A method for conducting suppression assays using small numbers of tissue-isolated regulatory T cells

The suppression assay is a commonly performed assay, measuring the ability of regulatory T cells (Treg) to suppress T cell proliferation. Most frequently, Treg are obtained from the peripheral blood or spleen. Lower yields are obtained by isolation from other tissues, rendering downstream suppression assays challenging to perform. Furthermore, the importance of suppressive subpopulations of Treg favours their isolation by fluorescent-activated cell sorting. Here we describe a method to isolate Treg from human tissues, using colorectal cancer tissue as an example. Treg suppressive capacity was further examined by expression of CCR5 to demonstrate the ability of our method to assess the suppressive capacity of regulatory T cell subsets.To optimise the standard suppression assay to achieve our research aims, the following modifications were made:
• Treg, isolated from tissues, were sorted directly into a well-plate.
• Responder T cells, which had been fluorescently-labelled prior to sorting, were added directly into the well-plate.
• Human Treg Suppression Inspector beads (Miltenyi Biotec Ltd., UK) provided a polyclonal stimulus for proliferation and were added to each well at a bead:lymphocyte ratio of 1:2.This method quantified the suppression of responder T cell proliferation by small numbers of strictly-defined Treg populations isolated from tissues.

Lymphocytes were isolated from human tissue samples by enzymatic and mechanical methods. Treg and Tconv were cell sorted from the tissue cell suspension by gating the lymphocytes forward scatter–side scatter region into a CD4–Live/Dead dot plot. CD4+ live cells were then gated into a CD127–CD25 dot plot. This last dot plot was used to define Treg (Streams 1 and 2) and Tconv cells (Stream 3). Treg were gated into a CCR5 histogram to define CCR5low and CCR5high Treg. Thus, specific Treg subsets were isolated for use in downstream suppression assays. Histograms depicting percentage Foxp3 expression by colorectal cancer-isolated Treg and Tconv show representative data from 9 separate experiments. Treg isolated from colorectal cancer therefore express high amounts of Foxp3 whereas Tconv do not.Figure optionsDownload as PowerPoint slide