Characterization of MHC Class II Compartments by Immunoelectron Microscopy
At present the best way to determine the precise intracellular localization of proteins, in a potentially semiquantitative way, is the combination of ultrathin cryosectioning and immunogold labeling. This paper focuses on the intracellular localization of MHC class II molecules, which are involved in the T helper response to exogenous antigens. Newly synthesized MHC class II heterodimers associate with invariant chain molecules, which in turn direct the MHC class II complex to the endocytotic route. Proteolytic digestion of the invariant chain frees MHC class II molecules so that they can bind antigenic peptides. Immunoelectron microscopy has been an important tool to identify the endocytotic compartments that are enriched in MHC class II and that are the potential sites of antigenic peptide binding. The methods that can be used to characterize MHC class II compartments (MIICs) in various antigen-presenting cells (APCs) are described in detail. In all APCs studies so far, MIICs are situated late in the endocytotic pathway and display lysosomal characteristics. Still, immunoelectron microscopy allows us to define subsets of MIICs, which can be distinguished by their morphology, accessibility to endocytotic tracers, and expression of invariant chain and HLA-DM. Different types of MIICs can be found that display internal vesicles (multivesicular), internal membrane sheets (multilaminar), or both. The multivesicular type of MIIC contains detectable invariant chain and is the primary site of antigen entry. The multilaminar MIIC is situated later in the endocytotic route and has lost most of the invariant chain antigenicity. These data suggest a sequential maturation of MIICs, which correlates with the degradation of invariant chain and the subsequent binding of antigenic peptides.
Kleijmeer
MJ
Department of Cell Biology, Faculty of Medicine and Institute for Biomembranes, Utrecht University, Heidelberglaan 100, Utrecht, 3584 CX, The Netherlands
Raposo
G
Geuze
HJ