Generation Of Autoantigens Research Articles

Granzyme serine proteases in inflammation and rheumatic diseases.

Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation.

Immunopeptidome mining reveals a novel ERS-induced target in T1D.

Autoreactive CD8+ T cells play a key role in type 1 diabetes (T1D), but the antigen spectrum that activates autoreactive CD8+ T cells remains unclear. Endoplasmic reticulum stress (ERS) has been implicated in β-cell autoantigen generation. Here, we analyzed the major histocompatibility complex class I (MHC-I)-associated immunopeptidome (MIP) of islet β-cells under steady and ERS conditions and found that ERS reshaped the MIP of β-cells and promoted the MHC-I presentation of a panel of conventional self-peptides. Among them, OTUB258-66 showed immunodominance, and the corresponding autoreactive CD8+ T cells were diabetogenic in nonobese diabetic (NOD) mice. High glucose intake upregulated pancreatic OTUB2 expression and amplified the OTUB258-66-specific CD8+ T-cell response in NOD mice. Repeated OTUB258-66 administration significantly reduced the incidence of T1D in NOD mice. Interestingly, peripheral blood mononuclear cells (PBMCs) from patients with T1D, but not from healthy controls, showed a positive IFN-γ response to human OTUB2 peptides. This study provides not only a new explanation for the role of ERS in promoting β-cell-targeted autoimmunity but also a potential target for the prevention and treatment of T1D. The data are available via ProteomeXchange with the identifier PXD041227.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

The gut microbiota posttranslationally modifies IgA1 in autoimmune glomerulonephritis.

Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila. IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1KI-CD89tg) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.

Muscle and Muscle-like Autoantigen Expression in Myasthenia Gravis Thymus: Possible Molecular Hint for Autosensitization

The thymus is widely recognized as an immunological niche where autoimmunity against the acetylcholine receptor (AChR) develops in myasthenia gravis (MG) patients, who mostly present thymic hyperplasia and thymoma. Thymoma-associated MG is frequently characterized by autoantibodies to the muscular ryanodine receptor 1 (RYR1) and titin (TTN), along with anti-AChR antibodies. By real-time PCR, we analyzed muscle-CHRNA1, RYR1, and TTN-and muscle-like-NEFM, RYR3 and HSP60-autoantigen gene expression in MG thymuses with hyperplasia and thymoma, normal thymuses and non-MG thymomas, to check for molecular changes potentially leading to an altered antigen presentation and autoreactivity. We found that CHRNA1 (AChR-α subunit) and AIRE (autoimmune regulator) genes were expressed at lower levels in hyperplastic and thymoma MG compared to the control thymuses, and that the RYR1 and TTN levels were decreased in MG versus the non-MG thymomas. Genes encoding autoantigens that share epitopes with AChR-α (NEFM and HSP60), RYR1 (neuronal RYR3), and TTN (NEFM) were up-regulated in thymomas versus hyperplastic and control thymuses, with distinct molecular patterns across the thymoma histotypes that could be relevant for autoimmunity development. Our findings support the idea that altered muscle autoantigen expression, related with hyperplastic and neoplastic changes, may favor autosensitization in the MG thymus, and that molecular mimicry involving tumor-related muscle-like proteins may be a mechanism that makes thymoma prone to developing MG.

Setting the Stage for Insulin Granule Dysfunction during Type-1-Diabetes: Is ER Stress the Culprit?

Type-1-diabetes (T1D) is a multifactorial disorder with a global incidence of about 8.4 million individuals in 2021. It is primarily classified as an autoimmune disorder, where the pancreatic β-cells are unable to secrete sufficient insulin. This leads to elevated blood glucose levels (hyperglycemia). The development of T1D is an intricate interplay between various risk factors, such as genetic, environmental, and cellular elements. In this review, we focus on the cellular elements, such as ER (endoplasmic reticulum) stress and its consequences for T1D pathogenesis. One of the major repercussions of ER stress is defective protein processing. A well-studied example is that of islet amyloid polypeptide (IAPP), which is known to form cytotoxic amyloid plaques when misfolded. This review discusses the possible association between ER stress, IAPP, and amyloid formation in β-cells and its consequences in T1D. Additionally, ER stress also leads to autoantigen generation. This is driven by the loss of Ca++ ion homeostasis. Imbalanced Ca++ levels lead to abnormal activation of enzymes, causing post-translational modification of β-cell proteins. These modified proteins act as autoantigens and trigger the autoimmune response seen in T1D islets. Several of these autoantigens are also crucial for insulin granule biogenesis, processing, and release. Here, we explore the possible associations between ER stress leading to defects in insulin secretion and ultimately β-cell destruction.

