[Study on ex vivo expansion of highly purified NK cells from human peripheral blood and changes in their function].
OBJECTIVE
To explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.


METHODS
NK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.


RESULTS
CD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).


CONCLUSIONS
High purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.
Li
Xiao-Hong
XH
Center of Hematology, PLA General Hospital, Beijing, China.
Ma
Jian
J
Wu
Xiao-Xiong
XX
Wang
Fei-Fei
FF
Li
Meng
M
Da
Wan-Ming
WM
Yu
Li
L
Gao
Chun-Ji
CJ
D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000494:pharmacology; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D007378:Interleukins / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D054353:Perforin / Q000378:metabolism
D019409:Interleukin-15; D007376:Interleukin-2; D007378:Interleukins; D054353:Perforin; D018664:Interleukin-12; D007371:Interferon-gamma; D053804:Granzymes