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PubMed2006

Variable dimerization of the Ly49A natural killer cell receptor results in differential engagement of its MHC class I ligand.

Natural killer (NK) cells play a vital role in the detection and elimination of virally infected and tumor cells. The Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I molecules on target cells. Previous crystal studies revealed that the Ly49A homodimer binds one MHC molecule in an asymmetric interaction, whereas the Ly49C homodimer binds two MHC in a symmetrical fashion. Moreover, the bound receptors adopt distinctly different homodimeric forms: a "closed state" for Ly49A and an "open state" for Ly49C. Steric clashes between MHC molecules would preclude the closed Ly49A dimer from engaging two MHC in the manner of the open Ly49C dimer. To determine whether individual Ly49 receptors can undergo a conformational switch enabling them to bind MHC in different ways, we carried out a solution NMR study of unbound Ly49A, aided by dipolar coupling technology. This study reveals that, in solution, unligated Ly49A adopts a symmetric, open-state, homodimer conformation similar to that seen previously for Ly49C. Hence, Ly49A can assume both closed and open states. To address whether the Ly49A dimer can bind two MHC molecules in solution, besides the binding of one MHC observed in the crystal, we carried out analytical ultracentrifugation experiments. Velocity sedimentation demonstrates that the Ly49A dimer can engage two MHC molecules in solution, in agreement with NMR results showing that unbound Ly49A exists predominantly in the open state.

Animals↗
PubMed2001

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation.

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (Ii), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.

Animals↗
PubMed2002

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Alleles↗
PubMed1988

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants.

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.

Animals↗
PubMed2002

The bovine papillomavirus oncoprotein E5 retains MHC class I molecules in the Golgi apparatus and prevents their transport to the cell surface.

During papillomavirus infection, the E5 protein localizes in the cell Golgi apparatus and other endomembrane compartments. Cells transformed by E5 do not express major histocompatibility class I complex (MHC I) on the cell surface, while cells transformed by the other transforming proteins E6 and E7 do. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Here we show that expression of bovine papillomavirus E5 causes the retention of MHC I in the Golgi apparatus, thus preventing its transport to the cell surface. We ascribe this effect to a failure of acidification of the Golgi apparatus, as similar effects are observed in control cells treated with the ionophore monensin. Treatment of E5-transformed cells with either beta- or gamma-interferon increases the synthesis of MHC I, showing that inhibition of MHC I expression by E5 is not irreversible. However, even after interferon treatment, MHC I, although increased in quantity, is not transported to the cell surface. E5 therefore affects MHC I at several levels, but prevention of MHC I transport to the cell surface appears to be the dominant effect. Lack of surface MHC I would have profound consequences for presentation of viral peptides to the immune system.

Animals↗
PubMed1997

Effects of exercise on the macrophage MHC II response to inflammation.

The purpose of this investigation was to determine the effects of different doses of exercise on the ability of Propionibacterium acnes (P. acnes) to induce major histocompatibility complex (MHC) II antigen expression on macrophages (M phi's). Pathogen-free male Balb/c mice were exercised on a treadmill moderately (MOD, 15-17 m/min, 5% grade, 30 min/day) or exhaustively (EXH, 15-40m/min, 5% grade, 2-4hr/day) for a period of 7 days during P. acnes-induced inflammation. A control group (CON) consisted of animals exposed to the treadmill environment and handling. Sub-optimal (0.03 mg/g b.wt., i.p.) and optimal (0.08 mg/g b.wt.) doses of P. acnes were used to increase M phi MHC II expression. Animals were sacrified on Day 7 and M phi's were harvested by peritoneal lavage. Direct immunofluorescent staining was performed by incubating peritoneal exudate cells (10[6]) with an FITC-labeled anti-mouse MHC II (I-A[d]) antibody. Basal expression of MHC II was not affected by exercise. There were no significant differences among the groups in the percentage of M phi's expressing MHC II at any dose of P. acnes. However, EXH significantly (p < 0.05) suppressed the expression (mean fluorescent intensity, MFI) of MHC II when compared to MOD (37.1+/-1.95 [mean+/-sem] vs 49.1+/-2.15, p < 0.05) at the suboptimal P. acnes dosage. At the optimal P. acnes dose, both MOD and EXH significantly suppressed (27+/-1.6, 25+/-2.2 and 41.5+/-3.2, for EXH, MOD, and CON, respectively, p<0.0001) P. acnes-induced M phi MHC II MFI. Plasma corticosterone was highly (r=-0.71, p = 0.001) inversely correlated with M phi MHC II expression. However, exercise failed to affect P. acnes-induced production of interferon-gamma. These data suggest that, dependent on the degree of stimulation, exercise can negatively affect M phi expression of MHC II, an effect that may be detrimental to the M phi's ability to present antigen to T lymphocytes.

