| Objective:To observe the amplification and cytotoxicity of NK cells cultured in IL-2and IL-15supplemented medium in vitro, and to compare the efficiency of Herceptin on NK cells amplification and cytotoxicity against multiple tumor cells in vitro. Methods:30patients with breast cancer in Tianjin Medical University Cancer Institute and Hospital from February2012to March2013were enrolled, PBMCs from those patients were isolated by Ficoll-Hypaque density gradient centrifugation and.cultured with the NK cells conditioned medium supplemented with IL-2and IL-15. Cells expansion and the distribution of different lymphocyte subsets were detected using flow cytometry after being cultured with IL-2and IL-15for15days. The expression levels of activating and inhibitory receptors on cell surface were compared during whole process by flow cytometry as well. The anti-tumor cytotoxicity against HLA-matched or mismatched cancer cells was measured using LDH cytotoxicity and CD107a release assay. A novel culture system for NK cells expansion were established, composing Herceptin, IL-2and IL-15. The cell expansion and distribution of different lymphocyte subsets (especially for NK cells) were detected by flow cytometry on Day0, Day5, Day10, Day15, as well as bothell surface receptors. The anti-tumor cytotoxicity against different cancer cells were measured using LDH cytotoxicity and CD107a release assay and the interaction between expansion products and cancer cells were observed using multi-dimensional workstation. Cytokine secretion (IFN-γ, TNF-α and TGF-β) was detected on Day15by ELISA when expansion products co-cultured with different cancer cells which were generated with or without Herceptin. Results:After expansion for15days, the expansion folds of total cells in two groups were37.6±26.31and44.82±6.84, respectively. The expansion folds of NK cells in two groups were89.60±57.17and102.81±94.06respectively. The expansion folds of CD3+CD56+NKT cells in two groups were257.01±149.73and362.74±208.86respectively and the expansion folds of T cells in two groups were37.18±16.84and43.20±14.75respectively. No significant difference of the total cell expansion and NK cells expansion folds was observed between the two groups.For group1, NKp30and NKp44on NK cells increased significantly than those on uncultured PBMCs, while there were no significant difference on T cells (P＜0.05). Activation receptors DNAM-1and NKG2D on both CD3+CD56+NKT cells and T cells significantly increased (P<0.05). However, CD16on NK cells significantly decreased than those on PBMCs (P<0.05). The cytotoxicity of total expansion cells, NK cells and CD3+CD56+NKT cells against HLA-mismatched tumor cells were significantly higher than that against HLA-matched tumor cells (P＜0.05). After Herceptin treatment, the cytotoxicity of expansion products against multiple cancer cells especially against HLA-mismatched and HER2+cancer cells, as well as the cytotoxicity of NK and CD3+CD56+NKT cells were higher than that without Herceptin (P＜0.05). Consistently, activating surface receptor, such as NKp44significantly increased after Herceptin treatment (P<0.05). Besides that, more potent adhension of NK and CD3+CD56+NKT cells were detected against HLA-mismatched and HER2+cancer cells in vitro (P<0.05). In addition, Herceptin treatment increased the secretion of IFN-y and TNF-a but inhibited the secretion of TGF-β(P<0.05). Conclusion:Not only NK cells but also CD3+CD56+NKT cells can be amplified dramatically after being cultured with IL-2and IL-15. The cytotoxicity of expanded products are mainly derived from NK cells mediated non HLA-restricted killing activity. Herceptin enhanced the cytotoxicity of NK cells by increasing activating receptors expressing on NK cells, upregulating cell adhension and promoting Thl cytokine secretion. |
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