The Relationship Between Anti-Cell Division Cycle and Apoptosis Regulator 1 Autoantibodies, Anti-Sp4 Autoantibodies, and Cancer in Anti-Transcription Intermediary Factor 1γ-Positive Dermatomyositis.

OBJECTIVE: The objective of this study was to describe the frequency, co-occurrence, and cancer association of anti-cell division cycle and apoptosis regulator 1 (anti-CCAR1) and anti-Sp4 in two large independent adult dermatomyositis (DM) cohorts. METHODS: Anti-transcription intermediary factor 1γ (anti-TIF1γ)-positive patients with DM from two independent cohorts were studied to determine the serologic overlap of anti-CCAR1 and anti-Sp4 autoantibodies measured by enzyme-linked immunosorbent assay. Associations between cancer-associated myositis (CAM) and antibody-defined subgroups within anti-TIF1γ-positive patients with DM were determined. RESULTS: A total of 305 anti-TIF1γ-positive patients with DM were studied: 169 patients from Johns Hopkins and 136 patients from Stanford. In each cohort, approximately one-third of anti-TIF1γ-positive patients with DM were anti-Sp4 positive, one-third were anti-CCAR1 positive, 20% were positive for both, and half of patients were negative for both. There was a strong association with CAM in patients lacking both these antibodies (Johns Hopkins, odds ratio [OR] 12.9 [95% confidence interval (CI) 3.6-89.5]; Stanford, OR 4.5 [95% CI 1.8-13.2]). The strongest negative association with CAM was found in patients with anti-Sp4 or anti-CCAR1 (Johns Hopkins, OR 0.07 [95% CI 0.01-0.27]; Stanford, OR 0.22 [95% CI 0.07-0.55]). CONCLUSION: Both anti-Sp4 and anti-CCAR1 autoantibody subgroups are negatively associated with CAM. Although the magnitude of this association is substantial, cancer occasionally occurs in patients positive for either specificity. Conversely, approximately half of anti-TIF1γ-positive patients with DM are negative for both antibodies (anti-Sp4/anti-CCAR1 negative), and thus this subgroup may warrant more intensive cancer surveillance.

Precise identification and tracking of HMGCR-reactive CD4+ T cells in the target tissue of patients with anti-HMGCR immune-mediated necrotising myopathy.

BACKGROUND: Anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR)-positive immune-mediated necrotising myopathy (IMNM) is characterised by the presence of IgG autoantibodies against HMGCR and a strong association with specific HLA-DR alleles. Although these findings implicate HMGCR-specific CD4+T-cells in the disease's pathogenesis, no such cells have been described. In this study, we aimed to identify and characterise HMGCR-reactive CD4+T-cells and assess their presence in affected muscle tissue from patients with anti-HMGCR+IMNM. METHODS: Peripheral blood mononuclear cells from patients with anti-HMGCR+IMNM (n=10) and dermatomyositis (DM; n=10) were stimulated with HMGCR protein and peptides identified using a natural antigen processing assay (NAPA; n=6). CD4+T-cell activation was assessed by CD154 upregulation via flow cytometry. T-cell receptor ß(TCR) sequencing was performed on paired HMGCR-reactive T-cells and muscle biopsy tissue (n=5). RESULTS: CD4+T-cell responses to HMGCR protein were higher in patients with anti-HMGCR+IMNM compared with DM (median 0.06 vs 0.00, p=0.0059). These responses were enriched in Th1-Th17 cells, and when present, they positively correlated with anti-HMGCR antibody levels (r2=0.89, p=0.0012). NAPA revealed convergent presentation of seven HMGCR core peptides, with substantial overlap in the peptide repertoires between patients. These HMGCR peptides elicited robust CD4+T-cell responses, with 9/10 anti-HMGCR+IMNM patients responding to at least one peptide, compared with 1/10 DM (p=0.0003). Analysis of HMGCR-reactive TCRs ß yielded antigen-reactive motifs that were enriched in muscle biopsies (projection score 0.03 vs 0.63, p=0.007). CONCLUSION: HMGCR-antigen-reactive CD4+T-cells are present in the circulation and target tissue of patients with anti-HMGCR+IMNM, suggesting an active role for these cells in the pathogenesis of anti-HMGCR+IMNM.

SOX-5 Transcription Factor: a Novel Psoriatic Autoantigen Preferentially Found in Women.

