Partial immune reconstitution following highly active antiretroviral therapy: can adjuvant interleukin-2 fill the gap?

Giulia Marchetti*, Fabio Franzetti and Andrea Gori

Institute of Infectious Disease and Tropical Medicine, University of Milan, ‘Luigi Sacco’ Hospital, Milan, Italy


* Corresponding author. Tel: +39-02-39043350; Fax: +39-02-3560805; Email: giulia.marchetti{at}unimi.it


    Abstract
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Highly active antiretroviral therapy (HAART) induces a substantial control of HIV viral replication, but it allows for only a partial immune reconstitution, thus prompting the rationale for the adjuvant use of immunomodulants. Based on its in vitro action as a major T cell growth factor, interleukin (IL)-2 has now been extensively investigated for its potential to correct the HIV-driven immune deficiencies, possibly translating into immunological control over HIV infection. Specific immunological end points have thus far been addressed within extensive Phase I/II trials, disclosing a broad insight into several aspects of the IL-2-mediated immune reconstitution allowing for interesting clinical speculation. Indeed, preliminary results indicate that adjuvant IL-2 induces a significant CD4 cell rescue in patients with no immune recovery following long-term HAART, thus standing as a valid and safe therapeutic option for these patients. Furthermore, in these patients, the IL-2-mediated immune reconstitution is characterized by a rise in both peripheral turnover and de novo T cell synthesis, with reversion of the skewed HIV-driven immunophenotypic pattern, a substantial increase in IL-7 production and in several markers of immune function. Combined, these findings indicate IL-2 has a beneficial effect in correcting the severe disruption in T cell homeostasis induced by HIV, through the interaction with T cells and cytokine microenvironment. However, whether or not these immunological effects translate into an actual immunological competency and therefore clinical benefit, still awaits demonstration from ongoing large, controlled clinical studies.

Keywords: HIV , HAART , immune reconstitution , immunotherapy , IL-2


    Rationale for immunotherapy in HIV infection
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 Abstract
 Rationale for immunotherapy in...
 Open issues: assessing the...
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By efficiently suppressing viral replication, highly active antiretroviral therapy (HAART) has dramatically decreased HIV-related morbidity and mortality rates.1,2 However, from an immunological standpoint, HAART allows for only a partial immune reconstitution. Indeed, ex vivo measures of immune function generally do not normalize and HIV-specific T cell responses almost invariably remain impaired.3,4 Furthermore, although HAART induces CD4 cell reconstitution in the majority of HIV-infected individuals, the degree of T cell recovery is quite heterogeneous among patients, and discordant viro-immunological responses have been described,5,6 raising critical issues in the clinical management of these patients. Even though several variables have been identified that correlate with the degree of immune restoration during HAART,69 the actual underlying mechanisms responsible for such an incomplete immune reconstitution are still poorly understood, and probably reflect the severe HIV-driven perturbations in T cell dynamics and homeostasis as well as the interaction between host and viral factors.10

A fascinating strategy aiming at the broadest immune reconstitution, possibly overcoming the limitations of HAART, consists of the adjuvant use of immunomodulants. Thus far, several immune-based approaches have been investigated, and include cytokines that stimulate cell-mediated immunity, like interleukin (IL)-2, IL-12, IL-7 and IL-15,11 immunosuppressive drugs aiming at the containment of the immune hyperactivation known to characterize HIV immunopathogenesis, such as corticosteroids, hydroxyurea, ciclosporin A, mycophenolate mofetil and thalidomide,12 and compounds selectively targeting HIV-specific immunity, thus potentially enhancing direct anti-HIV cytotoxicity (e.g. tucaresol and murabutide).13,14

Thus far, IL-2 represents the most promising immunomodulant approach, and extensive work has been carried out, indeed proving its efficacy in significantly expanding the CD4 cell pool.11 However, several issues on the role of IL-2 in HIV disease have yet to be clarified, and include its functional effect, the population that would mostly benefit from IL-2, the best treatment schedule, the IL-2-driven immune reconstitution, and, most importantly, its actual clinical benefit.


