IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4⁺ T lymphocytes from HIV-1–infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4⁺ T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Highly active antiretroviral therapy (HAART) has dramatically altered the HIV-1 pandemic, at least in the developed world. Most patients treated with HAART maintain clinically undetectable plasma viral loads with concomitant dramatic decreases in mortality and morbidity. However, persistently infected resting CD4⁺ T lymphocytes have been demonstrated in the peripheral blood of HIV-1–infected individuals even after treatment effectively suppressed most productive viral infection (1). It has been estimated that most HIV-1–infected individuals would require complete suppression of viral replication for many decades on current HAART regimens to possibly reach viral eradication (1). Therefore, new treatment strategies designed to purge the pool of latently infected cells are required.

Cytokine treatment has been proposed not only as an immune-adjunctive therapy to HAART, but also to activate latently infected CD4⁺ T lymphocytes and increase the turnover rate of the latent viral reservoir to promote viral clearance. Several cytokines, including IL-2, IL-7, and IL-15, which share a common γ chain, might have putative roles in improving control of HIV-1 infection; of these, IL-2 is the best characterized (2–4). Although recombinant human IL-2 (rhIL-2) plus HAART produced significant CD4⁺ T lymphocyte expansion without an increase in viral load (5–8), the effects of this combination on purging viral reservoirs is somewhat unclear (2, 3).

IL-7 is a nonredundant cytokine essential for thymopoiesis, which is produced by a number of cell types including stromal cells (9), normal and malignant B cells (10), thymic epithelial cells (11), intestinal epithelial cells (12), BM-derived fibroblast-like cells (13), keratinocytes, and dendritic cells (14). IL-7 has pleiotropic effects on T cell homeostasis, such as enhancement of mature T cell survival (15–17), expansion of both peripheral CD4⁺ and CD8⁺ T cells in T lymphocyte–depleted hosts (18), and mobilization of hematopoietic stem cells from the bone marrow to the peripheral circulation (19). Importantly, IL-7 enhances both HIV-1–specific CD8⁺ cytotoxic cellular activity of humans in vitro (20), as well as CD4⁺ T helper cell–dependent humoral responses and cytotoxic CD8⁺ T cell activity in mice immunized with HIV-1 envelope protein (21). Furthermore, Kaech et al. (22) demonstrated that, after a viral infection, a small population of effector CD8⁺ T cells had increased expression of the IL-7 receptor α. These cells are the precursors of the memory CD8⁺ T cell pool that confers protective immunity, and their survival was shown to depend on IL-7. Napolitano et al. (23) reported that patients with AIDS have higher plasma levels of IL-7 than do other HIV-1–infected subjects. IL-7 levels were inversely correlated with CD4⁺ T lymphocyte loss and directly correlated with plasma HIV-1 viral load. Moreover, Llano et al. (24), using the same IL-7 detection kit, demonstrated that higher IL-7 plasma levels occur in HIV-1–infected individuals than in healthy donors (9.4 ± 5.7 pg/ml and 3.6 ± 3.05 pg/ml, respectively). Importantly, when HIV-1–seropositive subjects were grouped by CD4⁺ T cell count — less than 200, 200–500, or greater than 500 — higher values of plasma IL-7 were detected as compared with healthy donors (12.26 ± 5.9, 6.67 ± 4.1, and 5.69 ± 2.7 pg/ml, respectively) (24). This probably represents a homeostatic response to lymphopenia, since the high levels of IL-7 decrease when the CD4⁺ T cell count increases after a HAART regimen is initiated. The authors hypothesized that IL-7 might play an important role in HIV-1 disease progression (23).

Of note, IL-7 was shown to be able to induce HIV-1 replication in vitro from PBMCs of HIV-1–infected individuals who were not treated with HAART (25–27), and to increase the expression of HIV-1 Tat mRNA in CD8-depleted PBMCs from chronically HIV-1–infected patients (25). Moreover, recent in vitro studies indicate that IL-7 has minimal effects on T cell phenotype, while inducing substantial expression of latent HIV-1 (28–30), which suggests that IL-7 might be used as an additive therapy to HAART, not only for its immunological effects but also to purge HIV-1–latent reservoirs. IL-2 has already been used in a few clinical trials in association with HAART, either alone or in combination with OKT 3 (2–4). However, whether or not the addition of these immunostimulatory therapies was able to induce significant expression from a true HIV-1 latent reservoir is controversial. Distinct cytokines play critical roles in the regulation of T cell homeostasis and thus they may be beneficial in addition to HAART by virtue of improving the impaired immune response. Furthermore, these cytokines could induce the replication of different HIV-1 viral strains or archives out of proviral latency. This hypothesis prompted us to assess IL-7 as a therapeutic agent in inducing activation of latent HIV-1, to investigate its action on quiescent human T lymphocytes in infected patients on HAART, and to examine further the molecular and virological characteristics of the viruses induced by both IL-7 and IL-2.

New clinical approaches, such as stimulation therapy (or more precisely, immune-activation therapy), are now being investigated to try to achieve long-term remission or viral eradication. In fact, it has been speculated that this strategy aims to increase the turnover of latent viral reservoirs through activation of quiescent infected cells, promoting cell death and accelerating viral clearance, especially when combined with intensification of antiretroviral therapy (4).

