In vitro Treg suppression assays

Abstract

Determining the activity of a regulatory T-cell population in vitro is often the first step in analyzing its function. To obtain reliable and reproducible results, it is critical to follow the protocol that is most applicable to your experimental question. We have outlined below a basic in vitro suppression assay as well as a variety of alternative/additional protocols that can be utilized alone or in combination as desired.

Fig. 1

Plate diagram for T_reg assay. T_regs are titrated into a T_conv cell proliferation assay starting at a 2:1 T_conv:T_reg ratio.

Fig. 2

T_reg-mediated suppression as measured by carboxyfluorescein succinimidyl ester (CFSE) dilution. T_conv were isolated from C57BL/6 mice and labeled with 5 μM CFSE. Cells were activated with anti-CD3 + anti-CD28 coated beads and cultured either alone or in the presence of T_regs at a 4:1 T_conv:T_reg ratio. After 72 h, proliferation was determined by CFSE dilution and flow cytometric analysis.

Fig. 3

T_reg-mediated suppression. T_reg cells were purified by FACS and mixed at different ratios with naïve wild type T_conv cells and anti-CD3 + anti-CD28 coated beads for 72 h. Proliferation was determined by [³H]-thymidine incorporation.

Fig. 4

Transwell plate setup. T_conv cell proliferation in the lower well of a Transwell plate can be suppressed T_reg cells in the top well of a Transwell plate when they are activated in the presence of T_conv cells. Proliferation of lower well T_conv cells is determined by [³H]-thymidine incorporation.

Fig. 5

T_conv/T_reg purification. (a) Murine splenocytes were processed and red blood cells lysed prior to staining with anti-CD4, anti-CD25 and anti-CD45RB antibodies. T_conv and T_reg were purified by FACS based on the profile shown. (b) Red blood cell depleted murine splenocytes were stained with anti-CD4 and anti-Foxp3 antibodies. In parallel, purified T_conv (CD4⁺CD25⁻CD45RB^hi) and T_reg (CD4⁺CD25⁺CD45RB^lo) were stained with anti-CD4 and anti-Foxp3 antibodies and % Foxp3+ cells were determined by flow cytometry.

Fig. 6

Ficoll gradient for T-cell purification. Depiction of lymphocyte layer following Ficoll separation of cord blood or PBMCs.