Research paperA novel and rapid method to quantify Treg mediated suppression of CD4 T cells

•CD25 is highly correlated with cell division as measured by CFSE.•A CD25/CD134-based assay simplifies and shortens assays of Treg suppression.•The CD25/CD134-based assay works for frozen samples and limited cell numbers.•The CD25/CD134-based assay can be utilized with bead based and APC based stimuli.

Measuring regulatory T cell suppression provides important insight into T cell dysfunction in autoimmune disease. However, to date, suppression assays are limited by the requirement for freshly isolated cells, and significant cell numbers. Here, we present a novel and rapid in vitro assay using effector T cell surface expression of both CD25 and CD134 as a surrogate marker of regulatory T cell-mediated suppression. This surface marker-based suppression assay works for frozen samples and for samples with limited cell numbers. It is also shorter taking two days to complete compared to the four days required for proliferation-based assays. Furthermore, this assay works with both in vitro expanded and natural Tregs, as well as anti-CD3/anti-CD28 bead-based and APC stimulation conditions. In conclusion, we have developed and validated a new suppression assay for cryopreserved samples with limited cell numbers that may be helpful to investigate T cell regulation in the context of infection or autoimmune diseases.