Synergistic anti-tumor effects with co-expression of GM-CSF and IFN-γ in murine tumors

We explored the potential therapeutic benefit of introducing GM-CSF, IFN-γ or a combination of both factors into CT26 tumor cells. CT26 cells secreting either GM-CSF or IFN-γ exhibited delayed tumorigenicity; however, cells expressing both GM-CSF and IFN-γ did not form tumors. Even when wild type CT26 cells were introduced into a distant site of mice that had been inoculated with CT26/GM-CSF/IFN-γ cells, no tumors were generated. Furthermore, when we injected GM-CSF + IFN-γ cells into animals bearing established tumors, the tumors were either rejected or their development was delayed, suggesting that synergistic effects were induced against these tumors via a systemic immune response. Histopathological examination of the tumors injected with cells expressing GM-CSF and IFN-γ combined showed necrosis and few signs of malignancy. The growth of tumors from mice treated with CT26/GM-CSF/IFN-γ cells exhibited a delay in tumor formation and no effects were seen in athymic nude mice, which are deficient in T lymphocytes, or in splenectomized nude mice, which are deficient in natural killer (NK) cells, respectively. Our data indicate a dual role for T and NK cells in mediating the anti-tumor activity of this therapy. Our results suggest that transduction of tumor cells with both GM-CSF + IFN-γ results in a powerful synergistic effect of the 2 cytokines that is of greater therapeutic benefit than transduction with either cytokine alone. Int. J. Cancer 77:907–912, 1998.© 1998 Wiley-Liss, Inc.     

Targeting of IL-2 and GM-CSF immunocytokines to a tumor vaccine leads to increased anti-tumor activity

Fusion proteins combining antibodies with cytokines such as IL-2 and GM-CSF appear to be promising reagents for tumor therapy. In this study, we combined such immunocytokines with the tumor vaccine ATV-NDV consisting of irradiated tumor cells infected with Newcastle disease virus (NDV). The two fusion proteins bsF-GMCSF and tsHN-IL2-GM-CSF, binding, respectively, to the viral fusion protein (F) or to hemagglutinin-neuraminidase (HN) expressed on the surface of the vaccine cells and containing GM-CSF or GM-CSF and IL-2-activities were produced by recombinant antibody technology. The purified molecules showed the expected binding specificity and biological activity inherent to the respective cytokine. Using a newly established in?vitro tumor neutralisation assay (TNA), we showed improved antitumoral effect through tumor growth inhibition when human peripheral blood mononuclear cells from healthy donors were stimulated with immunocytokine modified versus non-modified tumor vaccine cells. These effects induced by the fusion proteins, in the presence of a suboptimal T cell activation signal 1 provided by bsHN-CD3, occured only when these were bound to the tumor vaccine. Furthermore, it was shown that CD14+ monocytes could be activated by the GM-CSF cytokine fused within the recombinant proteins and that they contributed essentially to the antitumor effect in the TNA. The data presented here suggest an easy way for a broad clinical development and application of tumor-targeted IL-2- and GM-CSF-based immunocytokines based on the associated increase of anti-tumor activity mediated by T cells and monocytes.     

白细胞介素2锚定的exosomes的制备及其对膀胱癌细胞的诱导杀伤效应

目的 制备白细胞介素2(IL-2)锚定的肿瘤细胞来源的exosomes疫苗,探讨其体外诱导细胞毒性T淋巴细胞(CTL)的抗肿瘤效应.方法 构建含糖基化磷脂酰肌醇(GPI)信号肽序列与人IL-2的融合基因的pEGFP-N1-IL2gpi质粒,建立稳定表达GPI-IL-2的T24细胞系(T24/GPI-IL-2).采用超速和蔗糖梯度密度离心法提取Ex/GPI-IL-2(即T24/GPI-IL-2细胞培养液中提取的exosomes),并对其进行形态学观察和分子标志检测.通过混合淋巴细胞实验和细胞毒实验,观察Ex/GPI-IL-2对T细胞的增殖和诱导杀伤效应.结果 成功构建重组质粒pEGFP-N1-IL2gpi,建立了稳定表达GPI-IL-2蛋白的细胞系.Ex/GPI-IL-2为直径约30～90nm的类圆碟形小囊泡,表达细胞间黏附分子1(ICAM-1)、热休克蛋白70(HSP70)、MAGE-1和GPI-IL-2等免疫相关蛋白.Ex/GPI-IL-2经过树突状细胞负载后,能更有效地诱导T细胞的增殖和细胞毒效应(P＜0.05).结论 通过基因转染技术,可以将IL-2锚定到肿瘤细胞来源的exosomes上,获得的Ex/GPI-IL-2能够诱导CTL对肿瘤细胞产生更强的诱导杀伤效应,为以exosomes为基础的肿瘤免疫治疗提供了新的途径.

