树突状细胞诱导的CTL对神经胶质瘤细胞的体外杀伤作用研究
摘要
目的：研究负载肿瘤抗原的树突状细胞(DCs)活化的特异性细胞毒性T淋巴细胞(CTLs)对神经胶质瘤细胞的体外杀伤效应，探讨其用于临床治疗的可行性。方法：体外原代培养胶质瘤细胞，冻融法获取胶质瘤细胞抗原，联合应用粒细胞／巨噬细胞集落刺激因子、白细胞介素-4和肿瘤坏死因子-α等，对人外周血单核细胞进行体外诱导来获取DCs，负载肿瘤抗原后，激活自体T淋巴细胞，制备特异性CTLs，MTT法检测对胶质瘤细胞的体外杀伤效应。结果：自诱导第2天起，倒置显微镜下观察到部分细胞体积增大，形态由圆形变得不规则，成多形性，可见驼峰样或细刺状胞浆突起。DCs培养第10天，流式细胞仪检测DCs高表达CD_(1a)、CD_(80)、CD_(83)、CD_(86)、HLA-DR。同种异体混合淋巴细胞反应显示少量DCs即可明显刺激同种异体淋巴细胞增殖且DCs与淋巴细胞比例在1：10时淋巴细胞增殖最明显。负载胶质瘤抗原的DCs激活的CTLs对胶质瘤细胞的杀伤作用显著高于对K562细胞的杀伤作用(P＜0.01)，并且杀伤活性随着效靶比的增加而增加。负载胶质瘤抗原的DCs激活的CTLs对胶质瘤的杀伤活性显著高于未经抗原致敏的DCs刺激的CTLs对胶质瘤的杀伤作用(P＜0.01)。上述两组CTLs对K562细胞杀伤作用类似(P＞0.05)。未经抗原致敏的DCs刺激的CTLs对胶质瘤细胞和K562细胞杀伤无显著差别(P＞0.05) 结论：联合应用细胞因子rhGM-CSF、rhIL-4、rhTNF-α可从人外周血中诱导出典型的树突状细胞，将DCs负载胶质瘤抗原后，激活的CTLs在体外对胶质瘤细胞能产生高效而特异的杀伤作用，为临床应用DCs瘤苗奠定基础。
Objective To investigate the antitumor efficiency of the special cytotoxic T lymphocytes activated by dendritic cells loaded with glioma antigens in vitro and the feasibility of immunotherapy of glioma with dendritic cells for clinical application. Method Glioma cells were prepared by primary culture in vitro and tumour antigens were extracted by thawing and freezing. Peripheral blood monocytes were cultured to produce DCs with recombinant human granulocyte-macrophage colony - stimulating factor, recombinant human interleukin-4, recombinant human tumor necrosis factor- . After pulsed in vitro with glioma lysates, DCs were used to induce T lymphocytes into cytotoxic T lymphocytes for glioma cells. The cytotoxicity of CTLs to patient' s glioma cells was assayed by MTT. Result After culter for two days, cells began to increase in size and be charactered with dendritic morphology. After 10 days, more CDla , CD80, CD83, CD86, HLA-DR were detected by flow cytometer. Dendritic cells stimulated lymphocytes to increase greatly in Allo-MLRs. It was obvious when the proportion was 1 to 10. The CTLs could kill glioma cells markedly in vitro and the killing rate of glioma cells was more higher than that of K562 (P<0.01) .The ratio increased with the improvement of the proportion of effector cells to target cells. The CTLs were more effective to glioma than the CTLs cultured without glioma antigens (P<0.01) . Tow kinds of CTLs had similar effect to K562 (P>.05) .Conclusion The antigen specific CTLs killed glioma cells effectively and specifically, which might play a great role in clinical therapy.
Objective To investigate the antitumor efficiency of the special cytotoxic T lymphocytes activated by dendritic cells loaded with glioma antigens in vitro and the feasibility of immunotherapy of glioma with dendritic cells for clinical application. Method Glioma cells were prepared by primary culture in vitro and tumour antigens were extracted by thawing and freezing. Peripheral blood monocytes were cultured to produce DCs with recombinant human granulocyte-macrophage colony - stimulating factor, recombinant human interleukin-4, recombinant human tumor necrosis factor- . After pulsed in vitro with glioma lysates, DCs were used to induce T lymphocytes into cytotoxic T lymphocytes for glioma cells. The cytotoxicity of CTLs to patient' s glioma cells was assayed by MTT. Result After culter for two days, cells began to increase in size and be charactered with dendritic morphology. After 10 days, more CDla , CD80, CD83, CD86, HLA-DR were detected by flow cytometer. Dendritic cells stimulated lymphocytes to increase greatly in Allo-MLRs. It was obvious when the proportion was 1 to 10. The CTLs could kill glioma cells markedly in vitro and the killing rate of glioma cells was more higher than that of K562 (P<0.01) .The ratio increased with the improvement of the proportion of effector cells to target cells. The CTLs were more effective to glioma than the CTLs cultured without glioma antigens (P<0.01) . Tow kinds of CTLs had similar effect to K562 (P>.05) .Conclusion The antigen specific CTLs killed glioma cells effectively and specifically, which might play a great role in clinical therapy.