Animals↗
PubMed2005

Interplay among coactivator-associated arginine methyltransferase 1, CBP, and CIITA in IFN-gamma-inducible MHC-II gene expression.

Class II major histocompatibility (MHC-II) genes are prototype targets of IFN-gamma. IFN-gamma activates the expression of the non-DNA-binding master regulator of MHC-II, class II transactivator (CIITA), which is crucial for enhanceosome formation and gene activation. This report shows the importance of the histone methyltransferase, coactivator-associated arginine methyltransferase (CARM1/PRMT4), during IFN-gamma-induced MHC-II gene activation. It also demonstrates the coordinated regulation of CIITA, CARM1, and the acetyltransferase cyclic-AMP response element binding (CREB)-binding protein (CBP) during this process. CARM1 synergizes with CIITA in activating MHC-II transcription and synergy is abrogated when an arginine methyltransferase-defective CARM1 mutant is used. Protein-arginine methyltransferase 1 has much less effect on MHC-II transcription. Specific RNA interference reduced CARM1 expression as well as MHC-II expression. The recruitment of CARM1 to the promoter requires endogenous CIITA and results in methylation of histone H3-R17; hence, CIITA is an upstream regulator of histone methylation. Previous work has shown that CARM1 can methylate CBP at three arginine residues. Using wild-type CBP and a mutant of CBP lacking the CARM1-targeted arginine residues (R3A), we show that arginine methylation of CBP is required for IFN-gamma induction of MHC-II. A kinetic analysis shows that CIITA, CARM1, and H3-R17 methylation all precede CBP loading on the MHC-II promoter during IFN-gamma treatment. These results suggest functional and temporal relationships among CIITA, CARM1, and CBP for IFN-gamma induction of MHC-II.

Animals↗
PubMed1997

RFXAP, a novel subunit of the RFX DNA binding complex is mutated in MHC class II deficiency.

Major Histocompatibility Complex class II (MHC-II) deficiency is a disease of gene regulation that provides a unique opportunity for the genetic dissection of the molecular mechanisms controlling transcription of MHC-II genes. Cell lines from MHC-II deficiency patients have been assigned to three complementation groups (A, B and C) believed to reflect the existence of distinct essential MHC-II regulatory genes. Groups B and C, as well as an in vitro generated regulatory mutant representing a fourth group (D), are characterized by a specific defect in the binding activity of RFX, a multimeric DNA binding complex that is essential for activation of MHC-II promoters. RFX5, a subunit of RFX, was recently shown to be mutated in group C. We have now isolated a novel gene, RFXAP (RFX Associated Protein), that encodes a second subunit of the RFX complex. RFXAP is mutated in the 6.1.6 cell line (group D), as well as in an MHC-II deficiency patient (DA). This establishes that group D is indeed a fourth MHC-II deficiency complementation group. Complementation of the 6.1.6 and DA cell lines by transfection with RFXAP fully restores expression of all endogenous MHC-II genes in vivo, demonstrating that RFXAP is a novel essential MHC-II regulatory gene.

