Assess T-Cell Phenotype and Function in a Spheroid T-Cell Killing Assay

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: 

  • Provide bulk measurement of killing (e.g. imaging) without a deeper investigation into effects on cell marker expression or cytokine release
  • Require multiple workflows for quantification of different parameters, often necessitating multiple different instruments
  • Lack physiological relevance towards in vivo conditions
  • Involve lengthy, time-consuming workflows; requiring steps such as protocol optimization, fixation and repetitive washes

Here we demonstrate a simple, robust workflow for measuring T cell response in a spheroid T cell killing assay using the iQue® Advanced High Throughput Flow 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.


Application

Assay Concept

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. 

Key Advantages
  • 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

Enable 3D tumor analysis

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®.

Interrogate phenotype & function

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® advanced flow cytometry

Incucyte® 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. 

Unlock your productivity

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 kits

Incucyte® 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.

Streamline data acquisition

Rapid sampling speeds facilitate increased replication, multi-kit analysis and high-throughput quantification 


Figure 4. Pre-set gating templates in iQue Forecyt® provide automated phenotyping of T cell subsets

BT474 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. 

Ordering Information

iQue® Human T Cell Activation Kit

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Available Sizes

Catalog Numbers

1 x 96 well

90560

5 x 96 wells

90561

1 x 384 wells

90562

5 x 384 wells

90563

iQue® Human T Cell Killing Kit

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Available Sizes

Catalog Numbers

1 x 96 well

97060

5 x 96 wells

97061

1 x 384 wells

97062

5 x 384 wells

97063

iQue® Human T Cell Exhaustion Kit

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Available Sizes

Catalog Numbers

1 x 96 well

97069

5 x 96 wells

97070

1 x 384 wells

97071

5 x 384 wells

97072

iQue® Human T Cell Memory Kit

Platform: Compatible with iQue® 3/iQue® Screener Plus - VBR Configuration

Available Sizes

Catalog Numbers

1 x 96 well

97035

5 x 96 wells

97036

1 x 384 wells

97037

5 x 384 wells

97038

Additional characterization can be achieved through optional QPanel T Helper Kits, see individual application pages for details.

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Resources

Technical Resources

Protocol

T Cell Killing in Single Spheroids

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Product Guide

iQue® Human T Cell Activation Kit

Download Now

Product Guide

iQue® Human T Cell Killing Kit

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Product Guide

iQue® Human T Cell Exhaustion Kit

Download Now

Product Guide

iQue® Human T Cell Memory

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