Biomedical subjects
Publications and source records attributed to H M Grey.
X-ray crystallographic and peptide-MHC binding studies have begun to clarify the interaction between antigenic peptides and MHC proteins at the molecular level. At the same time, our understanding of the mechanisms of peptide-MHC interactions in physiologic cellular conditions has been significantly expanded by the isolation and characterization of naturally processed antigenic peptides.
The interaction between synthetic peptides and A2.1 class I MHC molecules has been investigated using an inhibition of Ag presentation assay and unbiased peptide sets derived of either viral or eucaryotic origin. For the various sets, strong binding (defined as significant inhibition at the 30 micrograms/ml level) was detected in 7 to 46% of the peptides tested, with an overall frequency of 26%. A set of self-peptides derived from human beta 2 microglobulin was also included in the study. In this case, strong binding was detected in 3 of 15 peptides (20%), thus formally demonstrating a lack of self-/non-self-discrimination at the level of class I molecules. When the whole A2.1-binding database of 105 peptides thus generated was examined by sequence analysis, a significant correlation was found with a recently proposed A2.1-binding motif, whereas no particular positive or negative association was detected between the capacity to bind A2.1 and three different class II alleles (DR1, DR5, and DR7). Finally, using this approach, several peptides capable of binding both A2.1 and multiple DR alleles have been identified, suggesting possible candidates for development of peptide vaccines eliciting both class I and class II restricted responses.
Using a dinitrophenylated and biotinylated peptide antigen, we have developed an affinity chromatography procedure to purify complexes of a given peptide species and a given class II major histocompatibility complex antigen away from class II molecules occupied by other peptides. We show that hen egg lysozyme peptide-I-Ed complexes purified according to this procedure have a greatly enhanced capacity to activate hen egg lysozyme-specific T cells but have lost the capacity to activate three different alloreactive T-cell hybridomas. These data demonstrate that the class II molecule in and of itself is not sufficient to activate alloreactive T cells. Rather, the data suggest that recognition of specific complexes formed between allo-class II and particular autologous peptides may be required. Alternatively, alloreactive T cells may be recognizing "empty" major histocompatibility complex molecules.
We have analyzed the stability of Ag-class II complexes on the surface of live APC. It was found that the disappearance of complexes formed by I-Ed and the hen egg lysozyme 107-116 peptide is twice as rapid on the surface of live, as compared to glutaraldehyde-fixed APC. Moreover, the addition of peptides with a high affinity for I-Ed could reduce the half-life of Ag-class II complexes on either live or fixed APC. The same effect was detectable using purified class II molecules adhered on plastic microtiter wells, and even in the case of complexes formed in vitro between radiolabeled Ag and purified class II molecules. This effect of accelerated dissociation appeared to be specific because only I-Ed binding peptides were able to accelerate the dissociation of the hen egg lysozyme 107-116/I-Ed complex either on the surface of cells or in purified form in solution, and high affinity I-Ed binders did not affect the half-life of purified OVA 323-339/I-Ad complexes. These results demonstrate that free ligand can influence the kinetics of dissociation of class II-peptide complexes, and therefore suggest that a mechanism other than the classical first order kinetics may be involved in this process.
Single amino acid substitutions of Ag and MHC were used to analyze the fine structure of the influenza hemagglutinin (HA)-derived epitope (HA 307-319) recognized in the context of DR7 molecules by a T cell clone. Putative T cell (HA 308, 310, 311, 313, and 316) and DR (HA 309, 312, and 317) contact residues of the Ag were identified by the use of single amino acid-substituted analogs that were tested for their T cell-activating and DR-binding capacities. The peptide-DR7-T cell interaction was further characterized by the use of a panel of 13 site-directed DR7 mutant transfectants analyzed for their capacity to present Ag to T cells, and for their purified mutant DR7 molecules to bind HA 307-319 or its single amino acid-substituted analogs. Eight mutants lost their Ag-presenting function, whereas only one had any decrease in peptide binding. Finally, for three of the mutants it was possible to correct the deleterious effects of mutation by using a particular single amino acid-substituted analog of the peptide molecule. The observed pattern of complementation led to a model that predicts that the Ag assumes an extended conformation, with a turn, in the binding groove, such that the following residues are in close proximity: DR 86-HA 309, DR 71-HA 312, DR 30-HA 314, and 315.
