Selective expansion of anti-tumor innate lymphocytes in long-term cultures after a single BCG pulse

Natural Killer (NK) cells are cytotoxic lymphocytes involved in the recognition of pathogen-infected and cancer cells. NK cells are very attractive as cell therapy tools because they are neither restricted by donor compatibility nor do they cause toxicity. Although their anti-tumor role has been long known, for development of NK-based therapies it is important to select the appropriate subpopulation. Similarly, non-MHC restricted T cells, in particular γδ T cells, have also been proposed as novel weapons against cancer. Here, we describe a new approach for production and characterization of anti-tumor innate lymphocyte cultures, containing mainly NK and γδ T cells, based on stimulation of peripheral blood mononuclear cells (PBMC) with BCG (Bacillus Calmette-Guérin), the tuberculosis vaccine, which is also successfully used to treat non-muscle invasive bladder cancer. Anti-tumor innate lymphocytes specifically proliferate from BCG-primed PBMC and can be cultured for weeks in low doses of IL12, IL15 and IL21. These cells kill a wide range of tumors and remain functional for weeks, with minimal manipulation. The phenotypic analysis of these cultures by multi-parametric flow cytometry is explained. Functional assays, including lymphocyte degranulation, cytokine production and radioactive isotope-free specific lysis experiments are also described.