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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Virology. Author manuscript; available in PMC Aug 15, 2009.
Published in final edited form as:
PMCID: PMC2593685
NIHMSID: NIHMS64583
Distinctive In Vitro Effects of T-Cell Growth Cytokines on Cytomegalovirus-Stimulated T Cell Responses of HIV-Infected HAART Recipients
Julie Patterson,1 Renee Jesser,1 and Adriana Weinberg1,2,3*
1Department of Pediatrics of the University of Colorado School of Medicine, Denver, CO
2Department of Medicine of the University of Colorado School of Medicine, Denver, CO
3Department of Pathology of the University of Colorado School of Medicine, Denver, CO
*Corresponding author: Adriana Weinberg, 4200 E. 9th Ave., Campus Box C227, Denver, CO 80262, Phone: 303-315-4624, Fax: 303-315-1787, Adriana.Weinberg/at/uchsc.edu
Functional immune-reconstitution is limited after HAART, maintaining the interest in adjunctive immune-modulators. We compared in vitro the effects of the γ-chain T-cell growth cytokines IL-2, IL-4, IL-7 and IL-15 on cytomegalovirus-stimulated cell-mediated immunity. IL-2 and IL-15 increased cytomegalovirus-specific lymphocyte proliferation in HAART recipients, whereas IL-4 and IL-7 did not. The boosting effect of IL-2 and IL-15 on proliferation correlated with their ability to prevent late apoptosis. However, IL-2 increased the frequency of cells in early apoptosis, whereas IL-15 increased the frequency of fully viable cells. Both IL-2 and IL-15 increased cytomegalovirus-induced CD4+ and CD8+ T cell proliferation and the synthesis of Th1 and pro-inflammatory cytokines and chemokines. However, only IL-2 increased the frequency of regulatory T cells and Th2 cytokine production, both of which have the potential to attenuate antiviral immune responses. Overall, compared to other γ-chain cytokines, IL-15 had the most favorable profile for boosting antiviral cell-mediated immunity.
Keywords: cytomegalovirus, cell-mediated immunity, HIV infection, HAART, T cell growth cytokines
Highly active antiretroviral therapy (HAART) decreases the incidence of opportunistic infections and increases CD4+ T cells in HIV-infected patients. However, reconstitution of adaptive immune responses is often incomplete as demonstrated by persistently poor responses to vaccines (Lange et al., 2003; Weinberg et al., 2006a) and the occasional occurrence of opportunistic infections, such as cytomegalovirus (CMV), herpes zoster, and disseminated tuberculosis in individuals with >200 CD4+ cells/µl (Jacobson et al., 1997; Komanduri et al., 2001; Lawn, 2005).
We previously reported that HAART recipients had multiple abnormalities of the CMV-specific cell-mediated immunity (CMI)(Weinberg et al., 2006b; Weinberg et al., 2001; Weinberg et al., 2003), including decreased proliferation of CMV-stimulated peripheral blood mononuclear cells (PBMC), which inversely correlated with apoptosis of both resting and CMV-stimulated PBMC (Weinberg et al., 2004). Both proliferation and apoptosis improved after in vitro treatment with the viral serpin crmA, which blocks caspase 8 and 9 activities, but not after cell death receptor blockade, suggesting that the mitochondrial apoptotic pathway was involved in the decreased CMV CMI. HIV infection has been shown to directly or indirectly increase apoptosis via the intrinsic mitochondrial pathway (Genini et al., 2001; Holm and Gabuzda, 2005), but other factors, such as insufficient induction of bcl-2 or analogous anti-apoptotic factors by T-cell growth cytokines may also contribute to this effect (Pahwa et al., 2006; Petrovas et al., 2004; Zaunders et al., 2003). Several cytokines, including the Th1 cytokines IL-2 and IL-15, the Th0 cytokine IL-7, and the Th2 cytokine IL-4, increase bcl-2 production and activate other cell survival mechanisms by signaling through the common γ-chain receptor (Adachi et al., 1996; Leonard, 2001). In addition, HIV infection has been associated with abnormal IL-2, IL-7 and IL-15 production and/or function, suggesting that exogenous addition of these cytokines may improve immune functions (d'Ettorre et al., 2002; De Paoli, 2001; Ostrowski SR, 2003; Sasson et al., 2006).
