Biology Reference
In-Depth Information
exclusion and protease inhibitor (PI) uptake. With both methods, a signi®cant
increase in the percentage of dead cells was observed in the ADCC samples
when compared with control samples. It is of interest to note that addition of
anti-HIV antiserum to NK from one HIV-infected patient mediated a signi-
®cant increase in the number of dead cells as determined by PI uptake. This
increase might re¯ect the increase in HIV-infected T cells in this patient (Jewett
et al., 1997).
The high percentage of dead cells and cells undergoing DNA fragmentation
in ADCC may be a re¯ection of depletion of both NK e¨ector cells and T cells
serving as target cells. To obtain the percentage of dead cells for each sub-
population, we labeled NK cells with ¯uoroscein isothiocyanate (FITC) after
puri®cation and reconstituted with gp120-coated target cells in the absence and
presence of anti-HIV antiserum. After overnight incubation, the level of DNA
fragmentation and the percentage of cells that had lost forward-angle light
scatter were calculated with two parameters, log green ¯uorescence and for-
ward-angle scatter. It was found that a signi®cant number of both NK cells and
T cells in the mixed lymphocyte population had decreased in size and showed
high levels of DNA fragmentation in the ADCC samples. NK cells incubated
with gp120-coated CEM target cells in the presence of anti-HIV antiserum
showed increased levels of DNA fragmentation in both the NK cells and the
CEM targets. These experiments indicated a total loss of both NK and T lym-
phocytes in the ADCC samples (see Table 9.3).
Highly puri®ed NK cells obtained from HIV-seronegative donors were cul-
tured with gp120-coated autologous CD4
T lymphocytes in the presence and
absence of anti-HIV antiserum and incubated overnight to induce antibody-
dependent NK cell cytotoxicity (ADCC). The NK samples were then subjected
to the 4-h
Cr release assay to assess their cytotoxic function against K562
target cells. Relative to control samples, a signi®cant inhibition of NK function
was observed in the samples where ADCC had taken place. Likewise, a sig-
ni®cant increase in the percentage of fragmented DNA was observed in the
ADCC samples when compared to control samples.
51
T A B L E 9.3. Properties of Normal NK Cells after Interaction with the ADCC
Target Cells
Properties
Control
Anti-HIV serum
gp120
gp120 and anti-HIV
CD16
ÿ
CD56
CD69
ÿ
ÿ
ÿ
Cytotoxicity
TNF-a secretion
ÿ
ÿ
ÿ
IFN-g secretion
ÿ
ÿ
ÿ
Fas
ÿ
ÿ
ÿ
DNA fragmentation
ÿ
ÿ
ÿ
Recovery
