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


METHODS
Culture 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.


RESULTS
After 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.


CONCLUSIONS
Wilms tumor is sensitive to human NK cells and is highly efficient for selective expansion of NK cells from PBMCs.
Peng
Bao-Gang
BG
Department of Hepatobiliary Surgery, First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan Road 2, Guangzhou 510080, Guangdong Province, China. pengbaogang@163.net
Liang
Li-Jian
LJ
He
Qiang
Q
Huang
Jie-Fu
JF
Lu
Ming-De
MD
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000628:therapy*; D002455:Cell Division; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D006801:Humans; D016219:Immunotherapy, Adoptive; D007680:Kidney Neoplasms / Q000473:pathology*; D007694:Killer Cells, Natural / Q000166:cytology* / Q000473:pathology*; D008113:Liver Neoplasms / Q000097:blood / Q000628:therapy*; D051379:Mice; D009000:Monocytes / Q000473:pathology*; D009396:Wilms Tumor / Q000473:pathology*