Due to the incomplete immune reconstitution resulting from HAART, cytokines, chemokines and other immune mediators have been administered to enhance immune responses of HIV-infected subjects (De Paoli, 2001; Katlama et al., 2002; Keh et al., 2006; Kuekrek et al., 2005; Lindemann et al., 2004; Martinez-Marino et al., 2004; Sereti et al., 2004; Sereti et al., 2002). In vivo administration of IL-2, in addition to HAART, consistently increased CD4+ T cell numbers, but generally failed to augment pre-existing or to replace previously lost antigen-specific CMI (De Paoli, 2001; Lindemann et al., 2004). IL-2 administration rarely increased responses to recall or neo antigens (Kuekrek et al., 2005; Levy et al., 2006; Nacsa et al., 2005; Valdez et al., 2003). Although the presence of IL-2 during primary antigenic exposure may be required for the development of immunologic memory (Weinberg and Merigan, 1988; Williams, Tyznik, and Bevan, 2006), IL-2 administration is also associated with an increase in the number of circulating regulatory T cells (Treg) and activation-induced cell death (AICD) in the presence of persistent antigen presentation (Anderson et al., 2005; Malek, 2004; Sereti et al., 2002), which may contribute to the limited effect of IL-2 on reconstitution of adaptive immunity of HIV-infected individuals (Nacsa et al., 2005).
IL-15 is a Th1 cytokine that shares many properties with IL-2, including β- and γ-chain cellular receptors (Cornish, Sinclair, and Cantrell, 2006; Dubois et al., 2003). However, IL-15 also has specific effects including a marked increase of memory CD8+ T cell survival(Sato et al., 2007). IL-15 has not been extensively studied in HIV-infected subjects, but IL-15 administration to SIV-infected macaques enhanced their immune reconstitution (Picker et al., 2006).
IL-7 induces lymphopoiesis and peripheral T cell homeostasis and prolongs survival of memory and effector CD4+ and CD8+ cells (Fry and Mackall, 2005; Picker et al., 2006; Rathmell et al., 2001). In addition, IL-7 may increase cytotoxicity (Lum et al., 2004) and antigen-specific IFNγ production (Jennes et al., 2002).
IL-4 also stimulates T cell growth and proliferation and, like IL-2 and IL-15, can prevent AICD (Kennedy and Celis, 2006). IL-4, however, is more likely to dampen than boost antiviral CMI (Wherry EJ, 2004).
The goal of this study was to compare the effect of γ-chain cytokines on apoptosis and CMV-specific CMI in HIV-infected individuals and to determine the function of the T cells expanded with the use of exogenous cytokines.
Subjects
This study enrolled 62 CMV-seropositive individuals, including 36 HIV-infected subjects on HAART and 26 HIV-uninfected controls, according to the norms of the Colorado Multiple Institutional Review Board. Other inclusion criteria for HIV-infected subjects were HAART, defined as ≥ 3 antiretrovirals including ≥ 2 classes, for ≥3 months; CD4+<100 cells/µl before HAART and ≥100 cells/µl at enrollment. At blood draws, the median duration of HAART was 18 months; the median CD4+ cell count was 340 cells/µl with a maximum of 1309 cells/µl; and the median and upper quartile plasma HIV RNA was <102.6 copies/ml with a maximum of 105.3.
Reagents
CMV or control antigens were prepared by glycine extraction and UV inactivation of CMV AD169- and mock-infected human lung fibroblasts, respectively. Although inactivated, the CMV-infected cell lysate stimulates both CD4+ and CD8+ T cells similar to the responses to other inactivated virions (Burgdorf et al., 2007; Larsson et al., 2002; Rutebemberwa et al., 2007). Recombinant human IL-2, IL-4, IL-7, and IL-15 were purchased from R&D Systems. Staining reagents were as follows: carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes), PerCP-conjugated anti-CD4 (anti-CD4-PerCP) and anti-CD8-PerCP mouse mAbs (Becton Dickinson); and anti-CD25-APC and anti-FoxP3-FITC (eBiosciences).
Specimen processing and cryopreservation
PBMC from heparinized blood, separated by Ficoll/Hypaque gradient (Sigma) centrifugation, were cryopreserved as previously described (Weinberg et al., 2000) at 107 cells/ml in10% dimethyl sulfoxide (Sigma)-containing fetal bovine serum (Gemini Bio-Products).
