Cancer Immunology, Immunotherapy

, Volume 67, Issue 4, pp 615–626 | Cite as

The role of interleukin-2, all-trans retinoic acid, and natural killer cells: surveillance mechanisms in anti-GD2 antibody therapy in neuroblastoma

  • Rosa Nguyen
  • Jim Houston
  • Wing K. Chan
  • David Finkelstein
  • Michael A. Dyer
Original Article


Although anti-disialoganglioside (GD2) antibodies are successfully used for neuroblastoma therapy, a third of patients with neuroblastoma experience treatment failure or serious toxicity. Various strategies have been employed in the clinic to improve antibody-dependent cell-mediated cytotoxicity (ADCC), such as the addition of interleukin (IL)-2 to enhance natural killer (NK) cell function, adoptive transfer of allogeneic NK cells to exploit immune surveillance, and retinoid-induced differentiation therapy. Nevertheless, these mechanisms are not fully understood. We developed a quantitative assay to test ADCC induced by the anti-GD2 antibody Hu14.18K322A in nine neuroblastoma cell lines and dissociated cells from orthotopic patient-derived xenografts (O-PDXs) in culture. IL-2 improved ADCC against neuroblastoma cells, and differentiation with all-trans retinoic acid stabilized GD2 expression on tumor cells and enhanced ADCC as well. Degranulation was highest in licensed NK cells that expressed CD158b (P < 0.001) and harbored a killer-cell immunoglobulin-like receptor (KIR) mismatch against the tumor-specific human leukocyte antigen (HLA; P = 0.016). In conclusion, IL-2 is an important component of immunotherapy because it can improve the cytolytic function of NK cells against neuroblastoma cells and could lower the antibody dose required for efficacy, thereby reducing toxicity. The effect of IL-2 may vary among individuals and a biomarker would be useful to predict ADCC following IL-2 activation. Sub-populations of NK cells may have different levels of activity dependent on their licensing status, KIR expression, and HLA–KIR interaction. Better understanding of HLA–KIR interactions and the molecular changes following retinoid-induced differentiation is necessary to delineate their role in ADCC.


Anti-GD2 antibody IL-2 Neuroblastoma NK cells Missing-self 



Antibody-dependent cell-mediated cytotoxicity


All-trans retinoic acid


Effector cell to target cell


Eagle’s minimum essential medium




Human leukocyte antigen




Killer-cell immunoglobulin-like receptor


Natural killer


Orthotopic patient-derived xenograft


Recombinant interleukin-2



We thank Merck Serono and the Children’s GMP, LLC for providing the Hu14.18K322A anti-GD2 antibody to conduct our studies. We also thank the Biological Resource Branch at the National Cancer Institute for providing rIL-2 used in the present study. We thank Drs. Jennifer Peters and Abbas Shirinifard for optimizing the automated microscopy protocol and optimizing the classifier for segmentation purposes in Fiji, respectively. We thank Dr. Dan Kaufman for reviewing the manuscript. We thank Nisha Badders for scientific editing.

Author contributions

RN: experimental design, conduction of experiments, analysis and interpretation of data, draft of the manuscript. JH: experimental design, conduction of flow cytometry experiments, review of the manuscript. WKC: experimental design, interpretation of data, review of the manuscript. DF: analysis of RNA sequencing data, review of the manuscript. MAD: experimental design, analysis and interpretation of data, review of manuscript, funding.


This work was supported, in part, by Cancer Center Support (CA21765) from the NCI, grants to M.A.D. from the NIH (EY014867 and EY018599 and CA168875). This research was supported by HHMI.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

Patient consented to donate biological material (blood and tumor) as part of an ongoing institutional protocol XPD09-234 MAST—MOLECULAR ANALYSIS OF SOLID TUMORS. This protocol was approved by the St. Jude Children’s Research Hospital IRB.

Animal source

All animals were handled according to IACUC approved policies.

