Cytoplasmic processing is a prerequisite for presentation of an endogenousantigen by major histocompatibility complex class II proteins

Biochemical and functional studies have demonstrated major histocompatibili ty complex (MHC) class II-restricted presentation of select epitopes derive d from cytoplasmic antigens, with few insights into the processing reaction s necessary for this alternate pathway. Efficient presentation of an immuno dominant epitope derived from glutamate decarboxylase (GAD) was observed re gardless of whether this antigen was delivered exogenously or via a cytopla smic route into human histocompatibility leukocyte antigen class II-DR4(+) antigen-presenting cells. presentation of exogenous as well as cytoplasmic GAD required the intersection of GAD peptides and newly synthesized class I I proteins. By contrast, proteolytic processing of this antigen was highly dependent upon the route of antigen delivery. Exogenous GAD followed the cl assical pathway for antigen processing, with an absolute requirement for en dosomal/lysosomal acidification as well as cysteine and aspartyl proteases resident within these organelles. Presentation of endogenous GAD was depend ent upon the action of cytoplasmic proteases, including the proteasome and calpain. Thus, translocation of processed antigen from the cytoplasm into m embrane organelles is necessary for class II-restricted presentation via th is alternate pathway. Further trimming of these peptides after translocatio n was mediated by acidic proteases within endosomes/lysosomes, possibly aft er or before class II antigen binding. These studies suggest that processin g of exogenous and cytoplasmic proteins occurs through divergent but overla pping pathways. Furthermore, two cytoplasmic proteases, the proteasome and calpain, appear to play important roles in MHC class II-restricted antigen presentation.