Unit

UNIT 3.11 Induction and Measurement of Cytotoxic T Lymphocyte Activity

  1. John Wonderlich1,
  2. Gene Shearer1,
  3. Alexandra Livingstone (JAM test)2,
  4. Andrew Brooks (CTL activity in vivo)3

Published Online: 1 MAY 2006

DOI: 10.1002/0471142735.im0311s72

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Wonderlich, J., Shearer, G., Livingstone, A. and Brooks, A. 2006. Induction and Measurement of Cytotoxic T Lymphocyte Activity. Current Protocols in Immunology. 72:3.11.1–3.11.23.

Author Information

  1. 1

    National Cancer Institute, Bethesda, Maryland

  2. 2

    Imperial College, London, United Kingdom

  3. 3

    University of Melbourne, Victoria, Australia

Publication History

  1. Published Online: 1 MAY 2006
  2. Published Print: APR 2006

Abstract

Cytotoxic T lymphocytes (CTL) kill target cells on the basis of cell-surface antigen recognition and are important in the host response to tumors, transplants, and viruses. This unit presents several protocols for generating and measuring CTL activity. The first describes generating CTL against some of the most commonly used target antigens. Two methods for the quantitation of CTL activity are described based on the two pathways used bt CTL to kill target cells. In one pathway, they release lytic granules containing perforin and granzymes, leading to apoptosis and target cell lysis. In a second pathway, they trigger apoptosis via Fas/Fas ligand interactions. In the chromium-release assay provided here, labeled antigenic targets are recognized and lysed, releasing radioactivity into the supernatant. In the JAM test protocol, CTL activity is determined by measuring degradation of radioactively labeled DNA in target cells that have undergone apoptotic cell death. Rather than measuring release of radioactivity, the JAM test measures the amount of DNA retained in target cells that are not killed by CTL. Two support protocols detail the generation of CTL precursors (CTLp) against antigens that require priming in vivo. A second set of support protocols describe the preparation of both stimulator and target cells for these responses using two representative antigens, trinitrophenyl and viruses. Finally, two alternate protocols illustrate how to determine total CTLp activity in a population that might express cytolytic activity. These protocols bypass MHC restriction and the original antigen specificity of CTLp by polyclonal stimulation of CTLp with mitogens followed by attachment of CTL to target cells and subsequent cytolysis.