Establishment of specific immunotherapy by activation of innate immunity

为了建立有效的化疗和免疫联合治疗，选择能有效诱导肿瘤细胞凋亡且对全身免疫活性影响较小的化疗药物。我们观察了抗癌药对人外周血单个核细胞(PBMC)的生长抑制活性、对胃癌细胞的诱导凋亡活性以及帕利特(1XL)和多西紫杉醇(TXT)对DC表面Toll样受体(TLR)-4mRNA表达的影响。我们进一步比较了肿瘤裂解物冲击的DC诱导的细胞毒性T淋巴细胞(CTL)和TXT诱导的细胞凋亡。采集健康志愿者PBMC，在含10%PCS的RPM1640培养液中，分别加入浓度为0.1xPP和10xPPC的抗菌药，通过细胞内Al？级别。1UNNEL法检测细胞凋亡率。未成熟的DC为…用含白细胞介素4(IL-4；50 ng/ml)和粒细胞巨噬细胞刺激因子(GM-SP50/M1)的无血清培养液(AIM V)培养5天，可获得更多的NED。然后用胃癌细胞MKN45的肿瘤细胞裂解液或TXT诱导的MKN45细胞凋亡液冲击DC 12小时，再加入OK-432(0.1KE/ml)继续培养30小时。用乳酸脱氢酶释放法检测CIL活性。虽然大多数抗癌药物对PBMC增殖的抑制作用呈剂量依赖关系，但TXT、多西氟尿苷和伊立贝烷即使在最高药物浓度下对PBMC也没有抑制作用。约60%的MKN45细胞在24小时后出现凋亡。1XL和TXT均治疗48小时。TXT可上调IDCs TLR-4mRNA的表达，但TXT不能上调IDCs TLR-4mRNA的表达，1×10~(-8)MCLI作用2小时后达高峰，凋亡细胞表现出与肿瘤细胞相似的杀伤活性。这些结果表明，TXT似乎是一种最佳的抗癌药物，用于胃癌患者的化疗和肿瘤特异性树突状细胞免疫治疗。然后，我们计划进行一项前瞻性的临床I/II期试验，使用TXT和5 v-DFUR的联合化疗，然后瘤内注射未成熟的DC。到目前为止，纳入了三名患者，免疫学和临床病理参数的分析正在进行中。较少

In order to establish an efficient combination therapy with chemotherapy and immunotherapy, selection of chemotherapeutic agents those effectively induce apoptosis on tumor cells and have less effect to systemic immunological activity. We investigated the growth inhibitory activity of anticancer agent on human peripheral blood mononudear cells (PBMC), apoptosis inducing activity on gastric cancer cells and expression of toll-like receptor (TLR)-4 mRNA on DCs by parlituel (1XL) and docetaxel (TXT). We further compared the cytotoxidty of cytothdc T lymphocytes (CTLs) induced by DCs pulsed with tumor lysate and apoptotic cells induced by TXT. PBMC were collected from healthy volunteers and cultured in RPM1640 with 10% PCS Anticanoer agents were added at concentrations of peak plasma concentration (PPC), 0.1xPP and 10xPPC concomitant with PHA Proliferating activity of PBMC was determined by measuring intracellular Al? levels. Apoptosis was evaluated by 1UNNEL assay. Immature DCs were obtai … More ned by culturing PBMCs in serum-free medium (AIM V) with interieukin-4 (IL-4 ; 5Ong/ml) and granulocyte-maaophage stimulating factor (GM-SP 5Ong/m1) for 5 days. Then DCs were pulsed with tumor cell lysate obtained from gastric cancer cells MKN45 or apoptotic MKN45 cells induced by TXT for 12hr and further cultured for 30hr following addition of OK-432 (0.1 KE/ml). CIL activity was determined by LDH-releasing assay. Although most of anticancer agents demonstrated the suppression activity on proliferation of PBMC in a dose dependent manner, TXT, doxifluridine and irinobecane did not show the suppressive activity on PBMC even in the highest drug concentration. About 60% of MKN45 cells demonstrated apoptosis after 24hr. and 48hr treatment of both 1XL and TXT. Expression of TLR-4 mRNA in iDCs was up-regulated by TXT, not by TXT, peaked at 2 hr after treatment at a concentration 1×10-8M Clis induced by DCs pulsed with tumor lysate and apoptotic cells showed similar killing activity to tarwt cells. These results suggest that TXT appears to be a optimal anticancer agent for a combination therapy with chemotherapy and tumor specific immunotherapy using dendritic cells in gastric cancer.We then planed a prospective clinical Phase I/II trial using a combination chemotherapy of TXT and 5 v-DFUR followed by intra-tumoral immature DC injection in patients with gastric cancer. So far, three patients were included and the analysis of immunological and clinicopathblogic parameters are now underway. Less