Induction of systemic immunity by expression of interleukin-23 in murine colon carcinoma cells

Interleukin-23 (IL-23), a novel cytokine composed of a newly identified p19 molecule and the p40 subunit of IL-12, can stimulate the proliferation in vitro of memory T cells. We examined whether Colon 26 murine colon carcinoma cells that were retrovirally transduced with the p19-linked p40 gene (Colon 26/IL-23) could produce antitumor effects in inoculated mice. The growth of Colon 26/IL-23 tumors developed in immunocompetent mice was significantly retarded and the tumors disappeared thereafter. Spleen cells from the mice that received Colon 26/IL-23 cells produced significant amounts of interferon-gamma, when they were cultured with irradiated Colon 26 but not irrelevant cells. Depletion of CD8(+) T cells suppressed the production of interferon-gamma. The mice that had rejected Colon 26/IL-23 tumors were resistant to subsequent challenge of parent but not irrelevant tumor cells. Colon 26/IL-23 tumors were not rejected in nude mice but the growth was retarded compared to parent tumors. Treatment of nude mice with anti-asialo GM(1) antibody did not influence the growth of Colon 26/IL-23 tumors. These data suggest that expression of IL-23 in tumors produces T cell-dependent antitumor effects and induces systemic immunity.     

Transduction of murine colon carcinoma cells with interleukin-15 gene induces antitumor effects in immunocompetent and immunocompromised hosts

We examined the antitumor effects caused by murine colon carcinoma cells (Colon 26) transduced with interleukin-15 (IL-15) gene. Although the in vitro proliferation rate of IL-15-secreting Colon 26 (Colon 26/IL-15) cells was not different from that of wild-type (wt) cells, small subcutaneous tumors of Colon 26/IL-15 cells that developed in syngeneic immunocompetent mice regressed spontaneously in contrast to tumors of wt cells. The mice that had eliminated tumors of Colon 26/IL-15 cells rejected wt cells when subsequently challenged. The survival of the mice that had been inoculated intraperitoneally with Colon 26/IL-15 cells was significantly prolonged compared with that of the mice injected with wt cells. However, in an experimental lung metastasis model, the survival of the mice inoculated with Colon 26/IL-15 cells remained the same as that of the mice inoculated with wt cells. The inoculation of Colon 26/IL-15 cells into immunocompromised nude or severe combined immunodeficient mice produced tumors, but the survival of the immunocompromised mice was significantly longer than that of the mice inoculated with wt cells. The nude mice inoculated with Colon 26/IL-15 cells also survived longer than the severe combined immunodeficient mice with Colon 26/IL-15 cells. Depletion of natural killer cells in nude mice with anti-asialo GM1 antibody did not influence the survival of the mice injected with Colon 26/IL-15 cells. Immunohistological examination revealed that CD31+ cells migrated into tumors of Colon 26/IL-15 cells that developed in immunocompetent and immunocompromised mice. Taken together, our results indicate that an inoculation of IL-15-producing tumor cells can produce antitumor effects that are mediated by a variety of immunocompetent cells.     

Expression of the interleukin-21 gene in murine colon carcinoma cells generates systemic immunity in the inoculated hosts

Interleukin-21 (IL-21) is a novel cytokine that can induce proliferation of activated T cells and maturation of natural killer (NK) cells. We therefore examined whether expression of the IL-21 gene in tumor cells could generate antitumor responses. Murine colon carcinoma Colon 26 cells that were transduced with the mouse IL-21 gene (Colon 26/IL-21) were rejected in syngeneic mice and the mice subsequently acquired protective immunity. The growth of Colon 26/IL-21 tumors developed in nude mice was retarded compared with that of parent tumors, and this growth suppression was not observed in nude mice that were treated with anti-asialo GM(1) antibody. Spleen cells from the mice that had rejected Colon 26/IL-21 cells showed cytotoxic activity to Colon 26 but not to irrelevant tumor cells, and produced larger amounts of interferon-gamma upon stimulation with irradiated Colon 26 cells. Spleen cells from Colon 26/IL-21-tumor- but not parent-tumor-bearing mice had lytic activity to YAC-1 cells. These data suggest that expression of IL-21 in tumors induces T- and NK-cell-dependent antitumor effects.     

