL-1β and IL-17 production from inflamed colon tissues. Together, our results support a novel mechanism in which chronic inflammasome activation promotes the development of intestinal inflammation.
CD177 participates in a novel mechanism for regulating neutrophil recruitment (P3093)
Authors: Wang Jun-Xia , Nigrovic Peter
Published: January 2023

Background: Neutrophils are the sine qua non of inflammatory arthritis, and contribute to pain, swelling, and tissue injury, rendering neutrophil migration to the joint a potential therapeutic target. We recently found that the murine neutrophil surface protein Ly6G modulates chemotaxis via interaction with β2 integrins. Whereas CD177 is a neutrophil-specific human protein in the same gene family, we sought to identify the role of CD177 in human neutrophil recruitment. Method: Flow cytometry was used to analyze the levels of cell surface proteins in circulating neutrophils and in neutrophils from inflamed joints. Fluorescence lifetime imaging microscopy/Fluorescence resonance energy transfer assay (FLIM/FRET) was used to quantify CD177-β2 integrin interactions. Result: In patients with rheumatoid arthritis, synovial neutrophils exhibit higher levels of surface CD177 expression comparing with blood neutrophils. Using FLIM/FRET, ~40% CD11a and ~20% CD11b β2 integrins interact with CD177 on surface of resting blood neutrophils. In vitro, a specific CD177 antibody inhibits LTB4-induced conformational activation of CD11b β2 integrin, which is correlated with an impaired neutrophil migration towards LTB4 in anti-CD177 treated cells in a transwell system. Conclusion: CD177 regulates neutrophil chemotaxis and may serve as a potential neutrophil-specific therapeutic target in neutrophilic inflammatory diseases such as arthritis.
Natural autoantibodies protect from autoimmune disease (P4050)
Authors: Chen Qing , Beardall Michael , Mannoor Kaiissar
Published: January 2023

Natural autoantibodies (NAAs) arise naturally without exogenous Ag stimulation. They constitute a substantial proportion of the normal Ab repertoire. They are mainly of the IgM class, unmutated, and typically polyreactive. It remains controversial whether NAA contribute to or protect from autoimmune diseases. Using site-directed transgenic mice expressing a prototypic NAA, named ppc1-5, we found that the expression of NAA in the autoimmune MRL-lpr mice protect from autoimmune manifestations. To demonstrate that the protection is, at least in part, via the secreted NAA per se, we administered purified ppc1-5 IgM to the wt MRL-lpr mice. We observed substantial reduction in proteinuria and kidney IC deposition, and prolonged survival in mice injected with ppc1-5 IgM as compared to those that received either PBS or control IgM. The ppc1-5 IgM injected mice had decreased levels of anti-Hep2 ANA and anti-dsDNA of the IgG3 subclass, although the total IgM and IgG levels did not alter significantly. We demonstrated that the ppc1-5 NAA, but not the control IgM, bound to apoptotic cells with a high capacity. Examination of cytokine production showed that the administration of ppc1-5 NAA significantly reduced the production of IFNgamma and IL-10 by CD4+ T cells. Taken together, our data demonstrate that the low-affinity, self-reactive NAAs can protect from lupus nephritis, and suggest that they may do so by promoting removal of apoptotic cells, and by regulating T-cell function.
The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing (P4054)
Authors: Kim Hang-Rae , Chang Soog-Hee , Kim Tae-Joo , Kim Young-Joo , Liu Yang , Min So-Youn , Park Min-Jung , Park Hyun-Sil , Nam Ki-Hoan , Seong Seung-Young , Kim Ho-Youn , Mohan Chandra
Published: January 2023

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R+ anti-DNA transgenic B cells by tracking 56R+B cells in mice without (B6.56R) or with the Sle1 locus (B6.Sle1.56R). Compared to B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA antibodies in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4+ T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at immunoglobulin heavy chain and light chain editing. Thus, the immunoglobulin heavy chains in Sle1.56R+ B cells are partnered more often with cationic light chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing B cell receptor revision, and possibly by shaping extrafollicular development of effector B cells, though the precise molecular mechanisms await further study.
Integrin ITGAM/CD11b negatively regulates BCR signaling to maintain autoreactive B cell tolerance (P4062)
Authors: Ding Chuanlin , Ma Yunfeng , Chen Xingguo , Cai Yihua , Liu Min , Hu Xiaoling , Yan Jun
Published: January 2023