Abstract：

Objective To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.Methods To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecuule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2. Results The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70,intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P ＜0.05). Conclusions GPI-IL-2 gene-modified tumor cells can make the exosornes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder trnssitional cell tumors.     

RGD修饰的腺病毒介导LIF和IL-24双基因共表达对MEG01白血病细胞的抑制作用

目的 利用RGD修饰改造白血病抑制因子（LIF）和白细胞介素 24（IL-24）双基因共表达腺病毒载体,以提高感染效率,探讨其对人白血病MEG01细胞的抑制作用。方法 同源重组法构建RGD修饰的表达LIF、IL 24的腺病毒载体Ad.RGD-LIF、Ad.RGD -IL24和Ad.RGD-LIF-IL24。在QBI-293A细胞中扩增腺病毒,检测病毒滴度。流式细胞仪检测各组腺病毒载体对MEG01细胞的感染效率。应用Western blotting检测目的基因LIF、IL-24在MEG01细胞中的表达。CCK-8法检测各组腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24、Ad.RGD-LIF-IL24感染MEG01细胞后对细胞增殖的影响。PE-AnexinV／7-AAD双染后,经流式细胞仪检测细胞凋亡的变化。Western blotting检测凋亡相关蛋白的表达。PI染色后,流式细胞仪检测目的基因表达对MEG01细胞周期的影响。实时定量PCR检测细胞周期调控基因p21和E2F1的表达。结果 成功构建了RGD修饰的腺病毒载体Ad.RGD-LIF、Ad.RGD-IL24和Ad.RGD-LIF-IL24。RGD修饰后的腺病毒能够显著增强对MEG01细胞的感染效率。CCK-8检测显示,与PBS组比较,携带单个目的基因的腺病毒载体Ad.RGD-LIF和Ad.RGD-IL24在第5 d对细胞生长的抑制率分别为29.2％和31.5％,均能够显著抑制MEG01细胞的生长;携带Ad.RGD-LIF-IL24双基因组的抑制率达42.5％,优于单基因组,差异均有统计学意义（P＜0.05）。凋亡检测显示,与PBS组（4.5％）和空病毒组Ad.RGD（7.4％）比较,Ad.RGD-IL24（20.9％）、Ad.RGD LIF（17.8％）均能够显著促进细胞凋亡,且Ad.RGD-LIF-IL24双基因组（29.7％）的促凋亡作用更强。Western blotting显示,LIF和IL-24能够提高促凋亡蛋白p53、Bax的表达,同时抑制抗凋亡蛋白Bcl-xl的表达。目的基因LIF、IL-24过表达会影响MEG01细胞周期的进程,使细胞阻滞在G2期。实时定量PCR显示,LIF和IL-24能够上调细胞周期调控基因p21的转录,抑制E2F1的表达。结论LIF和IL-24过表达的腺病毒载体在体外通过调节p53、Bax、Bcl-xl的表达,影响白血病细胞MEG01的生长,诱导其凋亡,并通过调控p21、E2F1的水平影响细胞周期的进程。     

CM—CSF及IL—4增强树突状细胞免疫功能

目的 探讨粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白介素－４（ＩＬ－４）对正常成人及大肠癌患者树突状细胞（ＤＣ）表现人白细胞抗原（ＨＬＡ）－ＤＲ及Ｂ７等免疫分子表达及其免疫功能的影响。方法 分离正常成人（ｎ＝１０）及大肠癌患者（ｎ＝１０）外周血ＤＣ,以ＧＭ－ＣＳＦ及ＩＬ－４联合刺激正常成人及大肠癌者ＤＣ,检测ＧＭ－ＣＳＦ及ＩＬ－４联合刺激前后ＤＣ表现ＨＬＡ－ＤＲ及Ｂ７表达水平及ＤＣ免疫功能变化。     

The immune system as anti-tumor sentinel: molecular requirements for an anti-tumor immune response.

