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In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-Ab and an antigenic peptide Ealpha52-68 covalently linked to its beta-chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, Ealpha52-68:I-Ab complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.

Here we demonstrate that keratinocyte growth factor (KGF) and FGFR2IIIb signaling can affect development and function of thymic epithelium (TE) and that αβ-lineage thymocytes contribute to intrathymic levels of KGF. Thymocyte expression of KGF is developmentally regulated, being undetectable in CD3−4−8− thymocytes and expressed at highest levels by mature CD4 or CD8 thymocytes. Exposure of thymocyte-depleted fetal thymic lobes to KGF resulted in reduced thymic epithelial expression of class II major histocompatibility complex (MHC), invariant chain (Ii), and cathepsin L (CatL) molecules involved in thymocyte-positive selection and also stimulated expression of the cytokines interleukin 6 (IL-6) and thymic stromal-derived lymphopoietin (TSLP), while having little effect on IL-7 or stem cell factor expression. Within intact fetal thymic organ culture (FTOC), exogenous KGF impairs the generation of CD4 thymocytes. Two lines of evidence point to responsiveness of the medullary TE compartment to KGF and FGFR2IIIb signaling. First, the medullary compartment is expanded in intact FTOC exposed to KGF in vitro. Second, in the RAG-deficient thymus, where the thymocytes do not express detectable levels of KGF message, the hypoplastic medullary TE compartment can be expanded by administration of recombinant KGF in vivo. This expansion is accompanied by restoration of the normal profile of medullary TE–associated chemokine expression in the RAG2−/−thymus. Collectively, these findings point to a role for KGF and FGFR signaling in the development and function of thymic epithelium.

Presentation of the Mtv-1 superantigen (vSag1) to specific V β -bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II αβ dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II–peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse–chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II– peptide complexes, as shown by using an antibody against an abundant endogenous class II–peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-Ab with a 17-amino acid peptide derived from the alpha chain of HLA-DR (DR alpha52-68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.