POS1066 SINGLE-CELL MULTI-OMIC EVALUATION OF DIFFERENCES IN T CELL POPULATIONS IN PROGRESSION OF SLE
Authors: A. Bylinska, M. Smith, R. Lu, B. Jones, C. Guthridge, M. C. Marlin, C. Wright, S. Macwana, W. Dejager, M. Beel, C. J. Lessard, C. Arriens, J. Merrill, J. A. James, J. M. Guthridge
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> 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. <h3>Objectives:</h3> Investigate whether alterations in T cell populations and activation of cellular pathways are dysregulated during autoimmunity development. <h3>Methods:</h3> 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). <h3>Results:</h3> 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 &lt;0.05). Furthermore, we identified a CD4+ T cell population (CTL) with elevated expression of cytotoxic markers <i>PRF1</i>, <i>GZMB</i>, <i>NKG7</i>, <i>CCL5</i> and transcription factors: <i>ZNF683</i>, <i>IKZF1</i>, <i>TBX21</i>, <i>ZEB2</i>. Individuals with that population express higher level of <i>IFN</i> related genes. Pathway analysis of CTL indicates upregulation of antiviral response, cellular cytotoxicity and exhaustion in ILE and SLE witth <i>IFNG, STAT3 and IL10</i> determined as activated upstream regulators. Olink assay confirmed these results and revealed <i>IFNB1</i> upregulation and viral response. TCR analysis indicates both CTL and CD8+ cytotoxic T cells have largest fraction of expanded clonotypes, with increased levels of <i>TRAV19</i>, <i>TRAV8</i>, <i>TRAV38</i>. Clonotypes similar transcriptionally, restricted to those two populations, are associated with higher expression of <i>TXNIP</i>, <i>TMSB4X</i>, <i>HLA</i>, <i>GZMB</i> and shared among ANA+ and ILE individuals. <h3>Conclusion:</h3> 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. <h3>REFERENCES:</h3> [1] Dorner, T. and R. Furie, <i>Novel paradigms in systemic lupus erythematosus.</i> Lancet, 2019 [2] Slight-Webb, S., et al., <i>Autoantibody-positive healthy individuals with lower lupus risk display a unique immune endotype.</i> J Allergy Clin Immunol, 2020 <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>