Differential effect of T cell growth cytokines on PBMC apoptosis and CMV-specific proliferation
To test the hypothesis that γ-chain receptor signaling is sufficient to decrease apoptosis and increase proliferation of PBMC from HIV-infected subjects, we examined the ex-vivo effect of IL-2, IL-4, IL-7 and IL-15 γ-chain cytokines on CMV-stimulated PBMC from HIV-infected and uninfected subjects. Although these cytokines have common γ-chain receptor activity, they differ with respect to their overall function: IL-2 and IL-15 have Th1 properties, IL-7 has Th0 properties and IL-4 has Th2 properties. The experiments using PBMC from healthy volunteers showed that all 4 cytokines significantly increased proliferation stimulated with mock-infected control antigen (average increase of 65240, 4802, 9251 and 105800 cpm for IL-2, IL-4, IL-7 and IL-15, respectively; not depicted), but also CMV-specific proliferation, assessed by the difference between 3H-thymidine incorporation in CMV and control-antigen-stimulated cultures (). IL-2, IL-7 and IL-15, but not IL-4, increased proliferation of control-antigen-stimulated PBMC from HIV-infected subjects (averages of 16300, 1836, 29780 and 97 cpm, respectively; not depicted). Only the Th1 cytokines IL-2 and IL-15 significantly increased CMV-specific proliferation of PBMC from HIV-infected subjects on HAART (). Although there was no apparent difference in the net effects of IL-4 and IL-7 on CMV-specific proliferation of PBMC from HIV-infected subjects, they resulted from different mechanisms: IL-4 failed to increase proliferation of either CMV or control antigen-stimulated PBMC, whereas IL-7 equally increased proliferation of CMV- and control antigen-stimulated PBMC.
Figure 1 Effect of T cell growth cytokines on CMV-specific proliferation and apoptosis of PBMC from HIV-infected and uninfected subjects. Data were derived from 36 HIV-infected subjects (median CD4=352 cells/µl; median plasma HIV RNA <400 copies/ml) (more ...)
In cytokine-untreated cultures, CMV-stimulated proliferation of PBMC from HIV-infected subjects was significantly lower compared with proliferation of PBMC from healthy individuals, with a mean difference±SEM of 30260±10350 cpm, p=0.008. To determine if exogenous addition of cytokines brought the CMV-specific proliferative responses of PBMC from HAART recipients closer to the levels observed in healthy subjects, we compared the CMV-specific cpm in cytokine-treated cultures of HIV-infected subjects with the CMV-specific cpm in cytokine-untreated cultures from healthy subjects. IL-2 and IL-15 reduced the gap between HIV-infected and uninfected subjects to 3808±13170 cpm (p=0.77) and 11000±17350 cpm (p=0.53), respectively. In contrast, exogenous IL-4 or IL-7 did not appreciably alter the CMV-specific proliferation difference between HIV-infected and uninfected subjects (p of 0.01 and 0.05, respectively).
IL-15 ex-vivo treatment significantly decreased apoptosis of CMV-stimulated PBMC from HIV-infected (mean difference±SEM of 27±4%, p<0.0001 paired T test) and healthy subjects (10±3%, p=0.006 paired T test). IL-2 marginally decreased apoptosis of CMV-stimulated PBMC from HIV-infected individuals (14±5%, p=0.06 paired T test), but not of PBMC from healthy subjects (p=0.13 paired T test). IL-4 and IL-7 tended to decrease apoptosis of PBMC from HIV-infected and uninfected subjects, but their effects did not reach statistical significance (p of 0.10 to 0.23, paired T test).
In cytokine-untreated cultures, apoptosis of CMV-stimulated PBMC from HIV-infected subjects was significantly higher compared with uninfected controls with mean difference±SEM Annexin V+ PBMC of 23±3% (p<0.0001, unpaired T test). IL-15 ex-vivo treatment reduced the gap in apoptosis between CMV-stimulated PBMC from HIV-infected vs. uninfected individuals to mean difference±SEM of 6±5% (p=0.28, unpaired T test). IL-2 treatment had a less potent effect on the excess apoptosis of HAART recipients compared with uninfected controls (mean difference of 8±4%, p=0.06, unpaired T test), whereas supplementation with exogenous IL-4 or IL-7 did not appreciably change the difference in apoptosis between CMV-stimulated PBMC cultures from HAART recipients vs. healthy subjects, which remained highly significant (p of 0.03 and 0.007, respectively). These data indicated that IL-15 had the most potent anti-apoptotic effect among the 4 cytokines evaluated in this study.
