Suppression of hepatocellular carcinoma by transplantation of ex-vivo immune-modulated NKT lymphocytes.
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 x 0(6) NKT cells per mouse). Group D mice did not undergo NKT cell transplantation. Group E mice were transplanted with 1 x 10(6) 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 IFNgamma, 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 IFNgamma and IL12, and of IL4. Ex-vivo modulation of NKT lymphocytes holds promise as a novel mode of immune therapy for HCC.
Margalit
Maya
M
Liver Unit, Department of Medicine, Hebrew University-Hadassah Medical Center, Jerusalem, Israel.
Shibolet
Oren
O
Klein
Athalia
A
Elinav
Eran
E
Alper
Ruslana
R
Thalenfeld
Barbara
B
Engelhardt
Dean
D
Rabbani
Elazar
E
Ilan
Yaron
Y
D019264:Adoptive Transfer; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D006528:Carcinoma, Hepatocellular / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D003602:Cytotoxicity, Immunologic; D004268:DNA-Binding Proteins / Q000378:metabolism; D003713:Dendritic Cells / Q000276:immunology*; D005260:Female; D017325:Hepatitis B Vaccines / Q000276:immunology*; D007111:Immunity, Cellular / Q000235:genetics / Q000276:immunology*; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology*; D008114:Liver Neoplasms, Experimental / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D050797:STAT4 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D015431:Weight Loss
D004268:DNA-Binding Proteins; D017325:Hepatitis B Vaccines; D050797:STAT4 Transcription Factor; C494091:Stat4 protein, mouse; D015534:Trans-Activators; D018664:Interleukin-12; D015847:Interleukin-4; D007371:Interferon-gamma