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.