A Simple Two-Step Method for the Isolation of Human CD4⁺CD25^+bright / FOXP3⁺ Regulatory T Cells Directly from Whole Blood.

Human CD4⁺CD25⁺ regulatory T cells (T_R) 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⁺ T_R are anergic, phenotypically CD25^+bright and express high levels of the transcription factor FOXP3. Peripheral blood T_R 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 T_R function and evaluate their therapeutic potential. Current methods for isolating T_R 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 T_R 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⁺CD25^neg T cell fractions using quantitative PCR. As T_R 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⁺CD25^neg 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⁺CD25^neg 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⁺CD25^neg T cells at a ratio of 1:1 almost completely eliminated detectable CD4⁺CD25^neg 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⁺ T_R in less time and with fewer steps than current isolation methods.