Peptides that bind with high affinity to major histocompatibility complex molecules could represent useful tools in treating class II-associated autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and multiple sclerosis. Although the concept has been validated in experiments with both purified receptor systems in vitro and cellular systems in vivo, many challenging problems need to be resolved before efficacious therapeutic agents are obtained.
We describe the establishment and validation, both at the biochemical and biological levels, of direct assays to measure the interactions between synthetic peptides and purified HLA class II molecules. The results of several independent approaches to define the structural requirements for the interaction of peptide-class II molecules are also described. Such approaches include random screening, sequence analysis, and site-directed mutagenesis. Finally, in vivo data illustrating the possibility that the competition at the level of the interactions between autoantigenic peptides and class II molecules could be a useful tool in the treatment of T cell-mediated autoimmune diseases are presented.
Blocking of the Ag presenting function of MHC by peptides capable of high affinity binding to this molecule has been proposed as a potential immunotherapeutic intervention in MHC-associated diseases. Recent studies have used this strategy to prevent the induction of experimental allergic encephalomyelitis (EAE) in mice. However, because of the close structural relationship between the inhibitor and encephalitogenic peptides, the results of these previous studies have been difficult to interpret with regard to whether MHC blockade was the mechanism by which the inhibitory peptides functioned. In our study, we have determined the capacity of unrelated peptides capable of binding with high affinity to IAs in inhibiting the induction of EAE in SJL/J mice after immunization with the autoantigenic peptide PLP 139-151. Prevention of the disease was accomplished by two methods: 1) when inhibitor was administered together with the encephalitogenic peptide at the time of immunization, as in previous studies, and 2) when inhibitor was administered at a separate site from the autoantigen 1 day before the immunization with that Ag. Inhibition was due to binding of the inhibitor to IAs, as evidenced by the fact that a control peptide incapable of binding to this MHC had no effect on the course of the disease. The finding that inhibitor could also be efficacious when administered at a separate site has implications for potential use of such a strategy to reverse ongoing autoimmune diseases. The inhibitor had to be present during the time of Ag stimulation, and had no long term inhibitory effects, in that a secondary immune response to the encephalitogenic peptide was not inhibited in animals given the inhibitory peptide before the induction of a primary response. This is compatible with the conclusion that MHC blockade was, in fact, the mechanism of the inhibition, rather than as a result of any long term suppressive effects on immunoreactive T cells. Finally, not only did administration of the inhibitory peptide lead to a prevention of the induction of EAE, but it could also be shown to decrease the T cell proliferative response in vitro to the autoantigen.
We have found that if core regions crucial for class II binding are incorporated in multiple copies in the same peptide molecule ("reiterative motifs"), marked enhancement of the binding capacity occurs. Isotype specificity (IAd vs IEd binding capacities) is retained in all three antigenic determinants so far analyzed (lambda rep 12-26, OVA 323-339, and hen egg lysozyme 105-120). The mechanism involved in such an effect is not clear, but experiments involving introduction of a peptide spacer between two repeated core regions do not support the notion that the effect is mediated by cross-linking of more than one MHC molecule, favoring the possibility that conformational effects or distinct subsites of interaction on the MHC molecule may be involved. Based on reiterative structures, a peptide molecule composed of only two different amino acids (Ala and His) has been produced that still retains a very high binding affinity. An 125I-radiolabeled form of this peptide has been used to demonstrate that the high binding detected is mediated by the same binding site involved in the interaction of IAd and OVA 323-339. Inhibition of Ag presentation studies further supports the immunologic relevance of the phenomena observed. Finally, we observed naturally occurring clustered binding sites in proximity of immunodominant protein regions, raising the possibility that the phenomenon might have a physiologic counterpart.
