T-Cell Characterization - Activation, Killing, Exhaustion and Memory
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.
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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.
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 iQue® 3 High-Throughput Screening (HTS) Cytometer.
Each activator resulted in a different CD3+ T cell protein expression and killing profile:
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:
* upper limit of detection reached
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.
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.
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 iQue® 3 HTS Cytometer.
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.
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Human T Cell Activation Cell and Cytokine Profiling 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
Human T Cell Mediated Killing Kit
97060
97061
97062
97063
Human T Cell Exhaustion Cell and Cytokine Profiling Kit
97069
97070
97071
97072
Human T Cell Memory Cell and Cytokine Profiling Kit
97035
97036
97037
97038
Additional characterization can be achieved through optional QPanel T Helper Kits, see individual application pages for details.
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