OBJECTIVE: Adaptive immunity mediates psoriatic disease pathogenesis. We aimed to identify novel psoriatic autoantigens and their phenotypic associations in deeply characterized patient cohorts. METHODS: Sera from psoriatic arthritis (PsA) patients were used for autoantibody discovery. Immunoprecipitations performed with cell lysates were on-bead digested, and autoantigens were identified by mass spectrometry. Prevalence and clinical features associated with anti-SRY-Box transcription factor-D (SOX-D) antibodies were determined by screening discovery cohorts of patients with PsA (n = 135), patients with psoriasis without PsA (n = 24), and healthy controls (n = 41). A PsA validation cohort (n = 325) and disease control samples of individuals with rheumatoid arthritis (RA; n = 66) and systemic lupus erythematosus (SLE, n = 66) were assayed for anti-SOX5 antibodies. Disease characteristics were compared by antibody status. Longitudinal data were analyzed using linear mixed-effects models with patient-specific intercept to ascertain associations. We also tested PsA sera for the recently described anti-ADAMTS-L5 autoantibody in PsA. RESULTS: The novel autoantigens identified were SOX-D transcription factors, with SOX-5 being the focus of this analysis. Anti-SOX5 antibodies were present in 8.9% (12 of 135) and 4.3% (14 of 323) of patients in the PsA discovery and validation cohorts, respectively, 12.5% of patients (3 of 24) in the psoriasis group, 2.4% (1 of 41) of healthy controls, and 7.6% (5 of 66) each of patients in the RA and SLE groups. Anti-SOX5 were associated with female sex in both PsA cohorts (discovery: 15.7% women, 2.6% men, P = 0.006; validation: 6.3% women, 1.4% men, P = 0.049). In a longitudinal analysis adjusted for sex, anti-SOX5 associated with biologic disease-modifying antirheumatic drug treatment (95% vs 61%; P = 0.001; n = 96) and with differences in estimated treatment effects by mechanism of action. Anti-ADAMTS-L5 autoantibodies were identified in 8 of 124 patients (6.5%) in the PsA group. CONCLUSION: SOX-D transcription factors are novel psoriatic autoantigens. Anti-SOX5 antibodies were preferentially found in women with PsA and associated with specific clinical and treatment characteristics, suggesting that anti-SOX5 antibodies may identify mechanistic subgroups. We independently validated anti-ADAMTS-L5 autoantibodies in PsA.

The Johns Hopkins Physician-Scientist Training Program to Enhance Institutional Retention and Entry Into Academic Positions: An Evaluation of Program Effectiveness and Outcomes.

PURPOSE: The Johns Hopkins Physician-Scientist Training Program (PSTP) was implemented to overcome well-documented challenges in training and retaining physician-scientists by providing physician-scientist pathway training for residents and clinical fellows. The program's core tenets include monthly seminars, individualized feedback on project proposals, access to mentors, and institutional funding opportunities. This study evaluated the effectiveness and outcomes of the PTSP and provides a framework for replication. METHOD: A query of institutional demographic data and bibliometric variables of the PSTP participants (2017-2020) at a single academic medical center was conducted in 2021. In addition, a voluntary survey collected personal and program evaluation information. RESULTS: Of 145 PSTP scholars, 59 (41%) were women, and 41 (31%), 8 (6%), and 6 (5%) of scholars self-identified as Asian, Hispanic, and Black, respectively. Thirty-three (23%) scholars received PSTP research support or career development microgrants. Of 66 PSTP graduates, 29 (44%) remained at Johns Hopkins as clinical fellows or faculty. Of 48 PSTP graduates in a post-training position, 42 (88%) were in academia, with the majority, 29 (76%), holding the rank of assistant professor. Fifty-nine of 140 available participants responded to the survey (42% response rate). The top-cited reason for joining the PSTP was exposure to mentors and administration (50/58 respondents, 86%), followed by seeking scholarly opportunities (37/58 respondents, 64%). Most scholars intended to continue a career as a physician-scientist. CONCLUSIONS: The PSTP provides internal research support and institutional oversight. Although establishing close mentor-mentee relationships requires individualized approaches, the PSTP provided structured academic pathways that enhanced participating scholars' ability to apply for grants and jobs. The vast majority continued their careers as physician-scientists after training. In light of the national evidence of a "leaky physician-scientist pipeline," programs such as the PSTP can be critical to entry into early academic career positions and institutional retention.

