Elena A. Armandola

A ROLE FOR HLA-DO AS A CO-CHAPERONE OF HLA-DM IN PEPTIDE LOADING OF MHC CLASS II MOLECULES

In B cells, the non‐classical human leukocyte antigens HLA‐DO (DO) and HLA‐DM (DM) are residents of lysosome‐like organelles where they form tight complexes. DM catalyzes the removal of invariant chain‐derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR–DM–DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM–DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high‐stability peptides. Thus, DO appears to act as a co‐chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.

SUBSTRATE SELECTION BY TRANSPORTERS ASSOCIATED WITH ANTIGEN PROCESSING OCCURS DURING PEPTIDE BINDING TO TAP

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules depends on translocation of cytosolic peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). Peptide transport by TAP is thought to include at least two steps: initial binding of peptide to TAP, and its subsequent translocation requiring ATP hydrolysis. These events can be monitored in peptide binding and transport assays. Previous studies have shown that the efficiency of peptide transport by human, mouse and rat transporters varies according to the C-terminals of peptide substrates in an allele and species-specific manner. However, it has not been clear during which step of peptide interaction with TAP selection occurs. We used an assay monitoring the peptide binding step to study the binding affinity of a library of 199 peptides for human TAP and the two major allelic rat TAP complexes. We observed a dominant influence of the C-terminus on peptide binding affinity for all transporters, and highly restrictive selection of peptides with aliphatic and aromatic C-terminals by rat TAP1/TAP2u complexes. The selectivity of peptide binding to rat TAP complexes is in full accordance with published data on selective peptide transport and on control of antigen presentation by rat TAP. These results strongly suggest that (i) peptide selection by TAP occurs exclusively in the initial binding step; (ii) all factors involved in peptide selection by TAP are present in insect cells.

Downmodulation of antigen presentation by H2-O in B cell lines and primary B lymphocytes

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory moleculeH2‐M, which helps to exchange the invariant chain‐derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2‐O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2‐M and selectively expressed in B cells. Using stable H2‐O ribozyme‐antisense transfectants, H2‐O overexpressing murine B cell lines, and H2‐O‐transgenic mice, we investigated the effects of H2‐O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levelsof H2‐O in the antigen‐presenting B cells. Thus, increased H2‐O expression downmodulated, whereas reduced H2‐O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2‐O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2‐O levels. Theintracellular location of H2‐O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2‐O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2‐O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.