T-Cell Characterization
T cells are fundamental in the coordination and execution of immune cell-mediated killing of infected or cancerous cells. Throughout the development of a T cell, variations in surface marker expression and cytokine release lead to important changes in function. Following antigen recognition, cytotoxic effector T cells become activated, inducing target-cell killing capabilities. Repeated stimulation can lead to loss of function due to T cell exhaustion and ultimately cell death. Post antigen clearance, some effector T cells will transition into long-term memory T cells which retain survival and proliferation properties. These provide lasting immunity through their ability to rapidly expand when re-exposed to a specific antigen.
A growing number of immunotherapies, for example bispecific antibodies, checkpoint inhibitors and CAR-T cells, are being developed, targeting various stages in T cell activation and differentiation pathways. The goal of these therapeutics is to enhance the natural power of the immune system to eliminate diseases such as cancer. Detailed characterization during the course of T cell development has the potential to offer greater insights leading to improved therapeutics.
Assay Concept
Figure 1. Illustration of the T cell characterization workflow principles. T cells are cultured alone or in the presence of labelled target cells. 10 µL samples are taken from the cell culture plate and used to feed into 4 different Human T Cell Biology Kits (T Cell Activation Kit, T Cell Mediated Killing Kit, T Cell Exhaustion Kit and T Cell Memory Kit). These kits can be mix and matched to suit user needs. Each kit contains a unique combination of antibodies, including basic T cell markers, for profiling cell surface marker expression, coupled with 2-plex Qbeads to quantify secreted cytokine concentration. Cell proliferation (optional) and viability can be measured simultaneously in each well.
Key Advantages
- Gain additional biological insight - Discern T cell phenotype and function in physiologically relevant cell models
- Unlock your productivity - High-throughput analysis with a suite of kits provides broad T cell characterization in minimal time
- Simultaneously quantify surface marker & cytokine expression - Multiplexed immunophenotyping and effector cytokine concentration read from a single well
- Flexible format and reduced sample consumption - A single assay plate feeds multiple, mix and match kits
Gain Additional Biological Insight
Figure 2. T cell response is dependent on mechanism of activation. PBMCs (120K/well) were co-cultured with Nuclight Green labelled Ramos cells (40K/well). Activation was induced with CD3/CD28 Dynabeads, CD3xCD19 BiTE antibody or Staphylococcal enterotoxin B (SEB). At 60h, 10 µL samples were analyzed using the T Cell Activation, T Cell Mediated Killing and T Cell Exhaustion Kits with the iQue3 system.
Each activator resulted in a different CD3+ T cell protein expression and killing profile:
- CD3/CD28 Dynabeads induced concentration-dependent T cell activation. The highest bead numbers saw the largest % expression of activation markers. This correlated with high levels of exhaustion markers.
- CD3xCD19 BiTE antibody mediated the greatest increase in target cell death with significantly lower levels of activation and exhaustion when compared to Dynabeads and SEB. This suggests BiTE-mediated immune cell killing could be sustained over a longer time period.
- SEB stimulation resulted in T cell activation and maximal killing even at the lowest concentrations. This resulted in high levels of exhaustion.
Unlock Your Productivity
Figure 3. Mechanism of activation directly affects T cell cytokine release.
Cytokine release from a co-culture assay containing Nuclight green labelled Ramos and PBMCs (3:1 ratio) was measured using Qbeads from the T Cell Activation, T Cell Mediated Killing and T Cell Exhaustion Kits. IFNɣ and TNFa
concentrations are measured in multiple kits, therefore the results from all kits have been averaged.
The effect of each activator on cytokine secretion was comparable to its effects on surface protein expression:
- CD3/CD28 Dynabeads induced a concentration-dependent increase in cytokine release across the range of bead numbers used.
- CD3xCD19 BiTE stimulated cytokine production was consistently lower when compared with beads or SEB (excluding the top concentration with Granzyme B). This suggests a high level killing of killing can be achieved with a reduced risk of toxicity (e.g cytokine release syndrome).
