Anti-leukemia activity of T cells impacted by dendritic cells added with sodium selenite / 中国实验血液学杂志

ABSTRACT

The study was purposed to explore the quantity, morphology and immunophenotype of dendritic cells (DC) acquired by co-cultivated system with 3 types of cytokines and sodium selenite (Se) from peripheral blood mononuclear cells (PBMNCs), and to investigate the effects of Se on inducing the cytotoxic T lymphocyte (CTL) to get specific anti-leukemic activity in vitro by DC pulsed with K562 cell frozen-thawed antigen (antigen cell loading). PBMNCs isolated from healthy donors were cultured in RPMI 1640 medium contained 10% FBS supplied with 3 cytokines (rhGM-CSF, rhIL-4, TNF-alpha) for 4 days, DCs harvested were divided into 4 groups, DCI DC alone; DCII DC + Se (adding 0.5 micromol/L of Se); DCIII DC + K562 (pulsed with lysed K562 cells); DCIV DC + Se + K562. Morphology of DCs was observed under microscope at day 7. The CD1a, CD40, CD83, and CD86 were detected by FCM. Cytotoxicity of T cells induced by DC were measured with LDH release test at day 12. The level of IL-12 in supernatant of cultured DCs were determined with ELISA. The results indicated that at 7th day DC in 4 groups showed characteristic morphology, the colony numbers of 4 groups were all higher than those before cultivation. There were no obvious differences of morphology and colony counts between DCI group and DCII group. The colony numbers of DCIII group and DCIV group increased, as well as the ratio of suspended cells enhanced. The expressions of CD1a, CD40, CD83 and CD86 in 4 groups of DC were significantly higher than those in PBMNC group (p < 0.01), the expressions of CD1a and CD40 in 4 groups of DC did not display significant difference (p > 0.05), the expressions of CD83 and CD86 in both DCIII group and DCIV group were all higher than those in DCI group and DCII group (p < 0.01), but their expressions of CD83 and CD86 in DCI and DCII were not significantly different (p > 0.05), as well as those in DCIII group and DCIV group. With the ratio of 251 between ET, killing rate of CTL on K562 cells in 4 DC groups were 15.3 +/- 2.3%, 26.3 +/- 3.7%, 28.2 +/- 4.5% and 36.2 +/- 3.7% respectively, all obviously higher than those of T cell group without being sensitized by DCs (5.9 +/- 2.4%) (p < 0.01), The CTL effect in DCIV group was the highest, which was higher than those in other 3 DC groups (p < 0.01); the effects in both DCII and DCIII group were also higher than that in DCI group (p < 0.01), but their difference between DCII and DCIII groups did not show significance (p > 0.05). The levels of IL-12 in supernatant of DCI, DCII, DCIII and DCIV groups were 257.0 +/- 64.2, 328.1 +/- 43.9, 323.0 +/- 53.5 and 353.9 +/- 46.2 pg/ml respectively, all significantly higher than that in supernatant of T cell alone group without being sensitized by DCs (35.27 +/- 27.1 pg/ml) (p < 0.01), The levels in DCII, DCIII and DCIV groups were all higher than that in DCI group (p < 0.01), but their levels between DCII, DCIII and DCIV groups were not of significant difference (p > 0.05). It is concluded that matured DCs can be successfully obtained from PBMNCs by a culture system contained rhGM-CSF, rhIL-4 and TNF-alpha with or without low-dose of Se (0.5 micromol/L) in vitro. Using K562 cell frozen-thawed antigen, DC express more adhesive molecules and co-stimulating molecules (CD83, CD86), and increase the secretion of IL-12, as well as the killing effects of CTL on special target cells. Low dose of Se did not showed effects on quantity and morphology of matured DC harvested, as well as their expression of mature phenotypes, it raised levels of IL-12 secreted by DCs, reaching the same level as using K562 cell frozen-thawed antigen, and it showed synergistic effect on induction of CTL with K562 cell frozen-thawed antigen.