Background: Systemic lupus erythematosus (SLE) is preceded by an at-risk stage of disease that can be marked by the presence of antinuclear antibodies (ANA) but the simultaneous absence of clinically overt disease across the distinct organ domains. The aim of this study was to create a detailed characterisation at the single-cell level in ANA+ individuals at-risk of developing SLE. Using integrative multi-omics (single-cell RNA-sequencing and CITEseq), we longitudinally profiled ANA+ at-risk individuals, identifying participants who developed clinical SLE, and signifying changes in the immunobiology of the at-risk stage.

Objectives: This study aimed to characterise at the single-cell level the immune profile of ANA+ at-risk indivuauls and to identify predictive features of progression to SLE.

Methods: Blood samples were obtained from patients from high disease activity SLE (n = 20), low disease activity SLE (n = 23), at-risk individuals before (n = 23) and after (n = 23) developing SLE (progressors), and at-risk individuals (n =23) who never progressed to SLE (non-progressors), and age- and sex-matched healthy donors (n = 17). SLE patients met 2012 SLICC classification criteria and disease activity was evaluated using BILAG-2004. Cryopreserved human peripheral blood mononuclear cells (PBMCs) were thawed; cell count and viability were assessed using acridine orange/propidium iodide fluorescence on a LUNA-FX7 cell counter (Logos Biosystems). Eight-sample pools were generated by combining equal numbers of viable cells, followed by viability-based sorting using 7-AAD dye (BioLegend, 420403). Sorted pools were loaded onto the 10x Genomics Chromium X (GEM-X 5’ Chip) according to the manufacturer’s protocol (CG000734), with 30,000–50,000 cells loaded per channel. Gene expression, feature barcoding, and V(D)J libraries were prepared using the Chromium GEM-X platform with Feature Barcode technology for Cell Surface Protein User Guide (CG000734). Final libraries were pooled and sequenced on the Illumina NovaSeq X system using a 2 × 150 bp configuration on a 25B flow cell. Clustering, differential abundance, and differential expression were performed by computational analysis.

Results: Pseudobulk analysis of the single-cell data highlighted the distinct transcriptomic profile of the ANA+ at-risk individuals against the healthy controls. Principal analysis identified the cellular composition at the single-cell level across the samples, which significantly varied among the patient groups and within the progressors and non-progressors. The largest differences in cellular abundance were observed in the myeloid and B cell compartments highlighting an expansion of specific cell subsets at baseline in the ANA+ at-risk individuals prior to the development of SLE. The highest effect size of differential expressed genes among the ANA+ at-risk individuals (progressors vs non-progressors) was also seen in myeloid and B cell subsets. Pathway analysis in those cell subsets revealed that type I and II interferon responses, complement activation, chemokine receptor binding were highly associated with progression to SLE, whilst negative regulation of other inflammatory and metabolic pathways appeared to reversely correlate with progression to SLE from the at-risk stage.

Conclusions: We created a detailed single-cell atlas of PBMCs from ANA+ patients at-risk of SLE highlighting subtle changes in cellular abundance and transcriptomic profile, which would be impossible with bulk techniques. Our novel findings have signified the pathogenesis of the ANA+ at-risk stage supporting the concept that disease onset occurs much earlier than clinically overt SLE. Additionally, this extensive immune resource of the ANA+ at-risk stage and progression to SLE can enable further interactive tools for subsequent investigations.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Antonios Psarras AstraZeneca, Liezel Tamon: None declared, Sinibaldo Arocha: None declared, Moustafa Attar: None declared, Karene Argoud: None declared, Md Yuzaiful Md Yusof GSK, Novartis, Alumis, Roche, Novartis, CSL Vifor, Aurinia, UCB, Rachael J.M. Bashford-Rogers: None declared, Alexander Clarke: None declared, Edward M. Vital AstraZeneca, Novartis, Pfizer, UCB, Abbvie, Aurinia, BMS, Janssen, Roche, Otsuka, Lilly, Dianthus, Ventus, CESAS, Medscape, Limbic, Roche, AstraZeneca.