Supplementary material

262_2017_2108_MOESM1_ESM.pdf (3.3 mb)
Supplementary material 1 (PDF 3365 KB)


  1. 1.
    Zent CS, Chen JB, Kurten RC et al (2004) Alemtuzumab (CAMPATH 1H) does not kill chronic lymphocytic leukemia cells in serum free medium. Leuk Res 28:495–507. CrossRefPubMedGoogle Scholar
  2. 2.
    Brentjens RJ, Davila ML, Riviere I et al (2013) CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5:177ra38. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Larkin J, Chiarion-Sileni V, Gonzalez R et al (2015) Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med 373:23–34. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Slamon DJ, Leyland-Jones B, Shak S et al (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792. CrossRefPubMedGoogle Scholar
  5. 5.
    Hurwitz H, Fehrenbacher L, Novotny W et al (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335–2342. CrossRefPubMedGoogle Scholar
  6. 6.
    Yu AL, Gilman AL, Ozkaynak MF et al (2010) Anti-GD2 Antibody with GM-CSF, Interleukin-2, and Isotretinoin for Neuroblastoma. N Engl J Med 363:1324–1334. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schulz G, Cheresh DA, Varki NM et al (1984) Detection of ganglioside GD2 in tumor tissues and sera of neuroblastoma patients. Cancer Res 44:5914–5920PubMedGoogle Scholar
  8. 8.
    Janeway CA Jr, Travers P, Walport M et al (2001) Immunobiology: The Immune System in Health and Disease. 5th edn. New York: Garland Science. The destruction of antibody-coated pathogens via Fc receptors. Accessed 6 May 2017
  9. 9.
    Kärre K, Ljunggren HG, Piontek G, Kiessling R (1986) Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319:675–678. CrossRefPubMedGoogle Scholar
  10. 10.
    Ruggeri L, Mancusi A, Capanni M et al (2007) Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value. Blood 110:433–440. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Henney CS, Kuribayashi K, Kern DE, Gillis S (1981) Interleukin-2 augments natural killer cell activity. Nature 291:335–338. CrossRefPubMedGoogle Scholar
  12. 12.
    Villablanca JG, Khan AA, Avramis VI et al (1995) Phase I trial of 13-cis-retinoic acid in children with neuroblastoma following bone marrow transplantation. J Clin Oncol 13:894–901. CrossRefPubMedGoogle Scholar
  13. 13.
    Stewart E, Shelat A, Bradley C et al (2015) Development and characterization of a human orthotopic neuroblastoma xenograft. Dev Biol 407:344–355. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Downing JR, Wilson RK, Zhang J et al (2012) The pediatric cancer genome project. Nat Genet 44:619–622. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Anders S, Pyl PT, Huber W (2015) HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169. CrossRefPubMedGoogle Scholar
  16. 16.
    André P, Biassoni R, Colonna M et al (2001) New nomenclature for MHC receptors. Nat Immunol 2:661. CrossRefPubMedGoogle Scholar
  17. 17.
    Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968) Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14:181–196PubMedPubMedCentralGoogle Scholar
  18. 18.
    Cederbrant K (2005) Natural killer cell assay. In: Vohr H-W (ed) Encyclopedic reference of immunotoxicology. Springer, Berlin, pp 469–472CrossRefGoogle Scholar
  19. 19.
    Sorkin LS, Otto M, Baldwin WM et al (2010) Anti-GD2 with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia. Pain 149:135–142. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Alderson KL, Luangrath M, Elsenheimer MM et al (2013) Enhancement of the anti-melanoma response of Hu14.18K322A by αCD40 + CpG. Cancer Immunol Immunother 62:665–675. CrossRefPubMedGoogle Scholar
  21. 21.
    Hank JA, Surfus J, Gan J et al (1994) Treatment of neuroblastoma patients with antiganglioside GD2 antibody plus interleukin-2 induces antibody-dependent cellular cytotoxicity against neuroblastoma detected in vitro. J Immunother Emphas Tumor Immunol 15:29–37CrossRefGoogle Scholar
  22. 22.
    Rossi AR, Pericle F, Rashleigh S et al (1994) Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12. Blood 83:1323–1328PubMedGoogle Scholar
  23. 23.
