Sheep MHC class II molecules. II. Identification and characterization of four distinct subsets of sheep MHC class II molecules

A panel of seven monoclonal antibodies, sequential immunoprecipitation and two-dimensional NEPHGE/SDS-PAGE analyses were used to identify and characterize subsets of sheep MHC class II molecules. Using sequential immunoprecipitation four distinct subsets of class II molecules were identified by the monoclonal antibodies SBU.II 28-1, 37-68, 38-27 and 42-20, while another monoclonal antibody, SBU.II 49-1, recognized all four subsets of class II molecules. These four subpopulations of sheep class II molecules displayed different two-dimensional gel profiles and, using splenocytes from four outbred sheep, the class II molecules recognized by SBU.II 28-1, 37-68 and 42-20 showed structurally detectable allelic polymorphism in their beta polypeptides, but no detectable variation in their alpha polypeptides. In contrast, the class II molecules recognized by SBU.II 38-27 showed allelic variation in both their alpha and beta polypeptides. Two-dimensional (2-D) gel analyses of non-glycosylated class II molecules immunoprecipitated by SBU.II 49-1 suggested that approximately 10-12 different class II molecules were expressed by a single sheep. The results of this study show that sheep express class II molecules that can be divided into four structurally and serologically distinct subsets, and provide additional evidence for the subdivision of the sheep MHC class II genetic region into at least three distinct subregions.     

Mutations of class II MHC molecules

It remains unclear how the tertiary interaction of T-cell receptor, la molecule and foreign antigen results in the extensive diversity of the helper T cell repertoire. Here Laurie Glimcher and Irwin Griffith focus on what has been learned about the relationship between structure and function of the la molecule from the use of mouse strains with mutations in the genes coding for these glycoproteins.     

Monoclonal antibodies to sheep lymphocytes. I. Identification of MHC class II molecules on lymphoid tissue and changes in the level of class II expression on lymph-borne cells following antigen stimulation in vivo

A rat monoclonal antibody is described that recognizes sheep MHC Class II antigen and appears to recognize a determinant that is non-polymorphic. This antibody precipitates polypeptides of apparent molecular weight 33,000 and 28,000/29,000 (under non-reducing conditions). The binding of this antibody was inhibited by an anti-human DP, DQ, DR beta-chain monoclonal (DA6.231) and its reactivity on murine cells suggests specificity for the I-E gene product. Using this antibody we have studied the distribution of Class II molecules in sheep and also the variations in Class II expression by cells in both peripheral and efferent lymph, produced as a result of in vivo antigen inoculation.     

Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules

Although buffaloes (Bubalus bubalis) are a major component of the livestock industry worldwide, limited progress has been made in the study of the mechanisms regulating the immune response to pathogens and parasites affecting their health and productivity. This has been, in part, attributable to the limited availability of reagents to study immune responses in buffalo. As reported here, a set of cross-reactive monoclonal antibodies (mAbs), developed against bovine, ovine and caprine leukocyte differentiation molecules (LDM) and major histocompatibility complex (MHC) molecules, were identified and used to compare expression of LDM in Italian and Egyptian buffalo. The results show most of the epitopes identified with the mAbs are conserved on LDM and MHC I and II molecules in both lineages of buffalo. Comparison of the composition of lymphocyte subsets between buffalo and cattle revealed they are similar except for expression of CD2 and CD8 on workshop cluster one (WC1) positive γδ T cells. In cattle, CD8 is expressed on a subset of CD2+/WC1- γδ T cells that are present in low frequency in blood of young and old animals, whereas, CD8-/CD2-/WC1+ γδ T cells are present in high frequency in young animals, decreasing with age. In the buffalo, CD2 is expressed on a subset of WC1+ γδ T cells and CD8 is expressed on all WC1+ γδ T cells. The availability of this extensive set of mAbs provides opportunities to study the immunopathogenesis of pathogens and parasites affecting the health of buffalo.     

Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness

Activation of phagocytes by lipopolysaccharide (LPS) causes synthesis and secretion of various mediators of inflammation. CD14, a glycosylphosphatidylinositol-anchored monocytic antigen serving as receptor for LPS, and members of the family of Toll-like receptors mediate cellular activation in response to LPS. Here we investigated whether expression of MHC class II molecules modified the response to LPS. Comparing LPS responsiveness of human and murine cells differing for expression of MHC class II molecules, we found that lack or a low level of expression of MHC class II molecules resulted in diminished secretion of pro-inflammatory cytokines following stimulation with LPS. Thus, expression of MHC class II molecules modifies LPS responsiveness, a finding suggesting that these molecules contribute to the pathogenesis not only of exotoxin-triggered toxic shock but also of endotoxin-triggered septic shock. Additionally to their role in antigen-specific immunity MHC class II molecules may influence the inflammatory response triggered by microbial constituents.     

Cross reaction of monoclonal antibodies to human MHC class I and class II products with bovine lymphocyte subpopulations

Peripheral blood lymphocytes (PBL) from cattle have been separated into T & B cell subpopulations using a panning technique. These T and B cell preparations have then been tested by direct and indirect complement mediated cytotoxicity tests with a series of monoclonal antibodies (Moabs) reacting with HLA class I and class II products.
The anti-class I monoclonals were monomorphic or non-reactive and where reactive killed both B and T cells.
The anti-class II monoclonals reacted only with B lymphocytes. There was no relationship between their reactivity pattern and any BoLA specificity.
This work was supported by the National Institutes of Health Grants AI21384 and CA38469.     

