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.
National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit (BRU), University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK ; Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Birmingham B15 2WW, UK.
Stephen T
ST
Ward
National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit (BRU), University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK ; Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Birmingham B15 2WW, UK.
Ka-Kit
KK
Li
National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit (BRU), University of Birmingham, Vincent Drive, Birmingham B15 2TT, UK.
Stuart M
SM
Curbishley
Regulatory T cells; immunosuppression; lymphocyte isolation; suppression assay