Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

alphavbeta5 integrin sustains growth of human pre-B cells through an RGD-independent interaction with a basic domain of the CD23 protein

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the alphavbeta5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg(172), Lys(173), and Cys(174) (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind alphavbeta5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the beta subunit of the alphavbeta5 integrin. The interaction between alphavbeta5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to alphavbeta5.     

HLA-E is the ligand for the natural killer cell CD94/NKG2 receptors

CD94/NKG2 is a recently described receptor present on natural killer (NK) cells and certain T cells that is composed of the CD94 chain covalently associated with a member of the NKG2 family of molecules. Both chains are glycosylated members of the C-type lectin superfamily. The CD94/NKG2 receptors are functionally heterogenous depending on which NKG2 family member is associated with CD94. Initially, it was thought that CD94/NKG2 receptors recognized a broad array of HLA-A, -B and -C (classical), as well as the nonclassical HLA-G, MHC class I molecules. Instead, recent data have suggested that this receptor is specific for HLA-E complexed with a peptide derived from the signal sequence (residues 3–11) of certain classical MHC class I molecules. Position 2 (residue 4) in the signal sequence derived peptides appears pivotal in determining whether the HLA-E/peptide complex confers resistance to NK-mediated lysis. The potential roles that the CD94/NKG2-HLA-E receptor ligand interaction might play in infection and tumor development are discussed.     

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.     

Dissection of the HLA-DR4 peptide repertoire in endocrine epithelial cells: strong influence of invariant chain and HLA-DM expression on the nature of ligands

Class II MHC (MHC II) expression is restricted to professional APCs and thymic epithelium but it also occurs in the epithelial cells of autoimmune organs which are the unique targets of the CD4 autoreactive T cells in endocrine autoimmune diseases. This specificity is presumably conditioned by an epithelium-specific peptide repertoire associated to MHC II at the cell surface. MHC II expression and function is dependent on the action of two main chaperones, invariant chain (Ii) and DM, whose expression is coregulated with MHC II. However, there is limited information about the in vivo expression levels of these molecules and uncoordinated expression has been demonstrated in class II-positive epithelial cells that may influence the MHC-associated peptide repertoires and the outcome of the autoimmune response. We have examined the pool of peptides associated to DR4 molecules expressed by a neuroendocrine epithelial cell and the consequences of Ii and DM coexpression. The RINm5F rat insulinoma cell line was transfected with HLA-DRB1*0401, Ii, and DM molecules in four different combinations: RIN-DR4, -DR4Ii, -DR4DM, and -DR4IiDM. The analysis of the peptide repertoire and the identification of the DR4 naturally processed ligands in each transfected cell were achieved by mass spectrometry. The results demonstrate that 1) the expression of Ii and DM affected the DR4 peptide repertoires by producing important variations in their content and in the origin of peptides; 2) these restrictions affected the stability and sequence of the peptides of each repertoire; and 3) Ii and DM had both independent and coordinate effects on these repertoires.     

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.     

Association of major histocompatibility complex II with cholesterol- and sphingolipid-rich membranes precedes peptide loading

Major histocompatibility complex class II protein (MHC II) molecules present antigenic peptides to CD4-positive T-cells. Efficient T cell stimulation requires association of MHC II with membrane microdomains organized by cholesterol and glycosphingolipids or by tetraspanins. Using detergent extraction at 37 degrees C combined with a modified flotation assay, we investigated the sequence of events leading to the association of MHC II with cholesterol- and glycosphingolipid-rich membranes (DRMs) that are distinct from tetraspanins. We find two stages of association of MHC II with DRMs. In stage one, complexes of MHC II and invariant chain, a chaperone involved in MHC II transport, enter DRMs in the Golgi stack. In early endosomes, these complexes are almost quantitatively associated with DRMs. Upon transport to late endocytic compartments, MHC II-bound invariant chain is stepwise proteolyzed to the MHC class II-associated invariant chain peptide (CLIP) that remains MHC II-bound and retains a preference for DRMs. At the transition between the two stages, CLIP is exchanged against processed antigens, and the resulting MHC II-peptide complexes are transported to the cell surface. In the second stage, MHC II shows a lower overall association with DRMs. However, surface MHC II molecules occupied with peptides that induce resistance to denaturation by SDS are enriched in DRMs relative to SDS-sensitive MHC II-peptide complexes. Likewise, MHC II molecules loaded with long-lived processing products of hen-egg lysozyme containing the immunodominant epitope 48-61 show a very high preference for DRMs. Thus after an initial mainly intracellular stage of high DRM association, MHC II moves to a second stage in which its preference for DRMs is modulated by bound peptides.     