Major histocompatibility complex (MHC) molecules are exposed to large quantities of self and nonself antigens. It is not known what fraction of MHC molecules needs to be occupied by antigen to induce a T cell response. A quantitative study of naturally processed antigen indicated that T cells could be activated when only 0.03 percent of the total I-Ed purified from antigen-presenting cells (APCs) was occupied with antigen. B cells and macrophages processed hen egg lysozyme (HEL) with different efficiencies, but similar degrees of occupancy were required for T cell stimulation. Higher occupancy was needed for I-Ed-transfected L cells, possibly reflecting the requirement for other accessory molecules for efficient APC-T cell interaction.
The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.
Previous work from our and other laboratories indicates that T cells recognize a complex between the MHC restriction element and peptide antigen fragments. This paper reviews the structural characteristics of the formation of such a complex. By analyzing in detail the interactions between purified IA(d) and IE(d) molecules and their peptide ligands, we found that some structural characteristics apply to both antigen-MHC interactions. In particular, we found: 1) each MHC molecule is capable of binding many unrelated peptides through the same peptide-binding site; 2) despite this permissiveness of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity (IA(d) or IE(d)); 3) IA(d) and IE(d) molecules recognize different and independent structures on the antigen molecule; 4) only about 10% of the single amino acid substitutions tested on two IA(d)- and IE(d)-binding peptides had significant effect on their MHC-binding capacities, while over 80% of these substitutions significantly impaired T cell recognition of the Ia-peptide complex; 5) based on the segregation between residues that are crucial for T cell activation and Ia binding, the easiest model for the antigen-Ia-T-cell-receptor complex pictures the antigen molecule sandwiched in a planar conformation between the MHC and the T cell.
A series of analogue peptides have been generated, using as a template the core region of the OVA 323-339 peptide identified as critical in determining binding to I-Ad. Several of these "core extended" peptides had increased affinities for the I-Ad molecule compared to the native sequence, and were able to inhibit activation of an I-Ad-restricted T cell hybridoma in vitro. The induction of a T cell proliferative response to a peptide antigen could be inhibited by co-administration of core-extended peptide with antigen in the same adjuvant emulsion. Furthermore, inhibition also occurred when the inhibitor molecule was delivered separately one day before immunization. Finally, the induction of the autoimmune disease, experimental allergic encephalomyelitis (EAE), in susceptible mice could be reduced by the administration of a core-extended peptide with high affinity for the appropriate class II molecule. These findings have implications for the use of MHC antagonists in the control and treatment of MHC-associated autoimmune conditions in humans.
The T cell hybridomas 231F1 and 12H5 constitutively secrete glycosylation-inhibiting factor (GIF) and glycosylation-enhancing factor (GEF), respectively, which lack affinity for OVA-coupled Sepharose. When the 231F1 and 12H5 cells were stimulated by OVA-pulsed syngeneic macrophages, however, GIF and GEF produced by the cells had affinity for OVA. Both the OVA-binding GIF from the 231F1 cells and OVA-binding GEF from the 12H5 cells bound to a mAb against TCR-alpha beta and a mAb against TCR-alpha, suggesting a serologic relationship between TCR and OVA-binding factors. However, the OVA-binding GIF and GEF bound to mAb 14-12 and 14-30, respectively. Because these mAb do not bind TcR alpha beta-chains, it appears that the Ag-binding factors are different from TCR itself. The OVA-binding factors from both 12H5 cells and 231F1 cells do not bind to urea-denatured OVA. The binding of the factors to OVA Sepharose was inhibited by a peptide corresponding to residues 307-317 (P307-317) in the native OVA, but not by the peptide corresponding to residues 323-339 (P323-339). Furthermore, the OVA-binding factors bound to P306-319-coupled Sepharose but not to P323-339-coupled Sepharose, and were recovered by elution of the former Sepharose at acid pH. The binding of OVA to anti-OVA antibodies was not inhibited by either peptide. Inasmuch as the 231F1 cells and 12H5 cells can be stimulated by P307-317 in the context of a MHC product, it appears that the Ag-binding factors and TCR-alpha beta on the cell sources of the factors may recognize the same epitope in the OVA molecules. The results also showed that Ag-binding factors and antibodies recognize distinct epitopes in the Ag molecules.
