Identification of HLA-DR as a ligand for TCR04 and TCR05. (A) Staining of Raji cells with tetramers of TCR02, TCR04, and TCR05 (sTCR). Streptavidin-PE alone was used as a negative control. (B) Comparison of soluble TCR04 staining as tetramers and monomers. (C) Histograms indicate loss of TCR04 binding after consecutive sorting rounds in the genome-wide CRISPR/Cas9 knockout screening. 3 d after transduction with the CRISPR/Cas9 library, the Raji cells were stained with sTCR04 and the cells with the lowest staining were sorted four times for further subculture. (D) Analysis of subclones derived from the genome-wide CRISPR/Cas9 knockout screening for contained sgRNAs and sTCR binding. The horizontal axis depicts the generated clones, whereas identified target genes are given on the left vertical axis. Red squares indicate genes targeted by the respective sgRNAs and lower panels indicate the MFI. (E) Histograms showing eGFP fluorescence of JE6.1 cells expressing TCR02, TCR04, and TCR05 induced by coculture with different Raji cell lines. DRA−/−: HLA-DRα-chain knockout (derived single-cell clone 1E11); 1E11 +DRA: HLA-DRα knockout clone transduced with a transgenic HLA-DRα-chain to rescue the phenotype. Cocultures and sTCR stainings were carried out n = 2–4 times.
Figure 2.

Identification of HLA-DR as a ligand for TCR04 and TCR05. (A) Staining of Raji cells with tetramers of TCR02, TCR04, and TCR05 (sTCR). Streptavidin-PE alone was used as a negative control. (B) Comparison of soluble TCR04 staining as tetramers and monomers. (C) Histograms indicate loss of TCR04 binding after consecutive sorting rounds in the genome-wide CRISPR/Cas9 knockout screening. 3 d after transduction with the CRISPR/Cas9 library, the Raji cells were stained with sTCR04 and the cells with the lowest staining were sorted four times for further subculture. (D) Analysis of subclones derived from the genome-wide CRISPR/Cas9 knockout screening for contained sgRNAs and sTCR binding. The horizontal axis depicts the generated clones, whereas identified target genes are given on the left vertical axis. Red squares indicate genes targeted by the respective sgRNAs and lower panels indicate the MFI. (E) Histograms showing eGFP fluorescence of JE6.1 cells expressing TCR02, TCR04, and TCR05 induced by coculture with different Raji cell lines. DRA−/−: HLA-DRα-chain knockout (derived single-cell clone 1E11); 1E11 +DRA: HLA-DRα knockout clone transduced with a transgenic HLA-DRα-chain to rescue the phenotype. Cocultures and sTCR stainings were carried out n = 2–4 times.

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