Immunity: New discovery of molecular mechanism of antigen cross-presentation

"Cross presentation" is an important concept in immunology. Antigen Presenting Cell (APC) stimulated by antigen, mainly dendritic cell (Dendritic cell, DC) can decompose antigen material, then assemble with MHC molecule, and finally express the antigen-MHC complex in Cell surface. The specific antigen-MHC complex of dendritic cells can interact with TCR on the surface of T cells, and cooperate with the interaction of auxiliary molecules to achieve the purpose of activating T cells. Among them, the MHC-I-antigen complex can activate CD8+ T cells, and the MHC-II-antigen complex can activate CD4+ T cells. Classical MHC-I antigen presentation relies on the antigen in the cytoplasm, and “cross-presentation” provides a path for the recognition of extracellular antigens by MHC-I.

"Cross presentation" is mainly realized by DC. It can mediate the presentation of non-DC infected pathogens and activate CD8+ T cells. Although this concept is very mature, the fine molecular mechanism has yet to be studied.

There are currently two hypotheses about the mechanism of cross-presentation: 1. Vacuole pathway. The hypothesis is that external pathogen molecules enter the endosome through endocytosis, and undergo lysosomal protease cleavage and fragmentation, after which the antigen material is assembled with the recovered MHC-I molecule in the endosome and released to the cell again Surface; 2. Endosome-cytoplasmic pathway. The cleaved antigen material is released from the endosome into the cytoplasm through the channel on the membrane, and then fragmented by the protease in the cytoplasm, and finally merges with the classical MHC-I pathway. At present, scholars generally believe that the second hypothesis is the main assembly method.

For the endosome-cytoplasmic pathway, the most important step is the release of antigen from the endosome to the outside of the cytoplasm. Previous studies have achieved the effect of inhibiting cross-presentation by using exotoxin A to inhibit the "endoplasmic reticulum-associated protein degradation element (ERAD)". Therefore, some important such proteins, including Sec61 and Derlin-1, are considered to be involved in cross-presentation. This critical step of presentation. However, this statement lacks direct evidence to support it. Recently, Sven Burgdorf's group from the University of Bonn in Germany published the latest research in the journal Immunity, which proved that Sec61 is involved in the key role of antigen release from endosomes to cytoplasm in cross-presentation.

First, the author repeated the previous results: they incubated mouse BMDC (bone marrow-derived DC) with OVA antigen, then added exotoxin A treatment (or no treatment), and then combined the treated DC with OT-I or OT-II is co-cultured and the subsequent activation of T cells (IL-2 expression) is detected. The results show that the treatment of exotoxin can significantly inhibit the activation of OT-I, but the activation of OT-II is not affected. This result indicates that DC has the effect of cross-presentation, and on the other hand, it indicates that the treatment of exotoxin specifically inhibits the process of cross-presentation.

Afterwards, the author used electroporation to transfer OVA-mRNA and GFP-mRNA into BMDC for intracellular expression. Then the treated DC was incubated with OT-I. The results showed that DC after electroporation can also stimulate the activation of OT-I, but this effect can no longer be inhibited by exotoxin A. Afterwards, the authors proved that Exotoxin A can inhibit the formation of MHC-I-OVA complex and the amount of OVA in the cytoplasm through the results of flow cytometry and WB. This result indicates that once the mature antigen material enters the cytoplasm, the inhibitory effect of exotoxin A is relieved.

Exotoxin A is known to inhibit the activity of many ERAD, including Sec61 and Derlin-1. First, the author knocked down the expression level of Derlin-1 by shRNA, and the results showed that this method could not inhibit the cross-presentation effect. After that, the author turned his attention to another protein, Sec61. Sec61 has two members, Sec61a1 and Sec61a2. Through RT-PCR analysis, the author found that the expression level of Sec61a1 in BMDC was higher than that of Sec61a2. In addition, after reducing the level of Sec61a1 in BMDC by electrotransformation of siRNA, the authors found that the cross-presentation effect of BMDC was significantly reduced. However, if OVA-mRNA is directly introduced into the cytoplasm at the same time, this inhibitory effect is relieved. In addition, direct stimulation of OT-I specific antigen peptides will not be negatively affected by Sec61a1 knockdown. This shows that Sec61a1 achieves the purpose of cross-presentation by regulating the release of antigen from endosomes.

Later, the author proved the fact that Sec61a1 exists on the surface of the endosomal membrane by means of electron microscopy.

To further prove the physiological function of Sec61a1. The author screened antibodies (scFv fragments) that specifically recognize Sec61a1 through phage surface display technology, and found a type of scFv that can specifically inhibit the recruitment of Sec61a1 to endosomes.

After that, the author introduced the specific scFv plasmid into BMDC through lentiviral transfection, and repeated the above experiment. Fluorescence imaging results show that the scFv can co-localize well with Sec61a1, and the scFv can significantly inhibit the content of OVA in the cytoplasm and the content of mature MHC-I antigen complex. The final activation experiment also showed that the scFv can inhibit the cross-presentation effect.

Finally, they used the DCs of TRIF-/- mice to find that TRIF protein is also involved in this cross-presentation process.

In summary, the author used a series of in vitro experiments to prove the important role of Sec61a1 in mediating DC cross-presentation.