Numbers of ARID3a ( AT - R ich Interaction D omain 3a ) -expressing B lymphocytes from patients with systemic lupus erythematosus (SLE) are associated with increased disease activity. Normally, ARID3a-expressing circulating naïve B cells are rare, but in SLE naïve B cells dramatically increase ARID3a expression. We found that in vitro stimulation of B lymphocytes from healthy individuals with a cocktail of cytokines and agonists induced ARID3a in a subset of activated naïve B cells and in IgD - CD27 - double negative B cells previously associated with autoimmunity. Single cell RNA-seq of isolated naïve B cells from ten SLE patients, with varying frequencies of ARID3a-expressing cells, revealed that ARID3a-associated genes included activation markers. Moreover, our data revealed the unexpected co-expression of the scavenger receptor CD68 with ARID3a, at both the transcript and protein level, in activated subsets of naïve B cells. Inhibition of ARID3a in stimulated B cell cultures blocked naïve B cell activation and CD68 expression. Together, these data identify ARID3a and CD68 as markers of naïve B cell precursors associated with autoimmunity in SLE.

Systemic lupus erythematosus (SLE) etiology involves complex interactions between genetic and environmental stimuli that lead to autoantibody production, chronic inflammation, and target organ involvement. Infections are considered likely environmental triggers of SLE because certain viruses and bacteria can stimulate the innate immune system, incite the production of autoreactive responses, and alter immune cell activation and regulation. Specifically, decades of research point to Epstein–Barr virus (EBV) contributing to lupus pathogenesis. EBV promotes the development of lupus-associated autoantibodies through molecular mimicry and epitope spreading, and EBV proteins can drive aberrant activation of B cells and cytotoxic T cells by mimicking the functional effects of CD40 and interleukin-10. In addition, EBV encodes several transcription factors and microRNAs that broadly disrupt the immune system by altering the activation of gene networks that regulate immune activity. In individuals who are inherently susceptible to immune dysregulation, such microbe-driven changes can enhance the onset or progression of autoimmune disease. Finally, some infections may protect against autoimmunity in animal models, highlighting the intricacy of the gene-environment interactions that influence SLE pathogenesis.

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538,194 single-cell and 142,881 single-nuclear profiles from kidney biopsies of 155 patients with LN and 30 preimplantation transplant biopsy controls, along with 327,326 single-cell blood profiles. We characterized key stromal and immune cell types and cell states; moreover, we distinguished cell states that were tissue specific from those that were also present in the blood. We observed that LN pathological features were associated with particular cell states. For example, after controlling for the effects of chronic tissue damage, we observed that expansion of glomerular and scar-associated macrophage populations correlated with increasing inflammatory disease activity. Scar-associated macrophages appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations support that therapeutic targeting of myeloid populations may offer a strategy to prevent renal inflammation and ongoing kidney damage in LN.

Objectives Lupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aimed to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. Methods We used single-cell RNA-sequencing (scRNA-seq) data of dissociated kidney samples from 156 patients with LN and 30 healthy individuals. We applied spatial transcriptomics (ST), utilising a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, to profile kidney samples acquired from 6 patients with LN and 2 controls. Results We generated a catalogue of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the National Institutes of Health chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4 T, GZMK+ CD8 T, B, and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with the expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are implied to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. Conclusions We suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

