Tut 5- IFN-y ELISpot and Assessment of T cell frequency and function Flashcards
In anti-viral immunity, what is a T cell epitope?
T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. In humans, professional antigen-presenting cells are specialized to present MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length, and non-classical MHC molecules also present non-peptidic epitopes such as glycolipids.
An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. For example, the epitope is the specific piece of the antigen to which an antibody binds. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.
The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. Conformational and linear epitopes interact with the paratope based on the 3-D conformation adopted by the epitope, which is determined by the surface features of the involved epitope residues and the shape or tertiary structure of other segments of the antigen. A conformational epitope is formed by the 3-D conformation adopted by the interaction of discontiguous amino acid residues. In contrast, a linear epitope is formed by the 3-D conformation adopted by the interaction of contiguous amino acid residues. A linear epitope is not determined solely by the primary structure of the involved amino acids. Residues that flank such amino acid residues, as well as more distant amino acid residues of the antigen affect the ability of the primary structure residues to adopt the epitope’s 3-D conformation. The proportion of epitopes that are conformational is unknown.
What is the role of the HLA proteins (encoded by the genes of the major histocompatibility complex, MHC) in anti-viral T cell-mediated immune responses?
HLAs corresponding to MHC class I (A, B, and C) which all are the HLA Class1 group present peptides from inside the cell. For example, if the cell is infected by a virus, the HLA system brings fragments of the virus to the surface of the cell so that the cell can be destroyed by the immune system. These peptides are produced from digested proteins that are broken down in the proteasomes. In general, these particular peptides are small polymers, about 9 amino acids in length. Give variation to the MHC complex.
MHC Class I molecules present peptides that are about _______aa long
MHC Class I: Peptides ~8-10 aa long
MHC Class II molecules present peptide antigens that are about _______aa long
MHC II: Peptides at least 13 aa long (~13-30)
After overnight incubation, cells are removed by ____________, and _________________ antibody added. What does this antibody bind to?
Subsequently, an enzyme conjugate is added. What does this reagent bind to?
Lastly, __________________________is added and will form an insoluble colorimetric precipitate. What does this reagent detect?
Removed by (Centrifuged at 1000g)
And ______antibody added (Biotinylated anti IFN-y antibody)
What does this antibody bind to? (Captured analyte e.g IFN-y)
What does this reagent bind to? (Secondary detection reagent (ALP conjugated streptavidin?)
Lastly__________is added (Chromogenic substrate e.g. BCIP)
What does this reagent detect? (Secondary detection reagent (ALP conjugated streptavidin?)
Why can SEB be used as a positive control in the IFN - 𝛄 ELISpot assay?
Staphylococcal enterotoxin B (SEB) is a powerful superantigen, inducing massive IFN-γ secretion by T cells. Thus SEB is suitable positive controls for cell viability, successful stimulation of cytokine secretion and overall T cell functionality.
How does SEB activate T cells? Draw a diagram of the interaction of SEB with the TCR?
A bacterial superantigen SEB exploits unique TCR proximal signaling processes in memory CD4 T cells to induce clonal anergy. SEB selectively targeted memory CD4 T cells in vivo and prevented helper function. These toxins bind directly to the MHC class II molecules on APCs and the variable β-chains of the TCR, and activate the endogenous pathways dependent upon immune synapse formation. CD28, a costimulatory receptor on T cells, has been recently identified as a superantigen receptor. Concerted interaction of the superantigen with all three receptors (CD28, MHC class II, and TCR) allows stable synapse formation resulting in exceptionally robust T cell responses, particularly Th1 cytokine induction, and lethality. Via this mechanism, SEB activates ∼20% of the T cell population, whereas exposure to normal Ags activates <0.01% of T cells.
Cells of which T cell subset are activated to secrete IFN - γ upon recognition of cognate peptide antigen?
CD8+
Why might the CD8+ fraction induce a higher magnitude response than the whole PBMC fraction?
Tregs that suppress the immune response would be present in PBMC sample but not pure CD8+ sample.
CD8 T-cell IFNy responses and activation can be independent of CD4 T cell activation especially in human immunodeficiency virus infected people who may suffer various degress of CD4 T cell deficiency. (T independent). CD8 t cells may undergo a greater extent of proliferation and activation by compensation for lack of CD4 T cells. HLA*0201 known to stimuate IFN-gamma responses in CD8 T cells. CD8 T cells activation occurs when cells specifically recognise short peptides (8-10 aa in length) derived from MHC 1 molecules. HLA in study are supertypes for binding.
What is the amino acid sequence of the CD8+ HLA-B*5501-restricted DENV NS5 T cell epitope?
WDVIPMV
Why the other three peptide truncations, and NS5 peptide 67, do not activate DENVspecific CD8+ T cells?
The truncations do no have the C terminus to bind to the P9 binding pocket in the MHC complex
Based on the data generated in this study, what can we conclude regarding the duration of DENV-specific T cell-mediated anti-viral immune responses?
CD8+ response is highly specific and will have a new response to each new strain of DENV
What is ELISpot?
The enzyme-linked immunospot (ELISpot) assay is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli.
How does ELISpot differ to ELISA?
The enzyme-linked immunospot (ELISPOT) assay allows characterization of single-cell immune responses through detection of secreted analytes. Although ELISPOT analysis shares similarities with ELISA, it has some essential differences. In general, the ELISPOT assay uses antibodies to capture and detect analytes of interest released by activated immune cells. Released analytes form specific antibody-antigen complexes and are visualized as spots of enzyme-substrate precipitates. These spots indicate both how many cells secrete the respective analyte and how much analyte is produced per individual cell.
Initially developed for the detection of antibody-secreting cells, ELISPOT assays are now frequently performed both in the context of clinical diagnostics and in research on T-cell responses, in particular antigen-specific T-cell subpopulations, as related to allergy, cancer, infections, or autoimmune diseases. The one spot-one cell principle allows sensitive detection of specific and rare immune cell subsets.
List things ELISpot can do
The ELISPOT assay enables detection of cytokine or effector molecule secretion on a single-cell level.
- ELISPOT analysis allows for ex vivo characterization of cell function, including antigen-specific T-cell monitoring.
- The ELISPOT assay has a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) or intracellular staining, facilitating measurement of very low numbers of analyte-producing cells (i.e., as low as 1 cell in 300,000).
- The ELISPOT assay facilitates high-throughput cell screening with low intra- and interassay variability.
- Because cells are not fixed or killed during the ELISPOT procedure, they can be further characterized in subsequent experiments.
Describe limitations of ELISpot.
- The ELISPOT assay is not applicable for analysis of whole blood but requires isolation of peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes, or other cell subsets.
- Detection of rare antigen-specific T cells is highly dependent on an appropriate stimulus.
- Currently, the ELISPOT/FluoroSpot assay only allows simultaneous detection of four analytes.
- The ELISPOT assay measures only the release of soluble analytes and does not allow for further cell phenotyping.
- Spot counts are strictly limited to membrane area per well.