Lymphocyte proliferation
This was performed using cryopreserved PBMC as previously described (Weinberg et al., 2001). Stimulation medium consisted of RPMI 1640 with glutamine (Gibco), 10% human AB serum (Nabi) and 1% antibiotics (Gibco). 106 PBMC/ml were stimulated with CMV antigen at a 1:200 final dilution in quadruplicate wells or culture tubes. Mock-infected antigen and pokeweed mitogen (Sigma) were used as negative and positive controls at final dilutions of 1:200 and 10 µg/ml, respectively. Recombinant human IL-2, IL-4, IL-7, and IL-15 in concentrations pre-determined to provide maximum stimulation (3.3 ng/ml, 2.5 ng/ml, 3.3 ng/ml and 2.5 ng/ml, respectively) were added to the stimulation medium. To measure proliferation, 6- day cultures maintained at 37°C and 5% CO2, were pulsed with 3H-thymidine (Perkin Elmer) for 6 h, harvested and counted using Unifilter plates (Perkin Elmer) and a microplate scintillation counter (Packard). CMV-specific proliferation was calculated by subtracting the median counts per minute (cpm) measured in the mock-infected control wells from the median cpm of the CMV-stimulated wells. For flow cytometric assays, cells were harvested, washed and stained as described below.
Apoptosis Assay
TACS Annexin V-FITC apoptosis detection kit (R&D Systems) was used for measurement of PBMC apoptosis. Samples containing 105 to 106 PBMC were centrifuged at 500g for 10 minutes, washed with cold phosphate-buffered saline (PBS), and resuspended in 100 µL of Annexin V Incubation Reagent containing Annexin V-FITC and propidium iodide (PI). After 15 min of incubation in the dark, 400 µL of binding buffer were added. Samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson).
T cell phenotypic characterization
Stimulated PBMC were washed in PBS and incubated with the appropriate surface marker mAb for 30 min at room temperature. For intracellular staining, PBMC were then permeabilized and fixed using Cytofix/Cytoperm (BD Pharmingen) for 20 minutes at 4°C, washed and stained with the appropriate reagents. After washing with PBS, PBMC were analyzed in real time with a FACSCalibur instrument and CellQuest software (BD Biosciences). The number of events collected was ≥10,000.
CFSE dilution assays
PBMC were labeled with 0.5 µM CFSE (Molecular Probes) in 0.1% bovine serum albumin PBS for 10 min at 37°C. Staining was quenched with cold 10% human AB serum in RPMI. An aliquot of PBMC was removed for baseline flow analysis and the remaining cells were stimulated with CMV or control antigen for 6 days, after which PBMC were washed in PBS, stained with CD4 and CD8 surface markers and analyzed by flow cytometry as described above.
Cytokine and chemokine detection
Inducible cytokines and chemokines were measured in the supernatants of 6-day CMV or mock-infected control antigen-stimulated PBMC using Human Cytokine Antibody Bead Kits (Biosource) according to the manufacturer’s instructions. CMV-specific production was expressed as the difference in concentration between CMV and mock-infected control antigen-stimulated cultures.
Statistical Analysis was performed using Instat3 and Prism4 (GraphPad) software. Two-tailed parametric T tests or ANOVA were used for the statistical analyses after verifying that the data were normally distributed. Significance was defined by p≤0.05.
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 (Fig 1). 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 (Fig 1). 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
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 (Fig 2).
Figure 2
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 (Fig 3).
Figure 3
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; Fig 4). 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
Figure 4
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; Fig 5). 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
Figure 5
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 (Fig 6). 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
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.
In vitro measures of CMV-specific CMI are significantly lower in HIV-infected subjects on HAART compared with healthy individuals. We sought to determine if any of 4 γ-chain cytokines IL-2, IL-4, IL-7 and IL-15 could boost CMV-specific CMI responses of HAART recipients. We found that IL-2 and IL-15 increased CMV-specific proliferation measured by 3H-thymidine incorporation and CFSE dilution. Furthermore, IL-2 and IL-15 in vitro supplementation substantially decreased the gap in proliferation between CMV-stimulated PBMC from HIV-infected subjects on HAART and healthy individuals. In contrast, IL-4 or IL-7 did not appreciably increase CMV-specific proliferation of PBMC from HIV-infected subjects. Interestingly, all 4 γ-chain cytokines increased CMV-specific proliferation of PBMC from healthy donors, underscoring some of the differences in immune functions between HIV-infected individuals on HAART and healthy subjects.
