Rabbit major histocompatibility complex. IV. Expression of major histocompatibility complex class II genes

The rabbit MHC class II DP, DQ, and DR alpha and beta chain genes were transfected into murine B lymphoma cells. The transfected cells expressed R-DQ and R-DR molecules on the cell surface but they did not express the R-DP genes either on the cell surface or at the level of mRNA. Northern blot analyses showed that the R-DP genes were expressed, albeit at low levels, in rabbit spleen. Similar analyses showed that the R-DQ and R-DR genes were expressed at high levels in rabbit spleen. A new monoclonal anti-rabbit class II antibody, RDR34, has been developed and shown to react with the R-DR transfected cells and not with the R-DQ transfected cells. The previously described monoclonal anti-rabbit class II antibody, 2C4, reacted with the R-DQ transfected cells and not with the R-DR transfected cells. Thus, 2C4 and RDR34 MAb's are specific for the R-DQ and R-DR molecules, respectively. Each of the antibodies reacted with approximately 50% of rabbit spleen cells as shown by immunofluorescent antibody studies.     

Monoclonal antibodies to human major histocompatibility complex class II antigens

The production and characterization of monoclonal antibodies to major histocompatibility complex (MHC) class II molecules have contributed significantly to delineating the number and structure of gene products of the D region of HLA. The "first generation" of class II monoclonal antibodies detected monomorphic determinants, often reacting with multiple class II loci gene products. Later generations of anti-class II monoclonal detected serologic specificities like those previously detected by human alloantisera. In addition, numerous antibodies have been generated against polymorphic HLA class II determinants which have not been previously described using human allosera. Biochemical analysis utilizing techniques such as radiolabeling and immunoprecipitation, one- and two-dimensional gel electrophoresis, Western blotting, limited N-terminal amino acid sequencing, and peptide mapping have localized the epitopes detected by polymorphic anti-class II monoclonals to various class II locus gene products. Such analyses with monoclonal antibodies have also identified class II gene products previously identified only with cellular reagents. The fact that several monoclonal antibodies with similar serologic specificity detect different class II locus gene products underscores the complexity of the HLA-D region and suggests some possible mechanisms for the generation of polymorphisms in this region.     

Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line.

Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post-implantation-stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well-defined class I product, we introduced the H-2Dd gene under control of the human beta-actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H-2Dd heavy chains and beta 2-microglobulin protein, but only small amounts of H-2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I-negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H-2Dd-transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly-defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post-implantation-stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post-translationally at the level of MHC class I assembly.     

Expression of major histocompatibility complex class II antigens on normal porcine intestinal endothelium

A novel monoclonal antibody (MIL 11) specific for an antigen expressed on porcine endothelial cells is described. The antigen recognized by MIL 11 is most strongly expressed in the intestine but is also expressed on the capillary endothelium of a wide range of tissues. Using two- and three-colour immunofluorescence microscopy we demonstrated the extensive coexpression of MIL 11 and major histocompatibility complex (MHC) class II antigens on normal porcine capillary endothelium in the intestine, trachea, thymus and small veins, while endothelium of large vessels and the heart were negative for MHC class II. In contrast to humans and rodents, available reagents do not detect MHC class II on the intestinal epithlium of pigs. However, porcine intestinal endothelium expressed both DR and DQ antigens. A population of strongly class II-positive cells was also detected immediately adjacent to the endothelium in the lamina propria. Three-colour immunofluorescence microscopy highlighted the close association between endothelium and intestinal CD4⁺ T cells. Lamina propria T cells were mainly MHC class II positive, whereas those in the epithelial compartment were MHC class II negative.     

Expressed major histocompatibility complex class II loci in fishes

Summary: Peptides derived from parasites are presented to T helper cells by major histocompatibility complex (MHC) class II αβ heterotrimeric cell-surface molecules. In mice and humans, the genes encoding these antigen-presenting molecules are known to be polymorphic and poly-genic. Multiple loci for MHC class II A and E genes are proposed to allow for an increased peptide-binding repertoire. The multigenic nature of expressed MHC class II loci and the differences between these loci in fishes are the focus of this review, Particular emphasis is placed on an evolutionary comparison of class II B loci, especially two class 11 B loci that have undergone dramatic changes from one another suggesting an ancestral gene duplication event that took place at an early stage in the evolution of teleosts, The number of functional class II αβ loci heterotrimers may have a profound impact on the organisms ability to battle constantly evolving parasitic infections.     

