Assembly of matched alpha/beta subunits to HLA class II peptide receptors.

Human antigen presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are composed of polymorphic α and ß subunits. The combination of polymorphic α- and ß-chains results in cis (encoded on the same chromosome) or trans (encoded on different chromosomes) combinations. Since some of the α-ß combinations may yield mismatched non-functional α-ß heterodimers, it is not entirely clear which type of HLA class II peptide receptors are found on the cell surface of antigen presenting cells. We have developed a combination of biochemical techniques for inspection of the assembly and intracellular transport of isotype matched and mismatched class II heterodimers.

Studying MHC class II peptide loading and editing in vitro.

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of major histocompatibility complex (MHC) class II molecules upon binding of antigenic peptides and the effect of DM on the interactions. Using purified soluble molecules, in this chapter, we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on the interactions. A simple qualitative method, Gentle SDS-PAGE Assay, would assess the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide-MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore surface plasmon resonance (SPR). We also have described an Intrinsic Tryptophan Fluorescence Assay for studying transient interactions of DM and MHC class II molecules.

Expression, purification and assembly of soluble multimeric MHC class II-invariant chain complexes.

Major histocompatibility class (MHC) II molecules are essential for running adaptive immune response. They are produced in the ER and targeted to late endosomes with the help of invariant chain (Ii) trimers. Ii trimerization may be induced by the Ii TM domain. To enable mechanistic and structural studies of MHC class II-Ii assembly, soluble forms of the complexes were expressed. We show that Ii trimerizes in the absence of the transmembrane part, prior to binding of α/ß chains. The biochemical analysis supports the suggestion that the MHC class II-Ii complexes are not necessarily trimers of trimers, but that the Ii trimer can also be occupied by one or two MHC class II complexes.

Invariant chain controls H2-M proteolysis in mouse splenocytes and dendritic cells.

The association of invariant (Ii) chain with major histocompatibility complex (MHC) class II dimers is required for proper antigen presentation to T cells by antigen-presenting cells. Mice lacking Ii chain have severe abnormalities in class II transport, T cell selection, and B cell maturation. We demonstrate here that H2-M, which is required for efficient class II antigenic peptide loading, is unexpectedly downregulated in splenocytes and mature dendritic cells (DCs) from Ii(-/-) mice. Downregulation reflects an increased rate of degradation in Ii(-/-) cells. Degradation apparently occurs within lysosomes, as it is prevented by cysteine protease inhibitors such as E64, but not by the proteasome inhibitor lactacystin. Thus, Ii chain may act as a lysosomal protease inhibitor in B cells and DCs, with its deletion contributing indirectly to the loss of H2-M.

Immunohistochemical techniques improve the diagnosis of myocarditis in cases of suspected sudden infant death syndrome (SIDS).

Immunohistochemical techniques have improved the diagnosis of myocarditis. In a post mortem study, eight specimens in each case of the formalin-fixed and paraffin-embedded hearts of 20 suspected cases of sudden infant death syndrome (SIDS) were investigated with traditional hematoxylin-eosin staining and immunohistochemical methods. The hematoxylin-eosin stained specimens were examined for myocarditis according to the Dallas criteria; only in one case was a myocarditis diagnosed. The subsequent definition of the major histocompatibility complex class II antigens (HLA-DP,DQ,DR and HLA-DR), known to be enhanced in cases of myocarditis, the quantification of leucocytes with leucocyte common antigen (LCA) and characterization and quantification of T-lymphocytes using a specific marker (CD-3) allowed the definite diagnosis of myocarditis in three additional cases, six cases were found with moderate changes and ten cases without signs of inflammation.

HLA-DMB expression by thyrocytes: indication of the antigen-processing and possible presenting capability of thyroid cells.