Amino Acid Sequence↗
PubMed2006

Regulation of MHC class II expression, a unique regulatory system identified by the study of a primary immunodeficiency disease.

Major histocompatibility complex class II (MHC-II) molecules are of central importance for adaptive immunity. Defective MHC-II expression causes a severe immunodeficiency disease called bare lymphocyte syndrome (BLS). Studies of the molecular defects underlying BLS have been pivotal for characterization of the regulatory system controlling the transcription of MHC-II genes. The precisely controlled pattern of MHC-II gene expression is achieved by a very peculiar and highly specialized molecular machinery that involves the interplay between ubiquitous DNA-binding transcription factors and a highly unusual, tightly regulated, non-DNA-binding coactivator called the MHC class II transactivator (CIITA). CIITA single handedly coordinates practically all aspects of MHC-II gene regulation and has therefore been dubbed the master controller of MHC-II expression. Several of the unusual features of the MHC-II regulatory system may be a consequence of the fact that CIITA originated from an ancient family of cytoplasmic proteins involved in inflammation and innate immunity. The function of CIITA in transcriptional regulation of MHC-II genes could thus be a recent acquisition by an ancestral protein having a role in an unrelated system.

Animals↗
PubMed1996

Autoimmunity associated with TGF-beta1-deficiency in mice is dependent on MHC class II antigen expression.

The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.

Animals↗
PubMed2005

Structure of a pheromone receptor-associated MHC molecule with an open and empty groove.

Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

Animals↗
PubMed2004

Endothelial expression of MHC class II molecules in autoimmune disease.

Major histocompatibility complex (MHC) class II molecules are up-regulated on endothelial cells in human allografts, and are thought to be involved in graft rejection. The MHC class II subtypes HLA-DR, DQ and DP regulate T cell dependent immune responses, and aberrant expression could be important in autoimmunity. Increased endothelial MHC class II expression has been demonstrated in several autoimmune diseases, including myocarditis with dilated cardiomyopathy, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Recent data suggest that there is an association between endothelial expression of MHC class II molecules and diffuse endothelial dysfunction, which may be part of the explanation of the increased risk of cardiovascular disease in patients with RA, SLE and other chronic inflammatory conditions. MHC class II transcription is in part genetically determined. Cytokine induced up-regulation of MHC class II molecules can be inhibited in vitro by antioxidants and different drugs, such as cyclosporin and statins. Research on the development of new treatments for systemic autoimmune diseases and cardiovascular disease should include evaluation of effects on endothelial activation, including MHC class II expression. This review also discusses the genetic basis of MHC class II expression and its implications for understanding MHC genotype associations with autoimmune diseases. Recent studies of interactions between endothelial cells and T cells are reviewed. Such interactions could be of major importance in the pathogenesis of autoimmune and vascular diseases.

Animals↗
PubMed1996

MHC class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice.

The common class I alleles (e.g., Kd and Db) within the H2g7 major histocompatibility complex (MHC) clearly contribute to autoimmune IDDM in NOD mice, but the mechanism by which this occurs has been controversial. One laboratory has reported that the peptide transporter encoded by the Tap1 gene within H2g7 is defective, and this contributes to IDDM by impairing MHC class I-mediated antigen presentation. If true, defective MHC class I-mediated antigen presentation should segregate with the H2g7 haplotype. NOD mice, related congenic stocks, and other control strains were used to test this hypothesis. H2g7-positive strains did not differ from those expressing other MHC haplotypes in ability to present MHC class I-restricted H3aa or H3ab minor histocompatibility (H) antigens to cytotoxic T-lymphocytes (CTL). The H2g7 haplotype was found to have a reduced capacity to mediate MHC class I-restricted presentation of the H47a minor H antigen. However, MHC class I-restricted presentation of H47a was found to be Tap independent. NOD mice and control strains also did not differ in ability to activate adenovirus-specific MHC class I restricted CTL. Thus, the H2g7 haplotype is not characterized by a Tap gene defect that only impairs the inductive phase of the immune response. In addition, MHC class I-restricted presentation of either minor H or adenoviral antigens was equivalent in male and female NOD mice. Therefore, while the class I alleles of the H2g7 haplotype exert diabetogenic functions in NOD mice, this is not elicited through a Tap gene defect. The absence of female-specific Tap gene defects also indicates this cannot account for the reduced male incidence of IDDM in some NOD mouse colonies.

