Improvement of NK cells functional activation after long-term IL-2 stimulation in vitro from ovarian neoplasia patients
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1
University of Campinas UNICAMP, Brazil
Introduction: NK cells are lymphocytes known by their ability to eliminate a variety of malignant cells without previous stimulation, in a process involving innate recognition by an array of stimulatory and inhibitory receptors. Similarly, the variant subset of NK-like T lymphocytes has been reported to eliminate tumor cells, but the targeting process might involve either innate or adaptive immune recognition. This study evaluated the functional activation of NK and NK-like T cells, the expression of activating receptors DNAM-1, NKp30 and NKp44 (pre, short- and long-term IL-2 stimulated), from blood and ascites of ovarian neoplasia patients.
Methods and Results: Blood and ascites were collected from 24 patients with ovarian neoplasias after signed consent: 11 benign (Bng), 6 malignant without metastasis (Mlg) and 7 malignant with metastasis (MlgMt). Ascites (Asc) was collected from 6 patients with ovarian neoplasia. Mononuclear cells were separated by Ficoll-Paque gradient. NK and NK-like T cells activation (pre, short- and long-term stimulated) were evaluated against K562 (1:1 ratio) by the expression of CD107a, analyzed by flow cytometry. Short-term stimulation with IL-2 (1000UI/ml) was conducted overnight in RPMI-1640 medium supplemented with FBS (10%) and L-glutamine (2mM). Long-term stimulation was conducted by a 21 day culturing process with SCGM CellGro medium supplemented with anti-CD3 (10ng/ml, first 5 days), IL-2 (1000UI/ml) and FBS (10%). NK functional activation of pre stimulated cells, seemed to be impaired as the disease develops, as assessed by the percentage median/variation (Bng=13.86/ 2.25-21.78; Mlg=9.57/ 6.48-12.96; MlgMt=7.73/ 3.65-10.06; Asc=6.99/ 1.68-21.68). Short-term stimulation increased NK cells activation (Bng=24.19/ 9.52-57.03; Mlg=24.49/ 8.69-52.37; MlgMt=17.59/ 9.28-35.63; Asc=33.54/ 3.83-43.50). Long-term stimulation increased NK cells activation significantly (p<0.001) (Bng=58.04/ 40.39-74.48; Mlg=46.61/ 33.89-54.76). The percentage of NK cells expressing the activating receptors DNAM-1, NKp30 and NKp44 increased significantly (p<0.05) after long-term stimulation. NK-like T cells showed no activation on pre, short- and long-term IL-2 stimulation. Statistical analysis inter groups was performed by Kruskal-Wallis test and intra groups by Mann Whitney test.
Conclusion: The functional integrity of NK cells was impaired as ovarian malignancies develop. Long-term stimulation resulted in a much higher number of functional NK cells compared to short-term, entitling this method for adoptive therapy. Long-term stimulation was particularly efficient to up-regulate DNAM-1 activating receptor on NK cells, representing a way to overcome down-regulation demonstrated on patients with ovarian carcinoma. The lack of activation of NK-like T cells (pre, short- and long-term IL-2 stimulated) suggests that these cells are not activated through innate pathway but through adaptive pathway.
Acknowledgements
Financial Support: FAPESP 2010/17202-7 and 2011/14520-0.
Keywords:
NK cells,
NK-like T cells,
IL-2,
NCRs,
Immunotherapy
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Innate immunity
Citation:
Silva
RF,
Petta
CA and
Guimarães
F
(2013). Improvement of NK cells functional activation after long-term IL-2 stimulation in vitro from ovarian neoplasia patients.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00496
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Received:
27 Mar 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Mr. Rodrigo F Silva, University of Campinas UNICAMP, Campinas, Brazil, rodrigoiverson@hotmail.com