What are T Cells?
By seeking out and destroying infected cells directly, the CD8+ subgroup of T cells play a critical role in the adaptive immune response. Every CD8+ T cell clone expresses a unique variant of a specialized receptor, the T cell receptor (TCR), that can recognize and bind to a specific antigenic peptide presented by MHC class I (MHC-I) molecules on the surface of target cells. Engaging infected or cancerous cells through this antigen:MHC-I complex causes CD8+ cells to secrete perforin and granzymes, leading to lysis of the target cell.
Tumor cells typically acquire extensive mutations in their genomes, including the genes of key regulatory and signaling proteins. When cleaved, processed and presented by the MHC-I molecule on the surface of antigen presenting cells, these mutated proteins can elicit a cellular immune response.
It is for this reason that T lymphocytes can be found inside tumors. Some cancer vaccines exploit this tumor targeting capacity of T cells by priming the cellular arm of the adaptive immune response to target cancer cells that are expressing proteins that are either mutated or expressed at abnormal levels.
Application Highlight: Detecting Cytolytic Activity of CD8+ T Cells Against Breast Cancer Cells
While in some contexts it is useful to quantify the number of antigen-specific CD8+ T cells in samples using assays such as ELISpot or flow cytometry, it is often critical to assess the functional cytotoxicity of these cells via killing assays. Measuring cytolytic activity via the chromium-51 release assay has long been the gold standard for evaluating CD8+ T cell responses.
In the assay shown below, SKBR-3 breast cancer cells expressing the HER2/Neu protein are pre-labeled with 51Cr. They are subsequently co-incubated with increasing amounts of a CD8+ T cell clone that expresses a TCR targeting an antigenic peptide of HER2/Neu, and target cell killing is detected by release of 51Cr into the medium.
This same assay is concurrently performed using the xCELLigence RTCA system without pre-labeling of the target cells. The RTCA system quantitatively detects the cytolytic activity of CD8+ T cells against the SKBR-3 target cells in a manner that is dependent on both time and number of CD8+ T cells added (left panel).
Side by side comparison with the 51Cr release assay shows that the sensitivity and dynamic range of the xCELLigence RTCA assay surpasses that of 51Cr (right panel). Moreover, the preclusion of radio-labeling, and the kinetic data provided by RTCA (including both the onset of cytolysis and the rate of tumor cell killing) make this assay especially attractive.
CD8+ T cell-mediated cytolysis of SKBR3 tumor cells. Upon CD8+ T cell addition, real-time impedance traces show a distinct drop in the number, size/shape, and/or attachment quality of SKBR3 tumor cells (left panel). This response is dose-dependent, with high effector:target cell ratios yielding the most substantial decrease (left panel). Plotting the percentage of tumor cell lysis, as determined by xCELLigence RTCA vs. a standard 51Cr release assay, demonstrates RTCA to be the more sensitive method (right panel). Figure adapted from J Vis Exp. 2012 Aug 8;(66):e3683.
Key Benefits of Using xCELLigence for Studying T Cell-mediated Cytolysis:
- Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
- Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
- Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
- Simple Workflow: Requires only the addition of effector cells to target cells (in the presence or absence of antibodies); homogeneous assay without additional sample handling.
- Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.
Explore Functional Potency Assays for Cancer Immunotherapy Research Download Handbook Cancer Immunotherapy Research Grant The research grant winner will be provided access to the xCELLigence Real Time Cell Analysis (RTCA) SP instrument, consumables, and consultation for up to 6 months. Apply by December 15, 2019. Learn More WEBINAR –The Next Generation of CAR-T Cells: New Techniques in Gene Editing and Rapid Assessment with Real Time Live Cell Analysis Watch our experts in CAR-T cell development as they discuss new techniques to further improve functionality.
Explore Functional Potency Assays for Cancer Immunotherapy Research
Cancer Immunotherapy Research Grant
The research grant winner will be provided access to the xCELLigence Real Time Cell Analysis (RTCA) SP instrument, consumables, and consultation for up to 6 months. Apply by December 15, 2019.
WEBINAR –The Next Generation of CAR-T Cells: New Techniques in Gene Editing and Rapid Assessment with Real Time Live Cell Analysis
Watch our experts in CAR-T cell development as they discuss new techniques to further improve functionality.Watch Now