CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope.

Löfgren's syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3-restricted manner. Using ELISPOT analysis, a greater number of IFN-γ- and IL-2-secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

Transglutaminase antibodies and celiac disease in children with type 1 diabetes and in their family members.

OBJECTIVES: We set out to determine the prevalence of tissue transglutaminase antibodies (anti-tTG) and celiac disease (CD) in children with newly diagnosed type 1 diabetes (T1D) and their first-degree relatives (FDR). The hypothesis was that the individuals with both diabetes and CD form a distinct subgroup in terms of human leukocyte antigen (HLA) class II genetics, islet autoantibodies, and clinical characteristics at diabetes diagnosis. SUBJECTS AND METHODS: This population-based observational study included 745 index children with T1D and their 2692 FDR from the Finnish Pediatric Diabetes Register. CD was ascertained by registers, patient records, and screening anti-tTG positive individuals for further testing. RESULTS: Among the index children, 4.8% had anti-tTG at diabetes diagnosis, and at the end of the study 3.2% had CD. Among the relatives, 2.9% had anti-tTG (4.8% mothers, 2.4% fathers, and 2.1% siblings), and 2.5% had CD (4.6% mothers, 2.1% fathers, and 1.4% siblings). Anti-tTG and CD associated with the HLA DR3-DQ2 haplotype. The usual female predominance of CD patients was observed in relatives (70%) but not among index children (46%). The index children with both diseases had a lower number of detectable islet autoantibodies than those with diabetes alone. CONCLUSIONS: The children with double diagnosis differed from those with diabetes alone in HLA genetics, humoral islet autoimmunity directed against fewer antigens, and in the lack of usual female preponderance among CD patients. Compared with 61% of the anti-tTG positive relatives, only 36% of anti-tTG positive index children developed CD implicating transient anti-tTG positivity at diagnosis of T1D.

Injections of Clostridium botulinum neurotoxin A may cause thyroid complications in predisposed persons based on molecular mimicry with thyroid autoantigens.

A woman with Hashimoto's thyroiditis, under replacement L-T4, repeatedly experienced, over a 10-year period, elevations of serum TSH after eyelid injections of Clostridium botulinum neurotoxin A (Btx). We hypothesized a link between Btx injections and TSH elevations via molecular mimicry, and aimed to verify our hypothesis. Using an in silico approach, we searched first for amino acid sequence homology between Btx and thyroid autoantigens, and next for HLA binding motifs within homologous segments. We found that (i) Btx and thyroid autoantigens share amino acid sequence homology; (ii) some homologous regions contain epitopes of both Btx and thyroid autoantigens; (iii) some of such regions contain HLA-DR3 and/or HLA-DR7 binding motifs, which predominate over other HLA-DRs. This is relevant because the patient's HLA-DR haplotype was DR3/DR7. In conclusion, clinical and bioinformatics data suggest a possible pathogenetic link between Btx and autoimmune thyroid diseases. Considering the wide and increasing medical and dermocosmetic use of Btx, and the frequently subclinical course of autoimmune thyroid diseases, we think that thyroid "complications" may pass frequently undetected in Btx-treated persons.

The binding of antigenic peptides to HLA-DR is influenced by interactions between pocket 6 and pocket 9.

Peptide binding to class II MHC protein is commonly viewed as a combination of discrete anchor residue preferences for pockets 1, 4, 6/7, and 9. However, previous studies have suggested cooperative effects during the peptide binding process. Investigation of the DRB1*0901 binding motif demonstrated a clear interaction between peptide binding pockets 6 and 9. In agreement with prior studies, pockets 1 and 4 exhibited clear binding preferences. Previously uncharacterized pockets 6 and 7 accommodated a wide variety of residues. However, although it was previously reported that pocket 9 is completely permissive, several substitutions at this position were unable to bind. Structural modeling revealed a probable interaction between pockets 6 and 9 through beta9Lys. Additional binding studies with doubly substituted peptides confirmed that the amino acid bound within pocket 6 profoundly influences the binding preferences for pocket 9 of DRB1*0901, causing complete permissiveness of pocket 9 when a small polar residue is anchored in pocket 6 but accepting relatively few residues when a basic residue is anchored in pocket 6. The beta9Lys residue is unique to DR9 alleles. However, similar studies with doubly substituted peptides confirmed an analogous interaction effect for DRA1/B1*0301, a beta9Glu allele. Accounting for this interaction resulted in improved epitope prediction. These findings provide a structural explanation for observations that an amino acid in one pocket can influence binding elsewhere in the MHC class II peptide binding groove.

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study.

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.

Point mutations in or near the antigen-binding groove of HLA-DR3 implicate class II-associated invariant chain peptide affinity as a constraint on MHC class II polymorphism.

During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II alphabeta dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations betaS11F, betaS13Y, betaQ70R, betaK71E, betaK71N, and betaR74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as alphaG20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

Molecular mimicry in diabetes mellitus: the homologous domain in coxsackie B virus protein 2C and islet autoantigen GAD65 is highly conserved in the coxsackie B-like enteroviruses and binds to the diabetes associated HLA-DR3 molecule.

It has been proposed that molecular mimicry between protein 2C (p2C) of coxsackie virus B4 and the autoantigen glutamic acid decarboxylase (GAD65) plays a role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). In this study we show that the amino acid sequence of p2C which shares homology with a sequence in GAD65 (PEVKEK), is highly conserved in coxsackie virus B4 isolates as well as in different viruses of the subgroup of coxsackie B-like enteroviruses. These are the most prevalent enteroviruses and therefore exposure to the mimicry motif will be a frequent event throughout life. Presentation of the homologous peptides by HLA molecules is essential for T-cell reactivity. Therefore, we tested whether the PEVKEK motif can bind to the IDDM-associated HLA-DR1, -DR3 and -DR4 molecules. Synthetic peptides with sequences derived from p2C and GAD65 did bind to HLA-DR3 but not to HLA-DR1 or -DR4. Replacement of amino acids within the motif showed that the PEVKEK motif binds specifically to HLA-DR3. Moreover, both p2C and GAD65 peptides bind in the same position within the peptide binding groove of the DR3 molecule which is an essential requirement for T-cell cross-reactivity. The results support molecular mimicry between p2C of coxsackie B-like enteroviruses and GAD65. However, this molecular mimicry may be limited to the HLA-DR3 positive subpopulation of IDDM patients.

HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules.

Major histocompatibility complex (MHC) class II-positive cell lines which lack HLA-DM expression accumulate class II molecules associated with residual invariant (I) chain fragments (class II-associated invariant chain peptides [CLIP]). In vitro, HLA-DM catalyzes CLIP dissociation from class II-CLIP complexes, promoting binding of antigenic peptides. Here the physical interaction of HLA-DM with HLA-DR molecules was investigated. HLA-DM complexes with class II molecules were detectable transiently in cells, peaking at the time when the class II molecules entered the MHC class II compartment. HLA-DR alpha beta dimers newly released from I chain, and those associated with I chain fragments, were found to associate with HLA-DM in vivo. Mature, peptide-loaded DR molecules also associated at a low level. These same species, but not DR-I chain complexes, were also shown to bind to purified HLA-DM molecules in vitro. HLA-DM interaction was quantitatively superior with DR molecules isolated in association with CLIP. DM-DR complexes generated by incubating HLA-DM with purified DR alpha beta CLIP contained virtually no associated CLIP, suggesting that this superior interaction reflects a prolonged HLA-DM association with empty class II dimers after CLIP dissociation. Incubation of peptide-free alpha beta dimers in the presence of HLA-DM was found to prolong their ability to bind subsequently added antigenic peptides. Stabilization of empty class II molecules may be an important property of HLA-DM in facilitating antigen processing.

[Analysis of peptides associated with class II MHC molecules, HLA-DR3: implication for the prediction of peptides useful for vaccines]. / Analyse des peptides s'associant aux molécules du CMH de classe II, HLA-DR3: implication pour la prédiction de peptides d'intérêt vaccinal.

