Panel A shows a series of flow cytometry plots. The first plot displays SSC-A over time, excluding initial events due to stream instability. The second plot shows FSC-A versus SSC-A to gate lymphocytes. The third and fourth plots gate single cells using FSC-A/FSC-H and SSC-A/SSC-H. The fifth plot excludes non-viable cells using FSC-W and Live/Dead Blue. The final plot selects CD3 positive, CD14/CD19 negative cells for analysis. Panel B presents Sanger sequencing chromatograms of the p.Y665F STAT5B region amplified from cDNA of PBMCs-derived freshly sorted TCR alpha beta and TCR V delta 2 cells, showing the nucleotide sequences. Panel C is a scatter plot summarizing the expression of various surface markers (CD69, CD26, KLRG1, TIGIT, PD-1, CD57, CD28, CD56, NKp80, and CD95) on PBMC-derived TCR V delta 2 cells from two healthy controls (black circles) and the patient (red circles). Panel D shows t-SNE plots of TCR V delta 2-negative cells from two healthy controls and the patient, distributed into clusters 1 to 14 based on the expression of multiple markers. Panel E is a heatmap characterizing the clustering of TCR V delta 2-negative cells from two healthy controls and the patient according to the expression of TCR V1, KLRG1, CD95, CD28, CD26, CD8, CD56, CD69, NKp80, TIGIT, PD-1, Pan gamma delta, and CD4.
Characterization of TCR Vδ2–negative cells from P1. (A) Gating strategy used to characterize T cells from P1 and HCs by multicolor flow cytometry analysis. The initial events of the acquisition were excluded due to stream instability observed during the first seconds of acquisition (SSC-A/Time). Lymphocytes were sequentially gated according to FSC-A/SSC-A, followed by singlets (FSC-A/FSC-H and SSC-A/SSC-H), and viable cells (FSC-W/excluded with LIVE/DEAD Blue). Then, CD3 cells were selected (CD3+/CD14−CD19−) and analyzed. (B) Sanger sequencing of p.Y665F STAT5B region amplified from cDNA of PBMC-derived freshly sorted TCR αβ (left) and TCR Vδ2 (right) cells. (C) Summary of CD69, CD26, KLRG1, TIGIT, PD-1, CD57, CD28, CD56, NKp80, and CD95 expression on PBMC-derived TCR Vδ2 cells from two HCs (black circles) and P1 (red circles). The data correspond to one experiment. (D) t-distributed stochastic neighbor embedding (t-SNE) of TCR Vδ2–negative cells from two HCs and P1 distributed in clusters 1 to 14 according to TCR Vδ1, KLRG1, CD95, CD28, CD26, CD8, CD56, CD69, NKp80, TIGIT, PD-1, pan-γδ, and CD4 expression. (E) Heatmap of clustering characterization of TCR Vδ2–negative cells from two HCs and P1 according to the expression of TCR Vδ1, KLRG1, CD95, CD28, CD26, CD8, CD56, CD69, NKp80, TIGIT, PD-1, pan-γδ, and CD4.