Secretion of interleukin-10 from murine colon carcinoma cells suppresses systemic antitumor immunity and impairs protective immunity induced against the tumors

Interleukin-10 (IL-10) is a T helper type 2 (Th2) cytokine that suppresses Th1-mediated, cell-mediated immune responses and reciprocally enhances antibody-mediated responses. Previous studies, however, demonstrated that forced expression of the IL-10 gene in tumor cells could unexpectedly produce antitumor effects. We then examined whether tumor-derived IL-10 could modulate systemic immune responses. Murine colon carcinoma (Colon 26) cells that were retrovirally transduced with the murine IL-10 gene (Colon 26/IL-10) were inoculated in syngeneic immunocompetent or T cell-defective nude mice. Growth of Colon 26/IL-10 tumors was augmented in immunocompetent and, to less extent, in nude mice compared with that of wild-type tumors developed in respective mice. Growth of wild-type tumors was accelerated to the same level as that of Colon 26/IL-10 tumors when wild type and Colon 26/IL-10 cells were respectively inoculated in different flanks of the same immunocompetent mice. This enhanced growth of wild-type tumors was not observed in nude mice. Immunocompetent mice that had rejected IL-2- or IL-12-secreting Colon 26 cells developed protective immunity and became completely resistant to wild-type Colon 26 cells subsequently challenged. However, some of the mice that had rejected IL-2 or IL-12 producers developed Colon 26/IL-10 tumors inoculated thereafter. The present study showed that production of IL-10 from tumor cells impaired T cell- and non-T cell-mediated systemic antitumor immunity in hosts.     

Antitumor effects produced by transduction of dendritic cells-derived heterodimeric cytokine genes in murine colon carcinoma cells

Interleukin (IL)-23 and IL-27 have been recently identified as members of the IL-12 heterodimeric cytokine family. The cytokines are released from activated dentritic cells and play a crucial role in cell-mediated immunity. Expression profiles of the cytokines' receptors suggest that target cells of the cytokines are distinct depending on T cell maturation and differentiation. We first examined whether murine colon carcinoma cells (Colon 26) expressing IL-27 (Colon 26/IL-27) could produce antitumor effects in syngeneic mice. The mice developed small Colon 26/IL-27 tumors and subsequently rejected them, whereas parent tumors continued to grow. Since Colon 26/IL-12 and Colon 26/IL-23 cells were also rejected in syngeneic mice, we then investigated the combinatory antitumor effects of the cytokines. We inoculated Colon 26/IL-12, Colon 26/IL-23, Colon 26/IL-27 cells or mixed populations of the cytokine producers into one flank of the mice that simultaneously received parent tumors in the other flank. The parent tumor growth influenced by the cytokine producers was similar to each other. Combinatory antitumor effects were not produced by the cytokine producers and application of the cytokines did not show any synergistic effects.     

Protective immunity is induced in murine colon carcinoma cells by the expression of interleukin-12 or interleukin-18, which activate type 1 helper T cells