IL-4 failed to enhance proliferation of CMV- or control antigen-stimulated PBMC from HIV-infected subjects, but enhanced CMV-stimulated proliferation of PBMC from healthy donors. This suggests that the IL-4 receptors or their downstream pathways may be saturated in cultured T cells from HIV-infected subjects. Others have noted a Th2 bias in the immune response of HIV-infected subjects before the advent of HAART (Wasik et al., 1997) and it is conceivable that this may not be reverted with therapy.
IL-7 had a similar proliferative effect on antigen-stimulated vs. unstimulated PBMC from HIV-infected subjects. This finding may not be unexpected considering that IL-7 is important for the survival of both naïve and memory T cells (Kondrack et al., 2003; Li, Huston, and Swain, 2003; Sun, Lehar, and Bevan, 2006; Vassena et al., 2007). However, IL-7 favored CMV-specific proliferation of PBMC from healthy individuals, suggesting that HIV infection alters the ability of PBMC to respond to IL-7. Several lines of investigation also support this conclusion: 1) HIV-infected patients have increased levels of circulating IL-7 associated with reduced IL-7 receptor-α expression (Sasson et al., 2006); 2) IL-7 increases Fas-mediated apoptosis in the context of HIV infection (Fluur et al., 2007); 3) IL 7 is not necessary for the survival of memory CD8+ cells (Klonowski et al., 2006), which represent an important contingent among the PBMC that proliferate in response to CMV stimulation in HIV-infected subjects. These and our data indicate that therapeutic use of IL-7 is unlikely to increase antigen-specific immune responses of HAART recipients.
The increase of CMV-specific proliferation of PBMC from HIV-infected subjects generated by IL-2 or IL-15 in vitro supplementation significantly correlated with the ability of each cytokine to protect the PBMC against late apoptosis. However, IL-15 had a more robust anti-apoptotic effect than IL-2 in PBMC cultures from HIV-infected and uninfected subjects. In addition, IL-15 arrested PBMC progression to apoptosis in earlier stages compared with IL-2, increasing the proportion of fully viable cells, whereas IL-2 prevented progression from early to late apoptosis, suggesting a mechanistic difference between the 2 cytokines. Since apoptosis is increased by in vitro antigenic stimulation (Weinberg, 2003), the ability of IL-15 to prevent apoptosis may account for its greater effect on CMV-induced proliferation by comparison with IL-2. This observation is also consistent with the notion that IL-15 has a unique role in prolonging T-cell survival and maintaining immunologic memory (Alpdogan et al., 2005; Berard et al., 2003; Picker et al., 2006; Schluns et al., 2002). The robust anti-apoptotic effect of IL-15 may allow HIV-infected patients with chronic antigenic stimulation to maintain high levels of antigen-specific T cells.
IL-2, but not IL-15 significantly increased the proportion of CD4+ Tregs in CMV-stimulated cultures from HIV-infected or uninfected subjects. This is in accordance with previous reports of increased in CD4+ Tregs in patients treated with IL-2 (Sereti et al., 2002). We also showed that IL-2 increased CD8+CD25+FoxP3+, which may also have Treg properties (Endharti et al., 2005). The ability of IL-15 to stimulate CMI without inducing excess Treg expansion may be an advantage for its use as an immune-stimulator in HIV-infected individuals.
IL-15, compared with IL-2, increased the production of a slightly broader array of Th1 cytokines and chemokines by CMV-stimulated PBMC from HIV-infected subjects, suggesting that IL-15 may have a more robust boosting effect on antiviral CMI. In contrast, IL-2, but not IL-15, significantly increased Th2 cytokine production by CMV-stimulated PBMC from HIV-infected subjects on HAART. Since Th2 cytokines have been previously shown to divert the immune response from Th1 to Th2 and IL-10 is also known to mediate Treg activity, the Th2-enhancing effect of IL-2 may attenuate antiviral defenses. This effect may also contribute to the failure of IL-2 administration to HAART recipients to boost their antigen-specific CMI when the antigen predominantly stimulates Th1 responses.
Taken together, our data indicate that IL-15 therapeutic administration is more likely than IL-2 or IL-7 to facilitate recovery of adaptive immune responses of HIV-infected individuals. Clinical studies are warranted to further assess its therapeutic value.
Acknowledgments
This work was supported by grant 1R21AI073121 and facilitated by the infrastructure and resources provided by the Colorado Center for AIDS Research Grant P30 AI054907. The authors thank Anna Vazquez for assisting in manuscript preparation.
Footnotes
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