引文
1 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature, 1998,392(6673):245-252
2 Cormier JN, Panell MC, Hackett JA, et al. Natural variation of the expression of HLA and endogenous antigen modulates CTLs recognition in an in vitro melanoma model. Int J Cancer, 1999,80:781
3 Matsui S, Ahlers JD, Vortmeyer AO, et al. A model for CD8~+ CTLs tumor immunosurveillance and regulation of tumor escape by CD4~+T cells through an effect on quality of CTLs. J Immunol, 1999,165:184
4 Chaux P, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer, 1997,72(4):619
5 Gabrilovich DI, Ciemik IF, Carbone DP. Dendritic cells in antitumor immune response. I. Defective antigen presentation in tumorbearing hosts[J].Cell Immunol, 1996;170(1):101-110
6 Ranieri E, Kierstead LS, Zarour H, et al. Dendritic cell/peptide cancer vaccines:clinical responsiveness and epitope spreading [J].Immunol Invest, 2000,29(2):121-125
7 Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids[J].Nat Med, 2000,6(3):332-336
8 Small EJ, Fratesi P, Reese DM, et al. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells[J]. J Clin Oncol, 2000, 18(23):3894-3903
9 巴德年主编 当代免疫学技术与应用 北京：北京医科大学中国协和医科大学联合出版社，1998 158
10 uting T, Wislson CC,Martin DM, et al. Autologous human monocyte-derived cells genetically modified to express melanoma antigen selicit cytotoxic Tcell responses in vitro:enhancement by contransfection of genesencoding the Thl-biasing cytokines IL-12 and INF-α.J Immunol, 1998,160(5):1139
11 Sigal LJ, Reiser H, Rock RL. Whe role of B7-1and B7-2 costimulation for the generation of CTLs responses in vitro. J Immunol, 1998,161(6):2740
12 Gabrilovich DI, Ciemik IF, Carbone DP. Dendritic cells in antitumor immune
response. I. Defective antigen presentation in tumorbearing host[J],Cell Immunol, 1996;170(1):101-110
13 Katsenelson NS, Shurin GV, Bykovskaia SN, et al. Human small cell lung arcinoma and carcinoid tumor regulate dendritic cell maturation and function. Mod Pathol, 2001,14(1):40
14 Kiertscher SM, Luo J, Dubinett SM, et al. Tumors promote altered maturation and early apoprosis of monocyte derived dendritic cells. J Immunol, 2001,164(3).:1269
15 Shortman K, Caux C. Dendritic cell and development:multiple pathways to natures djuvants. [J]Stem Cells, 1997,15(6):409 419
16 Nijman HW, Kleijmeer MJ, Ossevoort MA, et al. Antigen capture and major histocompatibility class Ⅱ compartments of fresh]y isolated and culture blood dendritic cells[J]. J Exp Med, 1995,182:163
17 Bancheresu J, Steinman RM, Dendritic cells and the control of immunity. Nature, 1998.392(6673):245
18 Schwarz SF, Divaris N, Kay C, et al. Mechanisms of tumor necrosis factor granulocyte-macrophage-colony-stimulating factor-induced dendritic cell development. Blood, 1993;82:3019
19 Roth MD, Gitilitz BJ, Kiertscher SM, et al. Granulocyte macrophage colony-stimulating factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and CD83+ cells in cancer patients. Cancer research, 2000,60(7):1934
20 Morse MA, Zhou LJ, Tedder TF, et al. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulatingfactor, interleukin-4, and tumor necrosis factor-α for use in cancer immunotherapy. Ann Surg, 1997; 226:6
21 Banchereau J, Steinman RM. Dendritic cells and the control of immumnity. Nature. 1998(392):245-52
22 Fu jii S, Fujimoto K, Shimizu K, et al. Presentation of tumor antigen by phagocytic dendritic cells clusters generated from human CD34+ hematopoietic progenitor cells:induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients. Cancer Res, 1999,59(9):2150-2158
23 Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-posiotive chronic myelogenous leukemia. Blood, 1997,89(4):1133-1142
24 Steiman RM, Nogueira N, Wetmer MD. Lumphokine enhances the expression and synthesis of 1a antigen on cultured mouse peritoneal macrophages. J Exp Med 1980;152:1248-1261
25 Steiman RM, Witmer MD. Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc Natl Acad Sci U S A 1978;85:5132-5136
26 Steiman RM, Gutchinov B, Witmer MD. Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice. J Exp Med 1983;157:613-627
27 Matsui S, Ahlers JD, Vortmeyer AO, et al. A model for CD8~+ CTLs tumor immunosurveillance and regulation of tumor escape by CD4~+T cells through an effect on quality of CTLs. J Immunol, 1999,165:184
