CELL INTELLIGENCE · 肿瘤细胞治疗研究

BACKGROUND: High-risk neuroblastoma (HR-NBL) is an aggressive tumor of the sympathetic nervous system with high risk of relapse and poor overall survival. Allogeneic hematopoietic cell transplant (allo-HCT) has been used previously in patients with HR-NBL; however, graft-versus-host disease (GVHD) and disease progression have limited clinical application. Ex vivo stimulated allogeneic natural killer (NK) cells represent an approach to enhance the graft-versus-tumor (GVT) effect without exacerbation of GVHD but have not shown efficacy in NBL. METHODS: Ex vivo stimulated NK cells from C57BL/6NCr (B6) mice were expanded with soluble interleukin-15 (IL-15) and IL-15 receptor alpha (IL-15R ) alone or with irradiated CD137L/CD54 + aggressive variant of the Neuro-2a murine neuroblastoma cell line (15-4P) at a 1:1 ratio for 10-12 days. Allogeneic NK cells were then analyzed for activation, proliferation, cytokine production, and cytotoxicity against two murine NBL cell lines, Neuro2a and NXS2, in the absence or presence of anti-T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). Lethally irradiated B6AJF1 mice received allo-HCT from B6 donors followed by NBL challenge after 7 days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4 days with/without infusions of 15-4P B6 NK cells on days 14, 21, and 28. In select experiments, T cell and NK cells were selectively depleted to establish contribution to the GVT effect. All groups were analyzed for tumor growth, GVHD and survival. RESULTS: Co-culturing NK cells with 15-4P results in 78-fold expansion with increased expression of Kiel-67 (Ki-67) and Natural Killer Group 2, Member D (NKG2D), NKp46, TNF-Related Apoptosis-Inducing Ligand (TRAIL) and TIM-3. 15-4P stimulated allogeneic NK cells showed enhanced cytotoxicity against NBL compared with IL-15 NK cells alone but was limited in part due to high expression of TIM-3 ligands on Neuro-2a compared with NXS2. The addition of TIM-3 blockade further enhanced NK cytotoxicity versus Neuro-2a, with enhanced 15-4P NK cell degranulation, Eomesodermin, TRAIL and Fas Ligand expression observed. In vivo, the combination of 15-4P stimulated allogeneic NK cells and TIM-3 blockade after allo-HCT resulted in prolonged survival against NBL with decreased tumor burden compared with NK cells or anti-TIM-3 alone. Depletion of NK cells, but not T cells, abrogated the GVT effect. CONCLUSION: Allo-HCT can be a platform for treating NBL using combination ex vivo stimulated allogeneic NK cell therapy with TIM-3 blockade to enhance the GVT effect without inducing GVHD.