We demonstrated that IL-7 was significantly more efficient than either IL-2 alone or even IL-2 in combination with phytohemagglutinin (PHA) in stimulating HIV-1 replication from CD8⁺ T cell/monocyte–depleted PBMCs. We also found that IL-7 showed a positive trend for inducing proviral reactivation from resting CD4⁺ T lymphocytes from HIV-1–infected patients on suppressive HAART. Moreover, different viral isolates were induced by IL-7 and PHA/IL-2, which strengthens the hypothesis that combination therapy may be more efficacious not only for the immunomodulatory effects but also to effectively stimulate different latent HIV-1 viral species.

IL-7 promotes HIV-1 replication more efficiently than PHA/IL-2 from patients’ PBMCs and resting CD4⁺ T lymphocytes. The ability of different cell activators to induce the expression of persistent HIV-1 was evaluated on specific cell populations from 14 well-characterized patients on virally suppressive HAART. All the patients involved in this study received a stable HAART regimen for over a year, with plasma viral RNA levels below 50 copies/ml (Table 1).

Table 1

Clinical characteristics of the patients in this study

As shown in Table 2, a total of 18 paired PBMC coculture reactions and 11 paired resting CD4⁺ T lymphocyte (CD4⁺, CD25^–/HLA-DR^–) cocultures were treated with IL-7 and PHA/IL-2 in parallel. Viral outgrowth was detected in 10 out of 18 (56%) PBMC cocultures treated with IL-7, whereas viral outgrowth was detected in only 4 out of 18 (22%) of those stimulated by PHA/IL-2 (χ² = 4.208, P < 0.05). Of note, the 4 patients’ cocultures that were positive for HIV-1 outgrowth with PHA/IL-2 were also positive for IL-7. However, unlike with IL-7, no viral outgrowth was detected from patients’ PBMC cocultures (5 cultures) when IL-2 was used alone, without combining with PHA.

Table 2

HAART-persistent viral outgrowth by cellular activation

When pure resting CD4⁺ T cells were stimulated with IL-7 or PHA/IL-2, HIV-1 replication was induced in 5 out of 11 (45.5%) IL-7–treated cultures and in 3 out of 11 (27.3%) of those stimulated with PHA/IL-2. Two additional IL-7–treated resting CD4⁺ T lymphocyte cocultures were compared with unstimulated cultures and with cultures stimulated with IL-2 alone. IL-7 induced HIV-1 replication in 2 resting CD4⁺ T cell cocultures, while both unstimulated and IL-2–treated cultures remained negative until the end of the study. The average first appearance of viral production occurred at 3.5 weeks of culture. IL-7–induced viral outgrowth (4.1 weeks on average) required 2 weeks longer than outgrowth induced by PHA/IL-2 (2 weeks on average). However, both IL-7 and PHA/IL-2 yielded comparable levels of virus.

In summary, these results indicate that IL-7 is more effective than PHA/IL-2 or IL-2 alone in inducing the expression of persistent viral reservoirs ex vivo from both PBMCs and resting CD4⁺ T lymphocytes of patients on fully virally suppressive HAART.

Phylogenetic analyses of the HIV-1 Env gp120 genes of IL-7–induced and PHA/IL-2–induced viral RNA. To investigate the phylogenetic relationships of replication-competent viruses and their proviral pool, we sequenced in entirety the viral envelope gp120 and obtained the genotype of the viruses induced by both PHA/IL-2 and IL-7 — as well as their corresponding proviruses — from 4 patients. HIV-1 virion RNA was isolated from coculture supernatants (Table 3), and proviral DNA was isolated from the same patients’ PBMCs and resting CD4⁺ T lymphocytes. Ten to fifteen clones for each proviral DNA or viral RNA sample were sequenced. Sequence contaminants were ruled out by comparison with common laboratory viral isolates.

Table 3

Viral outgrowth (p24 antigen value, pg/ml) during coculture

The predicted amino acid sequences of proviral DNA and viral RNA were compared within each patient. Initial studies first revealed that potential inactivating mutations (i.e., premature stop codons and/or frameshifts) were present in proviral DNA Env clones, as well as in PHA/IL-2–induced viruses. In 3 out of 4 patients (patients 1, 3, and 4), amino acid sequences of proviral DNAs exhibited high diversity and most of the amino acid differences were localized in the V1/V2 regions, including multiple amino acid substitutions and length polymorphisms. On the contrary, extremely high homogeneity was observed in the corresponding Env gene of virion-encapsidated RNA in 11 out of 12 coculture supernatants that were positive for HIV-1 p24 antigen. Rare single amino acid changes were observed and seemed to be recombinant forms of different cell-associated proviruses (data not shown).