Alternative exon usage in TRIM21 determines the antigenicity of Ro52/TRIM21 in systemic lupus erythematosus.

The origin and mechanisms of autoantigen generation in systemic lupus erythematosus (SLE) are poorly understood. Here, we identified SLE neutrophils activated in vivo by IFN as a prominent source of Ro52, also known as tripartite motif-containing protein 21 (TRIM21), a critical autoantigen historically thought to be primarily generated by keratinocytes in SLE. Different from mononuclear cells and keratinocytes, SLE neutrophils are enriched in several unique Ro52 species containing a core sequence encoded by exon 4 (Ro52Ex4) in TRIM21. Ro52Ex4 is the main target of anti-Ro52 antibodies and is found in 2 Ro52 variants (Ro52α and a potentially novel isoform termed Ro52γ) upregulated in SLE neutrophils. Further analysis of Ro52γ revealed a subset of autoantibodies against a unique C-terminal domain (Ro52γCT) generated from a frameshift due to the lack of exon 6 in Ro52γ. Antibodies to Ro52Ex4 and Ro52γCT distinguish SLE patient subsets characterized by distinct clinical, laboratory, treatment, and transcriptional profiles that are not discerned by the "classical" anti-Ro52 antibodies. These studies uncover IFN-activated neutrophils as a key source of unique immunogenic forms of Ro52 in SLE. Moreover, the finding of Ro52Ex4 and Ro52γCT as core targets of anti-Ro52 antibodies focus interest on Ro52γ as the potential isoform toward which immunological tolerance is initially lost in SLE.

The monocyte cell surface as a novel site of autoantigen generation in Rheumatoid Arthritis

Abstract Citrullination is recognized as a key pathogenic process in rheumatoid arthritis (RA), as evidenced by the formation of anti-citrullinated protein antibodies (APCAs) in the majority of patients; however, the mechanisms that result in citrullinated autoantigen generation are not fully understood. Although the citrullinating enzyme peptidylarginine deiminase IV (PAD4) is predominantly expressed by neutrophils and monocytes, the contribution of monocytes to the citrullinated autoantigen pool has been underexplored. In this study, we utilized multiple complementary methods including flow cytometry, immunofluorescence, and transmission electron microscopy, which revealed a predominantly extranuclear localization of PAD4 in monocytes with a fraction present on the cell surface. Surface PAD4 was enzymatically active and citrullinated both extracellular fibrinogen and endogenous surface proteins in a calcium dose–dependent manner. In addition, human monoclonal ACPAs cloned from patients with RA recognized fibrinogen citrullinated by monocyte-surface PAD4. Mass spectrometry analysis of citrullinated proteins from the cell surface fraction revealed CD11b to be a novel PAD4 substrate. Citrullinated CD11b was recognized by autoantibodies in 60% of ACPA+ RA patients compared to 6% of healthy controls (p=0.0021) and 0% of ACPA− RA patients (p≤0.001). Taken together, our study demonstrates that PAD4 is expressed on the surface of monocytes in an enzymatically active state that renders the monocyte surface a novel site of citrullinated autoantigen generation in RA. Supported by funds received from Bristol-Myers Squibb.

Endoplasmic Reticulum-Mitochondria Crosstalk and Beta-Cell Destruction in Type 1 Diabetes.

Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In response to inflammatory signals, beta-cells engage adaptive mechanisms where the endoplasmic reticulum (ER) and mitochondria act in concert to restore cellular homeostasis. In the recent years it has become clear that this adaptive phase may trigger the development of autoimmunity by the generation of autoantigens recognized by autoreactive CD8 T cells. The participation of the ER stress and the unfolded protein response to the increased visibility of beta-cells to the immune system has been largely described. However, the role of the other cellular organelles, and in particular the mitochondria that are central mediator for beta-cell survival and function, remains poorly investigated. In this review we will dissect the crosstalk between the ER and mitochondria in the context of T1D, highlighting the key role played by this interaction in beta-cell dysfunctions and immune activation, especially through regulation of calcium homeostasis, oxidative stress and generation of mitochondrial-derived factors.

