A MULTI-DIMENSIONAL APPROACH REVEALS A DYSREGULATED SYSTEMIC LUPUS ERYTHEMATOSUS IMMUNE RHEOSTAT WITH AN ABNORMAL IMMUNOREGULATORY RESPONSE AND REDUCED CTLA4 EXPRESSION IN EFFECTOR T CELLS

J. G. Yeo, K. Nay Yaung, A. Law, M. Wasser, T. Arkachaisri, J. Thumboo, A. Low, S. AlbaniSingHealth Duke-NUS Academic Medical Centre, Translational Immunology Institute, Singapore, Singapore KK Women’s and Children’s Hospital, Rheumatology and Immunology Service, Singapore, Singapore Duke-NUS Medical School, Duke-NUS Medical School, Singapore, Singapore Singapore General Hospital, Department of Rheumatology and Immunology, Singapore, Singapore  Background Systemic Lupus Erythematosus (SLE) immunopathogenesis involves a complex network of regulatory and effector cells with its balance constituting an immune rheostat that maintains immune homeostasis. Thus, the disease must be interrogated holistically to identify mechanistically and clinically relevant immune cell subsets for its pathogenesis. There is a crucial unmet need to examine the mechanism of tolerance loss in lupus and identify novel targets for potential therapeutic intervention to improve outcomes. Objectives To characterise the SLE immunome holistically and address our hypothesis that SLE is driven dually by an impaired immunoregulatory axis and perturbed immune effector system. Methods Forty-one peripheral blood mononuclear cell (PBMC) samples from 26 adult SLE patients and 27 age-matched healthy controls were studied with a 43 markers mass cytometry panel. The SLE patients (23 females) had a median age of 40 (interquartile range [IQR]: 28 to 54) years with a median SLEDAI 2K score of 4 (IQR: 0 to 6). Quality check, batch-effect correction, cell clustering, annotation and visualisation after t-distributed stochastic neighbour embedding (tSNE) dimensional reduction was done using our extended polydimensional immunome characterisation (EPIC) pipeline [1]. Frequencies are expressed as a percentage of total CD45 PBMC or ratio and described with median and IQR. Statistical significance is defined as p<0.05 (Mann-Whitney U test with Bonferroni correction). Results Our multi-parametric approach reveals multiple derangements in all the major immune lineages but predominantly in the CD4 T cell population (Figure 1). Interestingly, there were no significant differences in the memory (CD45RO) and naïve (CD45RA) Treg (CD3CD4CD25Foxp3CTLA) subsets between SLE and controls. Instead, an enrichment of an activated memory Treg-like T subset (CD3CD4CD45ROCD25Foxp3CTLAICOS) was found in SLE (SLE versus healthy: 0.86 [0.43 to 1.38]% versus 0.31 [0.24 to 0.42]%, p<0.01). These Treg-like cells do not express CD25, a phenotypic marker for natural Treg cells. Concurrently, there was a significant reduction in CTLA4 expressing naïve non-Treg CD4 T (effector) cells in lupus (SLE versus healthy: 0.59 [0.40 to 1.11]% versus 2.0 [0.9 to 2.9]%, p<0.01). This was accompanied by a significant reduction in the ratio of CTLA4 to CTLA4 naïve non-Treg TNFα effector T cells (SLE versus healthy, ratio of CTLA4 to total naïve non-Treg TNFα T cells: 0.14 [0.11 to 0.23] versus 0.33 [0.17 to 0.55], p<0.0001). Additionally, other significant changes such as activated IL4 T cell subset (CD3CD4CD45RAHLA-DRIL4) was increased in lupus (SLE versus healthy: 0.30 [0.19 to 0.52]% versus 0.07 [0.05 to 0.12]%, p<0.0001). Image/graph:Figure 1. (A) Cell types embedded tSNE plot. (B) Healthy and SLE immunomes (Density plots). (C) Expression embedded tSNE plots. (D) Differentially enriched CD4 T cell clusters in either healthy or SLE. Blue: significantly increased cell subsets in healthy. Red: significantly increased cell sub-sets in SLE. All plots are derived from a random sampling of 50,000 single-cell events, dimensionally reduced with 43 markers (n=23 healthy and 41 SLE). Conclusion There are multiple derangements in the immunoregulatory and effector axes in lupus consistent with its complex immunopathogenesis. The activated CD25 negative Treg-like subset indicates an impaired regulatory response in SLE. The reduced CTLA4 expression in the naïve non-Treg T cells in lupus suggests a perturbed negative feedback mechanism in the effector system. The validation of these results and delineation of the underlying disease mechanisms have translational therapeutic potential. Reference [1]Yeo JG, Wasser M et al. The Extended Polydimensional Immunome Characterisation (EPIC) web-based reference and discovery tool for cytometry data. Nat Biotechnol. 2020 Jun;38(6):679-684. Acknowledgements This research was supported by the National Research Foundation Singapore under its National Medical Research Council (NMRC) Centre Grant Programme (MOH-000988) and is administered by the Ministry of Health, Singapore’s NMRC. Other NMRC grant support CIRG21nov-0031 and CSAINV22jul-0008 is gratefully acknowledged. Disclosure of Interests None Declared. Keywords: Adaptive immunity, Systemic lupus erythematosus, Biomarkers DOI: 10.1136/annrheumdis-2023-eular.2990Citation: , volume 82, supplement 1, year 2023, page 140Session: Novel insights into disease taxonomy and immunophenotyping (Oral Presentations)