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.     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Expression of invariant chain (CD 74) and major histocompatibility complex (MHC) class II antigens in the human fetus.

During the initiation of an immune response, antigen-presenting cells employ MHC class II antigens as key molecules to present small peptides to CD4-positive lymphocytes. The invariant chain (Ii; CD74) plays a critical role in this process by influencing the expression and peptide loading of the MHC class II molecules. Therefore, coordinate expression of these molecules is believed to play an important role in antigen presentation. This study explores the expression of these molecules in fetal tissues. Formalin-fixed, paraffin-embedded multi-organ tissue blocks from aborted fetuses (age range 7-22 weeks) were immunostained for Ii/CD74 and MHC class II antigens using commercially available monoclonal antibodies for Ii/CD74 (LN2) and MHC class II antigens (LN3), respectively. Coordinate staining for Ii/CD74 and MHC class II antigens was seen in the skin, proximal renal tubules, tips of small intestinal mucosa, and cells of the reticuloendothelial system, including the spleen and thymus. Expression of Ii/CD74, but not of MHC class II antigens, was seen in pulmonary alveolar epithelium in all cases and in testicular Leydig cells (11 of 11 testes examined). The distribution and intensity of staining did not change significantly with age. In conclusion, this study describes distribution of Ii/CD74 and MHC class II antigens in human fetal tissues. Coordinate expression of Ii/CD74 and MHC class II antigens was identified in most fetal tissues, but there were also notable exceptions. In all cases this took the form of expression of Ii/CD74 in the absence of MHC class II expression. Discordance was particularly striking in pulmonary alveolar epithelium and testicular Leydig cells. This suggests that the Ii/CD74 molecule has functional roles in addition to its role in antigen presentation.     

Engineering protein for X-ray crystallography: the murine Major Histocompatibility Complex class II molecule I-Ad.

Class II Major Histocompatibility (MHC) molecules are cell surface heterodimeric glycoproteins that play a central role in the immune response by presenting peptide antigens for surveillance by T cells. Due to the inherent instability of the class II MHC heterodimer, and its dependence on bound peptide for proper assembly, the production of electrophoretically pure samples of class II MHC proteins in complex with specific peptides has been problematic. A soluble form of the murine class II MHC molecule, I-Ad, with a leucine zipper tail added to each chain to enhance dimer assembly and secretion, has been produced in Drosophila melanogaster SC2 cells. To facilitate peptide loading, a high affinity ovalbumin peptide was covalently engineered to be attached by a six-residue linker to the amino terminus of the I-Adbeta chain. This modified I-Ad molecule was purified using preparative IEF and one fraction, after removal of the leucine zipper tails, produced crystals suitable for X-ray crystallographic analysis. The protein engineering and purification methods described here should be of general value for the expression of I-A and other class II MHC-peptide complexes.     

Phage matrix for isolation of glioma cell membrane proteins

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.     