The hallmark of systemic lupus erythematosus (SLE) is loss of immunological tolerance and B cell hyperactivity. Recent studies demonstrated an association between the variant of the integrin-α-M (ITGAM, also called CD11b) gene and SLE susceptibility, severity of disease, and pathogenic anti-dsDNA antibody production. However, the cellular and molecular mechanism by which CD11b regulates disease pathogenesis is not understood. We found that autoreactive B cells defective of CD11b exhibited hyperproliferative responsiveness to B cell receptor (BCR) cross-linking. CD11b deficiency promoted cell cycle progression and B cell survival, which was associated with elevated anti-apoptotic gene and protein levels. In vivo engagement of BCR resulted in increased autoAb production and kidney Ig deposition in CD11b-deficient mice. CD11b-deficient autoreactive B cells had the decreased tyrosine phosphorylation including Lyn and CD22 with decreased SHP-1 recruitment but increased calcium influx. Colocalization analysis revealed that the inhibitory effect of CD11b is due to its cis-interaction with CD22. Interestingly, B cells transfected with wildtype CD11b or rs1143679 (R77H) lupus associated variant of CD11b suggest that this mutation completely impairs CD11b regulatory effect on BCR signaling. Taken together, these results reveal the negative regulatory role for CD11b in maintaining autoreactive B cell tolerance.
Vaccination against DLK-1 and DLK-2 as a cancer therapy (P4306)
Authors: Fabian Kellsye , Sabins Nina , Storkus Walter
Published: January 2023

Previous studies suggest that by improving Notch signaling in the tumor microenvironment (TME), one may rescue tumor infiltrating T lymphocyte function and inhibit tumor growth in vivo. Delta-like homolog 1 (DLK-1) and Delta-like homolog 2 (DLK-2) are homologous transmembrane proteins whose ectodomains may be shed from the cell surface by the action of the tumor necrosis factor alpha converting enzyme. Both the membrane-associated and soluble forms of DLK-1/-2 serve as antagonists of Notch receptor-mediated signaling. We report that DLK-1 is differentially expressed by vascular pericytes of murine renal cell carcinomas (RENCA) and have shown that vaccination of RENCA-bearing BALB/c mice with DLK-1-derived peptides or cDNA results in the activation of specific CD8+ T cells, the recruitment of these T cells into the TME and the normalization of tumor vasculature in vivo. However, DLK-1 and DLK-2 have been reported to counter-modulate each other’s expression. Here we show that DLK-1-based vaccination leads to the T cell-dependent loss of DLK-1 expression in RENCA tumors, but to an apparent compensatory increase in TME expression of DLK-2. We have recently defined H-2d class I-presented peptide epitopes recognized by CD8+ T cells and are currently evaluating the therapeutic benefits of vaccines targeting both DLK-1 and DLK-2 in our RENCA model. We believe that such vaccines will have significant translational value for the treatment of many solid, vascularized forms of cancer.
Infection-mimicking materials to regulate dendritic cell subsets and T cell in situ for cancer vaccination (P4324)
Authors: Ali Omar , Doherty Ed , Dranoff Glenn , Mooney David
Published: January 2023

Cancer vaccines typically depend on cumbersome and expensive manipulation of cells in the laboratory, and subsequent cell transplantation leads to poor lymph-node homing and limited efficacy. We have designed macroporous polymers mimicking key aspects of bacterial infection that may be used to directly control immune-cell trafficking and activation in the body. These systems have been applied as cancer vaccines in murine models of melanoma and glioma where they mediated tumor-specific responses eradicating large and distant tumors. These vaccines may be modified to present a variety of infectious cues with temporal control to finely tune the magnitude of immune responses to cancer cells. Consequently, this tool allows us to define a local and specific immune signature that correlates with vaccine efficacy. The results motivate clinical translation of immunologically active materials for cancer vaccination.
A role for the GITR pathway in anaphylaxis (P4326)
Authors: Murphy Judith , Merghoub Taha , Houghton Alan , Wolchok Jedd
Published: January 2023

