bluuebunny/crossref_metadata_2025 huggingface dataset:
VaxCelerate: the use of MTBhsp70-avidin as an adjuvant to rapidly generate self-assembling vaccines with biotinylated, antigen-specific peptides targeting emerging pathogens. (VAC6P.941)
Authors: Fishman Jordan , Moise Leonard , LeBlanc Pierre , Brauns Timothy , Berg Eric , Richer Daniel , Boyle Christine , De Groot Anne , Martin Bill , Baker David , Zeigler Brandon , Mais Dale , Taylor William , Coleman Russell , Shaw Warren , Gelfand Jeffrey , Poznansky Mark
Published: January 2023

Development of effective vaccines against emerging infectious diseases can take years to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. The VaxCelerate Project’s goal is to create a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 days. A self-assembling vaccine, consisting of a fusion protein M. tuberculosis MTBhsp70 and avidin (MAV), is at the core of the approach. Mixing the MAV with biotinylated pathogen specific immunogenic peptides yields a self-assembled vaccine (SAV). To minimize the time required, we used a distributed R&D model involving experts in protein engineering, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing. This approach was first tested using ovalbumin in C57Bl/6 mice, Flu (H1N1) specific peptides, and ultimately a Lassa fever virus (LFV) specific vaccine in transgenic HLA DR3 mice. Using a GLP validated assay we demonstrated that the MAV assembled LFV induced significantly increased class II peptide specific interferon-CD4+ T cell responses in transgenic mice compared to peptide or MAV alone controls. The use of an identical design for each vaccine may facilitate accelerated regulatory review and by developing safety assessment tools that are more relevant to human vaccine responses than current preclinical models.
Expression of microRNAs in response to a TLR dependent adjuvant PorB from Neisseria meningitidis (VAC6P.943)
Authors: Mosaheb Munir , Platt Andrew , Reiser Michael , Shaik-Dasthagirisaheb Yazdani , Toussi Deana , Liu Xiuping , Wetzler Lee
Published: January 2023

MicroRNAs (miRs) are small non-coding RNAs that are involved in post-translational modulation of gene expression. Not much is known about the role of miRs in vaccine adjuvant activity. In this study, we investigated miRs elicited by PorB, a TLR2 dependent adjuvant from N. meningitidis, in vivo and in vitro. Mice (WT) were immunized 3 times, 2 weeks apart with Ova +/- PorB. Splenic RNA was obtained 24 hours after each immunization and analyzed by microarrays. Bone marrow-derived macrophages (BMDM) from WT, MyD88 or TLR2 knock out mice were stimulated with PorB, RNA was isolated and miRs expression was determined by RT-PCR. The in vivo study revealed that miR505 and miR15a expression was increased, while miR689 and miR666 expression was decreased when PorB was included in the vaccine preparation. Interestingly the differential expression of these miRs were seen after the 2nd but not after the 1st or 3rd immunization, suggesting that the regulatory events driven by PorB occurred after the 2nd immunization. We also observed a high increase in the expression of miR155 upon stimulation with PorB in vitro, however the miR155 expression was dampened in BMDM from TLR2 knock out mice and completely abrogated in MyD88 knock out mice. PorB seems to be able to induce high level of miR155 in a TLR2 independent as well as TLR2 dependent mechanism. These data increase our understanding of the association of miR expression and PorB adjuvant activity.
A systems biology approach demonstrates that PO and ID BCG induce distinct CD4+ T cell molecular signatures. (VAC7P.970)
Authors: Xia Mei , Zhang Guang lan , Blazevic Azra , Brusic Vladimir , Hoft Daniel
Published: January 2023

Vaccination with Bacillus Calmette-Guérin (BCG) is and constitutes the most widely used tuberculosis (TB) control strategy in the world. Protective efficacy of BCG may be affected by the methods and routes of vaccine administration. Here we have used a systems biology approach to study innate and adaptive responses to oral (PO) and intradermal (ID) BCG vaccination in humans. 60 healthy adults vaccinated with PO and/or ID BCG was studied. Although both PO and ID BCG could induce systemic Th1 responses capable of IFN-γ production, ID BCG more strongly induced systemic Th1 responses, while significantly higher mucosal responses (TB-specific sIgA and bronchoalveolar lavage T cells) were induced by PO BCG vaccination. In order to identify early gene signatures that predict optimal mucosal and systemic immune responses, CD4+ memory T cells were isolated from both PO and ID BCG vaccinated volunteers pre- (Day 0) and post-vaccination (Day 7 and 56), and studied by Illumina BeadArray transcriptomal analysis. Notably, distinct gene expression profiles were identified both on day 7 and day 56 comparing the PO and ID BCG vaccinated groups by GSEA analysis. Correlations between specific expression patterns and mucosal vs. systemic immune responses are being studied. These data highlight the utility of systems biology approaches in studying differential mucosal and systemic immune responses.
Combined vaccination of subcutaneous BCG and intranasal HBHA with cholera toxin enhances early protective immunity against pulmonary M. tuberculosis infection (VAC7P.972)
Authors: Fukui Masayuki , Umemura Masayuki , Matsuzaki Goro
Published: January 2023

