Biology Reference
In-Depth Information
the breakdown of the cellular organelles and cell membrane and ultimately cell
death.
The granule-exocytosis pathway involves tight, calcium-independent, e¨ector/
target adhesions through the T-cell receptor-MHC/peptide molecules. Priming
and triggering of cell killing is calcium dependent. The next step is the release
of granules from the ``cores,'' including perforin and a number of granzymes,
particularly granzymes A and B (Spaeny-Dekking et al., 1998). These granules
act on the cellular membrane of the target cells, leading to a break in the
membrane and cell death.
The Fas/FasL pathway is independent of perforin and other granules
( Kajino et al., 1998). It is also triggered by the trimodal cross-linking of TCR/
MHC/antigen. It involves the cross-linking of the Fas ligand expressed on the
surface of the e¨ector CTL and the Fas molecule expressed on the surface of
the target cells, leading to a triggering of a cascade of mechanisms culminating
in apoptotic target cell death.
The most used method of measuring membrane integrity is the chromium
release assay (Segal and Snider 1989). In this assay, target cells expressing
MHC-restricted antigens are pulsed with radioactive
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Cr. The cells are then
plated in culture with autologous e¨ector CTL, either freshly isolated or fol-
lowing in vitro expansion. The CTL are added at di¨erent ratios to the target
cells, usually from 100:1 to 3.125:1. Target cells expressing an irrelevant antigen
are used as control in parallel wells. The culture period used is usually 4±6 h.
The encounter between the target and the CTL e¨ectors should commence
killing of the target within minutes of this incubation, leading to the release of
the chromium into the culture media. At the end of the incubation, the super-
natents are collected, and the amount of chromium released is measured using a
gamma counter, which measures the released
Cr. The data is then analyzed
by calculating the percentage of speci®c killing or lytic units. Percentage of
speci®c killing is calculated using the formula:
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amount of experimental chromium released ÿ spontaneously released chromium 100
amount of maximum chromium released
ÿ spontaneously released chromium
The spontaneous chromium released refers to the wells containing only target
cells, whereas the maximum release data refer to wells containing target cells
and detergents, such as 2.5% Triton, which has the capability of lysing all the
cells and releasing maximum amount of chromium. The antigen-speci®c CTL
killing is then calculated for each E:T ratio.
Other less routinely used studies of membrane integrity include non-
radioactive techniques such as the CARE-LASS, a ¯uorometric microassay
( Lichtenfels et al., 1994). In this assay, an acetoxymethyl ester of calcein, which
crosses cell membrane easily, is added to the target cells and is converted to the
polar ¯uorochrome calcein in the cytoplasm of intact cells. In the presence of
CTL killing, this ¯uorochrome is released and measured using an automated
¯uorescence scanner. Another closely related technique is the time-resolved