In patient 1, IL-7–induced and PHA/IL-2–induced viral genotypes were quite different from each other. However, within each specific cytokine-induced group, viral sequences were found to be homogeneous despite being from of PBMC and resting CD4⁺ T lymphocyte origin. In the full-length 300-AA region V1–V5 of HIV-1 gp120 we observed 5 single amino acid changes in 5 separate clones (2 in V1/V2, 1 in C2, 1 in V3, and 1 in the C3 region) and 4 amino acid deletions in another clone (in the C3 region), from a total of 8 clones derived from IL-7–induced viral RNA from resting CD4⁺ T lymphocytes. PBMC/IL-7–stimulated viruses contained 2 single amino acid changes in 2 of the 6 clones (1 in V3 and the other in the C3 region). There were 3 single amino acid changes (2 in V1 and 1 in the C3 region) scattered in 3 of the total 8 clones in resting CD4⁺ T cell/IL-2–stimulated viruses. Similarly, high homogeneity was observed in PBMC/IL-7–stimulated viruses for patients 2, 3, and 4, resting CD4⁺ T cell/IL-7–induced viruses for patients 3 and 4, and PBMC/IL-2–induced viruses for patients 2, 3, and 4. Surprisingly, these initial analyses suggested that certain restricted proviral quasispecies had been activated by each specific stimulator.

To further analyze in detail the genetic diversity of the recovered viruses by treatment with different cytokines, and to determine the potential importance in inducing divergent proviral archives, we generated rooted phylogenetic trees using the HIV-1 gp120 V1–V5 nucleotide sequences for proviruses and induced viruses from each of the 4 patients (Figure 1). We evaluated the phylogenetic relationships among intrapatient sequence sets of PBMC or resting CD4⁺ T lymphocyte proviral DNA and cell-free viral RNA induced by IL-7 or PHA/IL-2. Of note, the laboratory viral strain HXB2 segregated distantly from all patients’ sequences.

Figure 1

Phylogenetic analyses of HIV-1 envelope gene sequences of proviral DNA and cytokine-induced virion RNA from the same cell populations. The full-length of gp120 V1 to V5 regions were cloned and sequenced directly from proviral DNA of PBMCs and purified, resting CD4⁺ T lymphocytes, and IL-7 or PHA/IL-2–induced virion RNA for 4 patients (numbers 1–4, A–D). Proviral DNA sequences are presented by black stars (open stars for those strains from PBMC and filled for those of resting CD4⁺ T lymphocytes). IL-7–induced viral RNA sequences are indicated by blue squares (open squares for PBMC and IL-7–induced and filled squares for resting CD4⁺ T lymphocyte and IL–induced). PHA/IL-2–induced viral RNA sequences are indicated by red circles (open circles for those induced by PBMC with PHA/IL-2 and filled circles for those induced by resting CD4⁺ T- lymphocyte with PHA/IL-2). Growth of patient 4’s virus from PBMCs with PHA/IL-2 was from 10/04/2001. All sequences are shown with HXB2 (GenBank accession no. K03455) as the reference outgroup. Branch lengths are drawn in proportion to the number of nucleotide substitutions per site, and bootstrap probabilities (1,000 iterations) exceeding 70% for each node are noted.

The phylogenetic tree for patient 1 reveals an interesting intrapatient sequence relationship. There are 2 primary branches, as shown in Figure 1A. The top branch represents viral RNA from IL-7–stimulated cocultures, and the bottom branch illustrates viral RNA from PHA/IL-2–stimulated cocultures. Proviral DNAs from PBMCs and resting CD4⁺ T lymphocytes were detected on both branches. These data indicated that IL-7 and PHA/IL-2 induced the replication of distinct viruses that belonged to different subpopulations that were contained in the same proviral pool. Of interest, a similar phenomenon was observed for patient 2, as shown in Figure 1B. In patients 1 and 3 (Figure 1, A and C), IL-7 induced the replication of very similar HIV-1 isolates (i.e., segregated in the same branch of the phylogenetic tree) regardless of whether PBMCs or pure resting CD4⁺ T lymphocytes were stimulated, showing a highly provirus-specific effect. Interestingly, in patient 3, IL-7 and PHA/IL-2 induced the growth of 2 distinct viruses, on separate branches, solely from resting CD4⁺ T lymphocytes. This suggests a selective effect on a true latent HIV-1 reservoir not contaminated by the presence of activated CD4⁺ T lymphocytes in PBMCs, known to produce low levels of HIV-1 replication. We were able to show that the separation of these 2 distinct viruses in 2 separate branches could be found, in patient 3, even when the entire full-length HIV-1 genome was sequenced and analyzed (data not shown). In patient 3, envelope sequences also derived from full-length viral sequencing fit into the subbranches of the tree based on PHA/IL-2 versus IL-7 treatment (Figure 1C). Similarly, for patient 4, viral populations were again clustered tightly in quite separate subbranches, as shown in Figure 1D. Finally, intrastimulus viral quasispecies were also analyzed. IL-7–induced virus showed high homogeneity in 3 out of 4 patients, while much more diversity was observed in PHA/IL-2 virus populations (data not shown).

The segregation of these HIV-1 viral populations induced by diverse stimulators highlights that proviral reactivation by different cytokines may be from differing proviral quasispecies existing in the same or different latently infected CD4⁺ T lymphocyte subpopulations. These results suggest that more than one stimulator might be needed, in combination, to purge or deplete HIV-1 reservoirs in a more efficient and complete manner.

