Resting T regulatory cells as biomarkers for disease activity in Systemic Lupus Erythematosus patients: results from a monocentric cohort.

Systemic lupus erythematosus (SLE) has been traditionally considered a B cell–mediated disease, although increasing evidence supports a key role for T cells in its pathogenesis. Among T cell subsets, regulatory T cells (Treg) are CD4⁺ T cells characterized by the expression of FOXP3+, a key regulator for their function, that maintain immune homeostasis through immunosuppressive mechanisms, including CTLA-4–mediated regulation and anti-inflammatory cytokines secretion. Three major Treg subsets have been identified: (i) non-suppressive Treg with low FOXP3 expression and pro-inflammatory potential; (ii) activated Treg; (iii) resting Treg, characterized by FOXP3 and IL2RA expression, which remain quiescent but can differentiate into effector Treg upon antigen stimulation. Data from the literature demonstrated that Treg in SLE patients are quantitatively unbalanced, phenotypically remolded, and functionally impaired, due to several different mechanisms, such as defective IL-2/CD25–STAT5 signaling, co-receptor dysregulation, and increased apoptosis. All this leads to unstable FOXP3⁺ populations and expansion of atypical subsets, that fail to adequately restrain effector and B-cell responses. Indeed, in active SLE patients it has been demonstrated an increased frequency of Treg, leading to a paradox: preserved or increased total Treg numbers, but with suppressive compartment quantitatively and qualitatively insufficient. Objectives Objectives Given the inconsistent results in the literature, this study aims to evaluate distinct Treg subsets as biomarkers of disease activity and their association with SLE-related clinical manifestations. Consecutive SLE patients were enrolled. Clinical, demographic and laboratory data were collected according to 2019 ACR/EULAR criteria. We evaluated disease activity by applying the Systemic Lupus Erythematosus Activity Disease Index-2K (SLEDAI-2K). Accordingly, active disease was defined for a SLEDAI- 2k value ≥4, excluding only serology. For each patient we performed peripheral venous blood sampling and immunophenotyping analysis by flow-cytometry, after PBMC isolation. We included 53 SLE patients (M/F 3/50; median age 45 years, IQR 19.5; median disease duration 144 months, IQR 156). At the time of analysis, 25 patients (47.2%) were treated by glucocorticoids, 48 by hydroxychloroquine (90.6%), 25 (47.2%) by at least one immunosuppressive/biological drug. A median value of SLEDAI-2k equal to 2 (IQR 6) was registered. Based on SLEDAI-2k values, 26 patients (49.0%) had active disease; according to SLEDAI-2K items, the most frequent active manifestations were articular,cutaneous, and renal involvement (observed in 50.0%, 26.9%, and 15.5% of patients, respectively). When considering overall Treg cells, we observed a significantly higher median percentage in patients with active disease compared with non-active disease [active disease: 9.5 (IQR 6); non-active disease 6.1 (IQR 6); p=0.04]. The analysis of Treg subsets revealed a significantly higher proportion of resting Treg in patients with active SLE compared with those with inactive disease [1.38 (IQR 1.6) versus 0.75 (IQR 0.8), p=0.01; figure 1A], along with a significant positive correlation between resting Treg percentage and SLEDAI-2K values (p=0.003, R 0.4, r2 0.15, 95%CI 0.04-0.21; figure 1B). We also evaluated the expression of Tregs cells according to the different disease-related manifestations, focusing on joint, renal and skin involvement. We found a significantly higher percentage of resting Tregs cells in patients with active renal (median 2.0, IQR 2.8) and joint involvement (median 1.5, IQR 1.9) when compared with non-active disease (median 0.75, IQR 0.8; p=0.02 for renal involvement and p=0.03 for joint manifestations; figure 1C). This present study investigated the possible role of different Treg subset as biomarkers for disease activity in SLE patients, focusing on the identification of the most performant subset. While the percentage of overall Treg cells was significantly higher in patients with active disease, among their different subsets, only resting Treg showed a significant correlation with SLEDAI-2K scores, supporting their potential role as a biomarker of disease activity. We could hypothesize that active disease may impair the activation and differentiation of resting Treg into functional suppressive cells, potentially through mTOR pathway activation, with consequent defective autophagy and reduced suppressive ability. Consequently, total Tregs may be normal or increased, while their regulatory function is compromised, thereby perpetuating immune dysregulation and disease activity.