Figure 3.

In vitro expansion of FoxP3⁺ T2NKT cells. (A) Gating strategy to flow-sort T2NKT cell after double-step MACS enrichment. From left to right, cells were gated for lymphocytes based on FSC/SSC and singlets. Biotin FITC excluded cells labeled with TCRVα7.2, TCRVα24 or TCRγᵹ antibody. Cells were then gated for CD3⁺ CD4⁺ CD56⁺ CD161⁺. (B) Schematic map depicting each step of the expansion protocol. (C) Cell counting after 3 weeks expansion using Flow Cytometry Counting Beads. Counting beads were positive for PE-Cy7, cells were separated from MACSiBeads based on FSC. (D) Quality control analysis of T2NKT cell expansion after 3 weeks. Lymphocytes were separated from MACSiBeads and dead cells by FCS/SSC. MAIT cells, γᵹ T cells and iNKT cells were excluded by staining with Biotin FITC. Within the CD3⁺ T cell population, PLZF was highly expressed (80.7% of the total lymphocyte population). These cells were mainly CD56⁺ CD161⁺ (75.7% of the total lymphocyte population) and a subset expressed regulatory-like phenotype FoxP3⁺ CD25⁺ CD127^low (31% of the total lymphocyte population). (E) Summary table indicating absolute number of cells before expansion (day 0) and after expansion (day 21). Percentage of FoxP3⁺ cells after expansion is included.