MOESM3 of A strategy to protect off-the-shelf cell therapy products using virus-specific T-cells engineered to eliminate alloreactive T-cells

Additional file 3: Fig. S3. CMVSTs expressing iCHAR can still reduce activation of responder alloreactive T-cells that lack Fas expression. (A) Knockout of Fas in allogeneic PBMC using CRISPR technology. Freshly isolated PBMC were nucleofected with Cas9 and single guide RNAs (sgRNA) to Fas and rested overnight. PBMC were then co-cultured with CMVSTs and Fas expression on gated responder T-cells was measured on Day 8. (B) CMVSTs were co-cultured with PBMC that were knocked out for Fas. On Day 8, activation of gated responder T-cells was assessed by CD71 staining. CD8 and CD4 subsets were gated and analyzed separately. (C) Quantification of CD71+ T-cells for both CD8 and CD4 subsets on Day 8 (mean ± SEM, n = 3). Of note, the level of activation of allogeneic PBMCs that are knocked out for Fas was lower compared to when unmodified, which is likely due to the non-specific toxicity associated with electroporation and knockout impairing the allo-reaction. Significance was determined by paired two-tailed Student’s t-test. *p < 0.05 compared to NT CMVST condition. R = Responder, S = Stimulator.