bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT4 is dispensable for Th17 generation but essential for accumulation of pathogenic CD4 T cells in the CNS during EAE. (P4114)
Authors: McWilliams Ian , Harrington Laurie
Published: January 2023

Multiple Sclerosis is a demyelinating autoimmune disease of the central nervous system (CNS) and is studied using the mouse model experimental autoimmune encephalomyelitis (EAE). Studies utilizing EAE have shown critical pathogenic roles for Th1 and Th17 CD4 T cell subsets. STAT4 is an important Th1 transcription factor that, when activated by IL-12, results in the production of IFNγ, the hallmark Th1 cytokine. Deletion of STAT4 protects mice from EAE; in contrast, deletion of IL-12 and/or IFNγ does not ameliorate disease, revealing gaps in our current understanding of STAT4 biology during EAE. Given that STAT4-/- mice are resistant to EAE, we utilize bone marrow (BM) chimeric mice to provide a physiological environment in which to study the intrinsic role of STAT4 in CD4 T cells during EAE. Interestingly, we find that during EAE the in vivo frequency of STAT4-/- CD4 T cells is greatly reduced in the CNS of BM chimeric mice compared to the wild-type (WT) CD4 T cells. In addition, these STAT4-/- CD4 T cells are able to produce the IL-17 (the hallmark Th17 cytokine) and activate STAT3 via IL-23 signaling (a critical Th17 pathway associated with disease). Although the “pathogenic” Th17 phenotype is intact in STAT4-/- CD4 T cells, this is not sufficient to drive EAE in STAT4-/- mice, therefore, we posit that the defect in CD4 T cell accumulation resulting from either proliferation, cell death, or migration is the critical STAT4 mechanism driving EAE pathogenesis.
In vivo multi-photon molecular imaging technique visualizes immune and inflammatory cell cross-talks in metabolic diseases (P3231)
Authors: Nishimura Satoshi , Nagasaki Mika
Published: January 2023

To elucidate the underlying mechanisms of adult common diseases based on chronic inflammation, it is vital to examine the immune and inflammatory cell kinetics in living animals. Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy, and we assessed dynamic cellular interplay in diseased conditions. We found that large numbers of CD8+ effector T cells infiltrated into obese adipose, and these cells were essential for the initiation and development of adipose inflammation. In addition, we identified a novel B cell subset that is abundant in adipose tissue. Immunological or genetic B cell depletion, and B cell-specific Il10 deletion enhanced CD8+ T cell activation and adipose inflammation, and exacerbated insulin resistance in obesity. Adipose environmental factors supported B cell survival and IL-10 production. Adipose B cells also exhibited unique surface phenotypes, distinct from those of known regulatory B cell subsets. Our findings indicate that adipose B cells are a naturally occurring regulatory B cell subset that is essential for negative regulation of diet-induced adipose inflammation and maintenance of homeostasis within adipose tissue, and that B cell dysfunction pivotally contributes to the progression of inflammatory processes. Our results also clearly demonstrated the power of our imaging technique to analyze complex cellular interplays in inflammatory diseases, especially parenchymal and stromal cell cross talks, and to evaluate new therapeutic interventions against them.
Global miRNA expression profiles uncover serum miRNAs as novel biomarkers for diabetes staging in NOD mice (P3267)
Authors: Mi Qing-Sheng , Weiland Metthew , Qi Ruiqun , Zhou Li
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune diseases resulting from T cell-mediated pancreatic beta cell destruction. New biomarkers are urgently needed for earlier T1D risk prediction and progression. MicroRNAs (miRNAs) regulate beta cell and immune cell development and function, and are involved in T1D development. Recently, it has been discovered that serum contains large amounts of stable miRNAs derived from immune cells and other tissues/cells, and that serum miRNAs might serve as biomarkers for disease prediction and progression. Yet little is known about serum miRNAs in T1D. We hypothesize that serum miRNAs could be a novel class of blood-based biomarker for diabetes staging. Serum RNAs were isolated from nonobese diabetic (NOD) mice at different stages (3-4, 7-8, 16-19 weeks, and newly diagnosed diabetes) during diabetes development. Serum miRNA expression profiles were performed by real-time RT-PCR MicroRNA Arrays. Twenty-three miRNAs appeared to have specific expression patterns during diabetes development. Single qRT-PCR further confirmed that serum miR-150, miR-146b, and miR-215 are significantly upregulated starting at the stage of insulitis, destructive insulitis and diabetes, respectively. Our data highly suggest that serum miRNAs are potential novel biomarkers for diabetes prediction and staging, and that miR-150, miR-146b and miR-215 could be used as serum biomarkers for diabetes staging or therapy response in the NOD mouse.
Dual analysis of autoantibody and cytokine profiles improves the predictive power of biomarker discovery in lupus (P3277)
Authors: Bourdon David , Van Le Suzy , Liu Xiangdong , Thio Guene , Meng Lihao , Ramachandran Niro
Published: January 2023

We’ve developed ProtoPlex™, a Luminex® xMAP® bead-based assay capable of measuring human autoantibodies to our nearly 9,500 already purified proteins. Combining autoantibody and cytokine profiles yields statistically powerful data sets for disease relevant biomarker discovery efforts. Biomarker discovery workflows fall into two important areas: discovery and validation; yet, no single well-established discovery-to-validation workflow exists. There's growing demand among translational investigators for predictive markers of diseases such as cancer and autoimmune disorders. Clinical research aims to identify proteomic biomarkers leading to early detection and patient stratification, as well as provide prognostic information for guidance and monitoring of treatment efficacy. Given the importance of cytokine signaling in regulation of the immune system, and that aberrant signaling may lead to development of autoimmune diseases, we hypothesized that there is synergistic value in measuring both halves of the proteome in human serum samples: antigens and autoantibodies. Here we describe this novel approach, utilizing the ProtoPlex™Autoantigen Panel and Novex® inflammatory cytokine panels, to identify distinct marker sets for the chronic autoimmune disease Systemic Lupus Erythematosus.
A sensitive immuno-PCR assay to measure a pro-inflammatory cytokine in human patient serum (P3243)
Authors: Khalfin Yana , Asundi Aarati , Delarosa Donnie , DeForge Laura , Nelson Chris , Vandlan Richard , Meng Gloria , Loyet Kelly
Published: January 2023

