Cell surface HLA-DR-invariant chain complexes are targeted to endosomes by rapid internalization.

Class II molecules of the major histocompatibility complex (MHC) bind peptides derived from protein antigens delivered into endocytic compartments and present these peptides to CD4+ T cells. The precursors to functional MHC class II molecules loaded with peptides are complexes of the invariant chain associated with class II alpha beta heterodimers. Targeting of newly synthesized MHC class II molecules to endosomes is mediated by the invariant chain, but the intracellular transport route is not known. This study demonstrates that in a human B-cell line a large population of MHC class II-invariant chain complexes reaches endosomes by rapid internalization from the cell surface. Quantitation of cell surface MHC class II-invariant chain complexes and of their surface half-life revealed that 3000 complexes internalized per minute into endosomes. This highly efficient endocytosis was mediated by the cytoplasmic tail of the invariant chain. After internalization, the invariant chain dissociated from the MHC class II-invariant chain complexes. This pathway may represent an important mechanism for loading class II molecules with immunogenic peptides from several endocytic compartments.

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.

The alpha 1 domain of the HLA-DR molecule is essential for high-affinity binding of the toxic shock syndrome toxin-1.

Several exoproteins from the bacterium Staphylococcus aureus are highly potent polyclonal activators of T cells in the presence of cells bearing class II antigens of the major histocompatibility complex (MHC). These toxins, including the toxic shock syndrome toxin (TSST-1), act at nanomolar concentrations, bind directly to class II molecules, and do not require the processing typical of nominal antigen. Each toxin is capable of stimulating a subpopulation of peripheral T lymphocytes bearing particular V beta sequences as part of their alpha beta T-cell receptors. It is not known how these so-called 'superantigens' bind to class II and how this binding stimulates T cells. In this study, the different affinities of TSST-1 for human class II molecules DR and DP were exploited to define the region of a class II molecule necessary for high-affinity binding. Using chimaeric alpha- and beta-chains of DR and DP expressed at the surface of transfected murine fibroblasts and a binding assay with TSST-1, it was shown that the alpha 1 domain of DR is essential for high-affinity binding, and further that TSST-1 binding did not prevent subsequent binding of a DR-restricted antigenic peptide. This is compatible with a model of superantigen making external contacts with both class II and T cell receptor, and suggests that the V beta portion of the T-cell receptor interacts with the nonpolymorphic alpha-chain of DR.

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.