Differential major histocompatibility complex class II locus expression on human laryngeal epithelium

The survival of a laryngeal allograft will be dependent on the immunological composition of the donor larynx and, in particular, on the expression of major histocompatibility complex (MHC) class II antigens on professional and non-professional antigen-presenting cells. Laryngeal and tonsillar biopsies from normal individuals aged 18-78 years were processed and prepared for quantitative, multiple-colour immunofluorescence using mouse antihuman monoclonal antibodies to human leucocyte antigen (HLA)-DR, HLA-DQ and CD45. The laryngeal epithelium expressed HLA-DR locus products at variable levels, but expression of HLA-DQ was virtually absent. Tonsillar epithelial cells expressed HLA-DR at the basal layer only, while HLA-DQ was similarly not expressed. In contrast, both HLA-DR and -DQ locus products were present on lamina propria and intraepithelial leucocytes in both laryngeal and tonsillar mucosae, although at varying levels. The finding that laryngeal epithelial cells express MHC class II antigens has implications for the survival of laryngeal allografts and suggests that they may require significant immunomodulation. In addition, antigen presentation by epithelial cells has been hypothesized to contribute to the immunoregulatory function of mucosal tissues, and the finding that HLA-DQ locus products are only expressed at low levels by laryngeal epithelium raises questions about the repertoire of peptides to which the mucosal immune system can respond.     

Interferon-induced expression of class II major histocompatibility antigens in the major histocompatibility complex (MHC) class II deficiency syndrome

Class II antigens encoded by genes of the major histocompatibility complex (MHC) are expressed by a variety of cell types and have a vital role in the cellular interactions required for an effective immune response. We have analyzed the regulation of HLA-DR, DP, and DQ class II antigen expression on cells of different lineage from an immunodeficient patient with the MHC class II deficiency syndrome. T and B lymphocytes, monocytes, and fibroblasts, which initially expressed no class II antigens, were treated with inductive stimuli that normally lead to enhanced expression of class II antigens. Monocytes, but not fibroblasts, cultured for 48–96 hr in the presence of recombinant gamma interferon expressed all three types of class II antigens. In contrast, T lymphocytes did not express class II antigens following their exposure to a variety of stimuli, including activation with phytohemagglutinin and culture in the presence of interleukin-2, transformation by the retrovirus HTLV-1 or HTLV-2, or exposure to the demethylating agent 5-azacytidine. Similarly, class II antigens were not induced on B cells by cross-linkage of surface immunoglobulin molecules with anti-mu, exposure to Epstein-Barr virus, or treatment with soluble factors secreted by activated T cells. These results demonstrate that the regulation of class II MHC antigen expression by monocytes and lymphocytes is dissimilar and suggest that different regulatory genes are involved in the control of class II antigen expression by cells of different lineage.     

Conformational isomers of a peptide–class II major histocompatibility complex

Summary: The relative plasticity of peptide binding to class II major histocompatibility complex (MHC) molecules permits formation of multiple conformational isomers by the same peptide and MHC molecule; such conformers are specifically recognized by distinct subsets of T cells. Here, we review current knowledge and recent advances in our understanding of peptide–class II MHC conformational isomerism and the mechanisms that generate distinct MHC–peptide conformers. We focus on our studies of two T‐cell subsets, type A and B, which recognize distinct conformers of the dominant epitope of hen egg white lysozyme presented by I‐A^k. These conformers form via different pathways and in distinct intracellular vesicles: the type A conformer forms in late endosomes upon processing of native protein, while the more flexible type B conformer forms in early endosomes and at the cell surface. In this process, H2‐DM acts as a conformational editor, eliminating the type B conformer in late endosomes. Type B T cells constitute a significant component of the naïve T‐cell repertoire; furthermore, self‐reactive type B T cells escape negative selection and are present in abundance in the periphery. Ongoing studies should elucidate the role of type B T cells in immunity to pathogens and in autoimmune pathology.     

T cell development in a major histocompatibility complex class II-deficient patient

In this report we show that the major histocompatibility complex (MHC) class II-negative thymus of a bare lymphocyte syndrome (BLS) patient contains a reduced CD4⁺ CD8^? T cell population when compared to thymocytes derived from a MHC class II-expressing thymus. Of these CD4⁺ CD8^? BLS thymocytes, approximately only one third co-expressed the CD3 antigen, moreover at a lower expression level when compared to control thymocytes. This suggests a partial maturation of the CD4⁺ CD8^? T cells in the absence of MHC class II expression. Among the BLS thymocytes, CD4⁺ CD8⁺ thymocytes could easily be detected. Noteworthy, the number of CD4^? CD8⁺ thymocytes was significantly increased. CD4⁺ CD8^? T cells could also be found among the BLS peripheral blood mononuclear cells, albeit at reduced numbers. Despite the absence of peripheral MHC class II expression, the majority of these CD4⁺ CD8^? T cells co-expressed the CD45RO marker. In the BLS patient, thymocytes as well as peripheral CD4⁺ CD8^? T cells were not restricted in the use of the available T cell receptor (TcR) V gene family pool. However, the lack of detectable levels of thymic and peripheral MHC class II antigen expression in the BLS patient had altered the CD4^?skewing patterns of TcR V gene families which were present in normal individuals. In conclusion, the lack of MHC class II expression in the BLS patient does not completely inhibit the CD4+ CD8^? T cell development.     

Constitutive endocytosis and recycling of major histocompatibility complex class II glycoproteins in human B-lymphoblastoid cells.

Cleavable cell surface radiolabelling reagents were used to measure the endocytosis and recycling of class II major histocompatibility complex (MHC) glycoproteins in human B-lymphoblastoid cells. It is shown that mature class II alpha beta dimers on the cell surface are constitutively endocytosed and that recycling can be demonstrated even from small endosomal pools. Endocytosis was blocked when cellular ATP levels were depleted or when clathrin polymerization was inhibited, implicating clathrin-coated pits in the endocytic process. Taken together with earlier studies, these results suggest that mature class II MHC molecules are constitutively endocytosed and recycled from acidic peripheral endosomes which may enhance their capacity to bind and present T cell epitopes which do not require processing.     

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments

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-A^b with a 17-amino acid peptide derived from the α chain of HLA-DR (DRα52–68). 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.     

Isoelectric focusing of bovine major histocompatibility complex class II molecules

Serological approaches have been relatively unsuccessful in defining the allelic products of the bovine major histocompatibility (MHC) class II loci. We demonstrate that bovine class II allelic products can be characterized by precipitation with a polyclonal antiserum and separation using one-dimensional isoelectric focusing. Polymorphic beta chains were present in immunoprecipitates from both biosynthetically and surface-labeled lectin-stimulated bovine T cells. Precipitates from biosynthetically labeled but not surface-labeled T cells contained a basic invariant chain and a non-polymorphic structure. The non-polymorphic structure appears to be a beta chain. The polymorphic class II beta chain co-segregated with bovine MHC class I allelic products in a half-sibling family, providing evidence for linkage between bovine class I and class II loci. This approach to the biochemical analysis of the bovine class II structures should facilitate the investigation of the association between the bovine products and disease susceptibility.     

Conserved T-cell receptor class II major histocompatibility complex contact detected in a T-lymphocyte population.

T-cell receptor (TCR) interacts with an antigenic peptide deeply buried in the major histocompatibility complex (MHC) molecule. How class II MHC is contacted by TCR during antigen recognition remains largely elusive. Here we used a panel of I-Ek mutants to identify two I-Ek residues that were frequently contacted by TCR among a large pool of T cells specific for the same antigen. The restricted TCR interaction with I-Ek was independent of the antigen peptides. We also identified a dominant heteroclitic residue on I-Ek, beta81H, in which mutation led to increased recognition of antigens in individual T-cell clones. Moreover, both the conserved TCR-I-Ek interaction and the heteroclitic TCR-I-Ek recognition were detected in T lymphocytes freshly isolated from mice primed with the specific antigens. The identical TCR-I-Ek interaction in a heterogeneous T-cell population suggested the dominance of invariant TCR-class II MHC interaction.     

Marek's disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells

Many herpesviruses modulate major histocompatibility complex (MHC) expression on the cell surface as an immune evasion mechanism. We report here that Marek's disease virus (MDV), a lymphotrophic avian alphaherpesvirus, up-regulates MHC class II cell surface expression in infected cells, contrary to all other herpesviruses examined to date. This MDV-induced class II up-regulation was detected both in vitro and in vivo. This effect was not solely an indirect effect of interferon, which is a highly potent natural inducer of MHC class II expression, since MHC class II up-regulation in cultured primary fibroblast cells was confined to the infected cells only. MHC class II up-regulation was also observed in infected cells of the bursa of Fabricius during the lytic phase of MDV infection in birds and upon reactivation of MDV from latency in an MDV-transformed cell line. As MDV is a strictly cell-associated virus and requires activated T cells for its life cycle, this up-regulation of MHC class II in infected cells may contribute to virus spread within the infected host by increasing the chance of contact between productively infected cells and susceptible activated T cells.