Pro-inflammatory cytokines may be useful biomarkers for certain therapies in relevant patient samples. However, detection of low abundance cytokines in patient serum has been difficult for some cytokines, thus there is a need to develop a more sensitive assay than the traditional ELISA. Herein we developed an immuno-PCR assay that is in the low to sub pg/ml range to detect levels of a pro-inflammatory cytokine in human serum samples. Immuno-PCR combines the amplification power of PCR with the versatility of ELISA, resulting in improved sensitivity. Biotinylated anti-analyte bound to Streptavidin-coated tubes was used to capture analyte. The captured analyte was detected by anti-analyte conjugated to DNA, in combination with real-time PCR. This immuno-PCR method was found to be at least ~17-fold more sensitive than ELISA, with a lower limit of quantification of ~2 pg/ml in human serum. While this is a promising improvement, a greater fold-improvement over the ELISA detection limit may still be possible. Therefore, we are currently investigating conditions and variables to further improve the sensitivity of this immuno-PCR assay. This assay strategy may be helpful, not only for biomarker quantification, but also in numerous applications where a very sensitive assay is needed.
Therapeutic role of the novel checkpoint regulator VISTA in murine autoimmune disease models. (P5174)
Authors: Ceeraz Sabrina , Sergent Petra , Schned Alan , Burns Christopher , Noelle Randolph
Published: January 2023

Negative checkpoint regulators are critical in triggering the immune response and in the clinic, therapeutics targeting the CTLA-4 and PD-L1 pathway have been successful in treating human cancers and marginal therapeutic efficacy noted in autoimmune diseases. Our lab recently identified a novel checkpoint regulator termed V-region Immunoglobulin-containing Suppressor of T cell Activation (VISTA). VISTA is structurally distinct from other members of the Ig superfamily. This study aimed to determine the therapeutic efficacy of VISTA in autoimmunity is effective. To address this, we investigated the role of a VISTA Ig fusion protein (VISTA-Ig) in autoimmune disease models of Multiple Sclerosis (Experimental Autoimmune Encephalomyelitis (EAE)) and Systemic Lupus Erythematosus (SLE). In sick EAE mice, therapeutic treatment with VISTA-Ig suppressed disease severity compared with untreated mice in vivo. In the lupus prone mouse strain NZBW-F1 which closely resembles human SLE, the prophylactic and therapeutic effect of VISTA-Ig was studied. Prophylactic treatment with VISTA-Ig prevented the onset of proteinuria and weight loss compared with untreated mice and therapeutic treatment reversed proteinuria in sick mice. Clinical evaluation of kidneys from the VISTA-Ig treated group showed minimal to mild glomerulonephritis compared with untreated mice. In conclusion we demonstrate that VISTA-Ig is a novel effective therapeutic in treating established autoimmune diseases.
Role of IL-22 in tissue regeneration in autoimmunity (P5164)
Authors: Singh Bhagirath , Hill Thomas , Krougly Olga , Qian Alexander , Bellemore Stacey , Lee-Chan Edwin , Nikoopour Enayat , Hill David
Published: January 2023

The various cell types in our bodies are constantly regenerating but at a different rate. In autoimmune diseases cells once destroyed can regenerate. However, immune system once activated continues to target and destroy these cells. Thus, tissue regeneration remains a challenge in these diseases. Cytokines play a major role in immune regulation, inflammation, tissue injury and autoimmunity. We have shown that immunostimulation by mycobacterial adjuvants such as BCG vaccine as well as complete Freund’s adjuvant (CFA) can prevent the autoimmune process and can stimulate tissue regeneration. These adjuvants induce regulatory Th17 (Treg17) cells and stimulate expression of Regenerative (Reg) genes such as Reg1 and Reg2 in pancreatic islets. Th17 cells also produce Interleukin-22 (IL-22) that has been shown to stimulate This is probably mediated through STAT3/ERK signaling. Reg gene expression. Blocking of IL-22 prevented the expression of Reg genes in vivo. In this study we explored the cell types that express Reg genes following IL-22 treatment by using RT-PCR analysis and by histological staining. Our hypothesis is that the Reg gene expression drives the islet regeneration following tissue injury by autoimmunity in type 1 diabetes. These approaches offer alternatives to tissue transplantation in autoimmunity.
CTL-promoting effects of IL-21 counteract murine lupus and outweigh B cell stimulatory effects (P5147)
Authors: Rus Violeta , Nguyen Vinh , Tegla Cosmin , Chen Ching , Rus Horea
Published: January 2023

