bluuebunny/crossref_metadata_2025 huggingface dataset:
A case for research consortia to accelerate vaccine candidate discovery: identification of a stable antigenic HIV-1 Clade A Env candidate (P6370)
Authors: Fliing Steven , Hoffenberg Simon , Caulfield Michael , Parks Christopher , King C. , Burton Dennis , Ward Andrew , Wilson Ian , Moore John , Koff Wayne
Published: January 2023

IAVI’s NAC provides a multi-disciplinary platform to decipher the interaction of bNAbs with HIV Env and accelerate translation to development of vaccines capable of eliciting potent neutralizing abs. Recent successes highlight the value of a consortium to expedite progress. IAVI initiated Protocol G to identify neutralizing sera from HIV-1 infected patients. Using these samples, a cross-disciplinary team discovered abs with unprecedented potency against HIV. PG9 was isolated from a subject infected with HIV-1 clade A. Consortium members reasoned that Env sequences from the clade A infected donor could lead to immunogen discovery. Using bioinformatics, MRCA sequence was aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database and BG505 was identified with the highest homology. Soluble BG505 retains features of native trimer. While native Env trimer is the target for bnAbs, its instability impedes efforts to develop recombinant Env. Because BG505 retains features of native trimer, it might be optimal for immunogen design. NAC partners report construction of a soluble trimer from BG505 that binds PG9 with high affinity and show it is uniquely stable. Formulations of gp120 and trimers are under analysis for immunogenicity. Challenges remain in development including analysis of structure, immunogenicity, ab responses elicited; and production for human testing. A consortium approach of multidisciplinary experience will be required to expedite advancement of vaccine concepts.
Measuring immunological synapse and actin organization using the FlowSight imaging flow cytometer (P5018)
Authors: Pugsley Haley , Kong Raymond , Hall Brian , Vaidyanathan Shobana , Basiji David , Friend Sherree
Published: January 2023

Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse (IS) after T cell receptor stimulation are required for the antigen-specific response. One way to measure an IS is by fluorescently labeling the molecules that have been recruited to the synapse and imaging via microscopy. However, IS are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis FlowSight imaging flow cytometry platform to objectively collect imagery of large numbers of cells to assess the percentage of T cells involved in an organized IS. In this study, Raji B cells were loaded with Staphylococcal enterotoxin B (SEB) to make APCs. The SEB-loaded APCs were incubated with human T cells to create T cell-APC conjugates. In addition, we investigated the inhibition of actin by cytochalasin D and how that affects the T cell-APC conjugates. The T cells, APCs and conjugates were fluorescently labeled. Using the FlowSight imaging flow cytometry platform we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized IS in an objective and statistically significant manner.
Plasma membrane interactions that determine presentation of antigens by Antigen-presenting cells. (P5022)
Authors: Hari Aswin , Shi Yan
Published: January 2023

Antigen presentation is a critical process in host defense for initiating adaptive immune responses. The APC loads peptides derived from antigens onto major histocompatibility complex (MHC) molecules and exports them to the surface for T cells sampling. If the primary interaction between an APC and T cell is successful, i.e. the TCR binds an appropriate MHC-peptide complex, this leads to an adaptive immune response. Peptide antigens are presented on two types of MHC molecules, MHC I and MHC II via several distinct processing pathways. In turn, the MHC molecule dictates which type of T cell can be activated and thereby the nature of the adaptive immune response. Therefore, antigen loading onto MHC molecules is a critical determinant of the adaptive immune responses. Exogenous antigens can be presented on MHC I and II molecules. Antigen processing mechanisms have been elucidated in great detail, but the fundamental reason behind why an antigen is directed into a specific pathway is not known. One of the foremost aims for this project is to determine why particulate antigens are presented more often on MHC I and why soluble antigens are more efficiently presented on MHC II, by deciphering the interactions of plasma membrane with particulate versus soluble antigen. This fundamental sensing mechanism of the human body when elucidated can help in generating an antiviral response in diseases like HIV and in field of preventative medicine.
X-ray crystal structure of HLA-DQ2 in complex with CLIP1 and CLIP2 (P5026)
Authors: Nguyen Thanh Binh , Jayaraman Priya , Bergseng Elin , Madhusudhan Mallur Srivatsan , Sollid Ludvig M. , Kim Chu-Young
Published: January 2023

The class II MHC protein HLA-DQ2 is strongly associated with type 1 diabetes and celiac disease. CLIP (MHC class II-associated invariant chain peptide) occupies the peptide binding groove of all newly synthesized class II MHC proteins until HLA-DM replaces it with an antigen peptide. We have determined the atomic structure of HLA-DQ2-CLIP1 and HLA-DQ2-CLIP2 complex at 2.8 Ang and 2.2 Ang, respectively. Our crystal structure shows that MRMATPLLM of CLIP1 occupies the P1 to P9 pockets of HLA-DQ2. For CLIP2, PLLMQALPM occupies the P1 to P9 pockets of HLA-DQ2. We present our analysis of the MHC-peptide interaction in these two structures, and we highlight the structural similarities and differences compared to other class II MHC-CLIP complex structures reported to date.
Unexpected side reaction of lysine and arginine side chains preceding a photocleavable group in MHC-II UV-cleavable peptides. (P5028)
Authors: Negroni Maria , Stern Lawrence
Published: January 2023

