Distinct antigen MHC class II complexes generated by separate processing pathways.

Abstract

The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS‐unstable complexes with the purified MHC class II molecule I‐Ak (Ak). These complexes were also detected on the surface of antigen‐presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS‐stable, long‐lived Ak complexes containing *HEL peptides and, unexpectedly, full‐length *HEL. Both SDS‐stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA‐DM or proteases. SDS‐stable *HEL‐Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS‐unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full‐length protein as a product of antigen processing.

The Pathway of Antigen Uptake and Processing Dictates MHC Class II-Mediated B Cell Survival and Activation

“…Moreover, HEL processing involves an acidic compartment related to late endosome or the MIIC (18). Because the 46 -61 epitope is buried in HEL molecule, the requirement for its processing might be less sensitive to Ab (BCR)-mediated regulation.…”

“…BCRtargeted Ags access the endocytic compartments very efficiently, and this property contributes to their enhanced processing and presentation compared with uptake of fluid phase-added Ag. The processing of HEL involves the generation of HEL 46 -61 -I-A k complexes in late endosomal compartments (17,18). Both 3A9 T cells and the C4H3 mAb recognize the peptide-MHC complex (16,31).…”

“…T cells from 3A9 mice recognize the same epitope as C4H3, i.e., HEL 46 -61 -I-A k (11,13). This epitope is buried within HEL, and therefore must be processed in late endosomal compartments (17,18). This is expected to occur in compartments that are accessed by both fluid phase and BCR uptake of HEL.…”

MHC Class II Antigen Presentation by Dendritic Cells Regulated through Endosomal Sorting

“…Upon arrival at the cell surface, pMHCII complexes can be endocytosed only to recycle again to the plasma membrane via early endocytic compartments Watts 1990, 1992). The latter pathway is extremely rapid (Reid and Watts 1990) and potentially may provide opportunities for MHCII to also sample antigens that differ from those found in the late endosomal compartments, thereby contributing to the diversity in antigen presentation (Pinet et al 1995;Lindner and Unanue 1996;Griffin et al 1997;Pathak and Blum 2000). MHCII peptide loading at these alternate locations may differ for the requirements of DM or Ii (Vergelli et al 1997;Villadangos et al 2000) and is more prominent for some MHCII isotypes (van Lith et al 2010).…”

Cholera toxin increases intracellular pH in B lymphoma cells and decreases their antigen-presenting ability

“…To inhibit internalization, 10 mM NaN 3 4 mM NaF and 10 mM 2-deoxyglucose were included in the incubation. The mixture of these reagents has been shown to almost completely inhibit endocytosis [43]. Following incubation, the cells were washed with ice-cold MEM (Sigma Chemical Co.).…”