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

Abstract

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 negCD25 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 negFOXP3 neg and do not mediate suppression.