Genome-wide association studies have identified genetic polymorphisms at 11p15 associated with Systemic Lupus Erythematosus (lupus). Statistical fine mapping prioritizes a highly prevalent coding haplotype within the IRF7 gene. Analysis of ancient DNA confirms that this haplotype has persisted at high frequencies in the global population for millennia. The IRF7 risk haplotype is sufficient to increase nuclear localization of IRF7 and transcriptional activity downstream of pattern recognition receptor pathways. This risk haplotype increases IRF7 DNA binding strength and alters IRF7 DNA sequence specificity, resulting in genotype-dependent increases in IFN-α production in numerous biological systems, including monocytes and airway epithelial cells. CRISPR engineering of a homologous risk variant in mouse Irf7 results in both enhanced innate control of virus infection and increased autoantibody titers in a model of autoimmunity. Altogether, we establish a persistent and prominent genetic IRF7 haplotype that amplifies IRF7 activity in a manner that has immunological risks and benefits. HIGHLIGHTS:Genetic analysis using modern and evolutionary datasets identifies a persistent and highly prevalent lupus-associated coding haplotype in IRF7 at 11p15 The IRF7 lupus risk haplotype increases IFN-α production by monocytes and airway epithelial cells The IRF7 lupus risk haplotype increases IRF7 DNA binding strength and alters DNA sequence specificity A homologous lupus risk variant in mouse Irf7 enhances control of vesicular stomatitis virus and exacerbates autoantibody production.

Symptoms of acute SARS-CoV-2 infection often resolve quickly but are sometimes associated with persistent immune dysfunction. The factors that predispose individuals to compromised immune function have not been well defined. We investigated CD4+ T cell phenotype and function in a small cohort of individuals who recovered from mild to moderate SARS-CoV-2 infection without hospitalization and were divided into short or prolonged symptom duration groups. Five individuals with prolonged symptom duration showed marked downregulation of CD4 on CD3+CD8- T cells (CD4low group) and a poor response to TCR stimulation with the superantigen Staphylococcal enterotoxin B (SEB), as shown by weak upregulation of the activation markers CD134, CD25, CD279, and CD69. CD4 surface intensities recovered to normal levels in four of these individuals within 3-12 months. Selected cytokines (IL-1RA, IL-7, and VEGF) were elevated in individuals with low CD4, but plasma levels of anti-S1 IgG did not correlate with CD4 hyporesponsiveness. Bulk RNA sequencing of unstimulated and SEB-treated CD3+CD8- T cells revealed a > 50% reduction in the number of differentially expressed genes in the CD4low group compared to the same individuals after CD4 levels were recovered and a healthy control group. Analyses of differentially expressed genes in unstimulated CD4low cells suggested a response to IFN, while SEB-stimulated CD4low cells showed reduced functionality of T cell activation, differentiation, and glycolysis pathways. In summary, in some individuals, prolonged symptomatic recovery from SARS-CoV-2 infection was associated with evidence of IFN signaling and transient reduction in CD4 expression accompanied by attenuated TCR activation by SEB.

OBJECTIVE:Kidney survival is the ultimate goal in lupus nephritis (LN) management, but long-term predictors remain inadequately studied, requiring long-term follow-up. This study aimed to identify baseline and early longitudinal predictors of kidney survival in the Accelerating Medicines Partnership LN longitudinal cohort. METHODS:We performed time-to-event analyses of clinical, centrally scored histologic, and serological predictors of kidney function loss (sustained ≥40% estimated glomerular filtration rate [eGFR] decline or progression to end-stage kidney disease) in 172 LN patients with a median follow-up of 4.6 years (range 0.5-7.8). RESULTS:Kidney function loss occurred in 57 of 172 (33%) patients. Baseline lower eGFR, non-first kidney biopsy, and higher National Institutes of Health (NIH) chronicity index were associated with eGFR loss. Chronicity index was the strongest predictor, but no clear threshold defined higher risk. NIH activity index and International Society of Nephrology (ISN) class were not predictive. Proteinuria at 12 months was prognostic, with urine protein-to-creatinine ratio <0.7 g/g associated with lower risk; however, no single threshold ensured protection, and lower levels had better outcomes. Lack of complete clinical response at 3, 6, or 12 months predicted future eGFR loss, whereas partial response conferred intermediate risk. Serological markers were not associated with eGFR loss. CONCLUSION:Low baseline eGFR and chronic histologic damage, but not activity or ISN class, predicted eGFR loss. Proteinuria <0.7 g/g at one year was associated with better outcomes but did not ensure protection. Because proteinuria does not reflect intrarenal inflammation, these results suggest current response definitions serve better as prognostic indicators than true measures of treatment efficacy, and better biomarkers are needed.

