T-cell epitope mapping using the ELISPOT approach
Section snippets
Why ELISPOT: a highly sensitive measure of the clonal size of antigen-specific T-cell populations in vivo
During an immune response low-frequency antigen-specific naive T cells clonally expand. The overall clonal size reached defines the effector cell mass and therefore the magnitude of the acquired immune repertoire. During initial encounter with the antigen, naive T cells differentiate into memory cells with specific cytokine producing or killing effector function that defines the quality of the immune response. The scope of immune diagnostics therefore has to account for both clonal size and
Why use peptide libraries: a comprehensive approach to define the engaged T-cell repertoire
The ELISPOT assay takes advantage of the fact that memory T-cell populations do not secrete effector cytokine until they encounter cognate antigen [9]. To detect such T cells, the appropriate form of cognate antigen needs to be provided. Because T cells recognize processed antigen, the mode of antigen administration and the type of APC defines what determinant is presented to the test cell population in the recall assay. Soluble antigens added to cell culture are preferentially presented on
Validation of peptide library approach
The basic tenet of testing libraries using an IFN-γ ELISPOT approach is that peptide-induced IFN-γ production signifies in vivo-primed memory cells. In the experimental setting, this issue has been addressed by testing naive versus immunized mice. BALB/c mice were immunized with hen egg lysozyme (HEL) using adjuvants that induce either type 1 or type 2 immunity [8]. HEL is one of the best defined prototype antigens with respect to determinant hierarchy/immunodominance. Splenocytes were assayed
Predicted versus actually recognized T-cell determinants
An increasing number of T cell determinants have been established for the different MHC alleles empirically and considerable progress has been made toward an understanding of MHC–peptide binding motifs [28], [29], [30], [31], [32], [33] and making predictions as to candidate T-cell determinants. Such approaches have offered the potential to select candidate peptides for immune monitoring when it is necessary to limit (for feasibility purposes) the repertoire of test peptides (e.g., when using
Cytokine-specific antibodies
Several vendors sell IFN-γ antibodies that perform excellently in ELISPOT assays, including Pharmingen/BD, R&D Systems, Endogen, and Mabtech. Antibodies that have been produced for flow cytometry application have frequently been deaggregated, which results in poor ELISPOT assay performance; one must be careful to use ELISPOT-grade reagents. Some vendors have established that their product permits immune monitoring at single-cell resolution (recommended). Because spot sizes, even for a T-cell
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Viral T-cell epitopes – Identification, characterization and clinical application
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2022, Veterinary MicrobiologyDevelopment of a novel ZIKV vaccine comprised of immunodominant CD4+ and CD8+ T cell epitopes identified through comprehensive epitope mapping in Zika virus infected mice
2021, VaccineCitation Excerpt :These include 15 pools selected from P1 ~ P21, and another 15 pools selected from P22 ~ P42 (Fig. 1B, Fig. 3A). Based on the peptide-matrix set-up analysis as previously described [25], we identified 225 individual probable epitope candidates (from a total of 427 peptides). These peptides were used individually as stimuli in the next rounds of IFN-γ Elispot assay, from which magnitude of responses stimulated by 94 representative peptides were shown in Fig. 2 and the most strongest ones were all included.
Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries
2021, Journal of Immunological MethodsCitation Excerpt :To harness the high-throughput screening efficiency of the ELISpot assay and to further maximize the availability of limited clinical samples, we employed an in vitro polyclonal expansion procedure using a CD3/CD4 bi-specific monoclonal antibody (BSMAB) to generate CD8 T cells for testing of a large library of peptide in ELISpot (Wong and Colvin, 1991; Hayes et al., 2016). In this approach, epitopes are mapped in two sequential ELISpot assays using two timepoints of polyclonal CD8 T cell expansion from a single vial of cryopreserved peripheral blood mononuclear cells (PBMC); the first assay, after seven days of expansion, screens large peptide pools and the second assay, after an additional three days of expansion, tests individual peptides contained in the pools that scored positive in the first assay (Anthony and Lehmann, 2003; Tobery et al., 2001). We evaluated response specificities and magnitudes of in vitro polyclonal expanded CD8 T cell to HIV-1 Gag PTE 15-mer peptides designed by two different epitope mapping strategies: overlapping peptides designed from a subtype C consensus sequence (121 peptides) and peptides designed from worldwide circulating viral sequences that included variants of the same epitope (320 peptides).
T cell receptor sequence clustering and antigen specificity
2020, Computational and Structural Biotechnology JournalIdentification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein
2019, Microbes and Infection