Figure 2.

Two-step magnetic-activated cell sorting (MACS) strategy for enrichment of human T2NKT cells. (A) Two-step MACS strategy consisted first in a CD56 positive selection from human PBMC with CD56 REAlease microbeads. These beads were then detached from the cell surface after separation. In the second step, NKT cells were enriched using CD3 microbeads. The outcome of this procedure was a CD3⁺ CD56⁺ cell isolate enriched in NKT cells. (B) Summary table indicating the frequency of cells relative to preMACS values and absolute (Abs) cell numbers after each MACS step. (C) Gating of human leucocytes enriched for CD56⁺ CD3⁺ cells using the two-step MACS protocol. The sample was enriched for iNKT cells (2% of total CD3⁺ T cells) and T2NKT cells (5.3% of total CD3⁺ T cells). (D) Expression of the transcription regulator promyelocytic leukemia zinc finger (PLZF) in subsets of human lymphocytes. Differential expression of PLZF was observed between conventional T cells (blue line) and NK cells (black line). There was similar PLZF expression in T2NKT cells (red line) and NK cells, whereas iNKT cells (green line) showed higher expression. (E) A CD4⁺ FoxP3⁺ CD25⁺ CD127^low subpopulation of CD3⁺ CD56⁺ CD161⁺ TCRγδ^- TCRVα7.2^- TCRVα24^- T2NKT cells after the two-step enrichment process was identified in all 3 donors (one donor is shown as an example).