OBJECTIVEThis study was to expand the cytotoxic T lymphocytes (CTL) through inducing the differentiation of umbilical blood monomuclear cells (UBMNC) by using various combination of cytokines, and to investigate the functions of expanded CTL.

METHODSThe MNC were isolated by ficoll density gradient centrifugation. Then, the PHA-P, IFN-γ combined with IL-2, IL-15 and other cytokines were used for induction and expansion of the cord blood-derived CTL. The biological function of CTL was examined by phenotype analysis, cytotoxic tests and real-time fluorescence quantitative PCR.

RESULTSAfter expansion for 15 days, the cell number increased by 1522% ± 137%. The content of CD3(-)CD8(-) cells in uncultured cord blood MNC was 95%, and the CD3(+)CD8(+) CTL cells reached 82.77% in cultured cord blood MNC after expansion for 15 days. The expanded CTL cell showed the cytotoxic activity against K562 and HeLa cell line. The killing rate of MNC was 61.88 ± 1.08%. After expansion, the killing rate could reach to 90% with the average value of 90.33 ± 2.02%. The expanded CTL cells highly expressed some key cytokines, such as granzyme A, granzyme B, GM-CSF, granulysin, IFN-γ, TGF-β, TNF-α and perforin. Compared with the control group, the expression of IFN-γ and TGF-β significantly increased (P < 0.05), and the other factors dramatically increased (P < 0.01).

CONCLUSIONThe cord blood-derived CTL can be expanded by different combinations of cytokines. These protocols may provide alternative choices for CTL cell expansion in tumor adoptive immunotherapy.

Cytokines ; Fetal Blood ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granzymes ; Histocompatibility Antigens Class I ; Histocompatibility Antigens Class II ; Humans ; Immunotherapy, Adoptive ; Perforin ; Phytohemagglutinins ; T-Lymphocytes, Cytotoxic

This study was objective to explore the effect of IFN-γ on immunosuppressive capability of mesenchymal stem cells (MSC) derived from umbilical cord. The immunomodulating capability of MSC was changed by stimulating cell surface receptors like Toll-like receptors (TLR). The inhibition of T-lymphocyte proliferation by MSC was tested via cell co-cultures. Further RT-PCR and ELISA were performed to examine the expression changes in gene and protein level. The results showed that the IFN-γ could promote the immunosuppressive effect of umbilical cord derived MSC. IFN-γ-stimulated MSC could suppress the proliferation of T cells more effectively. IFN-γ stimulation up-regulated the expression of immunosuppressive genes like IDO1, COX2, HLA-G, and soluble suppressive proteins such as HLA-G, KYN, IL10, PGE2 of MSC. And the immuno suppression capability of IFN-γ-stimulated MSC was 2-7 folds higher than control in MSC and lymphocyte co-culture tests. It is concluded that IFN-γ can effectively enhance the immunosuppressive capability of MSC.
Cells, Cultured ; Humans ; Immunosuppression ; Interferon-gamma ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Umbilical Cord ; cytology