Measuring the Activity of Cytolytic Lymphocytes

Abstract

The protocols in this unit describe how to measure the activity of the two most common types of lymphocytes that have cytolytic activity: cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Measurement of CTL activity requires priming of cells, which can be performed either in vivo or in vitro. Priming leads to a population of differentiated, antigen-specific CTL. Protocols in which CTL are primed in vivo and in vitro are included in this unit. In contrast to CTL, the cytolytic activity of NK cells can be measured in the absence of antigen priming. In all of these protocols, single-cell suspensions of effector and target cells are prepared separately. The target cells are labeled with (51)Cr, and then the effector and target cells are co-cultured for several hours. The amount of target cell lysis is determined by measuring the amount of (51)Cr released into the culture medium. For all of the protocols, the effector cells are derived from mouse spleen. The target cells are either spleen cells from mice of a different MHC haplotype, virus-infected cells, tumor cells, or cultured cell lines. These protocols provide a methodological framework that can be adapted for measuring the activity of cytolytic lymphocytes in a variety of experimental paradigms.

The regulatory T cell effector molecule fibrinogen‐like protein 2 is necessary for the development of rapamycin‐induced tolerance to fully MHC‐mismatched murine cardiac allografts

“…Maximum lysis was determined by addition of Triton‐X100 (Sigma‐Aldrich) and spontaneous background release was assessed by incubating target cells alone. Per cent specific lysis was calculated with the following formula: per cent cytotoxicity = [(experimental release − background release)]/[(maximum lysis − background release)] × 100%, as previously described …”

“…Per cent specific lysis was calculated with the following formula: per cent cytotoxicity = [(experimental release À background release)]/[(maximum lysis À background release)] 9 100%, as previously described. 24 Infection with lymphocytic choriomeningitis virus strain WE Lymphocytic choriomeningitis virus (LCMV) WE was obtained as a gift from the laboratory of Dr Pamela Ohashi (Ontario Cancer Institute, Toronto, Canada) and was propagated on L929 cells. Virus was purified from virus containing supernatants by banding on isopycnic Renografin-76 (Sigma Aldrich, St Louis, MO) gradients as described previously.…”

Benefits of functional assays in personalized cancer medicine: more than just a proof-of-concept

“…Yet, several functional assays are being developed 160 , using various readouts. A so-called gold standard technique is the chromium 51 release assay, which has been used for decades 161 . Easier to implement, biophysical parameters such as real-time cellular impedance can be converted into cytolysis measurement to study the in vitro activity of most immunotherapy drug classes and immune effector cells 162 .…”