Immune modulation using monoclonal antibodies has a significant impact on the overall survival of patients with metastatic melanoma. Monoclonal antibodies that engage members of the tumor necrosis factor receptor (TNFR) superfamily have shown promising tumor protection in preclinical models. Glucocorticoid-induced TNFR-related protein (GITR) is a particularly potent target for tumor immunotherapy. A single dose of GITR agonist antibody results in 60% protection from established B16 melanoma tumors. Therefore, several doses of anti-GITR may be necessary in order to fully eradicate established tumors. Because GITR is expressed throughout the immune system, we investigated whether multiple doses of anti-GITR result in adverse immune-related events. We observed that three doses of anti-GITR cause anaphylaxis, a side effect that has not been reported using this or other TNFR superfamily agonist antibodies. Anaphylaxis was not observed upon the first or second doses of anti-GITR or in GITR knockout mice. Anti-GITR-induced anaphylaxis is associated with a severe drop in body temperature and elevated serum proinflammatory cytokine levels. We are further investigating the causes of toxicity resulting from repetitive anti-GITR treatment and whether this effect can be reversed or prevented while preserving the antitumor activity of anti-GITR, an important step given the translational potential of this therapy.
SA-4-1BBL and monophosphoryl lipid A as a novel adjuvant system with potent therapeutic efficacy against cancer in the absence of detectable toxicity and autoimmunity (P4334)
Authors: Dinc Gunes , Srivastava Abhishek K , Sharma Rajesh , Yolcu Esma , Zhao Hong , Shirwan Haval
Published: January 2023

Although subunit cancer vaccines based on tumor associated antigens (TAAs) are attractive as treatment modalities, they have limited therapeutic efficacy due to their weak immunogenicity and tumor immune evasion mechanisms. Adjuvants with potent immune stimulatory activities as component of TAA-based vaccines can overcome these limitations. In this study, we used the combination of a novel SA-4-1BBL costimulatory molecule and Toll-like receptor 4 (TLR 4) agonist monophosphoryl lipid A (MPL) with distinct mechanisms of action as a novel adjuvant system and HPV E7 as TAA in a mouse cervical cancer model. A single immunization resulted in eradication of 100% of established E7 expressing TC-1 tumors. The combined adjuvant therapy required CD8+ T cells and IFN-γ responses for efficacy. Importantly, the efficacy of the vaccine was achieved in the absence of detectable toxicity and autoimmunity as assessed by liver enzymes, blood urea nitrogen, alteration in the number of T cells, B cells, NK cells, NKT cells, macrophages, and dendritic cells, histology of major tissues, gross anatomical evidence of organ toxicity, and antibodies against ssDNA as a sign of autoimmunity. Taken together, these data demonstrate the utility of SA-41BBL/MPL as a novel adjuvant system for the development of therapeutic TAA-based subunit cancer vaccines in the absence of detectable toxicity and autoimmunity.
Influence of cytokine auto antibodies on serum IFNα activity in SLE (P6287)
Authors: Kuruganti Srilalitha , Deshpande Ashlesha , Chatham Winn , Walter Mark
Published: January 2023

The type-I interferon (IFN-I) family consists of 12 IFN-α subtypes as well as IFN-β and IFN-ω. IFN-α subtypes are thought to play a pivotal role in the pathogenesis of autoimmune diseases such as lupus by enhancing the production of anti-nuclear autoantibodies and driving end organ damage in patients with lupus nephritis. Despite the putative role of IFN-αs in disease, serum IFN-α activity levels are often low or undetectable in lupus patients. Auto antibodies (AAbs) against a single IFNα subtype (IFNα4) have been detected in lupus patients suggesting they may alter serum IFNα activity and disease severity. To better understand this relationship, we have measured total IFNα and IFNα subtype-specific AAbs levels in lupus patient serum. Our results thus far reveal an excellent correlation between IFNα activity and AAb levels, suggesting AAbs may provide an indirect marker of cytokine activity in some patients. Toward this end, we have determined the AAb profile for eleven cytokines implicated in lupus to understand their relationship to IFNα levels and overall disease severity. The resulting cytokine profile will be presented towards the goal of determining the best targets for anti- cytokine therapy in lupus.
NF-kB is involved in abnormal BAFF signaling in peripheral monocytes of patients with primary Sjögren’s syndrome. (P6274)
Authors: Yoshimoto Keiko , Tanaka Maiko , Kojima Masako , Ogata Hideko , Suzuki Katsuya , Kameda Hideto , Abe Tohru , Takeuchi Tsutomu
Published: January 2023