MHC class II molecules (MHCII) present peptides to CD4+ T cells, and HLA-DM (DM) catalyzes peptide exchange favoring binding of high affinity peptides. The effects of DM on peptide association and dissociation kinetics are not yet clear. In the absence of peptide, DR1 (a MHCII) is in equilibrium between inactive and active conformations. In order to understand the kinetics of this process and how it is affected by DM, we wanted to generate DR1 in the active conformation using a peptide carrying the photocleavable 3-amino-3-(2-nitrophenyl)-propionic acid residue, an approach previously used for other MHCI and MHCII-binding peptides. We used well-characterized variants of a peptide derived from influenza hemagglutinin (HA). The variants bind to DR1 with an affinity similar to the parent HA peptide. Surprisingly, photolysis of peptides did not result in peptide release. Mass spectrometry showed that the main photoproduct had a mass decrease of 2Da, resulting from photocleavage followed by unexpected religation to a rearranged peptide that can still bind DR1. Replacement of peptide lysine (or arginine) residues by methionine yielded the expected cleavage products. The methionine substituted peptides can be used for further studies on DR1-peptide binding kinetics and the effect of DM on the different rates of the reaction. These studies provide a caveat to routine use of photocleavable peptides in MHC-peptide exchange studies.
Substrate specificity of human ERAP1 haplotypes in vivo identify three functional classes and multiple fine specificity (P5034)
Authors: Reeves Emma , Edwards Christopher , Elliott Tim , James Edward
Published: January 2023

Recent genome wide association studies have identified single nucleotide polymorphisms within Endoplasmic Reticulum Aminopeptidase1 (ERAP1) as being associated with autoimmune diseases, cervical carcinoma and hypertension. ERAP1 performs a key final step in antigen processing, trimming N-terminally extended peptides to the final epitope for presentation by MHC I molecules. Here we show that ERAP1 sequenced from individuals is polymorphic, with multiple haplotypes identified. Characterization of the amino acid trimming capacity of the identified haplotypes reveal distinct profiles that correlate with the SNPs they possess. In addition, the properties of the amino acids preferentially trimmed vary between haplotypes. RP-HPLC and mass spectrometry analysis allows classification of the ERAP1 haplotypes into three functional groups; those that have a predominantly i) Normal, ii) Hypo or iii) Hyper-functional activity. These results indicate that the repertoire of peptides presented at the cell surface depends, not only on the MHC I, but also on the ERAP1 expressed within an individual. This therefore has important implications for predisposition to disease, the ability to combat cancer and infection, and also vaccine efficacy.
The impact of conformational entropy on epitope selection by MHC class II (P5010)
Authors: Ferrante Andrea
Published: January 2023

The interaction between pathogen-derived peptides and molecules of the Class II Major Histocompatibility Complex (MHCII) is a thermodynamically cooperative process, characterized by the conformational flexibility of the reactants. However, the contribution of conformational entropy to epitope selection by MHCII needs to be elucidated. Here, cycle-mutated peptides derived from influenza H3 HA306-319 are analyzed as they interact with the human MHCII allele HLA-DR1 (DR1) by isothermal titration calorimetry, fluorescence polarization, circular dichroism (CD) and molecular dynamics (MD) simulation. Peptide ligand to DR1 displays enthalpy/entropy compensation that results in similar free energy of binding, even though individual enthalpy and entropy terms change significantly across the series. CD analysis indicates that complexes, whose entropic contribution to the binding is the greatest, feature spectra that are consistent with an incomplete conversion to the compact form. These distinct binding modes correlate with the stability of the complex during DM-mediated peptide release and are supported by MD simulations. These results indicate that, at the molecular level, binding modes and thermodynamic binding signatures can be very different even for closely related peptides, and these signatures have a significant impact on the outcome of the epitope selection process.
Immunodeficiency and autoimmunity in H2-O deficient mice (P5016)
Authors: Chen Xinjian , Jensen Peter , Gu Yapeng
Published: January 2023

HLA-DO/H2-O is a highly conserved non-polymorphic MHC class II-like molecule whose physiological function remains unknown. The cell maturation-dependent DO expression in B-lymphocytes and DCs suggests a role H2-O in promoting presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. We report herein that H2-O-/- mice spontaneously develop high titers of IgG2a/c antinuclear antibodies (ANA) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1-/- mice with T and B cells from H2-O-/- or WT mice demonstrated that production of ANAs requires participation of CD4+ T cells from H2-O-/- mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O-/- mice exhibit a delayed generation of humoral immunity to model antigens (OVA and KLH), affecting all major T-dependent Ig classes including IgG2a/c. Consistently, presentation of all the natural Ag epitopes by H2-O-/- APC was inefficient as compared to WT APC. Thus, H2-O promotes immunity towards exogenous Ag while preventing autoimmunity. We propose that H2-O, through spatially and temporally inhibiting H2-M, enhances presentation of exogenous antigen by preventing nascent/newly-synthesized class II molecules from forming stable complexes with endogenous self-peptides.
The constant region affects antigen binding of anti-DNA antibodies by altering secondary structure (P4002)
Authors: Putterman Chaim , Xia Yumin , Janda Alena , Eryilmaz Ertan , Casadevall Arturo
Published: January 2023