Anti-Gephyrin Antibodies: A Novel Specificity in Patients With Systemic Sclerosis and Lower Bowel Dysfunction.

OBJECTIVE: Autoantibodies are clinically useful in phenotyping patients with systemic sclerosis (SSc). Gastrointestinal (GI) function is regulated by the enteric nervous system (ENS) and commonly impaired in SSc, suggesting that the SSc autoimmune response may target ENS antigens. We sought to identify novel anti-ENS autoantibodies with an aim to clinically phenotype SSc GI dysfunction. METHODS: Serum from a patient with SSc with GI dysfunction but without defined SSc-associated autoantibodies was used for autoantibody discovery. Immunoprecipitations performed with murine myenteric plexus lysates were on-bead digested, and autoantigens were identified by mass spectrometry. Prevalence was determined, and clinical features associated with novel autoantibodies were evaluated in a SSc cohort using regression analyses. The expression of gephyrin in human GI tract tissue was examined by immunohistochemistry. RESULTS: We identified gephyrin as a novel SSc autoantigen. Anti-gephyrin antibodies were present in 9% of patients with SSc (16/188) and absent in healthy controls (0/46). Anti-gephyrin antibody-positive patients had higher constipation scores (1.00 vs 0.50, P = 0.02) and were more likely to have severe constipation and severe distention/bloating (46% vs 15%, P = 0.005; 54% vs 25%, P = 0.023, respectively). Anti-gephyrin antibody levels were significantly higher among patients with severe constipation (0.04 vs 0.00; P = 0.001) and severe distention and bloating (0.03 vs 0.004; P = 0.010). Severe constipation was associated with anti-gephyrin antibodies even in the adjusted model. Importantly, gephyrin was expressed in the ENS, which regulates gut motility. CONCLUSION: Gephyrin is a novel ENS autoantigen that is expressed in human myenteric ganglia. Anti-gephyrin autoantibodies are associated with the presence and severity of constipation in patients with SSc.

Distinct Scleroderma Autoantibody Profiles Stratify Patients for Cancer Risk at Scleroderma Onset and During the Disease Course.

OBJECTIVES: We examined whether an array of scleroderma autoantibodies associates with risk of cancer and could be useful tools for risk stratification. METHODS: Scleroderma cancer cases and scleroderma controls without cancer from the Johns Hopkins Scleroderma Center and the University of Pittsburgh Scleroderma Center were studied. Sera were assayed by Lineblot and enzyme-linked immunosorbent assay (ELISA) for autoantibodies against centromere, topoisomerase 1, RNA polymerase (POLR) 3, PM/Scl, Th/To, NOR90, U3 RNP, Ku, Ro52, U1RNP, and RNPC3. Logistic regression models were constructed to examine whether distinct autoantibodies associated with overall cancer at any time and cancer-associated scleroderma (cancer occurring three years before and after scleroderma onset). The effects of having more than one autoantibody on cancer were further examined using random forest analysis. RESULTS: A total of 676 cases and 687 controls were studied. After adjusting for relevant covariates, anti-POLR3 (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.03-2.11) and monospecific anti-Ro52 (OR 2.19, 95% CI 1.29-3.74) were associated with an increased overall cancer risk, whereas anticentromere (OR 0.69, 95% CI 0.51-0.93) and anti-U1RNP (OR 0.63, 95% CI 0.43-0.93) were associated with lower risk. When examining risk of cancer-associated scleroderma, these immune responses remained associated with increased or decreased risk: anti-POLR3 (OR 2.28, 95% CI 1.33-3.91), monospecific anti-Ro52 (OR 2.58, 95% CI 1.05-6.30), anticentromere (OR 0.39, 95% CI 0.20-0.74), and anti-U1RNP (OR 0.32, 95% CI 0.11-0.93). Anti-Ro52 plus anti-U1RNP or anti-Th/To was associated with decreased cancer risk compared with anti-Ro52 alone. CONCLUSIONS: These data suggest that five distinct scleroderma immune responses, alone or in combination, may be useful tools to stratify the risk of cancer for scleroderma patients. Further study examining cancer risk in autoantibody subgroups relative to the general population is warranted.

