Culture and differentiation of cytokine-induced killer cells from umbilical cord blood-derived mononuclear cells

Abstract

Cytokine-induced killer cells (CIK) are cytotoxic T cells, which have both NK and T cell properties. These cells are characterized by potent, non-MHC-restricted cytotoxicity and reduced alloreactivity, which make them appealing for use in adoptive immunotherapy of cancer and virus infections. In this study, CIK cells were generated by stimulating umbilical cord blood-derived mononuclear cells (UCB-MNCs) with interferon-gamma (IFN-g) on day 0. Anti-CD3 antibody and interleukin-2 (IL-2) were added after 24 hours at four different experimental concentration combinations in order to identify the optimal cytokine amounts for CIK cell proliferation. Cells were collected at four time points over a 21-day period (day 0, 7, 14, 21) for analysis of cell marker presentation using flow cytometry, as well as transcription-level cytokine production using RT-PCR. The results showed that in the 21-day culture, the average final expansion levels of CD3⁺CD56⁺ CIK cell were in the range of hundredfold, accounted for 26% in the bulk culture. Most important, these cells strongly expressed granzyme B (80.87%), a potent factor involved in cell-mediated cytotoxicity. These CIK cells also transcriptionally overexpressed the three cytokine genes that produce IFN-g, tumor necrosis factor-alpha (TNF-a), and IL-2; these are key for immune cell mobilization against tumors as well as foreign pathogens. Our research establishes an effective cytokine concentration and time protocol for use in generation of CIK cells from UCB-MNCs, potentiating greater applications of CIK cell-adoptive immunotherapy in both research and clinical settings. Thus, the 3­^rd and 4^th experimental conditions both stimulated CIK cell differentiation with 50 ng/ml of anti-CD3 antibody, but with IL-2 concentrations of 500 U/ml and 1000 U/ml, respectively.