- Introduction
Viral and bacterial infections form a serious and constant threat to our well-being. Luckily for us, the human body has developed adequate countermeasures to fight these opportunistic and foreign invaders in the form of our immune system. Material from both intracellular and exogenous origin is continuously presented to the cells of our immune system, and in this way monitored for suspicious activity. The presentation of antigens is achieved through three different pathways (see sidebar), of which one is the so-called MHC class II pathway. Once a cell is recognized as being infected, it is eradicated by white blood cells to prevent further spreading of the infection.
- MHC class II pathway
The MHC class II pathway processes material taken up by the cell from its environment into small peptides (also called antigens), which are subsequently loaded on the MHC class II molecules from which this pathway derives its name. Once loaded, the class II molecules travel to the cell surface where they present the bound antigens to the CD4+ T-cells of our immune system (see figure). This complex cascade of events is supported by a number of specialized chaperone molecules, starting with the invariant chain in the endoplasmic reticulum. This chaperone binds the class II molecules directly after synthesis and temporarily occupies the empty peptide binding groove, while simultaneously targeting the formed complex to the cellular compartment where antigen loading has to take place, the MHC class II containing compartments (MIIC). In these lysosomal compartments, the invariant chain is degraded by proteases until only the part occupying the peptide-binding groove is left, which is called CLIP. A second dedicated chaperone, HLA-DM, helps to exchange CLIP for antigenic peptides, after which the loaded class II molecules are transported to the plasma membrane. HLA-DM plays a crucial role in this pathway, as without its presence the process of antigen presentation via MHC class II molecules is severely hampered. Therefore, we set out to develop an assay that would allow us to directly visualize the interaction between HLA-DM and class II molecules in living cells.
- Approach
We made stable cell lines expressing the class II molecule HLA-DR coupled to the cyan variant of GFP (CFP) and HLA-DM coupled to the yellow variant (YFP). Tagging these molecules with GFP did not prevent proper functioning and localization, and using confocal Fluorescence Resonance Energy Transfer (FRET) microscopy we could now directly determine if both molecules interacted and where this happened (see figure). As a control we created an additional cell line expressing HLA-DO/CFP and HLA-DM/YFP. HLA-DO is yet another dedicated chaperone expressed in B-cells, and known to continuously interact with HLA-DM. Using this FRET assay we studied the interaction between HLA-DR and HLA-DM under various conditions described to influence MHC class II antigen presentation.- Results
- Interaction between HLA-DR and HLA-DM were observed in MIICs. To test whether this interaction required the acidic pH present in MIIC, we added the compound chloroquine for two minutes to neutralize these compartments. Although pH sensitivity had been described in vitro, the interaction between HLA-DR and HLA-DM was not pH sensitive in living cells, indicating that additional factors support interaction in vivo. The MIIC have a special architecture and exist of a limiting membrane containing many smaller internal vesicles. To test wether this special structure is essential for proper interaction between HLA-DR and HLA-DM, we had to swell up the MIIC, as the native MIIC are too small to allow discrimination of internal vs limiting membranes using confocal microscopy. Long-term chloroquine is known to induce swelling of MIIC, and after 6h of treatment, electron microscopy revealed structures with a limiting membrane only. Surprisingly, HLA-DM/HLA-DR interaction was no longer observed on these swollen MIICs (see figure). As the swelling of MIIC occurs gradually, we repeated the experiment using only 3 hours of chloroquine treatment, and observed structures that were swollen but still had some remaining internal vesicles. FRET was observed only on the internal vesicles, but not on the limiting membrane (see figure). The internal structures of the MIIC thus form a special "MHC class II loading microdomain" that facilitates the interaction between HLA-DR and HLA-DM.
We confirmed these results using an alternative manner to swell the MIIC. By knocking down VPS34, a lipid kinase, we prevented the formation of internal structures. Again, no interaction between HLA-DR and HLA-DM was observed on the limiting membrane of these swollen MIICs. Interestingly, many bacteria survive intracellular in a so-called phagosome, which also contains a limiting membrane only. In addition, it is known that these bacteria secrete effector proteins that modulate lipid metabolism, for instance by targeting VPS34. We therefore infected our cells with Salmonella (see figure). Again, no interaction between HLA-DR and HLA-DM was observed on the membrane surrounding the bacterium, while interaction was not disturbed on normal MIIC within the same cell (see figure). Biochemical analysis further revealed that indeed no peptide loading takes place on these phagosomes. The absence of HLA-DR and HLA-DM interactions at the limiting membrane thus prevents local loading of MHC class II molecules in phagosomes, which may allow these bacteria to successfully evade the immune system (see figure).- Outlook
- The focus of our current research is to identify the factors that make up the MHC class II micro-loading domain on the internal vesicles of the multi-vesicular bodies.
- Reference
- W.Zwart and A. Griekspoor, et al. Immunity 2005 vol.22 pp.221-233. |
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Our immune system has developed three different pathways to counteract foreign entities that try to invade our body. The MHC class I pathway is functional in all nucleated cells, and continuously samples the intracellular protein content for signs of potential danger coming from within, like viral infections or tumorigenic signals. In contrast, the MHC class II pathway samples material that is taken up by the cell from the outside world, looking for pathogens like bacteria and parasites that live extracellularly. This pathway is only present in cells from the immune system. Finally, the CD1 pathway presents lipids from both endogenous and exogenous origin, and seems to have evolved specifically to counteract intracellularly living bacteria.
Most bacteria live outside the cell in our body where they are relatively easy detected by the immune system. A number of bacteria have therefore decided to look for a hide-out and have evolved to live intracelullarly. Pathogens like Salmonella typhimurium - the cause of Salmonella foodpoisoning -, Mycobacterium tuberculosis, and Mycobacterium leprae enter the cell, survive, and replicate in special organelles called phagosomes, out-of-sight for the cells of the immune system. To facilitate this process, these bacteria secrete special factors, called effector proteins, that modulate the normal cell machinery such that it helps the bacterium to enter and survive.