Effects of Gasdermin D in Modulating Murine Lupus and its Associated Organ Damage.

Gasdermin D (GSDMD) is the key executioner of an inflammatory cell death mechanism known as pyroptosis. Recent reports have also implicated GSDMD in other mechanisms of cell death, including apoptosis, necroptosis, and NETosis. Given the role of dysregulated cell death in autoimmune syndromes such as systemic lupus erythematosus (SLE), this study was undertaken in a murine lupus model to investigate whether GSDMD plays a pathogenic role in systemic autoimmunity by promoting inflammatory cell death, leading to increased generation of nuclear autoantigens and autoantibodies. An imiquimod-induced model of SLE was tested in GSDMD-/- mice (n = 30), with wild-type (WT) mice as controls (n = 34), on a C57BL/6 background. At the time of euthanasia, the mice were examined for serum autoantibodies, immune complex deposition, organ inflammation, immune dysregulation, and type I interferon responses. A model of pristane-induced lung injury in GSDMD-/- mice (n = 7), with WT mice as controls (n = 10), was used to confirm the pulmonary phenotype. Regulation of various mechanisms of cell death by GSDMD was investigated in the mice. Unexpectedly, GSDMD-/- mice developed enhanced mortality, more severe renal and pulmonary inflammation, and exacerbated autoantibody production in response to imiquimod. Pulmonary involvement was also more severe in the absence of GSDMD in mice with pristane-induced lung injury. Compared to WT mice, lack of GSDMD was associated with increased levels of circulating nuclear autoantigens (P < 0.01), anti-double-stranded DNA autoantibodies (P < 0.01), tissue immune complex deposition (P < 0.05), expansion of myeloid cell subsets (P < 0.05), and enhanced B cell activation and plasma cell differentiation (P = 0.001). Moreover, in the absence of GSDMD, enhanced autoantigen generation was associated with increased local induction of cell death in vivo. GSDMD negatively regulates autoantigen generation and immune dysregulation in response to tissue injury and may play previously unappreciated protective roles in systemic autoimmunity.

ANCA autoantigen gene expression highlights neutrophil heterogeneity where expression in normal-density neutrophils correlates with ANCA-induced activation

ANCA vasculitis is an autoimmune disease with increased expression of the autoantigen genes, myeloperoxidase (MPO) and proteinase 3 (PRTN3), but the origin and significance of expression is less distinct. To clarify this, we measured MPO and PRTN3 messenger RNA in monocytes, normal-density neutrophils, and in enriched leukocytes from peripheral blood mononuclear cells. Increased autoantigen gene expression was detected in normal-density neutrophils and enriched leukocytes from patients during active disease compared to healthy individuals, with the largest difference in enriched leukocytes. RNA-seq of enriched leukocytes comparing active-remission pairs identified a gene signature for low-density neutrophils. Cell sorting revealed low-density neutrophils contained mature and immature neutrophils depending on the presence or absence of CD10. Both populations contributed to autoantigen expression but the frequency of immature cells in low-density neutrophils did not correlate with low-density neutrophil MPO or PRTN3 expression. Low-density neutrophils were refractory to MPO-ANCA induced oxidative burst, suggesting an alternative role for low-density neutrophils in ANCA vasculitis pathogenesis. In contrast, normal-density neutrophils were activated by MPO-ANCA and monoclonal anti-PR3 antibody. Normal-density neutrophil activation correlated with MPO and PRTN3 mRNA. Increased autoantigen gene expression originating from the mature low-density and normal-density neutrophils suggests transcriptional dysregulation is a hallmark of ANCA vasculitis. Thus, the correlation between autoantigen gene expression and antibody-mediated normal-density neutrophil activation connects autoantigen gene expression with disease pathogenesis.

Introduction: Evolution of inflammatory arthritis from innate to adaptive immune mechanisms.

Introduction: Evolution of inflammatory arthritis from innate to adaptive immune mechanisms.

AAgAtlas 1.0: A Database of Human Autoantigens Extracted from Biomedical Literature.