The concept behind immune surveillance against cancer is that tumor cells continuously develop, but that there may not be clinical evidence of their presence because the immune system recognizes the cells as foreign and destroys them. A clear role for the immune system in preventing and/or eliminating tumors is emerging as insights into the molecular requirements for the induction and effector function of cytolytic T lymphocytes (CTL) have been gained. Using murine tumor rejection models, the role of particular molecular components of the immune system in controlling tumor growth has been defined. However, tumor rejection does not always occur spontaneously in vivo, indicating that defects in the generation or execution of an anti-tumor immune response may be common. Understanding defects when they arise should allow for development of new therapeutic approaches in tumor-bearing individuals. Many clinical studies are underway to test strategies to induce or heighten an antitumor immune response in cancer patients.     

苓甲抗癌复方提取物在荷瘤小鼠中抗肿瘤免疫应答机制

目的:初步探究苓甲抗癌复方提取物(LAM)在荷瘤小鼠中抗肿瘤免疫应答机制.方法:LAM治疗荷瘤小鼠,检测小鼠生长、瘤块体积与重量的变化;流式细胞术和ELISA分析小鼠脾脏中Treg细胞、NK细胞和各种细胞因子;Western blot分析LAM对Treg细胞相关蛋白作用.结果:LAM抑制荷瘤小鼠肿瘤的生长,引发肿瘤坏死,抑制肿瘤促增殖蛋白Ki67的表达,降低血液中CA153的含量;降低脾脏中Treg细胞比例和血清中TGF-β与IL-10的含量,提高IL-2、IFN-γ、穿孔素、颗粒酶B的含量和NK细胞的比例.结论:LAM抑制肿瘤的生长,并抑制Treg细胞的数量与功能,提高机体的免疫功能.     

Boerhaavia diffusa stimulates cell-mediated immune response by upregulating IL-2 and downregulating the pro-inflammatory cytokines and GM-CSF in B16F-10 metastatic melanoma bearing mice

Effect of Boerhaavia diffusa extract on the Cell Mediated Immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of Boerhaavia diffusa enhanced Natural Killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared to the metastatic tumor bearing control. Production of the cytokine IL-2 was significantly enhanced by the administration of Boerhaavia diffusa compared to the untreated metastatic tumor bearing control. Levels of GM-CSF and pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-a were significantly lowered by Boerhaavia diffusa administration compared to metastatic control. The gene expression level of IL-2, IL-1beta, IL-6, TNF-alpha and GM-CSF in B16F-10 cells also correlate the above result. These results indicate Boerhaavia diffusa could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.     

癌胚抗原与IL-2真核双表达质粒的构建及体外表达

目的：构建癌胚抗原（CEA）与白细胞介素2（IL－2）的真核双表达质粒，并检测其在体外的表达。方法：利用分子生物学技术，将CEA基因片段和IL－2基因片段连接于真核双表达质粒pIRES中，测定序列后，用脂质体包裹转染细胞，采用ELISA双抗体夹心法检测CEA和IL－2的表达。结果：核酸序列测定证实本实验所构建的质粒正确，质粒上所接入的CEA和IL－2的碱基序列与标准序列的同源性分别为99．8％和99．9％。该双表达质粒在体外转染细胞内后可表达CEA和IL－2分子。结论：实验所构建的pIRES-CEA-IL－2双表达质粒能在体外同时表达CEA和IL－2分子，说明本质粒具有在细胞水平同时表达两种生物大分子的活性，这一结果为其以后在整体动物水平的实验研究奠定了基础。     