28 Chaux P, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer, 1997,72(4):619
29 Zitvogel L, Mayoreomo JI, Tjandrawan T, et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells:dependence on T cells, B7 costimulation, and T helper cell 1 associated cytokines[J].J Exp Med, 1996,183(1):87-97
30 Ashley DM, Faiola B, Nair S, et al. Comparative accessory cell function of human eripheral blood dendritic cells and monocytes. J Exp Med, 1997;186(7):1177-1182
31 Cayeux S, Richter G, Becket C, et al. Direct and indirect T cell priming by dendritic cell vaccines[J]. Eur J Immunol, 1999,29:225
32 Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T. Results of a phaso Ⅰ clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001, 50(7): 337-44
33 Hsu F, Benike C, Fagnoni F, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med, 1996,
36(2): 52
34 Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nat Med, 1998, 86(4): 328
35 Salgaller ML, Tjoa BA, Lodge PA, et al. Dendritic cell-based immunotherapy of prostate cancer. Crit Rev Immunol, 1998,18(1):109
36 Hsu F, Benike C, Fagnoni F, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med, 1996, 36(2): 52
37 Krause SW, Neumann C, Soruri A, et al. The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells. Immunother, 2002, 25(5): 421
38 Tjoa BA, Simmons SJ, Bowes VA, et al. Evaluation of Phase Ⅰ/Ⅱ clinical trials in prostate cancer with dendritic cells and PSMA Deptides. Prostate, 1998, 36(6): 39
39 Scheinberg LC, Edelman FL, Levy WA. Is the brain "an immunologically privileged site?" 1 Studies based on intracerebral homotransplantations and isotransplantation to sensitized hosts. Arch Neurol, 1964, 11(1): 248
40 Schmidt-wolf IGH, Negrin RS, Kiem HP, et al. Use of a SCID mouse/Human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cells with potent antitumor cell actibity. J Exp Med. 1991; 174:139
41 Lu PH, Negrin RS.A novel population of expanded human CD3+ CD56+cells derived from T cells with potent in vivoantitumor activity in mice with severe combined immunodeficiency .J Immunol, 1994;153:1678
2 Cormier JN, Panell MC, Hackett JA, et al. Natural variation of the expression of HLA and endogenous antigen modulates CTLs recognition in an in vitro melanoma model. Int J Cancer, 1999,80:781
3 Matsui S, Ahlers JD, Vortmeyer AO, et al. A model for CD8~+ CTLs tumor immunosurveillance and regulation of tumor escape by CD4~+T cells through an effect on quality of CTLs. J Immunol, 1999,165:184
4 Chaux P, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer, 1997,72(4):619
5 Gabrilovich DI, Ciemik IF, Carbone DP. Dendritic cells in antitumor immune response. I. Defective antigen presentation in tumorbearing hosts[J].Cell Immunol, 1996;170(1):101-110
6 Ranieri E, Kierstead LS, Zarour H, et al. Dendritic cell/peptide cancer vaccines:clinical responsiveness and epitope spreading [J].Immunol Invest, 2000,29(2):121-125
7 Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids[J].Nat Med, 2000,6(3):332-336
8 Small EJ, Fratesi P, Reese DM, et al. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells[J]. J Clin Oncol, 2000, 18(23):3894-3903
9 巴德年主编 当代免疫学技术与应用 北京：北京医科大学中国协和医科大学联合出版社，1998 158
10 uting T, Wislson CC,Martin DM, et al. Autologous human monocyte-derived cells genetically modified to express melanoma antigen selicit cytotoxic Tcell responses in vitro:enhancement by contransfection of genesencoding the Thl-biasing cytokines IL-12 and INF-α.J Immunol, 1998,160(5):1139
11 Sigal LJ, Reiser H, Rock RL. Whe role of B7-1and B7-2 costimulation for the generation of CTLs responses in vitro. J Immunol, 1998,161(6):2740
12 Gabrilovich DI, Ciemik IF, Carbone DP. Dendritic cells in antitumor immune
response. I. Defective antigen presentation in tumorbearing host[J],Cell Immunol, 1996;170(1):101-110
13 Katsenelson NS, Shurin GV, Bykovskaia SN, et al. Human small cell lung arcinoma and carcinoid tumor regulate dendritic cell maturation and function. Mod Pathol, 2001,14(1):40
14 Kiertscher SM, Luo J, Dubinett SM, et al. Tumors promote altered maturation and early apoprosis of monocyte derived dendritic cells. J Immunol, 2001,164(3).:1269
15 Shortman K, Caux C. Dendritic cell and development:multiple pathways to natures djuvants. [J]Stem Cells, 1997,15(6):409 419
