CD8+ natural killer T (NKT) cells from EBV-associated tout patients are quantitatively and functionally impaired. EBV-induced CD8+ NKT cells drive syngeneic T cells into a Thl-bias response to suppress EBV-associated malignancies. IL-4-biased CD4+ NKT cells do not affect either syngeneic T cell cytotoxicity or Th cytokine secretion. Circulating mDC1 cells from patients with EBV-associated malignancies impair the production of IFN-γ by CD8+ NKT cells. In this study, we have established a human-thymus-SCID chimaera model to further investigate the underlying mechanism of EBV-induced CD8+ NKT cells in suppressing EBV-associated malignancies. In the human-thymus-SCID chimera, EBV-induced CD8+ NKT cells suppress EBV-associated malignancies in a manner dependent on the Th1-bias response and syngeneic CD3+ T cells. However, adoptive transfer with CD4+ NKT cells alone inhibits T cell immunity. Interestingly, CD4+ NKT cells themselves secrete high levels of IL-2, enhancing the persistence of adoptively transferred CD8+ NKT cells and T cells, thereby leading to a more pronounced T cell anti-tumour response in chimaeras co-transferred with CD4+ and CD8+ NKT cells. Thus, immune reconstitution with EBV-induced CD4+ and CD8+ NKT cells synergistically enhances T cell tumour immunity, providing a potential prophylactic and therapeutic treatment for EBV-associated malignancies. Cellular & Molecular Immunology. 2009;6(5):367-379.