CD4 and B-cell chronic lymphocytic leukemia: To analyze the frequencies of Treg subsets, we isolated CD4+ T-cells from HD PBMCs, cultured them for 72 h in the absence/presence of CLL-EVs, and identified conventional Tregs (CD4+ CD25+ FoxP3+ [31]) and Tr1 cells (CD49b+ LAG-3+ [32]) by flow cytometry.