16 Nijman HW, Kleijmeer MJ, Ossevoort MA, et al. Antigen capture and major histocompatibility class Ⅱ compartments of fresh]y isolated and culture blood dendritic cells[J]. J Exp Med, 1995,182:163
17 Bancheresu J, Steinman RM, Dendritic cells and the control of immunity. Nature, 1998.392(6673):245
18 Schwarz SF, Divaris N, Kay C, et al. Mechanisms of tumor necrosis factor granulocyte-macrophage-colony-stimulating factor-induced dendritic cell development. Blood, 1993;82:3019
19 Roth MD, Gitilitz BJ, Kiertscher SM, et al. Granulocyte macrophage colony-stimulating factor and interleukin 4 enhance the number and antigen-presenting activity of circulating CD14+ and CD83+ cells in cancer patients. Cancer research, 2000,60(7):1934
20 Morse MA, Zhou LJ, Tedder TF, et al. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulatingfactor, interleukin-4, and tumor necrosis factor-α for use in cancer immunotherapy. Ann Surg, 1997; 226:6
21 Banchereau J, Steinman RM. Dendritic cells and the control of immumnity. Nature. 1998(392):245-52
22 Fu jii S, Fujimoto K, Shimizu K, et al. Presentation of tumor antigen by phagocytic dendritic cells clusters generated from human CD34+ hematopoietic progenitor cells:induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients. Cancer Res, 1999,59(9):2150-2158
23 Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-posiotive chronic myelogenous leukemia. Blood, 1997,89(4):1133-1142
24 Steiman RM, Nogueira N, Wetmer MD. Lumphokine enhances the expression and synthesis of 1a antigen on cultured mouse peritoneal macrophages. J Exp Med 1980;152:1248-1261
25 Steiman RM, Witmer MD. Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc Natl Acad Sci U S A 1978;85:5132-5136
26 Steiman RM, Gutchinov B, Witmer MD. Dendritic cells are the principal stimulators of the primary mixed leukocyte reaction in mice. J Exp Med 1983;157:613-627
27 Matsui S, Ahlers JD, Vortmeyer AO, et al. A model for CD8~+ CTLs tumor immunosurveillance and regulation of tumor escape by CD4~+T cells through an effect on quality of CTLs. J Immunol, 1999,165:184
28 Chaux P, Favre N, Martin M, et al. Tumor infiltrating dendritic cells are defective in their antigen presenting function and inducible B7 expression in rats. Int J Cancer, 1997,72(4):619
29 Zitvogel L, Mayoreomo JI, Tjandrawan T, et al. Therapy of murine tumors with tumor peptide-pulsed dendritic cells:dependence on T cells, B7 costimulation, and T helper cell 1 associated cytokines[J].J Exp Med, 1996,183(1):87-97
30 Ashley DM, Faiola B, Nair S, et al. Comparative accessory cell function of human eripheral blood dendritic cells and monocytes. J Exp Med, 1997;186(7):1177-1182
31 Cayeux S, Richter G, Becket C, et al. Direct and indirect T cell priming by dendritic cell vaccines[J]. Eur J Immunol, 1999,29:225
32 Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T. Results of a phaso Ⅰ clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001, 50(7): 337-44
33 Hsu F, Benike C, Fagnoni F, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med, 1996,
36(2): 52
34 Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nat Med, 1998, 86(4): 328
35 Salgaller ML, Tjoa BA, Lodge PA, et al. Dendritic cell-based immunotherapy of prostate cancer. Crit Rev Immunol, 1998,18(1):109
36 Hsu F, Benike C, Fagnoni F, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med, 1996, 36(2): 52
37 Krause SW, Neumann C, Soruri A, et al. The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells. Immunother, 2002, 25(5): 421
38 Tjoa BA, Simmons SJ, Bowes VA, et al. Evaluation of Phase Ⅰ/Ⅱ clinical trials in prostate cancer with dendritic cells and PSMA Deptides. Prostate, 1998, 36(6): 39
39 Scheinberg LC, Edelman FL, Levy WA. Is the brain "an immunologically privileged site?" 1 Studies based on intracerebral homotransplantations and isotransplantation to sensitized hosts. Arch Neurol, 1964, 11(1): 248
40 Schmidt-wolf IGH, Negrin RS, Kiem HP, et al. Use of a SCID mouse/Human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cells with potent antitumor cell actibity. J Exp Med. 1991; 174:139
41 Lu PH, Negrin RS.A novel population of expanded human CD3+ CD56+cells derived from T cells with potent in vivoantitumor activity in mice with severe combined immunodeficiency .J Immunol, 1994;153:1678