CD4 − /CD8 − double-negative tumor-infiltrating lymphocytes expanded from solid tumor tissue suppress the proliferation of tumor cells in an MHC-independent way

Purpose Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4 − /CD8 − double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3 + CD4 − CD8 − TILs (double-negative TIL, DN-TILs) derived from solid tumor. Methods DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Results Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3 + TILs, which expanded more than 800-folds in 2 weeks, consisting of a mixture of alpha–beta (αβ) and gamma–delta (γδ) T-cell receptor (TCR)—expressing cells (with the majority being αβ-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. Conclusion Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL.