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.     

Kinetics of registry selection of chimeric peptides binding to MHC II

Major histocompatability complex type II proteins (MHC II) are alphabeta-heterodimeric glycoproteins that present peptides to the T cell receptor (TCR) of CD4(+) T-cells. This presentation may result in activation of these T-cells, depending on the nature of the peptide. Peptides interact specifically with MHC II with nine peptide amino acid positions, and the corresponding MHC II pocket positions are usually labeled P1-P9. However, the length of peptides binding to MHC II may be greater than nine amino acids, and therefore these peptides may potentially bind to the MHC II in more than one registry. To investigate the mechanism by which a long peptide binds to I-E(k), a murine MHC II, a chimeric peptide with two nonoverlapping registries, f-IAYLKQATKQLRMATPLLMR was designed. The IAYLKQATK peptide segment is based on moth cytochrome c 95-103 (MCC 95-103), and the QLRMATPLLMR segment is based on murine Ii CLIP 89-99 M90L (Ii CLIP 89-99 M90L). This chimeric peptide forms two isomeric complexes. The MCC and Ii CLIP registries dissociate from I-E(k) with t(1/2) values of >800 and 4.94 h, respectively. The registry composition of this MHC II/chimeric peptide complex was found to change as a function of time in approaching thermodynamic equilibrium: the results are consistent with a kinetic model that involves no intramolecular isomer interconversion. The model depicts uncorrelated binding to the MHC II determined by relative association rates to the two registries. This is followed by dissociation and subsequent rebinding, leading ultimately to a preponderance of the most stable complex. Similar results were obtained at pH 5.3. The behavior of this chimeric peptide approximates the binding of a 1:1 solution mixture of two peptides to MHC II, where the more stable complex is selected over time. We have also found that a chimeric peptide and a human MHC II, HLA-DR40401, form isomers with relative association rates to DR0401 at pH 5.3 of 15% for one isomer and 85% for the second isomer.     

Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles.

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.     

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.     

Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.     

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading

Background: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide ‘editor’, favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive.Results: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM.Conclusions: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.     

Kinetics of peptide binding to the class II MHC protein I-Ek

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.     

H2-Mbeta 1 and H2-Mbeta 2 heterodimers equally promote clip removal in I-A(q) molecules from autoimmune-prone DBA/1 mice

Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4(+) helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4(+) T cells. In contrast to the HLA-DM region in humans, the beta-chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta1 and Mbeta2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-A(q) alone or in the presence of H2-Malphabeta1 or H2-Malphabeta2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-A(q) molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-A(q) molecules. Moreover, I-A(q) cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-A(q) exhibits a high affinity for CLIP since virtually all I-A(q) molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.     

Cutting edge: editing of recycling class II:peptide complexes by HLA-DM.

HLA-DM catalyzes the exchange and selection of ligands for MHC class II molecules within mature endosomal/lysosomal compartments. Here, evidence is provided that DM edits peptides in early endosomes, thus influencing presentation via recycling class II molecules. Maximal class II-restricted presentation of an albumin-derived peptide, dependent on endocytosis and recycling class II molecules, was observed in cells lacking HLA-DM. DM editing of this epitope was observed in early endocytic compartments as shown using inhibitors of early to late endosomal transport. Editing was tempered by coexpression of HLA-DO, suggesting that DM:DO ratio may be important in guiding epitope editing in early endosomal compartments. Thus, HLA-DM appears to interact with, and edit epitopes displayed by, recycling class II molecules.     

Enhanced catalytic action of HLA-DM on the exchange of peptides lacking backbone hydrogen bonds between their N-terminal region and the MHC class II alpha-chain

The class II MHC homolog HLA-DM catalyzes exchange of peptides bound to class II MHC proteins, and is an important component of the Ag presentation machinery. The mechanism of HLA-DM-mediated catalysis is largely obscure. HLA-DM catalyzes exchange of peptides of varying sequence, suggesting that a peptide sequence-independent component of the MHC-peptide interaction could be involved in the catalytic process. Twelve conserved hydrogen bonds between the peptide backbone and the MHC are a prominent sequence-independent feature of the MHC-peptide interaction. To evaluate the relative importance of these hydrogen bonds toward HLA-DM action, we prepared peptide variants that lacked the ability to form one or more of the hydrogen bonds as a result of backbone amide N-methylation or truncation, and tested their ability to be exchanged by HLA-DM. We found that disruption of hydrogen bonds involving HLA-DR1 residues alpha51-53, a short extended segment at the N terminus of the alpha subunit helical region, led to heightened HLA-DM catalytic efficacy. We propose that those bonds are disrupted in the MHC conformation recognized by HLA-DM to allow structural transitions in that area during DM-assisted peptide release. These results suggest that peptides or compounds that bind MHC but cannot form these interactions would be preferentially edited out by HLA-DM.     

Distinct antigen MHC class II complexes generated by separate processing pathways.

The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (Ak). These complexes were also detected on the surface of antigen-presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS-stable, long-lived Ak complexes containing *HEL peptides and, unexpectedly, full-length *HEL. Both SDS-stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA-DM or proteases. SDS-stable *HEL-Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS-unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full-length protein as a product of antigen processing.     

Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.     

What to do with HLA-DO?

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.     

Antigen Presentation: Kissing cousins exchange CLIP

HLA-DM, a recent addition to the immunoglobulin family, is structurally most like class II molecules of the MHC. DM facilitates exchange of CLIP, a temporary filler of the groove of class II molecules, for antigenic peptides.     

Structural basis for the binding of an immunodominant peptide from myelin basic protein in different registers by two HLA-DR2 proteins

Susceptibility to multiple sclerosis (MS) is associated with certain MHC class II haplotypes, in particular HLA-DR2. Two DR beta chains, DRB1*1501 and DRB5*0101, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP 84-102) to MBP-specific T cells from MS patients. We have determined the crystal structure of HLA-DR2a complexed with MBP 86-105 to 1.9 A resolution. A comparison of this structure with that of HLA-DR2b complexed with MBP 85-99, reported previously, reveals that the peptide register is shifted by three residues, such that the MBP peptide is bound in strikingly different conformations by the two MHC molecules. This shift in binding register is attributable to a large P1 pocket in DR2a, which accommodates Phe92, in conjunction with a relatively shallow P4 pocket, which is occupied by Ile95. In DR2b, by contrast, the small P1 pocket accommodates Val89, while the deep P4 pocket is filled by Phe92. In both complexes, however, the C-terminal half of the peptide is positioned higher in the binding groove than in other MHC class II/peptide structures. As a result of the register shift, different side-chains of the MBP peptide are displayed for interaction with T cell receptors in the DR2a and DR2b complexes. These results demonstrate that MHC molecules can impose different alignments and conformations on the same bound peptide as a consequence of topological differences in their peptide-binding sites, thereby creating distinct T cell epitopes.     

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.     

Hydrogen bond integrity between MHC class II molecules and bound peptide determines the intracellular fate of MHC class II molecules.

MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability. In this study, we have used I-A(k), a class II molecule known to form strong pocket interactions with bound peptides, to probe the general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds contributed by positions 81 or 82 in the beta-chain of I-A(k) results in class II molecules that are internally degraded when trafficked through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs.     

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.     

HLA-DO as the Optimizer of Epitope Selection for MHC Class II Antigen Presentation

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.     

Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.