Development of chemokine-cocktail therapy against hyman malignant gliomas using lymphocyte-specific chemokines

淋巴细胞在人类恶性胶质瘤中经常被观察到，但其出现的机制尚不完全清楚。为了阐明恶性胶质瘤的肿瘤免疫，我们采用RT-PCR、Northern blot、免疫blot和免疫组化等方法分析了8种新型淋巴细胞特异性趋化因子在胶质瘤细胞系和胶质瘤组织中的表达，并考察了其与不同亚群淋巴细胞浸润的相关性。对于检测的8种趋化因子（LARC， TARC， ELC， SLC， PARC， LEC， HCC-2和SCM-1a），通过RT-PCR可以在所有12种胶质瘤细胞系中清楚地检测到LARC的表达。此外，在大多数胶质瘤细胞系中均可检测到TARC和SCM-1a的表达。然而，大多数趋化因子的表达水平较低，因此除了LARC在2个细胞系中表达外，Northern blot分析无法显示它们的表达。肉豆蔻酸佛酚酯可强烈诱导U87 MG细胞中LARC mRNA和LARC蛋白的表达。8个胶质母细胞瘤组织中有4个通过免疫印迹检测，33个样本中有9个（27.3%）通过免疫组织化学检测产生了LARC蛋白。有趣的是，LARC染色阳性与CD8-、CD4-和cd45r0阳性细胞的浸润显著相关（p<0.001）。虽然LARC的组成表达水平较低，但某些刺激可强烈诱导其表达，并在人恶性胶质瘤的肿瘤免疫中发挥重要作用。

Lymphocytes are frequently observed in human malignant glioma, the mechanism(s) underlying their appearance is not fully understood. To clarify tumor immunity in malignant gliomas, we analyzed the expression of 8 novel lymphocyte-specific chemokines in human glioma cell lines and glioma tissues by RT-PCR, Northern blot, immunoblot and immunohistochemistry, and examined the correlation with the infiltration of various subsets of lymphocytes. For the 8 chemokines examined (LARC, TARC, ELC, SLC, PARC, LEC, HCC-2, and SCM-1a), expression of LARC was clearly detectable in all 12 glioma cell lines by RT-PCR. Additionally, expression of TARC and SCM-1a was detectable in the majority of glioma cell lines. However, the expression level of most chemokines was low, so that Northern blot analysis could not demonstrate their expression with the exception of LARC in 2 cell lines. Expression of LARC mRNA and LARC protein was strongly induced by phorbol myristate ester in U87 MG cells. The production of LARC protein was demonstrated in 4 of 8 glioblastoma tissues by immunoblot, and 9 of 33 samples (27.3%) by immunohistochemistry. Interestingly, the positivist of LARC staining was significantly correlated with the infiltration of CD8-, CD4-, and CD45R0-positive cells (p<0.001).Although the constitutive expression level of LARC is low, certain stimulations could strongly induce its expression, and play a crucial role in the tumor immunity of human malignant glioma.