Panel A: A series of flow cytometry plots. The plots include FSC-A vs SSC-A, CD3 vs DAPI, SSC-A vs SSC-H, and FSC-A vs FSC-H. Panel B: Flow cytometry plots show Cell Trace versus TCR V delta 2. Panel C: A scatter plot summarizing the percentage of TCR V delta 2 cells from healthy controls (black circles) and P1 (red circles) that upregulated CD39, ICOS, CD137, and CD69 after 3.5 days of being challenged with HMBPP (0.4 nanomolar). Panel D: A heatmap showing transcript levels in P1 compared to healthy controls of TCR LGL leukemia-related genes reported to be upregulated. Panel E: A heatmap showing transcript levels reported to be downregulated in T cell LGL leukemia in P1 compared to healthy controls.
Proliferation and activation assays of TCR Vδ2 cells challenged with HMBPP in P1 vs. HCs. (A) Gating strategy to assess proliferation and activation of T cells, which were freshly isolated from PBMCs and challenged with HMBPP (0.4 nM). Lymphocytes were sequentially gated on FSC-A/SSC-A, followed by viable CD3 cells (CD3+/LIVE/DEAD excluded with DAPI). Singlets were selected on FSC-A/FSC-H and SSC-A/SSC-H, and the expansion of TCR Vδ2–positive cells was determined using a cell trace. (B) Flow cytometry plots of TCR Vδ2 cells from HC105, HC106, HC107, and P1 expanded for 3.5 days in the presence or absence of HMBPP (0.4 nM). PBMCs were cultured with or without HMBPP, and the expansion of total T cells was analyzed. (C) Summary of TCR Vδ2 cells from HCs (black circles) and P1 (red circles) that upregulated CD39, ICOS, CD137, and CD69 after 3.5 days of being challenged with HMBPP (0.4 nM). Medians are represented in each condition. The data correspond to one experiment. (D and E) Heatmap of TCR γδ T-LGL leukemia–related genes reported to be (D) upregulated or (E) downregulated. TCR γδ cells from P1 were isolated at five different time points.