Screening for reactivity of γδ TCRs. (A) γδ T cells were FACS-sorted from frozen PBMCs and analyzed via single-cell sequencing. UMAP plot showing the distribution of expanded γδ TCR clones in an HSCT patient. (B) Expression of T cell maturation and activation markers by scRNA sequencing. (C) Relative frequency of clones expressing TCR02 and TCR05 among the total TRD repertoire over time. (D) γ- and δ-chain CDR3 sequences of TCR02, TCR04, and TCR05. The amino acid exchange between TCR04 and TCR05 is marked in red. (E) Schematic representation of the JE6.1 cells containing a reporter gene of GFP fused to an NF-κB–responsive promoter. (F) Reactivity of JE6.1 reporter cells expressing different γδ TCRs against CMV-infected fibroblasts defined as mean fluorescence intensity (MFI) of eGFP. Statistical test: two-way ANOVA with Tukey’s multiple comparisons test; ****, P < 0.0001 (n = 3–4, error bars = SD of mean). (G) Representative histograms of JE6.1 GFP fluorescence after coculture with Raji cells. (H) Heatmap of reactivity of JE6.1 reporter cells expressing TCR02, TCR04, TCR05, and the Vγ9δ2 TCR towards leukemia cell lines. Each square represents the mean MFI of two to three independent experiments. Beads coated with TCR-engaging anti-CD3 and CD28 antibodies were used as a positive control. K562: erythromyeloblast; BL64 and Raji: Burkitt’s lymphoma; U937/KG-1/THP-1: myeloid leukemia; CEM: T cell lymphoma cell lines. Coculture experiments were repeated n = 2–4 times.