Background and Purpose: We revealed that BAFF induced a robust increase in the production of IL-6 by monocytes from patients with primary Sjögren’s syndrome(pSS), and that the expression levels of a BAFF receptor (BR3) , NF-IL-6 and NF-kB2 were enhanced in pSS monocytes compared to the control. The purpose of this study is to elucidate the possible abnormalities of a BAFF signaling pathway in pSS monocytes. Materials and Methods: The expression level of BR3 on monocytes in whole blood from pSS patients or normal control was analyzed by FACS. Peripheral monocytes were stimulated with soluble BAFF (sBAFF), and the production of IL-6 by the cells was measured by ELISA. The expression levels of IKK-alpha and IKK-beta were analyzed by western blotting. Results and Discussion: FACS analysis showed that the expression level of BR3 was significantly elevated in pSS monocytes compared to the control. Interestingly, the expression level of BR3 on pSS monocytes was positively correlated with the amount of IL-6 produced by sBAFF-treated monocytes. Moreover, NF-kB activation inhibitor suppressed the IL-6 production by sBAFF-treated pSS monocytes in a dose-dependent manner. In addition, phosphorylation levels of IKK-alpha and IKK-beta in pSS monocytes were enhanced upon stimulation with sBAFF. These data suggest that sBAFF acts through BR3 to induce the expression of IL-6 in monocytes, and that IKK-alpha and IKK-beta are involved in the signal transduction pathway triggered by sBAFF.
Immunosurgical resection of host tissues mediated by a novel dendritic cell-based vaccine proficient in the promulgation of TH1 immunity by exploitation of the p43/sCTLA-4 axis (P4248)
Authors: Halpert Matthew , Cloud-Carstens Julienne , Collinson-Pautz Matthew , Liang Dan , Konduri Vanaja , Klump Sherry , Bhardwaj Ratan , Berens Michael , Paulter Robia , Spencer David , Levitt Jonathan , Decker William
Published: January 2023

The current understanding of TH1 polarization and the central role dendritic cells (DC) play in skewing such a response engender hope that DC-based vaccines may ultimately be capable of promoting therapeutic efficacy against recalcitrant pathogens and neoplastic self. However, to date the development of such a vaccine has been unsuccessful presumably because the full panoply of signals required for promulgation of bona fide TH1 immunity remains uncharacterized. Recently, our group demonstrated that synergistic loading of DC with homologous MHC class I and II antigens generates a powerful TH1 response significantly stronger and more specific than that generated by alternatively-loaded DC. Using this approach, we developed ‘TH1 DC’ vaccines against both autologous prostate and seminal vesicle (SV) in the wild type mouse. The data indicate that vaccines generated by homologous antigenic matching can overcome peripheral tolerance, producing a clinical result reminiscent of surgical resection. Translation of this platform to cancer immunotherapy has shown preliminary success against established tumors in inbred mouse models as well as spontaneous canine oligodendrocytomas. We demonstrate that these results are in part dependent upon the newly-characterized AIMp1(p43)/sCTLA-4 axis and comment upon the important role of this axis in the development of TH1 immunity as well as its applicability to cancer immunotherapy.
A novel quinazolin-derived reactive oxygen species-inhibitor suppresses influenza A virus-induced inflammatory mediators and leads to enhanced survival in mice (P4214)
Authors: Gangappa Shivaprakash , De La Cruz Juan , Ganesh Thota , Diebold Becky , Cao Weiping , Smith Susan , Taylor Andrew , Singh Neetu , Hofstetter Amelia , Kumar Amrita , Katz Jacqueline , Sambhara Suryaprakash , Lambeth J. David
Published: January 2023

Acute lung injury (ALI) and its more severe form acute respiratory distress (ARD) can result from infectious agents, including several strains of influenza A virus. Recent evidence suggests that excessive production of reactive oxygen species (ROS) is a major contributor to ALI caused by influenza. We evaluated the effects of TG6-44, a novel inhibitor of ROS production, in in vitro and in vivo models of influenza A virus infection. In vitro, using the monocytic cells (THP-1) and human PBMC infected with influenza A virus (X31), we found that TG6-44 treatment decreased virus-induced ROS and inflammatory markers in THP1 (IL6, IFNγ , MCP1, TNFα , MIP1β) and in PBMC (IL6, IL8, TNFα , MCP1). Also, in influenza A virus-infected THP1 cells, TG6-44 treatment led to a reduction in virus-induced cell death as evidenced by decreased Caspase3 activation, decreased proportion of Annexin V+/PI+ cells, and increased Bcl2 phosphorylation. Notably, TG6-44-treatment decreased the proportion of THP1 cells expressing viral nucleoprotein and delayed its translocation into the nucleus. Moreover, mice infected with a lethal dose of influenza A virus (PR8) and given TG6-44 had both reduced levels of virus-induced inflammatory markers in lungs and a higher survival rate compared to controls. Taken together, our results demonstrate anti-inflammatory and anti-infective effects of TG6-44, and suggest ROS-inhibitors as valuable adjunct therapeutics to reduce ALI/ARD caused by influenza A virus infection.
Nanolipoprotein delivery enhances immunostimulatory properties of innate immune agonists and provides protection against lethal influenza challenge (P4218)
Authors: Weilhammer Dina , Alam Shabnam , Blanchette Craig , Fischer Nicholas , Loots Gabriela , Corzett Michele , Thomas Cindy , Hwang Mona , Lychak Cheri , Sant Andrea , Rasley Amy
Published: January 2023

