Supplementary Figure S2. Cytotoxic activity of NK cells from CD1-Foxn1nu mice. A, The MHC class II-deficient mouse lymphoma cell line, YAC-1, was used to test NK cell cytotoxicity. B, IL-2 (dark gray) or IL-15-activated (light gray) NK cells isolated from the spleen of CD1-Foxn1nu mice were plated with YAC-1 cells at an E:T ratio of 5:1 and 2.5:1 for the duration of 4 hours. Specific tumor lysis was significantly higher with NK cells activated with IL-15 than with IL-2. C, Xenoreactivity of murine NK cells against K562 and SJNBL46_X (PDX) with (+AB) and without antibody compared to YAC-1. D, CD107a expression was increased in IL-2- and IL-15-preactivated NK cells compared to resting NK cells after co-culture with YAC-1. E, To quantitate IFN-γ expression, NK cells were stimulated with IL-12/IL-18 in culture (red). Unstimulated NK cells served as controls (gray). IL-15 preactivation was associated with higher IFN-γ expression than was IL-2 or no preactivation. F, Expression analysis of perforin and granzyme B. The expression of perforin was slightly higher in IL-15 than in IL-2-activated NK cells; however, granzyme B was similar.
ARTICLE ABSTRACT
Immunotherapy with IL2, GM-CSF, and an anti-disialoganglioside (GD2) antibody significantly increases event-free survival in children with high-risk neuroblastoma. However, therapy failure in one third of these patients and IL2-related toxicities pose a major challenge. We compared the immunoadjuvant effects of IL15 with those of IL2 for enhancing antibody-dependent cell-mediated cytotoxicity (ADCC) in neuroblastoma.
We tested ADCC against neuroblastoma patient-derived xenografts (PDX) in vitro and in vivo and examined the functional and migratory properties of NK cells activated with IL2 and IL15.
In cell culture, IL15-activated NK cells induced higher ADCC against two GD+ neuroblastoma PDXs than did IL2-activated NK cells (P < 0.001). This effect was dose-dependent (P < 0.001) and was maintained across several effector-to-tumor ratios. As compared with IL2, IL15 also improved chemotaxis of NK cells, leading to higher numbers of tumorsphere-infiltrating NK cells in vitro (P = 0.002). In an orthotopic PDX model, animals receiving chemoimmunotherapy with an anti-GD2 antibody, GM-CSF, and a soluble IL15/IL15Rα complex had greater tumor regression than did those receiving chemotherapy alone (P = 0.012) or combined with anti-GD2 antibody and GM-CSF with (P = 0.016) or without IL2 (P = 0.035). This was most likely due to lower numbers of immature tumor-infiltrating NK cells (DX5+CD27+) after IL15/IL15Rα administration (P = 0.029) and transcriptional upregulation of Gzmd.
The substitution of IL15 for IL2 leads to significant tumor regression in vitro and in vivo and supports clinical testing of IL15 for immunotherapy in pediatric neuroblastoma.