We investigated the antitumor effects induced by the production of interleukin-12 (IL-12) or IL-18, which influence the function of T helper type 1 cells, in murine colon carcinoma cells (Colon 26). Retrovirally transduced cells with IL-12 genes that encoded both p35 and p40 (Colon 26/IL-12) lost their tumorigenicity when inoculated subcutaneously or intraperitoneally into syngeneic immunocompetent mice. Moreover, the mice that had rejected the Colon 26/IL-12 cells generated protective immunity to wild-type (wt) cells when subsequently challenged. Colon 26 cells transduced with the IL-18 gene (Colon 26/IL-18) could not form subcutaneous tumors in immunocompetent mice, and the mice became resistant to inoculated wt cells. Immunohistochemical analysis revealed that the numbers of blood vessels in Colon 26/IL-12 or Colon 26/IL-18 tumors were markedly reduced, and that the expression of adhesion molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 increased on the endothelium in the stroma of Colon 26/IL-12 tumors. The loss of tumorigenicity of Colon 26/IL-12 or Colon 26/IL-18 cells was not observed in immunocompromised mice. However, the survival days of the immunocompromised mice inoculated with Colon 26/IL-12 but not Colon 26/IL-18 cells were significantly longer than those inoculated with wt cells. The secretion of cytokines that stimulate T helper type 1 cells from tumor cells can thereby induce an antitumor response. However, the effector cells involved in these antitumor effects could differentially migrate to the tumors, and the inhibition of angiogenesis may partially contribute to the antitumor responses observed.     

Expression of interleukin-21 and -23 in human esophageal tumors produced antitumor effects in nude mice

Interleukin (IL)-21 induces proliferation of activated T and natural killer (NK) cells and enhances the lytic function of NK cells. IL-23, a heterodimer composed of p19 and p40 molecule, can also enhance T cell proliferation and interferon-gamma production from activated T cells; however, its effects on NK cells are currently unknown. We therefore examined whether human tumors expressing IL-21 or IL-23 could produce antitumor effects in inoculated nude mice. Esophageal tumor T.Tn cells were retrovirally transduced with the IL-21 gene (T.Tn/IL-21) or the p19-linked p40 genes (T.Tn/IL-23). The proliferation in vitro and the class I expression of the major histocompatibility complexes of these transduced cells were not different from those of parent cells. T.Tn/IL-21 and T.Tn/IL-23 cells developed small tumors in nude mice, but the tumors regressed spontaneously thereafter in contrast to parent tumors. Expressed IL-21 or IL-23 in human esophageal tumors can produce antitumor effects in a T cell-defective condition.     

Transduction of the IL-21 and IL-23 genes in human pancreatic carcinoma cells produces natural killer cell-dependent and -independent antitumor effects

We examined whether novel cytokines, interleukin (IL)-21 and IL-23, that were expressed in tumors could produce antitumor effects in the inoculated mice. Human pancreatic cancer AsPC-1 cells were retrovirally transduced with murine IL-21 or IL-23 (p19-linked p40) gene (AsPC-1/IL-21, AsPC-1/IL-23) and were injected into nude or severe combined immunodeficiency (SCID) mice. Although the proliferation in vitro of the transduced cells remained the same as that of parent cells, growth of AsPC-1/IL-21 and AsPC-1/IL-23 tumors developed in nude mice was retarded compared with that of parent tumors. Treatment of nude mice with anti-asialo GM(1) antibody temporally abrogated the growth retardation of AsPC-1/IL-21, but not AsPC-1/IL-23 tumors; however, the growth of AsPC-1/IL-21 tumors came to be retarded thereafter with the regeneration of natural killer (NK) cells. The growth of AsPC-1/IL-21 tumors developed in SCID mice was also retarded compared with parent tumors and the growth retardation was abrogated by treatment with anti-asialo GM(1) antibody. The growth of AsPC-1/IL-23 tumors in SCID mice was not different from that of parent tumors. Cytotoxic activity and secretion of interferon-gamma in response to AsPC-1 cells were induced in spleen cells of the mice bearing AsPC-1/IL-21 or AsPC-1/IL-23 tumors. When nude mice were injected with a mixed population of AsPC-1/IL-21 and AsPC-1/IL-23 cells, no synergistic effects were observed. These data collectively suggest that expression of IL-21 and IL-23 in tumors can produce NK cell-dependent and -independent antitumor effects in an alpha beta T cell-defective condition, respectively.     