To investigate the association between the proliferative and anti-apoptotic effects of ex-vivo cytokine treatment, we performed correlation analyses between the cytokine-induced increase in log10 CMV-specific cpm with the decrease of Annexin V+PI+% PBMC. Significant positive correlations were found for IL-2 and IL-15 (p=0.01 each) but not for IL-4 or IL-7. These results suggested that the lack of survival and proliferation of PBMC from HAART recipients in response to CMV stimulation might be due to a Th1 cytokine specific deficit that could be corrected by the exogenous administration of IL-2 and even more so of IL-15. IL-4 and IL-7 did not appreciably improve apoptosis or proliferation of CMV-stimulated PBMC from HAART recipients, therefore all subsequent experiments addressed differences and similarities between the effects of IL-2 and IL-15 only.
Effect of IL-2 and IL-15 on CMV-stimulated CD4+ and CD8+cell proliferation and apoptosis
It was previously reported that the effect of IL-15 was more prominent on CD8+ compared with CD4+ T cells. To determine if IL-15 preferentially elicited CD8+ cell proliferation in the presence of CMV antigenic stimulation and to understand how this compared with the effect of IL-2 on CMV-specific T cell proliferation, we used CFSE dye dilution, which enabled the differentiation between CD4+ and CD8+ cell proliferation. We used the CMV lysate as a stimulant after showing that this particulate antigen induces both CD4+ and CD8+ cell proliferation ().
Figure 2 CD4+ and CD8+ cell expansions in cultures stimulated with inactivated CMV antigen. The plots represent typical examples of CFSE-measured proliferation in HIV-infected (right 2 columns) and uninfected (left 2 columns) subjects. PBMC are labeled with CFSE (more ...)
The analysis of CMV or control antigen-stimulated PBMC cultures from 8 HIV-infected subjects showed that there were no appreciable differences in the effect of IL-2 or IL-15 on CD4+ vs. CD8+ cell expansions (p>0.5 for either cytokine). Both cytokines increased cell divisions in CMV or control antigen-stimulated cultures by approximately an order of magnitude. Compared with IL-2, IL-15 treatment generated higher CMV-specific proliferation of CD4+ (mean±SEM of 11±3% vs. 4±3%, p=0.05) and CD8+ cells (8±2% vs. 1±2%, p=0.05), suggesting that IL-15 may have a more discriminative antigen-enhancing effect compared with IL-2 ().
Figure 3 Effect of IL-2 and IL-15 on CMV or control-antigen stimulated expansion of CD4+ and CD8+ PBMC from HIV-infected subjects on HAART. The bars represent means and SEM of results derived from 8 HAART recipients (median CD4= 322 cells/µl; median plasma (more ...)
Neither IL-2 nor IL-15 exogenous administration appreciably changed viability of control antigen-stimulated PBMC from 6 donors equally distributed between HIV-infected and uninfected individuals (mean±SEM viability of 44±7%, 46±5% and 51±5% in untreated, IL-2-treated and IL-15-treated cultures, respectively; 0.12, ANOVA for repeated measures; not depicted). In contrast, in CMV-stimulated cultures, IL-15 exogenous administration significantly increased the percentage of viable T cells to 48±5% compared with 40±6% in cytokine-untreated cultures (p=0.005) and 43±5% in IL-2-treated cultures (p=0.002; ). In contrast, viability of IL-2-treated and cytokine-untreated T cells was not appreciably different. IL-2 treatment resulted in a significantly higher percentage of cells in early apoptosis (15±1%) compared with cytokine-untreated and IL-15-treated lymphocytes (13±1% each, p=0.05). IL-2 and IL-15 treatment equally decreased the percentage of T cells in late apoptosis from 38±6% in untreated cultures to 23±3% and 23±4% in IL-2- and IL-15-treated cultures, respectively (p=0.005 and 0.006, respectively).