We previously demonstrated an important role of the constant (C) region in the pathogenicity of anti-DNA antibodies (Ab) through modulation of immunoglobulin affinity and specificity. However, the mechanisms by which the C region affects the binding of autoantibodies to nuclear antigens (Ag) have yet to be elucidated. From a parent IgG3 anti-nuclear Ab, PL9-11, we generated IgG1, IgG2b, and IgG2a variants that share identical variable regions but that differ in their C region. The affinity of these Abs for histone antigens was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine the wavelength shift of the Ab panel upon the addition of DNA and histone Ags. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure of the PL9-11 panel corresponding to Ab-Ag interactions. SPR analysis revealed significant differences of histone 2A/2B binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the Ab isotype, while increasing salt concentrations lowered KD values. Circular dichroism analysis determined that changes in Ab secondary structure content differed between the IgG isotypes upon Ag binding. In conclusion, the Ag-binding affinity of anti-nuclear Abs is dependent on the particular C region. Ag binding effects on Trp fluorescence, as well as secondary structure shifts, were also C region dependent. Alteration of secondary structures influenced by C regions may explain differences in fine specificity of anti-DNA Abs between Abs with similar variable regions, as well as cross-reactivity of anti-DNA Abs with non-DNA Ags.
The blood—brain barrier is breached in murine neuropsychiatric Systemic Lupus Erythematosus (P4004)
Authors: Wen Jing , Wagshul Mark , Stock Ariel , Fleysher Roman , Branch Craig , Putterman Chaim
Published: January 2023

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the earliest manifestations in human SLE, and a major cause of morbidity and mortality in this disease. While the pathways underlying NPSLE are not fully understood, blood brain barrier (BBB) disruption and altered brain perfusion have been postulated to contribute to the pathogenesis. The purpose of this study was to evaluate BBB integrity, macromolecule content, and other indicators of neurological damage in MRL-lpr/lpr mice which spontaneously develop NPSLE, compared to the background control strain MRL/+. Female MRL-lpr/lpr and MRL/+ mice were imaged by MRI at 9-10 weeks of age, at which time major neuropsychiatric symptoms including depression and cognitive dysfunction were already present. Dynamic contrast enhancement MRI indicated increased gadolinium uptake in MRL/lpr mice in all regions of the brain, with the largest differences visualized in the hypothalamus where these changes were significant. Furthemore, MRL/lpr mice displayed significantly higher concentrations of albumin, total immunoglobulin, and antibodies to double stranded DNA in their cerebrospinal fluid. In contrast, whole brain analysis of magnetic transfer contrast and quantitative T2 imaging indicated no significant differences between the MRL-lpr/lpr and MRL/+ mice. Thus, a compromised blood brain barrier (particularly in specific regions) in lupus prone MRL/lpr mice may allow for the passage of circulating autoantibodies from the serum into the brain, and induction of some of the neuropsychiatric abnormalities observed in this mouse strain. Preventing the regional breakdown in barrier integrity may be a novel approach to treatment of neuropsychiatric involvement in SLE.
Understanding lupus pathology in FcγRIIB-/-yaa mice: autoantibodies and autoantigens (P4008)
Authors: Kole Hemanta , Scott Bethany , Bolland Silvia
Published: January 2023

FcγRIIB knock-out mice in B6 background develop a lupus like autoimmune disease. With the addition of the yaa (Y-chromosome autoimmune accelerator) gene, pathogenecity in these male mice is significantly enhanced and autoantibodies switch from nuclear to nucleolar specificity. High titer of λ-light chains immunoglobulin, and anti-RNA antibodies were found in R2-/-yaa serum. Also, R2-/-yaa serum selectively bound 5 major nuclear proteins compared to R2-/- serum. To characterize the autoantibodies produced by R2-/-yaa mice and the nucleolar antigens that react with these antibodies, we have produced hybridomas. Hybridoma H526 produced anti-nucleolar IgG2c-λ antibodies. The antibody binds RNA, a synthetic polyribonucleotide poly (GC)20 and a RNA-associated nucleolar protein, nucleolin. R2-/-yaa serum also found to cross-react with poly (GC)20, nucleolin and also with poly ADP-ribose (PAR). Simultaneous presence of nucleolin and PAR autoantibodies has been found to be associated with lupus. Further characterization of nucleolin modification and lupus development is in progress.
Sex differences in the effect of vitamin D on inflammatory heart disease: protective in women but damaging in men (P3069)
Authors: Stafford Katelyn , Cooper Leslie , Douglass Erika , Brandt Jessica , Bucek Adriana , Coronado Michael , Kew Richard , Fairweather DeLisa
Published: January 2023

An estimated 1 billion people worldwide have deficient or insufficient levels of vitamin D (VitD), while roughly 25% of individuals in the US are reported to have inadequate VitD levels. Considerable evidence indicates that VitD deficiency is associated with an increased risk of cardiovascular disease (CVD), yet it remains unclear whether low VitD is simply a biomarker of CVD or has a true pathologic role. The role of VitD deficiency in the pathogenesis of inflammatory myocarditis is unknown- a disease that is higher in men. We found that only 20% of myocarditis patients had low VitD levels. When we examined VitD receptor (VDR) deficient mice in a model of viral myocarditis, we found that VDR decreased myocarditis in females (p=0.007) but increased inflammation in males (p=0.006). Examination of cytokines in the heart found that VDR-/- male mice had significantly reduced IFN-γ and IL-1β and increased IL-17A, but no significant differences were found in females. Comparison of microarray data during myocarditis to published VitD response element genes revealed around 200 genes associated with proinflammatory (e.g. caspase-1) and profibrotic (e.g. TGF-β1, MMP) responses were increased in WT male mice, which was confirmed by qRT-PCR in VDR-/- mice. Our data confirms that VitD through the VDR promotes myocardial inflammation in males, providing a mechanism for how low VitD levels in men increase myocarditis. This work was funded by NIH R01 HL087033 and HL111938.
CD36 expression in chondrocytes is involve in the clearance of apoptotic cells (P3096)
Authors: González Elena , Ortiz-Navarrete Vianney
Published: January 2023