cGAS-STING信号通路调控抗肿瘤免疫应答的研究进展

作为胞质中的 DNA 感受器,环腺苷酸鸟苷酸合成酶（cyclic GMP-AMP synthase,cGAS）能够识别细胞质内的异常DNA,激活干扰素刺激基因（stimulator of interferon genes,STING）信号通路,介导下游的干扰素相关基因、炎性相关因子和趋化因子的产生,从而启动机体的免疫应答。STING蛋白作为DNA感受通路下游关键的接头分子,广泛表达于免疫细胞、肿瘤细胞和基质细胞等多种类型的细胞中,发挥感受胞质DNA和免疫防御的信号转导作用。STING蛋白激动剂作为新型激动剂已用于多种肿瘤的临床前研究和临床试验治疗,展示出诱人的应用前景。但是,也有文献报道该信号通路的过度激活在某些情况下对肿瘤的生长有一定的促进作用。本文对近年来cGAS-STING信号通路调控免疫应答以及STING激动剂在肿瘤免疫治疗中应用的相关研究进展作一综述,为靶向该通路的抗肿瘤免疫治疗的临床应用提供依据。     

Opposing effects of fibrosarcoma cell-derived IL-1 alpha and IL-1 beta on immune response induction

There is evidence that cell-associated IL-1 alpha supports immune response induction. Here we explored the impact of malignant cell-derived IL-1 on immunogenicity, immune response induction and tumor-induced immunosuppression using 3-methylcholanthrene-induced fibrosarcoma lines derived from wild-type (wt), IL-1 alpha-, IL-1 beta- or IL-1a beta-knockout (IL-1 alpha(-/-), IL-1 beta(-/-), IL-1 alphabeta(-/-)) C57BL6 mice. The wt, IL-1 alpha(-/-), IL-1 beta(-/-) and IL-1 alphabeta(-/-) fibrosarcoma lines express MHC class I molecules at a high level. The lines do not differ in their susceptibility toward NK cells, macrophages, and allogeneic CTL, or in their capacity as stimulators of an allogeneic response. However, IL-1 beta(-/-) tumors rarely grow in the syngeneic host, which is the consequence of a strong T helper and CTL response induction by IL-1 alpha-competent, IL-1 beta(-/-) tumors. On the other hand, IL-1 beta-competent, IL-1 alpha(-/-) tumors strongly assist CD11b(+)Gr-1(+) myeloid-derived suppressor cell and regulatory T cell expansion, which both suppress with high efficacy activated T helper cell proliferation and CTL lysis. In IL-1 alphabeta(-/-) tumors, the absence of IL-1 alpha becomes decisive, i.e. despite reduced suppressor cell recruitment, tumor growth was unimpaired due to inefficient immune response induction. Thus, sarcoma cell-derived IL-1 alpha and IL-1 beta do not act in concert. Induction of a strong immune response by IL-1 alpha demands therapeutic exploitation, which may become more efficient if systemic induction of immunosuppression by IL-1 beta can also be circumvented.     

肿瘤放疗对免疫功能的影响

肿瘤放疗能够影响机体免疫,最新研究显示适当的放疗方式能够通过调节肿瘤微环境、激活免疫细胞、释放死亡危险信号而激活免疫,产生旁观者或远位效应。随着放疗与免疫联合治疗临床评价指标研究及临床试验的开展,将有望改变传统的肿瘤治疗模式。     

Nonspecific immunotherapy with intratumoral lipopolysaccharide and zymosan A but not GM-CSF leads to an effective anti-tumor response in subcutaneous RG-2 gliomas

