Expansion and activation of natural killer cells from PBMC for immunotherapy of hepatocellular carcinoma.

aimTo induce efficient expansion of natural killer (NK) cells from peripheral blood mononuclear cells (PBMCs) using a culture of anchorage-dependent Wilms tumor cell lines, and to provide a reliable supply for adoptive immunotherapy of hepatocellular carcinoma.

methodsCulture expansion of NK cells was achieved using PBMCs cultured with Wilms tumor cells. Cytotoxicity was measured using a standard (51)Cr release assay and crystal violet staining technique. The proportions of CD3+, CD4+, CD8+, CD16+, and CD56+ cells were determined by flow cytometry.

resultsAfter PBMCs from healthy donors and hepatocellular carcinoma (HCC) were cultured with irradiated HFWT cells for 10-21 d, CD56+ CD16+ cells shared more than 50% of the cell population, and more than 80% of fresh HFWT cells were killed at an effector/target ratio of 2 over 24 h. NK-enriched lymphocyte population from HCC patients killed HCC-1 and 2 cells with sensitivities comparable to fresh TKB-17RGB cells. HCC cells proliferated 196-fold with the irradiated HFWT cells at 18 d. Stimulation by HFWT cells required intimate cell-cell interaction with PBMC. However, neither the soluble factors released from HFWT cells nor the fixed HFWT cells were effective for NK expansion. The lymphocytes expanded with IL-2 killed fresh HFWT target cells more effectively than the lymphocytes expanded with the 4-cytokine cocktail (IL-lbeta, IL-2, IL-4 and IL-6). IL-2 was the sole cytokine required for NK expansion.

conclusionWilms tumor is sensitive to human NK cells and is highly efficient for selective expansion of NK cells from PBMCs.