Establishment and Multidimensional Characterization of a Novel Epstein-Barr Virus (EBV)-Negative Human Type I Natural...

Natural killer T cells (NKT), a unique subset of T lymphocytes, remain incompletely understood in terms of their development and functional regulation. Well-characterized immortalized cell lines are critical tools for investigating NKT cell biology in vitro. We established and maintained a novel NKT cell line, NKT617, derived from the peripheral blood of a patient with aggressive large granular lymphocyte leukemia. Cell morphology was assessed using scanning electron microscopy and transmission electron microscopy. Flow cytometry characterized the cell phenotype, whereas T-cell receptor (TCR) clonal gene rearrangement determined lineage. RNA sequencing compared gene expression profiles of NKT617 with other hematological tumor cell lines, such as natural killer (NK) cells, T cells, B cells, and monocytes. Tumorigenic potential was evaluated via in vitro colony formation assays and in vivo zebrafish xenograft models. NKT617 has been continuously cultured for >2 years, exceeding 100 passages, demonstrating monoclonal immortalization. It proliferates in a low-dose recombinant human interleukin-2-dependent manner and grows in suspension culture as single cells and aggregates. Flow cytometric analysis showed that NKT617 exhibits an NK-cell phenotype CD56⁺, CD158a⁺, NKp46⁺, membrane-bound CD3^-, and T-cell features cytoplasmic CD3⁺, CD2⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺, with clonal TCRβ rearrangement. NKT617 was negative for B-cell and myeloid markers and positive for the TCRα chain (Vα24-Jα18), confirming its identity as a type I NKT cell. Transcriptome analysis showed shared markers with NK-cell and T-cell lines but a gene expression profile closer to NK cells. In vitro and in vivo assays confirmed its tumorigenic capacity. We report the first Epstein-Barr virus-negative human type I NKT cell line, NKT617, which offers significant potential for studying NKT cell development and advancing chimeric antigen receptor-based therapies. This cell line serves as a valuable tool for exploring NKT cell biology and developing targeted immunotherapies.