IL-7–mediated viral replication is in part dependent on activation of resting CD4⁺ T lymphocytes. Cytokine treatment can alter the activation state of the host cells and thus change patterns of viral replication. Our findings that IL-7 induced expression of latent virus effectively from patients’ PBMCs and resting CD4⁺ T lymphocytes suggested that this cytokine activated the latently infected peripheral resting CD4⁺ T lymphocytes harboring HIV-1 proviral DNA. The expression of various activation and proliferation markers, including CD25, CD69, HLA-DR, and Ki-67, were then assessed. Resting CD4⁺ T lymphocytes were purified from healthy donors’ peripheral blood and treated with IL-7 alone (20 ng/ml) for 14 days or PHA (5 μg/ml for 48 hours) plus IL-2 (20 ng/ml) for 3 days. As shown in Figure 2A, we observed an increased number of cells expressing the activation marker CD25 in IL-7–treated resting T cell populations, which was associated with an increase of the proliferation marker Ki-67, but not with an increase in HLA-DR or CD69 (data not shown). A high level of CD25 expression (36.2%) was observed on day 1 and increased at the following time points (71.4% on day 7 after treatment), as compared with untreated cells (data not shown). Ki-67 expression peaked on day 7 (13%) and was followed by a rapid decline (data not shown). Intracellular Ki-67 staining demonstrated the proliferation of IL-7–treated resting CD4⁺ T lymphocytes, in agreement with previous studies (31–36). Figure 2B illustrates the cell activation efficiency when treated with PHA and then IL-2. These results suggest that the partial activation and proliferation of the resting T lymphocyte population treated with IL-7 may contribute to the induction of HIV-1 replication out of latency.

Figure 2

Stimulatory patterns of resting CD4⁺ T lymphocytes by IL-7. (A and B) Detection of cell activation markers in IL-7–treated or PHA/IL-2–treated human resting CD4⁺ T lymphocytes. Freshly isolated resting CD4⁺ T lymphocytes stimulated with IL-7 or PHA/IL-2 were stained with FITC-conjugated anti–KI-67, anti–HLA-DR, and PE-conjugated anti-CD25. Three independent experiments were performed, of which one representative is illustrated. Gm, geometric mean; CV, coefficient of variance. (C) Effects of IL-7 on the cell cycle of human peripheral blood resting CD4⁺ T lymphocytes. IL-7 alone or PHA/IL-2–stimulated, initially resting CD4⁺ T lymphocytes are shown with the cell-cycle status indicated in each quadrant. DNA is depicted on the vertical axis and RNA is shown on the horizontal axis. Positions indicating stages in the cell cycle are illustrated in the third panel. The cell cultures either alone or in the presence of IL-7 were continued for a full 7 days. PHA/IL-2–stimulated cells were treated with PHA (5 μg/ml) for 48 hours and then IL-2 (10 ng/ml) for 3 days.

To further define the extent of cellular activation, we tested the effect of IL-7 on inducing cell-cycle progression. Highly purified resting CD4⁺ T lymphocytes were cultured in media alone or in the presence of IL-7 for 7 days. As shown in Figure 2C, an increase in cellular RNA was observed on day 3 after treatment and, on day 7, 17% of the cells progressed into the S and G₂/M phase of the cell cycle (data not shown). These data lead us to further suggest that IL-7–induced cellular proviral expression could be partially based on its effect on cell activation and cell-cycle progression.

To test whether IL-7–induced resting T lymphocyte activation was an incidental cross-reactive effect of autocrine-mediated release of secondary cytokines from the IL-7–stimulated resting CD4⁺ T lymphocytes, we used a proteomics microarray in coculture supernatants of both IL-7–treated and untreated resting T lymphocytes to test for the presence of 42 cytokines. We did not detect any significant difference in endogenous cytokine release (data not shown). These results suggest a potential direct effect of IL-7 in inducing resting CD4⁺ T lymphocyte activation without intermediate interaction of other released cytokine(s).

Infectivity of PHA/IL-2–induced and IL-7–induced viruses. Next, we examined the infectivity and replication-competence of both PHA/IL-2–induced and IL-7–induced viruses. The HIV-1_NL4-3 isolate was used as a positive control. The viruses were normalized by HIV-1 p24 antigen levels to an input concentration of 5 ng/ml per 10⁶ cells. The kinetics of HIV-1 replication was measured by HIV-1 p24 antigen detection in triplicate culture supernatants. As shown in Table 4, both PHA/IL-2–induced and IL-7–induced virions have comparable infectivity with the laboratory strain NL4-3. These data indicate that the cytokine-induced viruses are fully replication-competent and capable of perpetuating the spread of HIV-1 to uninfected cells.