Osteoarthritis (OA) is a chronic-degenerative arthropathy with high incidence and prevalence in which cartilage is destroyed due to extracellular matrix erosion (collagen and proteoglycan’s loss) and death of chondrocytes mainly by chondroptosis (program cell death) that could be clearance by neighboring chondrocytes. In order to confirm chondrocyte’s phagocytic capacity we performed a phagocytosis assay with Salmonella thyphimurium-GFP (moi 30) and we observed that after 30 min chondrocytes were able to phagocytose bacteria. We also found that 60% of chondrocytes, either from normal or OA tissue, were able to phagocyte 2 or more apoptotic chondrocytes. In addition, we found that less than the 5% of the chondrocytes express CD36 and the percentage of CD36 positive cells increases after the contact with apopotic cell. These data confirm that chondrocytes have the capacity to clearance apoptotic cells and that CD36 is involve in this process. Suggesting that chondrocytes could be able to eliminate apoptotic chondrocytes in the tissue in order to maintain homeostasis cartilage.
B lymphocytes regulate a pro-inflammatory T cell balance in human Type 2 diabetes (P3101)
Authors: DeFuria Jason , Jagannathan-Bogdan Madhu , Kuchibhatla Ramya , McDonnell Marie , Apovian Caroline , Nikolajczyk Barbara , Denis Gerald
Published: January 2023

Lymphocytes play key roles in the chronic inflammation critical for T2D pathogenesis. We have shown T2D patients have an elevated ratio of pro- to anti-inflammatory T cells, and B cells that produce a pro-inflammatory cytokine profile. Thus lymphocytes promote T2D-associated inflammation. Numerous studies implicate the pro-inflammatory CD4+ T cell balance in T2D pathogenesis, but mechanisms that underlie elevated CD4+ T cell inflammation are poorly understood. We explored the possibility that the T2D-associated changes we identified in B cell function regulate T cell inflammation. We show that B cells control the T2D-associated increase in Th17-mediated inflammation in T2D patients and in obese/insulin resistant mice. Surprisingly, the disease-associated ability of B cells to regulate T cell function is contact-dependent, despite evidence that B cell cytokines hyper-secreted in T2D patients activate T cells. In contrast, elevated activation of Th1 cytokines is B cell-independent. We conclude that both T cell-intrinsic and T cell-extrinsic (B cell-dependent) changes regulate T cell inflammation in T2D. These data indicate that B cell depletion may partially curb T2D-associated T cell inflammation, and thus disease pathogenesis; however, combinatorial treatments aimed at multiple inflammatory axes may be required for favorable clinical outcomes.
A2B adenosine receptors inhibit obesity-induced adipose tissue inflammation (P3109)
Authors: Csoka Balazs , Koscso Balazs , Toro Gabor , Kokai Endre , Nemeth Zoltan , Bai Peter , Hasko Gyorgy
Published: January 2023

Obesity promotes adipose tissue inflammation, which in turn causes insulin resistance in target organs by dysregulation of adipokine release and by increased classical and decreased alternative macrophage activation. Because A2B adenosine receptors are important regulators of macrophage activation, we examined the role of A2B adenosine receptors in diet-induced inflammation and insulin resistance. A2B receptor deletion impaired glucose metabolism in mice fed chow but not high fat diet. This impaired glucose homeostasis in A2B receptor deficient mice was paralleled by increased classical macrophage activation and inhibited alternative macrophage activation. The expression of alternative macrophage activation-specific transcriptions factors, including CCAAT/enhancer binding protein β, interferon regulatory factor 4, and peroxisome proliferator-activated receptor γ was decreased in A2B receptor deficient mice. Furthermore, in in vitro studies we found that activation of A2B receptors decreased free fatty acid-induced classical macrophage activation and increased IL-4-induced alternative macrophage activation. Therefore, therapeutic strategies targeting A2A and A2B receptors hold promise for preventing and/or treating obesity-related metabolic abnormalities.
The role of Ikaros in adipocyte differentiation of MDI-stimulated 3T3-L1 cells (P3111)
Authors: Park Bongkyun , Pyo Suhkneung
Published: January 2023

Adipogenesis is a tightly regulated cellular differentiation process, in which the preadipocytes are transformed into differentiated adipocyte cells. Ikaros has been known as a cell cycle regulator on lymphoid and myeloid cell differentiation. However, the precise role of Ikaros in adipogenesis is still unclear. We investigated the role of Ikaros in vitro in adipocyte differentiation of preadipocyte cell line, 3T3-L1. Western blot analysis showed that Ikaros expression was significantly increased by 2 days in MDI-treated 3T3-L1 cells, whereas the expression of c-Myc was markedly decreased after 2 days. Oil red O staining revealed that inhibition of Ikaros by siRNA suppressed lipid droplet accumulation. In addition, knockdown of Ikaros resulted in re-entry of cell cycle progression from G1 phase as well as inhibition of p21 and p27 expression, cell cycle arrest proteins. In contrast, c-Myc expression was increased. Immunoprecipitation analysis demonstrated that the interaction of Ikaros and c-Myc was significantly enhanced in MDI-treated 3T3-L1 cells, suggesting that the interaction between Ikaros and c-Myc is required for adipogenesis. Taken together, our data suggest that Ikaros may play a critical role in adipocyte differentiation by negatively regulating c-Myc expression.
Continuous immunosurveillance of oligodendrocyte antigens leads to the generation of effector T-cell pools that might contribute to CNS autoimmune disease (P3123)
Authors: Fabry Zsuzsanna , Harris Melissa , Hulseberg Paul , Ling Chagying , Karman Jozsef , Clarkson Benjamin , Sandor Adam , Nagy Andras , Sandor Matyas
Published: January 2023