Therapies targeting the innate immune system have the potential to provide transient, non-specific protection from a variety of infectious organisms. As such, we have employed a nanolipoprotein (NLP) platform to enhance the efficacy of synthetic innate immune agonists. NLPs are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. We demonstrate that the synthetic Toll-like receptor (TLR) ligands monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODNs) can be readily incorporated into NLPs. Further, conjugation of both MPLA and CpG ODNs to NLPs significantly enhances their immunostimulatory profiles in vivo compared to administration of agonists alone. Specifically, conjugation to NLPs promotes agonist trafficking to secondary lymphoid organs, enhances and prolongs stimulation of innate immune cells, significantly increases cytokine production, and enhances upregulation of myriad immunoregulatory genes in vivo. Importantly, enhancement of cytokine production by agonists conjugated to NLPs was also observed in primary human dendritic cells. Strikingly, BALB/c mice pretreated with NLP-CpG constructs prior to a lethal influenza challenge were protected from weight-loss induced death, whereas pretreatment with CpG alone had no effect on survival. Taken together, these data suggest that NLP:agonist conjugates potentially represent a novel therapeutic measure against emerging pathogens.
pSTAT3: A target biomarker to study the pharmacology of the anti-IL 21R antibody ATR-107 in human whole blood (P4194)
Authors: Zhu Ming , Pleasic-Williams Susan , Lin Tsung , Wunderlich David , Cheng John , Masferrer Jaime
Published: January 2023

IL-21 has been shown to play an important role in autoimmune diseases. ATR-107 is an antibody which directly targets the IL-21 receptor. To aid the clinical development of ATR-107, there is a need for understanding the mechanism of action of this antibody when assessing target engagement in human subjects. To determine ATR-107 biological activity and potency in human blood, its inhibitory function against IL-21 induced STAT3 phosphorylation in human peripheral T and B cells was measured. The data show that IL-21 induces STAT3 phosphorylation in a dose-dependent manner, consistent with its translocation to the nuclear. Using a flow cytometry based functional whole blood assay, the data also show that ATR-107 is a potent IL-21 pathway inhibitor. It competes with IL-21 for receptor binding in a competitive manner, but once it binds to the receptor it behaves like a non-competitive inhibitor, most probably due to the long observed koff. The dose-dependent inhibition observed with ATR-107 correlates inversely with the levels of receptor occupancy, both in ex vivo whole blood assays and directly in human blood when ATR-107 was given to healthy volunteers. Altogether, IL-21 induced pSTAT3 in T and B cells can be used as a biomarker to evaluate the target engagement of ATR-107 in human whole blood. The non-competitive feature of this antibody reveals by pSTAT3 assay may help with the translation of preclinical information and dose selection towards ATR-107 clinical efficacy.
SIGN-R1 confers susceptibility to West Nile Virus encephalitis (P6036)
Authors: Chandran Ramakrishna , Srivastava Ruchi , Cantin Edouard
Published: January 2023

DC-SIGN is a critical determinant of susceptibility to flaviviral infections. To study its role in immunity to West Nile Virus (WNV), we used mice that lacked the murine homolog SIGNR1. Following infection with a lethal dose of WNV, knockout (KO) but not wildtype (WT) mice were resistant to WNV; all WT mice succumbed by day 9 pi. CNS inflammation in WT and KO mice was similar. CD45high infiltrates in brains were similar at day 6-8 pi; macrophages and DCs were predominant in both KO and WT mice at day 6 pi but Ly6Chigh inflammatory macrophages was significantly increased in WT compared to KO brains (45% Vs 23%). T cell infiltrates were increased in WT compared to KO brains at day 8 pi, yet they did not protect. Analysis of T cell activation revealed increased CD62Llow CD44high effector CD4+ (40% Vs 10%) and CD8+T cells (35% Vs 5%) in the spleens of KO compared to WT mice. Enhanced T cell activation was supported by an increase in IFN-γ secreting T cells in the KO spleen. Higher levels of PD-1+ CD4+ and CD8+ T cells observed in WT compared to KO spleens possibly contributed to the reduced activation. Treatment of WT mice with a blocking mAb 22D1 and an RNA aptamer selected for binding to SIGNR1 that transiently reduces surface SIGNR1 expression significantly protected mice from mortality. These data suggest that SIGN-R1 plays an inhibitory role in activating WNV specific T cells and is a potential target for immunotherapeutic approaches for WNV and other flaviviral infections.
Deletion of Bim compromises the quality of CD8 T cell memory after acute infection (P6060)
Authors: Sharma Shalini , Sturm Oliver , Milasta Sandra , Dillon Christopher , Green Douglas , Thomas Paul
Published: January 2023

