Figure 2.
RhoG deficiency impairs NK and CD8+ T-cell cytotoxicity. (A) Target cell killing by primary NK cells from patient and healthy relatives upon coculture with K562 target cells. (B) Degranulation by primary NK cells from patient and healthy relatives assessed by CD107a surface exposure. (C) Target cell killing and degranulation in expanded CD8+ T cells from patient and normal donors upon coculture with P815 target cells coated with anti-CD3 mAb. (D) Killing and degranulation by NK-92 WT and RHOG KO cells upon coculture with K562 target cells for 24 hours. (E) siRNA-mediated RHOG KD in CD8+ T cells of healthy individuals. The efficiency of 2 siRNAs (#1 and #2) targeting RHOG compared with the control siRNA was calculated as a ratio between RhoG and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels (left). The effect of RHOG KD using 2 different siRNAs on degranulation of normal donor-derived CD8+ T cells shown as correlation between RhoG protein levels and degranulation efficiency (middle and right). (F) Effect of ITX3, an inhibitor of Trio-dependent RhoG activation, on killing capacity of primary T and NK cells. ND peripheral blood mononuclear cells (PBMC) were preincubated with ITX3 for 1 hour before the addition of target cells. Anti-CD3 monoclonal Ab-coated P815, and K562 cells were used to trigger T- and NK-cell cytotoxicity, respectively. (G) Degranulation assay showing reconstitution of impaired cytotoxic granules release in RHOG KO NK-92 cells upon transfection with plasmids encoding WT RHOG or GFP. All data are presented as the mean ± standard error of the mean (SEM). P values were calculated using a multiple Student t test and 2-way analysis of variance. *P < .05; **P < .01; ***P < .001; *****P < .0001.

RhoG deficiency impairs NK and CD8+ T-cell cytotoxicity. (A) Target cell killing by primary NK cells from patient and healthy relatives upon coculture with K562 target cells. (B) Degranulation by primary NK cells from patient and healthy relatives assessed by CD107a surface exposure. (C) Target cell killing and degranulation in expanded CD8+ T cells from patient and normal donors upon coculture with P815 target cells coated with anti-CD3 mAb. (D) Killing and degranulation by NK-92 WT and RHOG KO cells upon coculture with K562 target cells for 24 hours. (E) siRNA-mediated RHOG KD in CD8+ T cells of healthy individuals. The efficiency of 2 siRNAs (#1 and #2) targeting RHOG compared with the control siRNA was calculated as a ratio between RhoG and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels (left). The effect of RHOG KD using 2 different siRNAs on degranulation of normal donor-derived CD8+ T cells shown as correlation between RhoG protein levels and degranulation efficiency (middle and right). (F) Effect of ITX3, an inhibitor of Trio-dependent RhoG activation, on killing capacity of primary T and NK cells. ND peripheral blood mononuclear cells (PBMC) were preincubated with ITX3 for 1 hour before the addition of target cells. Anti-CD3 monoclonal Ab-coated P815, and K562 cells were used to trigger T- and NK-cell cytotoxicity, respectively. (G) Degranulation assay showing reconstitution of impaired cytotoxic granules release in RHOG KO NK-92 cells upon transfection with plasmids encoding WT RHOG or GFP. All data are presented as the mean ± standard error of the mean (SEM). P values were calculated using a multiple Student t test and 2-way analysis of variance. *P < .05; **P < .01; ***P < .001; *****P < .0001.

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