    Open issues: assessing the correct place and role of adjuvant IL-2 in HIV disease
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IL-2 is the major T cell growth factor, potently stimulating the proliferation and function of T-lymphocytes and natural killer cells, improving antigen processing, and promoting the production and release of other cytokines.15 Furthermore, as HIV infection significantly impairs IL-2 production, it was originally proposed that its exogenous administration could help restore immune function in HIV-positive patients. This assumption led to the development of therapeutic strategies aimed at modulating IL-2 signal strength for clinical benefit. Indeed, numerous controlled clinical studies in HIV-positive patients with widely varying CD4 counts have now demonstrated that, when combined with HAART, IL-2 results in a significant rise in CD4 cell count compared with HAART alone, with only transient or even no associated bursts of HIV plasma viraemia.1625 From a clinical standpoint, IL-2 was generally well tolerated with minor adverse effects, the most commonly experienced by patients being flu-like symptoms, nausea and asthenia which are usually self-limiting within a few days after the end of IL-2 cycles.26

Aside from the selective expansion of CD4 cells, several issues on the role of IL-2 in HIV disease have yet to be clarified, and include the role of IL-2 in functional immune enhancement, the population that would mostly benefit from IL-2, the best treatment schedule, and the IL-2-driven immune reconstitution. Above all, the key question remains whether the IL-2-mediated rise in CD4 cell count is indeed associated with an actual clinical benefit in terms of disease progression and death.

This latter issue is now being addressed in two large international, randomized clinical trials. More specifically, the International Study of Interleukin-2 in people with Low CD4+ T-Cell Counts on Active Anti-HIV Therapy (SILCAAT) was designed to investigate HIV-infected patients with CD4 cell counts of 50–299/mm3 over 4–6 years. However, SILCAAT was discontinued by the sponsor in October 2002, and is now been continued for the patients enrolled, under the sponsorship of the National Institutes of Health.27 The second worldwide clinical trial, Evaluation of Subcutaneous Proleukin (Interleukin-2) in a Randomized International Trial, ESPRIT, is a National Institutes of Health-supported study of 4000 HIV-positive individuals with CD4 cell counts of at least 300/mm3 over a 5 year period.28 Within the next few years, these studies could hopefully provide the most important results to clarify the clinical benefit of adjuvant IL-2 in HIV infection.

Thus, while clinical end points are now under investigation, specific immunological end points have been addressed in Phase I/II trials, aiming at answering the open questions relative to the role of IL-2 in HIV disease, possibly laying the premises for more focused clinical trials.1724

Clinical setting

Thus far, no clear-cut consensus has been reached on the population(s) of HIV-infected individuals who will benefit the most from IL-2 immunotherapy, with the fittest treatment schedules, allowing for the maximum benefit and the minimum side effects. As for the IL-2 regimen, following initial studies using continuous intravenous high dose administration, several lines of evidence have now converged to the conclusion that lower dosages given subcutaneously in an intermittent fashion are equally effective in expanding CD4 cell counts.29 Furthermore, low dose, intermittent regimens are much better tolerated and display only transient interference with HIV viral replication.

With respect to the cohort of HIV-positive patients, even though IL-2 has been proposed for patients with high-to-moderate CD4 counts, aiming at the maintenance of an appropriate CD4 cell compartment, the actual rationale and clinical impact of adjuvant IL-2 in individuals with a quantitatively preserved T cell compartment are indeed still controversial and have yet to be defined.