Impaired tumorigenicity of human pancreatic cancer cells retrovirally transduced with interleukin-12 or interleukin-15 gene

We examined the antitumor effect of locally secreted interleukin (IL)-12 or IL-15 on human pancreatic cancer cells (AsPC-1). We subcutaneously inoculated AsPC-1 cells retrovirally transduced with IL-12 or IL-15 cDNA into nude mice. Tumors derived from these cells showed retarded growth compared with those from wild-type (wt) cells. Nude mice inoculated intraperitoneally with the cytokine producers survived longer than those injected with wt cells. These cytokine producers were also tested for their tumor growth in severe combined immunodeficient mice. The tumor growth of IL-12 producers was similarly suppressed as found in nude mice, but the average tumor volumes of IL-15 producers were not statistically different from those of wt tumors. In nude mice that were administered anti-asialo GM1 antibody before the inoculation of the tumor cells, growth retardation of tumors of IL-12 producers remained the same as in untreated animals, but that of IL-15 producers was markedly reduced. Immunohistochemical analysis revealed that CD11b+ cells migrated into the tumors of cytokine producers and that the number of CD31+ endothelial cells within the tumors was not different between IL-12 producers and wt cells. Taken together with other data, it is possible that granulocytes are candidate cells for the IL-12-mediated antitumor effect, and that natural killer cells and gammadelta T cells are involved in the IL-15-induced antitumor effect. We did not observe synergistic effects of these cytokines to suppress subcutaneous tumors.     

Effects on apoptosis and cell cycle arrest contribute to the antitumor responses of interleukin-27 mediated by retrovirus in human pancreatic carcinoma cells

Interleukin (IL)-27, composed of p28 and Epstein-Barr virus-induced gene 3 (EBI3) subunits, has diverse functions in regulating immune systems. Human melanoma cells have been shown to express both IL-27 receptor subunits, and growth inhibition by IL-27 was detected. We investigated whether forced expression of the p28-linked EBI3 gene in human pancreatic carcinoma cells (AsPC1) by retroviral vector would produce IL-27-mediated antitumor effects and the related mechanisms. The data demonstrated that AsPC1 cells expressed both IL-27 receptor subunits, and tumor growth of AsPC1/IL-27 in mice was retarded compared with vector DNA-transduced tumors and survival of the mice was prolonged. Expression of cytokines such as interferon-γ, tumor necrosis factor-α and IL-1β in tumor specimens increased, while the secretion of IFN-γ and TNF-α from spleen cells of mice bearing IL-27-transfected tumors increased. Moreover, cell cycle arrest was induced in AsPC1/IL-27 inoculated mice with upregulated p21 expression and downregulated survivin expression. The appearance of apoptotic cells increased in tumor specimens of mice bearing IL-27-transfected tumors compared with the mice bearing DNA-transfected tumors by confirming the expression of apoptosis-related proteins and activated apoptotic pathways through detection of cleaved PARP. These results suggest that transfection of the IL-27 gene into human pancreatic carcinoma cells could produce antitumor effects in?vivo and induction of cell cycle arrest and apoptosis could be the mechanism of IL-27 action in tumor regression.     

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.     

T-cell-dependent antitumor effects produced by CD40 ligand expressed on mouse lung carcinoma cells are linked with the maturation of dendritic cells and secretion of a variety of cytokines

CD40/CD40 ligand (CD40L) interaction plays an essential role in cell-mediated immune responses. We examined whether expression of CD40L in murine lung carcinoma (A11) cells could produce antitumor effects. The proliferation rate in vitro of A11 cells transfected with the murine CD40L gene (A11/CD40L) was not different from that of parent cells; however, half of the immunocompetent mice inoculated with A11/CD40L cells did not form tumors and the growth of A11/CD40L tumors developed in the rest of mice was significantly retarded compared with that of parent tumors. Protective immunity was also induced in the mice that had rejected A11/CD40L cells. In T-cell-defective nude mice, these antitumor effects were not observed. Bone-marrow-derived dendritic cells (DCs), when cultured with A11/CD40L cells, formed clusters with the tumors and showed upregulated CD86 expression. Expression of the interleukin-23 (IL-23) p19, IL-12p35, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with A11/CD40L but not with A11 cells, and P40, the subunit of both IL-12 and IL-23, was secreted from the cocultured DCs. These data directly showed that the expression of CD40L in tumors facilitated the interaction between DCs and the tumors, enhanced the maturation of DCs, induced secretion of cytokines, and consequently produced T-cell-dependent systemic immunity.     

