Interleukin-18 Synergism with Interleukin-2 in Cytotoxicity and NKG2D Expression of Human Natural Killer Cells

  • Qi, Yuan-Ying (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Lu, Chao (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Ju, Ying (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Wang, Zi-E (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Li, Yuan-Tang (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Shen, Ya-Juan (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University) ;
  • Lu, Zhi-Ming (Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University)
  • Published : 2014.10.11


Natural killer (NK) cells play an important role in anti-tumor immunity. Interleukin (IL)-18 is an immunoregulatory cytokine that induces potent NK cell-dependent anti-tumor responses when administrated with other cytokines. In this study, we explored the effects of combining IL-18 and IL-2 on NK cytotoxicity as well as expression levels of the NK cell receptor NKG2D in vitro. Freshly isolated PBMCs were incubated for 48 h with IL-18 and IL-2, then CD107a expression on $CD3^-CD56^+$ NK cells was determined by three-colour flow cytometry to evaluate the cytotoxicity of NK cells against human erythroleukemia K562 cells and human colon carcinoma HT29 cells. Flow cytometric analysis was also employed to determine NKG2D expression on NK cells. The combined use of IL-18 and IL-2 significantly increased CD107a expression on NK cells compared with using IL-18 or IL-2 alone, suggesting that the combination of these two cytokines exerted synergistic enhancement of NK cytotoxicity. IL-18 also enhanced NKG2D expression on NK cells when administered with IL-2. In addition, blockade of NKG2D signaling with NKG2D-blocking antibody attenuated the up-regulatory effect of combining IL-18 and IL-2 on NK cytolysis. Our data revealed that IL-18 synergized with IL-2 to dramatically enhance the cytolytic activity of human NK cells in a NKG2D-dependent manner. The results appear encouraging for the use of combined IL-18 and IL-2 in tumor immunotherapy.