Autoantibodies are antibodies against host self-proteins (autoantigens), which play significant roles in homeostasis maintenance and diseases with autoimmune disorders. Numerous papers were published in the past decade on the identification of human autoantigens in different human diseases. However, there is no consensus collection with all the reported autoantigens yet. To address this need, previously we developed a human autoantigen database, AAgAtlas 1.0, by text-mining and manual curation, which collects 1126 autoantigens associated with 1071 human diseases. AAgAtlas 1.0 provides a user-friendly interface to conveniently browse, retrieve, and download human autoantigen genes, their functional annotation, related diseases, and the evidence from the literature. AAgAtlas is freely available online http://biokb.ncpsb.org/aagatlas/ . In this chapter, we make an introduction and provide a guide to the users of AAgAtlas 1.0 database.

The role of cancer-associated autoantibodies as biomarkers in paraneoplastic myositis syndrome.

The aim of this study is to provide a comprehensive overview of the current insight about the clinical utility of cancer-associated autoantibodies (CAAs) as biomarkers in paraneoplastic myositis syndrome (PMS). In addition, the possible mechanisms of the relationship between malignancy and myositis onset are discussed. It has become increasingly clear that a subgroup of the myositis-specific autoantibodies could be considered as CAAs because they are closely related to the PMS. Increased risk of cancer was found in patients with antitranscriptional intermediary factor 1-γ (TIF1-γ), antinuclear matrix protein-2 (NXP-2), anti3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) or antismall ubiquitin-like modifier 1-activating enzyme (SAE) antibodies. However, the diagnosing sensitivity and specificity of these CAAs for PMS are different among different cohort studies. Abnormally expressed or mutated autoantigen genes in tumor could possibly induce cross immunity against self-proteins and subsequently lead to the development of PMS. Anti-TIF1-γ, anti-NXP-2, anti-HMGCR and anti-SAE antibodies may act as CAAs in PMS. It is necessary to closely screen and monitor for cancer in patients with CAAs. The recent studies of the relationship between CAAs and PMS provided important new insights into the disease mechanisms.

Modulation of MS-like disease by a multi epitope protein is mediated by induction of CD11c+CD11b+Gr1+ myeloid-derived dendritic cells

Specific neutralization of the pathogenic autoimmune cells is the ultimate goal in therapy of Multiple Sclerosis (MS). However, the pathogenic autoimmunity in MS, can be directed against several major target antigens, and therefore targeting pathogenic T-cells directed against a single target antigen is unlikely to be effective. To overcome this multiplicity and the potential complexity of pathogenic autoreactivities in MS, we have put forward the concept of concomitant multi-antigen/multi-epitope targeting as, a conceivably more effective approach to immunotherapy of MS. We constructed an (Experimental Autoimmune Encephalomeylitis (EAE)/MS-related synthetic human Target Autoantigen Gene (MS-shMultiTAG) designed to encode in tandem only EAE/MS related epitopes of all known encephalitogenic proteins. The MS–related protein product (designated Y-MSPc) was immunofunctional and upon tolerogenic administration, it effectively suppressed and reversed EAE induced by a single encephalitogenic protein. Furthermore, Y-MSPc also fully abrogated the development of “complex EAE” induced by a mixture of five encephalitogenic T-cell lines, each specific for a different encephalitogenic epitope of MBP, MOG, PLP, MOBP and OSP. Strikingly, Y-MSPc was consistently more effective than treatment with the single disease-specific peptide or with the peptide cocktail, both in suppressing the development of “classical” or “complex” EAE and in ameliorating ongoing disease. Overall, the modulation of EAE by Y-MSPc was associated with anergizing the pathogenic autoreactive T-cells, downregulation of Th1/Th17 cytokine secretion and upregulation of TGF-β secretion. Moreover, we show that both suppression and treatment of ongoing EAE by tolerogenic administration of Y-MSPc is associated also with a remarkable increase in a unique subset of dendritic-cells (DCs), CD11c+CD11b+Gr1+-myeloid derived DCs in both spleen and CNS of treated mice. These DCs, which are with strong immunoregulatory characteristics and are functional in down-modulation of MS-like-disease displayed increased production of IL-4, IL-10 and TGF-β and low IL-12. Functionally, these myeloid DCs suppress the in-vitro proliferation of myelin-specific T-cells and more importantly, the cells were functional in-vivo, as their adoptive transfer into EAE induced mice resulted in strong suppression of the disease, associated with a remarkable induction of CD4 + FoxP3+ regulatory cells.These results, which highlight the efficacy of “multi-epitope-targeting” agent in induction of functional regulatory CD11c+CD11b+Gr1+myeloid DCs, further indicate the potential role of these DCs in maintaining peripheral tolerance and their involvement in downregulation of MS-like-disease.

