Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets.

OBJECTIVE: The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39(+) Treg on magnetic immunobeads. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4(+) T cells on AutoMACS using antibodies (Abs) specific for all lineage(+) cells. CD4(+)CD39(+) Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4(+)CD39(+) T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. RESULTS: The purity, recovery and viability of the separated CD4(+)CD39(+) T cells were satisfactory. The isolated CD4(+)CD39(+) T cell population consisted of FOXP3(+)CD25(+) T cells which hydrolyzed exogenous ATP and suppressed autologous CD4(+) T cell proliferation and of FOXP3(neg)CD25(neg) T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4(+)CD39(+), CD4(+)CD127(neg), CD4(+)CD49d(neg) or CD4(+)CD25(high) Treg. CONCLUSION: CD4(+)CD39(+) Treg capture on immunobeads led to a discovery of two CD39(+) subsets. Similar to CD39(+) Treg in the peripheral blood, half of these cells are CD25(+)FOXP3(+) active suppressor cells, while the other half are CD25(neg)FOXP3(neg) and do not mediate suppression.