3.4 FACTORES QUE AFECTAN LA CALIDAD DE CARTERA EN LAS
3.4.2 Factores de riesgo que asumen las entidades
The cytolytic activity o f PBMC from 27 H IV -1 ", 17 HIV-1 ^ CDCII, 15 H TV -l^ CDCIV and 11 AVI individuals is shown. In this assay the target cell, PS 15 was incubated with the effectors at the ratios indicated in the presence o f 1/xg/ml PHA. Arithmetic means were compared to the H IV -1 " control using the students t-test; * p < 0.001
T able 8.1 T he E ffect o f C D 16+ D epletion on C ytolytic Activity^ Part I n W ithout Activation PBMC CD16 Depleted H IV -1 - 10 8 .3 ± 1 .6 ^ 1.7±0.6**‘' HIV-1+ CDCII 6 2 0 .2 ± 5 .5 7 .4 ± 2 .3 * CDCIV 5 3 6 .5 ± 3 .8 2 8 .8 ± 6 .9 AVI 6 3 7 .5 ± 4 .8 1 3 .2 ± 0 .2 Part II n
After PHA Stimulation
PBMC CD16 Depleted
H IV -1 - 10 6 2 .6 ± 4 .7 4 5 .6 ± 2 .8 *
HIV-1 + 9 3 9 .7 ± 4 .5 2 1 .8 ± 5 .5 *
^ The cytolytic activity of PBMC before and after removal of CD 16^ NK cells was compared in freshly isolated PBMC (Part I) and after 3 days activation with PHA plus rIL-2 (Part II).
^ The percentage cytotoxicity at E:T 12:1 in the LDC assay is shown
^ The data were compared using the paired Students t-test, * p < 0 .0 5 , * * p < 0.005
rapidly after only 24 hours in culture (52%; E:T 12:1) reaching a peak of around 65% between days 2 and 3. Thereafter cytolytic activity slowly declined unless cultures were supplemented with IL-2 or restimulated. A comparison o f H IV -1~ and H IV -1^ individuals revealed that the initial early increase in cytolytic activity during the first three days was absent in H IV -1^ patients. This is illustrated in Fig. 8.2 which shows a representative example of one H IV -1~ and one H I V - 1 d o n o r . Although the starting level of killing is higher in this H IV -1^ individual compared to the HIV-1"" control (Control 10.2%, HIV-1 ^ 27.7% ; E:T 12:1), the activation associated increase in cytotoxicity is delayed. In fact, activation results in an initial drop in activity over the first three days, and reaches those seen in the seronegative only by day 5.
Activation with anti-CD3 also induces CTL activity in C D 3~ T cells (Jung, M artin & M uller-Eberhard 1987) probably through the action o f IL-2 on NK cells. As this could obscure any change in T cell mediated killing, the initial failure to increase cytotoxic killing was further investigated in a larger group o f patients after removal of NK cells. W hen C D 16^ cells were removed prior to stimulation, the cytotoxicity that subsequently developed in both H IV -1 " and HIV-1 ^ cultures was reduced (Table 8.1) but was substantially higher than CTL activity in unstimulated cultures. A comparison of cytotoxicity in 10 H IV -1 " and 10 HIV-l'*' individuals prior to culture and after 3 days activation with PHA plus IL-2 is shown in Fig. 8.3. From this it is clear that although initial levels of killing are higher in the H IV -1^ group (H IV -1" 8 .0 ± 1 .4 % ; HIV-1 + 2 8 .2 + 6 .1 ; E:T 12:1; p < 0 .0 0 1 ), activation over this short time period does not significantly increase the amount of cytotoxicity in the HIV-1 ^ group (34.7 ± 5.1 % ). Lymphocytes from H IV -1 " individuals on the other hand acquire high levels of killing after this short term stimulation
(6 8 .4 + 5 .2 % ; p < 0.001).