Table 4

Comparison of the replication competence of viruses

IL-7 and HIV-1 long terminal repeat transactivation. Finally, to evaluate and precisely isolate the potential differential effects of the stimulatory regimens on HIV-1 long terminal repeat–mediated (HIV-1 LTR–mediated) transcription, we performed gene reporter assays. These assays demonstrated that IL-7, at 6 hours after addition, induced HIV-1 LTR activity by only 2-fold at a concentration of 100 ng/ml, while at lower concentrations (i.e., 10–20 ng/ml), IL-7 demonstrated no effect. IL-2 alone (10 ng/ml), 6 hours after addition, did not have any effect on HIV-1 LTR activity. Both PMA and PHA (10 ng/ml), 6 hours after addition, significantly induced HIV-1 LTR activity by approximately 25-fold (data not shown). These results suggest that, at least in this specific HIV-1 LTR transactivation assay, IL-7 exerts only a modest effect on LTR-directed transcription, and thus this may not represent the molecular pathway involved with more robust stimulation of archival proviruses in the PBMCs of patients on virally suppressive HAART.

Of note, differences in specific induction of select proviruses by divergent cytokines could be related to the existence of diverse proviral quasispecies in distinct subpopulations of resting CD4⁺ T lymphocytes, such as CD4⁺/CD45RA⁺ naive and CD4⁺/CD45RO⁺ memory T lymphocytes, or to regulation of viral expression by specific cytokines, possibly through their effects on viral transcription. As such, we sequenced the LTRs of replication-competent viruses induced by IL-7 and PHA/IL-2 in certain individuals. Nonetheless, there were no specific nucleotide sequence differences in the LTR U3 regions from paired cytokine-induced viruses (i.e., PHA/IL-2–induced versus IL-7–induced) in the 2 patients analyzed (data not shown).

The addition of T cell–activating agents to potent antiretroviral therapy has been proposed as a possible strategy to purge cellular reservoirs in long-lived, HIV-1–infected quiescent CD4⁺ T lymphocytes. IL-2 administration is one of the most extensively studied immune-based therapies in HIV-1–infected individuals (7). IL-2 in combination with HAART has led to CD4⁺ T lymphocyte expansion, although it had modest effects on purging the HIV-1 reservoir either alone or in combination with OKT-3. IL-7 has been proposed as a possible supplement to HAART (28), not only for its immunomodulatory properties and its crucial role in T cell homeostasis, but also to deplete HIV-1 reservoirs by stimulating the pool of latently infected cells.

In the present study, we investigated the effects of IL-7 and PHA/IL-2 on HIV-1 replication in CD8⁺ T cell/monocyte–depleted PBMCs and resting CD4⁺ T lymphocytes freshly isolated from HIV-1–infected individuals on virally suppressive HAART and with plasma HIV-1 RNA levels lower than 50 copies/ml. We demonstrated that IL-7 is more effective than IL-2 in enhancing HIV-1 proviral reactivation, not only from CD8⁺ T cell/monocyte–depleted PBMCs but also from resting CD4⁺ T lymphocytes. IL-7 was able to induce HIV-1 replication in 56% of CD8⁺ T cell/monocyte–depleted PBMCs and in 45.5% of pure populations of resting CD4⁺ T lymphocyte cocultures. Importantly, PHA/IL-2 stimulation induced HIV-1 replication in only 22% and 27.3% of CD8⁺ T cell/monocyte–depleted PBMCs and resting CD4⁺ T lymphocyte cocultures, respectively. Moreover, in 6 patients when IL-7 was compared with IL-2 alone, we found that the latter did not induce HIV-1 replication in any of the coculture assays, while 3 out of 5 cocultures treated with IL-7 were positive for viral outgrowth (data not shown).

In a previous study in the pre-HAART era, IL-7 was shown to upregulate HIV-1 replication from PBMCs of HIV-1–infected individuals ex vivo, although these subjects were not on HAART, and thus the noise of high levels of viral load and continuous viral replication prevented further analysis regarding the possible effects on true HIV-1 latency (25). In our study, the effects of IL-7 on HIV-1 replication were tested in both CD8⁺ T cell/monocyte–depleted PBMCs and pure populations of resting CD4⁺ T lymphocytes, with complementary results. Moreover, our present data indicate that IL-7 had a direct effect on resting CD4⁺ T lymphocyte–associated viral outgrowth. This rules out the possibility that contaminating cells in the CD8⁺ T cell/monocyte–depleted PBMC cocultures may have interfered with the induction of HIV-1 replication, either directly or through the release of cytokines or chemokines. Furthermore, potentially interfering secondary cytokines were not detected in IL-7–treated resting CD4⁺ T cell coculture supernatants, via a proteomics approach. Thus, these results strongly suggest that IL-7 alone is more efficient than PHA/IL-2 in inducing HIV-1 replication directly from latently infected cells of HIV-1–infected subjects on HAART. In future studies, it will be interesting and possibly important to combine IL-7 with other cytokines including, but not limited to, IL-2.

