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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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      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\u03b1) alone or with irradiated CD137L\/CD54\n                      <jats:sup>+<\/jats:sup>\n                      aggressive variant of the Neuro-2a murine neuroblastoma cell line (15\u20134P) at a 1:1 ratio for 10\u201312\u2009days. 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\u2009days to mimic tumor relapse. Select groups received anti-TIM-3 starting on day 9 for every 4\u2009days with\/without infusions of 15\u20134P 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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Co-culturing NK cells with 15\u20134P 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\u20134P 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\u20134P NK cell degranulation, Eomesodermin, TRAIL and Fas Ligand expression observed. In vivo, the combination of 15\u20134P 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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2024-010239","type":"journal-article","created":{"date-parts":[[2025,12,12]],"date-time":"2025-12-12T10:45:19Z","timestamp":1765536319000},"page":"e010239","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":2,"title":["TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell 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