IL-21 promotes autoimmunity in the chronic graft versus host disease (cGVHD) model through both B and CD4 T cell intrinsic mechanisms. To determine the importance of IL-21R signaling in CD8 T cells in murine lupus, we assessed the effect of IL-21 administration on autoimmune parameters in the DBA-into-F1 model of cGVHD. In addition, the effect of IL-21R deficiency on CD8 T cells was assessed in the B6-into-F1 model of acute (a) GVHD using IL-21R sufficient and deficient donor (d) cells. cGVHD mice that received rmIL-21 exhibit attenuated autoimmune parameters with respect to host B cell expansion, anti-ssDNA autoAb production and renal disease along with enhanced dCD8 expansion and donor anti-host CTL activity. Phenotype of aGVHD induced with IL-21R-/- donor cells converted from an acute to a chronic pattern characterized by increased autoAb production, decreased host B cell elimination, impaired CTL activity and decreased dCD4 and dCD8 cell expansion. In both models, IL-21R signaling on dCD8 cells resulted in increased dCD8 expansion, granzyme B level and IFNg expression. These results suggest that lack of IL-21/IL-21R interaction on CD8 T cells converts acute to chronic GVHD, while in cGVHD, IL-21 administration counteracts the humoral component by enhancing CTL generation and host B cells elimination. Therefore, IL-21R signaling on CD8 T cells attenuates disease phenotype in murine lupus, thus opposing the enhancing effects of IL-21R signaling on CD4 T cells and B cells.
Suppression of IL-17 and GM-CSF production in T cells by immunomodulator ameliorates the pathogenesis of autoimmune neuroinflammation (P5150)
Authors: McMickle Anthony , Xie Li , Chen Jing , Awar Nadia , Nady Soad , Sredni Benjamin , Drew Paul , Yu Shiguang
Published: January 2023

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a common animal model for MS. A number of studies provide evidence that IL-17-producing T cells (Th17 cells) play a dominant role in the pathogenesis of MS and EAE. Recent work by others further indicated that the ability of Th17 cells to induce EAE is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF). Therefore, targeting Th17 cells and/or inhibiting production of GM-CSF has potential therapeutic options for EAE and MS. Our current studies demonstrated that AS101 (a small, non-toxic organotellurium compound used in the treatment of cancer), inhibits Th17 cell differentiation and reduces GM-CSF production in T cells by inhibiting activation of the transcription factors Stat3, RORγt and NFAT, and up-regulating activation of ERK and SOCS3 expression. Furthermore, administration of AS101 in vivo reduces the severity of chronic EAE and inflammatory cell infiltration into the CNS. These studies will provide insights concerning the possible use of AS101 in clinical treatment of MS.
Blockade of CSF-1 and IL-34 in mouse model of inflammatory arthritis and colitis show a pathogenic role for IL-34 (P5160)
Authors: Lin WeiYu , Delarosa Donnie , Young Judy , Lee Dave , Jeet Surinder , Huang Zhiyu , DeVoss Jason , Chen Yongmei , Wu Yan , Caplazi Patrick , Austin Cary , Martin Flavius
Published: January 2023

Colony-stimulating factor1 (CSF1) and IL34 signaling through their receptor (CSF1R) promotes monocyte and macrophage (Mf) differentiation, proliferation and migration. Mf activation through CSF1/IL34 and CSF1R signaling is associated with a broad spectrum of pathologies. Therapeutic monoclonal antibodies (mAb) against CSF1 and CSF1R or specific inhibitors of CSF1R kinase activity have been tested in the animal models and some human trials. To date there are no reports on IL34 or dual CSF1 and IL34 blockade as a therapeutic strategy for targeting myeloid driven diseases. Here we describe neutralization of both CSF1 and IL34 using specific mAb in collagen induced arthritis (CIA) and DSS-induced colitis mouse models. The efficacy of the dual blockade was superior to the single blockade of CSF1 or IL34 in both models. In CIA treatment was started at day 31 post disease induction and continued for 7 weeks. Anti-CSF1 plus anti-IL34 combination therapy reduced arthritis severity comparable to anti-TNF therapy. Mechanistic studies showed reduction of Mf and monocytes in the joint of dual Ab treated group. Dual blockade of CSF1 and IL34 decreased colon histology score in colitis mice. The mice with colitis produced higher CSF1 and IL34 in serum and colon tissue compared with the controls. In conclusion, blocking CSF1 and IL34 by specific mAb could be an effective therapy for myeloid driven inflammatory disease.
An evidence for a potential linkage between Acanthamoeba infections and multiple sclerosis (P4551)
Authors: Massilamany Chandirasegaran , Jamerson Melissa , Madayiputhiya Nandakumar , Nandakumar Renu , Marciano-Cabral Francine , Sejbaek Tobias , Illes Zslot , Reddy Jay
Published: January 2023

We recently observed that Acanthamoeba castellanii (ACA), a free-living amoeba, has the potential to trigger central nervous system autoimmunity by generating cross-reactive T cells for multiple myelin antigens, namely, proteolipid protein (PLP) 139-151 and myelin basic protein (MBP) 89-101. Additionally, the homology model derived for human leukocyte antigen-DR2, complexed with MBP 85-99 and its mimicry epitope, predicts the possibility of generating cross-reactive T cells for human MBP 85-99 in Acanthamoeba-exposed individuals. These observations raise a question whether prior exposure to ACA infection can trigger multiple sclerosis (MS). To address this hypothesis, we extracted DNA from cerebrospinal fluid obtained from MS patients and individuals with other neurological disorders, and subjected them to PCR that amplified a 500 bp fragment of the small subunit 18S rDNA using ACA-specific primer set. We confirmed the identity of PCR products by sequencing. To provide additional evidence for a linkage of ACA to MS pathogenesis, we asked whether anti-human PLP antibody can detect amoebic rhodanese-related sulfurtransferase (RST), since this protein contains the mimicry epitope ACA 83-95 for PLP 139-151. Through western blotting and LC/MS analyses, we noted that PLP antibody binds RST, suggesting that anti-Acanthamoeba immune responses have the potential to target myelin antigens. Collectively, the data suggest a linkage of ACA infection to MS pathogenesis.
Weak TCR signaling due to repeated immunization with antigen is the key for peripheral TCR revision generating aiCD4 T cell: a novel self-organized criticality theory (P4537)
Authors: Shiozawa Shunichi , Uto Kenichi , Tsumiyama Ken
Published: January 2023