The XIST lncRNA is a sex-specific reservoir of TLR7 ligands in SLE.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a dramatic sex bias, affecting 9 times more women than men. Activation of Toll-like receptor 7 (TLR7) by self-RNA is a central pathogenic process leading to aberrant production of type I interferon (IFN) in SLE, but the specific RNA molecules that serve as TLR7 ligands have not been defined. By leveraging gene expression data and the known sequence specificity of TLR7, we identified the female-specific X-inactive specific transcript (XIST) long noncoding RNA as a uniquely rich source of TLR7 ligands in SLE. XIST RNA stimulated IFN-α production by plasmacytoid DCs in a TLR7-dependent manner, and deletion of XIST diminished the ability of whole cellular RNA to activate TLR7. XIST levels were elevated in blood leukocytes from women with SLE compared with controls, correlated positively with disease activity and the IFN signature, and were enriched in extracellular vesicles released from dying cells in vitro. Importantly, XIST was not IFN inducible, suggesting that XIST is a driver, rather than a consequence, of IFN in SLE. Overall, our work elucidated a role for XIST RNA as a female sex-specific danger signal underlying the sex bias in SLE.

Association of Anti-CCAR1 Autoantibodies With Decreased Cancer Risk Relative to the General Population in Patients With Anti-Transcriptional Intermediary Factor 1γ-Positive Dermatomyositis.

OBJECTIVE: To describe the disease specificity, clinical phenotype, and risk of cancer in dermatomyositis (DM) patients with autoantibodies against cell division cycle and apoptosis regulator protein 1 (anti-CCAR1). METHODS: The frequency of anti-CCAR1 autoantibodies was measured by enzyme-linked immunosorbent assay in the serum of DM patients from 2 independent cohorts (Johns Hopkins and Stanford), with patients with several other rheumatic diseases and healthy controls used as comparators. Clinical features and the risk of cancer incidence relative to that in the general population were determined in anti-CCAR1-positive DM patients. RESULTS: Anti-CCAR1 antibodies were significantly associated with anti-transcriptional intermediary factor 1γ (anti-TIF1γ) antibodies present in the serum of patients with DM: 80 (32%) of 252 anti-TIF1γ-positive DM patients versus 14 (8%) of 186 anti-TIF1γ-negative DM patients were positive for anti-CCAR1 antibodies (P < 0.001). Anti-CCAR1 antibodies were not detected in any of the 32 serum samples from healthy controls, and were present at very low frequencies in the sera of patients with other rheumatic diseases: 1 (2.3%) of 44 patients with anti-hydroxymethylglutaryl-coenzyme A reductase-positive necrotizing myopathy, 1 (2.3%) of 44 patients with inclusion body myositis, and 3 (6.5%) of 46 patients with systemic lupus erythematosus were positive for anti-CCAR1 antibodies. Upon examining data on occurrence of cancer from the onset of DM onward, the observed number of cancers diagnosed in anti-TIF-1γ-positive DM patients was significantly greater than expected in both cohorts, with a standardized incidence ratio (SIR) of 3.49 (95% confidence interval [95% CI] 2.39-4.92) in the Johns Hopkins cohort and a SIR of 4.54 (95% CI 3.04-6.52) in the Stanford cohort (each P < 0.001). DM patients who were both anti-TIF1γ positive and anti-CCAR1 positive had lower SIRs for cancer, with a SIR of 1.78 (95% CI 0.77-3.51) (P = 0.172) in the Johns Hopkins cohort and a SIR of 1.61 (95% CI 0.44-4.13) (P = 0.48) in the Stanford cohort. CONCLUSION: Anti-CCAR1 autoantibodies are specific for anti-TIF1γ-positive DM. Their presence in anti-TIF1γ-positive patients attenuates the risk of cancer to a level comparable to that seen in the general population.

Cumulative disease damage and anti-PM/Scl antibodies are associated with a heavy burden of calcinosis in systemic sclerosis.

