T Cell Exhaustion | Cell and Cytokine Profiling
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.
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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).
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 analyzed using the iQue® Human T Cell Exhaustion Kit on the iQue® 3 High-Throughput Screening (HTS) Platform.
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.
Figure 4. Pre-determined gates on iQue Forecyt® enable automatic phenotyping of exhausted T cell subsets.
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.
If you use magnetic beads to induce T cell exhaustion, you do not need to remove them prior to collecting your samples on the iQue® High-Throughput Screening (HTS) Cytometer. Once you perform the analysis, the cells will be gated out from the smaller sized beads, then only the iQue Qbeads® that detect the cytokines TNFα and IFNγ will be analyzed via a pre-gated template to rapidly obtain results.
The kit can show reversal of T cell markers of exhaustion and functional capability to produce elevated levels of IFNγ and TNFα over exhausted cells, but that direct measurement of cytotoxicity would be better shown by examining that functional property with our iQue® Human T or NK cell Killing Kit or other method to directly measure cytotoxicity.
Due to the sensitivity of the iQue® HTS platform and the ability to perform assay miniaturization, you can detect just a few thousand of the exhausted T cells using the iQue® Human T Cell Exhaustion Kit. We recommend setting up your assay plates with a minimum of 1 x 106 cells/mL and transferring 10 µL of that sample into your assay plate, which represents 10,000 cells. You may need to optimize your assay conditions or collect more cell sample if your analysis requires greater cell counts. The iQue® data acquisition protocol can be easily modified to acquire a larger sample volume if needed.
Compatible with iQue® 3 / iQue® Screener Plus - VBR configuration
1 x 96 well
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5 x 96 wells
97070
1 x 384 wells
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5 x 384 wells
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Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.
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