Fig 4. ITK knock-down induces bona-fide suppressive T_REG.

Human naïve T cells treated with NTC or sd-ITK and differentiated under T_REG polarizing conditions (T_REG-polarized) were labeled with CVT, and CD4⁺CD25^Neg responder T cells were labeled with CFSE (T-responder). T_REG and T-responder were cultured in the presence of anti-CD3/CD28 activation beads. Flow cytometry analysis was performed after 4 days. (A) Division index for each T_REG-polarized/T-responder ratio was calculated as the average number of cell divisions of the original CFSE-labeled population versus the undivided CFSE^high cells using the FlowJo proliferation platform. (B) Representative flow cytometry plots using a 1:1 T_REG-polarized/T-responder ratio. N = 4 donors. Median ± IQR, *p<0.05, ***p<0.001 (multiple t-tests; see Methods).