The autoimmune disease theory explains that autoreactive clones derive from restricted clones either slipped from thymic selection or reactivated from tolerance, which cannot explain varieties of autoantibody exceeding 140 specificities seen in SLE. We instead show that autoreactive clones newly emerge via de novo TCRalpha but not beta revision at periphery from thymus-passed non-autoreactive clones (PLoS ONE 4(12):e8382,2009). This autoreactive autoantibody-inducing CD4 (aiCD4) T cell stimulates B cells to generate autoantibodies and helps to generate CD8 CTL via antigen cross-presentation and cause SLE. We studied how aiCD4 T cell is produced. Methods: Balb/c mice were immunization 8x with staphylococcus enterotoxin B (SEB), signaling molecules and their phosphorylation were assayed using western blot and antibodies. Results: The chromatin of CD4 T cell V,J region was not acetylated/ H3K4me3-lated and open, thereby facilitating the access of RAG1/2. Upon repeated immunization 8x with SBE, RAG1/2 was up-regulated but CD3zeta, ZAP70, LAT, SLP-76, PLCgamma1 and NFAT1/2 were down-regulated in splenic Vbeta8+CD4 T cell. Instead, re-activation of RAG1/2 was inhibited when TCR signal was augmented vice versa by adding PMA+ionomycin, indicating that loss of TCR signal reactivates RAG1/2 at peripheral lymphoid organ in spleen. Conclusion: Weak TCR signaling due to repeated immunization with antigen re-expresses RAG1/2 at periphery to induce aiCD4 T cell and cause SLE.
Disruption of transient receptor potential melastatin 2 attenuate lipopolysaccharide-induced heme oxygenase-1 expression in mouse macrophages through p38 mitogen-activated protein kinase (P5202)
Authors: Fang XiangMing , Qian XiaoWei
Published: January 2023

Transient Receptor Potential Melastatin 2 (TRPM2) is a member of transient receptor potential channels. TRPM2 mediated Ca2+ entry is an important mechanism for lipopolysaccharide (LPS)-induced cytokine and reactive oxygen species (ROS)-induced chemokine production in monocytes/macrophages. However, whether TRPM2-mediated Ca2+ entry contributes to intracellular protective protein Homo oxygenase-1 (HO-1) production in response to LPS remained unknown. Bone marrow-derived macrophages were isolated from TRPM2 knock-out (KO) mice and wild-type (WT) mice and cultured. After 100ng/ml LPS stimulation, the protein levels of HO-1 and its related signaling molecules p38 MAPK and Nrf2 were measured using immunobloting. Furthermore, in LPS stimulated WT macrophages, the p38 MAPK levels were detected after chelating Ca2+, and the HO-1 levels were evaluated after inhibition of p38 MAPK signaling. In TRPM2 KO macrophages, the expression levels of HO-1 were significantly reduced after LPS stimulation. The levels of phosphorylation of p38 MAPK and Nrf2 nuclear translocation showed the same manner when compared to those in WT macrophages. Chelation of calcium obviously inactivated LPS-induced phosphorylation of p38 MAPK. Inhibition of p38 MAPK signaling completely suppressed HO-1 expression. TRPM2 plays an important role in controlling HO-1 expression in macrophages and thereby may protect against inflammation and tissue oxidant injury.
Pregnancy establishment is influenced by CCR7 and CCR9 (P6360)
Authors: Teles Ana , Zenclussen Ana
Published: January 2023

Pregnancy establishment and maintenance represents a challenge for the maternal immune system. It has to be alert against pathogens while tolerating paternal alloantigens in fetal structures. The maternal immune system is not only aware of these alloantigens, but it also actively tolerates them to ensure the survival of the fetus throughout pregnancy. Regulatory T cells (Treg) are reportedly involved in this process. They are known to act in both paracrine and cell-contact dependent ways and therefore their cellular localization is essential for their functional ability of suppressing specific immune responses. Chemokines are known coordinators of leukocyte migration and our aim was to understand how these molecules influence Treg recruitment at the feto-maternal interface and interfere with pregnancy success. We found that both CCL21 and CCL25 chemokine ligands are present in the uterus before and during pregnancy and their ligands CCR7 and CCR9 are expressed in Tregs from the uterus and lymph nodes draining the uterine region. CCR7 mediated the homing of Treg to the uterus while CCR9 determined the maturation state of uterine DCs and presence of T effector cells, and both critically interfered with implantation if genetically deleted. Our data provide new insights on the mechanisms of tolerance acquisition during pregnancy.
Diesel exhaust particle induction of IL17A contributes to severe asthma (P6373)
Authors: Brandt Eric , Butsch Kovacic Melinda , Lee Gerald , Gibson Aaron , Acciani Thomas , LeCras Timothy , Budelsky Alison , Khurana Hershey Gurjit
Published: January 2023

