[Establishment of culture system for selective amplification of CD8+ T cells in vitro and analysis of its functional and molecular phenotypes from tumor-infiltrating lymphocytes in malignant pleural/ascites].

Abstract

Objective To explore the culture method of mass amplification for tumor-infiltrating lymphocytes (TILs) from malignant pleural/ascites in vitro, and identify the function and molecular phenotype of these amplified cells. Methods The pleural/ascites fluid was extracted under aseptic conditions, and lymphocytes were isolated by density gradient centrifugation. Then TILs were amplified by the program based on combined IFN-γ, OKT3 and IL-2, and the cell morphology and growth rate were recorded. The molecular phenotypes of the amplified lymphocytes were analyzed by Flow cytometry, and the killing ability against tumor cells was detected by CCK-8 assay. Results In this culture program, TILs remained in good condition until the 26th day, and the proliferation rate began to decrease on the 30th day. The proportions of CD4^-CD8⁺ and CD8⁺CD56⁺ T cells gradually increased as cell culture time extended while the proportions of CD4⁺CD25⁺ T cells decreased gradually. Unlike the proportions prior to amplification, the proportions of SLAMF7, CD45RO, PD-1 and granzyme B positive cells in T lymphocyte subpopulation were significantly increased, meanwhile, the expression of exhausted T-cell marker CD57 was also gradually increased. The cytotoxicity of amplified CD8⁺ T cells from TILs was significantly stronger than that from PBMC, and the cytotoxicity reached the peak at the effect-target ratio of 10:1 and was significantly different among tumor cell types. Conclusion A culture program for TILs amplification from cancerous thoracic/ascites is established. The method is simple and efficient. The effector cells are mainly CD8⁺ T lymphocytes with active phenotype.