Immunosurveillance of antigens from the CNS is tightly regulated in order to avoid autoimmune disease, such as multiple sclerosis. To test how CNS antigens are recognized by the peripheral immune system, we created transgenic mice having constitutive or inducible expression of EGFP-fused model neoantigens in myelinating glial cells. We examined the level of systemic priming to CNS neoantigens and compared it to priming from non-immune privileged gut tissue. We report here that the level of the EGFP-triple peptide in both the constitutive and inducible models is sufficiently high in the CNS to induce neoantigen-specific T cell priming in the periphery; however, there is no recruitment of these cells to the CNS. Immunosurveillance of CNS antigens in the peripheral immune tissues is enhanced during neuroinflammation, during which time activated neoantigen-specific T cells accumulate in the CNS. These data suggest that CNS antigens are surveilled in the periphery, leading to the generation of effector T cell pools. Additional inflammatory signals from the CNS induce the migration of effector T cells, which form microclusters with dendritic cells and autophagic oligodendrocytes that leads to the amplification of CNS inflammation. Increased immunosurveillance and inflammation might contribute to the chronic phase of CNS autoimmune disease. These new models can help us dissect the mechanisms of disease initiation and maintenance that is of clinical importance.
CD8+ T cell exhaustion in sarcoidosis contributes to disease severity (P3137)
Authors: Richter Kyra
Published: January 2023

Sarcoidosis is a multisystem granulomatous disease of unknown etiology. Though patients develop inflammation at active sites of involvement, an anergic phenotype dominates peripherally. Recent reports suggest that dysfunction in the CD4+ T cell compartment contributes to the anergic responses, yet little is known about the role of CD8+ T cells. Chronic inflammation studies demonstrate that persistent antigen stimulation leads to CD8+ T cell exhaustion. Furthermore, an increase in CD8+ exhausted T cells has been shown to correlate with disease severity in multiple chronic inflammatory diseases. Therefore we hypothesized that CD8+ T cell exhaustion contributes to sarcoidosis disease severity. We report that expression of inhibitory receptors including PD-1 are upregulated on CD8+ T cells in sarcoidosis subjects with active disease, whereas cytokine production, proliferation, and cytotoxicity are impaired. In addition, we assessed CD8+ T cell function during active disease and upon disease improvement. Improvement of disease correlated with reversal of the exhausted phenotype. These results demonstrate that CD8+ T cell exhaustion contributes to immune dysregulation in sarcoidosis. Importantly, restoration of CD8+ T cell function results in disease resolution indicating that CD8+ T cell exhaustion may correlate with disease severity and thus, exhausted CD8+ T cells may be a potential biomarker to predict disease outcome.
Lack of macrophage/monocyte derived β-endorphin contributes toward sensations of pain/discomfort in Irritable Bowel Syndrome (P3153)
Authors: Hughes Patrick , Moretta Melissa , Lim Amanda , Grasby Dallas , Bird Daniel , Bampton Peter , Andrews Jane , Zola Heddy , Krumbiegel Doreen
Published: January 2023

Introduction: Neuroimmune interactions are increasingly implicated in Irritable Bowel Syndrome (IBS). Immune derived β-endorphin (β-end) inhibits colonic sensory afferents in health (Hughes et. al. Gut 2012), but little is known of the immunocyte responsible for β-end secretion in humans and whether this is altered in IBS patients. Methods: β-end and TNF-α staining was assessed by flow cytometry analysis of CD4, CD8, CD19, CD14 or CD33 labelled PBMC that were unstimulated or stimulated with PMA/ionomycin or LPS and compared between 9 healthy and 21 IBS patients. β-end immunoreactivity was assessed in CD3 or CD68 labelled colonic biopsies from 7 healthy and 10 IBS patients. Results: In PBMC β-end was observed in unstimulated monocytes and LPS stimulated macrophages, but not unstimulated or PMA/ionomycin stimulated T or B cells, and was decreased in IBS patients relative to healthy subjects. Most β-end stained cells in colonic biopsies were CD68 positive, while only a low proportion were CD3 positive, and IBS patients had decreased numbers of CD68+ve cells. Intracellular levels of β-end negatively correlated with TNF-α in monocytes, and positively correlated with macrophage numbers in colonic biopsies. Discussion: Monocyte / macrophages are the predominant immune derived source of β-end in human subjects, and this is lower in IBS patients. The loss of immune derived β-end potentially contributes toward the sensations of pain / discomfort in IBS.
Human mast cells secrete mitochondrial DNA that has inflammatory actions, is increased in the serum of children with autism, and is inhibited by luteolin (P3155)
Authors: Theoharides Theoharis , Zhang Bodi , Asadi Shahrzad , Weng Zuyi
Published: January 2023

