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X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (alpha beta)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple alpha beta heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the alpha beta and (alpha beta)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (alpha beta)2 complex displays thermal and pH stability similar to that of the alpha beta complex, both being denatured by SDS at temperatures above 50 degrees C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (alpha beta)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.

We have previously identified an intracellular compartment involved in the association between processed lysozyme and IAk major histocompatibility complex class II molecules (called the lysozyme-loading compartment (LLC)). Here, we show that the LLC polypeptide composition analyzed by two-dimensional gel electrophoresis shares similarities with that of early endosomes, but not with that of late endosomes. The transferrin receptor, a well known marker for both early and recycling endosomes, colocalizes with IAk molecules in LLC. Moreover, both transferrin and fluid-phase markers have access to LLC after 15 min of internalization. In the presence of concanamycin B, SDS-stable dimer formation and transport of class II molecules out of LLC are impaired. In contrast, nocodazole treatment has no effect. These results suggest that LLC is a specialized compartment of the recycling pathway involved in lysozyme loading and in the targeting of lysozyme-major histocompatibility class II complexes toward the cell surface.

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II-invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II-associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild-type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.

In this report we report the identification of novel molecular associations involving the MHC class II (Ia) Ag expressed on the surface of Ag-presenting B lymphocytes. Biosynthetically radiolabeled murine B cells were incubated for 2 h in the presence or absence of T lymphocytes before treatment with the cleavable cross-linking reagent dimethyl-3,3'-dithiobispropionimidate. Anti-Ia immunoprecipitates of solubilized cell extracts revealed novel cross-linked products of Mr 90,000 to 95,000 which, upon cleavage of the cross-linker, could in part be resolved into native Ia and other structures of approximately Mr 67,000. The detection of the cross-linked products was significantly enhanced in B cells that had been co-cultured with T lymphocytes, but not with other cell types, and co-culture with various monoclonal T cell lines resulted in different levels of enhancement. Detection of the 90- to 95-kDa cross-linked products appeared to be independent of the foreign Ag for which the T cells were specific and could be enhanced when either cell type was replaced by a plasma membrane fraction, indicating that it resulted from direct cell-cell contact. These results suggest that some proportion of the Ia glycoproteins expressed on the surface of B cells become associated with other structures of Mr 67,000 upon Ag-non-specific interactions between T and B lymphocytes.

Four human class II molecules, one FA, one DC1, and two DR-like molecules, were isolated from DR2 and DRw6 homozygous cell lines by means of a variety of monoclonal antibodies and were compared with each other by two-dimensional (2-D) gel electrophoresis. Anti-DR2 or anti-DR3 + 5 + w6 sera immunoprecipitated two distinct light chains (L1 and L2) and one heavy chain (H1) from a DR2 or DRw6 homozygous cell line, respectively. One or both of these two class II molecules were also immunoprecipitated by "DR-specific" monoclonal antibodies and were considered to constitute a "DR" family of molecules. Three DC1-specific monoclonal antibodies, SDR4.1, Tu22, and PLM5, immunoprecipitated a set of heavy (H2) and light (L3) chains distinct from those of two DR-like molecules. The heavy chains of the DC1 antigens from DR2 and DRw6 cell lines were indistinguishable, whereas the light chains were structurally distinct from each other. A fourth molecule, FA, was identified by a novel monoclonal antibody and was also detected by two additional antibodies, Tu39 and SG171, that blocked the SB-specific T-cell proliferative response. The FA light chain (L4) was distinct from those of the former three antigens on both cell lines, whereas the FA heavy chain was indistinguishable from the DC1 heavy chain (H2) in this 2-D gel analysis. Thus, four light chains and two heavy chains were isolated from both DR2 and DRw6 homozygous cell lines. A possible gene-antigen organization of the DC-like antigens was also discussed.