Most interestingly, stronger evidence has been brought about for the use of IL-2 in patients who fail to restore the circulating CD4 cell compartment following long-term HAART, despite a marked reduction in virus load (immunological non-responders, INRs),5,6,30,31 and possibly in HAART-naive individuals with advanced disease. Indeed, results have been reported from several controlled studies evaluating IL-2 in INRs.2124 In particular, the safety, tolerability and immunological benefit of IL-2 plus HAART versus HAART alone was recently investigated in 22 INRs (CD4 cells ≤ 200/mm3; HIV-RNA ≤ 50 copies/mL following at least 12 months of stable HAART) enrolled in a controlled randomized trial.23 In this study, a low-dose prolonged intermittent IL-2 regimen was specifically chosen, consisting of three IL-2 cycles (one cycle: 3 x 106 IU once a day subcutaneously at days 1–5 and 8–12) for an overall duration of 10 weeks. The low dose prolonged intermittent IL-2 regimen was chosen with the specific aim of balancing tolerance with efficacy, on the basis of both ex vivo lymphocyte growth patterns observed in patients with cancer, displaying maximum increase after 2–3 weeks of treatment,3235 and in vivo data showing sustained immunological response and significantly lower toxicity when using low IL-2 dosages.21,29 The IL-2 regimen adopted proved safe, with only mild side effects, consisting of low-grade fever, mild constitutional symptoms, and reversible nodule at the site of IL-2 injection. Importantly, no substantial interference with HIV replication was seen. In terms of immunological efficacy,21,22,24 IL-2 resulted in a significant increase in CD4 cells > 100% versus baseline levels, sustained up to 1 year of follow-up, whereas HAART patients experienced a much less relevant and slower rise in CD4 cells (Figure 1a and b). Furthermore, clinical records of the patients enrolled suggested that the accelerated IL-2-driven CD4 cell gain might indeed be effective in preserving an adequate cellular immunity, as no HIV-related clinical events were observed among IL-2-treated patients, whereas more than 30% of HAART-alone patients presented minor opportunistic infections.23 Taken together, these findings demonstrate that low-dose prolonged intermittent IL-2 immunotherapy in INRs may exert an immunological effect in INRs, with a possible role also in preventing the onset of HIV-related clinical events, thus outweighing the IL-2-related side effects. However, results from larger ongoing clinical studies are needed to confirm these data.27,28



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Figure 1. Longitudinal analysis of absolute (a) and percentage CD4 (b) and CD8 (c and d) recovery kinetics, cell turnover (e and f) and TREC content (g and h) in HIV+ patients undergoing either IL-2 immunotherapy plus HAART (IL-2 patients) or HAART alone (HAART controls). The figure shows the pattern of T cell dynamics following adjuvant IL-2 in patients who fail to restore CD4 cell compartment, despite complete control of HIV-viraemia.23 Black arrowhead indicates the end of three IL-2 cycles. During the study period, IL-2 patients experienced a significant and sustained increase in both absolute and percentage CD4 cells (a and b), whereas CD8 significantly increased only in absolute value at 48 weeks follow-up (c and d). HAART alone resulted in a slower rise in both CD4 and CD8 cells, reaching significance only in absolute value after 48 weeks (a–d). At week 10, in IL-2 patients CD4 TRECs displayed a tendency to increase (P=0.08) (g), together with a significant rise in CD4 cell turnover (e), whereas CD8 TRECs and cell turnover remained stable (g and e). HAART controls although displaying an equal suppression of CD4 and CD8 cell turnover (f) did not show any changes in CD4 and CD8 TRECs (h).50

 
Based on these encouraging results in INRs, the use of adjuvant IL-2 has also been recently proposed for individuals initiating HAART with severe CD4 cell depletion of less than 100 cells/mm3, ‘advanced naive’ patients, due to the quite inefficient immune reconstitution described in these individuals.36 By favouring a rapid CD4 cell rescue, the use of IL-2 in this specific clinical setting could significantly shorten the time frame at highest risk of HIV-related clinical events, even though several doubts remain on the actual responsiveness to IL-2 of highly immunocompromised patients23 (Marchetti, G., Bandera, A., Molteni, C., Meroni, L., Moroni, M., Franzetti, F. and Gori, A., unpublished data).

Furthermore, the use of IL-2 has also been recently proposed within structured treatment interruption (STI) protocols during both chronic disease and primary HIV infection (PHI).37 In particular, preliminary data reported that in chronically infected patients with CD4 cells > 500/mm3, the administration of IL-2 before HAART interruption results in a longer time before CD4 drop to the threshold for restarting therapy, indeed suggesting that, by prolonging the time off-therapy, an IL-2-based intervention may indeed enhance an HAART-sparing strategy.38,39 Moreover, IL-2 has also been proposed within STI protocols in patients treated with HAART during PHI, with the additional rationale here to selectively expand HIV-specific T cell clones, which have been shown to characterize the very early phases of HIV infection, and to be preserved by HAART initiation during PHI.40 Controlled studies evaluating IL-2 in these populations are ongoing41,42 (Marchetti, G., Cesari, M., Bandera, A., Trabattoni, D., Molteni, C., Schenal, M., Meroni, L., Moroni, M., Galli, M., Franzetti, F., Clerici, M. and Gori, A., unpublished data). These data, albeit preliminary, allow for the intriguing speculation of a possible role of IL-2 in sustaining a normal and functional CD4 cell compartment also in the context of STI protocols. Furthermore, for toxicity sake, the possibility of sparing antiretrovirals for longer time frames by administering IL-2, thus containing the heavy HAART-related long-term toxicity, is by itself an argument strong enough to justify the reversible IL-2-induced side effects.