We hypothesized that IL-7 and PHA/IL-2 stimulation might induce the replication of different viral strains. As such, the HIV-1 envelope gp120 V1–V5 region was sequenced and analyzed. Interestingly, the phylogenetic trees revealed that in patients 1 and 2 there were 2 primary branches showing distinct segregation of sequences with regard to PHA/IL-2–induced and IL-7–induced viruses (Figure 1, A and B). Patient 3 revealed distinct PHA/IL-2–induced versus IL-7–induced virions from solely resting CD4⁺ T lymphocytes (Figure 1C). In patient 4, 2 viral populations were also clustered tightly in separate sub-branches (Figure 1D). Cytokine-induced replication-competent viruses were contained in the corresponding proviral pool of PBMCs and/or pure resting CD4⁺ T lymphocytes. These results strongly indicated that different cytokines had induced divergent proviral DNA expression from the CD4⁺ T lymphocyte proviral archive. It remains a formally possible hypothesis that the existence of diverse proviral quasispecies may occur in distinct subpopulations of resting CD4⁺ T lymphocytes, such as CD4⁺/CD45RA⁺ naive and CD4⁺/CD45RO⁺ memory T lymphocytes. In addition, regulation of viral expression by specific cytokines, possibly through their effects on viral transcription or at other sites of the lentiviral life cycle, may have led to viral strain–specific induction.

Resting CD4⁺ T lymphocytes are considered a major HIV-1 reservoir (1). Thus, we investigated whether IL-7 had an effect on the activation or proliferation of these cells, which might be involved in the induction of HIV-1 from latency. We observed an increased expression of CD25 in the IL-7–treated resting CD4⁺ T lymphocyte population in accordance with previous results, which further demonstrated that CD4⁺/CD25⁺ T cells actively produce virus in vitro (37). The expression of T cell activation markers CD69 and HLA-DR was not modified by IL-7. Moreover, IL-7 induced an increase of the proliferation marker Ki-67 in resting CD4⁺ T lymphocytes, as well as their progression into S and G₂/M phases of the cell cycle.

In the interest of evaluating the potential use of IL-7 in humans, in vivo studies in a SCID-hu mouse model (28) and in macaques (31) have been performed, and the immunological and virological effects of IL-7 were assessed. In SIV-infected monkeys, a remarkable increase in CD4⁺ T cells, particularly CD4⁺ and CD8⁺ naive T cells, was demonstrated. Furthermore, the absolute number of CD4⁺ HLA-DR⁺ cells was augmented and correlated with an increase of the absolute number of Ki-67–positive cells (31). Surprisingly, IL-7 did not alter the plasma viral load levels, in accordance with a previous study by Fry et al. (38), in which antiretroviral therapy was administered. On the other hand, in the SCID-hu mouse model, IL-7 effectively stimulated the pool of latently HIV-1 infected cells while having minimal effects on T cell phenotype (28). Of note, IL-7 has been used in combination with an HIV-1–specific immunotoxin, with some success, to activate and deplete viral residual disease, also in SCID-hu mice (39).

The present studies provide direct evidence that exogenous IL-7 upregulated the expression of HIV-1 in latently infected cells from HIV-1–infected individuals on suppressive HAART, more effectively than did PHA/IL-2 or IL-2 alone. In previous studies, 1 cytokine alone rarely had robust effects on activating proviruses from latency (30). Of importance, different viral quasispecies from the same cellular latency pool were induced. The present findings suggest that cytokine combinations might be required in addition to HAART to stimulate diverse HIV-1 proviral quasispecies from latency. Not only is IL-7 alone more potent than PHA/IL-2, but it is important to point out that PHA cannot be used in a regimen for humans. Intensification therapy added to HAART, to attempt to inhibit viral spread of stimulated virions, and the immunomodulatory effects of IL-2 and IL-7, should now be taken into consideration (40), as they may act synergistically in controlling cytokine-induced reactivation of HIV-1. Further studies are needed to now rationally design clinical protocols to deplete or purge latent HIV-1 residual disease with these agents (41).

The authors wish to thank Anne Malin and Ruth Brower for clinical assistance with patients in this study, Rita M. Victor and Brenda O. Gordon for their excellent secretarial support, Hui Zhang for helpful discussions, and especially the patients who volunteered for these studies. This work was supported in part by US Public Health Services grants AI43289 and NS41864 (to R.J.Pomerantz), and the Center for AIDS research (CFAR) AI45008 at the University of Pennsylvania.

Joseph Kulkosky’s present address is: Department of Biology, Chestnut Hill College, Philadelphia, Pennsylvania, USA.

Nonstandard abbreviations used: HAART, highly active antiretroviral therapy; LTR, long terminal repeat; PHA, phytohemagglutinin; rhIL-2, recombinant human IL-2.

Conflict of interest: The authors have declared that no conflict of interest exists.

