T Cell Activation
Activation of naive T cells by an antigen and costimulatory signals initiates clonal expansion of CD4+ helper and CD8+ cytotoxic T cells. In addition to T cell proliferation, a variety of signaling pathways are activated, leading to expression of functional cell surface markers and the release of cytokines.
There is a growing market of therapies that focus on using the immune system to target cancer, including:
The ability to monitor and quantify activation profiles allows for a greater understanding of how T cells react to certain stimuli, which in turn can provide insight into how well drugs will be able to harness the power of the immune system.
Explore the iQue® High-Throughput Screening (HTS) Platform
Buy now - iQue® Human T Cell Activation Kit
The iQue® Human T Cell Activation Kit assesses the activation status of T cells while providing information about their health and their role in cytokine secretion. This one-wash assay requires minimal hands-on time and measures cells and beads. This kit provides a multiplexed approach to measuring T cell activation, proliferation and cytokine release in a single well.
Whether you are discovering a new therapeutic antibody, developing a specific checkpoint inhibitor or evaluating CAR T-cell function, driving your research forward hinges on the ability to rapidly evaluate more samples in a biologically relevant, reproducible and cost-effective way.
The iQue® HTS Platform utilizes a fixed wide dynamic range allowing for the collection of both the phenotypes and functional analysis of secreted cytokines simultaneously, eliminating the discrepancy of different time points or the need to split samples for subsequent analysis.
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Discern T cell activation status in physiologically relevant cell modelsSee example data below
Multiplexed immunophenotyping and effector cytokine concentration read from a single wellSee example data below
Pre-determined gates on iQue Forecyt® enable automatic phenotyping of T cell subsetsSee example data below
Simple protocol where cells and supernatant are taken as one sample allowing for a streamlined workflow. No labelling, or compensation optimization steps requiredSee example data below
Different T cell phenotypes are profiled for the expression of 3 activation markers: CD69 (early), CD25 (late), and HLA-DR (even later). The 2 effector cytokines (IFNγ 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 possible but is not included in this illustration.
Figure 1. Illustration of Human T Cell Activation Kit assay principles.
PBMCs were treated with a panel of Kinase inhibitors (20 µM) for one hour. CD3/CD28 Dynabeads were then added to stimulate activation for 24 hours. 10 µL samples were analyzed using the T Cell Activation Cell and Cytokine Profiling Kit and the iQue® HTS platform.
Figure 2. Kinase inhibitor screen highlights variation in human T cell activation profiles.
(A) Plate view of CD4+CD69+ gating strategy. Green box identifies media only control. Red box identifies cyclosporine control. Yellow boxes identify the compounds shown on graphs B-D (1: Ruxolitinib; 2: Bisindolylmaleimide I; 3: AZD 7762).
(B, C) Expression of CD69, CD25 and HLA-DR on CD4+ and CD8+ cells.(D) Release of IFNγ and TNFα cytokines from PBMCs.
SKOV-3 cells were seeded (4K/well) in a co-culture assay with PBMCs (10K/well) from 3 donors. Activation was induced by CD3/CD28 Dynabeads. Cells were lifted following the adherent cell lifting protocol after 4 days and analyzed using the T Cell Activation Cell and Cytokine Profiling Kit. (A-C) Expression of markers of early (CD69), late (CD25) and even later (HLA-DR) T cell activation. (D) IFNγ release by each donor.
Figure 3. Quantify variability in activation profile and cytokine release from PBMCs isolated from different donors.
Ramos cells (40 K/well) were seeded with PBMCs from three different donors at a 3:1 effector-to-target ratio. CD3/CD28 Dynabeads were added to induce T cell activation (top concentration ratio of 4 Dynabeads: 1 PBMC, 1 in 2 serial dilution). After 60 hours, cells were analyzed using the iQue® Human T Cell Activation Kit. (A) Positive (4:1 Dynabeads) and negative (no Dynabead) controls were used to confirm positions of the templated gates. (B) Heat map shows the % of CD8 cytotoxic T cells positive for activation marker CD25 expression with each PBMC donor (n=3) . (C) Data from B plotted as concentration response curves.
Figure 4. Pre-defined gates on iQue Forecyt® enable automatic phenotyping of human T cell subsets.
High-Throughput Screening (HTS) by Cytometry’s use in Cancer Immunotherapy, with the potential to predict on-target off-tumor effects.
A Guide to HTS Cytometry Assays and Workflows
The iQue® Human T Cell Activation Kit uses a multiplexed approach to perform activated T cell immunophenotyping and cytokine detection simultaneously. The kit provides the appropriate antibodies for the identification of activated T cells as well as iQue Qbeads® for the detection and quantification of secreted IFN and TNF. Both components are added to each assay well together with a viability dye and incubated with the cell and supernatant sample mixture. A preset gating strategy then separates the fluorescent signals from cells and beads and auto populates the accompanying template in order to express the data for both T cell populations and cytokine concentrations.
The iQue® Human T Cell Activation Kit allows for a single assay plate to be used for multiple measurements during a time course. Only a tiny volume (10 µL) of cell and supernatant sample is needed to perform analysis using the T Cell Activation kit, meaning that once each sample has been taken (aseptically in a hood) the assay plate can be returned to the incubator ready for the next sample in your time course. Before taking a sample it is recommended to gently triturate the contents to ensure even sampling however this will not affect the cells nor will the small sample volume.
The iQue® Human T Cell Activation Kit can be used in combination with the iQue® Human T Cell Companion Kits which allow the measurement of up to 6 human cytokines in addition to those already included in the T Cell Activation kit. Sartorius also offers a range of both cytokine and chemokine detection iQue Qbeads® so that customers can make their own additional panels to be multiplexed with the T Cell Activation kit. A single 10 µL sample can be used to analyze up to 30 secreted proteins and customers can choose from over 50 analytes to quantify. And, if your particular analyte is not offered in our current portfolio, iQue Qbead® Devscreen reagents allow the flexibility to attach your own capture antibodies or target proteins for a more bespoke, personalized solution.
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