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.     

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.     

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.     

Functional reconstitution of class II major histocompatibility complex molecules, I-A alpha k and I-A beta k

Genomic DNA fragments carrying the genes, I-A alpha k and I-A beta k, encoding the class II major histocompatibility complex (MHC) molecule I-Ak were inserted into a transducing vector pRSVneo. This vector was introduced into a B lymphoma line expressing the class II molecules (H-2d). The resultant transformants expressed I-Ak molecules on the membrane surface. These transformants were capable of presenting antigen to I-Ak restricted helper T (Th) cells and were susceptible to lysis by class II specific killer T cells. The vector pR-A alpha kA beta k which contains both I-A alpha k and I-A beta k genes will provide a useful tool to analyze function of Ia molecules in cellular interaction, recognition, regulations and stimulation.     

Functional and molecular characterization of I-A kappa beta mutants is consistent with the predicted three dimensional structure of class II MHC molecules

Class II MHC (Ia) molecules have been shown to be critical as restriction elements in the T helper/inducer cell recognition of antigen. Efforts to determine the role of allelic variation in MHC restricted antigen presentation have included the use of serologically selected mutants to correlate structural variations in Class II molecules with changes in the antigen presenting function of Ia bearing cells. Such studies have revealed that serologically selected mutations tend to occur in a single immunodominant region and that even a single amino acid substitution can alter T cell recognition of Ia molecules. We report here the characterization of two more serologically selected Class II A beta chain mutations. Each is due to a single base change which alters a single amino acid. One of these mutations is in the third hypervariable region (amino acid 64--glutamine to arginine) and alters the antigen presenting function. The second mutation at amino acid 48, though a relatively non-conservative change (arginine to cysteine), has no effect on APC phenotype. Such a result would be predicted based on comparisons made with the proposed three dimensional crystallographic structure of Class I molecules and models proposed for Class II molecules based on Class I structure. The amino acid change at position 48 is in a portion of the molecule that is most likely unavailable to bind antigen or interact with T cell receptor whereas the mutation at amino acid 64 is on an exposed face of the alpha helix, a region which could affect interaction with either antigen and/or the T cell receptor.     

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.     

The impact of the non-classical MHC proteins HLA-DM and HLA-DO on loading of MHC class II molecules

Summary: Peptide binding to classical major histocompatibility complex (MHC) class II molecules is known to be determined by the properties of the class ii peptide binding groove but recently it turned out to be co-controlled by the activity of the non-classical MHC molecules HLA-DM and HLA-DO: HLA-DM functions as a mediator of peptide exchange. In addition, HLA-DM is a chaperone for MHC class II molecules in endosomal and lysosomal loading compartments because it stabilizes the empty MHC class Ii peptide binding groove and keeps it receptive for peptide loading until appropriate peptide ligands are captured. Since HLA-DM favors the generation of high-stability peptide-MHC class Ii complexes by releasing low-stability peptide ligands, DM activity affects the peptide repertoire presented on the ceil surface of antigen-presenting cells. HLA-DO is expressed mainly in B cells and binds tightly to HLA-DM thereby modulating its activity Together, HLA-DM and HLA-DO are critical factors in shaping the MHC class Il-associated self or foreign peptide repertoire of antigen presenting cells and, hence, govern initiation or prevention of an immune response.     

Characterization of the interaction of a TCR alpha chain variable domain with MHC II I-A molecules.

The alphabeta TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR alpha chain variable domain (Valpha4.2-Jalpha40; abbreviated to Valpha4.2) with the MHC class II molecule I-Au. Valpha4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the Valpha domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4alpha) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the beta chain of I-Au. Furthermore, the binding of Valpha4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble Valpha4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. Valpha4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2(u) and H-2(d) haplotypes. The binding of Valpha4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR alpha chains. This Valpha4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.     

HLA class II peptide-binding and autoimmunity

The HLA class II locus is located in the 6p21.3 region on the short arm of chromosome 6 and encompasses approximately 700 kb. It consists of over 30 gene loci including the major class II structural genes DP, DQ and DR. While autoimmune disease correlates to specific DP, DQ or DR alleles have been documented, due to the strong linkage disequilibrium between the different HLA alleles, especially between the DR and DQ, the precise identification of susceptible MHC alleles for a number of autoimmune diseases remains elusive.     

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.     

