Gannagé Monique, Münz Christian
Viral Immunobiology, Institute of Experimental Immunology, University Hospital of Zürich, Zürich, Switzerland.
Methods Enzymol. 2009;452:403-21. doi: 10.1016/S0076-6879(08)03624-0.
Major histocompatibility complex (MHC) class I and II molecules can both present cytosolic and nuclear antigens to CD8(+) and CD4(+) T cells, respectively. However, MHC class I displays proteasomal, whereas MHC class II molecules display lysosomal, degradation products. One pathway by which intracellular antigens gain access to lysosomal degradation is macroautophagy. Therefore, MHC class II presentation of antigens that are targeted to autophagosomes can be used to investigate regulation events of the macroautophagy pathway. We fuse antigens to Atg8/LC3 for targeting to autophagosomes, because this ubiquitin-like protein is selectively coupled to autophagosome membranes, and the portion that is coupled to the inner autophagosome membrane is degraded with this membrane in lysosomes. The localization of these fusion antigens in MHC class II loading compartments can be visualized by immunofluorescence and electron microscopy, and used as a measure of autophagic amphisome generation. In addition, MHC class II presentation of autophagosome-targeted antigens can be monitored by CD4(+) T cell recognition and indicates completion of macroautophagy. Together these immunological assays are well suited to investigate autophagic flux and analyze experimental conditions and physiological perturbations for their influence on macroautophagy.
主要组织相容性复合体（MHC）I类和II类分子分别可将胞质和核抗原呈递给CD8(+)和CD4(+) T细胞。然而，MHC I类展示蛋白酶体降解产物，而MHC II类分子展示溶酶体降解产物。细胞内抗原进入溶酶体降解的一条途径是巨自噬。因此，靶向自噬体的抗原的MHC II类呈递可用于研究巨自噬途径的调控事件。我们将抗原与Atg8/LC3融合以靶向自噬体，因为这种类泛素蛋白选择性地与自噬体膜偶联，且与自噬体内膜偶联的部分会在溶酶体中随该膜一起降解。这些融合抗原在MHC II类装载区室中的定位可通过免疫荧光和电子显微镜观察到，并用作自噬性两性体生成的指标。此外，靶向自噬体的抗原的MHC II类呈递可通过CD4(+) T细胞识别来监测，并表明巨自噬的完成。这些免疫学检测方法一起非常适合研究自噬通量，并分析实验条件和生理扰动对巨自噬的影响。
