OBJECTIVE:Osteoarthritis (OA) involves systemic inflammation, yet peripheral blood immunophenotypes and their underlying epigenetic landscapes remain poorly defined. We characterized cellular and chromatin accessibility profiles in knee OA and radiographic progressors (RPs). METHODS:We performed multimodal immunophenotyping using cytometry by time-of-flight mass spectrometry (CyTOF) on peripheral blood samples from 21 patients with knee OA and 11 healthy controls (HCs). To confirm findings, we developed DNA methylation-based imputation models and applied them to two independent validation cohorts (n = 723). Finally, single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) was performed to interrogate chromatin accessibility landscapes in a patient subset. RESULTS:CyTOF identified significant expansions of CD8+ central memory T cells, CD4+ Treg cells, and nonclassical monocytes in patients with OA versus HCs (all P ≤ 0.05). These expansions were robustly confirmed in the large-scale validation cohorts (all P < 0.0001). Conversely, RPs displayed validated reductions in circulating CD4+ and CD8+ central memory pools. scATAC-seq revealed extensive epigenetic remodeling, particularly within monocytes and Treg cells. Paradoxically, unsupervised clustering indicated that epigenetically defined proinflammatory clusters were depleted in OA blood. Pathway analysis revealed that these depleted clusters possessed activated, migratory phenotypes, whereas the expanded circulating cells among patients with OA displayed quiescent, nonmigratory epigenetic signatures. CONCLUSION:Knee OA is characterized by validated systemic expansions of specific Treg cell and monocyte subsets. Single-cell epigenetic profiling suggests epigenetically distinct subpopulations driven by age, OA, comorbid conditions, or migration from circulation to the periphery.

Abstract Introduction Post Acute Sequelae of COVID-19 (PASC), also called Long COVID, is an infection-associated chronic syndrome. Despite proposed viral persistence mechanisms, no therapeutic benefit was observed in randomized placebo-controlled trials of nirmatrelvir/ritonavir (NMV/r) in adults with Long COVID, including the Selective Trial of Paxlovid for PASC (STOP-PASC). This systems immunology analysis aimed to characterize immune profiles of participants during clinical trial intervention, identify biomarkers associated with patient-reported outcomes, and investigate potential mechanisms underlying Long COVID. Methods We performed comprehensive immunological profiling of 152 STOP-PASC trial participants using plasma proteomics (Olink® Explore HT 5400 panel), autoantigen arrays, viral serology, and microclot assays at baseline, day 15, and week 10. We assessed associations between immune features and patient-reported outcomes. We also conducted meta-analysis of nine independent Long COVID proteomics cohorts (n = 590 total samples) to identify conserved inflammatory signatures. Results NMV/r treatment at day 15 compared with baseline induced transient changes in plasma proteins that normalized by week 10, primarily impacting myeloid cell/monocyte, lysosome, and complement activation pathways. Cardiovascular symptoms were negatively associated with SARS-CoV-2 antibody levels at baseline. No widespread differences in autoantibody profiles, Epstein-Barr virus (EBV) reactivation, or microclotting were observed between STOP-PASC Long COVID participants, pre-pandemic controls, and individuals without Long COVID. Meta-analysis of publicly available Olink® data from Long COVID cohorts identified a conserved 60-protein Long COVID Signature (LCS) score revealing multi-compartment immune activation involving monocyte, neutrophil, and T/NK cell modules. Conclusion These findings advance our understanding of Long COVID immunology and may help direct future proteomic biomarker endpoints for Long COVID clinical trials. Funding Source Pfizer Topic Categories Computational and Systems Immunology (COMP)

