[Expansion and cytokine secretion profile of human valpha24(+) NKT cells from different sources].
This study was purposed to investigate the phenotype, in vitro expansion and cytokine secretion profile of Valpha24(+) NKT cells from cord blood (CB), peripheral blood (PB), and granulocyte colony stimulating factor-mobilized peripheral blood mononuclear cells (G-PBMNCs). Fresh mononuclear cells (MNCs) were isolated by the method of gradient centrifugation and then cultured with alpha-GalCer (100 ng/ml), IL-2 (50 U/ml), IL-15 (50 ng/ml) for 12 days. Valpha24(+) NKT cells were purified by anti-Vbeta11 TCR McAb and goat anti-mouse IgG magnetic beads. The phenotype and purity of Valpha24(+) NKT cells were determined by flow cytometry. Cytokine production was analyzed by ELISA. The results showed that Valpha24(+) NKT cells in CB, PB and G-PBMNCs were expanded by 221.5 (95 - 501), 456.5 (101 - 2207), and 756.38 (82 - 20373)-fold respectively. After stimulation by phorbol-12-myristate-13-acetate (PMA) for 24 hours, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells from CB and PB were 180.33 (144.67 - 2253.48) vs 190.67 (110.07 - 6060.16) ng/ml, 864.33 (401.33 - 3386.67) vs 508.49 (253.82 - 8840.00) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.503 +/- 0.642 vs 0.455 +/- 0.562 respectively. After expansion of Valpha24(+) NKT cells from G-PBMNCs, IL-4 and IFN-gamma produced by Valpha24(+) NKT cells at day 9 and day 12 were 139.08 (7.62 - 606) vs 89.3 (0 - 729.2) ng/ml, 14264.8 (1168 - 18059) vs 14488 (1041 - 18261) ng/ml respectively, with IL-4/IFN-gamma ratio of 0.0531 +/- 0.1081 vs 0.0376 +/- 0.1148 respectively. It is concluded that in presence of IL-2 and IL-15, alpha-GalCer can facilitate the rapid short-term expansion of Valpha24(+) NKT cells from CB, PB, and G-PBMNCs. Valpha24(+) NKT cells from G-PBMNCs show much high potential of expansion in comparison to the counterparts from CB or PB (p < 0.05). The activated Valpha24(+) NKT cells can secrete IFN-gamma and IL-4 in large amounts, with IFN-gamma in particular.
Zhai
Wei-Hua
WH
National Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Disease Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.
Huang
Yong
Y
Wang
Mei
M
Zhou
Zheng
Z
Zhai
Wen-Jing
WJ
Zhang
Rong-Li
RL
Zhang
Ping
P
Han
Ming-Zhe
MZ
D018929:Cell Culture Techniques; D005312:Fetal Blood / Q000166:cytology; D005699:Galactosylceramides / Q000494:pharmacology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D015847:Interleukin-4 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008213:Lymphocyte Activation; D055611:Natural Killer T-Cells / Q000378:metabolism*
D005699:Galactosylceramides; D019409:Interleukin-15; D007376:Interleukin-2; C493154:alpha-galactosylceramide; D015847:Interleukin-4; D007371:Interferon-gamma