T-cell epitope mapping using the ELISPOT approach

The ELISPOT assay is particularly well suited to measure both clonal size and effector function of low-frequency antigen-specific T-cell populations directly ex vivo. Typically, an ELISPOT assay is performed with a freshly obtained sample (or cryopreserved sample) using less than 24h of culture. Additionally, this assay allows for the simultaneous analysis of hundreds of variables in parallel from a single tissue specimen. Because of these capabilities, this assay has found widespread use in the context of direct ex vivo immune diagnostic monitoring in humans. Herein we describe the rationale, methodology, and useful hints for performing T-cell epitope mapping using ELISPOT analysis, and review typical results of such an analysis.     

Use of combinatorial peptide libraries for T-cell epitope mapping

T lymphocytes play important roles not only in infectious diseases and autoimmunity, but also in immune responses against tumors. For many of these disorders, the relevant target antigens are not known. Designing effective methods that allow the search for T-cell epitopes is therefore an important goal in the areas of infectious diseases, oncology, vaccine development, and numerous other biomedical specialties. So far, the strategies used to examine T-cell recognition have been based largely on mapping T-cell epitopes with overlapping peptides from known proteins or with entire proteins, e.g., from a specific virus, bacterium, or human tissue. These approaches are tedious and have a number of limitations. It is, for example, almost impossible to isolate T cells that infiltrate an organ or infectious site and identify their specificity unless one already has a concept as to which antigens may be relevant. During recent years, a number of laboratories have developed less biased approaches that employ either the selection of putative T-cell epitopes based on the prediction of binding to certain major histocompatibilty complex (MHC) molecules and peptide or protein libraries that have been generated in expression systems, e.g. phage, or rely on combinatorial peptide chemistry. The latter technique has been refined by a number of laboratories including ours. Bead-bound or, preferably, positional scanning synthetic and soluble combinatorial peptide libraries allow the identification of T-cell epitopes within complex mixtures of proteins even for T cells that have been expanded from an organ infiltrate with a polyclonal stimulus. The practical steps that are involved in the latter method are described in this article.     

Interaction of dendritic cells with mycobacteria: where the action starts

Dendritic cells (DC) are the major antigen-presenting cells in the induction of cellular responses to intracellular pathogens, such as mycobacteria. Recent studies have shown that they also play a critical role in the regulation of immune responses. The interaction of DC with microbial antigens may be the controlling factor in the development of a Th1-orientated protective immunity. Analysis of the innate response of DC to mycobacteria and the involvement of the DC receptors in antigen recognition have highlighted the pivotal role of these cells in T-cell activation. Mycobacteria-infected DC have an enhanced capacity to release pro-inflammatory cytokines and chemokines and are potent inducers of interferon-gamma-producing cells in vivo. Therefore, DC manipulation for maximal antigen presentation and Th1 cytokine production may form the basis of a new generation of vaccines, with improved efficacy against mycobacterial infections.     

Anti-alpha 1-6 epitope, specificity of a T-cell hybrid-secreted factor: affinity- and ion-exchange chromatographic separation

We have studied the specificity of the products of a "T-cell" hybridoma, Th 1, a fusion product of AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized mice, which has been shown to affect the anti-Dx but also the anti-SRC response from culture supernatants and ascitic fluids. The anti-Dx-affecting material was separated from unspecific effector molecules by Sephadex affinity chromatography combined with HPLC DEAE chromatography and gel filtration. The activities of fractions were tested for their effects on anti-Dx, anti-SRC, and anti-SSS-III IgM responses. We show that the anti-Dx response-affecting material binds to Sephadex. Its Ig contamination can be reduced by two DEAE chromatographies at pH 6 and 8.1. At pH 8.1 it starts eluting with 0.13 M NaCl, but is still contaminated with materials that affect the anti-SRC and to a smaller extent the anti-SSS-III response. On gel filtration it localizes in the area of 100-40 kDa. The effects of the active material on anti-Dx IgM varied from suppression to enhancement. The details of that effect are largely unknown but three other findings further confirm the Dx specificity of Th 1 products. The growth of Th 1 in mice induces the production of anti-Dx IgA, detectable in their sera with ELISA. The priming of mice with Th 1 products affects the magnitudes of anti-Dx IgM PFC responses to the subsequent immunization with Dx with or without the product. The binding to Dx of material from in vivo active fractions can be verified in the ELISA with an antiserum produced against Th 1.     

Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses

In the past few years, the technical breakthrough in generating MHC-peptide tetramers has revolutionized the analysis of T-cell responses. The major advantage of this technique over currently available methods is the ability of these tetramers to label T lymphocytes according to their antigenic specificity. The present review describes some technical aspects of tetramers generation and discusses some of the numerous possibilities opened up by this new technology.     

Pepitope: epitope mapping from affinity-selected peptides

Identifying the epitope to which an antibody binds is central for many immunological applications such as drug design and vaccine development. The Pepitope server is a web-based tool that aims at predicting discontinuous epitopes based on a set of peptides that were affinity-selected against a monoclonal antibody of interest. The server implements three different algorithms for epitope mapping: PepSurf, Mapitope, and a combination of the two. The rationale behind these algorithms is that the set of peptides mimics the genuine epitope in terms of physicochemical properties and spatial organization. When the three-dimensional (3D) structure of the antigen is known, the information in these peptides can be used to computationally infer the corresponding epitope. A user-friendly web interface and a graphical tool that allows viewing the predicted epitopes were developed. Pepitope can also be applied for inferring other types of protein-protein interactions beyond the immunological context, and as a general tool for aligning linear sequences to a 3D structure. AVAILABILITY: http://pepitope.tau.ac.il/     

Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.     

Dendritic cell immunotherapy: mapping the way

Dendritic cells (DCs) are the professional antigen-presenting cells of the immune system, with the potential to either stimulate or inhibit immune responses. Exploiting the immune-regulatory capacities of dendritic cells holds great promise for the treatment of cancer, autoimmune diseases and the prevention of transplant rejection. Although early clinical trials indicate that DC vaccines can induce immune responses in some cancer patients, careful study design and use of standardized clinical and immunological criteria are needed.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

Peptide epitope mapping in vaccine development: Introduction

Protection from infectious disease by the host immune response requires specific molecular recognition of unique antigenic determinants of a given pathogen. An epitope is an antigenic determinant which: 1) specifically stimulates the immune response (either B or T cell mediated); and 2) is acted upon by the products of these protective mechanisms. In B cell immunity, antibodies produced from stimulation by specific epitopes recognize and bind to these same antigenic structures. Identification of protective epitopes is extremely valuable to successful vaccine development. In order to be protective these antibodies must, in addition to recognition and binding, interfere with some vital step in pathogenesis such as adherence or toxin action. Protein B cell epitopes are frequently composed of the side chains (R-groups) of the amino acids found at solvent-exposed surfaces. These epitopes are classified as continuous (also linear or sequential) if composed of a single antibody-recognizing element located at a single locus of the primary structure. They are discontinuous (or assembled) if more than one physically separated entity is involved. T cell epitopes are peptides on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells) that are bound to major histocompatibility proteins; the T cell recognizes this peptide-MHC complex. Received 12 August 1996/ Accepted in revised form 03 November 1996     

Eimeria tenella: B-cell epitope mapping following primary and secondary infections

Immunisation against coccidiosis has become more reliable and effective with improved administration techniques for new vaccines. On the other hand, an ideal coccidial vaccine should contain both B- and T-cell immunogenic epitopes. Fine specificity of B-cell epitopes recognised by antibodies prepared following primary and secondary infections with Eimeria tenella were studied using "PepScan" techniques. Mapping of B-cell epitopes within an antigenic sequence from E. tenella showed that four distinct types of epitopes were recognised by the host immune system during the primary and secondary infections with the parasite. These observations demonstrated that new epitopes are also involved in induction of antibody responses following the secondary infection.     