PT006 / #521 Topic: AS15 - Lupus Nephritis-Clinical POSTER TOUR 02: RECENT INSIGHTS ON THE PATHOGENESIS OF LUPUS NEPHRITIS 23-05-2025 10:00 AM - 10:40 AM Lupus nephritis (LN) treatment decisions are commonly guided by histopathological classifications based on the ISN/RPS and NIH activity and chronicity indices. Since LN class and activity may shift over time, treatment adjustments are often necessary. However, repeated kidney biopsies are invasive and impractical, highlighting the need for noninvasive biomarkers to inform LN classification and guide therapy. In this study, we analyzed serum proteomic profiles to identify noninvasive biomarkers reflective of histological class, activity, and chronicity indices. This study recruited 196 SLE patients with lupus nephritis (LN) as part of the AMP RA/SLE network. Each patient underwent a kidney biopsy evaluated by a renal pathologist for LN classification using the ISN/RPS system and NIH activity and chronicity indices. Serum samples were collected at biopsy to explore noninvasive biomarkers. High-throughput proteomic analysis was conducted using the Olink Explore HT platform to identify protein expression patterns linked to LN class, activity, and chronicity. Multivariate logistic regression, adjusted for age, gender, and genetic ancestry, along with random forest algorithms, were used to pinpoint potential biomarkers to guide LN treatment decisions. Compared to healthy controls, LN patients upregulated multiple pathways related to the innate and adaptive immune systems, including TNF, IL-10, efferocytosis, and antigen processing and presentation pathways. Patients with pure proliferative LN (class III or IV) showed further upregulation in B cell receptor signaling, Th1/Th2 differentiation, neutrophil degranulation, Th17 differentiation, and leukocyte chemotaxis pathways compared to those with minimal disease (class I/II), membranous (V), or mixed proliferative (III/IV+V) LN. Machine learning models using a decision-tree-based boost algorithm achieved high accuracy for distinguishing healthy controls (95.3% [86.9%-99%]) and LN patients (99.5%, [97% - 100%]), as well as advanced sclerosing (class VI), compared to other classes (AUC, 0.85 ± 0.11; accuracy, 88.1% ± 0.7%). When distinguishing membranous vs pure proliferative classes, the ML model showed a modest prediction performance with an AUC of 0.75 ± 0.06 with a cross-validation accuracy of 71.1% ± 0.6%. When compared to healthy controls, there are 862 upregulated proteins, including interferons, IL-10, and lymphocyte surface receptors, shared among patients with membranous, proliferative, and mixed classes and 92 downregulated proteins, including C2, C4, and C8 (Figure 1C). In addition, the expression of 398 and 2252 proteins was associated with the NIH activity and chronicity indices, respectively (Figure 1D). Specifically, proteins involved in IL-18, TNF, and IL-1 pathways and intracellular proteins from multiple organ systems with prominent enrichment in immune cells positively correlated with the activity index (Figure 1D). Interestingly, proteins enriched in interferon, growth factor and neurotrophin receptor pathways and intracellular proteins from multiple organ systems, particularly the nervous system, correlated with the chronicity index (Figure 1E). Figure 1. This study revealed that lupus nephritis (LN) patients exhibited significant upregulation of immune pathways, including TNF and IL-10, compared to healthy controls, particularly in proliferative LN. A machine learning model effectively distinguished LN patients from healthy controls and showed moderate performance in differentiating membranous from proliferative LN. Proteomic analysis identified proteins associated with NIH activity and chronicity indices, underscoring the potential of serum proteomics as a noninvasive tool for LN classification and monitoring.

O056 / #121 Topic:AS23 - SLE-Diagnosis, Manifestations, & Outcomes ABSTRACT CONCURRENT SESSION 09: SLE THERAPY – REVISITING OLD DRUGS AND UNLOCKING HIDDEN POTENTIAL OF NEW MEDICATIONS 24-05-2025 10:40 AM - 11:40 AM Patients with features of systemic lupus erythematosus (SLE) who do not have sufficient criteria to be classified can be designated as having incomplete lupus (ILE). This is a common condition seen in clinical practice and it has further significance as a group that has high risk of progression to SLE. Identification and treatment of those at risk has the potential to reduce the severity and incidence of SLE. Based on previous studies, hydroxychloroquine (HCQ) was chosen as an intervention for a randomized, double-blind, placebo-controlled trial to determine whether the rate of accumulation of clinical and immunologic features of SLE as defined by the 2012 SLICC criteria could be reduced. ILE was defined as ANA positivity with one or 2 additional criteria from