IL-27基因对Eca109细胞在裸鼠体内的成瘤抑制作用及其机制

背景与目的:细胞因子为主的肿瘤生物治疗已成为肿瘤研究领域热点之一。本研究观察白细胞介素(interleukin,IL)-27基因转染人食管癌Eca109细胞株后在裸鼠体内的成瘤抑制作用及其机制。方法:以逆转录病毒为载体,采用基因转染的方法用G418梯度筛选法建立转染IL-27基因的Eca109细胞,RT-PCR检测其基因导入情况,ELISA法检测IL-27的分泌和其诱导外周血单个核细胞(peripheral blood mononuclear cells,PBMC)产生γ-干扰素(interferon,IFN)的能力,MTT法观察Eca109/IL-27细胞生长情况。将Eca109/IL-27、Eca109/LXSN和Eca109细胞接种于裸鼠皮下,观察其成瘤性、移植瘤的生长情况并计算抑瘤率。流式细胞技术检测移植瘤组织肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)中CD16、FasL的表达和肿瘤细胞上Fas的表达。结果:RT-PCR示Eca109/IL-27细胞中有IL-27p28和IL-27EBI3亚基基因表达,Eca109/LXSN和Eca109细胞中未见表达(P<0.01),从而成功建立稳定转染的Eca109/IL-27细胞株,ELISA检测Eca109/IL-27中IL-27的分泌量高于Eca109/LXSN和Eca109细胞(P<0.01)。IL-27基因转染不影响人食管癌细胞株的体外生长(P>0.05),Eca109/IL-27诱导PBMC产生IFN-γ含量高于Eca109/LXSN和Eca109[(56.28±1.61)pg/mL vs.(12.70±0.82)pg/mL]和(11.06±0.64)pg/mL,P<0.01),Eca109/IL-27移植瘤体积较Eca109和Eca109/LXSN减小,瘤重减轻,有抑瘤作用(P<0.05);Eca109/IL-27细胞接种的肿瘤组织TIL中CD16阳性率升高,FasL的表达增高(P<0.05),肿瘤细胞表面Fas表达增加(P<0.05)。结论:IL-27基因修饰Eca109细胞在裸鼠体内产生了抑制肿瘤生长的作用,其机制可能是通过活化自然杀伤细胞,以Fas/FasL的途径产生的。     

Loss of tumorigenicity of human breast cancer cells engineered to produce IL-2, IL-4 or GM-CSF in nude mice.

Human breast cancer cells (OCUB-M), retrovirally transduced with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-2 (IL-2) or IL-4 gene were examined for their antitumor activities in nude mice. Although cell proliferation rates in vitro of these cytokine-producing cells were not significantly different from that of wild-type cells, nude mice that were subcutaneously inoculated with cytokine-producing cells did not develop tumors in contrast to mice that were injected with wild-type cells. Injection of GM-CSF-producing cells into the vicinity of growing wild-type tumors retarded subsequent growth of wild-type tumors. Histological examination of tumors which received GM-CSF-producing cells revealed marked infiltration of mononuclear cells around the tumors. Irradiation of cytokine-producing cells diminished their proliferation capacity but production of cytokine(s) was retained. Therefore, inoculation of irradiated cytokine producer cells into growing tumors can be used as a therapeutic maneuver for breast cancer.     

