Cytokine Release Assay for Neoantigen Validation

The Cytokine Release Assay (CRA) is a critical assay for validating neoantigens, assessing their ability to activate T cells, and inducing immune responses. This assay helps determine whether a neoantigen can stimulate T cells to release cytokines, indicating a functional immune response.

Principle of Cytokine Release Assay

  1. The Cytokine Release Assay  assesses T cell activation by measuring cytokine secretion (IFN-γ, IL-2, TNF-α).

  2. When T cells recognize a neoantigen presented by antigen-presenting cells (APCs), they become activated and release cytokines.

  3. These cytokines can be detected using ELISPOT, ELISA, Luminex, or flow cytometry.

Model Description

Neoantigen pulsed antigen presenting cells are co-cultured with the responder cells leading to the effective antigen presentation and the activation of the responder cells.

Readouts

Responder cell response by cytokine secretion (IFN-γ, IL-2, TNF-α) using ELISA, ELISpot and intra-cellular cytokine staining.

Add-On Services

Evaluation of cytokine release, T cell activation, proteomics, and transcriptomics.

Key Components

A. T Cells (Responder Cells):

  • Autologous PBMCs (from patient) or TILs (tumor-infiltrating lymphocytes).

  • Can also use CD8+ or CD4+ T cell-enriched fractions.

B. Antigen-Presenting Cells (APCs):

  • Dendritic Cells (DCs) (derived from monocytes) or autologous PBMCs.

  • Can also use T2 cells (HLA-A2+ TAP-deficient cells) for MHC class I-restricted peptides.

C. Neoantigen Peptides:

  • Synthetic peptides (25-30 amino acids long) covering predicted neoepitopes.

  • Can be loaded onto APCs for presentation to T cells.

D. Co-Culture System

  • APCs are pulsed with neoantigen peptides and incubated before adding T cells.

  • T cells and APCs are co-cultured in a 96-well plate. It ensures effective antigen presentation and T cell stimulation.

E. Cytokine Detection Methods

  1. ELISA → Measures IFN-γ, IL-2, TNF-α, etc.

  2. ELISpot Assay → Detects single T cells secreting cytokines (high sensitivity).

  3. Multiplex Bead-Based Assays (Luminex, MSD) → Quantifies multiple cytokines simultaneously.

  4. Intracellular Cytokine Staining (ICS) + Flow Cytometry → Identifies cytokine-producing CD8+/CD4+ T cells.

← Assays