In order to characterize peptide binding motifs of MHC class II molecules HLA-DR3, we have sequenced pool or single peptides eluted from the binding groove. Anchor residues identified are in agreement with peptide binding and sequencing studies reported by different groups. Four positions seem to be dominant (i, i + 3, i + 5, i + 8) while 2 secondary positions (i + 1, i + 2) could cooperate to facilitate binding. According to all the criteria define here and the literature, we propose an anchor motif specific for DR3, which has been tested on the sequence of an antigen from Schistosoma mansoni. Three out of 6 putative epitopes identified share common sequences with immunodominant regions determined in humans and by experimental immunizations in animal models. Extended to other alleles, this approach could be suitable to define potentially immunodominant peptides useful for vaccines.

The impact of DR3 microvariation on peptide binding: the combinations of specific DR beta residues critical to binding differ for different peptides.

HLA-DR molecules are a group of highly polymorphic glycoprotein heterodimers that present peptide antigens to T lymphocytes for immune surveillance. To assess the significance of limited polymorphism on the functional differentiation of DR molecules, the binding of several immunogenic peptides to the DR3 microvariants [DR(alpha, beta 1*0302) and DR(alpha, beta 1*0301)] and to mutants of these DR3 molecules was examined. This analysis has shown that each residue (DR beta 26, DR beta 28, DR beta 47, and DR beta 86), which differentiates these two DR3 molecules, contributes to their functional distinction and that the relative contribution of each residue varies for different peptide/DR3 complexes. For example, DR beta 28 and DR beta 86 controlled the mycobacterium tuberculosis 65-kD heat shock protein peptides 3-13 and 4-15 (HSP) binding specificity to DR (alpha, beta 1*0301). [HSP does not bind to DR(alpha, beta 1*0302)], whereas DR beta 26, DR beta 28, and DR beta 86 controlled the influenza hemagglutinin peptide 306-318 (HA) binding specificity to DR(alpha, beta 1*0302). [HA does not bind to DR(alpha, beta 1*0301).] In comparison, DR beta 86 alone controlled the binding level difference of sperm whale myoglobin peptide 132-151 (SWM) and of myelin basic protein peptide 152-170 (MBP) [both bind to DR(alpha, beta 1*0301) at levels five times greater than to DR(alpha, beta 1*0302)] to the DR3 molecules. Although not critical, additional DR beta residues influenced the binding level of individual peptides of each of the DR3 molecules and, again, the combinations of these residues differed for different peptide/DR3 complexes. These data showed that individual DR residues vary in their relative contribution to the interaction between a specific DR molecule and different peptides and that limited polymorphism can create substantial differences in the peptide binding profiles among DR molecules.

The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3.

A complex between HLA-DR3 and a fragment of invariant chain called CLIP was isolated from a human cell line defective in antigen presentation and its X-ray crystal structure determined. Previous data indicate that this complex is an intermediate in class II histocompatibility maturation, occurring between invariant chain-DR3 and antigenic peptide-DR3 complexes. The structure shows that the CLIP fragment binds to DR3 in a way almost identical to that in which antigenic peptides bind class II histocompatibility glycoproteins. The structure is the substrate for the loading of antigenic peptides by an exchange process catalysed by DM.

Mutational analysis of two DR alpha residues involved in dimers of HLA-DR molecules.

Crystallographic analysis of HLA-DR1 molecules reveals a "dimer of dimers" with two reciprocal salt bridges between Glu 88 and Lys 111 of the two DR alpha chains. To determine whether these amino acids are critical for Ag presentation, we generated a panel of human B cell transfectants expressing DR alpha chains with mutations at residues 88, 111, or both. The mutant DR alpha chains, paired with endogenous DR3 beta chain, form cell surface dimers that retain epitopes recognized by a panel of anti-DR3 Abs. Replacement of Glu 88 with Ala (88A) selectively eliminates the ability to activate an alloreactive (anti-DR3) T cell clone. Mutant DR molecules with Lys substituted for Glu 88 (88K) fail to activate an alloreactive, an Ag-specific, and a peptide-specific T cell line. The DR alpha 88 mutants bind an exogenously supplied DR3-specific peptide and the mutant DR molecules migrate as dimers on SDS-PAGE, implying that their defective Ag presentation is not due to an inability to bind antigenic peptides. In contrast, substitution of Lys 111 with either Ala (111A) or Glu (111E) does not abrogate Ag presentation. Further, the defect introduced by Glu 88 to Lys mutation (88K) is not overcome by compensatory Lys to Glu mutation at position 111 (111E). Taken together, these results indicate an important functional or structural role for position 88 of the DR alpha chain, but argue against a requirement for interaction between DR alpha 88 and 111 during Ag-specific T cell stimulation.