IL-2-driven immune reconstitution

The hallmark of IL-2-driven immune reconstitution in HIV infection is the significant and sustained increase in CD4 cells. However, the specific immunological pathways involved in the IL-2-mediated CD4 cell expansion still remain somehow controversial, as well as the relationships with the pathogenetic mechanisms underlying the HIV-mediated immune deficits. Several hypotheses have been proposed, that include the contribution of peripheral proliferation and survival of existing cells,43,44 the rate of T cell apoptosis,45 de novo T cell synthesis by the thymus,23,46,47 or a combination of these factors. However, a comprehensive model of the immune pathways and T cell homeostatic balance featuring IL-2-mediated immune reconstitution is still missing.

These issues, combined with the description of IL-2-non-responders48 indicate that the IL-2-driven immune reconstitution is indeed quite a complex process, resulting from protean interactions with several mechanisms of T cell homeostasis translating in multiple immunological benefits, which go far beyond the mere quantitative expansion of CD4 cells. In particular, the possibility that IL-2 directly stimulates de novo T cell synthesis has important implications since this synthesis could favour the reconstitution of a more comprehensive immunity.

In this perspective, a deeper understanding of the effect of IL-2 on CD4 and CD8 cell dynamics and homeostasis, could provide an innovative framework for the most targeted and clinically advantageous IL-2-based strategies. Recently, several groups have extensively addressed this issue by combining T cell immune phenotyping, in vivo and ex vivo measurement of T cell proliferation and apoptosis, quantification of neothymopoiesis by T cell receptor excision circles (TRECs), and plasma IL-7 measurements, indeed allowing for a comprehensive picture of T cell immune recovery following adjuvant IL-2 in comparison with HAART alone.23,43,46,4952

Effect of IL-2 on T-lymphocyte proliferation and survival
Both enhanced T cell turnover and survival have been called upon as major mechanisms regulating the size of the peripheral T-lymphocyte pool following IL-2 immunotherapy.43 As for T cell proliferation, notwithstanding its role as the main T cell growth factor leading to the proliferation and differentiation of the whole T lymphocyte compartment, the hallmark of IL-2-driven immune reconstitution is the selective expansion of CD4 cells, ultimately resulting in the relative outgrowth of this subset. In particular, in INRs, IL-2 was shown to affect differentially the CD4 and CD8 cell subsets, with a preferential action on CD4 cells. Indeed, whereas HAART controls displayed a constrained yet parallel CD4 and CD8 cell recovery, IL-2 resulted in a more significant rise in CD4 than CD8 cell count (Figure 1ad), supporting the different regulation of CD4 and CD8 cell recovery during IL-2 immunotherapy. Since the peripheral T cell pool is maintained by both proliferation and renewal, the contribution of cell turnover and neothymopoiesis to both CD4 and CD8 cell subsets was also examined. IL-2 administration was associated with a significant rise in Ki67-proliferating CD4 cells, whereas no changes were seen in the CD8 cell pool (Figure 1e and f). Interestingly enough, at IL-2 discontinuation, a significant reduction was observed in CD4 cell turnover to below pre-treatment values (Figure 1e). Conversely, in HAART controls, the frequency of proliferating CD4 and CD8 was closely matched at all time-points (Figure 1f). These data confirm that CD4 and CD8 cells possess inherent biological differences,53 that become relevant under both endogenous and exogenous perturbations of physiological homeostasis like HIV infection itself or exposure to specific immune stimuli, such as IL-2.

Similar to the effect on peripheral T cell turnover kinetics, IL-2 was also associated with a significant long-term reduction in the percentage of activated CD38 CD8 cells,19,50 with possible implications in the containment of the immune activation-induced detrimental effects on HIV pathogenesis.10 Conversely, the CD8 activation status remained constantly elevated in HAART controls.