the SLICC 2012 list. Males and females 15 to 49 years of age were eligible for enrollment. After baseline evaluation including ophthalmologic exam, participants were randomized 1:1 to HCQ or placebo. Evaluations at 3-month intervals included clinical and laboratory measures as well as patient-reported outcomes (PROs). Treatment was continued for 24 months, but if SLICC criteria were satisfied sooner, patients exited the study. Ophthalmologic exams were carried out at conclusion of treatment. A total of 187 ILE patients were randomized at 7 sites in the USA. After excluding 7 patients found to have SLE criteria at baseline when pending laboratory data were completed, 180 patients were available for analysis; 92 were randomized to HCQ and 88 received placebo. The mean age was 33 years, 91.1% were female and 74.4% were White individuals. At randomization, 65.6% had 2 SLICC criteria; the remainder had 3 SLICC criteria. The most common manifestations involved skin and joints. SLE per criteria developed in 24 participants (13.3%) during the trial who were terminated early and 40 (22%) developed additional SLICC criteria. The primary outcome was the rate of acquisition of SLICC criteria analyzed via a generalized linear mixed-effects model, with an embedded ordinal logistic regression, comparing the changes over time for the 2 arms. This showed similar slopes in the 2 groups (P=0.72). The odds of progressing to a higher SLICC score relative to the previous score was 14% smaller for every 3-month increase in time for the HCQ group and 18% smaller for the placebo group, a difference which was not statistically significant (P=0.69). A key secondary outcome was time to progression to SLE. Using a Cox proportional hazards regression model, the hazard of progressing to SLE was 10% higher for the HCQ group than for placebo, which was not a statistically significant difference (P=0.81). Adverse events were similar in the 2 groups and no serious adverse events related to use of HCQ were recorded. Five individuals were excluded from entry due to abnormal ophthalmologic findings; none developed during the trial. The SMILE results do not endorse the use of HCQ to prevent accumulation of SLICC SLE criteria. However, the definition of ILE used in SMILE does include individuals who are at risk for progressive disease and may be useful in future studies of preventive therapies. Other ongoing analyses will determine whether autoantibodies, inflammatory mediators or PROs were related to progressive illness or use of HCQ.

Background:Autoimmune disease patients on immunosuppressants exhibit reduced humoral responses to primary COVID-19 vaccination. Booster vaccine responses and the effects of holding immunosuppression around vaccination are less studied. We evaluated the efficacy and safety of additional vaccination in mycophenolate mofetil/mycophenolic acid (MMF/MPA)-, methotrexate (MTX)-, and B cell-depleting therapy (BCDT)-treated autoimmune disease patients, including the impact of withholding MMF/MPA and MTX. Methods:In this open-label, multicenter, randomized trial, 22 MMF/MPA-, 26 MTX-, and 93 BCDT-treated autoimmune disease patients with negative or suboptimal antibody responses to initial COVID-19 vaccines (BNT162b2, mRNA-1273, or AD26.COV2.S) received a homologous booster. MMF/MPA and MTX participants were randomized (1:1) to continue or withhold treatment around vaccination. The primary outcome was the change in anti-Wuhan-Hu-1 receptor-binding domain (RBD) concentrations at 4 weeks post-additional vaccination. Secondary outcomes included adverse events, COVID-19 infections, and autoimmune disease activity through 48 weeks. Results:Additional vaccination increased anti-RBD concentrations in MMF/MPA and MTX patients, irrespective of whether immunosuppression was continued or withheld. BCDT-treated patients also demonstrated increased anti-RBD concentrations, albeit lower than MMF/MPA- and MTX-treated cohorts. COVID-19 infections occurred in 30-46% of participants, were predominantly mild, and included only two non-fatal hospitalizations. Additional vaccination was well-tolerated, with low frequencies of severe disease flares and adverse events. Conclusion:Additional COVID-19 vaccination is effective and safe in immunosuppressant-treated autoimmune disease patients, regardless of whether MMF/MPA or MTX is withheld. Trial Registration. ClinicalTrials.gov (NCT#05000216).