Alleles↗
PubMed2006

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation.

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.

Animals↗
PubMed1993

Regulation of MHC class I and beta 2-microglobulin gene expression in human neuronal cells. Factor binding to conserved cis-acting regulatory sequences correlates with expression of the genes.

MHC class I molecules are coexpressed with beta 2-microglobulin (beta 2-M) on many somatic cells. However, these proteins are normally not present on cells of the central nervous system (CNS). Cells derived from human neuroblastomas were used as a model for investigating the molecular basis for the paucity of MHC class I and beta 2-M gene expression in neural cells and for the induction of these genes by two cytokines, IFN-gamma, and TNF-alpha. These cytokines independently increased MHC class I and beta 2-M cell surface expression on the neuroblastoma cell lines. IFN-gamma or TNF-alpha also increased MHC class I and beta 2-M steady-state RNA levels and the expression of MHC class I and beta 2-M CAT reporter constructs transiently transfected into the neuroblastoma cell lines, indicating that the cytokines acted by increasing the transcription of these genes. MHC class I and beta 2-M genes share two conserved regulatory elements, an NF kappa B-like site and the IFN consensus sequence, that act as a constitutive enhancer and an IFN-responsive element, respectively. Low MHC class I and beta 2-M gene expression in these cells was accounted for by undetectable to low factor binding activity specific for the above regulatory elements of these genes. TNF-alpha increased factor binding activity specific for the NF kappa B-like elements and IFN-gamma increased factor binding activity specific for the IFN consensus sequence elements of the MHC class I and beta 2-M genes, but not vice versa. Taken together, our results indicated that IFN-gamma and TNF-alpha increased MHC class I and beta 2-M gene expression in the neuroblastoma cell lines by inducing factor binding to the regulatory elements present in both genes.

Base Sequence↗
PubMed1996

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha.

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.

Adjuvants, Immunologic↗
PubMed1997

Immunodominance of a single CTL epitope in a primate species with limited MHC class I polymorphism.

MHC class I molecules play a crucial role in immunity to viral infections by presenting viral peptides to cytotoxic T lymphocytes. One of the hallmarks of MHC class I genes in outbred populations is their extraordinary polymorphism, yet the significance of this diversity is poorly understood. Certain species with reduced MHC class I diversity, such as the cotton-top tamarin (Saguinus oedipus), are more susceptible to fatal viral infections. To explore the relationship between this primate's limited MHC class I diversity and its susceptibility to viruses, we infected five cotton-top tamarins with influenza virus. Every tamarin recognized the same immunodominant CTL epitope of the influenza nucleoprotein. Surprisingly, this nucleoprotein peptide was bound by Saoe-G*08, an MHC class I molecule expressed by every cotton-top tamarin. Two tamarins also made a subdominant response to an epitope of the matrix (M1) protein. This peptide appeared to be bound by another common MHC class I molecule. With the exception of an additional subdominant response to the polymerase (PB2) protein in one individual, no other influenza-specific CTL responses were detected. In populations or species with limited MHC class I polymorphism like the cotton-top tamarin, a dependence on shared MHC class I molecules may enhance susceptibility to viral infection, since viruses that evade MHC class I-restricted recognition in one individual will likely evade recognition in the majority of individuals.

Animals↗
PubMed1998

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules.

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

Amino Acid Sequence↗