Fig 1: NKD2-mediated ORAI1+ vesicle trafficking is crucial for the effector function of primary T cells(A) Representative histograms showing levels of NKD2 protein in primary T cells transduced with lentiviral vectors encoding scrambled sgRNA (control), NKD2 sgRNA #1, and NKD2 sgRNA #3, together with those encoding Cas9. Cells were permeabilized and stained with anti-NKD2 Ab. The bar graph (right) shows average (±SEM) from four independent experiments.(B) Representative traces showing averaged SOCE from primary control (73 cells) and NKD2 KO (KO sgRNA #1, 82 cells; KO sgRNA #3, 71 cells) T cells after TCR stimulation using anti-CD3 Abs, followed by ionomycin treatment in the presence of external solution containing 2 mM Ca2+. Bar graph (right) shows averaged baseline-subtracted SOCE (±SEM) from three independent experiments.(C) Representative histograms showing levels of newly inserted PM-resident ORAI1 protein in primary control and NKD2 KO effector T cells after TCR stimulation. Cells were transduced as described above to induce deletion of NKD2 and stimulated and stained as described in Figure 3B for detection of newly inserted PM-resident ORAI1. Bar graph (right) shows average (±SEM) of pooled technical replicates from three independent donors.(D) Representative flow plots showing expression of IFN-?, IL-2, and TNF in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells after re-stimulation with anti-CD3 and anti-CD28 Abs. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments.(E) Representative histograms showing levels of T-bet in control, NKD2 KO (sgRNA #1), and NKD2 KO (sgRNA #3) cells. Bar graph shows means ± SEM of pooled technical replicates from two independent experiments.*p < 0.05; **p < 0.005; ***p < 0.0001.See also Figure S6.