Mast cells participate through cytokine release in acquired and innate immunity, as well as inflammation involved in the pathogenesis of autism, atopic dermatitis and psoriasis, but the trigger is unknown. The peptide neurotensin (NT), found in the brain, gut and skin, is significantly increased in the serum of autistic children (n=20; 3 years old) along with significantly more mitochondrial (mt)DNA for Cytochrome B (p=0.0002), as compared to normally developing controls (n=12); these findings may characterize an autistic endophenotype since over 50% of patients have “allergic-like” symptoms and brain inflammation. Here we report that human LAD2 cells stimulated by NT and other triggers secrete mt components, including mtDNA, extracelluarly without cell death. Purified LAD2 mt components further trigger release of histamine, IL-1β, IL-8, PGD2 and TNF from mast cells, as well as vascular endothelial growth factor (VEGF) from human keratinocytes and human microvascular endothelial cells, and IL-6 from human microglial cells. When mtDNA from LAD2 cells was injected ip in rats, it was detected in rat serum within 4 hrs indicating that extravascular mtDNA could enter the systemic circulation. Pretreatment with the natural flavonoid luteolin inhibited mediator secretion in vitro. Such mt components secreted from stimulated live mast cells may act as “innate pathogens” contributing to the pathogenesis of inflammatory diseases and may serve as targets for novel treatments.
Type III hereditary angioedema: diagnostic and management challenges (P3173)
Authors: Issa-El-Khoury Karine , McCusker Christine , Alizadehfar Reza , Lejtenyi Christine , Ben-Shoshan Moshe
Published: January 2023

INTRODUCTION: Type III hereditary angioedema (HAE-III) is a heterogeneous entity presenting with normal C1-INH levels and is often difficult to diagnose. CASE PRESENTATION:A 13-year-old previously healthy girl presented to our emergency department with facial angiodema. Medications included Naproxen for tooth pain and oral combined contraceptives for menometrorrhagia that she had been taking for 6 months. She initially developed pruritic plaques on her legs that resolved within 5 days. On the day of presentation, she developed progressively painful lips’ swelling. There were no systemic symptoms. Angioedema resolved within 24 hours and did not recur over a three-month follow-up period. Family history was notable for recurrent angioedema in her mother and two maternal aunts. RESULTS:Investigations revealed normal tryptase level, C3 and C4 levels, C1 esterase inhibitor level and functional protein assay. DISCUSSION:Given the family history and clinical presentation, the diagnosis is consistent with HAE-III. In contrast to other forms of HAE, normal C4 levels do not rule out this entity. NSAIDs have also been implicated in its pathogenesis and could have contributed to her symptoms. Recent studies suggest that HAE-III may be associated with urticaria. In this case, prophylactic medication such as tranexamic acid or bradykinin inhibitors will be determined according to attacks’ frequency and severity. Clinicians should maintain high level of awareness of this rare disease.
The micro-RNA signature of Lupus Nephritis: a new view on the disease pathogenesis (P3084)
Authors: Costa-Reis Patricia , Russo Pierre , Sullivan Kathleen
Published: January 2023

Introduction: MicroRNAs (miRNAS) are responsible for post-transcriptional gene silencing. Our hypothesis is that dysregulated miRNAs modify gene expression programs in lupus nephritis (LN), leading to sustained pathology. Methods: RNA was extracted from 18 kidney specimens of LN patients and 6 controls, human kidney cell lines and primary monocytes. Direct digital detection of over 700 miRNAs was performed and confirmed by qRT-PCR. Results: A LN-specific miRNA signature was identified, reflecting cell proliferation and inflammation, according to pathway analysis. When compared to normal tissue, a significant decrease in miR-26a and miR-30b was found in LN (p=0.002; p=0.005, respectively). The results were confirmed by qRT-PCR. miR-26a was associated with crescents in kidney specimens, a classic proliferative sign (p=0.04). Human kidney tubular, mesangial and endothelial cells expressed miR-26a and miR-30b. These miRNAs were increased in primary monocytes from lupus patients and in monocytes exposed to immune complexes, a stimulus for terminal differentiation. Conclusions: We identified, for the first time, decreased miR-26a and miR-30b expression in LN. These miRNAs have been implicated in the regulation of proliferation and our finding of an association with crescents and increased expression in monocytes from lupus patients, which are known to be more differentiated and more prone to apoptosis, is consistent with a role in the regulation of pathologic proliferation in LN.
A small shared epitope-mimetic compound potently accelerates osteoclast-mediated bone damage in autoimmune arthritis (P3090)
Authors: Fu Jiaqi , Ling Song , Liu Ying , Hannah Margaret , Gilon Chaim , Holoshitz Joseph
Published: January 2023