The factors that promote IL17A production during the pathogenesis of severe asthma remain undefined. Diesel exhaust particles (DEP) are a major component of traffic related air pollution and are implicated in asthma exacerbation. We assessed the impact of DEP exposure on asthma severity in asthmatic children from the Greater Cincinnati Pediatric Clinic Repository. Among high DEP-exposed children with allergic asthma, 32.2% of the children had severe asthma, compared to only 14.2% in the low DEP-exposed group. Similarly, Balb/c mice co-exposed to DEP and house dust mite extract (HDM) markedly demonstrated enhanced airway inflammation and hyperresponsiveness (AHR) compared to HDM alone. Exposure to DEP alone did not induce classic features of asthma but generated a Th17 response. DEP and HDM co-exposures enhanced pulmonary Th2 and Th17 cell numbers compared to HDM alone. High DEP-exposed children with allergic asthma had elevated serum IL17A levels compared with low DEP-exposed children. Finally, IL17A neutralization alleviated DEP-induced exacerbation of AHR. In conclusion, Th17 cells contribute to DEP-mediated exacerbation of allergen-induced asthma. Neutralization of IL17A may be a useful therapeutic strategy to counteract the asthma promoting effects of traffic related air pollution especially in severe asthmatics.
Racial disparities in the vitamin D-mediated innate immune response following supplementation (P3379)
Authors: Reyes Rachel , Rafison Brandon , Hur Andy , Joyce Peter , Modlin Robert , Liu Philip , Adams John
Published: January 2023

We have previously demonstrated that innate (Toll-like receptor 2/1) and adaptive (IFN-γ) immune signals converge on a common vitamin D-dependent antimicrobial pathway in the human macrophage against Mycobacterium tuberculosis, including activation of CYP27B1 which converts 25D into the active 1,25D hormone as well as expression of antimicrobial peptides cathelicidin and human beta defensin 4. This antimicrobial activity is dependent on the level of extracellular 25D in culture. Therefore, we asked if this antimicrobial response could be recapitulated using sera from 25D deficient subjects before and after vitamin D repletion of the host in vivo. Serum has been collected prospectively from 67 Hispanic/Latino, 12 black and 21 non-Hispanic white, vitamin D-insufficient/deficient (8-29 ng/ml) before and after treatment with 500,000 IU vitamin D3. The mean total serum 25D in all three ethnic groups was 20ng/ml before and rose significantly (p<0.001) after vitamin D3 repletion, with a mean fold-change of 2.0, 2.9 and 3.0 in white, Hispanic and blacks, respectively. Despite the most substantial rise in total 25D in the minority populations, the ability of the sera from white donors to increase IFN-γ-stimulated macrophage cathelicidin was 3-fold (p<0.01) and 2-fold greater (p<0.05) than in cells conditioned in African and Hispanic/Latino American sera, respectively. These data suggest an ethnic/racial disparity in response to vitamin D supplementation.
Increased IL-22-producing cells contribute to liver fibrosis through promoting Th17 migration in chronic HBV patients (P3363)
Authors: Zhang Zheng , Zhao Juanjuan , Fu Yangxin , Wang Fu-Sheng
Published: January 2023

Interleukin-22 is markedly upregulated in chronic HBV (CHB) infection; however, its functional role in patients with liver fibrosis (LC) remains obscure. We screened mRNA expression in HBV-infected liver tissue vs normal control and found IL-22-associated pathway was significantly changed. We further found that intrahepatic IL-22+ cell counts were significantly increased in HBV-infected patients, especially in LC patients. Further analysis indicated that IL-22 could be produced by various liver-infiltrating lymphocytes, but preferentially produced by IL-22+IL-17a+IFN-γ- Th17 cells in LC patients, which was also positively associated with liver fibrosis severity in CHB patients. We also found human HSCs express high levels of IL-22R. IL-22 exposure increased pSTAT3 expression and fibrotic protein production, and also prevented soluble TRAIL-mediated HSC apoptosis in vitro. Blockade of IL-22 in a HBV-transgenic mice model with T cell-mediated liver fibrosis significantly restricted liver fibrosis progression, and Th17 migration and CCL20 expression. IL-22-treated HSCs secreted CCL20, and blockade of CCL20 preferentially reduced the migration of Th17 cells in vitro. These data suggest that the increased IL-22-producing cells contribute to liver fibrosis through promoting Th17 migration in chronic HBV patients, and may facilitate the rational development of immunotherapeutic strategies targeting IL-22 for the amelioration of liver fibrosis in chronically HBV-infected subjects.
Phosphorylation and the regulation of CD40L mRNA stability by polypyrimidine tract binding protein (P1394)
Authors: Saifuddin Ali , Yang Alex , Covey Lori
Published: January 2023

The expression of the CD40 ligand (CD40L) is controlled at the posttranscriptional level by an activation-induced program of regulated mRNA stability that is dependent on the formation of a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex (Complex I) that binds to the 3’UTR of the CD40L mRNA. PTB is expressed as two major spliced isoforms in T cells, PTB-1 and PTB-4, which differ by the inclusion of exon 9 in PTB-4. To understand how these sites influence the function of PTB, mutants were generated at Ser 306 and Tyr 308 in the parent PTB-4 and introduced into Jurkat T cells expressing RNAi-mediated downregulated levels of PTB. Decreased PTB is known to result in an unstable CD40L transcript. We found that mutant proteins were expressed at relatively equal levels to each other and to that of exogenously-expressed wildtype PTB-4. Also, the distribution into the nuclear and cytoplasmic fractions was highly similar to endogenous PTB. We report the effect of the PTB mutations on the stability and cellular distribution of CD40L RNA. Also, comparing the functional activity of PTB-4 with PTB-1, which lacks the targeted phosphorylation sites, revealed distinct differences. Because we have previously demonstrated that PKC affects the PTB-mediated stability of CD40L mRNA, identifying the role of specific phosphorylation sites broadens our understanding of how events during T cell activation regulate expression of this critical co-stimulatory molecule.
T cell correlates of solid protective immunity following immunization of macaques with a live-attenuated trachoma vaccine (P4403)
Authors: Caldwell Harlan , Olivares-Zavaleta Norma , Carmody Aaron , Whitmire Bill , Kari Laszlo
Published: January 2023

