CHARACTERIZATION OF MEMORY T CELL SUBSETS DURING FLARES AND DISEASE QUIESCENCE IN LUPUS

O003 / #470 Topic: AS01 - Adaptive Immunity SCIENTIFIC HYBRID SESSION: BASIC TRACK PRESENTATIONS - OUTSTANDING ABSTRACT PRESENTATIONS 23-05-2025 9:00 AM - 10:00 AM Background/Purpose Around 70% of Systemic Lupus Erythematosus (SLE) patients follow a relapsing-remitting pattern of disease, with unpredictable flares of disease activity followed by variable periods of disease quiescence. CD4 + T cell subsets have been shown to play an important role in driving the autoantibody production which causes flares in SLE, however the precise T cell changes that accompany flares are unknown. Here, we characterized the antigen-experienced memory T cell compartment at various phases of disease to gain insight into this question. Methods CITE-seq was used to assess the transcriptomic profiles of CD4 + memory T cells in flaring (change in the clinical SLEDAI-2K > 0 in the last month prompting a change in therapy) and quiescent (clinical SLEDAI = 0 for at least a year, prednisone dose < 10) SLE patients. CD4 + memory T cells were isolated from previously archived PBMCs, stained with oligo-conjugated antibodies against surface proteins, and then partitioned, barcoded, and sequenced. Samples from 15 distinct patients at 2 separate clinical visits spaced at least 1 year apart were examined. TCR sequencing was performed to assess clonotype expansion/contraction. The longitudinal nature of our data permitted examination of transcriptional changes both between and within patients. Results Integration of the gene and surface protein expression data led to identification of 23 unique immune populations (Figure 1A). Samples from flaring patients were more enriched for T follicular helper (Tfh), T peripheral helper (Tph), and Th1 cells. Conversely, quiescent patients were more enriched for central memory T cells (TCMs), specifically TCM1/2/6, compared to flaring patients (p < 0.05) (Figure 1B,C). There was no difference in the proportion of exhausted Treg cells between flaring and quiescent patients at baseline suggesting that exhaustion of this subset is a consistent feature of SLE, potentially contributing to an inherent level of immune dysregulation regardless of clinical flare status. Differential gene expression analyses at baseline showed a high prevalence of IFN-induced genes in most identified cell subsets within the genes that were upregulated in flaring compared to quiescent patients, with a few exceptions (eg, Th1). TCR repertoire analyses of samples at baseline revealed a higher proportion of expanded clonotypes in various clusters, such as Th17, in flaring patients (Figure 2A), which was not seen in quiescent patients (Figure 2B). Similarly, clonal overlap among subsets was more pronounced in the flaring patient samples (Figure 2C) than in the quiescent samples (Figure 2D). This overlap indicates that these T cells have shared antigen specificity, suggesting cell plasticity or differentiation from a common precursor – which will be investigated by trajectory pathway analyses. Figure 1: A) UMAP depicting 23 annotated clusters. B) Bar chart of normalized weighted proportions of cells across clusters at baseline based on disease stams: Flaring (F, orange) and Quiescent (Q, blue). C) Boxplots showing distribution of proportional percentages of subsets for each patient at baseline based on disease status. Each dot represents an individual patient’s data point. To assess for statistical difference, Mann-Whitney U test was used, with p-values adjusted for multiple comparisons using the Benjamini-Hochberg method (*, adjusted p < 0.05). Figure 2: Left: Stacked barchart representing the distribution of clonally expanded T cells in each of the identified clusters at baseline (visit 1) in (A) flaring and (B) quiescent patients. Right: Chord diagrams representing clonal overlap between the clusters at baseline in (C) flaring and (D) quiescent patients. The width of each chord indicates the normalized proportion of unique or shared clones between groups which highlights the extent of clonal overlap. Conclusions Although flaring patients demonstrate expansion of relatively few T cell subsets (Tfh, Tph, Th1 cells), analysis of TCR clonotypes suggests that T cells involved in the autoimmune response are found in several additional T cell subsets (Th17, Th2), indicating that there is substantial functional diversification/plasticity in the response. These findings are absent in quiescent patients, which instead show enrichment of TCMs expressing diverse TCRs. Our findings suggest that expanded populations of antigen-specific T cells can be seen in flaring patients, and that further characterization of these cells may provide insight into the specificity of the T cells that support autoantibody production in lupus flares.