Immunogenicity of tumor peptides: importance of peptide length and stability of peptide/MHC class II complex

Nonameric P815AB, a cytotoxic-T-lymphocyte-defined minimal core peptide encoded by the murine mastocytoma gene P1A, fails to initiate CD4⁺ cell-dependent reactivity in vivo to class-I-restricted epitopes when mice are administered peptide-pulsed dendritic cells. Effective immunization requires T helper effects, such as those mediated by coimmunization with class-II-restricted (helper) peptides or by the use of recombinant interleukin-12 (rIL-12). Although P815AB does possess class-II-restricted epitopes, they are likely suboptimal, resulting in poor affinity and/or stability of MHC/P815AB complexes and inadequate activation of the antigen-presenting cell function of dendritic cells. The present study has examined a series of longer, P815AB-centered peptides (11–14 amino acids in length, all P1A-encoded) for their ability to initiate CD4⁺ and CD8⁺ cell-mediated responses to the nonamer in vivo, their ability to bind class II MHC in vitro, and their ability to assemble class II molecules stably. By means of a class-I-restricted skin test assay in mice receiving peptide-pulsed dendritic cells, we found that a 12-mer and a 13-mer effectively immunized against the core P815AB peptide, and that this correlated with IL-2 production in vitro by CD4⁺ cells in response to the nonamer. In vitro studies, involving affinity-purified class II molecules, showed that the capacity to assemble class II molecules stably, more than the affinity for class II MHC, correlated with the ability of the different P815AB peptides to prime the host to the core peptide seen by the T cells. Received: 25 February 1999 / Accepted: 14 April 1999     

Assembly of HLA DR1 molecules translated in vitro: binding of peptide in the endoplasmic reticulum precludes association with invariant chain.

MHC class II molecules usually bind peptides in the endocytic pathway, but can also present endogenous peptides from newly synthesized proteins in a chloroquine-insensitive manner, suggesting that peptide binding might occur in the endoplasmic reticulum (ER). We used in vitro translation of HLA-DR1 class II molecules in the presence of microsomes to study peptide binding in the ER. Formation of functional class II molecules in vitro depends on formation of disulfide bridges in alpha and beta chains. The class II alpha beta heterodimers made by in vitro translation resemble class II molecules synthesized in cells in (i) their reactivity with conformation-specific antibodies, (ii) their assembly with Ii chain homotrimers, (iii) the generation of SDS-stable dimers upon peptide binding and (iv) their specificity of peptide binding. The assembly of class II molecules occurs via an alpha beta intermediate and can occur post-translationally, but only in intact microsomes. Class II alpha beta heterodimers are able to bind peptides in ER-derived microsomes, a process that precludes subsequent association of class II molecules with Ii chain. This mechanism might explain presentation of endogenous peptides by class II molecules.     

Breadth of the CD4+ T cell response to Anaplasma marginale VirB9-1, VirB9-2 and VirB10 and MHC class II DR and DQ restriction elements

MHC class II molecules influence antigen-specific CD4+ T lymphocyte responses primed by immunization and infection. CD4+ T cell responses are important for controlling infection by many bacterial pathogens including Anaplasma marginale and are observed in cattle immunized with the protective A. marginale outer membrane (OM) vaccine. Immunogenic proteins that comprise the protective OM vaccine include type IV secretion system (T4SS) proteins VirB9-1, VirB9-2 and VirB10, candidates for inclusion in a multiepitope vaccine. Our goal was to determine the breadth of the VirB9-1, VirB9-2 and VirB10 T cell response and MHC class II restriction elements in six cattle with different MHC class II haplotypes defined by DRB3, DQA and DQB allele combinations for each animal. Overlapping peptides spanning each T4SS protein were tested in T cell proliferation assays with autologous antigen-presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified; three representing two or more epitopes in VirB9-1, ten representing eight or more epitopes in VirB9-2 and seven representing seven or more epitopes in VirB10. Of the eight DRA/DRB3 molecules, four presented 15 peptides, which was biased as DRA/DRB3*1201 presented ten and DRA/DRB3*1101 presented four peptides. Four DQA/DQB molecules composed of two intrahaplotype and two interhaplotype pairs presented seven peptides, of which five were uniquely presented by DQ molecules. In addition, three functional mixed isotype (DQA/DRB3) restriction elements were identified. The immunogenicity and broad MHC class II presentation of multiple VirB9-1, VirB9-2 and VirB10 peptide epitopes justify their testing as a multiepitope vaccine against A. marginale.     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.