Compelling evidence indicates that T cells recognize complexes formed by major histocompatibility complex-encoded molecules and antigenic peptide fragments. This is based largely on the ability of small synthetic peptides to substitute for naturally processed antigen in stimulating T cells. Naturally processed fragments of exogenous antigen are thought to arise by limited proteolytic degradation of native antigen inside acidic compartments of antigen-presenting cells, but until now no physiologically processed antigen has been directly analysed. Here we report the characterization of physiologically processed antigen eluted from mouse class II major histocompatibility complex I-Ed molecules. The antigenic material corresponds to a previously described antigenic determinant of hen egg lysozyme (HEL 107-116) and has a relative molecular mass Mr of about 2,000. HPLC analysis identified at least two or three separate molecular species, suggesting limited, albeit significant, heterogeneity of naturally processed peptides. Finally, under our experimental conditions, it was calculated that a substantial proportion (10-40%) of I-Ed molecules were occupied by these HEL-derived antigenic determinants.
The present study investigated the possibility that protein Ag fragments in the form of peptides could serve as the priming Ag in the generation of a MHC class I-restricted immune response. Trypsin-digested chicken ovalbumin (OVA-TD) fragments were used as the model Ag. The results demonstrate the peptides within OVA-TD, when injected into C57BL/6 mice, could prime T cells which lysed H-2b Ia-EL4 target cells in an OVA-TD-specific manner. In contrast to priming with OVA-TD, immunization of mice with intact OVA did not lead to generation of CTL against OVA-TD or OVA. Furthermore, target cells sensitized with intact OVA failed to be recognized by OVA-peptide-specific CTL indicating that the target cells serving as APC were unable to generate the relevant peptide determinants recognized by the T cells. These results support the idea that the processing pathway within APC for class I-restricted T cells may differ from that used for class II-restricted T cells. Using OVA-TD-specific CTL clones (phenotypically Thy 1+, CD8+, CD4-, Pgp-1+) isolated from primed animals to screen OVA-TD fractions separated by HPLC, two T cell peptide determinants were identified corresponding to OVA sequences 111-122 and 370-381. Both determinants were recognized by CTL clones in the context of the H-2Db molecule.
Here we have demonstrated that denatured, but not native protein antigens can interact with Ia molecules. Thus, the failure of native antigens to be recognized as such by T cells appears to be at least in part due to a deficient antigen/Ia interaction. These results also support previous observations that some T cells can recognize denatured antigens without a further processing requirement. Moreover, a striking correlation was observed between the in vitro binding pattern of denatured proteins and the pattern of restriction of T cell responses elicited by immunization with the native antigen, raising the possibility that an unfolding step may actually occur early during in vivo processing and influence the final outcome of Ia-restricted T cell responses.
In an attempt to define some of the conformational requirements for binding of the antigenic peptide OVA 323-336 to purified IAd molecules, three distinct experimental approaches were applied. First, the effect of introducing proline or glycine residues within the region of OVA 323-336 crucial for its IAd binding capacity was analyzed. In most instances these substitutions had little or no effect, suggesting that neither alpha-helical nor beta-sheet regular structures may be strictly required for productive interaction with MHC molecules. Some of the same substitutions were also found to have no effect on the capacity of the peptide to stimulate OVA 323-336 specific T cell hybridomas, suggesting that regular structures such as alpha-helices or beta-sheets may not be strictly required for T cell stimulation, either. Second, we introduced, within the OVA 323-336 molecule, structural modifications predicted to alter its dipole characteristics and stabilize helical structures. No improvement of the IAd binding capacity was detected following these structural alterations. Surprisingly, some but not others of these analogs displayed increased antigenicity for OVA 323-336 specific T cell hybridomas. Third, a panel of analogs of OVA 323-336 were synthesized in which the crucial IAd binding core region was linked to non-native sequences of differing conformational propensities. When 22 such analogs were tested for IAd binding, it was found that these non-native sequences could drastically influence the binding capacity, but no correlation was found between their effect and their alpha-helical, beta-sheet, or beta-turn conformational propensity as calculated by the Chou and Fasman algorithm. In summary, all the data presented herein suggest that, at least in the case of OVA 323-336 and IAd, the propensity of the antigen molecule to form secondary structures such as alpha-helices, beta-sheets, or beta-turns does not correlate with its capacity to bind MHC molecules.