Galpin/DA/N2 IIIBenv 
each of 32 HIV-positive patients such that target/stimulators for each patient had the 
correct MHC for antigen presentation. These EB V-transformed cell lines were 
transduced with HIV-IT (V), and tested by Western blot analyses to verify HIV-1 env 
expression. These cell lines were also transduced with an HIV-1 gag expressing vector. 
PBMC were isolated from whole blood samples of patients by a Ficoll-Hypaque gradient. 
Isolated PBMC were cultured with irradiated autologous HIV-1 env-expressing 
EB V-transformed cell lines for 7 to 10 days. After this in vitro stimulation, the cultures 
were used as effectors in a -^Cr-release assay where net specific lysis of 
HIV-1 env-expressing cells was measured. 
Most adults have been exposed to EBV, thus, when the patient PBMC are stimulated 
in vitro with an EB V-transformed cell line, the EBV-specific CTL are efficiently 
activated. Two approaches were taken to address this issue. First, differing ratios of 
responder PBMC to EBV stimulator cells (R:S ratio) were used in the in vitro culture step 
which lowers background lysis. Secondly, excess unlabeled EBV-targets were 
incorporated into the assay to compete out the EBV-specific lysis of the labeled target. 
Both approaches proved to be useful in controlling the masking effect that EBV-specific 
lysis exhibited. 
Thirty two patients with CD4 + counts from 260 to greater than 1 ,000 were tested for 
HIV-1 env-specific lysis after in vitro stimulation. Twenty six patients had a net specific 
lysis greater than 20%, 5 had net specific lysis between 10 - 20% only one patient 
consistently had no HIV-1 env-specific lysis (net lysis less than 10% at 10 different time 
points). Thirty-one of these patients have been tested for HIV-1 gag responses, with 30 
demonstrating net specific lysis greater than 20%. Additional experiments indicated that 
the measured lysis was not attributable to natural killer cell activity. In a single patient, 
the CTL were shown to be CD8 + . Further characterization of the CD4 + and CD8 + 
components of this CTL response in humans is underway. 
Recombinant DNA Research, Volume 17 
[889] 