Interleukin-23 and interleukin-27 exert quite different antitumor and vaccine effects on poorly immunogenic melanoma

Recent studies revealed that two novel interleukin (IL)-12-related cytokines, IL-23 and IL-27, have potent antitumor activities. However, the antitumor effects were mainly evaluated in relatively highly immunogenic tumors and have not been fully evaluated against nonimmunogenic or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of IL-23 and IL-27 on poorly immunogenic B16F10 melanoma and found that the antitumor responses mediated by IL-23 and IL-27 were clearly different. In syngeneic mice, mouse single-chain (sc) IL-23-transfected B16F10 (B16/IL-23) tumors exhibited almost the same growth curve as B16F10 parental tumor about until day 20 after tumor injection and then showed growth inhibition or even regression. In contrast, scIL-27-transfected B16F10 (B16/IL-27) tumors exhibited significant retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect of B16/IL-23 was mainly mediated by CD8+ T cells and IFN-gamma whereas that of B16/IL-27 mainly involved natural killer cells and was independent of IFN-gamma. We also found that antitumor effects of B16/IL-23 and B16/IL-27 were synergistically enhanced by treatment with IL-18 and IL-12, respectively. Furthermore, B16/IL-23-vaccinated mice developed protective immunity against parental B16F10 tumors but B16/IL-27-vaccinated mice did not. When combined with prior in vivo depletion of CD25+ T cells, 80% of B16/IL-23-vaccinated mice completely rejected subsequent tumor challenge. Finally, we showed that the systemic administration of neither IL-23 nor IL-27 induced such intense toxicity as IL-12. Our data support that IL-23 and IL-27 might play a role in future cytokine-based immunotherapy against poorly immunogenic tumors.     

Antitumor effects of interleukin-2 gene-modified fibroblasts in an orthotopic colon cancer model.

We transduced the interleukin-2 (IL-2) gene into murine fibroblasts BALBCL7 or murine colon cancer CT26 using a retroviral vector. BALBCL7 transduced with IL-2 gene secreted 748 pg/ml of IL-2, whereas IL-2 gene-modified CT26 secreted 1,167 pg/ml of IL-2 (48 h incubation, 1x10(6)/ml). Then, we inoculated gene-modified BALBCL7 and/or CT26 cells into BALB/c female mice, and observed the tumor growth. The tumor growth was inhibited in mice inoculated with parental CT26 plus IL-2 gene-modified BALBCL7, compared with that in mice given parental CT26 alone (P<0.01). Moreover, we investigated the cytotoxic activity of spleen cells derived from mice treated with gene-modified cells, and performed phenotypic analysis of the effector cells. The killer cells derived from mice inoculated with IL-2 gene-modified BALBCL7 plus parental CT26 showed higher cytotoxic activity than those from mice inoculated with CT26 alone. The cytotoxic activity was almost completely blocked by anti-CD8 antibody (Ab), and partially blocked by anti-asialo GM1 Ab. Next, we inoculated CT26 tumor tissue into murine cecum orthotopically, and treated the animals with gene-modified BALBCL7 plus parental CT26. The tumor size in the cecum was significantly decreased, compared with parental CT26 alone (P<0.01).     

Antitumor Effects of Interleukin-2 Gene-modified Fibroblasts in an Orthotopic Colon Cancer Model