One important feature of T-cell memory formation is the process of cell death during the contraction phase of the immune response. CD8 T-cell contraction after acute infections is mediated by the intrinsic apoptosis pathway, regulated by the interaction of anti-apoptotic BCL2 with pro-apoptotic Bim. In a murine model of influenza A virus infection, we found that Bim-deficient animals experienced a total ablation of the CD8+ T cell contraction phase leading to the significant accumulation of antigen-specific cells. While these cells displayed typical memory phenotypes, in vivo adoptive transfer and in vitro functional studies show that Bim-deficient memory CD8 T cells are non-responsive to stimulation by antigenic peptides and fail to expand properly on secondary challenge, indicating an intrinsic defect of this spent effector population. These functional deficiencies begin to emerge even at the peak of the primary response. Taken together, these results demonstrate that efficient memory generation entails the generation of a distinct, productive memory lineage that is capable of evading Bim-mediated apoptosis.
Highly stable CD8 populations are maintained by systemic, low level division across multiple clones during MCMV memory inflation. (P6067)
Authors: Smith Corinne , Turula Holly , Snyder Christopher
Published: January 2023

Murine cytomegalovirus (MCMV) establishes a persistent/latent infection that induces a characteristic “memory inflation” of CD8 T cells specific for some viral epitopes. The inflationary CD8 populations are maintained at high numbers for life in spite of the fact that inflationary effectors turnover with a half-life of 45-60 days. The current model to describe memory inflation suggests that waves of memory CD8s divide in the lymph nodes to produce effectors that migrate to the tissues to control viral activity. We used an adoptive transfer of two congenically marked OT1 populations to show that, in spite of the constant turnover, two inflationary populations were maintained in parallel with minimal fluctuation over time. Likewise, within the endogenous antigen population, different clones identified by V-beta staining divided at a similar frequency. Division of antigen specific CD8s occurred at a low level systemically, including lymph nodes, spleen and non-lymphoid organs, as measured by Ki67 expression and BrdU incorporation. Although the frequency of division was the highest in draining lymph nodes, large numbers of dividing cells were still found in non-lymphoid organs, even when lymphocyte egress was blocked from lymph nodes with FTY720 treatment. These results suggest that inflationary populations are maintained by systemic, stable, low-level division across multiple clones and that division is not restricted to small numbers of cells dividing in the lymph nodes.
Increased frequency of Tim-3+ T cells is associated with symptomatic WNV disease clinical outcome (P6085)
Authors: Lanteri Marion , Diamond Michael , Govero Jennifer , Pinto Amelia , Law Jacqueline , Chew Glen , Wu Shiquan , Niki Toshiro , Inglis Heather , Hirashima Mitsuomi , Busch Michael , Norris Philip , Ndhlovu Lishomwa
Published: January 2023

