首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.  相似文献   

2.
We generated transgenic mice that expressed hen egg-white lysozyme (HEL) under a class II MHC promoter. The A7 line expressed HEL with a point mutation in the Asp(52) residue, the main anchor amino acid responsible for the selection of the chemically dominant family of peptides (52-60) by I-A(k) molecules. Mice expressing HEL with Ala(52) were completely unresponsive when immunized with the same protein, i.e., HEL A52. However, the same mice immunized with wild-type HEL elicited T cells that recognized a conformation of the 52-61 core sequence uniquely different between Asp(52) and Ala(52) containing peptides. Importantly, some T cells also recognized the HEL A52 peptide given exogenously but not the same peptide processed from HEL A52 protein. Thus, a core MHC anchor residue influences markedly the specificity of the T cells. We discuss the relevance of these findings to autoimmunity and vaccination with altered peptides.  相似文献   

3.
We have analyzed the interaction of the hen egg-white lysozyme (HEL) peptide 107-116 with the MHC class II molecule I-Ek, using truncated and single residue substitution analogues to measure activation of I-Ek-restricted, 107-116-specific T cell hybridomas and competition for Ag presentation by I-Ek molecules. These results have been compared with previous findings on the interaction of the same peptide with the I-Ed molecule. Stimulation of T cell hybridomas by truncated peptides defines the sequence 108-116 as the minimum epitope necessary for activation of both I-Ek- and I-Ed-restricted T cell hybridomas. Substitution analysis pinpoints three residues (V109, A110, and K116) in the sequence 108-116 as being critical for binding to I-Ek molecules and demonstrates the involvement of most other residues in recognition by T cells. Results previously obtained for binding of HEL 107-116 to I-Ed molecules indicated that peptide residues R112, R114, and K116 were critical for interaction with I-Ed. Comparison of these results indicates a difference in the likely MHC contact residues between the HEL sequence 108-116 and I-Ed or I-Ek molecules, suggesting that the same HEL peptide assumes a different conformation in the binding site of these two MHC molecules. This in turn affects residues interacting with the specific T cell receptor. According to the hypothetical tridimensional structure predicted for class II molecules, the difference in MHC contact residues observed within the sequence 108-116 can be related to polymorphic amino acids in the binding site of I-Ek and I-Ed molecules. A search through published binding data for a common pattern in this and other I-Ek-binding peptides has permitted us to derive a possible motif for predicting peptide binding to I-Ek molecules. This putative motif was tested by determining binding to I-Ek of an unbiased panel of about 150 synthetic peptides. Binding data indeed demonstrate the presence of this motif in the majority of good binders to I-Ek molecules.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
Association between the class II major histocompatibility complex (MHC) and the class II invariant chain-associated peptide (CLIP) occurs naturally as an intermediate step in the MHC class II processing pathway. Here, we report the crystal structure of the murine class II MHC molecule I-A(b) in complex with human CLIP at 2.15A resolution. The structure of I-A(b) accounts, via the peptide-binding groove's unique physicochemistry, for the distinct peptide repertoire bound by this allele. CLIP adopts a similar conformation to peptides bound by other I-A alleles, reinforcing the notion that CLIP is presented as a conventional peptide antigen. When compared to the related HLA-DR3/CLIP complex structure, the CLIP peptide displays a slightly different conformation and distinct interaction pattern with residues in I-A(b). In addition, after examining the published sequences of peptides presented by I-A(b), we discuss the possibility of predicting peptide alignment in the I-A(b) binding groove using a simple scoring matrix.  相似文献   

7.
We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.  相似文献   

8.
The protein hen egg white lysozyme (HEL) contains two segments, in tandem, from which two families of peptides are selected by the class II molecule I-Ak, during processing. These encompass peptides primarily from residues 31-47 and 48-63. Mutant HEL proteins were created with changes in residues 52 and 55, resulting in a lack of binding and selection of the 48-63 peptides to I-Ak molecules. Such mutant HEL proteins donated the same amount of 31-47 peptide as did the unmodified protein. Other mutant HEL molecules containing proline residues at residue 46, 47, or 48 resulted in extensions of the selected 31-47 or 48-62 families to their overlapping regions (in the carboxyl or amino termini, respectively). However, the amount of each family of peptide selected was not changed. We conclude that the presence or absence of the major peptide from HEL does not influence the selection of other epitopes, and that these two families are selected independently of each other.  相似文献   

