New anti-tumor immunotherapy with intratumoral injection of IL-2+IL-12 gene-modified dendritic cells.

1. 我们研究了肺癌患者肿瘤浸润淋巴细胞中产生IL-2和IL-12的辅助T型1细胞（Th1）的比例，IL-2和IL-12通常激活树突状细胞（dc）。1)应用流式细胞术分析肺癌患者肿瘤浸润淋巴细胞中Th1与Th2的比值。2)与外周血相比，肿瘤组织中th1 - th2比值显著升高。小肿瘤（直径小于3cm）和鳞状细胞癌的Th1.2比例较高，提示其具有DCs激活的优势。我们利用树突状细胞的抗原呈递能力，开发了肿瘤特异性细胞毒性T淋巴细胞（CTL）。1)诱导dcs：从与人肺腺癌细胞系（PC16）匹配的HLA-A2阳性健康供体中分离出单个核细胞，产生特异性免疫反应。用GM-CSF （1000U/ml）和IL-4 （1000U/ml）孵育5 d，然后用肿瘤裂解物和TNF-α (1000U/ml)孵育2 d。2)在含有IL-2和固相抗cd3刺激的培养基中，用dc刺激细胞发展成针对pc14的CTL数天。刺激3周后，淋巴细胞数量增加约100倍，发展为CTL。3)CTL的抗肿瘤细胞毒活性我们采用ELISA法分离IFN-γ测定CTL在混合肿瘤和CTL培养中的细胞毒活性。dc刺激诱导的CTL显示出最高的γ-IFN浓度，并被抗hla - a2抗体显著阻断。综上所述，由于肺癌组织中有大量Th1通过有利的细胞因子分泌激活dc，提示肺癌患者使用自体dc进行CTL治疗的可能性。

1. We investigated the proportion of T helper type 1 cells (Th1) producing IL-2 and IL-12, which usually activates dendritic cells (DCs), in tumor infiltrating lymphocytes of lung cancer patients.1) The ratio of Th1 to Th2 in tumor infiltrating lymphocytes of lung cancer patients were analyzed with flow cytometry.2) The Th1-to-Th2 ratio was significantly elevated in the tumor tissues compared with peripheral blood. The cases of small size tumor (diameter was less than 3cm) and squamous cell carcinoma were suggested to have the advantage of DCs activation because of their higher proportion of Th1.2. We developed the tumor specific cytotoxic T lymphocytes (CTL) with using the antigen-presenting abilities of DCs.1) Induction of DCsMononuclear cells were separated from HLA-A2 positive health donors matching with human lung adenocarcinoma ell lines (PC16) because of the production of specific immuno-reaction. DCs were induced by incubating for five days with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) and following for two days with tumor-lysates and TNF-α (1000U/ml).2) Development to CTL against PC14Autologous lymphocytes were stimulated with DCs for a couple of days in medium containing IL-2 and solid-phase anti-CD3 stimulation. After three week stimulation lymphocytes increased their number about a hundred times and developed to CTL.3) Anti-tumor cytotoxic activity of CTLwe evaluated the cytotoxic activity of CTL in mixed tumor and CTL culture using IFN-γ secration assay by ELISA.CTL induced with the stimulation with DCs showed the highest γ-IFN concentration and significantly blocked by anti-HLA-A2 antibody.In conclusion, because there are a lot of Th1 activating DCs by favorable cytokine secretion in lung cancer tissues, the possibility of CTL therapy using autologous DCs in lung cancer patients were suggested.