Cancer Immunotherapy: T Cell-Mediated Cytolysis

ACEA Biosciences xCELLigence

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.

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:
  1. Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
  2. Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
  3. Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
  4. 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.
  5. Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.
T Cell-mediated Cytolysis – Supporting information:

  • Adherent target cells tested:
    TIII melanoma, SK-BR3, HCC1419, MCF-7, BT20, 15-12RM, OAW42, HLA-negative NCI-ADR-RES cells, murine 4T1 mammary gland tumor cells, BCSC (breast cancer stem cell), MSC (mesenchymal stem cell), BT20, HCC1419

 

  • T Cell-mediated Cytolysis Publications:
  1. T cells contribute to tumor progression by favoring pro-tumoral properties of intra-tumoral myeloid cells in a mouse model for spontaneous melanoma. Lengagne R, Pommier A, Caron J, Douguet L, Garcette M, Kato M, Avril MF, Abastado JP, Bercovici N,Lucas B, Prévost-Blondel A. PLoS One. 2011;6(5):e20235. (INSERM, France)
  2. Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing the Cr51 release assay to the xCELLigence system. Erskine CL, Henle AM, Knutson KL. J Vis Exp. 2012 Aug 8;(66):e3683. (Mayo Clinic, USA)
  3. Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells. Henle AM, Erskine CL, Benson LM, Clynes R, Knutson KL. J Immunol. 2013 Jan 1;190(1):479-88. (Mayo Clinic, USA)
  4. CD47 in the tumor microenvironment limits cooperation between antitumor T-cell immunity and radiotherapy. Soto-Pantoja DR, Terabe M, Ghosh A, Ridnour LA, DeGraff WG, Wink DA, Berzofsky JA, Roberts DD. Cancer Res. 2014 Dec 1;74(23):6771-83. (National Institutes of Health, USA)
  5. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells. Peper JK, Schuster H, Löffler MW, Schmid-Horch B, Rammensee HG, Stevanović S. J Immunol Methods. 2014 Mar;405:192-8. (University of Tübingen, Auf derMorgenstelle, Germany)
  6. Targeting specificity of dendritic cells on breast cancer stem cells: in vitro and in vivo evaluations. Nguyen ST, Nguyen HL, Pham VQ, Nguyen GT, Tran CD, Phan NK, Pham PV. Onco Targets Ther. 2015 Jan 30;8:323-34. (Vietnam National University, Vietnam)
  7. A simple in vitro method for evaluating dendritic cell-based vaccinations. Pham PV, Nguyen NT, Nguyen HM, Khuat LT, Le PM, Pham VQ, Nguyen ST, Phan NK. Onco Targets Ther. 2014 Aug 18;7:1455-64. (Vietnam National University, Vietnam)