Biology Reference
In-Depth Information
assays (ELISA), radioimmunoassay, or IFN-g-sensitive cell lines, which mea-
sure secreted IFN-g in culture supernatants, to the ELISPOT assay, which uses
an immobilized, high-protein binding, anti-IFN-g antibody-coated membrane
to capture secreted IFN-g as it is secreted ( Larsson et al., 1999). The number of
cells secreting IFN-g is then determined by adding biotinylated secondary cap-
ture antibody followed by the use of avidin-bound biotinylated horseradish
peroxidase and color reagent. The number of spots counted indicates the num-
ber of cells producing INF-g, and HIV-speci®c spots are determined by sub-
tracting the number of spots in control, nonstimulated culture wells.
More recently, focus is shifting toward measuring intracellular IFN-g and
other cytokine levels using ¯ow cytometry (Prussin, 1997; Prussin and Met-
calfe, 1995; Vikingsson et al., 1994). This has the advantage of giving more in-
formation on the cell types producing the cytokines, and other markers such as
activation or (CD69, CD25) apoptosis (7AAD, propridium iodide) can be
measured at the same time. The technique involves stimulation of whole blood
or isolated PBMC with peptides or recombinant viruses, mainly vaccinia. This
stimulation could be done for a short or long duration depending on the
methods used. For example, peptide stimulation is usually done for a period of
4±6 h in the presence of costimulatory molecules (anti-CD28 and anti-CD49d),
whereas viral stimulation is usually done for 18±24 h corresponding with the
periods of peak cytokine productions. Irrespective of the method of stimulation
used, within the last 4 h of stimulation, secretion of cytokines from the cells is
inhibited by using monensin or brefeldin A, which, act to prevent the exocytosis
of produced cytokines. This step is followed by the ®xing and permeabilization
of the cells followed by staining with relevant conjugated antibodies speci®c for
surface and intracellular molecules. Cellular analysis is then performed using
¯ow cytometry ( Yamamura et al., 1995). Figure 6.3 shows an example from
our lab where CD3
/CD8
cells were gated and the proportion of these cells
that are CD69
and produce IFN-g are calculated.
The main disadvantage of all these methods is that they do not measure
direct cellular killing but, rather, indicate the ability of these cells to recognize
and act against HIV-speci®c antigens and produce cytokines.
Measurements of DNA Fragments
These studies rely on the ability of DNA to break down into fragments during
cellular apoptosis. Within minutes of CTL target cell contact, nuclear damage
leading to double-stranded DNA break followed by cleavage of DNA into
small oligonucleotides is noted (Duke, et al., 1988). This step is important in the
ultimate cell lysis and the extent of this damage has been shown to correspond
to the e½ciency of CTL lysis of the cells. DNA fragmentation is not peculiar to
CTL lysis, and other causes of cell death have the same consequence. The
method essentially involves the collection of released DNA fragments using a
DNA harvester or detergent lysates and quantifying them. The data derived has
