Application
T Cell Exhaustion
Cell and Cytokine Profiling
T Cell Exhaustion
T-cell exhaustion is a broad term used to describe T cell dysfunction resulting from chronic stimulation. Exhausted T cells present with a distinct phenotype including overexpression of inhibitory markers such as PD-1, LAG-3 and TIM-3 as well as impairment in their ability to release pro-inflammatory cytokines (IFNγ and TNFα). Exhaustion commonly occurs in the tumour microenvironment where T cells suffer a loss of their cytotoxic function and become ineffective in their ability to kill cancerous cells.
There is much interest in reversing or overcoming the state of exhaustion, particularly in the production of CAR-T cells by including checkpoint inhibitor antibodies. Being able to simulate T cell exhaustion problem-solution scenarios will provide further insight into this phenomena.
Assay Concept
Multiplexed approach to measure T cell exhaustion, proliferation and cytokine release in a single well.
Figure 1. Illustration of iQue® Human T Cell Exhaustion Kit assay principles. T cell phenotypes are profiled for the expression of 3 exhaustion markers: PD-1 (early), LAG-3 (mid), and TIM-3 (late). The 2 effector cytokines (IFNg and TNFα) are also quantified using 2-plex Qbeads in a sandwich immunoassay format in the same well. Simultaneous measurement of T cell proliferation or encoded target cells is optional (not included in this illustration).
Key Advantages
Gain additional biological insight into T cell exhaustion status in physiologically relevant models
Simultaneously quantify surface protein & cytokine expression
Figure 2. Exhausted T cells overexpress inhibitory receptors and produce low concentrations of effector cytokine TNFα
Non-exhausted (i.e. no prior stimulation) or pre-exhausted CD3+ T cells were seeded at 200K/well. Exhausted T cells had been stimulated with Dynabeads CD3/CD28 (1:1 bead-to-cell ratio) and re-stimulated every 2-3 days (5 stimulations total) prior to seeding. In-well activation was induced with varying densities of Dynabeads CD3/CD28. Control wells contained no Dynabeads. At 24h, 10uL samples were analysed using the iQue® Human T Cell Exhaustion Kit on the iQue® 3.
Gain additional biological insight into T cell activation status in physiologically relevant models
Figure 3. LAG-3 overexpression persists on exhausted T cells over 3 days
Non-exhausted (i.e. no prior stimulation) or pre-exhausted CD3+ T cells were seeded at 200K/well. Exhausted T cells had been repeatedly stimulated (5 times total, every 2-3 days) with Dynabeads CD3/CD28 (1:1 bead-to-cell ratio). In-well, activation was induced with Dynabeads CD3/CD28. Every 24h, 10uL samples were analysed using the iQue® Human T Cell Exhaustion Kit.
Easy to use and interpret; pre-defined gating strategy
Figure 4. Pre-determined gates on iQue Forecyt® enable automatic phenotyping of exhausted T cell subsets.
Minimal sample manipulation: low volume, one wash, no dilution
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Simple protocol where cells and supernatant are taken as one sample allowing for a streamlined workflow.
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No labelling, or compensation optimization steps required.
Figure 5a. Easy to follow protocol for the analysis of T cell phenotypes and cytokine release using the iQue® Human T Cell Exhaustion Kit.
Figure 5b. Easy to follow modified protocol to allow the inclusion of target cells in the assay. Allowing the analysis of T cell phenotypes and cytokine release using the iQue® Human T Cell Exhaustion Kit.
Ordering Information
| iQue® Human T Cell Exhaustion Kit | ||
|---|---|---|
| Platform | Compatible with iQue® 3 / iQue® Screener Plug - VBR configuration | |
| Available Sizes | Catalog Numbers | Order Now |
| 1 x 96 well | 97069 | iQue® Human T Cell Exhaustion Kit |
| 5 x 96 wells | 97070 | |
| 1 x 384 wells | 97071 | |
| 5 x 384 wells | 97072 | |
Technical Resources
Documents
iQue® Human T Cell Exhaustion Kit Manual
Adherent Cell Lifting Protocol