After a five-year period of steady annual cases, the 2012 West Nile virus (WNV) season was marked by a 4-fold resurgence with 5387 diagnosed infections, 2734 neuroinvasive cases and 243 deaths. Currently, no treatment or approved human vaccine is available for WNV infection. Understanding factors contributing to anti-WNV immunity and protection is therefore mandated. The present study investigates the surface expression patterns of negative T cell regulatory molecules over the course of WNV infection in a cohort of 32 WNV+ blood donors enrolled in longitudinal follow-up after initial WNV RNA+ blood donation (index) in those with asymptomatic (n=24) or symptomatic (n=8) disease outcome. We observed increased frequencies of Tim-3+CD4+ T cells during acute WNV infection (<90 days post-index) compared to uninfected controls (GEE P<0.0001). WNV+ donors had elevated frequencies of Tim-3+PD-1+ events in CD4+ and CD8+ T cells (P<0.05). Frequencies of Tim-3+ events in CD4+ and CD8+ T cells were higher in symptomatic than in asymptomatic subjects in the acute phase of infection (P=0.0009 and 0.033 on day 14 post-index, respectively), and symptomatic subjects had higher frequencies of Tim-3+CD4+ T cells throughout infection (GEE P<0.005). Our study highlights key mechanisms of immune vulnerability and reveals that T cell negative regulation is linked to symptomatic WNV disease and may serve as a novel target for improving protective responses or dampening inflammation and symptoms.
Induction of pathogenic Th17 cells by inducible salt sensing kinase SGK1 (P1145)
Authors: Wu Chuan , Yosef Nir , Thalhamer Theresa , Zhu Chen , Xiao Sheng , Kishi Yasuhiro , Regev Aviv , Kuchroo Vijay
Published: January 2023

Th17 cells are highly proinflammatory cells that are critical for clearing extracellular pathogens and induction of multiple autoimmune diseases. IL-23 plays a critical role in stabilizing and endowing Th17 cells with pathogenic effector functions. IL-23 has been shown to reinforce the Th17 phenotype by increasing expression of IL-23 receptor (IL-23R). However, the molecular mechanism by which IL-23 sustains the Th17 response and induces pathogenic effector functions is unclear. Here we used unbiased transcriptional profiling of developing Th17 cells to construct a model of their signaling network and identify major nodes that regulate Th17 development. We identified serum glucocorticoid kinase-1 (SGK1) is critical for regulating IL-23R expression and for stabilizing the Th17 cell by deactivation of Foxo1, a direct repressor of IL-23R expression. SGK1 has been shown to govern Na+ transport and homeostasis. We show that a modest increase in salt (NaCl) concentration induces SGK1 expression, promotes IL-23R expression and enhances Th17 cell differentiation in vitro and in vivo, ultimately accelerating the development of autoimmunity. The loss of SGK1 resulted in abrogation of Na+-mediated Th17 differentiation in an IL-23-dependent manner. These data indicate that SGK1 is critical for the induction of pathogenic Th17 cells and provides a molecular insight by which an environmental factor such as a high salt diet could trigger Th17 development and promote tissue inflammation.
Characterization of the in vivo function of RGC-32 in T-cells (P1151)
Authors: Tegla Cosmin , Cudrici Cornelia , Nguyen Vinh , Danoff Jacob , Vlaicu Sonia , Rus Violeta , Badea Tudor , Rus Horea
Published: January 2023

We have previously shown that RGC-32 is involved in cell cycle regulation in vitro. To define the in vivo role of RGC-32, we generated RGC-32 knockout mice. These mice develop normally and do not spontaneously develop overt tumors. To assess the effect of RGC-32 deficiency on cell cycle activation in T cells we determined the proliferation rate of CD4 and CD8 from spleens of RGC-32-/- mice compared to wild type (WT) mice. CD4 T cells from RGC-32-/- mice display a significant increase in 3H-thymidine incorporation when compared with WT mice after stimulation with anti-CD3/anti-CD28. In addition, both CD4 and CD8 T cells from RGC-32-/- displayed a significant increase in the proportion of proliferating Ki67+ cells, indicating that in T cells, RGC-32 has an inhibitory effect on cell cycle activation. Furthermore, Akt and FOXO1 phosphorylation induced in CD4 from RGC-32-/- were significantly higher indicating that RGC-32 inhibits cell cycle activation by suppressing FOXO1 activation. To further examine the effect of RGC-32 on T cell functions we investigated the effect of RGC-32 on T regulatory (Treg) cells differentiation. A significantly lower percentage of CD4+ FoxP3+ Treg cells was found in unstimulated cells from RGC-32-/- compared to WT mice. Significantly fewer CD4+ FoxP3+ Treg cells are induced by TGF-β in RGC-32-/- compared to WT mice. Thus, RGC-32 is involved in controlling cell cycle in vivo, and in addition seems to play a role in the differentiation of Treg cells.
mTOR regulates CD4 and CD8 effector T cell differentiation via serum- and glucocorticoid-regulated kinase 1 (P1161)
Authors: Heikamp Emily , Patel Chirag , Collins Sam , Waickman Adam , Scharma Archna , Naray-Fejes-Toth Aniko , Fejes-Toth Geza , Sen Jyoti , Horton Maureen , Powell Jonathan
Published: January 2023

