In Vitro Functional Analysis of Regulatory T cells: Focus On Proliferation And Differentiation

Regulatory T (Treg) cells play key roles in maintaining immune homeostasis. Abnormalities in Treg cell function are associated with various diseases, including autoimmune diseases, organ transplant rejection, infection, and cancer. Manipulating Treg cells is considered a potential therapeutic strategy for treating inflammatory diseases, including autoimmune disorders, solid organ transplant rejection, graft-versus-host disease, and allergic diseases. Classic in vitro Treg cell suppression assays mainly assess the inhibitory effect of Treg cells on effector T (Teff) cell proliferation. However, in addition to proliferation, T cell differentiation is important for T cells to perform their immune functions. Therefore, this protocol provides a modified in vitro Treg cell suppression assay for investigating the effects of Treg cells on the proliferation and differentiation of Teff cells. In this protocol, the preparation of mouse Treg cells and naive T cells is first introduced, followed by the co-culture strategy for studying the regulatory effects of Treg cells on the proliferation and differentiation of Teff cells, including Th1 and Th17 cells, and finally, the staining strategy for flow cytometry after co-culture is presented. This in vitro functional analysis of Treg cells can be used to investigate the effects of Treg cells on T cell proliferation and polarization.