T Cell Killing
Cell and Cytokine Profiling
T Cell Killing
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 are able to 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, BiTEs, CARs and Trucks. Harnessing the power of the immune system is seen as a powerful tool in fighting cancer and has shown promising results in the immuno-oncology field. 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 utlizes the iQue® Human T Cell Killing Kit for simultaneous, high throughput analysis of target cell killing, T cell phenotypic and activation marker expression, 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.
Figure 1. Illustration of iQue® Human T Cell Killing Kit assay principles.
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.
Gain biological insights
Assess T cell killing in physiologically relevant models
Figure 2. CD3xCD19 Bispecific T Cell Engager (BiTE) antibody causes concentration dependent killing, marker expression and cytokine release over a 6 day time course.
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.
Increase your productivity
Simultaneous measurement of marker expression and secreted cytokines in a mixed cell and bead assay format
Figure 3. Immunocult CD3/CD28/CD2 activator causes concentration and time dependent alterations in T cell activation, exhaustion and killing markers.
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.
Streamline data analysis
Real time data analysis and novel visualization tools enable rapid generation of quantitative readouts
Figure 4. Pre-determined gates on iQue Forecyt® enable automatic phenotyping of T cell subsets
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) summarised as concentration response curves.
Save time and precious sample
Collapse traditional workflows into one miniaturized, multiplex assay
Figure 5. Easy to follow protocol for the analysis of T cell phenotypes and cytokine release using the iQue® Human T Cell Killing Kit.
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 optimisation or dilutions and includes only a single wash step; minimizing time to results.