T-cell-based immunogenicity assay for evaluating human antigen-specific responses

Determining the antigen specificities of the endogenous T-cell repertoire is important for screening naturally occurring or therapy-induced T-cell immunity and may help identify novel targets for T-cell-based therapies. This assay enables the rapid, sensitive, and high-throughput expansion of antigen-specific T cells from human PBMCs using peptide stimulation and detecting antigen-specific effector cytokine formation.

Principle of the Assay

  1. Target cells (PBMCs) are stimulated with adjuvant solution containing peptides.

  2. Restimulate again with the peptides and treat the cells with protein transport inhibitors.

  3. Antigen-induced IFN-γ, TNF-α and IL-2 production by CD4+ and CD8+ T cells are measured using flow cytometry.

Model Description

PBMCs are stimulated with peptide-containing adjuvant solution, and restimulated with peptides. Antigen-induced IFN-γ, TNF-α, and IL-2 production in CD4+ and CD8+ T cells is assessed by flow cytometry.

Readouts

Intracellular expression of IFN-γ, IL-2 and TNF-α usig flow cytometry.

Add-On Services

Evaluation of cytokine release, proteomics, and transcriptomics.

Key Components of the Assay

a. Peripheral Blood Mononuclear Cells (PBMCs)

  Primary source of T cells, isolated from human blood.

b. Antigen (Peptides)

  Target antigen(s) used to stimulate T-cell responses.

  Can be synthetic peptides, whole proteins, or pathogen-derived antigens.

c. Stimulation Conditions

  Primary Stimulation: PBMCs are exposed to the antigen to activate T cells.

                 Restimulation (Boosting Step): Enhances antigen-specific T-cell expansion.

Controls: Negative (unstimulated PBMCs) and positive (mitogens like PHA, anti-CD3/CD28) controls ensure assay validity.

d. Detection Methods

  Flow Cytometry: Measures activation markers (CD69, CD25) and intracellular

                 cytokine production (IFN-γ, TNF-α, IL-2).

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