Perspectives of New Advances in the Pathogenesis of Vitiligo: From Oxidative Stress to Autoimmunity.

Vitiligo is an autoimmune cutaneous disease in which melanocytes are destroyed by CD8+ T cells resulting in disfiguring white spots. From the very beginning of the disease, oxidative stress plays a significant role in promoting the onset of vitiligo, as noted by many studies. Multiple factors lead to the overproduction of reactive oxygen species (ROS), and collaboratively cause ROS accumulation in vulnerable melanocytes. However, ROS are responsible for melanocyte damage manifested by the level of molecules, organelles, and cells, and the generation of autoantigens, through different pathways related to the dysregulation of melanocytes. Recent studies have shown that presentation of autoantigens is mediated by innate immunity, which bridges the gap between oxidative stress and adaptive immunity. The recruitment of CD8+ T cells induced by cytokines and chemokines guarantees the final destruction of epidermal melanocytes. Moreover, emerging concerns regarding regulatory T cells and resident memory T cells help explain the reinstatement and relapse of vitiligo. Here, we provide new perspectives in the advances in understanding of this disease pathogenesis and we attempt to find more interrelationships between oxidative stress and autoimmunity.

IRE1α Implications in Endoplasmic Reticulum Stress-Mediated Development and Pathogenesis of Autoimmune Diseases.

Inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) is the most prominent and evolutionarily conserved endoplasmic reticulum (ER) membrane protein. This transduces the signal of misfolded protein accumulation in the ER, named as ER stress, to the nucleus as “unfolded protein response (UPR).” The ER stress-mediated IRE1α signaling pathway arbitrates the yin and yang of cell life. IRE1α has been implicated in several physiological as well as pathological conditions, including immune disorders. Autoimmune diseases are caused by abnormal immune responses that develop due to genetic mutations and several environmental factors, including infections and chemicals. These factors dysregulate the cell immune reactions, such as cytokine secretion, antigen presentation, and autoantigen generation. However, the mechanisms involved, in which these factors induce the onset of autoimmune diseases, are remaining unknown. Considering that these environmental factors also induce the UPR, which is expected to have significant role in secretory cells and immune cells. The role of the major UPR molecule, IRE1α, in causing immune responses is well identified, but its role in inducing autoimmunity and the pathogenesis of autoimmune diseases has not been clearly elucidated. Hence, a better understanding of the role of IRE1α and its regulatory mechanisms in causing autoimmune diseases could help to identify and develop the appropriate therapeutic strategies. In this review, we mainly center the discussion on the molecular mechanisms of IRE1α in the pathophysiology of autoimmune diseases.

Selective inhibition of gelatinases mitigates T-cell receptor signaling in CD4+ T-cells resulting in reduced clinical severity in a murine model of Multiple Sclerosis.

Abstract Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components. The gelatinases, MMP-2 and MMP-9, have functions essential for normal physiological processes such as leukocyte trafficking, but their dysregulation is associated with various pathologies including Multiple Sclerosis (MS). During MS, CD4+ T-cells activated in the periphery penetrate the blood-brain barrier (BBB), recruit immune cells, initiate destruction of the myelin sheath, and cause axonal loss. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. In EAE, gelatinases are required for penetration of the BBB and generation of auto-antigens. Recent studies have demonstrated that they contribute to normal intracellular function of various cell types including antigen activated T-cells; however, their intracellular role in immune cell activation during EAE pathogenesis is not known. In our studies, we demonstrate that gelatinases are important for effective T-cell receptor (TCR) signaling. Upon activation, CD4+ T-cells lacking gelatinase activity demonstrated a reduced ability to express early activation markers, proliferate, produce cytokines, and undergo metabolic reprogramming. However, in the absence of TCR stimulation, CD4+ T-cells have no proliferative defects. In addition, RNA-seq analysis highlight key proteins impacted by gelatinase inhibition that are associated with the TCR signaling. Furthermore, EAE mice demonstrated reduced clinical severity following gelatinase inhibition. These results emphasize the importance of the gelatinases as therapeutic targets in CD4+ T-cell mediated autoimmune diseases.

Viral Impact in Autoimmune Diseases: Expanding the "X Chromosome-Nucleolus Nexus" Hypothesis.

Viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. We get some insight by considering demands a virus places on host cells. Viruses not only require production of their own proteins, RNA and/or DNA, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. Since the nucleolus is a major site of RNA processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral RNA and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. Polyamines are at high levels in nucleoli to assist in RNA folding. The size and activity of nucleoli increase directly with increases in polyamines. Nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive X chromosome, leading to expression of previously sequestered DNA. Sudden expression of a large concentration of Alu elements from the disrupted inactive X can compete with RNA transcripts containing intronic Alu sequences that normally maintain nucleolar structural integrity. Such disruption of nucleolar activity can lead to misfolded RNAs, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. Many autoantigens in lupus are, at least transiently, components of the nucleolus. Considering these effects of viruses, the “X chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive X by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered Alu elements, which can fragment the nucleolus, leading to generation of autoantigens.

Abstract LB-225: Expression- and immune-profiling of neuroblastoma associated with Opsoclonus-myoclonus-ataxia syndrome (OMAS) to identify features of auto- and tumor immunity

Abstract Background: Neuroblastoma (NB) is a pediatric cancer arising from the primordia of the autonomic nervous system. In spite of aggressive treatment, five year survival rates remain around 50% for high risk NB. OMAS is an autoimmune disorder that affects 2-3% of children with NB, causing ataxia, myoclonic jerks, and disordered mood/behavior. In contrast to high risk NB, survival rates among children with OMAS exceed 90%. The OMAS causative antigen is unknown, there are no molecular markers of active disease, and the nature of the immune process is poorly understood. We hypothesize that the distinctive immune response in OMAS patients is responsible for superior NB-tumor outcomes, and that deeper understanding of immune response in OMAS may provide insights into mechanisms of effective anti-NB tumor immunity. Methods: We have undertaken a molecular study of a large cohort of OMAS NB samples that were collected as part of a Children’s Oncology Group clinical trial for OMAS therapy. Fifty four children were enrolled in the trial, from which tumor, blood and cerebrospinal fluid were collected at time of diagnosis. We compared RNAseq gene expression profiles of OMAS NB to low- and high-risk NB without OMAS, to identify differentially expressed genes in OMAS that may drive anti-neuronal reactivity or that improve antitumor immunity. In parallel, we are investigating the immune profile of OMAS, to determine whether any distinctive features of OMAS immunity may be linked to improved tumor outcomes. We used T cell receptor β-chain sequencing of tumor genomic DNA to profile tumor-infiltrating lymphocytes in OMAS NBs and control NBs without OMAS. Results: Initial analysis shows that OMAS samples cluster together, and separately from low- and high-risk NB without OMAS. B and T cell gene signatures dominate the differentially expressed genes and drive the clustering. This strongly implies increased tumor infiltration in OMAS samples. We have identified several intriguing candidate autoantigen genes, which we are now testing for recognition by OMAS patient serum antibody repertoires. While we predicted a clonal, antigen driven response in OMAS patient samples, β-chain repertoires from these patients exhibit no clonal dominance, but rather comprise diverse repertoires, suggesting that the antitumor response may not be dominated by the OMAS associated antigen. Nevertheless, comparison of CDR3 amino acid sequence repertoires using Jaccard Index reveals detectable similarity of OMAS T-cell repertoires. Conclusion: We find the strongest feature of OMAS NB so far to be its immune signal. Current work aims to determine the activation state and subtype of tumor infiltrating T cells in OMAS, and to test whether candidates from RNAseq affect T cell activation and differentiation. Furthermore, B cells are essential for OMAS, since treatment with anti-CD20+ monoclonal antibody is crucially beneficial in achieving remission from OMAS. Autoantibody production is presumed essential. However, recent work on other autoimmune diseases, like lupus and multiple sclerosis, supports a critical cellular function for B cells as antigen presenting cells (APCs) in those diseases. Ongoing experiments, including BCRseq and MHC typing from OMAS associated NBs, will help address questions about critical B cell functions in OMAS. Citation Format: Miriam I. Rosenberg, Jessica A. Panzer, Tal Sagiv, Pedro A. de Alarcon, Nir Friedman, Igor Ulitsky, John M. Maris. Expression- and immune-profiling of neuroblastoma associated with Opsoclonus-myoclonus-ataxia syndrome (OMAS) to identify features of auto- and tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-225. doi:10.1158/1538-7445.AM2017-LB-225