We transduced the interleukin-2 (IL-2) gene into murine fibroblasts BALBCL7 or murine colon cancer CT26 using a retroviral vector. BALBCL7 transduced with IL-2 gene secreted 748 pg/ml of IL-2, whereas IL-2 gene-modified CT26 secreted 1,167 pg/ml of IL-2 (48 h incubation, 1×10⁶/ml). Then, we inoculated gene-modified BALBCL7 and/or CT26 cells into BALB/c female mice, and observed the tumor growth. The tumor growth was inhibited in mice inoculated with parental CT26 plus IL-2 gene-modified BALBCL7, compared with that in mice given parental CT26 alone ( P < 0.01). Moreover, we investigated the cytotoxic activity of spleen cells derived from mice treated with gene-modified cells, and performed phenotypic analysis of the effector cells. The killer cells derived from mice inoculated with IL-2 gene-modified BALBCL7 plus parental CT26 showed higher cytotoxic activity than those from mice inoculated with CT26 alone. The cytotoxic activity was almost completely blocked by anti-CD8 antibody (Ab), and partially blocked by anti-asialo GM1 Ab. Next, we inoculated CT26 tumor tissue into murine cecum orthotopically, and treated the animals with gene-modified BALBCL7 plus parental CT26. The tumor size in the cecum was significantly decreased, compared with parental CT26 alone ( P < 0.01).     

IL-27基因修饰树突状细胞活化的特异性CTL对人食管癌裸鼠移植瘤生长增殖的影响

研究IL-27基因转染树突状细胞（DC）体内诱导免疫杀伤食管癌细胞的效能及其机制。方法:基因转染的方法建立表达IL-27基因的树突状细胞（DC/IL-27）;构建人食管癌细胞裸鼠移植瘤模型,皮下注射食管癌细胞抗原致敏、IL-27基因修饰DC（DC/IL-27-Ag）活化的特异性CTL后观察荷瘤裸鼠抑瘤率;TUNEL法检测荷瘤裸鼠肿瘤细胞的原位凋亡;流式细胞术检测移植瘤细胞的细胞周期、凋亡率。结果:RT-PCR显示DC/IL-27细胞中有IL-27 p28和EBI3亚基基因表达提示转染成功;免疫接种DC/IL-27-Ag活化的CTL组的抑瘤率为58.28%,明显高于其他组,差异有统计学意义（P<0.01）。TUNEL法检测显示,DC/IL-27-Ag活化的CTL组凋亡率明显高于其他组（P<0.01）,流式细胞术（FCM）显示肿瘤组织内细胞增殖指数为（23.92±1.60）%,显著低于其他组,差异有统计学意义（P<0.01）;细胞凋亡率为（32.78±0.83）%,显著高于其他组,差异有统计学意义（P<0.01）。结论:DC/IL-27-Ag可活化特异性CTL,在裸鼠体内产生了抑制肿瘤生长的作用,显著抑制食管癌细胞增殖、促进其凋亡,为DC应用于食管癌的免疫治疗提供了理论和动物实验依据。      

Acquired immunity in nude mice induced by expression of the IL-2 or IL-4 gene in human pancreatic carcinoma cells and anti-tumor effect generated by in vivo gene transfer using retrovirus.

We have examined the anti-tumor effect in nude mice caused by human pancreatic cancer cells (AsPC-1) modified to secrete IL-2 or IL-4. Loss of tumorigenicity of cytokine-producing, but not wild-type, cells was observed despite their unaltered in vitro proliferation rates; and these anti-tumor effects were dependent on the amount of cytokine released. Wild-type cells inoculated into mice which had rejected IL-2- or IL-4-producer cells showed significant growth retardation, while no retardation was detected when unrelated human colon carcinoma cells were inoculated. Histological examination of regressing IL-2- or IL-4-producing AsPC-1 tumors in nude mice revealed infiltration by CD11b-, but not CD90-, positive cells around the tumors. Treatment of nude mice with anti-asialoGM(1) antibody did not affect loss of tumorigenicity. Mice injected i.p. with IL-2- or IL-4-producing AsPC-1 cells did not die, in contrast to mice inoculated with wild-type cells. Injection of retrovirus-bearing IL-2, but not beta-galactosidase, gene into mice which had wild-type cells in the peritoneal cavity also significantly prolonged survival. Thus, expression of the IL-2 or IL-4 gene in AsPC-1 cells may generate tumor-specific acquired immunity, even in mature T cell-deficient conditions. An anti-tumor response can be induced by in vivo transfer of the IL-2 gene.