OBJECTIVES: Ectopic calcification (calcinosis) is a common complication of SSc, but a subset of SSc patients has a heavy burden of calcinosis. We examined whether there are unique risk factors for a heavy burden of calcinosis, as compared with a light burden or no calcinosis. METHODS: We reviewed the medical records of all patients in the Johns Hopkins Scleroderma Center Research Registry with calcinosis to quantify calcinosis burden using pre-specified definitions. We performed latent class analysis to identify SSc phenotypic classes. We used multinomial logistic regression to determine whether latent phenotypic classes and autoantibodies were independent risk factors for calcinosis burden. RESULTS: Of all patients, 29.4% (997/3388) had calcinosis, and 13.5% (130/963) of those with calcinosis had a heavy burden. The latent phenotypic class with predominantly diffuse skin disease and higher disease severity (characterized by pulmonary hypertension, interstitial lung disease, cardiomyopathy, severe RP, gastrointestinal involvement, renal crisis, myopathy and/or tendon friction rubs) was associated with an increased risk of both a heavy burden [odds ratio (OR) 6.92, 95% CI 3.66, 13.08; P < 0.001] and a light burden (OR 2.88, 95% CI 2.11, 3.95; P < 0.001) of calcinosis compared with the phenotypic class with predominantly limited skin disease. Autoantibodies to PM/Scl were strongly associated with a heavy burden of calcinosis (OR 17.31, 95% CI 7.72, 38.81; P < 0.001) and to a lesser degree a light burden of calcinosis (OR 3.59, 95% CI 1.84, 7.00; P < 0.001). CONCLUSIONS: Calcinosis burden is associated with cumulative SSc-related tissue damage. Independent of disease severity, autoantibodies to PM/Scl are also associated with a heavy burden of calcinosis.

Subsets of Idiopathic Inflammatory Myositis Enriched for Contemporaneous Cancer Relative to the General Population.

OBJECTIVE: This study investigates cancer risk in idiopathic inflammatory myopathy (IIM) relative to the general population. METHODS: We conducted a single-center, retrospective cohort study of IIM patients and malignancy. Myositis-specific and -associated autoantibodies were determined by Euroimmun line blot, enzyme-linked immunosorbent assay, and immunoprecipitation. We calculated standardized prevalence ratios (SPRs) and adjusted for calendar year, age, sex, race, and ethnicity by comparing observed cancers in IIM patients versus expected cancers in the general population using the Surveillance, Epidemiology, and End Results registry. RESULTS: Of 1,172 IIM patients, 203 (17%) patients with a cancer history were studied. Over a median follow-up of 5.2 years, the observed number of IIM patients diagnosed with cancer was increased 1.43-fold (SPR 1.43 [95% confidence interval (95% CI) 1.15-1.77]; P = 0.002). Within 3 years of IIM symptom onset, an increased SPR was observed for anti-transcription intermediary factor 1γ (anti-TIF1γ)-positive patients for ovarian and breast cancer (ovarian SPR 18.39 [95% CI 5.01-47.08], P < 0.001; breast SPR 3.84 [95% CI 1.99-6.71], P < 0.001). As expected, anti-TIF1γ positivity was associated with a significantly elevated SPR; however, only 55% (36 of 66) of all cancers within 3 years of dermatomyositis onset were observed in anti-TIF1γ-positive patients. Other myositis-specific autoantibodies, including anti-Mi-2, anti-small ubiquitin-like modifier activating enzyme (SAE), and anti-nuclear matrix protein 2 (NXP-2), accounted for 26% (17 of 66) of cancers diagnosed within 3 years of dermatomyositis onset. No cancer association, positive or negative, was observed for patients with antisynthetase, anti-melanoma differentiation-associated protein 5 (anti-MDA-5), or anti-hydroxymethylglutaryl-coenzyme A reductase (anti-HMGCR) antibodies. CONCLUSION: In a tertiary referral center population, anti-TIF1γ was most strongly associated with breast and ovarian cancer. Patients with antisynthetase, anti-MDA-5, or anti-HMGCR antibodies had the same cancer risk as the general population.

IMMUNOEDITING IN AUTOIMMUNE RHEUMATIC DISEASES.

The striking association of specific autoantibodies with distinct disease phenotypes and trajectories in human autoimmune rheumatic diseases provides a powerful opportunity to interrogate disease mechanism. In scleroderma, a subgroup of patients with autoantibodies to POLR3 have coincident onset of cancer and scleroderma. The majority of these patients have genetic changes (somatic mutations and loss of heterozygosity) in the POLR3A gene in their matched cancers, coupled with immune responses directed against the mutated and wild type autoantigen. In some individuals with scleroderma or dermatomyositis where specific immune responses mark a high risk of emergent cancer, cancer does not emerge. Such patients have a broader immune response that targets additional autoantigens, suggesting that the breadth and magnitude of the immune response regulates cancer, and that the rheumatic diseases provide a unique window into natural immunoediting of cancer in humans. This has implications for prediction and therapy in both autoimmunity and cancer.

