T-cells are indispensable for an effective and specific immune response. The different T-cell subtypes have distinct critical functions. Some activate antibody-producing B-cells, others mainly activate phagocytes and kill cancerous and infected cells. Furthermore, T-cells assure self-tolerance and the downregulation of immune responses after clearance on an infection. Due to the importance of T-cells and the devastating effects of a misdirected or unwanted T-cell response, they are the focus of various basic and clinical research studies.
The activation of T-cells is antigen-specific and requires recognition of peptides that are bound to HLA molecules (peptide-HLA) by the T-cell receptor. To examine T-cell responses towards peptides, peripheral blood mononuclear cells (PBMC) cultures are a favored model. Here exogenous peptides of interest are loaded on vacant HLA complexes on antigen-presenting cells (APC) and presented to T-cells. The peptide-HLA mediated activation of T-cells triggers production and release of activation markers such as cytokines that can be quantified via ELISpot.
The enzyme-linked immune absorbent spot (ELISpot) assay determines the frequency of antigen-specific T-cells by detecting released activation markers such as cytokines and further effector molecules. In the ELISpot assay, T-cells are activated with antigens in PBMC culture, which allows binding to HLA molecules on antigen-presenting cells and presentation to T-cells. The best-known ELISpot assay is the interferon-gamma (IFN-γ) test, a marker for type 1 immune responses. Nonetheless, ELISpot can be also used to distinguish different subsets of activated T-cells by detecting characteristic cytokines and molecules. The method is remarkably robust, accurate and sensitive, and enables quantitative single cell analysis of low-frequency T-cells.
Monitor T-cell activation and identify effector lineages
In response to antigen-specific activation, the different subtypes of CD4 and CD8 T-cells release characteristic cytokines such as IFN-γ and TNF-α (cellular, type 1 immune response) or IL-4 and IL-5 (humoral, type 2 response). These effector molecules direct the route of the immune response substantively and ensure an antigen-adjusted and appropriate response. Consequently, investigating the activation of T-cell subtype characteristic cytokines is crucial to understand T-cell mediated responses and to evaluate an anticipated success of a vaccination strategy or immunotherapy.
ELISpot assays allow the quantification of subtypes-characteristic activation markers either individually or in parallel at single cell level. Therefore, they are perfectly suited to monitor antigen-mediated T-cell responses.
T-cell activation is mandatory for effective and long-lasting vaccine-induced immunity. Ideally, the vaccination strategy induces antigen-specific activation and proliferation of effector CD4 and/or CD8 T-cells and subsequently the differentiation of memory T-cells. The latter guarantees a prompt and protective response upon antigen reencounter during an infection. Testing for the development of vaccination-induced memory T-cells by cultured ELISpot assays helps to evaluate the success of a vaccine strategy.
The loss of tolerance to self-antigens triggers the development of autoimmune diseases. A promising tool to suppress autoimmunity is the application of tolerizing peptides as modulators of T-cell responses. These short synthetic peptides can inhibit harmful autoreactive T-cells and induce regulatory T cells (Tregs) that help to restore self-tolerance. Testing for peptide-mediated modulation of T-cells by detecting T-cell characteristic cytokines can help to forecast in vivo studies.
Agonistic or antagonistic monoclonal antibodies (mAbs) can target T-cell co-receptors that function as checkpoint molecules in immunity. These mAbs can effectively turn the immune response against self-tumor antigens, and are considered as promising tools in cancer treatment. Preclinical testing of new mAbs in PBMC culture can assess mAb-modulated T-cell responses and help predict outcomes of immunotherapy.
Identify T-cell or coincident T-cell and B-cell epitopes
T-cell epitope identification
T-cell epitope mapping describes the search for the peptide sequences of an antigen that elicit a T-cell response when presented on an HLA/MHC molecule. Precise identification of immunogenic epitopes is facilitated by testing of peptide libraries. An overlapping peptide library is set up by fragmentation of the original protein into overlapping peptides of equal length, and peptides are analyzed for immunogenicity using a combination of T-cell immunogenicity prediction tools and functional assays such as ELISpot.
We typically apply a two-step approach: Initially, mini pools containing equal number of peptides are tested for T-cell activation, followed by individual validation of each peptide within a positive mini pool.
Identification of coincident T-cell and B-cell epitopes
B-cells and T-cells can respond cooperatively to the same antigen, and in some cases a B-cell epitope may simultaneously function as T-cell epitope. Our approach allows for the identification of peptides that are recognized by both B-cell and T-cell receptors.
To identify coincident T-cell and B-cell epitopes, we combine peptide microarray-based antibody profiling with ELISpot assays: Serum samples are screened with PEPperCHIP® Peptide Microarrays for B-cell receptor epitopes, and identified hit peptides are validated by T-cell immunogenicity prediction tools. Promising candidates are then further evaluated using ELISpot assays with PBMCs form the same donors.
Download application note: Discovery of Overlapping Immunogenic B-cell and T-cell Epitopes
Autoreactive T lymphocytes are key players in allergy and autoimmune diseases. They can act both as regulatory and effector cells. Identification of T-cell epitopes in organ/tissue-specific targets of destruction can help to elucidate pathology and can help to improve T-cell targeted therapy.