Expression of HLA class II molecules on thyrocytes is a characteristic feature of autoimmune thyroid disease and may lead the thyroid cells to present autoantigens to CD4+ T lymphocytes. Since HLA-DM is a critical molecule in class II-restricted antigen processing and presentation, we assessed the expression of HLA-DMB, -invariant chain (Ii), class II transactivator (CIITA) and DRA in an untransformed, pure thyrocyte strain HTV-59A. Here we report that both HLA-DMB mRNA and the protein are expressed in thyrocytes and that CIITA expression is enhanced by interferon-gamma (IFN-gamma) treatment and occurs before DMB, Ii and DRA up-regulation, suggesting CIITA expression is a requirement for antigen processing in thyrocytes. These results indicate that thyrocytes are capable of antigen processing and possibly antigen presentation to T cells.

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain.

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.

The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules.

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.

Crystal structure of mouse H2-M.

H2-M (HLA-DM in humans) resides in an acidic endosomal compartment, where it facilitates the loading of antigenic peptides into the peptide-binding groove of class II MHC. The crystal structure of a soluble form of H2-M has been solved to 3.1 A resolution, revealing a heterodimer with structural similarities to the MHC family of proteins. In contrast to its antigen-presenting cousins, the membrane distal alpha helices of H2-M pack closely together, occluding most of the binding groove except for a single large pocket near the center. The structure of H2-M has several unique features that may play a role in its function as a molecular chaperone and peptide exchange factor.

Purification and characterization of endogenous peptides extracted from HLA-DR isolated from the spleen of a patient with rheumatoid arthritis.

We have purified HLA-DR from the spleen of a patient with rheumatoid arthritis. The patient had Felty's syndrome and was heterozygous for the DR4Dw4 antigen. We have isolated endogenous peptides from purified HLA-DR molecules. The peptides were purified by reverse phase HPLC and the major peaks were subjected to N-terminal sequencing. The peptides were derived from a variety of proteins: human serum albumin, human erythroid protein 4.1, 60S ribosomal proteins L31 and L35, VCAM-1, human immunoglobulin lambda chain and cathepsin-S. A peptide corresponding to the sequence of human serum albumin (HSA) residues 106-120 was synthesized and shown to bind to HLA-DR4Dw4 (IC50 = 1.41 microM). We have confirmed and refined current ideas about the structural motif for the binding of peptides to HLA-DR and HLA-DR4Dw4.

Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Purification of the complex of cathepsin L and the MHC class II-associated invariant chain fragment from human kidney.

The complex of cathepsin L and the fragment of the MHC class II-associated invariant chain was purified from human kidney. M(r) of the complex, as determined by gel filtration, is about 40,000. Both components were identified by amino acid and sequence analyses. The bound invariant chain fragment is almost identical to the additional segment found in p41, but not in the p31 form of the invariant chain. The complex has significantly enhanced stability at neutral and slightly alkaline pH, and reduced proteolytic activity against the synthetic substrate Z-Phe-Arg-MCA compared to free cathepsin L. The complex exhibits no enzymatic activity against the protein substrate azocasein. For the first time, the invariant chain was found in a complex with a protein, which was not an MHC molecule.

HLA-D antigens and Paget's disease of bone.

HLA-A, -B, -C, -DR, and -DQ antigens were determined in 25 Ashkenazi Jews with Paget's disease of bone. HLA-DR2 was more frequent in the Pagetic patients compared with 57 healthy controls of the same ethnic origin. This finding concurs with a previous report and raises the possibility that HLA-DR2 may be associated with Paget's disease of bone, probably by predisposing the bone cells to viral infection.

Different staphylococcal enterotoxins bind preferentially to distinct major histocompatibility complex class II isotypes.

The stimulation of T cells by staphylococcal enterotoxins (SE) is strictly dependent on major histocompatibility complex (MHC) class II-bearing cells. The interaction between SE and MHC class II molecules was studied on the human B cell lymphoma Raji and its MHC class II-negative variant RJ 2.2.5. Affinity purification with SEA and SEB matrix allowed the isolation of HLA-DR-like molecules from detergent lysates of 125I surface-labeled Raji cells, but not from RJ 2.2.5 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also revealed preferences in the binding of other SE such as SED, SEE and toxic shock syndrome toxin 1 to DR-like molecules, SEC2 to HLA-DQ-like molecules and SEC3 to DR- and DQ-like molecules. Preadsorption of the different MHC class II MHC isotypes confirmed the preferential binding of SEA to DR and of SEC2 to DQ. The implications of these findings for the understanding of SE-induced T cell activation and the potency of SE as a tool in the study of MHC class II antigens are discussed.

