Direct γδ TCR interaction with HLA-DR. (A and B) Analysis of the interaction between TCR04 and TCR05 with HLA-DRB1*03 presenting CLIP via surface plasmon resonance. (A) Sensorgram showing the interaction of injected TCR02, TCR04, TCR05 (2.5 µM), and HBS-EP (blank) with captured HLA-DR (capture level ∼440 RU). (B) Single-cycle kinetics of injected TCR04 (1:2 dilution ranging from 12.5 to 200 nM) over ∼290 RU captured HLA-DR and TCR05 (438–7,000 nM) over ∼488 RU captured HLA-DR. The curves were fit using a 1:1 binding model. n = 3. (C) Transfer of proliferation dye eFluor 670 upon γδ TCR activation. Dot plots show fluorescence of Raji cells prestained with eFluor 670 (e670^high) and cocultured with JE6.1 cells expressing the indicated γδ TCRs (e670^neg or 670^low). Histograms show JE6.1 GFP fluorescence after 24 h of coculture, pregated on JE6.1 cells (T cells). The experiment was repeated at least three times. (D) Streptavidin-APC staining to detect transferred biotin after a coculture of γδ TCR-expressing JE6.1 cells with biotinylated Raji cells. (E) HLA-DR staining of JE6.1 cells expressing the indicated γδ TCRs after 5 min coculture with Raji WT cells. (F) Cumulative results of all HLA-DR transfer for all cocultured JE6.1 cell lines as median fluorescence intensity of HLA-DR-APC, gated on CD19^‒. All transfer experiments were repeated two times. Error bars represent SD of mean.