T Cell Killing – Cell and Cytokine Profiling
Cytotoxic T lymphocytes (CTLs) are an essential part of the immune system and have a crucial role in the elimination of infected and malignant cells. CTLs are a functional (CD8+) subset of the immune system and induce apoptosis in target cells using perforin to form holes in the target cell membrane through which granzymes can enter.
Immune cell mediated cytotoxicity can be induced by a variety of therapeutic drugs such as monoclonal antibodies, bispecific T cell engagers (BiTEs), chimeric antigen receptors (CARs) and Trucks. Harnessing the immune system’s power is seen as a powerful cancer fighting tool and has shown promising results in immuno-oncology. Traditional techniques used to investigate T cell phenotype and cytotoxic function often:
- Require separate assays and instrumentation for cytokines, cytotoxicity and marker expression
- Use labor intensive, indirect measurements of cytotoxicity (e.g., Chromium release assay)
- Necessitate manual analysis and compilation of data from multiple sources
- Involve lengthy, time-consuming workflows requiring steps such as protocol optimization, fixation and repetitive washes
The iQue® Human T Cell Killing application utilizes the iQue® Human T Cell Killing Kit for simultaneous, high-throughput analysis of T cell phenotypic and activation marker expression, target cell killing and quantification of secreted effector proteins and cytokines. Combining the power of iQue® Advanced High-throughput Flow Cytometry with real-time data analysis using integrated iQue Forecyt® software provides a simplified solution to enhance immuno-therapeutic drug discovery processes.
Solutions for T Cell Killing
iQue® Human T Cell Killing Kit
The Human T Cell Mediated Killing Kit was designed for ease of use in multiplexing cell phenotype and function markers along with bead-based, secreted protein profile measurements in the same assay. This one-wash assay requires minimal hands-on time to provide the following biological insights:
- Live immune cells are distinguished from dead cells by staining with a fluorescent membrane integrity dye.
- Cells are immunophenotyped by staining with a fluorescent antibody panel to separate CD3+ T cells, CD3- non-T cells, CD8+ T cytotoxic cells, and CD3+ CD8- T helper cells.
- The panel also includes 2 different T cell functional markers, CD25 and PD-1.
- Optimized workflow enables the measurement of pro-inflammatory effector secreted cytokine IFNγ and pro-apoptotic serine protease Granzyme B in the same assay well.
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iQue® 3 Advanced Flow Cytometry Platform
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® Advanced Flow Cytometry 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.
Key Advantages of T Cell Killing
Assess T cell killing in physiologically relevant models
See example data below
Simultaneous measurement of marker expression and secreted cytokines in a mixed cell and bead assay format
See example data below
Real-time data analysis and novel visualization tools enable rapid generation of quantitative readouts
See example data below
Collapse traditional workflows into one miniaturized, multiplex assay
See example data below
Example Data for iQue® Human T Cell Killing
Obtain biological understanding on T cell killing
CD8+ T cell status can be profiled for the expression of activation marker CD25 and exhaustion marker PD-1. The 2 effector cytokines (IFNγ and Granzyme B) 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 iQue® Human T Cell Killing Kit assay principles.
Incucyte® Nuclight Green labeled Ramos cells were seeded (15 K/well) in a co-culture assay with PBMCs (75 K/well). Activation was induced by CD3xCD19 BiTE antibody. Cells were triturated before samples of cells and supernatants were analyzed using the iQue® Human T Cell Killing Kit. (A) Target viability decreases over the 6-day time course in a concentration dependent manner. (B) Expression of T cell activation and (C) exhaustion markers on CD8+ cells increases up to day 3 and then begins to decline. (D) IFNγ and (E) Granzyme B release.
Figure 2. CD3xCD19 Bispecific T Cell Engager (BiTE) antibody causes concentration dependent killing, marker expression and cytokine release over a 6-day time course.
Simultaneously assess marker expression and secreted cytokines
Incucyte® Nuclight Green labeled Ramos cells were seeded (15 K/well) in a co-culture assay with PBMCs (75 K/well). Activation was induced by Immunocult CD3/CD28/CD2, highest concentration used is equivalent to the recommended stimulation (25 µL per 1e6 cells/mL). Cells were triturated before samples of cells and supernatants were taken and analyzed using the iQue® Human T Cell Killing Kit. (A) Expression of late (CD25) T cell activation on CD8+ cells. (B) Expression of (PD-1) T cell exhaustion on CD8+ cells. (C) IFNγ and (D) Granzyme B release.
Figure 3. Immunocult CD3/CD28/CD2 activator causes concentration and time dependent alterations in T cell activation, exhaustion and killing markers.
Real-time analysis and innovative visualization tools for rapid quantitative results
PBMCs (120K/well) from three different donors were co-cultured with Incucyte® Nuclight Green labeled Ramos cells (40K/well) and activated with a range of densities of Dynabeads CD3/CD28. Analysis was performed on day 3 using the iQue® Human T Cell Killing Kit. (A) Overlay plot shows difference in CD25 expression between positive (4:1 Dynabead to PBMC ratio) and negative (no Dynabeads) controls. (B) Heat map shows CD25 expression on PBMCs from each donors in response to Dynabeads. Donor 2 showed enhanced sensitivity at lower Dynabead densities compared to donors 1 and 3. (C) Data from (B) summarized as concentration response curves.
Figure 4. Pre-determined gates on iQue Forecyt® enable automatic phenotyping of T cell subsets
Streamline traditional workflows into a miniaturized, multiplex assay
Cell marker expression data and supernatant cytokine concentrations are acquired from a single well using a mixed cell and bead-based assay. This streamlined workflow requires no additional optimization or dilutions and includes only a single wash step; minimizing time to results.
Figure 5. Easy to follow protocol for the analysis of T cell phenotypes and cytokine release using the iQue® Human T Cell Killing Kit.
T Cell Killing Technical Resources
Featured Resources
T Cell Killing Frequently Asked Questions
The iQue® Human T Cell Killing Kit uses a multiplexed approach to perform analysis of killer T cell phenotypes and functions at different stages in a single, high-content assay including activation marker (CD25), exhaustion marker (PD-1), cell membrane integrity, cell count, secreted pro-inflammatory cytokine IFNγ and pro-apoptotic serine protease Granzyme B. 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 data for T cell populations and cytokine concentrations.
The assay has been validated using a minimum seeding density of 1 x 106 cells/mL, which is therefore recommended for the start of the assay. However, if you anticipate a rare cell event/subpopulation, then the number of cells must be increased to accommodate this to make sure the data is statistically relevant. Depending on the number of cells added, it is also important to ascertain whether cytokine levels can still be detected. If cytokine levels are too high to be measured against the linear part of the standard curve, it may be necessary to dilute the sample.
The iQue® Human T Cell Killing Kit is supplied with iQue QBeads® for the detection of IFNγ and Granzyme B. However, it only allows for measurement of these cytokines when they are secreted into the supernatant.
The total assay time is approximately three hours, with a hands-on time of approximately 30 minutes.
Samples may be fixed with certain fixatives (e.g., 1% PFA). However, it is important to understand how fixation may affect biological outcomes. The use of methanol for fixation is highly discouraged as it affects bead-based cytokine detection. Fixation and further wash steps may cause cell loss, affect the final event acquisition and warrant additional optimization. If significant cell loss is observed, perform the fixation in a cell-repellent plate (e.g., Greiner® #651970 or Greiner® #781970), which may reduce cell loss due to fixation or fixation-related cell cross-linking to the well bottom.