These results suggest that lymphocytes with CTL function are pre-activated in the blood of HIV-1'*’ patients but cannot be efficiently activated by TcR ligation unlike the resting C D 8‘*’ T cells present in H IV -1" individuals. The failure of lymphocytes from HIV-1 ^ individuals to acquire cytolytic activity after activation in vitro was due to the rapid death of the responding cells. Activated cell cultures from HIV-1'*' donors adjusted for viable cells showed high levels o f killing which were
51 % Cr Release 100 n 80 -
HIV-1-
HIV-1+
6 0 - 40 2 0 - 0 1 2 3 4 5 6 7Time (days)
Figure 8.2 Time course of cytolytic activity after stimulation in vitroPBMC from one H IV-1“ and one HIV-1 ^ individual were cultured in the presence o f anti-CD3 plus rIL-2 and the cytolytic activity measured daily using the LDC assay against P815 target cells. The percentage cytotoxicity at E:T 12:1 is shown.
The cytolytic activity o f each well was measured at an equivalent o f E:T 12:1 without re-adjusting for cell viability after activation
51
% Cr Release 8 0 1 6 0 -HIV-1-
H V-1+
Day 0
Day 3
Figure 8.3 A com parison of cytotoxicity before and after activation in vitro
PBMC were depleted o f C D 16^ , NK ceils and assayed before and after stimulation with anti-CD3 plus rIL-2 using the LDC assay against P815 target cells. The arithmetic mean ± SEM from 10 H IV -1 ~ and 10 HIV-1 ^ individuals at E:T 12:1 is shown.
The cytolytic activity o f each w ell was measured at an equivalent o f E:T 12:1 without re-adjusting for cell viability after activation
comparable to those seen in the seronegative group (H IV -1 " 58.3 ± 2 .3 % , HIV-1 ^ 5 6 .0 ± 1 .1 % ; E:T 12:1).
In conclusion, H IV -1^ individuals have high levels o f circulating CTL even in the later stages of HIV-1 disease. These cells are, however, unresponsive and after short-term stimulation in vitro this activity is lost due to the death o f the responding cells.
H IV -1 specific CTL activity (i) Freshly Isolated PBMC
To determine if the CTL activity measured in the LDC assay was directed against the HIV-1 virus, 10 HIV-1 ^ patients were assayed for specific CTL activity in freshly isolated PBMC. Cytolytic killing (> 1 0 % at E:T 25:1) was detected in 4 out o f 6 asymptomatic patients but was absent in all CDCIV patients tested (n = 4 ). However, CTL activity against PS 15 target cells in the LDC assay was present in all individuals and was highest in the CDCIV patients. This is illustrated in Fig. 8.4 which shows a representative HIV-1 ^ patient (A) who has CTL activity against P815 (17%) and H IV -1 gag (27%) but does not kill the NK target, K562, or the uninfected control cell lines. In contrast, the CDCIV patient shown (B) has activity only against P815 in the lectin dependent system (37%) but has < 5 % HIV-1 specific killing. No HIV-1 specific CTL were detected when PBMC from H IV -1 " donors were investigated (n = 2 ).
(ii) Lymph Node Suspensions
Using the LDC assay the cytotoxic activity of fresh lymph node suspensions was measured in 6 HIV-1 ^ individuals. When whole lymph node suspensions were used as effectors, CTL activity was low in all individuals (< 5 % at E:T 50:1). However, purified C D 8^ lymphocytes prepared by negative selection o f E"^ cells from two individuals did show detectable CTL activity (34.2% & 10.4% at E:T 50:1). An H IV -lgag specific CTL assay was then performed using lymph node suspension from one of these individuals both unstimulated and after co-culture with autologous PHA blasts for 5 days (Fig. 8.5). The unstimulated cells showed no HIV
lg<3g specific killing and were cytolytic only against the P815 target cells. However, after co-culture, low levels o f H IV -1 gag specific CTL were detected (13% at E:T