The DNA sensors AIM2 and IFI16 are SLE autoantigens that bind neutrophil extracellular traps.

Background: Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded (ds)DNA of various origins. Here, we investigate the ALR absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context. Methods: AIM2 autoantibodies were measured by immunoprecipitation in SLE and control subjects. Neutrophil extracellular traps were induced in control neutrophils and combined with purified ALR proteins in immunofluorescence and DNase protection assays. SLE renal tissues were examined for ALR-containing NETs by confocal microscopy. Results: AIM2 autoantibodies were detected in 41/131 (31.3%) SLE patients and 2/49 (4.1%) controls. Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16, and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We found that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling. Conclusions: Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE. Funding: These studies were funded by NIH R01 DE12354 (AR), P30 AR070254, R01 GM 129342 (JS), K23AR075898 (CM), K08AR077100 (BA), the Jerome L. Greene Foundation and the Rheumatology Research Foundation. Dr. Antiochos and Dr. Mecoli are Jerome L. Greene Scholars. The Hopkins Lupus Cohort is supported by NIH grant R01 AR069572. Confocal imaging performed at the Johns Hopkins Microscopy Facility was supported by NIH Grant S10 OD016374.

Systemic lupus erythematosus (SLE or lupus for short) is an autoimmune disease in which the immune system attacks healthy tissue in organs across the body. The cause is unknown, but people with the illness make antibodies that stick to proteins that are normally found inside the cell nucleus, where DNA is stored. To make these antibodies, the immune system must first 'see' these proteins and mistakenly recognise them as a threat. But how does the immune system recognise proteins that are normally hidden inside cells? During infection, a type of immune cell called a neutrophil releases DNA from its nucleus to form structures called neutrophil extracellular traps, or NETs for short. The role of these NETs is to capture and kill pathogens, but they also expose the neutrophil's DNA and the proteins attached to it to other immune cells. It is therefore possible that other immune cells interacting with NETs during infection may contribute to the development of lupus. Two proteins of interest are AIM2 and IFI16. These proteins form large, shield-like structures around strands of DNA, and previous work has shown that some people with lupus make antibodies against IFI16. Antiochos et al. wondered whether IFI16 and AIM2 might stick to NETs, exposing themselves to the immune system. Examining the blood of people with lupus revealed that one in three of them made antibodies that could stick to AIM2. Those people were also more likely to have antibodies that could stick to IFI16 and to strands of DNA. Using microscopy, Antiochos et al. also found AIM2 and IFI16 on NETs in the kidneys of some people with lupus. Further investigation showed that the presence of AIM2 and IFI16 prevents NETs from breaking down. If proteins like AIM2 and IFI16 can stop NETs from breaking down, they could allow the immune system more time to develop antibodies against them. Further investigation could reveal whether this is one of the causes of lupus. A clearer understanding of the antibodies could also boost research into diagnosis and treatment.

IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function.

BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).

Presence and Implications of Anti-Angiotensin Converting Enzyme-2 Immunoglobulin M Antibodies in Anti-Melanoma-Differentiation-Associated 5 Dermatomyositis.

OBJECTIVE: Patients with anti-melanoma-differentiation-associated 5 (anti-MDA5)-positive dermatomyositis (DM) share several striking similarities to patients with SARS-CoV-2. Our objective was to assess the prevalence of anti-angiotensin converting enzyme-2 (ACE2) immunoglobulin M (IgM) antibodies, found in patients with severe SARS-CoV-2, in two independent anti-MDA5-positive DM cohorts. METHODS: Anti-ACE2 IgM antibodies were assayed by enzyme-linked immunosorbent assay (ELISA) in two anti-MDA5-positive DM cohorts: a predominantly outpatient North American cohort (n = 52) and a Japanese cohort enriched for new-onset disease (n = 32). Additionally, 118 patients with SARS-CoV-2 with a spectrum of clinical severity were tested for anti-MDA5 antibodies by ELISA. RESULTS: Five of fifty-two (9.6%) North American patients and five of thirty-two (15%) Japanese patients were positive for anti-ACE2 IgM. In the North American cohort, all five patients with anti-ACE2 IgM antibodies had proximal muscle weakness, had interstitial lung disease, were significantly more likely to receive pulse dose methylprednisolone (80% vs 30%, P = 0.043), and had worse forced vital capacity (median 59% predicted vs 78%, P = 0.056) compared with the anti-ACE2-IgM-negative group. In the Japanese cohort, all five anti-ACE2-IgM-positive patients also required pulse dose methylprednisolone, and three of five (60%) patients died. Japanese patients with anti-ACE2 IgM had significantly worse oxygenation, as defined by a lower partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ratio (233 vs 390, P = 0.021), and a higher alveolar-arterial oxygenation gradient (91 vs 23 mm Hg, P = 0.024) than the IgM-negative group. CONCLUSION: We describe anti-ACE2 IgM autoantibodies in two independent cohorts with anti-MDA5-positive DM. These autoantibodies may be biomarkers for severe disease and provide insight into disease pathogenesis.

