BACKGROUND AND PURPOSEIn osteosarcoma (OS) patients, only a limited number of drugs are active and the regimens currently in use include a combination of at least two of these drugs: doxorubicin, cisplatin, methotrexate and ifosfamide. Today, 30-40% of patients still die of OS highlighting the urgent need for new treatments. Invariant NKT (iNKT) cells are a lymphocyte lineage with features of both T and NK cells, playing important roles in tumour suppression. Our aim was to test whether the cytoxicity induced by cisplatin, doxorubicin and methotrexate against OS cells can be enhanced by iNKT cell treatment. EXPERIMENTAL APPROACHiNKT cells were purified from human peripheral blood mononuclear cells by cell sorting (Va24Vb11 + cells) and used as effector cells against OS cells (U2-OS, HOS, MG-63). Cell death (calcein-AM method), perforin/granzyme B and Fas/FasL expressions were determined by flow cytometry. CD1d expression was analysed at both the gene and protein level. KEY RESULTSiNKT cells were cytotoxic against OS cells through a CD1d-dependent mechanism. This activity was specific for tumour cells, because human CD1d + mesenchymal stem cells and CD1d -osteoblasts were not affected. iNKT cell treatment enhanced drug-induced OS cell death in a concentration-dependent manner and this effect was reduced in CD1d-silenced OS cells. CONCLUSION AND IMPLICATIONSiNKT cells kill malignant, but not non-malignant, cells. iNKT cell treatment enhances the cytotoxicity of anti-neoplastic drugs against OS cells in a CD1d-dependent manner. The present data encourage further studies on the use of iNKT cells in OS therapy.