1. Siliciano, JD, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 2003. 9:727-728.
   View this article via: PubMed CrossRef Google Scholar
2. Chun, TW, et al. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nat. Med. 1999. 5:651-655.
   View this article via: PubMed CrossRef Google Scholar
3. Stellbrink, HJ, et al. Effects of interleukin-2 plus highly active antiretroviral therapy on HIV-1 replication and proviral DNA (COSMIC trial). AIDS. 2002. 26:1479-1487.
4. Kulkosky, J, et al. Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy. J. Infect. Dis. 2002. 186:1403-1411.
   View this article via: PubMed CrossRef Google Scholar
5. Emery, S, et al. Pooled analysis of 3 randomized, controlled trials of interleukin-2 therapy inadult human immunodeficiency virus type 1 disease. J. Infect. Dis. 2000. 182:428-434.
   View this article via: PubMed CrossRef Google Scholar
6. Pandolfi, F, et al. Low-dose IL-2 reduces lymphocyte apoptosis and increases naive CD4 cells in HIV-1 patients treated with HAART. Clin. Immunol. 2000. 94:153-159.
   View this article via: PubMed CrossRef Google Scholar
7. Pett, SL, Emery, S. Immunomodulators as adjunctive therapy for HIV-1 infection. J. Clin. Virol. 2001. 22:289-295.
   View this article via: PubMed CrossRef Google Scholar
8. Tambussi, G, et al. Efficacy of low-dose intermittent subcutaneous interleukin (IL)-2 in antiviral drug-experienced human immunodeficiency virus-infected persons with detectable virus load: a controlled study of 3 IL-2 regimens with antiviral drug therapy. J. Infect. Dis. 2001. 183:1476-1484.
   View this article via: PubMed CrossRef Google Scholar
9. Namen, AE, et al. B cell precursor growth-promoting activity. Purification and characterization of a growth factor active on lymphocyte precursors. J. Exp. Med. 1988. 167:988-1002.
   View this article via: PubMed CrossRef Google Scholar
10. Benjamin, D, et al. Human B cell lines constitutively secrete IL-7 and express IL-7 receptors. J. Immunol. 1994. 152:4749-4757.
    View this article via: PubMed Google Scholar
11. Fernandez, E, et al. Establishment and characterization of cloned human thymic epithelial cell lines. Analysis of adhesion molecule expression and cytokine production. Blood. 1994. 83:3245-3254.
    View this article via: PubMed Google Scholar
12. Watanabe, M, et al. Interleukin 7 is produced by human intestinal epithelial cells and regulates the proliferation of intestinal mucosal lymphocytes. J. Clin. Invest. 1995. 95:2945-2953.
    View this article via: JCI PubMed CrossRef Google Scholar
13. Ryan, DH, Nuccie, BL, Ritterman, I, Liesveld, JL, Abboud, CN. Cytokine regulation of early human lymphopoiesis. J. Immunol. 1994. 152:5250-5258.
    View this article via: PubMed Google Scholar
14. Kroncke, R, Loppnow, H, Flad, HD, Gerdes, J. Human follicular dendritic cells and vascular cells produce interleukin-7: a potential role for interleukin-7 in the germinal center reaction. Eur. J. Immunol. 1996. 26:2541-2544.
    View this article via: PubMed CrossRef Google Scholar
15. Vella, AT, Dow, S, Potter, TA, Kappler, J, Marrack, P. Cytokine-induced survival of activated T cells in vitro and in vivo. Proc. Natl. Acad. Sci. U. S. A. 1998. 95:3810-3815.
    View this article via: PubMed CrossRef Google Scholar
16. Vella, A, Teague, TK, Ihle, J, Kappler, J, Marrack, P. Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: STAT6 is probably not required for the effect of IL-4. J. Exp. Med. 1997. 186:325-330.
    View this article via: PubMed CrossRef Google Scholar
17. Boise, LH, Minn, AJ, June, CH, Lindsten, T, Thompson, CB. Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division. Proc. Natl. Acad. Sci. U. S. A. 1995. 92:5491-5495.
    View this article via: PubMed CrossRef Google Scholar
18. Komschlies, KL, et al. Administration of recombinant human IL-7 to mice alters the composition of B-lineage cells and T cell subsets, enhances T cell function, and induces regression of established metastases. J. Immunol. 1994. 152:5776-5784.
    View this article via: PubMed Google Scholar
19. Grzegorzewski, KJ, et al. Mobilization of long-term reconstituting hematopoietic stem cells in mice by recombinant human interleukin 7. J. Exp. Med. 1995. 181:369-374.
    View this article via: PubMed CrossRef Google Scholar
20. Carini, C, Essex, M. Interleukin 2-independent interleukin 7 activity enhances cytotoxic immune response of HIV-1-infected individuals. AIDS Res. Hum. Retroviruses. 1994. 10:121-130.
    View this article via: PubMed Google Scholar
21. Bui, T, Dykers, T, Hu, SL, Faltynek, CR, Ho, RJ. Effect of MTP-PE liposomes and interleukin-7 on induction of antibody and cell-mediated immune responses to a recombinant HIV-envelope protein. J. Acquir. Immune Defic. Syndr. 1994. 7:799-806.
    View this article via: PubMed Google Scholar
22. Kaech, SM, et al. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 2003. 12:1191-1198.
23. Napolitano, LA, et al. Increased production of IL-7 accompanies HIV-1-mediated T-cell depletion: implications for T-cell homeostasis. Nat. Med. 2001. 7:73-79.
    View this article via: PubMed CrossRef Google Scholar
