Using single-cell CITE-Seq and high-paramter flow cytometry to characterize human regulatory T cells

Regulatory T cells (Tregs) are an essential population of T cells that are critical for maintenance of peripheral tolerance. Imbalanced Treg frequency or dysregulation of Treg function can lead to the development of autoimmune diseases. Thus, the modulation of Treg frequency or function has become an important therapeutical approach to treat autoimmune disorders. Despite increased study, whether distinct populations of Tregs with unique functions exist is unclear. Here, we used a combination of single-cell CITE-Seq (scCITE-Seq) and high parameter flow cytometry to resolve the heterogeneity of Tregs in humans. Using magnetic cell enrichment in conjunction with fluorescence-activated cell sorting (FACS), we isolated Tregs and performed downstream scCITE-Seq. By using more than a hundred antibody-oligo conjugates, we identified distinct populations of Tregs, uncovered the proteomics signature associated with these populations, and revealed donor-to-donor variability in the peripheral Treg compartment. The protein expression pattern and antigen density on Tregs revealed by scCITE-Seq correlated to that of high dimensional flow cytometry. Trajectory analysis segregated Treg subsets into distinct differentiation pathways with unique proteomic signatures ranging from naïve, activated to memory Tregs. These findings improve our understanding of the heterogeneity of Tregs and provide a single-cell proteomic atlas for dissecting the complex roles of Tregs in health and disease.