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), features heterogeneous renal pathology and reliance on invasive biopsies for diagnosis, prognosis, and treatment selection. Current peripheral clinical markers inadequately capture disease activity and progression. Here, we performed comprehensive serum proteomic profiling of over 5,000 proteins in the large, longitudinal Accelerating Medicines Partnership Rheumatoid Arthritis/SLE cohort of 270 LN patients and 63 healthy controls. Machine learning identified distinct molecular signatures that classified LN versus controls, differentiated histological classes, and delineated activity- and chronicity-associated pathways, including inflammatory cytokine, PI3K/AKT, TGFb, and complement/coagulation pathways. An increase in VSIG4, CD27, HAVCR1, and LAIR1 consistently emerged as top biomarkers across multiple clinical contexts, and early decreases in these markers at 3 months were associated with complete treatment response at 1 year. By resolving coordinated serum protein modules linked to key inflammatory, PI3K/AKT, TGFb, and complement pathways, these signatures mechanistically connect circulating proteomic perturbations to intrarenal immune activation, tissue injury, and repair in LN. These findings demonstrate that serum proteomics reflect complex intrarenal immunopathology and offer a promising noninvasive "liquid biopsy" approach to refine LN classification and guide personalized management, potentially reducing the need for repeated invasive biopsies and improving therapeutic decision-making.

Lupus nephritis (LN), a severe manifestation of Systemic Lupus Erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538K single-cell and 140K single-nuclear profiles from kidney biopsies of 155 LN patients and 30 pre-implantation transplant biopsy controls, along with 325K single-cell blood profiles overlapping many of these patients. We identified key tissue cell types and cell states, and immune cell states; we were able to determine cell states that were tissue specific, and those that were present in the blood. We observed that LN pathological features are significantly associated with cell states using differential gene expression and Covarying Neighborhood Analysis (CNA). These analyses revealed broad changes in cell states associated with irreversible chronic tissue damage. After controlling for the effects of ongoing tissue damage, we observed that expansion of key glomerular and Scar Associated Macrophages (SAMs) populations tracked with increasing inflammatory disease activity. SAMs appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations strongly support that therapeutic targeting of myeloid populations may offer an as-of-yet unproven strategy to prevent renal inflammation and ongoing kidney damage in LN.

Background: Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases. Methods: Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering. Results: This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally. Conclusion: We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.

Objectives We assessed the role of a systemic lupus erythematosus causal, hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in Sjögren’s disease (SjD). Methods Association between NCF1-H90 and SjD was assessed in case-control cohorts. Peripheral blood mononuclear cells (PBMCs) and minor salivary gland (MSG) from patients with Sjögren's Syndrome type A antigen (SSA)+ SjD were assessed using cytometry by time-of-flight and single-cell RNA sequencing, respectively. Results The NCF1-H90 allele was associated with increased risk for SjD in Chinese and European Americans (Pmeta = 1.15E-55, odds ratio [OR] = 2.58), exhibiting a more robust association in patients with SSA+ SjD (OR = 3.37 in Chinese and OR = 2.60 in European Americans). In a longitudinal observational cohort, patients with homozygous H90 SjD at baseline had lower levels of complement C4 and higher scores on labial salivary gland biopsy, European Alliance of Associations for Rheumatology (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI), and EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI). These patients with H90 SjD sustained elevated ESSDAI and ESSPRI scores during follow-up years with decreased survival. The 0, 1, and 2 copies of H90 carriage in SjD PBMCs exhibited dose-dependent decreases in switched memory B cells, which might be recruited by fibroblasts into MSG, and further differentiated into long-lived Immunoglobulin G (IgG+) plasma cells, resulting in elevated serum SSA+ IgG levels and greater disease severity. In a retrospective belimumab-treated SSA+ female cohort, homozygous H90 patients showed significantly greater improvement in ESSDAI, C4 levels, and serum SSA+ IgG levels, supporting the role of NCF1-H90 as a predictive biomarker for enhanced response to B-cell-targeted therapy. Conclusions Low NCF1 activity increases the risk of severe SSA+ SjD by driving switched memory B-cell trafficking and IgG+ plasma cell differentiation, revealing targetable pathways in SSA+ SjD.