We have recently proposed that the shared epitope (SE) may contribute to rheumatoid arthritis (RA) pathogenesis by acting as a ligand that activates pro-arthritogenic signal transduction events. In order to examine this hypothesis, here we determined the arthritogenic effect of a novel small SE-mimetic compound, c(HS4-4). In this study, we show that c(HS4-4), containing the SE primary sequence motif QKRAA, interacted with the SE receptor calreticulin, potently activated reactive oxygen species (ROS) and nitric oxide (NO) production and markedly facilitated OC differentiation and function in vitro. The pro-osteoclastogenic potency of c(HS4-4) was 100,000 to 1,000,000-fold higher than the potency of a recently described linear SE peptidic ligand. When administered at low-nanogram doses to collagen-induced arthritis (CIA) mice, c(HS4-4) significantly facilitated arthritis onset, increased disease incidence and severity, augmented bone destruction, and increased OC abundance in synovial tissues and osteoclastogenic propensities of bone marrow-derived cells. In conclusion, c(HS4-4), a small SE-mimetic compound with drug-like properties, potently activates OC-mediated bone damage and disease severity in inflammatory arthritis. These findings support the hypothesis that the SE acts as signal transduction ligand that activates an arthritogenic pathway, and attest to the druggability of this pathway.
Akt activation by CD40 controls plasma cell and memory B cell differentiation but not germinal center formation by autoreactive B cells (P4032)
Authors: Lee Sang-Ryul , McCray Suzanne , Clarke Stephen
Published: January 2023

A hallmark of adaptive immunity is the formation of germinal centers that generate long-lived plasma cells and memory B cells. These events are undesirable by autoreactive B cells since they would lead the production of pathogenic autoantibodies and memory B cells that would accrue over a lifetime, but is unknown at a molecular level how this is achieved. To address this question we examined the ability of B cells specific for the SLE-specific self-antigen Sm to respond to antigen in vivo in the presence of functional T cells. Sheep red blood cells (SRBC) have an antigen that binds anti-Sm B cells and therefore immunization of anti-Sm mice with SRBCs provides antigen for anti-Sm B cell activation and activation of functional cognate T cells. We find that anti-Sm B cells from germinal centers in response to SRBC immunization, but fail to generate splenic or bone marrow plasma cells. Secondary, immunization shows the absence of memory B cell formation. BCR and CD40 signaling appears normal in anti-Sm B cells with the exception of an Akt signal by CD40. Our data suggest that Akt activation by CD40 signaling controls the formation of plasma cells and memory B cells by autoreactive B cells of the type activated in murine and human SLE. Experiments are underway to test this possibility.
The role of peritoneal B-1a cells in spontaneous and induced autoimmune hemolytic anemia. (P4038)
Authors: Scatizzi John , Theofilopoulos Argyrios , Kono Dwight
Published: January 2023

Previous studies have suggested peritoneal B-1a cells play a vital role in the development of anti-erythrocyte antibodies (AEA), but this remains controversial. While studying the Lbw2 lupus-susceptibility locus on chr 4, we discovered a subcongenic line, Lbw2-SE (NZB strain with 20 Mb NZW), that was deficient in peritoneal B-1a cells. NZB mice spontaneously develop autoimmune hemolytic anemia (AIHA) and exhibit an expansion of peritoneal B-1a cells, therefore we used this congenic to study the role of B-1a B cells in AEA production. Compared to NZB, Lbw2-SE mice had decreased total number of peritoneal B cell subsets (B-1a, B-1b, and B-2) but no differences in the number of splenic B cell subsets and a slight delay in AEA incidence similar to Lbw-2 congenics. To precisely map the B-1a-affecting variant, additional subcongenics were generated of which two (SEA & SED) had the low B1a phenotype. They, however, did not overlap suggesting the responsible mutation was located outside of the Lbw2-SE interval. The SEA and SED sublines had AIHA similar to NZB mice, while the SEB and SEC sublines had normal numbers of B-1a cells yet lower incidences of AIHA. Interestingly, after administering a TLR3 agonist to induce AEAs in young mice, the SEA and SED sublines with fewer B-1a cells demonstrated resistance to the development of AEAs. Our data indicate that B-1a cells are not required for the development of AEAs, but can enhance induction of AIHA under environmental conditions.
Autoantibody induction by DNA-containing immune complexes requires HMGB1 with TLR2/miR-155 pathway (P4040)
Authors: Xiong Sidong , Wen Zhenke , Xu Wei
Published: January 2023

Anti-dsDNA autoantibody has been reported as a central pathogenic factor of SLE. However, the mechanisms of anti-dsDNA antibody production still remain unclear. Recent evidence indicates that circulating DNA-containing immune complexes, one of the hallmarks of SLE, might be involved in the autoantibody production. Here we mainly explored the potential role and underlying mechanisms of DNA-containing immune complexes in the anti-dsDNA antibody production. We found that these immune complexes provoked anti-dsDNA antibody production primarily through their HMGB1 component, whose level was highly and positively correlated with the anti-dsDNA antibody level in SLE patients. Furthermore, the HMGB1-mediated autoantibody production was in a TLR2/MyD88 dependent pattern, and modulated by the miR-155 via targeting Ets-1. Our results firstly reported the crucial role of HMGB1 component of DNA-containing immune complexes in the autoantibody production, which was mediated and regulated by TLR2/MyD88/miR-155/Ets-1 pathways. These findings may provide novel mechanistic insight into the persistent anti-dsDNA antibody production and new clues for the development of therapeutic strategies for SLE.
NF-κB inducing kinase as a key player in regulating autoreactive B cell maturation (P4048)
Authors: Lee Sang-Ryul , Ramsdell Clifton , McCray Suzanne , Diz Ramiro , Clarke Stephen
Published: January 2023