Candidate epitope identification using peptide property models: application to cancer immunotherapy

Peptides derived from pathogens or tumors are selectively presented by the major histocompatibility complex proteins (MHC) to the T lymphocytes. Antigenic peptide-MHC complexes on the cell surface are specifically recognized by T cells and, in conjunction with co-factor interactions, can activate the T cells to initiate the necessary immune response against the target cells. Peptides that are capable of binding to multiple MHC molecules are potential T cell epitopes for diverse human populations that may be useful in vaccine design. Bioinformatical approaches to predict MHC binding peptides can facilitate the resource-consuming effort of T cell epitope identification. We describe a new method for predicting MHC binding based on peptide property models constructed using biophysical parameters of the constituent amino acids and a training set of known binders. The models can be applied to development of anti-tumor vaccines by scanning proteins over-expressed in cancer cells for peptides that bind to a variety of MHC molecules. The complete algorithm is described and illustrated in the context of identifying candidate T cell epitopes for melanomas and breast cancers. We analyzed MART-1, S-100, MBP, and CD63 for melanoma and p53, MUC1, cyclin B1, HER-2/neu, and CEA for breast cancer. In general, proteins over-expressed in cancer cells may be identified using DNA microarray expression profiling. Comparisons of model predictions with available experimental data were assessed. The candidate epitopes identified by such a computational approach must be evaluated experimentally but the approach can provide an efficient and focused strategy for anti-cancer immunotherapy development.     

Fine specificity of neonatal lymphocytes to an abundant malaria blood-stage antigen: epitope mapping of Plasmodium falciparum MSP1(33)

Cord blood T cells have been reported to respond to a variety of exogenous Ags, including environmental allergens and various viruses and parasites, as demonstrated by enhanced proliferation and cytokine secretion. This finding is evidence that Ags in the maternal environment transplacentally prime and result in fetal development of memory T cells. Some studies suggest these neonatal T cell responses may arise by nonspecific activation of T cells that express TCRs with low binding affinity, thus lacking fine lymphocyte specificity. To address this question, we examined malaria Ag stimulation of human cord and adult blood mononuclear cells in samples from residents of a malaria endemic area in Kenya. We constructed overlapping 18-mer peptides derived from sequences contained in dimorphic alleles of the C-terminal 33-kDa fragment of Plasmodium falciparum merozoite protein 1. This study identified a dominant T cell epitope for one MSP1(33) allele (MAD20) and two T cell epitopes for the second allele (K1); these epitopes were nonoverlapping and allele specific. In a given donor, peptide-specific proliferation and IFN-gamma secretion were highly concordant. However, IL-10 and IL-13 secretion were not correlated. Importantly, the fine specificity of lymphocyte proliferation and cytokine secretion in cord and adult blood mononuclear cells was similar. Cord blood cells obtained from malaria-infected pregnant women were 4-fold more likely to acquire a peptide-specific immune response. We conclude that the fetal malaria response functions in a fully adaptive manner and that this response may serve to help protect the infant from severe malaria during infancy.     

T cell epitope identification for bovine vaccines: an epitope mapping method for BoLA A-11

T cell responses play an important role in immunity to parasites and other microbial agents of infectious diseases, therefore a number of T cell-directed vaccines are in development. Computer-driven algorithms that facilitate the discovery of T cell epitopes from protein and genome sequences are now being used to accelerate preclinical studies of human vaccines. Similar tools are not yet available for predicting T cell epitopes for animal vaccines, but there may be sufficient data available to begin the process of compiling the algorithms. We describe the construction of a novel mathematical 'matrix' that describes the properties of bovine major histocompatibility complex (BoLA) system antigen (BoLA) A-11 peptide ligands, developed for use with EpiMatrix, an existing T cell epitope-mapping algorithm. An alternative means of developing BoLA matrices, using the pocket profile method, is also discussed. Matrices such as the one described here may be used to develop T cell epitope-mapping tools for cattle and other ruminants. Epitope-mapping algorithms offer a significant advantage over other methods of epitope selection, such as the screening of synthetic overlapping peptides, because high throughput screening can be performed in silico, followed by ex vivo confirmatory studies. Furthermore, using epitope-mapping algorithms, putative T cell epitopes can be derived directly from genomic sequences, allowing researchers to circumvent labor-intensive cloning steps in the genome-to-vaccine discovery pathway.     

Human CD30: structural implications from epitope mapping and modeling studies

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.