Fig. 2.

TCR-NK-92 in vitro validation.

(a,b) Dot plots showing correct folding of two transgenic TCRs (Radium-1 TCR and DMF-5 TCR). Surface expression of aCD3 and aVb3 TCR antibodies in NK-92, CD3-NK-92 and TCR(Radium-1)-NK-92 (left panel) and expression of aCD3 antibody and pMHC dextramer in NK-92, CD3-NK-92 and TCR(DMF-5)-NK-92 (right panel) (representative of three experiments). (c,d) Phosphorylation of the key components of TCR signaling cascade, ERK, CD3zeta, SLP76 and ZAP70 overtime in TCR-NK-92 (either DMF-5 TCR or Radium-1 TCR) was detected by phospho-specific flow cytometry upon stimulation with APCs loaded with either pMelan-A or pTGFβRII peptide. TCR-induced phosphorylation was measured at 5, 15 min after addition of APCs to TCR-NK-92 cells, following a 30 s centrifugation to bring the cells together. (n = 2) Error bars represent SD. (e) Outcome of ViSNE analysis on TCR-NK-92, NK-92 and T cells, either naïve or activated. (f) Bar graphs showing the level of IFNγ and TNFα produced by TCR(Radium-1)-NK-92 and CD3-NK-92 co-cultured with K562:HLA-A2 that were loaded with either irrelevant peptide (pMelan-A) or cognate peptide (pTGFβRII) or used as negative control (no peptide). (n = 3). Error bars represent SEM. (g) Dot plots showing frequency of activated TCR(Radium-1)-NK-92 cells. Detection of CD107a expression in TCR-NK-92 cells cultured in the presence of cognate peptide (pTGFβRII). (n = 4). Error bars represent SEM.