Background: A loss of tolerance to self-antigens leads to increased levels of autoantibodies against nuclear components (ANAs) prior to clinical disease onset. However, only about 4-8% develop autoimmune disease. Patients with incomplete lupus erythematosus (ILE) exhibit some clinical symptoms with most never progressing to Systemic Lupus Erythematosus (SLE). Exact mechanisms involved in T cell dysregulation and progression of autoimmune disease remain unclear. Objectives: Investigate whether alterations in T cell populations and activation of cellular pathways are dysregulated during autoimmunity development. Methods: PBMCs from 64 subjects, divided evenly among ancestry (African, European American) and disease group: healthy (ANA-), healthy with autoantibodies (ANA+), ILE, SLE, were sorted with microfluidic flow cytometer to remove dead cells and used for multiomics single-cell analysis with 5’scRNA-seq/137-plex Total-seq, BCR/TCR repertoire to identify distinct disease-associated clusters, differential gene signatures and dysregulated pathways. Cell counts were confirmed via CyTOF. Serum soluble biomarkers levels were obtained via Olink Proximity Extension Assay (Explore HT). Results: We obtained profiles for ~650,000 cells across all PBMCs. Differences in T cell fractions were observed by disease group. Analysis of differentially expressed genes revealed the importance of metabolic processes, such as autophagy and oxidative phosphorylation; downregulation of mitochondrial dysfunction in ANA+ and upregulation of MAPK and receptor kinase signaling in ILE and SLE. Pathway analysis indicates downregulation of TNFR Signaling in SLE compared to ILE and cytokine storm signaling in ANA+ compared to ANA-. These finding were confirmed by protein. Gene set enrichment analysis of serum soluble biomarkers indicated upregulation of T cell activation, proliferation, antigen presentation, MAPK cascade and receptor kinase signaling in ILE and SLE. We observed upregulation of MAP2K6 and MAP3K5 proteins in ILE compared to ANA+ (non-parametric test; pad <0.05). Furthermore, we identified a CD4+ T cell population (CTL) with elevated expression of cytotoxic markers PRF1, GZMB, NKG7, CCL5 and transcription factors: ZNF683, IKZF1, TBX21, ZEB2. Individuals with that population express higher level of IFN related genes. Pathway analysis of CTL indicates upregulation of antiviral response, cellular cytotoxicity and exhaustion in ILE and SLE witth IFNG, STAT3 and IL10 determined as activated upstream regulators. Olink assay confirmed these results and revealed IFNB1 upregulation and viral response. TCR analysis indicates both CTL and CD8+ cytotoxic T cells have largest fraction of expanded clonotypes, with increased levels of TRAV19, TRAV8, TRAV38. Clonotypes similar transcriptionally, restricted to those two populations, are associated with higher expression of TXNIP, TMSB4X, HLA, GZMB and shared among ANA+ and ILE individuals. Conclusion: Dysregulation of signaling in T cell activation appears to be manifesting in increased oxidative phosphorylation, dysregulation of MAPK kinases or alterations in apoptotic pathways and might be suggestive of a preclinical autoimmunity development trajectory and associated with clonal expansion. Alterations of these processes vary by ancestral background, reflecting the heterogeneity of SLE presentation. REFERENCES: [1] Dorner, T. and R. Furie, Novel paradigms in systemic lupus erythematosus. Lancet, 2019[2] Slight-Webb, S., et al., Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype. J Allergy Clin Immunol, 2020 Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1A. UMAP projection of distinct T cell clusters. B. T cell density for total population, by ancestry and disease groups. C. Pathway analysis of CD8+, CD4+ T cells with most distinct differences between ANA+ compared to ILE. D. Expression of cytotoxic markers across T cell clusters with CTL population highlighted. E. Fractions of CTL by disease group. F. Pathway analysis of CTL. G. Clonal expansion by T cell population (blue – singleton, orange – clonotypes with 2 cells, green – >3 cells)

O040 / #227 Topic: AS15 - Lupus Nephritis-Clinical ABSTRACT CONCURRENT SESSION 06: LUPUS NEPHRITIS – CLINICAL OUTCOMES, PREDICTION AND THERAPY 23-05-2025 1:40 PM - 2:40 PM Kidney survival is the ultimate outcome in lupus nephritis (LN), but predictors remain inadequately studied due to the need for long-term follow-up. This study aimed to identify clinical and histological predictors of kidney survival in LN. The Accelerating Medicines Partnership (AMP) enrolled patients undergoing a clinically indicated (UPCR >0.5) kidney biopsy with resultant histology class II, III, IV, and/or V lupus nephritis (LN). Clinical and demographic features were collected from the time of diagnostic biopsy. Response was defined at 1 year for patients with baseline UPCR >1. Histological features were centrally scored. Kidney function loss was defined as a sustained 40% decline in estimated glomerular filtration rate (eGFR) or