Effect of Mycobacterium bovis bacille de Calmette et Guérin (BCG) vaccine on preventing adult pulmonary tuberculosis has been reported to be limited. Therefore, development of a novel effective vaccination strategy against pulmonary tuberculosis has become an international research priority. We have previously reported that intranasal administration of a recombinant mycobacterial Ag, heparin-binding heamagglutinin adhesin (HBHA), combined with mucosal adjuvant cholera toxin (CT) into mice enhanced Th1-type immune response and suppressed extrapulmonary bacterial dissemination after M. tuberculosis (Mtb) infection in the lung. Here, we further report effects of the mucosal HBHA+CT vaccine on murine experimental pulmonary Mtb infection. Combination of subcutaneous BCG priming vaccine followed by the mucosal HBHA+CT vaccine as a booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14. The combination of subcutaneous BCG and the mucosal HBHA+CT vaccine was more effective than the BCG vaccine alone in induction of the early protective immunity. Further, the mucosal HBHA+CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. The results indicate that the combination of BCG priming vaccine and mucosal HBHA+CT boosting vaccine might be a good candidate for a new vaccine strategy against pulmonary tuberculosis.
Accelerated development of a T Cell epitope-driven biodefense vaccine for Burkholderia (VAC7P.957)
Authors: Liu Rui , Sangare Kotou , Desrosiers Joe , Martin Bill , Moise Leonard , Groot Annie
Published: January 2023

Background: Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM) are Category B pathogens. Burkholderia cepaciae (BC) causes chronic lung infections in cystic fibrosis patients. We are developing an epitope-driven multi-pathogen vaccine against all three Burkholderia species. Methods: Immunoinformatics tools were used to identify immunogenic consensus sequences (ICS) that are enriched for promiscuous and highly conserved CD4+ T cell epitopes in all three Burkholderia species. Top scoring 70 ICS were tested for binding affinity to 7 Class II HLA alleles in binding assays. Immunogenicity of the ICS was tested in human PBMCs, and in Balb/C (peptide immunization) and HLA DR3 transgenic mice (DNA-prime/peptide-boost immunization). Results: 100% of the ICS bound to at least two Class II HLA alleles for which they were predicted. The immunoinformatic predictions were validated in 77.3% of the HLA-peptide binding assays. Significant IFN-γ response was induced by all peptides in at least one human donor as measured by ELISpot assay. In contrast, 28.6% and 11% of the ICS peptides elicited significant IFN-γ responses in splenocytes from vaccinated Balb/C and DR3 transgenic mice as measured by ELISpot assay, respectively. Conclusions: These results illustrate the power of immunoinformatics tools for rapid selection of highly conserved epitopes for biodefense vaccines. Differences in human and murine immune responses may be due to ineffective delivery of the vaccine immunogen.
Priming protective BVDV-specific immunity using multi-component mosaic antigens (VAC7P.980)
Authors: Mwangi Waithaka , Fang Xin , Lokhandwala Shehnaz , Bray Jocelyn , Waghela Suryakant
Published: January 2023

Bovine viral diarrhea virus (BVDV), a Flavivirus, plays a key role in bovine respiratory disease complex, which leads to pneumonia and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits of the virus. There are diverse BVDV strains and thus, current vaccines contain type 1 and 2 viruses (BVDV-1 & 2). Modified live virus (MLV) vaccines are superior to killed virus, but they are susceptible to antibody neutralization and complement-mediated destruction that limits their efficacy in neonates. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which represent determinants conserved among BVDV strains and these antigens are the main targets involved in protection. We confirmed that a proto-type adenovirus-vectored vaccine expressing these antigens was recognized by mAbs, sera, and T cells raised against diverse BVDV strains. In a poof-of-concept efficacy study, the vaccine induced significantly higher IFN-γ spot forming cells (p<0.05), T cell proliferation (p<0.05), and SN titers (p<0.05) against BVDV-1 & 2 as compared to immune responses induced by a commercial MLV vaccine. Following challenge, the calves immunized with the proto-type and the MLV vaccines had mild clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate protective motifs from diverse BVDV strains.
Modulating cathepsin cleavage sites in HIV gp120 envelope protein alters the cellular immune response. (VAC7P.989)
Authors: Frey Blake , Sui Yongjun , Wang Yichuan , Hogg Alison , Yu Bin , Berman Phillip , Berzofsky Jay
Published: January 2023

A major goal in HIV vaccine development is to identify antigens that can elicit long-term, protective antibody and cellular responses. Limited success with native recombinant HIV gp120 proteins suggested altering antigen processing as an approach to improve the immunogenicity of the HIV gp120 protein. Cathepsins S and D are important proteases which play critical roles in antigen processing. Here, we have utilized recombinant HIV gp120 proteins with mutations at the cathepsin S site (T322T323) and D cleavage site (L181Y182) within HIV gp120 to investigate the effects of these mutations on MHC class I/II presentation to CD8+/4+ T cells. We have found that altering the cathepsin S site in the minimal epitope, IGPGRAFYTT from T322T323 to V322V323 might improve CD8+ T cell recognition to this epitope in the V3 domain. We are investigating the mechanisms by which this mutation might enhance the CD8 response. It is possible this mutation can increase the binding affinity by changing the anchor residue with the MHC I molecule, or it can prevent degradation of the minimal epitope, or both. In a mouse model, we found that gp120 elicited antigen-specific CD4+ T cell responses against the V1V2 domain. Currently, we are investigating whether the altered cathepsin sites in gp120 affect antigen-specific CD4+T cell responses to the V1V2 regions. As antigen processing plays an important role in immunogenicity, we speculate that HIV may exploit this process to escape recognition.
Predicting T cell responses to multiple antigens in lymph nodes (VAC7P.990)
Authors: Ziraldo Cordelia , Gong Chang , Kirschner Denise , Linderman Jennifer
Published: January 2023