- SEB induced high level cytokine release even at low concentrations.
* upper limit of detection reached
Simultaneously Quantify Surface Protein & Cytokine Expression
Figure 4. T memory cell response is affected by method of stimulation. PBMCs (120K/well) and Ramos cells (3:1 E:T ratio) were co-cultured with 3 different activators. 10 µL samples were analyzed at 60h using the T Cell Memory Kit.
(A) Schematic showing surface marker expression changes on T cells as they develop into different T memory phenotypes. From naive (TN) to Stem Cell Memory (TSCM) to Central Memory (TCM) to Transitional Memory (TTM) to Effector Memory (TEM) to Terminal Effector (TTE) T cells through to cell death.
(B), (C) and (D) Graphs showing % of CD3+ cells expressing proteins from each stage of the T cell memory development. IL-10 release is also shown. Teal bars are controls with no activator. Grey bars represent 3 ascending concentrations of each activator (light to dark): CD3/CD28 Dynabeads (30K, 120K and 480K beads/well); CD3xCD19 BiTE (0.12, 1.1 and 10 ng/mL) and SEB (1.2, 11 and 100 ng/mL).
Activation with BiTE antibody saw the lowest proportion of cells in the later stages of T memory cell development (i.e. TEM and TTE) and greater amounts in the earlier stages (TN, TSCM and TCM) when compared to Dynabead and SEB stimulation. This suggests that stimulation with BiTE antibody induces a greater level of killing (as shown in Figure 3) and also results in a higher percentage of T memory cells that have retained their self renewal potency enabling a stronger response to antigen.
Flexible Format and Reduced Sample Consumption
Figure 5. Protocol for the T cell characterization workflow.
Use cell and bead based kits to analyze activation, killing, exhaustion and memory in a combination that suits user needs.
T Cell Characterization Can Be Performed Following Co-Culture With Adherent Target Cell Lines
Figure 6. The effects of T cell stimulation can be measured following co-culture with adherent cell lines. PBMCs (25K/well) were co-cultured with adherent AU565 cells (5K/well). T cell activation was induced with SEB. At 60h, target and effector cells were lifted for endpoint analysis with the T Cell Activation, T Cell Mediated Killing and T Cell Exhaustion Kits and the iQue3.
SEB activation caused a concentration-dependent increase in target cell killing. This was accompanied by high expression of both activation (CD69, CD25 and HLA-DR) and exhaustion (PD-1, LAG-3 and TIM-3) markers on CD3+ T cells across all SEB concentrations used.
The activation of naïve T cells by an antigen and costimulatory signals initiates clonal expansion of both CD4+ helper and CD8+ cytotoxic T cells. In addition to T cell proliferation, a variety of signaling pathways are activated, leading to the expression of functional cell surface markers and the release of cytokines.
Ordering Information
Platform: Compatible with iQue3/iQue Screener PLUS VBR configuration | ||
| Available Sizes | Catalog Numbers | |
1 x 96 well | ||
5 x 96 wells | ||
1 x 384 wells | ||
5 x 384 wells | ||
Platform: Compatible with iQue3/iQue Screener PLUS VBR configuration | ||
| Available Sizes | Catalog Numbers | |
1 x 96 well | ||
5 x 96 wells | ||
1 x 384 wells | ||
5 x 384 wells | ||
Platform: Compatible with iQue3/iQue Screener PLUS VBR configuration | ||
| Available Sizes | Catalog Numbers | |
1 x 96 well | ||
5 x 96 wells | ||
1 x 384 wells | ||
5 x 384 wells | ||
Platform: Compatible with iQue3/iQue Screener PLUS VBR configuration | ||
| Available Sizes | Catalog Numbers | |
1 x 96 well | ||
5 x 96 wells | ||
1 x 384 wells | ||
5 x 384 wells | ||
Additional characterization can be achieved through optional QPanel T Helper Kits, see individual application pages for details.