Trachoma, caused by Chlamydia trachomatis, is the world’s leading cause of preventable blindness for which a vaccine is needed. We previously described a live-attenuated trachoma vaccine (LATV) in non-human primates (Kari L. 2011 JEM 208: 2217-23). Vaccinated macaques were either solidly or partially protected following challenge with virulent trachoma organisms. Our current goal was to characterize immune responses that correlated with solid protective immunity. Here, we preformed intramuscular boosting of LATV vaccinated macaques and studied the T cell recall response in PBMC by flow cytometry. Both solidly and partially protected macaques exhibited a strong PBMC CD8+ T recall response following intramuscular immunization. Notably, however; only CD8+ T cells from solidly protected macaques strongly proliferated in response to chlamydial soluble antigen (SA) prepared from chlamydial infected cells. The SA contained proteins known to be secreted by chlamydiae into the host cytosol making them logical antigenic targets of cytotoxic CD8+ T cells. The SA expanded CD8+ T cells expressed IFN-γ, granzyme B, and α4β7 integrin implicating them as effectors important to the development of solid protective immunity at the ocular mucosae. Identifying the effector function and antigens recognized by these CD8+ T cells will be the focus of future studies as this information will provide insights into the design of more conventional immunotherapeutic approaches for trachoma control.
Co-delivery of adjuvant and subunit antigens via a nanoparticle platform induces tissue-associated and systemic adaptive immune responses (P4409)
Authors: Dunkle Alexis , Blanchette Craig , Boone Tyler , Corzett Michele , Fischer Nicholas , Hoeprich Paul , Driks Adam , Rasley Amy
Published: January 2023

Vaccination is a critical countermeasure against infection for both biodefense and public health. As many pathogens are encountered at mucosal interfaces, a delivery system that induces robust mucosal immunity is paramount. Here, we investigate a nanolipoprotein particle (NLP) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a novel vaccine platform using disparate model antigens. Co-administration of MPLA-NLPs with ovalbumin significantly enhances the expansion of OVA-specific T cells in mice in vivo after a single intranasal inoculation, indicating that MPLA-NLPs function as efficient adjuvants. To combine the adjuvant effects with antigen delivery, we engineered MPLA-NLPs to accommodate attachment of His-tagged proteins. Intranasal administration of MPLA-NLPs conjugated to Bacillus anthracis antigens induces specific T cell responses in lung-draining lymph nodes and significant antibody responses in serum and bronchoalveolar lavage. Experiments to assess the immunogenicity of multiple antigens in combination on a single particle and to compare between co-administered formulations are ongoing. These data further support a role for NLPs as a flexible vaccine platform that allows co-delivery of antigen and adjuvant to lung-associated immune tissues and promotes robust adaptive immune responses.
Culture conditions promoting innate immunity and homeostatic proliferation generate highly enriched, MUC1- or HER2/neu-specific CD4+ and CD8+ T cells (P4419)
Authors: Cohen Peter , Pathangey Latha , McCurry Dustin , Gorman Jessica , Viso Carole , Gendler Sandra , Disis Mary
Published: January 2023

Although tumors from melanoma patients can often provide a source of therapeutically active T cells for adoptive therapy, it has historically proved challenging to employ peripheral blood as the T cell source for any type of cancer. We have observed nonetheless that HER2-specific T cells can be Ag-driven and enriched in culture when PBMCs from HER2-vaccinated breast cancer patients are treated with exogenous IL12. We hypothesized that stimulating innate immunity could further improve PBMC cultures by activating the DC subpopulation more effectively than adding exogenous rIL12. Bulk PBMC exposure to optimized TLR agonists produced vast quantities of IL12 and IL23, and upregulated HLA-DR, B7.1 and CD40 in the DC subpopulation, effects not produced by exogenous IL12. Subsequent IL7 exposure emulated homeostatic proliferation, selectively causing the Ag-driven T cell subset both to proliferate faster and to strongly resist apoptosis. Ag-specificity within two weeks approached 80-100% of both CD4+ and CD8+ T cells for recall Ags and 1-15% for a wide array of MUC1- and HER2-derived peptides, including the ability to distinguish glycoforms. Culture-expanded T cells retained a young CD28+/CD56- phenotype, uniformly expressed ROR-γ during culture, variably produced IL-2 and/or IL-17, and uniformly expressed T-bet and secreted IFN-γ when reexposed to Ag. Such T1/T17 bipotency may be ideal for anti-tumor adoptive therapy and highly efficient for use in Ag discovery.
Molecular mechanism of the Vγ9Vδ2 T lymphocytes anergy. (P4385)
Authors: Poupot Mary , Fruchon Severine , Boissard Frederic , Aruebo Marie-Eve , Cendron Delphine , El Faqihi-Olive Fatimah , Pont Frederic , Bardouillet Laure , Mekaouche Mourad , Ingoure Sophie , Sicard Helene , Dubreuilh Guy , Fournie Jean-Jacques
Published: January 2023