Immune responses to CCAR1 and other dermatomyositis autoantigens are associated with attenuated cancer emergence.

BACKGROUNDThe temporal clustering of a cancer diagnosis with dermatomyositis (DM) onset is strikingly associated with autoantibodies against transcriptional intermediary factor 1-γ (TIF1-γ). Nevertheless, many patients with anti-TIF1-γ antibodies never develop cancer. We investigated whether additional autoantibodies are found in anti-TIF1-γ-positive patients without cancer.METHODSUsing a proteomic approach, we defined 10 previously undescribed autoantibody specificities in 5 index anti-TIF1-γ-positive DM patients without cancer. These were subsequently examined in discovery (n = 110) and validation (n = 142) cohorts of DM patients with anti-TIF1-γ autoantibodies.RESULTSWe identified 10 potentially novel autoantibodies in anti-TIF1-γ-positive DM patients, 6 with frequencies ranging from 3% to 32% in 2 independent DM cohorts. Autoantibodies recognizing cell division cycle and apoptosis regulator protein 1 (CCAR1) were the most frequent, and were significantly negatively associated with contemporaneous cancer (discovery cohort OR 0.27 [95% CI 0.7-1.00], P = 0.050; validation cohort OR 0.13 [95% CI 0.03-0.59], P = 0.008). When cancer did emerge, it occurred significantly later in anti-CCAR1-positive compared with anti-CCAR1-negative patients (median time from DM onset 4.3 vs. 0.85 years, respectively; P = 0.006). Cancers that emerged were more likely to be localized (89% of anti-CCAR1-positive cancers presenting at stage 0 or 1 compared with 42% of patients without anti-CCAR1 antibodies, P = 0.02). As the number of additional autoantibody specificities increased in anti-TIF1-γ-positive DM patients, the frequency of cancer decreased (P < 0.001).CONCLUSIONAs the diversity of immune responses in anti-TIF1-γ DM patients increases, the likelihood of cancer emerging decreases. Our findings have important relevance for cancer risk stratification in DM patients and for understanding natural immune regulation of cancer in humans.TRIAL REGISTRATIONNot applicable.FUNDING SOURCESThe NIH, the Donald B. and Dorothy L. Stabler Foundation, and the Huayi and Siuling Zhang Discovery Fund.

Learning and Predicting from Dynamic Models for COVID-19 Patient Monitoring.

COVID-19 has challenged health systems to learn how to learn. This paper describes the context, methods and challenges for learning to improve COVID-19 care at one academic health center. Challenges to learning include: (1) choosing a right clinical target; (2) designing methods for accurate predictions by borrowing strength from prior patients' experiences; (3) communicating the methodology to clinicians so they understand and trust it; (4) communicating the predictions to the patient at the moment of clinical decision; and (5) continuously evaluating and revising the methods so they adapt to changing patients and clinical demands. To illustrate these challenges, this paper contrasts two statistical modeling approaches - prospective longitudinal models in common use and retrospective analogues complementary in the COVID-19 context - for predicting future biomarker trajectories and major clinical events. The methods are applied to and validated on a cohort of 1,678 patients who were hospitalized with COVID-19 during the early months of the pandemic. We emphasize graphical tools to promote physician learning and inform clinical decision making.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

OBJECTIVES: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. METHODS: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. RESULTS: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. CONCLUSIONS: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.

Advances at the Interface of Cancer and Systemic Sclerosis.

The interface between systemic sclerosis (SSc) and cancer has offered valuable insights into our understanding of SSc disease pathogenesis. Defining SSc subgroups both temporally and serologically has been instrumental in stratifying cancer risk, with autoantibodies to RNA polymerase 3 (RNApol3), RNA polymerase I large subunit (RPA194), RNA Binding Region Containing 3 (RNPC3), and centromere identifying subgroups at increased or decreased risk of cancer. Clinically, improved subgrouping of SSc patients provides the opportunity to detect cancer at earlier stages of disease while increasing our efficiency of cancer assessment. Additional studies are needed to define the optimal approach to cancer screening in SSc, and validation studies in different cohorts will be needed to confirm all findings.