T cells carrying receptors specific to an epitope are primed and differentiate in lymph nodes when they encounter their peptide-MHC counterpart displayed on dendritic cells. These epitopes are subunits of proteins derived from pathogens, but very few of the epitopes from any given pathogen elicit a measureable cell-mediated immune response. For infectious diseases lacking a protective vaccine, concurrent cellular immunity to several peptides may be necessary to target multiple stages of disease. In this work, we expand upon our previously developed computational model of T cell priming and differentiation in LNs to explore simultaneous induction of memory for multiple antigens. The model incorporates key parameters describing cell interactions, including pMHC-TCR binding parameters, cognate T cell frequency, and fraction of pMHCs specific to a TCR. Our results point to the importance of binding thresholds and pMHC fractions in determining immunodominance hierarchies. These findings are relevant to design of multi-subunit vaccines, where the aim is to simultaneously induce memory to several peptides.
Therapeutic vaccination against shingles broadens the VZV-specific CD4+ T cell response (VAC7P.994)
Authors: Laing Kerry , Russell Ronnie , Dong Lichun , Haas Juergen , Stern Michael , Johnston Christine , Wald Anna , Koelle David
Published: January 2023

Life-long latent neuronal infection with varicella zoster virus (VZV) is a legacy of childhood chickenpox. VZV reactivation occurs in about 1/3 of persons to cause shingles. Live, attenuated VZV (vOka)—the only licensed therapeutic vaccine—moderately reduces both the incidence and the severity of shingles. The partial level of protection is poorly understood but may plausibly derive from variation in T cell boosting. Twelve subjects (>50 years old) were vaccinated with vOka and blood obtained on days 0, 14, and 28. Ex vivo ICC analyses confirmed boosting of vOka-reactive T cells: the mean increase in frequency of IFNγ+IL2+ CD4+ T cells was 74% by day 14 (p=0.02). We enriched and expanded VZV-specific CD4+ T cells in an unbiased manner and defined the specificity and breadth of the resulting polyclonal CD4+ T cell lines using each of the 70 known VZV proteins. Prior to boosting, the median number of detectable responses was 9 ORFs (range 3-10). Vaccination increased the median breadth to 17 ORFs (range 9-20) at day 28. Prevalent targets before and after boosting were glycoproteins gE, gI, and gH, and the tegument proteins encoded by ORF12 and ORF44. While new responses were noted post-vaccination, baseline responses rarely disappeared. T cell breadth was well-maintained during VZV latency and further increased by therapeutic vaccination. In addition to CD4+ T cell magnitude, specificity and breadth are now accessible as candidate immune correlates of vaccine efficacy.
The αβ-double negative T cells in lymphoma patients: the predictive role and the functional attitude. (VAC8P.1003)
Authors: De Tullio Giacoma , Iacopino Pasquale , Guarini Attilio
Published: January 2023

Mutual interactions between the host immune system and lymphoma cells can either promote or control lymphomagenesis.A subset of CD4-CD8- double-negative T cells(DNTs) has been recently described to be involved in anti-tumor immunity acting as regulatory T cells and/or cytotoxic T cells.The aim of this study was to assess the role of DNTs on clinical outcome and progression of Lymphoma patients (pts).Peripheral blood(PB) and bone marrow(BM) samples of 46 Lymphoma pts,with non-Hodgkin’s(nHL) and classical Hodgkin Lymphoma(HL),and 16 PB of 16 healthy donors (hd),were selected and prospectively collected at diagnosis, at different time of therapy and at the time of relapse or progression.DNTs were purified and characterized for their phenotype and for tolerogenic or cytotoxic attitude with functional studies.The percentage of DNTs in BM (2.367+0.5891)of pts was lower than in PB(3.421+0.981) and decreased(p=0.006)in pts with untreated lymphoma as compared with hd,seeming to be modulated by disease relapse/progression or disease treatment.In HL αβ-DNTs were significantly increased as compared with other histotypes(p=0.0001).Interestingly,after ex vivo expansion,DNTs acquired a immunomodulatory cytokine profile.Our study demonstrated that αβ-DNTs could play an important role in both the development and the progression of lymphomas.Finally,it is likely that ex-vivo expanded DNTs exert an anti-tumor activity suggesting their possible use as a new strategy for adoptive immune-therapy.
Dynamics of the CD8+ T cell response to a model of acute viral infection (VIR4P.1017)
Authors: Emerson Ryan , DeWitt William , Lindau Paul , Desmarais Cindy , Makar Karen , Warren Edus , McElrath Juliana , Wald Anna , Robins Harlan
Published: January 2023

During an acute viral infection T cells specific to viral antigens, as determined by the T-cell receptor (TCR), activate and expand. To determine the breadth of immune response to infection, and to understand recruitment to immunological memory, characterizing the TCR repertoire before, during, and after a model of acute viral infection is necessary. Here we use the yellow fever vaccine (YFV) to model acute viral infection. Blood was collected from 9 subjects at 0, 14, and 90 days after YFV administration. In addition to isolating PBMC for all time points, we sorted for YFV-specific effector T-cells at day 14 and for central and effector memory T-cells in 6 of 9 subjects at day 90. For each sample, gDNA was extracted and the TCR repertoire was assayed by high-throughput TCRβ sequencing. Expanded T cell clones can specifically bind a pathogen-derived epitope (specific), or receive proliferation signals without antigen-specificity (bystander). YFV-specific clones were identified by enrichment in the YFV-specific day 14 sort vs. day 14 PBMC; in each subject, we estimate that the antigen-specific immune response comprises approximately 103 T cell clones. Bystanders were identified as clones expanding from day 0 to day 14 PBMC that are not YFV-specific. Many more YFV-specific clones were recruited to memory at day 90 than bystander clones (40% vs. 3%), indicating that antigen specificity, rather than just proliferation, is a major determinant of memory recruitment.
The transcription factor ZBTB32 negatively regulates CD8+ T cell responses during acute and chronic viral infection (VIR4P.1010)
Authors: Shin Hyun Mu , Kapoor Varun , Welsh Raymond , Berg Leslie
Published: January 2023