Microvariation creates significant functional differences in the DR3 molecules.

Two DR3 molecules differ by four amino acids whose side chains point into the DR antigen-binding groove. To begin to assess the role of microvariation on DR3 function, DRB1*0302 residues were replaced with DRB1*0301 residues at beta-chain positions 26, 47, 86, and 47 plus 86. Murine fibroblast cell lines expressing DR(alpha, beta 1*0301), DR(alpha, beta 1*0302), and the four mutant 0302 molecules were examined for alloproliferative DR(alpha, beta 1*0302)-specific TLC stimulation and peptide binding. Changing position 26 had the most profound effect on T-cell recognition (seven of nine TLCs did not respond). Two TLCs did not respond to the mutant 0302V86 molecule and four TLCs that did respond to this mutant lost responsiveness when positions 47 and 86 were mutated together. These data suggest that each of these variant residues, including position 47, influence T-cell recognition. Surprisingly, none of the mutations had an effect on the absolute binding of HA 307-319 (DR[alpha, beta 1*0302] specific) and HSP 3-13 (DR[alpha, beta 1*0301] specific); however, the mutant 0302 molecules changed at position 86 (glycine to valine) consistently bound HA 307-319 at significantly higher levels than DR(alpha, beta 1*0302). These data for position 86 are in contrast to other DR molecules and indicate that peptide contact residues for a specific DR molecule cannot be predicted based on binding results obtained with other DR molecules. These data suggest that each of these variant groove residues, although not accessible to the TCR, contribute to the significant functional differences between the DR3 microvariants through subtle influences on the DR3-peptide complex.

Assembly and intracellular transport of HLA-DM and correction of the class II antigen-processing defect in T2 cells.

MHC class II molecules expressed in T2 cells fail to acquire a normal complement of endocytically generated peptides. The defect is repaired by introducing HLA-DMA and HLA-DMB cDNA expression vectors, determined by the restoration of SDS stability of class II alpha beta dimers, restoration of a normal conformation for HLA-DR3 as detected by a monoclonal antibody, and by a reduction in class II-associated invariant chain peptides. The intracellular distribution of class II and invariant chain molecules is also restored to that of wild-type cells. The HLA-DMA and HLA-DMB products appear to form a heterodimer that, although transported at least to the medial Golgi, is not expressed at the cell surface. These findings are consistent with HLA-DM functioning intracellularly to facilitate class II-restricted antigen processing.

Intramolecular charge heterogeneity in purified major histocompatibility class II alpha and beta polypeptide chains.

Major histocompatibility (MHC) class II antigens are heterodimeric cell surface glycoproteins consisting of an alpha and a beta chain. Although one-dimensional SDS-polyacrylamide gel electrophoresis analysis of purified MHC class II antigens shows a single diffuse band for each chain, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of this heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mature protein. To investigate the role of the three N-linked oligosaccharides of the alpha and beta chains in determining the isoelectric point of each chain, affinity-purified MHC class II antigens from human and rat sources were deglycosylated using asparagine amidase. The complete enzymatic removal of all three N-linked oligosaccharides was confirmed by SDS-polyacrylamide gel electrophoresis as well as by four different lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of the HLA DR2 chains which lack the transmembrane and cytoplasmically exposed regions in Escherichia coli. Two-dimensional electrophoresis of these single chains also reveal multiple banding patterns. The two-dimensional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimension, treatment of the proteins with alkaline phosphatase to remove any potential phosphorylation, or preincubation in the presence of iodoacetamide. Multiple forms of recombinant alpha and beta chains were also observed in Tris-glycine-urea gels which merged into a single band in the presence of SDS. In addition, partially fractionated bands from preparative isoelectric focusing gels, when refocused, showed an identical number of multiple spots spanning the same range of isoelectric points. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the observed charge heterogeneity is independent of glycosylation and phosphorylation of the proteins.