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.     

Reconstruction of a pathway of antigen processing and class II MHC peptide capture

Endocytosed antigens are proteolytically processed and small amounts of peptides captured by class II MHC molecules. The details of antigen proteolysis, peptide capture and how destruction of T-cell epitopes is avoided are incompletely understood. Using the tetanus toxin antigen, we show that the introduction of 3-6 cleavage sites is sufficient to trigger a partially unfolded conformation able to bind to class II MHC molecules. The known locations of T-cell epitopes and protease cleavage sites predict that large domains of processed antigen (8-35 kDa) are captured under these conditions. Remarkably, when antigen is bound to the B-cell antigen receptor (BCR), processing can trigger a concerted 'hand-over' reaction whereby BCR-associated processed antigen is captured by neighbouring class II MHC molecules. Early capture of minimally processed antigen and confinement of the processing and class II MHC loading reaction to the membrane plane may improve the likelihood of T-cell epitope survival in the class II MHC pathway and may help explain the reciprocal relationships observed between B- and T-cell epitopes in many protein antigens and autoantigens.     

An empirical method for the prediction of T-cell epitopes

Identification of T-cell epitopes from foreign proteins is the current focus of much research. Methods using simple two or three position motifs have proved useful in epitope prediction for major histocompatibility complex (MHC) class I, but to date not for MHC class II molecules. We utilized data from pool sequence analysis of peptides eluted from two HLA-DR13 alleles to construct a computer algorithm for predicting the probability that a given sequence will be naturally processed and presented on these alleles. We assessed the ability of this method to predict know self-peptides from these DR-13 alleles, DRB1 ^*1301 and *1302, as well as an immunodominant T-cell epitope. We also compared the predictions of this scoring procedure with the measured binding affinities of a panel of overlapping peptides from hepatitis B virus surface antigen. We concluded that this method may have wide application for the prediction of T-cell epitopes for both MHC class I and class II molecules.     

Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles

We introduced previously an on-line resource, RANKPEP that uses position specific scoring matrices (PSSMs) or profiles for the prediction of peptide-MHC class I (MHCI) binding as a basis for CD8 T-cell epitope identification. Here, using PSSMs that are structurally consistent with the binding mode of MHC class II (MHCII) ligands, we have extended RANKPEP to prediction of peptide-MHCII binding and anticipation of CD4 T-cell epitopes. Currently, 88 and 50 different MHCI and MHCII molecules, respectively, can be targeted for peptide binding predictions in RANKPEP. Because appropriate processing of antigenic peptides must occur prior to major histocompatibility complex (MHC) binding, cleavage site prediction methods are important adjuncts for T-cell epitope discovery. Given that the C-terminus of most MHCI-restricted epitopes results from proteasomal cleavage, we have modeled the cleavage site from known MHCI-restricted epitopes using statistical language models. The RANKPEP server now determines whether the C-terminus of any predicted MHCI ligand may result from such proteasomal cleavage. Also implemented is a variability masking function. This feature focuses prediction on conserved rather than highly variable protein segments encoded by infectious genomes, thereby offering identification of invariant T-cell epitopes to thwart mutation as an immune evasion mechanism.     

Flow cytometric analysis of peptide binding to major histocampatibility complex class I for hepatitis C virus core T-cell epitopes

BACKGROUND/METHODS: To characterize the repertoire of T-cell epitopes on the hepatitis C virus (HCV) core protein, we studied major histocompatibility complex (MHC) class I binding of 75 decapeptides on 20 human B-cell lines and murine spleen cells using a flow cytometric assay. The results were compared with MHC class I stabilization on T2 cells, the SYFPEITHI algorithm, and known T-cell epitopes from the literature. RESULTS: Binding of peptides proved to be specific for MHC class I molecules. We observed peak fluorescence signals at positions amino acids (aa) 35-44, aa 87-96, aa 131-140, and aa 167-176 in virtually all HLA-A2-positive cell lines. These sites corresponded to T-cell epitopes predicted by SYFPEITHI and the positions of known T-cell epitopes, whereas T2 stabilization was at variance for two peptides. The assay was applied to HLA-A2-negative cells and murine spleen cells without further modification, and identified additional peptides, corresponding to known T-cell epitopes. CONCLUSIONS: Peptide binding to different MHC class I alleles can be mapped rapidly by a flow cytometric assay and enables a first orientation on the sites of possible T-cell epitopes. Application of this assay to HCV core suggests a rather limited repertoire of epitopes in the Caucasoid population.     

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Troybodies and pepbodies

All antibodies (Abs) with effector function are produced in mammalian cells, whereas bacterial production is restricted to smaller targeting fragments (scFv and Fab) without effector functions. In this project, we isolated different peptides that bind one of several Ab effector molecules. We have developed bacterial expression vectors for direct cloning of these peptides as fusions to scFv and Fab, and have obtained targeting fragments that also have the ability to bind Ab effector molecules. Some of these fusions (pepbodies) may also initiate Ab effector functions. We have also genetically inserted T-cell epitopes into Abs with specificity for antigen-presenting cell (APC) surface molecules to target the Ab-T-cell epitope fusions (Troybodies) to APCs. The approach is to exchange loops in Ig constant domains with single copies of well-defined T-cell epitopes. We have shown that a number of such T-cell epitopes are loaded on to MHC class II on APCs and are presented to specific T-cells. An increase in T-cell activation of up to four orders of magnitude is achieved compared with synthetic peptide. Our current goal is to identify all the loops in all Ig constant domains that may be loaded with T-cell epitopes to produce a multi-vaccine.     

Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression

Simian immunodeficiency virus (SIV) infection of the rhesus macaque is currently the best animal model for AIDS vaccine development. One limitation of this model, however, has been the small number of cytotoxic T-lymphocyte (CTL) epitopes and restricting major histocompatibility complex (MHC) class I molecules available for investigating virus-specific CTL responses. To identify new MHC class I-restricted CTL epitopes, we infected five members of a family of MHC-defined rhesus macaques intravenously with SIV. Five new CTL epitopes bound by four different MHC class I molecules were defined. These included two Env epitopes bound by Mamu-A*11 and -B*03 and three Nef epitopes bound by Mamu-B*03, -B*04, and -B*17. All four restricting MHC class I molecules were encoded on only two haplotypes (b or c). Interestingly, resistance to disease progression within this family appeared to be associated with the inheritance of one or both of these MHC class I haplotypes. Two individuals that inherited haplotypes b and c separately survived for 299 and 511 days, respectively, while another individual that inherited both haplotypes survived for 889 days. In contrast, two MHC class I-identical individuals that did not inherit either haplotype rapidly progressed to disease (survived <80 days). Since all five offspring were identical at their Mamu-DRB loci, MHC class II differences are unlikely to account for their patterns of disease progression. These results double the number of SIV CTL epitopes defined in rhesus macaques and provide evidence that allelic differences at the MHC class I loci may influence rates of disease progression among AIDS virus-infected individuals.     

Prediction and Molecular Modeling of T-cell Epitopes Derived from Placental Alkaline Phosphatase for use in Cancer Immunotherapy

Abstract

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYF- PEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.     

The relationship between predicted peptide-MHC class II affinity and T-cell activation in a HLA-DRbeta1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4+ T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide-DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC-peptide-TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-gamma) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide-DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

Design and use of conditional MHC class I ligands

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.     

Towards the in silico identification of class II restricted T-cell epitopes: a partial least squares iterative self-consistent algorithm for affinity prediction

MOTIVATION: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. RESULTS: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 peptides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r(pred) = 0.593 and r(pred) = 0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. AVAILABILITY: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHCPred.     

Microbial superantigens: from structure to function

Superantigens are highly potent immune stimulators with a unique ability to interact simultaneously with MHC class II molecules and T cell receptors, forming a trimolecular complex that induces profound T-cell proliferation and massive cytokine production. Recent structural studies have provided a wealth of information regarding these complex interactions, and it is now emerging that, despite their common 3-D architecture, superantigens are able to crosslink MHC class II molecules and T cell receptors in a variety of ways.     

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.     

The relationship between predicted peptide–MHC class II affinity and T-cell activation in a HLA-DRβ1*0401 transgenic mouse model

The HLA-DRB1*0401 MHC class II molecule (DR4) is genetically associated with rheumatoid arthritis. It has been proposed that this MHC class II molecule participates in disease pathogenesis by presenting arthritogenic endogenous or exogenous peptides to CD4⁺ T cells, leading to their activation and resulting in an inflammatory response within the synovium. In order to better understand DR4 restricted T cell activation, we analyzed the candidate arthritogenic antigens type II collagen, human aggrecan, and the hepatitis B surface antigen for T-cell epitopes using a predictive model for determining peptide–DR4 affinity. We also applied this model to determine whether cross-reactive T-cell epitopes can be predicted based on known MHC–peptide–TCR interactions. Using the HLA-DR4-IE transgenic mouse, we showed that both T-cell proliferation and Th1 cytokine production (IFN-γ) correlate with the predicted affinity of a peptide for DR4. In addition, we provide evidence that TCR recognition of a peptide–DR4 complex is highly specific in that similar antigenic peptide sequences, containing identical amino acids at TCR contact positions, do not activate the same population of T cells.     

Genetic approaches for the induction of a CD4+ T cell response in cancer immunotherapy

Recently, it has become more and more obvious that not only CD8+ cytotoxic T lymphocytes, but also CD4+ T helper cells are required for the induction of an optimal, long-lasting anti-tumor immune response. CD4+ T helper cells, and in particular IFN-gamma-secreting type 1 T helper cells, have been shown to fulfill a critical function in the mounting of a cancer-specific response. Consequently, targeting antigens into MHC class II molecules would greatly enhance the efficacy of an anti-cancer vaccine. The dissection of the MHC class II presentation pathway has paved the way for rational approaches to achieve this goal: novel systems have been developed to genetically manipulate the MHC class II presentation pathway. First, different genetic approaches have been used for the delivery of known epitopes into the MHC class II processing pathway or directly onto the peptide-binding groove of the MHC molecules. Second, several strategies exist for the targeting of whole tumor antigens, containing both MHC class I and class II restricted epitopes, to the MHC class II processing pathway. We review these data and describe how this knowledge is currently applied in vaccine development.     

Roles for asparagine endopeptidase in class II MHC-restricted antigen processing

The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.