The transcription factor, ZBTB32, is not expressed in resting naïve CD8+ T cells, but is upregulated following TCR stimulation in the presence of cytokines IFNγ, IL-12 or IL-2. To investigate the role of ZBTB32 in the development of anti-viral CD8+ T cells, Zbtb32-/- mice were infected with LCMV-Armstrong or LCMV-Clone 13. Following infection with LCMV-Armstrong, Zbtb32-/- mice exhibited normal viral clearance, but generated increased numbers of virus-specific CD8+ T cells, relative to wild-type mice, resulting in an increased memory cell population. The Zbtb32-/- CD8+ memory T cell population showed rapid proliferation and enhanced protective memory potential to secondary challenge. In contrast, upon infection with LCMV-Clone 13, 70% of Zbtb32-/- mice succumbed to a fatal disease starting at day 9 post-infection, and exhibited severe immune pathology in the lung compared to WT mice. Examination of the surviving mice indicated reduced LCMV-clone 13 viral titers in Zbtb32-/- mice. Gene expression analysis showed up-regulation of Eomes, Cd27, Pvr and Cd7 in virus-specific Zbtb32-/- CD8+ T cells. ChIP assays confirmed that ZBTB32 bound to the regulatory regions of these genes and recruited HDAC1 and HDAC2 to promote repressive histone modifications. These data indicate that ZBTB32 acts an epigenetic regulator and negatively regulates T cell responses and memory generation during acute and chronic LCMV infection.
An ultrafast refolding protocol for producing cell surface immunoreceptors and viral receptors (VIR5P.1029)
Authors: Zhai Lu , Wu Ling , Li Feng , Xu Bin
Published: January 2023

Functional cell surface immunoreceptors and viral receptors are often a cumbersome and time-consuming process and a bottleneck step that limits detailed biochemical and functional characterization of this important class of molecules. Here we introduce an ultrafast procedure that can be used to refold inclusion-body-based immunoreceptor and viral receptors within one day from the point of harvesting culture, a speed equivalent to soluble protein production. The protocol utilizes modular on-column Ni-NTA purification followed by efficient on-column refolding. We demonstrated the utility of this method for multiple immunoreceptors and viral surface receptors, including host immunoreceptors MHC class I chain A (MICA) and NKp46 and viral receptors UL144 and UL146. The method can be easily adapted into high throughput format for structure biology, chemical biology, and proteomics screenings.
Distant homolog identifies novel viral immune evasins from herpesvirus proteome (VIR5P.1031)
Authors: Xu Bin , Wu Ling , Zhai Lu , Li Feng
Published: January 2023

Viruses have evolved a number of mechanisms to avoid host immune responses. Herpesviruses have been known as a rich source in viral evasion genes including homologs mimicking the structures and functions of host immune proteins. Using a rapid high throughput computational structural mimicry analysis, we have screening the whole proteome of human herpesviruses that includes nearly 2,000 proteins. We discovered a number of novel distant viral homologs in herpesvirus proteome that are previously uncharacterized. This includes homologs involving MHC class I pathways and NK immune defense, or chemokine and cytokine mimics. Sequence identities between viral mimics and native host ligands/receptors can be as low as 9-25%. Two novel distant homologs are of particular interests as they are predicted to be involved in MHC class I modulation. These novel homologs are being expressed and produced and are undergone further characterization to determine their structural and functional characteristics and their roles in viral immune evasion. From these herpesviral homologs and their predicted structures, we hypothesize that herpesviruses utilize a variety of binding mechanisms to regulate a class of key MHC I molecules including MICA/B.
Cytomegalovirus and early-life irradiation synergize to erode protective immunity in aging (VIR5P.1034)
Authors: Pugh Jason , Sukhina Alona , Smithey Megan , Nikolich-Zugich Janko
Published: January 2023

Susceptibility to infectious disease increases with age, making it one of the leading causes of death in people over 65. The immune aging process may be accelerated by the presence of life-long latent infections, such as cytomegalovirus, that put a constant burden on the immune system. Theories of biological aging also suggest that immune cells may have defective functions in old age because of a lifetime of self-renewal, and DNA damage related senescence, among other factors. Sub-lethal whole-body irradiation results in dose-dependent death of immune cells throughout the body, forcing some peripheral self-renewal of surviving immune cells. We exposed mice to various sub-lethal irradiation doses (0Gy - 4Gy) in youth, and followed them for life. Some mice were also given life-long murine cytomegalovirus (MCMV) to reflect the most common latent virus in human populations. At the end of life, mice were vaccinated against, and then infected with, a potentially lethal dose of West Nile Virus (WNV). Neither MCMV, nor any dose of sub-lethal irradiation, independently altered survival following vaccination and WNV infection. However, mice that received a combination of 4Gy of irradiation in youth, and life-long MCMV infection, were less likely to survive WNV challenge. Mechanisms and implications for radiobiology, immune aging, and vaccine efficacy will be discussed.
Dysregulated immune responses in lupus-prone mice facilitate Th1-cell immunity against influenza A (BA12P.103)
Authors: Slight-Webb Samantha , Crowe Sherry , Anderson Jourdan , James Judith
Published: January 2023