Upon activation with phosphoantigens (PAgs), human and non-human primates circulating TCRVγ9Vδ2 T lymphocytes release pro-inflammatory cytokines, mediate tumor cell cytotoxicity and proliferate through an IL2-dependent clonal expansion. Together, these features make it versatile and attractive candidates for new cancer immunotherapies (Kabelitz, D et al., 2007, Canc.Res., 67(1):5-8). Despite a potent and rapid response of the TCRVγ9Vδ2 T lymphocytes in vitro and in vivo, repeated stimulations with PAgs desensitize selectively this T cell lineage in humans and non-human primates (Cendron, D. et al., 2007, Eur.J.Immunol., 37(2):549-65; Sicard, H. et al., 2005, J.Immunol., 175(8):5471-80). Thus, we investigated the gene expression pattern associated with this PAg-selective anergy of these lymphocytes. TCRVγ9Vδ2 T cells were isolated from blood samples from PAgs-injected macaques (3 repeated injections) for transcriptome studies. After the third stimulation, in addition to some genes corresponding to the inhibition of cell cycle, a significant gene signature emerged from the desensitized γδ T cells transcriptome involving the PPARα and CARM1 pathways, based on up-regulation of both EP300, HSD17B4, PRKAR2B and RB1 genes while RARA and PRKCA (PKCα) were down-regulated. Moreover, inhibition of proliferation and cytokine production by PPARα agonists-treated human TCRVγ9Vδ2 T lymphocytes confirmed the implication of PPARα pathway in the γδ T cells desensitization by PAgs.
Tolerance induction using Ag-ECDI-PLGA prevents autoimmunity in the BDC2.5 AT model for T1D (P5208)
Authors: Prasad Suchitra , Xu Dan , Miller Stephen
Published: January 2023

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the infiltration and destruction of insulin producing β-cells of the pancreatic islets by autoreactive T-cells. Treatment with autoantigenic peptide(s) chemically crosslinked with ECDI to splenocytes or other cellular carriers (Ag-ECDI-SP) can prevent and/or reverse murine autoimmune diabetes. Due to the inherit limitations of cellular therapy in a clinical setting including the sourcing and production of Ag-ECDI-SP, here we explore the use of biodegradable poly(lactic-co-glycolic acid) (PLGA) micro-particles as substitutes for splenic donor cells. Treatment with p31-ECDI-PLGA protected against disease in the BDC2.5 adoptive transfer model of T1D. Mice tolerized with p31-ECDI-PLGA mice had reduced levels of total infiltrating BDC2.5 CD4 T cells in the pancreas when compared to mice treated with the non-diabetogenic MOG35-55-ECDI-PLGA control. Additionally, BDC2.5 T cells isolated from the pancreas of p31-ECDI-PLGA treated mice had reduced levels of inflammatory cytokines, such as IFN-gamma, TNF-alpha and IL-17. These data demonstrate the efficacy of an antigen specific therapy to modulate a highly pathogenic autoimmune response using an FDA approved biodegradable PLGA microspeheres. Thus, this carrier system provides a more viable alternative to the cell based approach that can be readily translated in clinical trials.
Pharmacological repression of RORγ is therapeutic in the collagen-induced arthritis experimental model (P5214)
Authors: Chang Mi Ra , Lyda Brent , Kamenecka Theodore , Griffin Patrick
Published: January 2023

The nuclear receptor RORγ has been shown to play an essential role in the development of Th17 cells. Previously, it was reported that the potent and selective RORγ inverse agonist SR2211 was effective at modulating RORγ activity in cell culture. More recently we demonstrated that the RORγ inverse agonist SR2211 can repress expression and production inflammatory cytokines in Th17 cells and LPS stimulated RAW264.7 cells. Here we present in vivo efficacy of SR2211 in an experimental model of arthritis, the murine collagen induced arthritis or CIA model. Significantly reduced Joint CIA scores were observed in SR2211 treated mice as compared with vehicle in the acute phase of the model. CIA represents an autoimmune environment characterized by the accumulation of inflammatory cells into the synovium leading to the destruction of joints. In this model cytokines have been shown to be important regulators of synovial inflammation. Administration of SR2211 in CIA mice led to an increase in IFNγ in ex vivo stimulated draining lymph nodes. As IFNγ has been shown to be beneficial for the treatment of rheumatoid arthritis. In previous work, patients treated with IFNγ showed significant therapeutic potential of RA the data presented suggest the potential of targeting RORγ to repress inflammatory T cell function and macrophage activation in arthritis. Thus, SR2211 has potential utility in the treatment of inflammatory disease.
CRAC channel inhibitors block cytokine production with a pattern distinguishable from cyclosporine (P5216)
Authors: Ramos Stephanie , Butler Lori , Kimbler Trevor , Cao Jianguo , Grey Jonathan , Rogers Evan , Wang Zhijun , Whitten Jeffrey , Roos Jack , Stauderman Kenneth , Velicelebi Gonul
Published: January 2023

Engagement of the T cell receptor (TCR) triggers complex signaling cascades that result in various effector T cell responses. A critical component of TCR signaling is store-operated calcium entry (SOCE) that occurs through the activation of calcium release-activated calcium (CRAC) channels. Activated CRAC channels cause an increase in cytoplasmic calcium, which results in the transcription of calcineurin/NFAT-dependent genes involved in T cell proliferation and cytokine production. Both CRAC channel inhibitors and calcineurin inhibitors are known to inhibit T cell function. However, the relative impact of CRAC channel inhibitors and calcineurin inhibitors on Th1, Th2, Th17, and Treg functions are not known. Using anti-CD3/anti-CD28 to stimulate cytokine production by peripheral blood mononuclear cells (PBMCs), we demonstrate that novel CalciMedica CRAC channel inhibitors have a pattern of cytokine inhibition that is different from the calcineurin inhibitor cyclosporine (CsA). CRAC channel inhibitors inhibit Th1 and Th17 cytokines (IFNγ and IL-17) more potently than Treg and Th2 cytokines (IL-10 and IL-4), while CsA inhibits IL-10 more potently than IFNγ, IL-4, and IL-17. These results suggest that CRAC channel inhibitors and CsA have different potencies against different effector T cell subsets, and that CRAC channel inhibitors may achieve a more favorable balance of effects on pro- versus anti-inflammatory pathways in immune disorders.
Identification and characterization of T cell epitopes expressed on the surface of cancer cells. (P2002)
Authors: Rozanov Dmitri , Reddy Ashok , David Larry , Burrows Gregory , Rantala Juha , Levin Trevor , Wilmarth Philip , Anur Pavana , Chiotti Kami , Spellman Paul
Published: January 2023

