POS1066 SINGLE-CELL MULTI-OMIC EVALUATION OF DIFFERENCES IN T CELL POPULATIONS IN PROGRESSION OF SLE

• Published in: Annals of the rheumatic diseases Vol. 83; no. Suppl 1; p. 944
• Main Authors: Bylinska, A., Smith, M., Lu, R., Jones, B., Guthridge, C., Marlin, M. C., Wright, C., Macwana, S., Dejager, W., Beel, M., Lessard, C. J., Arriens, C., Merrill, J., James, J. A., Guthridge, J. M.
• Format: Journal Article
• Language: English
• Published: Kidlington BMJ Publishing Group Ltd and European League Against Rheumatism 01.06.2024; Elsevier B.V; Elsevier Limited
• Subjects: Adaptive immunity; Antigen presentation; Antiviral drugs; Apoptosis; Autoantibodies; Autoantigens; Autoimmune diseases; Autoimmunity; Autophagy; Biomarkers; CD4 antigen; CD8 antigen; Cell activation; Cell density; Cytokine storm; Cytokines and Chemokines; Cytotoxicity; Down-regulation; Genes; Immunological tolerance; Kinases; Lupus; Lymphocytes; Lymphocytes T; MAP kinase; Microfluidics; omics; Oxidative phosphorylation; Phosphorylation; Scientific Abstracts; Signal transduction; Systemic lupus erythematosus; T cell receptors; Transcription factors; Tumor necrosis factor receptors; omics; Adaptive immunity; Cytokines and Chemokines
• DOI: 10.1136/annrheumdis-2024-eular.6154
• ISSN: 0003-4967

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, 2020Figure 1.A. 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)Acknowledgements:NIL.Disclosure of Interests:None declared.