9.
Class II MHC molecules on the surface of an APC present immunogenic peptides derived mainly from exogenous proteins to CD4+ T cells. During its transport to the cell surface, class II molecules intersect the endocytic pathway where they acquire peptides derived from endocytosed proteins. However, class II-restricted presentation of endogenously derived peptides can also occur. The current studies were undertaken to examine the ability of different types of APC to generate and present four different T cell determinants derived from an endogenous, nonsecreted, truncated form of hen-egg white lysozyme (HEL[1-80]-Kk). This was compared with the ability of these APC to generate the same determinants from exogenous HEL. All the peptides derived from endogenous HEL[1-80]-Kk tested, were presented by B cells to HEL-specific T cell hybridomas with an efficiency similar to presentation of the same determinants from exogenous HEL. In contrast, an I-Ak-bearing rat fibroblast was unable to generate the HEL peptide 25-43 from exogenous HEL, but could efficiently produce it from endogenous HEL[1-80]-Kk. The results indicate first, that peptides derived from an endogenous Ag can be presented by MHC class II molecules with an efficiency comparable to that of the presentation of the exogenous Ag. Second, that Ag-presenting B cells can generate the same repertoire of antigenic peptides from endogenous Ag as those generated from the exogenous protein. And third, that in contrast to B cells, certain "nonprofessional" APC can generate, from an endogenous protein, T cell determinants distinct from those generated after endocytosis of the exogenous protein. These results suggest that processing of exogenous and endogenous Ag by different APC take place in different intracellular compartments.  相似文献   

10.
Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.  相似文献   

11.
The epitopes recognized by pathogenic T cells in systemic autoimmune disease remain poorly defined. Certain MHC class II-bound self peptides from autoimmune MRL/lpr mice are not found in eluates from class II molecules of MHC-identical C3H mice. Eleven of 16 such peptides elicited lymph node cell and spleen cell T cell proliferation in both MRL/lpr (stimulation index = 2.03-5.01) and C3H mice (stimulation index = 2.03-3.75). IL-2 and IFN-gamma production were detected, but not IL-4. In contrast to what was seen after immunization, four self peptides induced spleen cell proliferation of T cells from naive MRL/lpr, but not from C3H and C57BL/6.H2(k), mice. These peptides were derived from RNA splicing factor SRp20, histone H2A, beta(2)-microglobulin, and MHC class II I-A(k)beta. The first three peptides were isolated from I-E(k) molecules and the last peptide was bound to I-A(k). T cell responses, evident as early as 1 mo of age, depended on MHC class II binding motifs and were inhibited by anti-MHC class II Abs. Thus, although immunization can evoke peripheral self-reactive T cells in normal mice, the presence in MRL/lpr mice of spontaneous T cells reactive to certain MHC-bound self peptides suggests that these T cells actively participate in systemic autoimmunity. Peptides eluted from self MHC class II molecules may yield important clues to T cell epitopes in systemic autoimmunity.  相似文献   

12.
The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.  相似文献   

13.
Circular dichroism (CD) spectra of class II MHC peptides revealed the alpha-helical conformation of superantigen-binding peptides I-A beta b(60-90), I-A beta b(65-85), and I-A alpha b(51-80), but not the nonbinding peptide I-A beta b(80-100). These CD spectra provide biophysical evidence for the alpha-helicity of class II MHC molecular binding sites for the superantigen, staphylococcal enterotoxin A (SEA). Alanine-substituted analogs of the SEA binding-site peptide, I-A beta b(65-85), were used to implicate beta-chain residues 72 and 80 in class II MHC-SEA binding. The data support SEA binding away from the class II antigen binding cleft along the faces of the alpha-helices.  相似文献   

14.
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.  相似文献   

15.
Directed evolution of a single-chain class II MHC product by yeast display   总被引:1,自引:0,他引:1  
Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.  相似文献   

16.
KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.  相似文献   

17.
We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-A(k) molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.A(k) line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.  相似文献   

18.
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.  相似文献   

19.
R Lindner  E R Unanue 《The EMBO journal》1996,15(24):6910-6920
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.  相似文献   

20.
The binding of antigenic peptide to class II MHC is mediated by hydrogen bonds between the MHC and the peptide, by salt bridges, and by hydrophobic interactions. The latter are confined to a number of deeper pockets within the peptide binding groove, and peptide side chains that interact with these pockets are referred to as anchor residues. T cell recognition involves solvent-accessible peptide residues along with minor changes in MHC helical pitch induced by the anchor residues. In class I MHC there is an added level of epitope complexity that results from binding of longer peptides that bulge out into the solvent-accessible, T cell contact area. Unlike class I MHC, class II MHC does not bind peptides of discrete length, and the possibility of peptide bulging has not been clearly addressed. A peptide derived from position 24-37 of integrin beta(3) can either bind or not bind to the class II MHC molecule HLA DRB3*0101 based on a polymorphism at the P9 anchor. We show that the loss of binding can be compensated by changes at the P10 position. We propose that this could be an example of a class II peptide bulge. Although not as efficient as P9 anchoring, the use of P10 as an anchor adds another possible mechanism by which T cell epitopes can be generated in the class II presentation system.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号