T-Cell Killing in Spheroids
T cells are critical for control and execution of the adaptive immune response against infected or cancerous cells. Development of immunotherapies to enhance the T cell response against cancer, such as bispecific antibodies, checkpoint inhibitors and CAR-T cells, is continually on the rise. These act through targeting of different stages in the T cell activation and differentiation pathways; for example to increase T cell activation or the frequency of self-renewing memory cells, or to reduce the level of T cell exhaustion.
A crucial stage in the development of novel therapeutics is to characterize their function using in vitro assays. Much of this work, particularly in the field of flow cytometry, has been conducted using non-adherent cells or cell monolayers (2D). However, increasing evidence supports the use of advanced 3D tumor cell cultures, such as spheroids and organoids, as models that more closely reflect the in vivo scenario to enable improved clinical translation.
Traditional techniques for measuring T cell response often:
Here we demonstrate a simple, robust workflow for measuring T cell response in a spheroid T cell killing assay using the iQue® High-Throughput Screening Cytometer and associated suite of validated reagents. The user can choose to combine one or more iQue® kits, including the iQue® Human T Cell Activation, iQue® Human T cell Killing, iQue® Human T cell Exhaustion and iQue® Human T cell Memory Kits, to generate a range of outputs as desired. Enhance your screening and profiling studies by combining the power of the iQue® and the automated, integrated analysis for instantaneous pharmacological readouts for the effects of novel immunotherapies on T cell response.
Use promo code SPRING24 for buy one, get one free on our entire line of RUO growth factors and cytokines before September 30.
Figure 1a. Illustration of the iQue® spheroid T cell killing assay principles.
Spheroids are formed in ULA plates prior to addition of immune cells (PBMCs or T cells). Multiple 10 µL supernatant samples can be taken from a single assay plate to allow kinetic cytokine quantification. At assay endpoint, spheroids are dissociated to create a single cell suspension containing immune cells and target cells. Cells and cytokines are analyzed using the iQue® T cell characterization kits (Activation, Killing, Exhaustion and Memory). Each kit contains a unique combination of antibodies, including basic T cell markers, for profiling cell surface marker expression, coupled with 2-plex iQue Qbeads® to quantify secreted cytokine concentration. Cell proliferation (optional) and viability can be measured simultaneously in each well.
Enable 3D tumor analysis - Evaluate T cell response to solid tumors using a reproducible, easy to follow protocol for gentle dissociation of spheroids
Interrogate phenotype & function - Discern T cell phenotype and function during tumor cell killing in 3D advanced cell models
Unlock your productivity - Rapid sampling speeds facilitate increased replication, multi-kit analysis and high-throughput quantification
Streamline data acquisition - Remove bottle necks with plate level analysis and novel visualization tools, decreasing time to actionable data
Evaluate T cell response to solid tumors using a reproducible, easy to follow protocol for gentle dissociation of spheroids
Figure 1b. Assay Workflow - Protocol for analysis of T cells in a spheroid killing assay using the iQue® HTS platform.
Discern T cell phenotype and function during tumor cell killing in 3D advanced cell models
Figure 2a. Quantify cell subsets and cytokine release during T cell killing of tumor spheroids using iQue® High-Throughput Screening (HTS) by CytometryIncucyte® Nuclight green labeled MDA-MB-231 cells (2.5K/well) were seeded in ULA plates with 2.5% Matrigel® and incubated for 72h to promote spheroid formation. Unlabelled PBMCs were added at a 5:1 effector-to-target ratio (E:T) and activation was induced by ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator. On days 1, 3 and 7 identical plates were dissociated and T cell phenotypes analyzed using the iQue® Human T Cell Killing Kit. (A) Target cell viability decreases from day 1 to day 7 at the highest concentrations of Immunocult. (B) Expression of T cell activation and (C) exhaustion markers increased from day 1 to day 3, then decreased by day 7. (D) Release of Granzyme B, a protease directly involved in inducing T cell mediated tumor cell death, increased with Immunocult concentration.
Figure 2b. Investigate T cell function over time with low volume supernatant sampling for kinetic cytokine analysis BT474 (2.5K/well) spheroids were formed for 72 hours before PBMCs (12.5K/well) and CD3/CD28 Dynabeads were added. Supernatant samples (10 µL) were taken on days 1, 4 and 8 and cytokine concentrations were analyzed using iQue Qbeads®. Concentrations of cytokines indicative of T cell activation, IFNγ and TNFα, increased both over time and with Dynabead density. The release of Granzyme B protease, directly involved in target cell killing by cytotoxic lymphocytes was also increased. Production of IL-10, a cytokine with links to the frequency of T memory cell formation increased up to day 4 then decreased by day 8.
Rapid sampling speeds facilitate increased replication, multi-kit analysis and high-throughput quantification
Figure 3. Fast run times facilitate broad phenotype analysis using multiple T cell characterization kitsIncucyte® Nuclight green labeled BT474 (2.5K/well) spheroids were formed for 72 h before PBMCs (12.5K/well) and CD3/CD28 Dynabeads were added. Cells from replicate plates were analyzed on days 1, 4 and 8 using the iQue® Human T Cell Activation, Killing and Exhaustion kits. (A) Plate view plot of CD25+ cells in the live CD3+ population. (B) Target cell viability decreased over time. (C) Expression of early activation marker CD69 initially increased on day 1 then decreased by day 4. (D) Later activation marker, CD25, expression was enhanced on days 4 and 8. (E) Expression of PD-1, an early indicator of T cell exhaustion, was greatest on day 4.
Figure 4. Pre-set gating templates in iQue Forecyt® provide automated phenotyping of T cell subsetsBT474 spheroids (2.5K/well) were incubated with PBMCs at a 5:1 effector-to-target ratio (E:T) and activation was induced by CD3/CD28 Dynabeads. Analysis was performed at 24h using the iQue® Human T Cell Memory Kit. (A) Plot highlighting gating of Tscm (stem cell memory) and Tte (terminal effector) using the template provided in the kit. (B) Heat map and (C) concentration response curves exported directly from iQue Forecyt® software highlight the shift from the Tscm (stem cell memory) early phenotype towards the later stage Tte (terminal effector) phenotype with increasing Dynabead concentration. This shift is accompanied by a loss in the T cells’ self renewal potency.
iQue® Human T Cell Activation Kit
Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration
Catalog Numbers
1 x 96 well
90560
5 x 96 wells
90561
1 x 384 wells
90562
5 x 384 wells
90563
97060
97061
97062
97063
97069
97070
97071
97072
97035
97036
97037
97038
Additional characterization can be achieved through optional QPanel T Helper Kits, see individual application pages for details.
Request a Demo
Outlines methods for single cell dissociation of ACM, such as spheroids and organoids for surface marker analysis using flow cytometry.
A combined workflow approach of iQue® High-Throughput Screening (HTS) by Cytometry and Incucyte® Live-Cell Analysis.
Perform comprehensive, multiplexed analysis of cells, beads and secreted proteins all from the same cells at the same timepoint.
Two High-Throughput Screening by Cytometry solutions to address problems with traditional methods for measuring T cell response in 3D models
T Cell Killing in Single Spheroids
iQue® Human T Cell Killing Kit
iQue® Human T Cell Exhaustion Kit
iQue® Human T Cell Memory
iQue® Human T Cell Companion Kits
Please select your country so we can show you products that are available for you.
The content of our website is always available in English and partly in other languages. Choose your preferred language and we will show you the content in that language, if available.