Influenza viruses are a primary cause of respiratory tract infections causing the highest number of hospitalizations and mortalities out of any viral respiratory pathogen. In patients with systemic lupus erythematosis (SLE), infections remain a major cause of morbidity and early mortality. Despite the availability of vaccines, only antigenically related infections are prevented and vaccines may be less effective in some SLE patients. Whether infectious disease risk results from SLE-associated innate and adaptive immune defects or from immunosuppressive medication effects (or both) remains unknown. Using a murine model of SLE, we show MRL-lpr/lpr mice infected with influenza A PR8/34 H1N1 have delayed morbidity and decreased cellular infiltration into the lung compared to MRL/MPJ and C57BL/6 control mice. In addition, we show that lupus-prone mice accumulate more pulmonary IFNγ and TNFα virus-specific CD4+ and CD8+ T cells to clear infection. However, MRL-lpr/lpr mice have increased levels of IL-10 compared to controls limiting pulmonary inflammation. We further show that influenza virus did not increase production of lupus-specific autoantibodies or total IgG in SLE mice. These data suggest that the activated immune response of lupus-prone mice assists in the control of influenza A virus without worsening of disease symptoms.
Intricate interplay of cytokines determine the progression of SLE-like disease in BXSB.Yaa mice (BA12P.104)
Authors: Jain Shweta , Adkins Elisabeth , McPhee Caroline , Christianson Gregory , Sproule Thomas , Shin Dong-Mi , Roopenian Derry , Morse Herbert
Published: January 2023

Systemic lupus erythematosus (SLE) is a complex multi-organ disease that affects mostly females. It is caused by several environmental, genetic and stochastic factors. BXSB.Yaa is a recombinant inbred strain of mice with major manifestations that parallel those of human SLE, thereby making it a model system to investigate genetic and non-genetic determinants of disease initiation and progression. The Yaa mutation in male results due to translocation of a 4Mb chromosomal region, including Tlr7 gene, from X to the Y chromosome, making them susceptible to disease. The disease pathogenesis involves an intricate interplay of key cytokines such as IL-21, IL-6, Type I interferon (IFN) and IL-10. Enhanced expression of IL-21 by CD4-T (Tfh) cells expands autoreactive B cells that differentiate into plasmablasts and secrete autoantibodies. IL21 also promotes the development of CD8-T cells and NK cells which retard early disease development. Gene expression profiles of cells from young IL-21R deficient BXSB.Yaa mice revealed an “IFN signature” similar to human SLE females. Purified plasmacytoid dendritic cells (pDCs) from male mice produced higher levels of Type-I IFN than females. Type I IFN further stimulates IL6 producing B cells that drives differentiation of CD4 Tfh cells. IL6 also contributes to accelerated mortality in BXSB.Yaa mice. Moreover, BXSB.Yaa mice express IL10 and ablation of IL10 in these mice results in accelerated mortality.
CD275/ICOSL-independent germinal centers and autoantibody in autoimmune-prone RasGRP1-deficient mice (BA13P.125)
Authors: Barrington Robert , Buhlmann Janet , Wang Xiaogan , Bartlett Amber , Lim Bing , Smith William
Published: January 2023

Lymphopenia results in favorable microenvironments for the expansion and activation of autoreactive lymphocytes. Mice deficient in guanine nucleotide exchange factor Rasgrp1 (KO) are T lymphopenic early in life and develop a lymphoproliferative disorder with features of human SLE. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development as well as during germinal center responses, suggesting both T cell-independent and -dependent mechanisms are responsible. To understand whether T cells are involved in the breach of tolerance in germinal centers, we examined T effector cells in KO mice. In addition to the expected increase in Th1 cells, the frequency and number of Th17 cells were also elevated in KO mice. Th17 cells were positive for CXCR5 and Bcl-6, localized to germinal centers and upregulated levels of CD278/ICOS coordinately with the appearance of germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278-CD275 interactions were required for the development of Tfh17 cells and for autoantibody, we crossed KO and CD275-deficient mice. Interestingly, KO mice lacking CD275 formed germinal centers with an elevated frequency of Th17 cells and similar titers of autoantibodies. From these data, we suggest that CD278-CD275 interactions are not required for germinal centers and autoantibody production in mice lacking RasGRP1.
IL-13Rα1 plays a role in CD4+ T cell stimulation by antigen presenting cells and affects CNS autoimmunity (BA14P.213)
Authors: Khairallah Marie-Therese , Ellis Jason , Chen Weirong , Miller Mindy , Ukah Tobechukwu , Zaghouani Habib
Published: January 2023

Multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are immune-mediated inflammatory diseases of the central nervous system (CNS) triggered by activation of myelin-reactive Th1 and Th17 cells. Both IL-4 and IL-13 cytokines are regarded as anti-inflammatory cytokines against the disease but the mechanism by which these cytokines exercise modulatory function remains largely unknown. Since the heteroreceptor (HR) comprising IL-4 receptor alpha (IL-4Rα) and IL-13Rα1 is shared by both cytokines, we envisioned that IL-4 and IL-13 utilize the HR to drive disease modulation. The findings indicate that mice, in which the heteroreceptor has been made non-functional by gene deletion exhibited earlier onset and more severe disease symptoms when induced for EAE with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. In addition, MOG-specific T cell responses had increased IL-17 but reduced IFNγ production relative to heteroreceptor sufficient mice. Intriguingly, the HR-deficient mice had increased T regulatory cells relative to HR-sufficient animals. These findings suggest that the HR plays a role in the development of Tregs and regulate their function toward pathogenic cells in a subset specific manor.
Absence of IFNγ increased brain pathology in EAE-susceptible DR3DQ8 HLA transgenic mice through secretion of IL-17 and induction of pathogenic monocytes/microglia into the CNS (BA14P.202)
Authors: Mangalam Ashutosh , Luo Ningling , Luckey David , Papke Louisa , Smart Michele , Giri Shailendra , Rodriguez Moses , David Chella
Published: January 2023

Among all the genetic factors linked with multiple sclerosis (MS), MHC class-II molecules show the strongest association. Generation of HLA class-II transgenic mice has helped to elucidate the role of HLA class-II gene in MS. We have shown that the human HLA-DR3 (DRβ1*0301) gene predisposes to proteolipid protein (PLP)-induced EAE, whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DR3DQ6 double transgenic mice by producing anti-inflammatory interferon gamma (IFNγ). To further confirm the role of IFNγ in protection, we generated DR3DQ8 mice lacking IFNγ (DR3DQ8.IFNγ-/-). Immunization with PLP91-110 peptide caused atypical EAE in DR3DQ8.IFNγ-/- mice characterized by ataxia and spasticity, hallmarks of brain-specific disease. Severe brain specific inflammation and demyelination in DR3DQ8.IFNγ-/- mice with minimal spinal cord pathology further confirmed brain-specific pathology. Severe disease in DR3DQ8.IFNγ-/- mice was due to increased encephalitogenicity of CD4 T cells and its ability to produce higher levels of IL-17 and GM-CSF compared to DR3DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFNγ in inflammatory and demyelinating disease through down regulation of IL-17 and induction of neuro-protective factors in the brain by monocytes/microglial cells.
Differential regulation of T cell responses and disease severity by mycoplasma and its superantigen in autoimmune diabetic mouse (BA15P.228)
Authors: Mu Hong-Hua , Nourian Maziar
Published: January 2023

Infection has been extensively documented as a serious problem causing high mortality and morbidity in diabetic populations. M. arthritidis produces a potent superantigen (SAg) MAM, which has been shown to be at least partially responsible for the septic shock induced in certain susceptible mouse strains. In the present study, we have investigated how diabetic immune cells respond to the M. arthritidis and MAM SAg. By using two well-defined animal models of diabetes, STZ and NOD mice, we found that STZ mice infected with M. arthritidis developped severe lethal toxicity and the immune cells predominantly produces pro-inflammatory cytokines TNFalpha, IL-6, IL-1β, chemokines CCR2/4 and Tbx21, a Th1-related transcription factor; while MAM-activated T cells from NOD mice display a mixture of Th1 and Th17 profiles which are associated with the elevated levels of ICOS molecule and a unique transcription factor LSF (tcfcp2). Since ERK, a signaling protein of MAPK family, is reported to be responsible for the phosphorylation of LSF in T cell activation, thus we propose a novel ERK-LSF-ICOS signaling cascade that is required for the Th1/Th17 development in NOD mice in response to MAM. Our findings further suggest that the inhibition of these molecules might be potentially therapeutic in the alleviation of mortality and morbidity in diabetic individuals with severe infections.
In autoimmune-prone NOD mice, altered DC subsets induce aberrant activation of CD4 T cells, yet DCIR2+ DCs can still induce tolerance. (BA15P.218)
Authors: Tarbell Kristin , Price Jeffrey , Rahir Gwendoline , Beauchamp Nicole , Zhao Yongge , Dong Matthew
Published: January 2023

Stimulation of CD4+ T cells by antigen targeted in vivo to dendritic cells (DCs) in the absence of inflammation usually leads to tolerance. However, DCs are altered in autoimmune-prone mice such as non-obese diabetic (NOD) mice, a model for type 1 diabetes. We observed altered DC responses in NOD mice to innate immune stimuli such as CpG, which may alter the ability of these DCs to induce tolerance. In addition, determining which antigen presenting cell populations are capable of inducing tolerance in the context of chronic autoimmunity is critical for antigen-specific treatments for autoimmune disease. We recently reported that in NOD mice, DEC-205+ DCs induce effector responses, not tolerance, in autoreactive CD4+ T cells. We now show that DCIR2+ DCs, distinct from DEC-205+ DCs, can be tolerogenic in NOD mice. Beta cell antigen targeted via chimeric antibody to DCIR2+ DCs delays disease induction in a transfer model of type 1 diabetes. DCIR2+ DC stimulation induces partial deletion of autoreactive CD4+ T cells and remaining cells are unable to produce IFN-γ. These disparate T cell outcomes after targeting antigen to DEC-205+ or DCIR2+ DCs are underpinned by differential gene expression in antigen-specific T cells early after stimulation. In conclusion, we have characterized defects in DCs in NOD mice, and the resulting altered T cell responses with the goal of identifying ways to induce tolerance even in the context of autoimmunity.
The transcription factor Bcl-3 represses the function of regulatory T cells leading to the development of colitis (BA15P.223)
Authors: Reissig Sonja , Nikolaev Alexey , Waisman Ari , Hoevelmeyer Nadine
Published: January 2023