    Brodin P, Lakshmikanth T, Johansson S et al (2009) The strength of inhibitory input during education quantitatively tunes the functional responsiveness of individual natural killer cells. Blood 113:2434–2441. CrossRefPubMedGoogle Scholar
  24. 24.
    Joncker NT, Fernandez NC, Treiner E et al (2009) NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model. J Immunol 182:4572–4580. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Howlander N, Noone A, Krapcho M et al (2011) SEER Cancer Statistics Review, 1975–2009. National Cancer Institute, BethesdaGoogle Scholar
  26. 26.
    Navid F, Sondel PM, Barfield R et al (2014) Phase I trial of a novel anti-GD2 monoclonal antibody, Hu14.18K322A, designed to decrease toxicity in children with refractory or recurrent neuroblastoma. J Clin Oncol 32:1445–1452. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA (1982) Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J Exp Med 155:1823–1841CrossRefPubMedGoogle Scholar
  28. 28.
    Hank JA, Robinson RR, Surfus J et al (1990) Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2. Cancer Res 50:5234–5239PubMedGoogle Scholar
  29. 29.
    Ribeiro RC, Rill D, Roberson PK et al (1993) Continuous infusion of interleukin-2 in children with refractory malignancies. Cancer 72:623–628CrossRefPubMedGoogle Scholar
  30. 30.
    Ladenstein R, Poetschger U, Gray J et al (2016) Toxicity and outcome of anti-GD2 antibody ch14.18/CHO in front-line, high-risk patients with neuroblastoma: final results of the phase III immunotherapy randomisation (HR-NBL1/SIOPEN trial). J Clin Oncol 34(15_suppl):Abstract 10500Google Scholar
  31. 31.
    Atkins MB, Gould JA, Allegretta M et al (1986) Phase I evaluation of recombinant interleukin-2 in patients with advanced malignant disease. J Clin Oncol 4:1380–1391. CrossRefPubMedGoogle Scholar
  32. 32.
    Lode H, Siebert N, Kietz S et al (2013) Long-term continuous infusion of anti-GD2 antibody CH14.18/CHO in relapsed/refractory neuroblastoma patients. J Immunother Cancer 1(1_suppl):Abstract P244CrossRefGoogle Scholar
  33. 33.
    Delgado DC, Hank JA, Kolesar J et al (2010) Genotypes of NK Cell KIR Receptors, Their Ligands, and Fc Receptors in the Response of Neuroblastoma Patients to Hu14.18-IL2 Immunotherapy. Cancer Res 70:9554–9561. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Venstrom JM, Zheng J, Noor N et al (2009) KIR and HLA genotypes are associated with disease progression and survival following autologous hematopoietic stem cell transplantation for high-risk neuroblastoma. Clin Cancer Res 15:7330–7334. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Marsh SGE, Parham P, Barber LD (2000) The HLA factsbook. Academic Press, San DiegoGoogle Scholar
  36. 36.
    Jin HJ, Nam HY, Bae YK et al (2010) GD2 expression is closely associated with neuronal differentiation of human umbilical cord blood-derived mesenchymal stem cells. Cell Mol Life Sci 67:1845–1858. CrossRefPubMedGoogle Scholar
  37. 37.
    Zou Z, Nomura M, Takihara Y et al (1996) Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: a novel cDNA family encodes cell surface proteins sharing partial homology with MHC class I molecules. J Biochem 119:319–328CrossRefPubMedGoogle Scholar
  38. 38.
    Ho EL, Carayannopoulos LN, Poursine-Laurent J et al (2002) Costimulation of multiple NK cell activation receptors by NKG2D. J Immunol 169:3667–3675. CrossRefPubMedGoogle Scholar
  39. 39.
    Reynolds CP, Kane DJ, Einhorn PA et al (1991) Response of neuroblastoma to retinoic acid in vitro and in vivo. Prog Clin Biol Res 366:203–211PubMedGoogle Scholar
  40. 40.
    Schug TT, Berry DC, Shaw NS et al (2007) Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell 129:723–733. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Rosa Nguyen
    • 1
  • Jim Houston
    • 2
  • Wing K. Chan
    • 3
  • David Finkelstein
    • 4
  • Michael A. Dyer
    • 2
  1. 1.Department of OncologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Developmental NeurobiologySt. Jude Children’s Research HospitalMemphisUSA
  3. 3.The James Comprehensive Cancer CenterThe Ohio State UniversityColumbusUSA
  4. 4.Department of Computational BiologySt. Jude Children’s Research HospitalMemphisUSA

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