Fig. 1.

CD4 and TCR expression levels are important for HLA tetramer binding to HEK293T cells transiently transfected with a TCR complex. (A) Schematic representation of transient transfection of HEK293T cells with combinations of different plasmids. (B) HEK293T cells were transiently transfected with mouse CD3-GFP and TCR-Ametrine. 24h later cells were analyzed for GFP and Ametrine signal by flow cytometry. (C) Cells in B were also stained for surface expression of CD3 and TCRb and analyzed by flow cytometry. Data shows cells gated on viable, GFP+, Ametrine ​+ ​singlets. (D-E) HEK293T cells were transfected with GAD (D), or HA (E) TCR plasmid together with CD3 ​± ​hCD4 plasmids and stained simultaneously with both GAD-specific and HA-specific tetramers (TMR). Flow cytometry data shows cells gated on viable, GFP+, Ametrine+, CD3⁺ singlets. (F-G) Flow cytometry plots of CD4⁺ GAD (F), and CD4⁺ HA (G) TCR expressing HEK 293T cells, gated on viable, GFP+, Ametrine+, CD3⁺ singlets, comparing tetramer binding to surface expression of the TCR complex (CD3e). ∗∗P ​< ​0.01 by Mann-Whitney test (n ​= ​5/group). Data is representative of three independent experiments.