Two phase 1 studies report promising outcomes with chimeric antigen receptor (CAR) T cell therapies in patients with systemic lupus erythematosus, but the next stage of development must address key pitfalls around comparator arms and ancillary immune-modulating treatments.

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.

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).

No theragnostic biomarkers exist for systemic lupus erythematosus (SLE) to enable a precision medicine approach. Baseline serum IgA2 anti-double-stranded DNA (dsDNA) antibody levels are associated with response to combination belimumab after rituximab therapy in SLE (BEAT-lupus trial, ISRCTN 47873003). Analysis of the CALIBRATE trial (NCT02260934) confirms that baseline IgA2 anti-dsDNA antibody levels are specifically associated with response to belimumab after rituximab (odds ratio [OR] = 16.9, confidence interval [CI]: 2.8-101, compared to rituximab alone-CALIBRATE and BEAT-lupus combined data). IgA2 anti-dsDNA antibody levels decrease alongside IgA2 expression in plasmablasts only after this combination treatment. Increased serum B cell-activating factor (BAFF) levels are associated with rising IgA2 anti-dsDNA antibody levels after rituximab. IgA2 plasmablasts have increased BAFF receptor and interleukin (IL)-10 expression compared to IgA1 plasmablasts and have a distinct integrin profile implicating a gut mucosal origin. These findings validate IgA2 anti-dsDNA antibodies as a theragnostic biomarker of response and provide mechanistic insight into the selective targeting of IgA2+ B cells by combination belimumab after rituximab in SLE.

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)

Objectives Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Significant morbidity and early mortality necessitate early intervention. This study harnessed SLE-associated immune dysregulation to create a Lupus Classification Risk Index (LCRII) and Lupus Disease Activity Immune Index (LDAII) that identified individuals at risk for SLE classification and disease activity.Methods The LCRII was developed from 84 military personnel who developed classified SLE (≥4 American College of Rheumatology criteria) versus matched healthy controls, which was confirmed in 56 lupus blood relatives who developed SLE versus 154 matched unaffected relatives and 77 unrelated controls. The LDAII was informed by SLE patient visits with low (n=132) or active (n=179) disease and 48 matched controls. Data from blood samples assessed for circulating SLE-associated autoantibody specificities and soluble immune mediators informed the LCRII and LDAII. Random forest modelling guided the selection of informative analytes.Results An LCRII informed by 32 or 17 log-transformed/standardised mediators, weighted by their correlation to SLE-associated autoantibodies, differentiated pre-SLE individuals before reaching disease classification (area under the curve (AUC) ≥0.79, p<0.0001; effect size ≥1.1), even before the appearance of clinical criteria (AUC ≥0.74, p<0.0001; effect size ≥0.9). The LCRII-32, LCRII-17 and select mediators, MCP-3/CCL7, TNFRII, stem cell factor (SCF), IL-1α, IP-10/CXCL10 and TGF-β differentiated renal and serositis classification criteria (p<0.05). An LDAII informed by 26 or 13 log-transformed/standardised mediators, weighted by their correlation to SLE-associated autoantibodies or disease activity (hybrid Systemic Lupus Erythematosus Disease Activity Index; hSLEDAI), differentiated SLE patients with low (hSLEDAI <4) or active (hSLEDAI ≥4) disease (AUC >0.6, p ≤0.002, effect size ≥0.4), including clinical/serologic active versus quiescent disease (AUC ≥0.7, p<0.0001, effect size ≥0.6). The LDAII-26, LDAII-13 and select mediators MCP-1/CCL2, TNFRII, SCF, IL-2Rα, IL-10 and TGF-β differentiated renal and serositis manifestations.Conclusions We have conceptualised two immune mediator-informed indexes, the LCRII that predicts SLE from months to years before clinical presentation, and the LDAII that analogously predicts active disease in SLE to distinguish patients who would benefit from early intervention.

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.