Association of class II DNA restriction fragments with responsiveness to Ambrosia artemisiifolia (short ragweed)-pollen allergen Amb a V in ragweed-allergic patients.

Human IgE and IgG antibody responsiveness to the short ragweed-pollen allergen Amb a V (formerly known as Ra5) has been found to be strongly associated with HLA-D specificities Dw2 and DR2 in ragweed-allergic white individuals. To study the molecular basis of these associations, restriction fragment length polymorphism (RFLP) mapping was performed on a group of 45 white ragweed-allergic patients with full-length HLA-DR beta, DQ beta, and DQ alpha cDNA probes. The data on 41 of these subjects were used for the purposes of statistical analysis. With the DR beta probe, we found that the presence of three polymorphic restriction fragments correlated with responsiveness to Amb a V and with the DR2 specificity, namely, a 6.5 kb Eco RI fragment, a 9.4 kb Hind III fragment, and a 2.2 kb Hind III fragment. The presence of four fragments detected with the DQ beta probe correlated with responsiveness to Amb a V and with Dw2 specificity: a 2.3 kb Eco RI fragment, a 13.0 kb Pst I fragment, a 2.9 kb Taq I fragment, and a 5.2 kb Eco RV fragment. The DR beta Eco RI 6.5 kb and the DQ beta Eco RI 2.3 kb fragments were studied in detail; the concordant presence of these fragments was even more strongly associated with responsiveness to Amb a V. Fifteen of 17 responders had both fragments, whereas only one of 24 nonresponders had both fragments (p = 5 x 10(-7). This is the first time that such an association has been found between a person's immune response to a well-defined antigen and a set of HLA class II DNA restriction fragments.

Analysis of DR and DQ gene products of the DR4 haplotype in patients with IDDM: possible involvement of more than one locus.

Fifteen DR4-bearing haplotypes from twelve patients with insulin-dependent diabetes mellitus (IDDM) were analyzed serologically, cellularly, and biochemically. The HLA-Dw composition of these DR4-positive haplotypes was Dw4 (46%), Dw14 (22%), and Dw10 (33%). The biochemical analysis by two-dimensional electrophoresis (2D-PAGE) of the DR beta chains showed that each Dw specificity is characterized by a specific DR4 beta chain that appears to be identical in normal and diabetic individuals. Analysis of DQ beta chains in the DR4-bearing haplotypes revealed that certain Dw specificities such as Dw4 are characterized by the presence of either the DQw7 (formerly DQw3.1) or DQw8 (formerly DQw3.2) alleles, which generate the Dw4.1 or Dw4.2 subtypes, respectively. Others such as Dw14 and Dw10 are characterized by the presence of the DQw8 allele. In our sample of 12 patients the Dw4.2 (Dw4, DR4 beta I-4 DQw8) and Dw10 (Dw10, DR4 beta I-1, DQw8) subtypes were predominant. It is concluded that individual DR beta and DQ beta gene products from the DR4-bearing haplotype of IDDM patients are identical to those of normal control subjects and that Dw14 as well as Dw10 are involved in disease susceptibility. We suggest that disease susceptibility may be influenced by more than one locus within the HLA-D region.

A human lymphoblastoid B line with an abnormally large MHC class II beta chain.

We have examined the MHC class II beta chains in lymphoblastoid cell lines from over 200 individuals and describe one line which possesses, in addition to normal beta chains, a species of beta chain of unusually high Mr and abnormal pI which appears to be a product of the DR locus. This abnormality in Mr, detected by SDS-gel electrophoresis, was apparent only in the presence of mercaptoethanol and was shown to be due to difference in polypeptide chain length rather than to extra glycosylation.