  1. Aktas E, Kucuksezer UC, Bilgic S, et al (2009). Relationship between CD107a expression and cytotoxic activity. Cell Immunol, 254, 149-54.
  2. Atzpodien J, Reitz M (2005). Metastatic renal carcinoma longterm survivors treated with s.c. interferon-alpha and s.c. interleukin-2. Cancer Biother Radiopharm, 20, 410-6.
  3. Chaix J, Tessmer MS, Hoebe K, et al (2008). Cutting edge: priming of NK cells by IL-18. J Immunol, 181, 1627-31.
  4. Clark JM, Kelley B, Titze J, et al (2013). Clinical and safety profile of high-dose interleukin-2 treatment in elderly patients with metastatic melanoma and renal cell carcinoma. Oncology, 84, 123-6.
  5. Du G, Ye L, Zhang G, et al (2012). Human IL18-IL2 fusion protein as a potential antitumor reagent by enhancing NK cell cytotoxicity and IFN-gamma production. J Cancer Res Clin Oncol, 138, 1727-36.
  6. Gaffen SL, Liu KD (2004). Overview of interleukin-2 function, production and clinical applications. Cytokine, 28, 109-23.
  7. Hu YX, Li M, Jia XH, et al (2013). HPV16 CTL epitope peptideactivated dendritic cell and natural killer co-culture for therapy of cervical cancer in an animal model. Asian Pac J Cancer Prev, 14, 7335-8.
  8. Huergo-Zapico L, Acebes-Huerta A, Lopez-Soto A, et al (2014). Molecular bases for the regulation of NKG2D ligands in cancer. Front Immunol, 5, 106.
  9. Konjevic G, Jurisic V, Jovic V, et al (2012). Investigation of NK cell function and their modulation in different malignancies. Immunol Res, 52, 139-56.
  10. Kuppala MB, Syed SB, Bandaru S, et al (2012). Immunotherapeutic approach for better management of cancer--role of IL-18. Asian Pac J Cancer Prev, 13, 5353-61.
  11. Marcais A, Viel S, Grau M, et al (2013). Regulation of mouse NK cell development and function by cytokines. Front Immunol, 4, 450.
  12. Muntasell A, Magri G, Pende D, et al (2010). Inhibition of NKG2D expression in NK cells by cytokines secreted in response to human cytomegalovirus infection. Blood, 115, 5170-9.
  13. Ni J, Miller M, Stojanovic A, et al (2012). Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors. J Exp Med, 209, 2351-65.
  14. Rajbhandary S, Zhao MF, Zhao N, et al (2013). Multiple cytotoxic factors involved in IL-21 enhanced antitumor function of CIK cells signaled through STAT-3 and STAT5b pathways. Asian Pac J Cancer Prev, 14, 5825-31.
  15. Robertson MJ, Kirkwood JM, Logan TF, et al (2008). A doseescalation study of recombinant human interleukin-18 using two different schedules of administration in patients with cancer. Clin Cancer Res, 14, 3462-9.
  16. Segawa S, Goto D, Yoshiga Y, et al (2011). Involvement of NK 1.1-positive gammadeltaT cells in interleukin-18 plus interleukin-2-induced interstitial lung disease. Am J Respir Cell Mol Biol, 45, 659-66.
  17. Shen Y, Lu C, Tian W, et al (2012). Possible association of decreased NKG2D expression levels and suppression of the activity of natural killer cells in patients with colorectal cancer. Int J Oncol, 40, 1285-90.
  18. Son YI, Dallal RM, Lotze MT (2003). Combined treatment with interleukin-18 and low-dose interleukin-2 induced regression of a murine sarcoma and memory response. J Immunother, 26, 234-40.
  19. Son YI, Dallal RM, Mailliard RB, et al (2001). Interleukin-18 (IL-18) synergizes with IL-2 to enhance cytotoxicity, interferon-gamma production, and expansion of natural killer cells. Cancer Res, 61, 884-8.
  20. Song H, Hur DY, Kim KE, et al (2006). IL-2/IL-18 prevent the down-modulation of NKG2D by TGF-beta in NK cells via the c-Jun N-terminal kinase (JNK) pathway. Cell Immunol, 242, 39-45.
  21. Srivastava S, Pelloso D, Feng H, et al (2013). Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin. Cancer Immunol Immunother, 62, 1073-82.
  22. Tang F, Sally B, Ciszewski C, et al (2013). Interleukin 15 primes natural killer cells to kill via NKG2D and cPLA2 and this pathway is active in psoriatic arthritis. PLoS One, 8, e76292.
  23. Tarhini AA, Millward M, Mainwaring P, et al (2009). A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer, 115, 859-68.
  24. Tonn T, Schwabe D, Klingemann HG, et al (2013). Treatment of patients with advanced cancer with the natural killer cell line NK-92. Cytotherapy, 15, 1563-70.
  25. Tse BW, Russell PJ, Lochner M, et al (2011). IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms. PLoS One, 6, e24241.
  26. Vivier E, Tomasello E, Baratin M, et al (2008). Functions of natural killer cells. Nat Immunol, 9, 503-10.
  27. Wong JL, Berk E, Edwards RP, et al (2013). IL-18-primed helper NK cells collaborate with dendritic cells to promote recruitment of effector CD8+ T cells to the tumor microenvironment. Cancer Res, 73, 4653-62.
  28. Yu DP, Han Y, Zhao QY, et al (2014). CD3+ CD4+ and CD3+ CD8+ lymphocyte subgroups and their surface receptors NKG2D and NKG2A in patients with non-small cell lung cancer. Asian Pac J Cancer Prev, 15, 2685-8.
  29. Yuan CH, Yang XQ, Zhu CL, et al (2014). Interleukin-7 enhances the in vivo anti-tumor activity of tumor-reactive CD8+ T cells with induction of IFN-gamma in a murine breast cancer model. Asian Pac J Cancer Prev, 15, 265-71.
  30. Zafirova B, Wensveen FM, Gulin M, et al (2011). Regulation of immune cell function and differentiation by the NKG2D receptor. Cell Mol Life Sci, 68, 3519-29.
  31. Zhang C, Zhang J, Niu J, et al (2008). Interleukin-12 improves cytotoxicity of natural killer cells via upregulated expression of NKG2D. Hum Immunol, 69, 490-500.
  32. Zhao L, Wang WJ, Zhang JN, et al (2014). 5-Fluorouracil and interleukin-2 immunochemotherapy enhances immunogenicity of non-small cell lung cancer A549 cells through upregulation of NKG2D ligands. Asian Pac J Cancer Prev, 15, 4039-44.