NR3C1 deletion renders VSTs resistant to steroids without altering their phenotype or function. (A) Representative fluorescence-activated cell sorter (FACS) plots showing the percentage of apoptotic cells (annexin V⁺) and alive or dead cells (live/dead stain) in control Cas9 vs NR3C1 KO (exon 2) VSTs after culture with or without dexamethasone (Dexa; 200 μM) for 72 hours (n = 4). Inset values indicate the percentage of annexin V and alive/dead cells from each group. (B-C) Graph summarizing the percentage of live cells (B) and the absolute cell number (C) between control Cas9 and NR3C1 KO VSTs treated with or without 200 μM dexamethasone for 72 hours (n = 4). Statistical significance is indicated as **P ≤ .01. Bars represent mean values with standard deviation. (D) FACS plots showing the distribution of CD4 and CD8 (upper plots) and the frequency of CD62L and CCR7 (lower plots) in control Cas9 or NR3C1 KO VSTs that were treated with or without Dexa (200 μM) for 72 hours (n = 3). Inset values indicate the percentage of T cells expressing CD62L and/or CCR7 in each treatment group. These FACS plots were pregated on CD3⁺CD45RA⁻ T cells. (E-F) Bar graphs showing the percentage of IFN-γ, TNF-α, or IL-2 production by CD8⁺ (E) and CD4⁺ (F) VSTs treated with control Cas9 (green), NR3C1 KO (blue), or NR3C1 KO plus dexamethasone (Dexa; 200μM; red) in response to 6 hours of stimulation with viral PepMix (n = 3). The functional analysis of the Cas9+Dexa group was not performed due to the absence of viable cells resulting from the lymphocytotoxic effect of steroids. The bars represent mean values with SD. NS, not significant.