B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DO␣ encompasses the binding site for HLA-DM. More specifically, mutation of residue DO␣41 on an exposed lateral loop of the ␣1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR͞DO molecules, we confirmed the role of the ␣ chain and established that a second DM-binding region is located C-terminal to the DO␣80 residue, most probably in the ␣2 domain. Interestingly, after mutation of a buried proline (␣11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DO␣ allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel ␤-strands of the DO␣␤ heterodimer.antigen processing ͉ MHC ͉ class II ͉ HLA-DR C lassical MHC class II proteins are highly polymorphic heterodimers expressed at the surface of antigen-presenting cells where they bind antigenic peptides derived from endocytosed antigens (1). The ␣ and ␤ subunits associate in the endoplasmic reticulum (ER) with the nonpolymorphic invariant chain (Ii), and the complex is sorted to endocytic compartments where Ii is degraded. A small fragment [class II-associated Ii peptide (CLIP)] of the Ii is protected inside the groove and must be released before the binding of antigenic peptides (2). HLA-DM (DM), a nonpolymorphic intracellular chaperone, is responsible for CLIP removal and also for editing the peptide repertoire to favor those of higher class II-binding affinity (3).Class II-restricted antigen presentation in B lymphocytes is tightly regulated to ensure specificity of the activation process. These cells express another nonclassical class II molecule called HLA-DO (DO; H2-O in mice) that modulates the presentation of antigens in the endocytic pathway (4). Recently, it was shown that transfection of DO in class II transactivator (CIITA) ϩ cells caused the accumulation of classical class II molecules associated with CLIP (5, 6). These results clearly showed the inhibitory role of DO on class II-restricted antigen presentation.The precise molecular mechanism by which DO inhibits the catalytic activity of DM remains to be clarified. DO is found mostly in endosomes but is retained in the ER of DM Ϫ cells. Interestingly, DO-bound DM is not sequestered there but rather allows the complex to egress from the ER (7). Trafficking of DO͞DM to and inside the endocytic pathway is regulated by sorting signals located in the cytoplasmic tails of both molecules (8, 9). The need for DO to access peptide-loading compartments and to mod...

SummaryHuman leucocyte antigen (HLA)-DO is a non-classical major histocompatibility complex class II molecule which modulates the function of HLA-DM and the loading of antigenic peptides on molecules such as HLA-DR. The bulk of HLA-DO associates with HLA-DM and this interaction is critical for HLA-DO egress from the endoplasmic reticulum. HLA-DM assists the early steps of HLA-DO maturation presumably through the stabilization of the interactions between the N-terminal regions of the a and b chains. To evaluate a possible role for HLA-DM in influencing the conformation of HLA-DO, we made use of a monoclonal antibody, Mags.DO5, that was raised against HLA-DO/DM complexes. Using transfected cells expressing mismatched heterodimers between HLA-DR and -DO chains, we found that the epitope for Mags.DO5 is located on the DOb chain and that Mags.DO5 reactivity was increased upon cotransfection with HLA-DM. Our results suggest that HLA-DM influences the folding of HLA-DO in the endoplasmic reticulum. A mutant HLA-DO showing reduced capacity for endoplasmic reticulum egress was better recognized by Mags.DO5 in the presence of HLA-DM. On the other hand, an HLA-DO mutant capable of endoplasmic reticulum egress on its own was efficiently recognized by Mags.DO5, irrespective of the presence of HLA-DM. Taken together, our results suggest that HLA-DM acts as a private chaperone, directly assisting the folding of HLA-DO to promote egress from the endoplasmic reticulum.