CIITA M1-RNA抑制HeLa细胞表面MHC II类抗原的表达

探讨MHC II类转录激活因子(CIITA)的M1-RNA对细胞表面MHC II类分子表达的抑制。M1-RNA是核糖核酸酶P的催化活性单位,设计并克隆针对CIITA第452、629位点的M1-RNA(分别为M1-452-GS、M1-629-GS)及其相应的CIITA靶基因,分别插入pUC19、pGEM-7zf(+)载体,进行细胞外切割活性筛选。将细胞外切割作用明显的M1-629-GS亚克隆入psNAV载体(psNAV-M1-629-GS,pA629)并稳定转染HeLa细胞株,流式细胞术检测经典的MHC II类抗原(HLA-DR、-DP、-DQ)的表达,RT-PCR检测CIITA的mRNA水平。在重组人γ干扰素诱导下,pA629阳性HeLa细胞株表面HLA-DR、-DP抗原表达分别降低了83.03%及89.91%;同时CIITA的mRNA含量明显减少(P<0.05)。CIITA的M1-RNA抑制了自身mRNA含量,从而阻止其调控的MHC II类分子的表达,为移植物抗宿主病的研究提供了一种新方法。     

The function of the invariant chain in antigen presentation by MHC class II molecules

The existence of different pathways of antigen presentation by class I and class II molecules raises two fundamental questions. (1) Why are class II, but not class I molecules, transported to the endosomal compartment where the exogenous antigen is met? (2) What are the mechanisms that prevent class II molecules from being occupied and blocked by endogenous peptides before they reach endosomes? These and other questions were discussed at the recent 7th International HLA/H-2 Workshop. In round table discussions particular attention was paid to the class-II-associated invariant chain, because it was considered that this molecule was a likely candidate to explain some of the differences of class I and class II molecules in antigen presentation.     

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.     

Immunologically discrete conformation isomers of I-A locus-equivalent class II molecules detected in Lewis rats

Monoclonal antibodies MRC-OX6 and MRC-OX3 were used to define biosynthetically inter-related subsets of I-A-equivalent class II molecules from Lewis rat spleen cells. MRC-OX6 was shown to recognize specifically multiple forms of the class II molecule arising along its maturation pathway from the origin of polypeptide chain synthesis, the rough endoplasmatic reticulum, through the Golgi compartment to the plasma membrane. Three MRC-OX6-reactive polypeptide chain complexes were distinguished: (a) an early complex composed of immature components of the polymorphic alpha,beta heterodimer in noncovalent association with immature proteins of the invariant gamma-chain group p40, p33 (gamma), p28, p20; (b) a biosynthetic intermediate comprising the subunits alpha,beta and gamma all of them being extensively glycosylated and sialylated. The latter constituent is referred to as p36; (c) a cell surface structure consisting of the mature alpha,beta heterodimer devoid of the mature invariant chain p36. A two-dimensional (2D) separation pattern exhibited by an MRC-OX6-specific immunoprecipitate from spleen cells labeled for 4 h represents a superposition of these three MRC-OX6-reactive polypeptide chain complexes. In contrast, MRC-OX3 recognizes exclusively a fully mature alpha,beta heterodimer devoid of protein precursor forms and devoid of any invariant chains. Considering the previous observation that the respective alpha and beta subunits of MRC-OX6 and MRC-OX3-specific molecules comigrate on 2D O'Farrell gels combined with the data of this investigation, it is suggested that the mature MRC-OX6-specific alpha,beta heterodimer and the mature MRC-OX3-specific alpha,beta heterodimer originate from the same biosynthetic intermediate. We propose that before entering the plasma membrane an MRC-OX6-reactive molecule composed of fully glycosylated alpha, beta and p36 releases its post-translationally processed gamma chain (p36), giving rise to two conformation isomers, one alpha,beta heterodimer still being reactive with MRC-OX6 and one alpha,beta heterodimer which has lost the serologic MRC-OX6 and gained the MRC-OX3 specificity. Both alpha, beta heterodimers are expressed on the cell surface as two distinct subsets of I-A-equivalent class II molecules.     

Sheep MHC class II molecules. I. Immunochemical characterization

The physicochemical features, biosynthesis and glycosylation of sheep class II molecules were investigated using a panel of seven monoclonal antibodies. The class II molecules recognized by different monoclonal antibodies could be differentiated using SDS-PAGE. Two monoclonal antibodies, SBU.II 42-20 and 49-1, reacted with dissociated sheep class II molecules and recognized epitopes on class II alpha and beta polypeptides, respectively. The structure of the sheep class II heterodimer differed from that of mouse and man in that it was unstable in the presence of 1% SDS at 20 degrees and, following reduction, sheep beta polypeptides displayed a marked increase in MW, resulting in the apparent co-migration of reduced alpha and beta polypeptides on SDS-PAGE. This phenomenon was not seen using sheep class II molecules synthesized in the presence of tunicamycin. Pulse-chase analyses of biosynthetically labelled sheep class II molecules suggested the rapid association and glycosylation of sheep class II alpha and beta polypeptides during synthesis. Both alpha and beta polypeptides of sheep class II molecules carried N-linked oligosaccharides of MW 6,000 and 3,000, respectively. However, unlike human class II oligosaccharides, these were exclusively of the complex or sialylated type.