Experimental Oncology Unit (UNONEX), Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, São Paulo, Brazil.
Mol Cancer. 2009 Dec 7;8:116. doi: 10.1186/1476-4598-8-116.
CD1d-restricted iNKT cells are protective against murine melanoma B16F10-Nex2 growing subcutaneously in syngeneic C57Bl/6 mice as inferred from the fast tumor development in CD1d-KO in comparison with wild type animals. CD1d glycoproteins are related to the class I MHC molecules, and are involved in the presentation, particularly by dentritic cells (DC), of lipid antigens to iNKT cells. In the present work we attempted to identify the endogenous lipid mediator expressed in melanoma cells inducing such immunesurveillance response and study the possibility of protecting animals challenged with tumor cells with lipid-primed DC.
Crude cytosolic and membrane fractions from in vivo growing melanoma contained iNKT-stimulating substances. Lipids were then extracted from these cells and one of the fractions (i.e. F3A) was shown to prime bone marrow-derived dendritic cells (BMDC) to stimulate iNKT murine hybridoma (DN32D3) cells to produce IL-2. The active fraction was analyzed by electrospray ionization-mass spectrometry (ESI-LIT-MS) and both iGb3 and iGb4 were identified along with GM3. When iGb3 was incubated with BMDC and tested with DN32D3 cells, IL-2 was equally produced indicating iNKT cell activation. GM3 consistently inhibited this response. To assess the antitumor response-induced by iGb3, a cytotoxicity assay in vitro was used with [3H]-thymidine labeled B16F10-Nex2 cells. At target/effector (iGb3-activated iNKT) cell ratio of 100(-1)-100(-4) tumor cell lysis was shown. The antitumor activity in vivo was tested in mice challenged i.v. with B16F10-Nex2 cells and treated with iGb3- or alpha-galactosylceramide-primed DCs. A 4-fold lower tumor load in the lungs was observed with either treatment.
Our results show the expression of globo and isoglobohexosylceramides in murine melanoma B16F10-Nex2. The expression of iGb3 and its precursor, iGb4, on tumor cells may prime an effective iNKT cell-dependent antitumor response, modulated negatively by GM3 which is also produced in these cells. iGb3-primed BMDC exerted a significant iNKT cell-mediated anti-tumor activity in mice challenged with melanoma cells.
CD1d 限制性 iNKT 细胞可预防 C57Bl/6 同基因小鼠皮下生长的黑色素瘤 B16F10-Nex2，这可从 CD1d-KO 与野生型动物相比肿瘤快速生长推断出来。CD1d 糖蛋白与 I 类 MHC 分子有关，参与脂质抗原向 iNKT 细胞的呈递，特别是树突状细胞 (DC)。在本工作中，我们试图鉴定在诱导这种免疫监视反应的黑色素瘤细胞中表达的内源性脂质介质，并研究用脂质预刺激的 DC 保护受肿瘤细胞攻击的动物的可能性。
来自体内生长的黑色素瘤的粗质胞浆和膜部分含有可刺激 iNKT 的物质。然后从这些细胞中提取脂质，其中一个部分 (即 F3A) 显示可刺激骨髓来源的树突状细胞 (BMDC) 刺激 iNKT 鼠杂交瘤 (DN32D3) 细胞产生 IL-2。通过电喷雾电离-质谱 (ESI-LIT-MS) 分析活性部分，鉴定出 iGb3 和 iGb4 以及 GM3。当 iGb3 与 BMDC 孵育并用 DN32D3 细胞测试时，同样产生了 IL-2，表明 iNKT 细胞被激活。GM3 始终抑制这种反应。为了评估 iGb3 诱导的抗肿瘤反应，使用 [3H]-胸苷标记的 B16F10-Nex2 细胞进行体外细胞毒性测定。在靶细胞/效应细胞 (iGb3 激活的 iNKT) 比为 100(-1)-100(-4)时观察到肿瘤细胞溶解。在静脉内接受 B16F10-Nex2 细胞攻击的小鼠中测试体内抗肿瘤活性，并接受 iGb3 或 alpha-galactosylceramide 预刺激的 DC 治疗。用这两种治疗方法，在肺部观察到肿瘤负荷降低了 4 倍。
我们的结果表明，在小鼠黑色素瘤 B16F10-Nex2 中表达了 globo 和 isoglobohexosylceramides。肿瘤细胞上 iGb3 和其前体 iGb4 的表达可能引发有效的 iNKT 细胞依赖性抗肿瘤反应，GM3 可负性调节该反应，该物质也在这些细胞中产生。用 iGb3 预刺激的 BMDC 在接受黑色素瘤细胞攻击的小鼠中发挥了显著的 iNKT 细胞介导的抗肿瘤活性。
