Presentation of antigens internalized through the B cell receptor requires newly synthesized MHC class II molecules.

Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their alpha- or beta-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak beta-chain is required for the presentation of the hen egg lysozyme determinant (46-61) following BCR internalization, but that dependence is not seen for the (34-45) determinant derived from the same protein. The tail of the beta-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II beta-chains control the loading of determinants produced after extensive Ag processing.

Actin microfilaments control the MHC class II antigen presentation pathway in B cells.

Newly synthesised major histocompatibility complex class II molecules associate with invariant chains (Ii) to form nonameric complexes. These complexes are transported to endosomes, where proteolytic enzymes generate alphabeta class II dimers associated with nested Ii-derived peptides. These peptides are then exchanged with antigen peptide, and mature class II molecules reach the cell surface. The role of the actin cytoskeleton in the transport and maturation of class II molecules has not been studied. We show here that upon treatment with cytochalasin D (cyto D), the rate of Ii degradation is drastically reduced in B cells. Cyto D treatment also leads to a delayed appearance of stable forms of class II molecules, and a reduced presentation efficiency of antigen determinants requiring newly synthesised class II molecules. Under such conditions, we found that invariant chain fragments and class II molecules are accumulated in early and late endosomal compartments, whereas the leupeptin protease inhibitor induces their accumulation in lysosomal compartments. The addition of cyto D to leupeptin blocks the delivery of class II/invariant chain complexes to lysosomes, and further inhibits degradation of Ii. The dynamics of the actin cytoskeleton can therefore control the meeting point between newly synthesised class II molecules and lysosomal proteases, involved in Ii degradation and antigen peptide loading.

HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4.

For efficient entry into target cells, certain T cell-tropic HIV-1 isolates require both CD4 and the coreceptor CXCR4. However, the molecular interactions among CD4, CXCR4, and the HIV-1 envelope glycoproteins are only now being elucidated. Here we show that the binding of soluble gp120 from one macrophage-tropic and four T cell-tropic viruses to a CD4+, but not to a CD4-, T cell line, decreased the binding of an mAb specific for CXCR4 to its epitope, implying an interaction among gp120, CD4, and CXCR4. To confirm such an interaction, we conducted double- and triple-color confocal laser scanning microscopy on CD4+/CXCR4+ cells and determined the extent of CD4 and CXCR4 colocalization by a semiquantitative analysis. In the absence of gp120, a low level of constitutive colocalization between CD4 and CXCR4 was observed. Treatment with T cell-tropic-derived gp120 and, to a lesser extent, macrophage-tropic-derived gp120, increased the colocalization of CD4 with CXCR4, and triple staining indicated that gp120 was associated with the CD4-CXCR4 complexes. Cocapping of the gp120-CD4-CXCR4 complexes at 37 degrees C resulted in the cointernalization of a proportion of the gp120-CXCR4 complexes into intracellular vesicles. These data demonstrate that the binding of gp120 to CD4+ T cells induces the formation of a trimolecular complex consisting of gp120, CD4, and the HIV-1 coreceptor molecule CXCR4.

Selective modulation of the major histocompatibility complex class II antigen presentation pathway following B cell receptor ligation and protein kinase C activation.

We noticed that B cell receptor ligation or phorbol 12-myristate 13-acetate treatment induced intracellular vesicles containing major histocompatibility complex (MHC) class II and invariant chain (Ii), and increased the amount of transmembrane p12 Ii fragments coimmunoprecipitated with class II molecules. To determine the influence of protein kinase C activation on the MHC class II presentation pathway, we analyzed the subcellular distribution of Ii, the induction of SDS-stable forms of class II molecules, and their ability to present different antigens. Ii chains visualized with luminal and cytoplasmic directed antibodies appeared in early endosomal compartments accessible to transferrin in response to phorbol 12-myristate 13-acetate treatment, whereas transmembrane Ii degradation products equivalent to the p12 Ii fragments were colocalized with the B cell receptors internalized after cross-linking. Protein kinase C activation delayed in parallel the formation of SDS-stable forms of class II molecules and reduced the presentation of antigenic determinants requiring newly synthesized class II alphabeta-Ii complexes. These data indicate that B cell activation affects Ii processing and MHC class II peptide loading in endosomal compartments intersecting the biosynthetic pathway.

Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway.

We have generated mice lacking H2-M complexes, critical facilitators of peptide loading onto major histo-compatibility complex class II molecules. Ab molecules in these mice matured into stable complexes and were efficiently expressed at the cell surface. Most carried a single peptide derived from the class II-associated invariant chain; the diverse array of peptides normally displayed by class II molecules was absent. Cells from mutant mice presented both whole proteins and short peptides very poorly. Surprisingly, positive selection of CD4+ T cells was quite efficient, yielding a large and broad repertoire. Peripheral T cells reacted strongly to splenocytes from syngeneic wild-type mice, no doubt reflecting the unique peptide complement carried by class II molecules in mutant animals.