progression to end-stage kidney disease (ESKD). A Cox proportional hazard model was employed to identify predictors. We included 172 patients with a median follow-up time of 4.6 years (range 0.5-7.8), of whom 153 (89%) had >3 years follow-up. Clinical and demographic features are summarized in Table 1. A third of patients (56/172) developed eGFR loss with a median time to event of 2.6 years (range 0.13-7.1). Predictors of eGFR loss at time of biopsy included lower eGFR (especially eGFR <30 ml/min, HR 5.4), repeat biopsy status (HR 2.5), and NIH Chronicity Index (histological damage, HR 1.3 per unit) (Table 1). Sex, race, age, BMI, proteinuria, ISN class, NIH Activity Index, C3, C4, and anti-dsDNA were not associated with eGFR loss. Among histological features, an NIH Chronicity Index >2 (HR 2), glomerulosclerosis (HR 1.9), interstitial fibrosis (HR 2), tubular atrophy (HR 1.9), and interstitial inflammation (HR 2-but p=0.06), but not fibrous crescents or any of the NIH Activity Index glomerular features, were associated with eGFR loss (Table 1). Lack of clinical response at 3, 6, or 12 months was associated with future eGFR loss (Figure 1). Partial response at 12 months had higher risk of eGFR loss compared to complete response (HR 10.7), but lower than no response (HR 19). Reductions of UPCR >25% at 3 and >50% at 6 months were protective (HR 0.44-but p=0.11 and 0.3, respectively). Four patients with UPCR <0.5 at 1 year developed eGFR loss (1.8/100 person-years). Table 1. Association of baseline clinical, demographic, and histological features with GFR loss. Figure 1. Association of longitudinal clinical features with GFR loss Low baseline eGFR and histological damage, but not activity or ISN class, predicted eGFR loss. Improvement of UPCR was associated with lower risk of eGFR loss, though eGFR loss still occurred in patients in clinical remission (UPCR <0.5) at 1 year. Acknowledgments: This work was funded by the Plank Family Foundation and the Jerome L. Greene Foundation. The Hopkins Lupus Cohort is supported by NIH R01-DK-134625. Additionally, this research was supported by the Accelerating Medicines Partnership® Rheumatoid Arthritis and Systemic Lupus Erythematosus (AMP® RA/SLE) Network, a public-private partnership involving AbbVie Inc., Arthritis Foundation, Bristol Myers Squibb Company, Foundation for the National Institutes of Health, GlaxoSmithKline, Janssen Research and Development, LLC, Lupus Foundation of America, Lupus Research Alliance, Merck & Co., Inc. Sharp & Dohme Corp., National Institute of Allergy and Infectious Diseases, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Pfizer Inc., Rheumatology Research Foundation, Sanofi, and Takeda Pharmaceuticals International, Inc. The AMP Network aims to develop new methods for identifying and validating promising biological targets for diagnostics and drug development. Funding was provided through grants from the National Institutes of Health (UH2-AR067676, UH2-AR067677, UH2-AR067679, UH2-AR067681, UH2-AR067685, UH2-AR067688, UH2-AR067689, UH2-AR067690, UH2-AR067691, UH2-AR067694, and UM2-AR067678).

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying noninvasive, blood-based immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation and identify correlates of renal parameters. Unbiased analysis identified 3 immunologically distinct groups of patients that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at 1 year. Patients with enriched circulating granzyme B+ T cells showed more active disease and increased numbers of activated CD8+ T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive but with chronic renal injuries. The major immunologic axes of variation could be distilled down to 5 simple cytometric parameters that recapitulate several clinical associations, highlighting the potential for blood immunoprofiling to translate to clinically useful noninvasive metrics to assess immune-mediated disease in LN.

Rheumatoid arthritis (RA) is a systemic autoimmune disease currently with no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in at-risk populations prior to clinical onset is crucial to establishing effective prevention strategies. Here, we applied multimodal single-cell technologies (mass cytometry and CITE-Seq) to characterize the immunophenotypes in blood from at-risk individuals (ARIs) identified through the presence of serum antibodies against citrullinated protein antigens (ACPAs) and/or first-degree relative (FDR) status, as compared with patients with established RA and people in a healthy control group. We identified significant cell expansions in ARIs compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5lo naive B cell population was expanded in ACPA-positive FDRs. Further, we uncovered the molecular phenotype of the CCR2+CD4+ T cells, expressing high levels of Th17- and Th22-related signature transcripts including CCR6, IL23R, KLRB1, CD96, and IL22. Our integrated study provides a promising approach to identify targets to improve prevention strategy development for RA.