The complex of alpha and beta chains of HLA-D membrane antigens has been isolated from a lymphoblastoid homozygous B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1), by an exclusively chemical two-step procedure and characterized by electrophoresis as well as isoelectric focusing in polyacrylamide gel. Cells were gained using long term cultivation in large scale, the crude membrane by differential centrifugation. The proteins of the crude membrane were then solubilized in NP-40, pH 5.0. The first purification step for HLA-D antigens consisted in an ion-exchange chromatography on carboxymethyl cellulose using the solubilization buffer. By this procedure the complex of proteins with relative molecular masses of Mr approximately 34 000 and Mr approximately 29 000 was in a high percentage not bound to the carboxymethyl cellulose. The bound fraction contained the HLA-A, -B and -C antigens and a component with Mr approximately 31 000 corresponding to the well known Ii-fraction. The bound proteins could be recovered from the column by a sodium chloride gradient. The proteins not bound to the carboxymethyl cellulose were precipitated with acetone, dissolved, dialysed against SDS buffer, pH 7.2 and then submitted to the second purification step, the Sephacryl S-300 chromatography. By this procedure the corresponding complex could be further separated from higher and lower molecular proteins. The complex was used as the starting material for the separation of alpha and beta chains. Amino-acid sequences established of the isolated chains have already been communicated.

Three class II molecules were isolated from a homozygous DRw6 human B lymphoblastoid cell line using the monoclonal antibodies L243 (L203), L227, LKT 111, and Genox 3.53. Two of the antigens appeared to employ the same heavy chain but expressed different light chains. The two light chains were separated after denaturation using L227 and LKT 111. One or both of these two molecules carried the DRw6 and MT2 determinants. The third class II antigen expressed the DC1 determinant. It was composed of a heavy and light chain different from the DR-like antigen subunits. The antibodies L243, L227, and LKT 111 did not preclear the cell lysate of the DC1 antigen recognized by Genox 3.53. However, a xenoanti-DR serum immunoprecipitated both the DR-like and the DC1 antigens. Thus, in total, one cell line can express at least two class II heavy chains and three class II light chains. This observation was not unique to this cell line.

In a previous communication we described the preparation of the alpha/beta-chain complex of HLA-D membrane antigens from a homozygous lymphoblastoid B cell line, H2LCL (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1). In this paper we present the separation of the alpha- and beta-chains in quantitative scale using hydroxylapatite chromatography with a pH 7.15 phosphate buffer, containing sodium dodecyl sulfate. An alternative isolation procedure is electrophoresis, taking advantage of the different isoelectric points of the alpha- and beta-chains. The relative molecular masses, the isoelectric points and the N-terminal sequences are discussed and compared with the results of other investigators. Remarkable is the polymorphism of both the chains, especially the beta-chains. The importance of the previously described three step purification procedure for the preparation of these antigens for sequence studies has been pointed out.

Radioactive Ia.4 molecules were prepared from 3H- or 14C-labeled splenocytes, selected PEL, or bronchoalveolar macrophages (M phi). Studies in the accompanying paper indicated that incorporation into Ia.4 in these 3 populations is due to B cells, T cells, and macrophages, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to isolate the 58,000 m.w. form of Ia.4. The eluted molecule was then reduced, and the resultant alpha and beta subunits were isolated by a second separation on SDS-PAGE. Alpha (or beta) chains from 1 population labeled with 3H-amino acids was mixed with alpha (or beta) chains obtained from a population representing a different cell type that was labeled with 14C-amino acids and the mixture was digested with trypsin. Double-label (3H/14C) comparative peptide mapping was performed using high-pressure liquid chromatography to separate the peptides. Eighteen to 20 peaks of radioactivity were resolved from alpha chains, and 12 to 15 from beta chains. No reproducible differences were observed when comparing alpha or beta chains of T cells and macrophages, or those of B cells and macrophages. These results indicate that the primary structure of Ia.4 molecules is identical on the 3 cell types in question. The implications of having a T cell bearing the same Ia that it recognized on a macrophage in conjunction with antigen is discussed.