Intracellular transport of MHC class II molecules.

MHC class II molecules associate, during biosynthesis, with peptides derived from endocytosed antigen. Here, Jacques Neefjes and Hidde Ploegh describe the intracellular transport of MHC class II molecules and its relationship to the binding of peptides in endosomal compartments. They discuss alternative routes for the delivery of antigen to sites at which peptides associate with MHC class II molecules and raise the possibility of cell type-specific differences in the handling of MHC class II molecules, and hence in antigen presentation.     

Endogenous antigen presentation by MHC class II molecules

T cell recognition of antigen requires that a complex form between peptides derived from the protein antigen and cell surface glycoproteins encoded by genes within the major histocompatibility complex (MHC). MHC class II molecules present both extracellular (exogenous) and internally synthesized (endogenous) antigens to the CD4 T cell subset of lymphocytes. The mechanisms of endogenous antigen presentation are the subject of this review. Isolation and amino acid sequencing of peptides bound to the class II molecule indicate that a very high proportion (70–90%) of the total peptides presented by the class II molecule are in fact derived from the pool of proteins that are synthetized within the antigen-presenting cell (APC). This type of sequence information as well as the study of model antigens has indicated that proteins expressed in a diversity of intracellular sites, including the cell surface, endoplasmic reticulum and cytosol can gain access to the class II molecule, albeit with different efficiencies. The main questions that remain to be answered are the intracellular trafficking patterns that allow colocalization of internally synthesized antigens with the class II molecule, the site(s) within the cell where peptide: class II molecule complex formation can take place and whether presentation of ‘foreign’ as well as ‘self’ antigens takes place by mechanisms that vary from one cell type to another or that vary with the metabolic state of the APC. If such variability exists, is would imply that the array of peptides displayed by class II molecules at the cell surface has similar variability, a possibility that would impact on self tolerance and autoimmunity.     

Accessory molecules for MHC class II peptide loading

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.     

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.     

Presentation of cytosolic antigens via MHC class II molecules

Major histocompatibility (MHC) class II molecules function to present antigenic peptides to CD4 T lymphocytes. The pathways by which these molecules present exogenous antigens have been extensively studied. However by contrast, far less is known about the processing and trafficking of cytosolic antigens, which can also serve as an alternative source of ligands for MHC class II molecules. Self-proteins, tumor antigens, as well as viral proteins found within the cytosol of cells, can be presented via MHC class II molecules, resulting in the activation of specific CD4 T cells. Studies have begun to reveal unique steps as well as some similarities in the pathways for cytosolic and exogenous antigen presentation. Recent developments in this area are summarized here.     

Improved methods for predicting peptide binding affinity to MHC class II molecules

Major histocompatibility complex class II (MHC‐II) molecules are expressed on the surface of professional antigen‐presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC‐II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T‐cell epitopes. We here present updated versions of two MHC–II–peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC–peptide binding affinity data obtained from the Immune Epitope Database covering HLA‐DR, HLA‐DQ, HLA‐DP and H‐2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2 .     

The peptide-binding strategy of the MHC class II I-A molecules

Despite the importance of murine major histocompatibility complex (MHC) class II I-A molecules for immunological research, the overall peptide-binding specificities of I-A and the homologous human HLA-DQ molecules remain unresolved. Here, Boris Reizis and colleagues review current evidence suggesting that DQ/I-A molecules bind peptides with a different hierarchy of anchor positions than has been found in the well-characterized DR/I-E proteins.     

Peptides recognized by class I restricted T cells also bind to MHC class II molecules

The mechanisms of antigen recognition employed by both class I and class II MHC-restricted T cells are very similar, yet many of the T cell determinants described to date are recognized in the context of a single class of MHC molecules, and generally with only one or a very few different MHC alleles. To determine whether this might be due to a structural difference between class I and class II restricted T cell determinants, peptides previously shown to be recognized in the context of MHC class I proteins by mouse or human CD8+ T lymphocytes were tested for their capacity to bind to HLA-DR molecules on the surface of B lymphoblastoid cell lines (B-LCL). Four out of five class I restricted T cell determinants tested bound to a panel of B-LCL, and the binding was inhibited by anti-HLA-DR mAb. The peptides did not bind to the class II-negative B-LCL RJ2.2.5 nor to mouse L cells, but did bind to L cells transfected with HLA-DR1.     

The wide diversity and complexity of peptides bound to class II MHC molecules

The identification and quantitation of peptides selected by class II MHC molecules during natural processing of proteins is of key importance in understanding the repertoire and distribution of T cells. The examination of peptides selected by class II MHC molecules has depended greatly on mass spectrometry, a powerful technique that identifies and sequences peptides in complex mixtures with great sensitivity and precision. Such analysis has resulted in the identification of several factors, including the repertoire of peptides selected by MHC molecules during natural processing of proteins, motifs important for selection of processed peptides, conformational isomers of peptide-MHC complexes, and post-translational changes to the peptides.     

The role of MHC class II molecules in susceptibility and resistance to autoimmunity

The mechanism by which particular MHC class II alleles mediate susceptibility to a given autoimmune disease is unknown. During the past year, reports have indicated that the effects of MHC class II alleles which protect against type I diabetes in the nonobese diabetic mouse strain may, in some cases, be due to negative selection of diabetogenic T cell receptors and, in other cases, to positive selection of other T cells with a suppressive action on the diabetic process. Progress towards understanding the mechanisms of susceptibility continues to lag.