The regulation of autoreactive B cells is crucial in immune systems to prevent developing autoimmune diseases in human and animal models. A critical peripheral B cell tolerance mechanism is exclusion of autoreactive B cells from the mature repertoire due to a failure to compete with non-autoreactive B cells. B cell activation factor (Baff) is one of the most important factors to discriminate autoreactive and non-autoreactive B cells in periphery. Therefore, the level of Baff expression in vivo is significant, since excess Baff can override exclusion and lead to autoimmunity, and may contribute to some human autoimmune diseases. We show here using B cells with different affinities for Sm, a self-antigen targeted in systemic lupus erythematosus (SLE), that the Baff-induced level of NF-κB inducing kinase (NIK) is reduced commensurate with the affinity for Sm and independently of Baffr levels. Reduced NIK differentially affect downstream signaling pathways, proportionally decreasing Akt activation and its repertoire inclusion function, but leaving intact NF-κB2 activation and its survival function. Thus, NIK functions as a key player to regulate entry into the mature B cell repertoire and preferentially excluding autoreactive B cells.
Potent therapeutic activity of a novel Salmonella-based cancer vaccine (P4314)
Authors: Xu Xin , Hegazy Wael , Gao Xiuhua , Guo Linjie , Courtney Amy , Kurbanov Suhrab , Hensel Micheal , Metelitsa Leonid
Published: January 2023

Inadequate antigen delivery and insufficient activation of innate immunity are among major factors responsible for the failure of cancer vaccines. We recently reported a new type of an attenuated Salmonella-based oral vaccine that uses the evolutionary conserved SPI2 locus-encoded molecular machinery of Salmonella to express and translocate survivin into the cytosol of antigen-presenting cells. In order to maximize the vaccine therapeutic potential, we tested a series of SPI2 promoter/effector combinations, performed codon optimization of survivin cDNA, and used a synthetic NKT-cell ligand, 7WD8-5 as an adjuvant. We found the combination of sifB promoter with sseJ effector could enhance vaccine immunogenicity 5 fold that resulted in a much stronger antitumor activity in A20 murine lymphoma model compared with the original vaccine. The vaccine therapeutic efficacy was further enhanced by combination with 7WD8-5, resulting in complete tumor regression. The improved vaccine was also effective against established CT26 colon carcinoma tumors. Antibody depletion experiments demonstrated that the anti-tumor activity depends on both CD8 and CD4 T cells, but not NK cells. Importantly, we were able to visualize tumor infiltration with CD8 T cells that accompanied tumor regression. Therefore, antigen delivery via SPI2-regulated secretion system of Salmonella may serve as a foundation for effective cancer vaccines.
Vascular endothelial growth factor expression is associated with B cell activating factor production stimulated with tumor necrosis factor-α in MH7A human synovial cells (P6280)
Authors: Moon Eun Yi , Lee Geun-Hee , Lee Jae-Wook , Lee Jiyoung , Yang Donghee , Go Ahreum
Published: January 2023

Rheumatoid arthritis (RA), one of autoimmune diseases, is characterized by histological hallmarks such as hyperplasia of the synovial lining cells, mononuclear cell infiltration, angiogenesis, and osteoclastic bone destruction. Angiogenesis is mediated through expression of vascular endothelial growth factor (VEGF) in RA synovium. VEGF expression is enhanced by TNF-α, the main pro-inflammatory cytokine in RA. B cell activating factor (BAFF) which plays a role in maturation and maintenance of B cells is also associated with autoimmune RA. Here, we investigated whether BAFF could regulate VEGF expression in MH7A synovial cells that is established by transfection with the SV40 T antigen (Miyazawa et al., 1998). When MH7A cells were treated with TNF-α, data showed that TNF-α increased the expression of BAFF and VEGF as judged by western blotting, RT-PCR and luciferase promoter assay. Inhibition of BAFF expression with BAFF-siRNA decreased VEGF expression. In addition, BAFF and VEGF expression was mediated by nuclear translocation of c-Fos activated by the treatment with TNF-α as judged by the transfection of MH7A cells with c-fos-siRNA. In conclusion, our results suggest for the first time that angiogenesis in RA could be controlled by BAFF-mediated VEGF expression.
Bcr/Abl-induced deregulation of the IL-33/ST2 pathway in CD34(+) progenitors from chronic myeloid leukemia patients (P6284)
Authors: Gombert Jean-Marc , Levescot Anaïs , Flamant Stéphane , Jacomet Florence , Basbous Sara , Féraud Olivier , Bonnet Marie-Laure , Giraud Christine , Roy Lydia , Barra Anne , Turhan Ali , Guilhot François , Herbelin André
Published: January 2023

Although it is generally acknowledged that cytokines regulate normal hematopoiesis in an autocrine/paracrine fashion, their possible role in chronic myeloid leukemia (CML) and resistance to imatinib mesylate (IM) treatment remain poorly investigated. Here, we report that CD34(+) progenitors from CML patients at diagnosis are selectively targeted by the cytokine/alarmin IL-33. Indeed, CML CD34(+) progenitors up-regulate their cell surface expression of the IL-33-specific receptor chain ST2, proliferate and produce cytokines in response to IL-33, conversely to CD34(+) cells from healthy individuals. Moreover, ST2 overexpression is normalized following IM therapy, while IL-33 counteracts in-vitro IM-induced growth arrest in CML CD34(+) progenitors via re-activation of the STAT5 pathway. Clinically, CML is associated with high circulating levels of soluble ST2, commonly used as a functional signature of IL-33 signaling in vivo. Taken together, our results support the hypothesis that the IL-33/ST2 pathway facilitates Bcr/Abl-induced leukemogenesis and contributes to IM resistance in CML patients.