**5.1. Flow cytometry analysis of T regulatory (Treg) cells differentiation and function**

To study the effect of CML extracellular vesicles on the differentiation of thymic regulatory T cells (tTreg), we have employed a previously described *in vitro* assay of tTreg differentiation on JAWS II immature dendritic cells [80]. Briefly, during the step of preactivation of thymocytes on αCD3-coated plates, we added extracellular vesicles to allow their uptake or binding solely by thymocytes. Thymocytes were additionally stained with a violet proliferation dye 450, which would allow to track their proliferation and distinguish them from JAWS II cells. After 24 h of preactivation, thymocytes were washed and co-cultured with JAWS II cells. Afterwards, tTreg differentiation using multicolor flow cytometry was analyzed. This analysis allowed to track viability, proliferation (**Figure 8A**), differentiation of tTreg, as well as their phenotype (**Figure 8B**). Extracellular vesicles can also potentially influence suppressive activity of mature, already differentiated tTreg. To study this phenomenon, we sorted tTreg and cultured them, first with CML extracellular vesicles for 24 h and afterwards with conventional T cells (responder cells) stimulated for proliferation, to observe differences in suppressive activity manifested by decreased proliferation. Labeling of responder cells with CFSE allowed to track their proliferation after coculture with tTreg, which have been preincubated with either CML-derived or control extracellular vesicles. This in turn allowed to assess differences in tTreg suppressive activity due to influence of EVs. Combination with additional staining of surface receptors such as CD4 or CD8 enabled to distinguish between suppression towards helper (CD4+) or cytotoxic (CD8+) T cells (**Figure 8C**).