Purpose Nonspecific stimulation of cells of the immune system may be useful in generating an anti-tumor response for a variety of cancers and may work synergistically with currently available cytotoxic therapies. In this study we examined the response of syngeneic rat gliomas to treatment with several nonspecific stimulators of dendritic cells and macrophages alone or in combination with radiation therapy. Experimental design RG-2 gliomas were implanted subcutaneously and treated with intratumoral (IT) injections of the toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and zymosan A (ZymA) and the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF). Combination treatment with IT LPS and single-fraction external beam radiotherapy (EBRT) was also evaluated. Results Treatment with IT LPS and ZymA delayed tumor growth compared to saline controls. Multiple doses of both substances were superior to single doses, and led to complete tumor regression in 71% (LPS) and 50% (ZymA) of animals. GM-CSF showed no anti-tumor effects in this study. Combinations of IT LPS and EBRT appeared to have a synergistic effect in delaying tumor growth. Rechallenge studies and IT LPS treatment of RG-2 tumors in nude rats suggested the importance of T cells in this treatment paradigm. Conclusions Direct IT treatment with the TLR ligands LPS and ZymA are effective in generating an anti-tumor response. These treatments may synergize with cytotoxic therapies such as EBRT, and appear to require T cells for a successful outcome.     

The immune anti-tumor effects of GM-CSF and B7-1 gene transfection are enhanced by surgical debulking of tumor

Malignant mesothelioma (MM) is a solid tumor largely unresponsive to conventional therapies. Immunological gene therapy shows promise in murine models and human clinical trials; however, the role of surgery in combination with gene therapy has not been widely studied. The aim of this study was to determine if debulking surgery improved the effectiveness of gene therapy in a murine MM model. Mice were subcutaneously inoculated with the MM cell line, AC29, at two different sites, 4 days apart, to allow a surgical and distal site tumor to develop. Once tumors were established, the surgical site tumor was debulked and vaccination of syngeneic tumor transfectants encoding genes for IL-4, IL-2, GM-CSF, B7-1 or allogeneic MHC molecules commenced at a site away from both tumors, and tumor growth was measured. Neither debulking surgery nor gene therapy alone delayed tumor growth. However, there was a clear delay of tumor growth when debulking surgery was combined with vaccination of tumor transfectants expressing B7-1 or high levels of GM-CSF. Combinations of these two transfectants did not lead to a synergistic effect. This study demonstrates that debulking surgery can augment the immunostimulatory effects of immunological gene therapy and can delay tumor growth. This has implications for the future design of human gene therapy trials for solid tumors such as MM.     

Role of interleukin-2 (IL-2) in cancer-related immune deficiency: in vitro response to IL-2, production of IL-2, and IL-2 receptor expression in patients with advanced cancer

The aims of the investigation were 1) to determine if there are defects in interleukin-2 (IL-2) regulation on either phytohemagglutinin (PHA)-activated or non-PHA-activated peripheral blood mononuclear cells (PBMC) in cancer patients to ascertain the role of IL-2 in this disease; 2) to carry out preliminary experiments for a direct quantitative evaluation of endogenous IL-2 production by PBMC cultures; and 3) to evaluate the IL-2 receptor expression by PBMC of cancer patients. An assessment of lymphocyte subsets was also performed with monoclonal antibodies in a selected group of patients. A total of 170 patients entered the study. Cancer sites were larynx (48), breast (44), lung (30), colorectal(23), and gynecologic (25). Staging showed in the former three cancer sites predominantly localized or only locally advanced disease and in the latter two sites disseminated disease. PBMC cultures were performed with microtiter plate technique and 3H-thymidine uptake evaluation using polyclonal mitogens, IL-2, and a monoclonal antibody against IL-2 receptor. Our results provided evidence that the cancer patients exhibit a T-cell functional immunodepression, which, to some extent, progresses during tumor growth so that the localized disease shows a low-grade defect and advanced disease a high-grade defect. Our data also clearly suggest that IL-2 deficiency is the primary factor involved in this functional immune impairment. We found no significant defect in the IL-2 receptor expression by PBMC of cancer patients. Our data also seem to support the in vivo therapeutic administration of IL-2 and lymphokine-activated killer cells to cancer patients.     