Figure 4 Comparative effect of IL-2 and IL-15 on apoptosis of CMV-stimulated PBMC from HIV-infected subjects on HAART. Panel A shows a typical scatter plot. Live, early apoptotic and late apoptotic cells were measured by the % events in lower left, lower right (more ...)
Effect of IL-2 and IL-15 on the frequency of regulatory T cells (Tregs)
IL-2 was previously shown to increase CD4+CD25+FoxP3+ Tregs. We hypothesized that a diverse effect of IL-2 vs. IL-15 on Tregs may contribute to the differential effect of these cytokines on apoptosis and antigen-specific proliferation of CMV-stimulated PBMC. The effect of IL-2 and IL-15 on the frequency of Tregs was investigated using PBMC from 8 HIV-infected subjects on HAART (median CD4+ numbers =340 cells/µl and plasma HIV RNA<400copies/ml; ). We limited these experiments to HIV-infected donors because these constitute the target population for a potential therapeutic application. IL-2-treated cultures of CMV-stimulated PBMC had higher frequencies of CD4+CD25+FoxP3+ Tregs compared with untreated (mean difference=5%, p=0.03) or with IL-15-treated cultures (mean difference=4.6%, p=0.04). IL-2 also increased the frequency of CD8+CD25+FoxP3+ cells compared with untreated (mean difference=8%, p=0.03) or with IL-15-treated cultures (mean difference=7.4%, p=0.05). Addition of exogenous IL-15 did not appreciably increase the frequency of CD4+CD25+FoxP3+ or CD8+CD25+FoxP3+ Tregs compared with untreated cultures (p=0.4 for either comparison).
Figure 5 Comparative effect of IL-2 and IL-15 on CD4+ and CD8+ Treg frequencies in PBMC from HIV-infected subjects on HAART. Panel A shows a typical scatter plot. Panel B presents the data from 8 subjects (median CD4=340 cells/µl; median plasma HIV RNA<400copies/ml). (more ...)
Effect of IL-2 and IL-15 on cytokine and chemokine production of CMV-stimulated PBMC from HIV-infected subjects
Cytokine production was used to assess additional functional characteristics of CMV-stimulated PBMC. Th1 and Th2 cytokines and chemokines were measured in supernatants of CMV or control antigen-stimulated PBMC from 22 HIV-infected subjects on HAART with median CD4+ cells=315 cells/µl and median plasma HIV RNA<400 copies/ml (). The data, expressed as differences between CMV or control antigen-stimulated cultures treated with IL-2, IL-15 or no cytokine, showed that both IL-2 and IL-15 significantly increased the Th1 cytokines IFNγ, TNFα and the chemokine RANTES (p<0.05, paired T tests). For MIP-1α, only the boosting effect of IL-15 reached statistical significance (p=0.04). In contrast, IL-2, but not IL-15, significantly increased the production of Th2 cytokines including IL-4, IL-5, IL-10 and IL-13 (p<0.01, paired T tests). IL-2 had its largest effect on IL-5 production, which increased from mean±SEM of 5±0.7 pg/ml in untreated cultures to 114±34 in IL-2-treated PBMC cultures, followed by IL-13, IL-10 and IL-4. The amount of IL-4 measured in culture supernatants was small and so was the difference between untreated and IL-2 treated PBMC. However, the consistency of the effect of IL-2 across all Th2 cytokines together with the fact that there was no IL-4 detected in the culture medium or in the supernatant of the PBMC stimulated with control antigen suggests that these differences were real. The biologic significance of such small differences is difficult to ascertain.
Figure 6 Comparative effect of IL-2 and IL-15 on CMV-stimulated cytokine and chemokine production of PBMC from HIV-infected subjects on HAART. Bars represent means and SEM of cytokine or chemokine concentrations in CMV-stimulated PBMC culture supernatants after (more ...)
The effect of IL-2 and IL-15 on PBMC from 4 healthy individuals (data not depicted) was consistent with the findings of HIV-infected subjects. Both cytokines significantly increased the secretion of TNFα, RANTES and IL-10, but only IL-2 significantly increased IL-5 and IL-13 production.