The proto-oncogene Bcl-3 (B cell leukemia-3) is a member of the IκB family of NF-κB inhibitors and was originally identified as a component of a chromosomal rearrangement in some B cell lymphomas. In contrast to other IκB proteins, Bcl-3 associates with p50 and p52 homodimers and is predominantly a nuclear protein involved in regulating nuclear NF-κB activity. To understand more about the effects of Bcl-3 on T cells, we have generated a new mouse model, where Bcl-3 is overexpressed specifically in T cells. We could show that mice overexpressing Bcl-3 solely in T cells developed spontaneous colitis accompanied by a massive cell infiltration into the colon. Interestingly, by analyzing regulatory T cells we could demonstrate that these cells were not able to suppress an inflammatory response. Due to the suppressive dysfunction, Tregs of Bcl-3 overexpressing mice expressed less CD25, Foxp3 as well as less of the co-inhibitory molecule CTLA-4. Hence, the loss of suppression of Bcl-3 overexpressing Tregs might result in the development of severe spontaneous colitis. Our data emphasize a novel pathway in the maintenance of viability and suppressive function of Tregs in the gut that critically involves Bcl-3 as a negative regulator, qualifying this protein as an interesting pharmacological target for the treatment of IBD.
Autocrine il-6 drives skin-derived mesenchymal stem cell trafficking via regulating voltage-gated ca2+ channels (CAM5P.243)
Authors: Wang Honglin , Ke Fang , Zhang Lingyun , Liu Zhaoyuan , Yan Sha
Published: January 2023

Mesenchymal stem cells (MSCs) have demonstrated promising therapeutic potential for a variety of diseases including autoimmune disorders. A fundamental requirement for MSC-mediated in vivo immunosuppression is their effective trafficking. However the mechanism underlying MSC trafficking remains elusive. Here we report that skin-derived MSCs (S-MSCs) secrete high levels of interleukin-6 (IL-6). Disruption of the il6 or its signaling transducer gp130 blocks voltage-gated Ca2+ channels (VGCC) critically required for cell contraction involved in the sequential adhesion and de-adhesion events during S-MSC migration. IL-6 directly regulates CaV2.3 encoded by cacna1e of R-type Ca2+ channels through the JAK3/STAT3 pathway. Deletion of il6 or silencing CaV2.3 gene leads to a severe defect in S-MSC’s trafficking and immunosuppressive function in vivo. Thus, this unexpected requirement of autocrine IL-6 for activating CaV2.3 Ca2+ channels uncovers a previously unrecognized link between the IL-6 signaling and the VGCC, and provides novel mechanistic insights for the trafficking and immunomodulatory activities of S-MSCs.
Effect of adiponectin on a disintegrin and metalloproteinase with thrombospondin motifs-9 gene expression in human chondrocytes (CCR5P.257)
Authors: Yaykasli Kursat , Dogan Mustafa , Hatipoglu Omer , Yaykasli Emine , Kaya Ertugrul , Ozsahin Mustafa , Uslu Mustafa
Published: January 2023

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) is a family of peptidases. They comprise 19 members, and have several vital functions in organism. ADAMTS-9 with aggrecanolytic activity is responsible for the degradation of the articular cartilage components like aggrecan. Adiponectin is the most abundantly secreted adipokines (adipocytokines), and the characteristic of adiponectin is controversial. It was assumed that adiponectin has anti-inflammatory effect. However, recent studies showed the inflammatory features of adiponectin. It was aimed to evaluate the effect of adiponectin on ADAMTS-9 gene expression in human chondrocytes. Human articular chondrocytes were cultured and exposed by adiponectin at 1, 4, 8 and 12 µg/ml doses. At the end of the incubation, total RNA was reverse-transcribed by random primer. The expression levels of the ADAMTS-9 and β-actin genes were determined using real-time polymerase chain reaction. The ADAMTS-9 gene expression was found to increase after adiponectin incubation at 12 µg/ml dose. The interleukin-1β induced ADAMTS-9 gene up-regulation and the increased serum level of ADAMTS-9 in patients with osteoarthritis (OA) were reported before. Similarly, the putative involvement of adiponectin in OA and rheumatoid arthritis (RA) was demonstrated. Together with these findings, our results suggesting that adiponectin may involve in the degradation of aggrecan by increasing ADAMTS-9 gene expression.
IL-10 and IL-24 expression is regulated by distinct pathways in macrophages and NK cells (CCR5P.252)
Authors: Dabitao Djeneba , Christian Hedrich , Huska Jason , Bream Jay
Published: January 2023

Homologous gene in clusters can be coordinately regulated by shared genomic elements to synchronize biologic functions. Within the Il10 locus which includes Il19, Il20 and Il24, only IL-10 and IL-24 are preferentially expressed by Th2 cells. The extent to which IL-24 is regulated in other IL-10-producing cell types and its role in immunity remains unclear. To determine if these genes share regulatory pathways, we examined IL-24 expression in macrophages and NK cells which are key sources of IL-10. In macrophages, maximal LPS-induced IL-24 expression requires IL-4 and Stat6 while IL-10 induction is largely independent of Stat6. Likewise, in NK cells, IL-10 expression is independent of IL-4 but IL-24 is strongly induced by IL-4 in combination with IL-2 and is dependent on Stat6. We identified 4 putative Stat-binding elements in the Il24 gene and found that each site binds IL-4-induced Stat6 in macrophages and NK cells. Interestingly, these sites also bound Stat4 in IL-12-stimulated NK cells but not in macrophages. In NK cells, IL-24 expression required type I IFN signaling regardless of the stimulation while IL-10 expression required type I IFNs only in macrophages. Histone modifications in Il24 were dependent on IL-4/Stat6 in macrophages but not in NK cells. Our results indicate that although IL-10 and IL-24 are co-expressed in macrophages and NK cells, they are governed by distinct signaling pathways suggesting they are unlikely to share genomic regulatory elements.
Crystal structures of FasL:DcR3 and LIGHT:DcR3 complexes reveal the molecular basis for the broad specificity of DcR3 (CCR5P.255)
Authors: Liu Weifeng , Zhan Chenyang , Nathenson Stanley , Almo Steven
Published: January 2023