Human-human hybridomas for the study of anti-tumor immune response in patients with colorectal cancer

Human-human hybridoma technology was evaluated for the study of humoral immune reactions of colorectal cancer patients against their own tumors. Six fusions were carried out with lymphocytes from mesenteric lymph nodes from patients with colorectal cancer, using the human B-lymphoma cell line LICR-LON-HMy-2 as fusion partner. A total of 294 wells with cell growth were obtained. Supernatants from 26 of these reacted in enzyme-linked immuno-sorbent assay (ELISA) with one or more colon cancer cell lines. Cells from only one of these wells (D 4213) could be cloned. The clone was shown to produce antibody which by immunocytochemical analysis reacted with a panel of colon cancer cell lines and melanoma cell lines but not with several other cancer cell lines or normal human leukocytes. By immunohistochemical analysis on formalin-fixed paraffin-embedded tissue this antibody reacted strongly with antigen expressed by autologous and allogeneic colorectal cancers. Faint staining could occasionally be observed on normal colon epithelium. D4213 is a hybrid cell line since it is tetraploid and produces kappa and lambda light chains as well as gamma and mu chains, whereas HMy-2 produces only kappa and gamma chains. The study produces only kappa and gamma chains. The study suggests that patients may possess B cells producing antibody reactive with their own malignant cells.     

Induction of anti-tumor immunity by lyophilized myeloma cells secreting GM-CSF

We have previously demonstrated that a low dose of live myeloma FO cells induced a cellular immunity against tumor without additional modulating factors. In the present study, lyophilized myeloma FO cells were used to induce anti-tumor immunity. In a myeloma vaccination model, immunization with lyophilized myeloma FO cells alone induced a slight response. However, the immunity was dramatically enhanced by myeloma FO cells transfected with a recombinant adenovirus, Adv-1/GM-CSF. The immunocytochemical staining of Adv-1/GM-CSF transfected myeloma FO cells confirmed that more than 90% of cells were positive with GM-CSF expression. Results of sandwich ELISA showed the amount of secreted GM-CSF was 240 ng/24 h per 10(6) cells. Immunization with lyophilized myeloma FO cells secreting GM-CSF prevented the tumor growth in 60% of BALB/c mice. Antibodies against myeloma FO cells were found in the sera of immunized mice. Tumor-specific T-cell response was also evaluated using cytotoxic T lymphocyte assay. In conclusion, lyophilized myeloma FO cells secreting GM-CSF can be used as a potent vaccine to induce strong and protective anti-tumor immunity.     

INDUCTION OF IMMUNE RESPONSE BY IL-6 GENEMODIFIED LEUKEMIA CELLS

ＩＮＤＵＣＴＩＯＮＯＦＩＭＭＵＮＥＲＥＳＰＯＮＳＥＢＹＩＬ６ＧＥＮＥＭＯＤＩＦＩＥＤＬＥＵＫＥＭＩＡＣＥＬＬＳＣａｏＸｕｅｔａｏ曹雪涛ＧｅＬｉｎｇｆｕ葛林阜ＪｕＤｉａｎＷｅｎ鞠佃文ＹｕＹｉｚｈｉ于益芝ＴａｏＱｕｎ陶群ＺｈａｎｇＷｅｉｐｉｎｇ章卫...     

Glycolipid-anchored IL-12 expressed on tumor cell surface induces antitumor immune response

Systemic or local administration of cytokine has been used as a mode to enhance the antitumor immune response induced by many cancer vaccines. We have investigated whether the expression of cytokines on the tumor cell surface as a glycolipid (GPI)-anchored form will be effective in inducing antitumor immune response using a GPI-anchored interleukin (IL)-12 (GPI-IL-12) as a model. GPI-IL-12-induced the proliferation of concanavalin A-activated T cells and induced IFN-gamma secretion by activated and allogeneic T cells, indicating that the membrane-expressed IL-12 can stimulate T cells. GPI-IL-12 expressed on the tumor cell surface prevented tumor growth in mice in a highly tumorigenic murine mastocytoma model. These results suggest that the cell surface-expressed GPI-IL-12 can be effective in inducing antitumor immune response, and GPI-anchored cytokines expressed on the tumor cell surface may be a novel approach to deliver cytokines at the immunization site during vaccination against cancer. Furthermore, purified GPI-anchored cytokines can be used to quickly modify tumor membranes by the protein transfer method to express the desired cytokines for vaccine development.