Fig. 6.

Downregulation of surface MHC I molecules after specific knockouts in imDCs and mDCs. Monocytes were subject to CRISPR/Cas9 knockout of TAP1, tapasin, ERp57, VAPA, BAP31, or ESYT1, which were differentiated into imDCs and maturated into mDCs. (A) Exemplary immunoblots of imDCs (Left) or mDCs (Right) TAP1 or tapasin (Tsn) knockouts. Relative protein abundance of MHC I in control and knockout cells is shown. GAPDH was used as loading control. (B and C) Knockout or control DCs were stained with anti-HLA-A, -B, -C antibody, and the surface presentation was analyzed as median fluorescence intensity (FI) by flow cytometry. Normalized median FI ± SD was plotted. (B) imDCs are shown in light coloring and (C) mDCs in strong coloring. Median FI was normalized to control moDCs. Normalized median FI ± SD was plotted. Symbols represent results of one donor; imDCs are depicted in circles (n = 8) and mDCs in triangles (n = 6). (D) HLA-A2 [imDCs are depicted in circles (n = 5) and mDCs in triangles (n = 4)] or (E) HLA-A3 [imDCs are depicted in circles (n = 4) and mDCs in triangles (n = 4)] specific donors were treated with ribonucleoprotein complexes, and the surface presentation of the knockouts (KO) was analyzed by flow cytometry at imDC (light coloring) and mDC (strong coloring) stages. Results were normalized to control treated cells. Statistical analysis of all flow cytometry data was performed by the t test with correction for multiple comparisons. ***P ≤ 0.0001 and **P ≤ 0.001 and *P ≤ 0.01.