Table 1.
Experimental approaches to measure antigen-specific T cells.
| Assay | Feature/Parameter measured/Readout | Assay requirements | Advantages | Disadvantages |
|---|---|---|---|---|
| T cell spot-based assays (ELISPOT & FluoroSpot) | Cytokine secretion | 15 x 104 – 1 x 106 PBMCs/condition | * Provides quantitative (number of secreting cells) and qualitative (type of response based on the cytokine secreted) information at a single cell level. | * Limited number of cytokines detected (currently up to four cytokines). |
| 24 – 72 hours incubation (2, 3) | * Can be performed directly ex vivo (no expansion required). | * Labour intensive (4). | ||
| * Objective enumeration of cytokine-secreting cells using automated readers (although spots need to be validated by the human eye to exclude potential artifacts or spots which have not been accurately counted). | * Bulk culture of PBMCs which does not allow determination of the type of cytokine-secreting cell. | |||
| * Cells can be harvested for downstream analyses (e.g., transcriptional profiling or T cell cloning) (5). | * Cannot assess the phenotype of antigen-specific cytokine-secreting cells. | |||
| * Good reproducibility in individual or across different laboratories (6, 7). | * Requires in vitro culture. | |||
| * Whole antigens and synthetic peptides (in single or peptide pool format) can be used for stimulation. | ||||
| * Does not require previous knowledge of HLA restriction or epitopes targeted when whole antigens or overlapping peptides are employed. | ||||
| * Highly sensitive (6–8). | ||||
| * Can be performed with fresh and cryopreserved PBMCs. | ||||
| * Validation and assay harmonisation have been conducted (7). | ||||
| * Successfully employed in multiple laboratories (9–13). | ||||
| Dye-dilution assays (e.g. CFSE) | T cell proliferation | 1 – 2 x 106 PBMCs/condition | * Flow-cytometry based assay that provides quantitative information (number of cells proliferating) and enables phenotypic characterisation of antigen-specific T cells when additional cell surface and intracellular markers are used. | * Limited functional information (ability of cells to proliferate in response to antigen in vitro). |
| 7 days incubation (14) | * Can be performed directly ex vivo (no expansion required). | * Poor reproducibility due to variable background proliferation (14). | ||
| * Can be combined with flow cytometric sorting allowing transcriptomic, TCR analyses and cloning of autoreactive T cells at a single-cell resolution (15) | * Each sample must be analysed individually. | |||
| * Whole antigens and synthetic peptides (in single or peptide pool format) can be used for stimulation. | * Subjective gating. | |||
| * Does not require previous knowledge of HLA restriction or epitopes targeted when whole antigens or overlapping peptides are employed. | * Sensitive to bystander proliferation (which may overestimate the number of antigen-specific T cells) or bystander suppression caused by regulatory cytokines (IL-10 and TGF-beta) masking cells capable of proliferating (8). | |||
| * Easy to perform (8). | * Extrapolation required to calculate precursor frequency. | |||
| * Can be performed with fresh and cryopreserved PBMCs. | * Requires in vitro culture. | |||
| * Validation and assay harmonisation have been conducted (6) | ||||
| * Successfully employed in multiple laboratories (16–18) | ||||
| Peptide-MHC multimers | Frequency of epitope-specific T cells | 1 – 2 x 106 PBMCs/condition (HLA-class I) | * Allows detection and phenotyping of antigen-specific T cells directly ex vivo and estimation of their frequency. However, some studies claim in vitro expansion is required to increase the assay detection limit which, consequently, could alter the phenotype of these cells (19) | * Isolation and enumeration are challenging as this assay relies on low-affinity pMHC-TCR interactions. Standard pMHC tetramer staining is the most used method despite recent evidence demonstrating that it fails to detect antigen-specific T cells with very low affinities. The use of optimised protocols has been shown to increase the assay detection limit with results comparable to parallel functional assays (20). |
| 10 – 20 x 106 PBMCs/condition (HLA-class II) | * Physical detection of antigen-specific T cells does not rely on a particular effector function, and therefore is not influenced by sample preparation or cryopreservation methods (20). | * Limited reproducibility across different laboratories (21, 22). | ||
| No incubation required (20) | * The use of higher order multimers enhances the ability to recover a greater number of antigen-specific T cells (especially those with low-affinity TCRs) (20, 23, 24) and simultaneous detection of multiple epitope-specific T cells is possible using combinatorial MHC multimers (19, 25, 26). | * Does not provide functional information, although flow cytometric sorting combined with RNA transcriptional profiling enables characterisation of functional phenotypes. | ||
| * Can be combined with magnetic bead separation or flow cytometric sorting for isolation of peptide-specific T cells (20). | * Relies upon HLA-restricted presentation of selected β-cell epitopes and therefore requires previous knowledge of the HLA type of the individuals assessed and epitopes targeted. | |||
| * Compatible with enrichment methods to enhance detection of rare cell populations (27). | * A limited repertoire of epitope specificities can be tested in a single biological sample due to limitations in the number of available fluorochromes (25). | |||
| * Good reproducibility in individual laboratories (21, 22). | * Subjective gating. | |||
| * Allows identification of epitope specificities. | * Labour intensive. | |||
| * Does not require in vitro culture. | ||||
| * Can be performed with fresh and cryopreserved PBMCs. | ||||
| * Validation and assay harmonisation have been conducted (22, 28) | ||||
| * Successfully employed in multiple laboratories (23, 27, 29). | ||||
| Activation-induced marker (AIM) assay | Frequency of antigen-specific T cells | 5 – 10 x 106 PBMCs/condition | * Detection of antigen-specific T cells based on upregulation of TCR stimulation-induced surface markers by flow cytometry. | * Lack of sensitivity due to variable background response. |
| 16 – 24 hours incubation (30) | * Phenotype-agnostic method which allows identification of antigen-specific T cells with a variety of effector or regulatory functions (30–32). | * Does not provide functional information. | ||
| * Additional information about responding T cells can be obtained using multiparameter flow cytometry. | * Subjective gating. | |||
| * Can be performed directly ex vivo (no expansion required). | * Labour intensive. | |||
| * Compatible with enrichment methods to enhance detection of rare cell populations (33). | * Requires previous knowledge about the kinetics of the AIM markers used. | |||
| * Can be combined with flow cytometric sorting for isolation of antigen-specific T cells (33, 34). and downstream applications (e.g., single-cell transcriptional profiling). | * An accurate set of AIM markers is required for the identification of the complete pool of antigen-specific T cells. | |||
| * Detection of responses to a wide array of antigenic targets, including whole antigens and synthetic peptides (in single or peptide pool format). | * Validation and assay harmonisation initiatives are lacking. | |||
| * Does not require previous knowledge of HLA restriction or epitopes targeted when whole antigens or overlapping peptides are employed. | ||||
| * Can be performed with fresh and cryopreserved PBMCs. | ||||
| * Successfully employed in multiple laboratories (34–38). |