NKT cells are a regulatory subset of T lymphocytes with immune modulatory effects and an important role in anti‐tumor immunity. The feasibility of “ex‐vivo education” of NKT cells has recently been demonstrated. To evaluate the anti‐tumor effect of ex‐vivo immune‐modulated NKT lymphocytes in a murine model of hepatocellular carcinoma. Athymic Balb/C mice were sublethally irradiated and transplanted with human Hep3B HCC. NKT cells prepared from immunocompetent Balb/C mice were pulsed ex vivo with HCC‐derived antigens (Group A), Hep3B cells (group B) or BSA (group C), and adoptively transferred into HCC harboring mice (1 × 0⁶ NKT cells per mouse). Group D mice did not undergo NKT cell transplantation. Group E mice were transplanted with 1 × 10⁶ NKT cells from HBV‐immunized donors. Mice were followed for tumor size and weight. To determine the mechanism of the anti‐tumor effect, intrasplenic lymphocyte populations were analyzed by FACS for NKT, CD4+ and CD8+ lymphocyte subpopulations; STAT 1, 4 and 6 expression in splenocytes was assessed by Western blot, and serum cytokine levels were measured by ELISA. Adoptive transfer of NKT cells pulsed with HCC‐derived antigens (group A) and NKT cells from immunized donors (group E) resulted in complete disappearance of tumors within 4 weeks and attenuated weight loss (6.5% and 7% in groups A and E, respectively). In contrast, mice in groups B, C, and D developed large, necrotic tumors and severe weight loss (21%, 17% and 23% weight loss in groups B, C, and D, respectively). NKT/CD4 and CD8/CD4 ratios were significantly increased in groups A and E (12.3 and 17.6 in groups A and D, respectively, compared to 6.4, 4.8 and 5.6 in groups B, C and D, respectively, for the NKT/CD4 ratio; 41 and 19.8 in groups A and E, respectively, compared to 6.5, 11.8 and 3.2 in groups B, C, and D, respectively, for the CD8/CD4 ratio). Expression of the transcription factor STAT4 was evident in group A, but not in groups B‐D. Serum IFNγ, IL12 and IL4 levels were increased in groups A and E. Adoptive transfer of NKT lymphocytes exposed ex vivo by HCC‐derived antigens loaded on dendritic cells and NKT cells from immunized donors led to suppression of HCC in mice. NKT‐mediated anti‐tumor activity was associated increased NKT and CD8+ T lymphocyte numbers, increased expression of STAT4, a marker for IL‐12 activity and elevated serum levels of the proinflammatory cytokines IFNγ and IL12, and of IL4. Ex‐vivo modulation of NKT lymphocytes holds promise as a novel mode of immune therapy for HCC.

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