The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of T cell differentiation. mTOR integrates various signals in the immune microenvironment, including costimulatory ligands, cytokines, and the availability of nutrients to determine the outcome of T cell differentiation. Recently, we identified the serum- and glucocorticoid-regulated kinase 1 (SGK1) as a downstream target of mTORC2 that is a critical regulator of CD4 effector differentiation into Th1 and Th2 subsets. Specifically, SGK1 promotes Th2 differentiation by negatively regulating the NEDD4-2 E3 ligase mediated destruction of JunB. Simultaneously, SGK1 represses the production of IFN-γ by controlling the expression of the long isoform of TCF-1. Consistent with these functions, mice lacking SGK1 specifically in T cells fail to generate a Th2 response and are resistant to experimentally induced asthma. Likewise, such mice generate robust levels of IFN-γ in response to vaccines and more readily reject tumors. In addition, loss of SGK1 in CD8 T cells promotes memory differentiation by regulating the expression of CD127, CD62L, and eomesodermin. In summary, our findings reveal a novel role for SGK1 as an essential component of the mTOR pathway that guides differentiation of both CD4 and CD8 T cells. Targeting SGK1, therefore, may be a useful strategy for fine-tuning immune responses by manipulating T cell differentiation.
Androgens modulate anti-tumor immunity through interference with the T lymphocyte differentiation program (P1169)
Authors: Arredouani M. Simo , Kissick Haydn , Dunn Laura , Sanda Martin
Published: January 2023

Androgen modulation of immunity to tumor antigens have implications on designing novel prostate cancer immunotherapy regimens. However, the mechanisms governing androgen action are largely unknown. Herein we compared mouse castrate and non-castrate CD4 T cell transcriptomes and revealed major perturbations in gene regulation. Ingenuity pathway analysis identified dysregulations in key pathways, including Th cell differentiation, TCR signaling, Interferon signaling, and p38 MAPK signaling. Interestingly, nearly half of the genes that constitute the T cell differentiation pathway were affected. These genes include key transcription factors such as STAT1, Tbet, Foxp3, ROR-γ, costimulatory molecules CD40L and ICOS, and T helper cytokines such as IFN-γ, IL-17, TNF-α, and IL- 2. Transcription factors driving Th1 and Th17 differentiation as well as Th1 and Th17 cytokines and costimulatory receptors were upregulated. More CD4 T cells from castrated mice produced IFN-γ than did non-castrated ones. Conversely, exposure of activated CD4 T cells to IL-12 in the presence of androgens inhibited IFN-γ production and reduced Th1 differentiation genes like T-bet and IL-12-Rβ. This was concurrent with inhibition of STAT4 phosphorylation and upregulation of Ptpn22 and Ptpn1, both of which exhibit the ability to dephosphorylate STAT proteins. This suggests androgens’ negative effects on Th1 differentiation might be partly induced through Ptpn22- and Ptpn1-dependent dephosphorylation of STAT4.
Temporally-defined inflammatory pathways modulate distinct mechanisms of effector and regulatory CD4 T cell responses. (P1189)
Authors: Thompson Lucas , Lai Jen-Feng , Valladao Andrea , Thelen Tennille , Urry Zoe , Ziegler Steven
Published: January 2023

Potentially autoreactive T cells are controlled by both cell-intrinsic and dominant trans-suppressive mechanisms. One of the major mediators of dominant suppression of immune responses are regulatory CD4+ T cells that express the transcription factor Foxp3. These cells can arise during thymic T cell development or be induced in the periphery from naïve CD4+ T cells. The factors that drive development of induced regulatory T cells can represent a critical choice for health or autoimmunity in the case of potentially self-reactive CD4 T cells. Numerous studies have implicated a role for enhanced effector differentiation and suppressed induction of regulatory T cells in response to antigen in the context of inflammatory signals. Here we show that inflammatory stimuli can drive expansion of functional Foxp3+ regulatory T cells in vivo, largely driven by type I interferon signals. Furthermore, naïve CD4+ T cells that are bystander-activated by interferon in the absence of cognate antigen exhibit tolerance, reduced effector function, and enhanced induction of Foxp3 upon subsequent antigen encounter. These data suggest that transient bystander activation imposes a dynamic state of responsiveness in naïve CD4+ T cells, in which the nature of subsequent differentiation is shaped by temporal coordination of antigen/TCR and inflammatory signals. This work provides an interesting new framework for potential antigen-specific therapeutic immunization strategies.