The invariant chain induces compact forms of class II molecules localized in late endosomal compartments

Abstract

The invariant chain (Ii) binds to newly synthesized major histocompatibility complex (MHC) class II molecules and is targeted to an acidic compartment where it is degraded. To evaluate its role on the conformation and the subcellular distribution of murine MHC class II molecules we have established stable L cell transfectants expressing class II IAk heterodimers alone or in conjunction with p31 and p41 Ii chains. In these cells, class II molecules were present under three forms: alpha beta heterodimers bearing high mannose carbohydrate moieties, and fully glycosylated alpha beta heterodimers that are sensitive or resistant to sodium dodecyl sulfate dissociation at 20 degrees C. The latter class II molecules called compact heterodimers, were here highly induced in Ii-positive cells. Using in situ iodination of endosomal compartments, class II heterodimers were detected in late endosomal compartments essentially as compact forms in Ii-positive cells, and as non-compact forms in Ii-negative cells. Using confocal microscopy, IAk molecules were located in compartments distinct from early endosomes labeled with transferrin, but partially coincident with vesicles containing fluid-phase markers, and highly coincident with compartments containing large amounts of cathepsins B, D, H, and L in Ii-positive and Ii-negative cells. At the ultrastructural level, class II molecules were mostly present in multivesicular bodies, even without Ii expression. But Ii chains were needed to induce an efficient presentation of the hen egg lysozyme antigen and were sufficient to promote a major conformational change of the late endosomal, and/or lysosomal resident, class II molecules. Ii molecules are presumably playing a chaperoning function favoring the association of peptides with class II molecules in endosomal compartments.

Selective Modulation of the Major Histocompatibility Complex Class II Antigen Presentation Pathway following B Cell Receptor Ligation and Protein Kinase C Activation

“…Our results show that different mechanisms of PKC activation decreased the endosomal degradation of MHC class II associated Ii chains and induced an intracellular accumulation of p12 Ii protein fragments previously identified in MHC class II-Ii transfectants (20) and equivalent to the p10 Ii protein fragment SLIP (23,42). This is correlated with a selective modulation of antigen presentation, and a regulation of vesicular traffic as shown for the uptake of transferrin (43).…”

“…PMA treatment also triggers the accumulation of CLIP containing p12 Ii fragments previously identified in fibroblast transfectants (20) and analogous to p10 cytoplasmic derived fragments obtained in leupeptintreated B cells (23,42). The p12 Ii fragments contain in their cytoplasmic portion two dileucine based targeting motives able to direct Ii chain and Ii chimeric constructs to endosomal compartments (13,14).…”

“…The cytoplasmic Ii fragments identified in endosomal compartments after PKC activation are probably equivalent to cytoplasmic derived SLIP Ii protein fragment associated with intracellular class II molecules (23,42) and to the p12 Ii fragments identified in ␣␤-Ii-transfected fibroblasts (20). To test this hypothesis, we performed Western blotting with ␣Cyt.Ii, ␣Lum.Ii, and ␣CLIP antisera in total cell lysates from leupeptin (100 M)-and PMA (50 ng/ml)-treated cells (Fig.…”

The Effect of B Cell Receptor Signaling on Antigen Endocytosis and Processing

“…This approach allows us to monitor antigenic peptide -class II complex levels directly (by following C4H3 binding by flow cytometry), instead of relying on T cell stimulation (a readout which is vulnerable to changes in many factors, e.g., expression of costimulatory molecules, which are not be related to the level of specific antigenic peptide -class II complexes). The immunological relevance of this general approach is indicated by the observation that class II-restricted presentation of HEL internalized via both BCR-mediated or fluid phase endocytosis is absolutely dependent upon both newly synthesized MHC class II molecules as well as the class II chaperone HLA-DM (Bertolino et al, 1991;Humbert et al, 1993;St-Pierre and Watts 1990). Therefore, this model system this represents a good model system in which to evaluate the effect of BCR signaling on the cell biology of MHC class II-restricted antigen processing and presentation.…”

Expression of Major Histocompatibility Complex Class II Molecules in HeLa Cells Promotes the Recruitment of AP-1 Golgi-specific Assembly Proteins on Golgi Membranes

“…The cells were lysed, and MHC II were first immunoprecipitated with the L243 monoclonal antibody specific for ␣␤ dimers. The ␣␤ dimers associated with Ii or Ii⌬16 were subsequently immunoprecipitated with the DA6.147 monoclonal antibody recognizing both the precursor and mature forms of MHC II (18). These immunoprecipitates were dissociated with 10% SDS, and the biotinylated material was fractionated on streptavidinagarose beads as described previously (19).…”