In our search for better ways to stimulate anti-tumor T cell-mediated immune response we optimized powerful technique known as “reverse” immunogenetics, in which MHC I-bound peptides are recovered and sequenced using highly sensitive mass spectrometry. Using this technique we have identified more than 3,500 peptides eluted from the surface of 20 breast carcinoma cell lines of 4 different subtypes. By using sequence data for each breast carcinoma cell line we predicted bound peptide consensus sequence for a particular allele of HLA-A, -B, and C molecules. Next, we determined the frequency of the identification of both peptide and protein that is associated with this peptide in subtype-specific and all analyzed cell lines. This allowed us to associate some MHC I-presented peptides/proteins with a specific subtype of breast cancer as well as shared peptides/proteins. Some of the identified proteins are tumor specific and immunogenic. For example, Aldolase A, which is MHC I-presented in many cell lines, has been identified by Serex analysis in lung cancer and breast cancer patients. Lastly, using gene expression data for 60 breast carcinoma and normal cell lines as well as for 708 tumors and 329 normal tissues we have sorted all identified proteins in accordance with the level of their expression in non-transformed and transformed cells. This arrangement allowed us to identify, for the first time, several tumor specific genes that could be good targets for T cell-based therapy.
Memory to self-antigens in Systemic Lupus Erythematosus (P4020)
Authors: Chen Yunying , Wermeling Fredrik , Petersen Sven , Kaiser Ylva , Karlsson Mikael
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease associated with auto-IgG antibodies, which are produced due to defective clearance of apoptotic cells (AC) and loss of tolerance to self-antigens. SLE follows flare and remission courses; disease symptoms can come rapidly and unpredictably, suggesting the presence of immune memory to self-antigens in patients. However, experimental evidence and molecular mechanisms behind this are not known. We set up a mouse model by injecting normal mice with syngenic AC weekly for 4 weeks that breaks tolerance and induces auto-IgG production. One month later the serum auto-IgG drops to pre-immune level, these mice can rapidly respond to a single AC injection and produce increased auto-IgGs. This memory-like response is transferable into naïve mice. Serum level of auto-IgG subclass IgG2, but not IgG1, is dramatically increased in the memory response compared to that in the primary response, which contribute to pathogenic effects. The memory auto-IgGs are produced via both germinal center and plasmablast pathways. Spectratyping shows that B-cell repertoire is more skewed in memory response than that in pre-immune and primary response. These observations demonstrate an inducible immune memory to self-antigen in normal mice and establish a model for study of SLE flare-like response in mice. In addition, this study suggests that the formation of immune memory to self-antigens contributes to the disease relapse in SLE patients.
Detection and characterization of autoreactive B cells in patients with Rheumatoid Arthritis (P4024)
Authors: Titcombe Philip , Barsness Laura , Giacobbe Lauren , Gillespie Emily , Peterson Erik , Mueller Daniel
Published: January 2023

B cell tolerance to self antigen is critical for preventing several autoimmune diseases, including Rheumatoid Arthritis (RA), but how tolerance is maintained or broken in physiologic settings is not well understood. Our lab recently developed a novel protein-tetramer enrichment strategy for detecting and analyzing rare self antigen-specific B cells within the endogenous polyclonal repertoire of mice. We have now adapted this strategy to study human peripheral blood B cells specific for two different cyclic citrullinated peptides (CCP) that mimic the self-protein epitopes targeted by autoantibodies in a majority of RA patients. RA patients with epitope-specific anti-CCP IgG serum antibodies (Ab+ RA) display an increased frequency of CCP-specific B cells compared to Ab- RA patients and healthy controls. Ab+ RA patients also demonstrate an increased frequency of CCP-specific B cells that have undergone isotype-switching and memory cell differentiation (IgD- CD27+), and their numbers correlate with anti-CCP antibody levels. Future work tracking self antigen-specific B cells in humans will focus on identifying factors important for preserving or restoring B cell tolerance.
Extracellular Granzyme B: a key player in aneurysm pathogenesis (P3072)
Authors: Granville David , Ang Lisa , Bleackley Chris , Zhao Hongyan
Published: January 2023

Granzyme B (GZMB) is a pro-apoptotic protease that is released by cytotoxic immune cells and internalized by target cells in a perforin-dependent manner. However, GZMB contributes to aortic aneurysm pathogenesis through a perforin-independent, extracellular process involving extracellular matrix proteolysis. In vitro and in vivo studies have identified decorin, a key regulator of collagen organization, fibrillogenesis and collagen tensile strength, as a GZMB substrate. We hypothesized that GZMB contributes to aortic aneurysm through the degradation of decorin leading to reduced collagen organization and tensile strength resulting in aortic rupture. To induce aneurysm, apoE-/- mice were implanted with osmotic minipumps that constitutively released angiotensin II. Mice were injected with the GZMB inhibitors, serpin A3N or anti-GZMB neutralizing antibody. A dose-dependent reduction in the frequency of aneurysm rupture was observed in mice that received inhibitor. Reduced GZMB and decorin proteolysis were observed in inhibitor-treated mice. Adventitial collagen from serpin-treated mice exhibited higher fibre density and reduced fibril size irregularity. GZMB was also elevated in human aortic aneurysm. GzmB was expressed in lymphocytes in the intraluminal thrombus and in mast cells in the aneurismal aortic adventitia. In summary, extracellular GZMB contributes to decorin degradation resulting in reduced collagen organization and loss of aortic wall integrity leading to aneurysm.