Requirements for γδ TCR activation by HLA-DR. (A) GFP fluorescence of JE6.1 cells cocultured with either Raji WT cells, the HLA-DRB knockout (DRB^−/−), or DRB^−/− transduced with a range of different DRB subtypes. Statistical test: two-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001 (n = 3, error bars = SD of mean). (B) MFI of eGFP of JE6.1 cells cocultured with either Raji WT cells, DRB^−/− Raji cells, or DRB^−/− with restored HLA-DRB expression by gene repair (HDR). Each square represents the mean MFI of two to three independent experiments. (C) Multiple alignment of TCR δ-chain position 44–119. Compared are TCR05 with the CDR- and HV4-exchange mutants. (D) GFP fluorescence of JE6.1 cells expressing different hybrid versions or sequence chimeras of TCR05 with Raji cell lines. TCR05_g8*: a Vγ8-chain with the original CDR3γ from the TCR05. TCR05_CDR1d: CDR1 exchanged with the Vδ3-CDR1; TCR05_CDR2d: CDR2 exchanged with a Vδ2-CDR2; TCR05_HV4d: HV4 exchanged with Vδ2-HV4. Statistical test: two-way ANOVA with Tukey’s multiple comparisons test; *, P < 0.05; **, P < 0.005 (n = 3, error bars = SD of mean). (E and F) Coculture of primary B cells with JE6.1 reporter cells expressing TCR02/04/05. After MACS isolation, primary B cells from five healthy donors and from an archived sample of patient 15/02, from whom TCR02/04/05 were identified, were cocultured with JE6.1 reporter cells expressing all three γδ TCRs at a ratio of 1:1. Separation of JE6.1 and B-cells via CD19 staining (E) and GFP reporter fluorescence of JE6.1 cells after coculture (F).