A Simple Two-Step Method for the Isolation of Human CD4+CD25+bright / FOXP3+ Regulatory T Cells Directly from Whole Blood.
Authors: Guilbault Benoit , Fairhurst Maureen , Crellin Natasha , Levings Megan K. , Eaves Allen C. , Thomas Terry E. 
Published: April 2020

Abstract
Human CD4+CD25+ regulatory T cells (TR) have the ability to suppress T cell responses and have recently been shown to play a critical role in peripheral tolerance and regulation of immune responses. CD4+CD25+ TR are anergic, phenotypically CD25+bright and express high levels of the transcription factor FOXP3. Peripheral blood TR are rare and must be highly enriched for their suppressor function to be detected in vitro. This, combined with the lack of a unique marker that distinguishes them from activated T cells, makes it difficult to study TR function and evaluate their therapeutic potential. Current methods for isolating TR are cumbersome and time-consuming, and generally require three steps: Ficoll™ density centrifugation to isolate mononuclear cells; subsequent immunomagnetic T cell enrichment; and finally FACS sorting. The objective of this study was to develop a more simple technique to isolate highly purified TR from whole blood without using FACS sorting. Three steps were reduced to two by replacing the Ficoll™ separation and immunomagnetic T cell enrichment with a single antibody-mediated buoyant density centrifugation (RosetteSep®) to enrich CD4 T cells directly from whole blood. A cocktail of bi-specific antibodies was used to selectively bind unwanted cells to red cells causing them to pellet when centrifuged over Ficoll™. Purified CD4 T cells were recovered at the plasma-Ficoll™ interface and then separated using EasySep® column-free magnetic separation to select CD25 expressing cells. The EasySep® separation conditions were optimized for maximal CD4+CD25+bright T cell purity and recovery. The purified cells were analyzed for CD25, GITR, CD62L, HLA-DR and CTLA-4 expression. FOXP3 mRNA levels in purified CD4+CD25+ T cell fractions were compared to corresponding CD4+CD25neg T cell fractions using quantitative PCR. As TR are anergic and therefore not responsive to TCR stimulation, the purified CD4+CD25+ T cell fractions were assessed for anergy in response to anti-CD3/CD28 coated beads. Suppression activity of purified CD4+CD25+ T cells was assessed by measuring their ability to reduce the proliferative response of CD4+CD25neg T cells to CD3/CD28 beads. T cell proliferation was quantified by measuring dilution of the fluorescent dye CFSE with flow cytometry. Purified CD4+CD25+ T cell suspensions were 94 ± 3% (n=6) CD4+CD25+, 83 ± 7% (n=6) CD4+CD25+bright, 89 ± 2% (n=4) GITR+ and CD62L+ (92%, 95%; n=2). The purified cell suspensions were also highly enriched for CTLA-4 and HLA-DR expressing cells. FOXP3 mRNA levels in the purified CD4+CD25+ T cell fractions from two donors were found to be 64 and 124 times higher than for the corresponding CD4+CD25neg T cell fractions. The purified CD4+CD25+ T cells displayed low or undetectable proliferative responses (n=4) to CD3/CD28 beads suggesting most cells in the purified fractions were anergic. Mixing purified CD4+CD25+ T cells with CD4+CD25neg T cells at a ratio of 1:1 almost completely eliminated detectable CD4+CD25neg T cell proliferation (95 ± 3 % reduction, n=3), and suppression of proliferation continued to be detected when cells were mixed at a ratio of 1:10 (25 ± 6% reduction, n=3). In conclusion, combining RosetteSep® CD4 T cell enrichment with EasySep® CD25 positive selection yields highly pure CD4+CD25+bright / FOXP3+ TR in less time and with fewer steps than current isolation methods.