Decoy receptor 3 (DcR3) is a secreted TNF receptor discovered in human beings but not in mice. DcR3 is able to neutralize three TNF ligands, LIGHT, FasL and TL1A and further modulate the immune response. For example, neutralization of LIGHT by DcR3 can manipulate the co-stimulatory signal triggered by LIGHT:HVEM interaction or the cell death signal resulted from LIGHT:LTβR interaction. Upon ligation with FasL, DcR3 can block the cell apoptosis pathway commenced by FasL:Fas interaction. Besides, DcR3 binds to TL1A, which can interrupt its interaction with DR3 to boost T cell immunity or induce cell apoptosis in different cell types. Although the remarkable ability of DcR3 to neutralize three different ligands has already been realized, the mechanisms which support the broad specificity of DcR3 remain to be fully defined. Here I present the novel crystal structures of the FasL:DcR3 and LIGHT:DcR3 complexes. Along with the TL1A:DcR3 structure solved in our lab previously, the whole set of the structures of TNF:TNFR complexes engaged by DcR3 was solved. Our results show that the loops of the ligands are the major determinants for the recognition of DcR3. By appropriate conformational alterations of the flexible loops but not the rigid beta-strand sheets, the diverse residues on the loops of different ligands are able to capture the cognate residues on DcR3 by polar contacts and hydrophobic interactions, thus enable the different TNF ligands to bind to the same DcR3.
Efficacy of selective RAR-related orphan receptor gamma (RORγ) inverse agonists in psoriasis and asthma models (CCR6P.278)
Authors: Nellore Kavitha , Bejugam Mallesham , Damarla Ravi Krishna Babu , Hegde Pragathi , Dodheri Samiulla , Mukherjee Subhendu , Rao Narasimha , Lakshminarasimhan Anirudha , Ramanathan Anuradha , Balasubramanian Wesley , Mattegunta Suresh Babu , Mahalingam Natarajan , Samajdar Susanta , Subramanya Hosahalli
Published: January 2023

Th17 cells play a key pro-inflammatory role in a variety of autoimmune diseases. The nuclear hormone receptor RORγ controls the differentiation of Th17 cells and expression of IL-17. RORγ inverse agonists were designed using a combination of approaches including structure and knowledge based methods. Compounds were screened in a RORγ radio-ligand binding assay using 3H 25-Hydroxycholesterol, as well as in a cell based reporter assay to demonstrate inverse agonism. Compounds from multiple structural classes have been identified, with IC50 in the range of 5 - 500 nM in binding assay. Co-crystal structures of RORγ in complex with known as well as in-house compounds were utilized to optimize the lead compounds. Compounds from the lead series demonstrated good activity (< 500 nM) in reporter assay. Several compounds demonstrated > 10 fold selectivity against RORα as well as other nuclear receptors. Compounds from multiple series have shown significant inhibition of IL-17 release from differentiated mouse and human Th17 cells. Lead compounds have shown good pharmacokinetic properties in mice. Efficacy of lead compounds have been demonstrated in IMQ induced Psoriasis model as well as in intranasal OVA/LPS induced asthma model. In summary, we have identified structurally diverse small molecule inverse agonists of RORγ and have demonstrated efficacy in Th17 mediated efficacy models.
Acute alcohol intoxication suppresses early Il-17 response during pneumococcal pneumonia. (CCR6P.273)
Authors: Trevejo-Nunez Giraldina , Chen Kong , Kolls Jay
Published: January 2023

In animal models, acute alcohol intoxication suppresses the host immune responses against S. pneumoniae. Il-17 plays essential roles in host defense against extracellular pathogens including S. pneumoniae. We demonstrated that Il-17R signaling is critical for the clearance of S. pneumoniae by blocking the Il-17 receptor with monoclonal antibody. We thus hypothesize that alcohol impairs pneumococcal lung clearance by affecting Il-17 production and Il-17R related genes in the lung. We used a model of acute alcohol intoxication in mice followed by S. pneumoniae pneumonia. Alcohol and control group had similar bacterial burden in the lungs at 5 hours, however, the former group failed to clear pneumococci at 18h. Il-17a production in the lung was decreased in the alcohol group at 5 hours, suggesting that the impaired clearance is, at least in part, due to the suppression of Il-17 production. Gene expression analyses showed that both Il17a and Il17f transcripts were down-regulated in alcohol mice, suggesting the suppression is at transcriptional level. This notion is supported by decreased expression of Il-17 upstream genes such as Il1b and Il23. Consequently, downstream genes such as Csf3 and Cxcl1, which are critical in mobilizing and recruiting bacterial phagocytes, were down regulated at 5 hours compared to control mice. Taken together, this study provides a novel mechanism by which alcohol impairs host defense against bacterial infection.