Human Treg Deep Phenotyping by Flow Cytometry: Advancing Immunosafety Assays in Autoimmune and Healthy Populations 2305384

Abstract Introduction The FDA 2025 roadmap for reducing animal testing in preclinical safety studies has accelerated the need for robust human in vitro models in Immunosafety. Regulatory T cells (Tregs) are central to immune tolerance and play a critical role in autoimmune disease pathogenesis and therapeutic response. This study aimed to develop a comprehensive flow cytometry panel for deep phenotyping of human Tregs, facilitating their characterization in both healthy and autoimmune populations. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from consented donors. A 13-color flow cytometry panel was designed for compatibility with Treg isolation kits. The panel was tested with PBMCs isolated from healthy donors and autoimmune donors with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), or type I diabetes (T1D). Additionally, Tregs were purified and expanded using CD3/CD28 stimulation and IL-2 supplementation and expanded Tregs were tested side-by-side with PBMCs from matched donors. High-dimensional analysis was performed using FlowJo plugins (UMAP, FlowSOM) to validate gating strategies and marker expression. Results The panel reliably identified Treg subsets, with Helios and FoxP3 distinguishing thymic-derived Tregs. High-dimensional analysis confirmed equivalence between conventional gating and unsupervised clustering. Compared to healthy donors, Helios was significantly upregulated in SLE donors. ICOS was significantly downregulated in medicated SLE and RA donors, and CD95 was significantly downregulated in SLE and RA donors. LAG3 staining showed no difference between healthy and autoimmune donors. Conclusion The deep phenotyping Treg flow cytometry panel and Treg expansion protocols enhance the Immunosafety toolbox for human in vitro and in vivo immune toxicity testing. These methods support the transition to complex in vitro human models and provide valuable tools for future studies and regulatory assessments. Funding Source n/a Topic Categories Basic Autoimmunity (BA)