Fig. 1

Two CDR3δ peptides, GTM and OT3, with tumor binding specificity served as probes to screen the protein ligands of human γδ TCRs. a and b. Immunohistochemical staining of a tumor tissue array with the biotin-labeled GTM peptide a and biotin-labeled OT3 peptide b followed by HRP-conjugated streptavidin. The binding was visualized using DAB as the substrate (brown) (400×). Cancer: tumor tissue; paracancerous: paracarcinoma tissues. Scale bar, 50 μm. GTM and OT3 can specifically bind to several tumor cell lines. Fifty-three types of human tumor cell lines were stained with biotinylated (bio)-GTM c or (bio)-OT3 peptide d and APC-conjugated streptavidin. The binding activity was measured by flow cytometry. The flow data were standardized by the ratio between the mean fluorescence intensity of the peptide conjugate and the secondary antibody alone. The binding strength of the synthetic peptide was compared with that of normal human peripheral blood mononuclear cells (PBMCs). MST analysis of the binding activities of the GTM peptide e and OT3 peptide f to known γδ TCR-recognized stress-inducible ligands. The Kd value for each protein was calculated. g Schematic diagram of OT3- and GTM-γδ CAR molecules. A single-chain Vγ9-Vδ2 or Vγ4-Vδ1 structure, which is composed of a Vδ2 TCR with the tumor-specific CDR3δ sequence OT3 or Vδ1 TCR with the tumor-specific CDR3δ sequence GTM, forms the extracellular region of the γδ CAR. The transmembrane and intracellular segments of a classical third-generation CAR (CD8-CD28-4-1BB-CD3ζ) were linked to the Vγ-Vδ sequence. γδ CARs were cloned and inserted into the lentiviral vector cFUGW. Flow cytometry was used to assess the expression of the activation marker CD69, the secretion of TNF-α h and the cytotoxicity-related molecules granzyme B and perforin i in γδ CAR-T cells after coculture with OVCAR-8 cells in vitro. The statistical data are representative of three independent experiments and are expressed as the means ± SDs. *, p < 0.05; **, p < 0. 01. Cytotoxicity of OT3 CAR-T cells j and GTM CAR-T cells k in different tumor cells in vitro. Cytotoxicity was analyzed with an LDH cytotoxicity detection kit. The data are representative of three independent experiments and are expressed as the means ± SDs. OT3 CAR-αβ T cells exhibit strong cytotoxic activity against tumors in an NSG mouse model. OVCAR-8-transplanted NSG mice were treated with GFP-αβ T cells or OT3 CAR-αβ T cells. Tumor growth in every mouse at each time point was measured by assessing the radiance of the tumor cells l. The tumor growth curve m and survival curve n of each group are shown