24. Llano, A, et al. Interleukin-7 in plasma correlates with CD4 T-cell depletion and may be associated with emergence of syncytium-inducing variants in human immunodeficiency virus type 1-positive individuals. J. Virol. 2001. 21:10319-10325.
25. Smithgall, MD, Wong, JG, Critchett, KE, Haffar, OK. IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells. J. Immunol. 1996. 156:2324-2330.
    View this article via: PubMed Google Scholar
26. Chene, L, et al. Thymocyte-thymic epithelial cell interaction leads to high-level replication of human immunodeficiency virus exclusively in mature CD4(+) CD8(–) CD3(+) thymocytes: a critical role for tumor necrosis factor and interleukin-7. J. Virol. 1999. 73:7533-7542.
    View this article via: PubMed Google Scholar
27. Moran, PA, Diegel, ML, Sias, JC, Ledbetter, JA, Zarling, JM. Regulation of HIV production by blood mononuclear cells from HIV-infected donors: I. Lack of correlation between HIV-1 production and T cell activation. AIDS Res. Hum. Retroviruses. 1993. 9:455-464.
    View this article via: PubMed Google Scholar
28. Scripture-Adams, DD, Brooks, DG, Korin, YD, Zack, JA. Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype. J. Virol. 2002. 76:13077-13082.
    View this article via: PubMed CrossRef Google Scholar
29. Soares, MV, et al. IL-7-dependent extrathymic expansion of CD45RA+ T cells enables preservation of a naive repertoire. J. Immunol. 1998. 161:5909-5917.
    View this article via: PubMed Google Scholar
30. Unutmaz, D, KewalRamani, VN, Marmon, S, Littman, DR. Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes. J. Exp. Med. 1999. 189:1735-1746.
    View this article via: PubMed CrossRef Google Scholar
31. Nugeyre, MT, et al. L-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques. J. Immunol. 2003. 171:4447-4453.
    View this article via: PubMed Google Scholar
32. Steffens, CM, Managlia, EZ, Landay, A, Al-Harthi, L. Interleukin-7-treated naive T cells can be productively infected by T-cell-adapted and primary isolates of human immunodeficiency virus 1. Blood. 2002. 99:3310-3318.
    View this article via: PubMed CrossRef Google Scholar
33. Armitage, RJ, Namen, AE, Sassenfeld, HM, Grabstein, KH. Regulation of human T cell proliferation by IL-7. J. Immunol. 1990. 144:938-941.
    View this article via: PubMed Google Scholar
34. Welch, PA, Namen, AE, Goodwin, RG, Armitage, R, Cooper, MD. Human IL-7: a novel T cell growth factor. J. Immunol. 1989. 143:3562-3567.
    View this article via: PubMed Google Scholar
35. Fukui, T, et al. IL-7 induces proliferation, variable cytokine-producing ability and IL-2 responsiveness in naive CD4+ T-cells from human cord blood. Immunol. Lett. 1997. 59:21-28.
    View this article via: PubMed CrossRef Google Scholar
36. Yssel, H, Schneider, PV, Lanier, LL. Interleukin-7 specifically induces the B7/BB1 antigen on human cord blood and peripheral blood T cells and T cell clones. Int. Immunol. 1993. 5:753-759.
    View this article via: PubMed CrossRef Google Scholar
37. Borvak, J, et al. Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection. J. Immunol. 1995. 155:3196-3204.
    View this article via: PubMed Google Scholar
38. Fry, TJ, et al. IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates. Blood. 2003. 101:2294-2299.
    View this article via: PubMed CrossRef Google Scholar
39. Brooks, DG, et al. Molecular characterization, reactivation and depletion of latent HIV. Immunity. 2003. 19:413-423.
    View this article via: PubMed CrossRef Google Scholar
40. Ducrey-Rundquist, O, Guyader, M, Trono, D. Modalities of interleukin-7 induced human immunodeficiency virus permissiveness in quiescent T lymphocytes. J. Virol. 2002. 76:9103-9111.
    View this article via: PubMed CrossRef Google Scholar
41. Kulkosky, J, et al. Genotypic alteration of HAART-persistent HIV-1 reservoirs in vivo. Virology. 2003. 314:617-629.
    View this article via: PubMed CrossRef Google Scholar
42. Otero, M, et al. Peripheral blood dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART. AIDS Res. Hum. Retroviruses. 2003. 19:1097-1103.
    View this article via: PubMed CrossRef Google Scholar
43. Toba, K, Winton, EF, Koike, T, Shibata, A. Simultaneous three-color analysis of the surface phenotype and DNA-RNA quantitation using 7-amino-actinomycin D and pyronin Y. J. Immunol. Methods. 1995. 182:193-207.
    View this article via: PubMed CrossRef Google Scholar
44. Wang, FX, et al. Emergence of autologous neutralization-resistant variants from preexisting human immunodeficiency virus (HIV) quasispecies during virus rebound in HIV type 1-infected patients undergoing highly active antiretroviral therapy. J. Infect. Dis. 2002. 185:608-617.
    View this article via: PubMed CrossRef Google Scholar
45. Casado, G, et al. Near full-length genome characterization of an HIV type 1 CRF05_DF virus from Spain. AIDS Res. Hum. Retroviruses. 2003. 8:719-725.
46. Thompson, JD, Higgins, DG, Gibson, TJ. CLUSTAL W. improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994. 22:4673-4680.
    View this article via: PubMed CrossRef Google Scholar
47. Saitou, N, Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1997. 4:406-425.