Association of systemic lupus erythematosus autoantibody diversity with breast cancer protection.

BACKGROUND: Epidemiologic data suggest that patients with systemic lupus erythematosus (SLE) have a lower risk of breast cancer than women in the general population. In light of mechanistic studies suggesting that anti-DNA antibodies have anti-cancer effects, we sought to examine breast cancer risk in autoantibody strata in a well-characterized SLE cohort. METHODS: SLE patients without a cancer diagnosis prior to entry in the Hopkins Lupus Cohort were studied (N = 2431). Overall and site-specific cancer incidence was calculated in racial strata and compared with the US Surveillance, Epidemiology and End Results (SEER) registry. Breast cancer incidence was further examined in autoantibody subsets. Patients were considered positive for an autoantibody if they were ever positive for a specificity during their disease course. RESULTS: Patients with SLE had a 37% lower risk of breast cancer (SIR 0.63, 95% CI 0.39-0.95). The risk of HPV-associated cancers (SIR 4.39, 95% CI 2.87-6.44) and thyroid cancer (SIR 2.27, 95% CI 1.04-4.30) was increased. Cancer risk varied by race, with breast cancer protection occurring in non-African Americans (SIR 0.29, 95% CI 0.11-0.63) and the increased risk of HPV-associated cancers occurring in African Americans (SIR 7.23, 95% CI 4.35-11.3). Breast cancer risk was decreased in patients ever positive for anti-dsDNA (SIR 0.55, 95% CI 0.29-0.96), anti-La (SIR 0.00, 95% CI 0.00-0.78), and lupus anticoagulant (SIR 0.37, 95% CI 0.10-0.94). Patients who were positive for fewer (0-2) SLE autoantibodies did not have a lower risk of breast cancer (SIR 0.84, 95% CI 0.47-1.39), but patients with 3+ autoantibodies had a 59% decreased risk (SIR 0.41, 95% CI 0.16-0.84). CONCLUSIONS: Positivity for multiple SLE autoantibodies was associated with a lower risk of breast cancer, supporting the hypothesis that a highly diversified immune response may exert an anti-cancer effect against some cancers. Validation of racial differences in cancer risk in SLE is required to determine whether cancer screening strategies should be targeted to racial subgroups.

Autoantibodies targeting telomere-associated proteins in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune fibrotic disease affecting multiple tissues including the lung. A subset of patients with SSc with lung disease exhibit short telomeres in circulating lymphocytes, but the mechanisms underlying this observation are unclear. METHODS: Sera from the Johns Hopkins and University of California, San Francisco (UCSF) Scleroderma Centers were screened for autoantibodies targeting telomerase and the shelterin proteins using immunoprecipitation and ELISA. We determined the relationship between autoantibodies targeting the shelterin protein TERF1 and telomere length in peripheral leucocytes measured by qPCR and flow cytometry and fluorescent in situ hybridisation (Flow-FISH). We also explored clinical associations of these autoantibodies. RESULTS: In a subset of patients with SSc, we identified autoantibodies targeting telomerase and the shelterin proteins that were rarely present in rheumatoid arthritis, myositis and healthy controls. TERF1 autoantibodies were present in 40/442 (9.0%) patients with SSc and were associated with severe lung disease (OR 2.4, p=0.04, Fisher's exact test) and short lymphocyte telomere length. 6/6 (100%) patients with TERF1 autoantibodies in the Hopkins cohort and 14/18 (78%) patients in the UCSF cohort had a shorter telomere length in lymphocytes or leukocytes, respectively, relative to the expected age-adjusted telomere length. TERF1 autoantibodies were present in 11/152 (7.2%) patients with idiopathic pulmonary fibrosis (IPF), a fibrotic lung disease believed to be mediated by telomere dysfunction. CONCLUSIONS: Autoantibodies targeting telomere-associated proteins in a subset of patients with SSc are associated with short lymphocyte telomere length and lung disease. The specificity of these autoantibodies for SSc and IPF suggests that telomere dysfunction may have a distinct role in the pathogenesis of SSc and pulmonary fibrosis.