By confirming the selective activity of IL-2 on CD4 cells through a significant stimulation of peripheral proliferation during the period of IL-2 administration,43,45,49 these findings underline the efficacy of IL-2 in enhancing the most rapid and significant expansion of the highly exhausted CD4 cell pool in HIV disease. However, following cycle administration, IL-2 immunotherapy proved efficacious in lowering immune activation and CD4 cell proliferation, allowing for the speculation that the CD4 cell expansion during IL-2 follow-up is mainly maintained by increased survival of existing cells.44 Given these findings, decreases in turnover and prolonged survival in the periphery seem to be major mechanisms sustaining the IL-2-driven CD4 cell rescue in the long-term.

The possibility of immunotherapy to correct individual HIV-driven immune alteration, by exploiting the specific effects of different immunomodulants on T cell dynamics is indeed a fascinating and novel perspective in the treatment of HIV infection. In particular, the possibility that IL-2 contributes to the containment of T cell hyperactivation and proliferation allows us to view IL-2 treatment in HIV infection predominantly as immunotherapy targeting and reversing the HIV-driven generalized immune activation, with important pathogenetic and clinical implications.

Effect of IL-2 on T-lymphocyte apoptosis
Based on its critical role in the in vitro regulation of peripheral T cells,54,55 several groups have investigated the effect of IL-2 immunotherapy on T cell apoptosis rates, with somehow discordant results.45,56,57 In particular, the relative outgrowth of peripheral T cell turnover vis-à-vis to the actual CD4 cell gain during IL-2 cycles, give enough support to the hypothesis that elevated death rates accompany and indeed counterbalance the heightened T cell proliferation, still resulting in net CD4 cell expansion. According to the same model, it is also likely that the decreased T cell proliferation and activation observed in the IL-2 follow-up along with enhanced T cell survival might also lead to a parallel long-term reduction in T cell apoptosis.45

Taken together, these data indicate that the IL-2-driven consecutive balances in terms of death rates, proliferation and survival of the different subsets of T-lymphocytes determines the final effect on the T cell counts and phenotype. Indeed, immune phenotypic analysis of individual T cell subsets showed a discrepant pattern of recovery between IL-2 patients and HAART-alone controls. In particular, while HAART alone resulted in a preferential rescue of memory CD4 cells, with no changes in the naive compartment, IL-2 induced a significant and sustained expansion of both naive and memory CD4 cells,21,22,52,58 indeed reinforcing the potential of IL-2 to correct the HIV-driven imbalance within the naive/memory T cell pool, ultimately contributing to an overall rejuvenation of the T cell pool.

Effect of IL-2 on de novo T cell synthesis and IL-7/IL-7 receptor (IL-7R) system
As the IL-2-driven expansion of CD4 cells bearing a naive phenotype may indeed be derived from both peripheral expansion of existing cells and de novo T cell synthesis, the impact of neothymopoiesis on both CD4 and CD8 cell subsets was also studied. As for other parameters, CD4 and CD8 cells displayed a dichotomist trend, with a temporary tendency toward a rise in CD4 TRECs soon after IL-2 administration, possibly to an even higher extent in consideration of the high IL-2-driven proliferative dilution (Figure 1g and h).59 Conversely, no changes were detected within CD8 cells and in CD4 and CD8 TRECs in HAART controls (Figure 1g and h). These data indicate that the rise in naive CD4 cells induced by IL-2 does indeed reflect an increase in de novo T cell synthesis, proving the possible ability of IL-2 to stimulate thymic output, thus favouring the reconstitution of a most comprehensive immunity with a broad T cell receptor repertoire, which in turn could translate into a more efficient response to neoantigens.

Altogether, these data point to enhanced proliferation and prolonged survival of existing cells as the main mechanism responsible for IL-2-mediated CD4 cell recovery, and also suggest a boost of CD4 neothymopoiesis. Conversely, IL-2 failed to show an effect on both CD8 cell proliferation and activation in the periphery, thus implying that CD8 cells might follow different homeostatic dynamics,60 possibly peripheral distribution.

Aiming at a further understanding of the homeostatic effect of IL-2, the in vivo interactions between IL-2 and the IL-7/IL-7R system were also investigated,51 given the pivotal role of IL-7 in regulating both central and peripheral T cell homeostasis, and its effect on T cell survival and function.61 Compared with HAART alone, IL-2 induced a significant and sustained rise in IL-7 plasma levels, with no changes in IL-7R surface expression (Figure 2), indeed suggesting that in vivo IL-2 boosts IL-7 production without down-modulating its specific receptor, possibly preserving and enhancing IL-7-mediated T-lymphocyte homeostatic regulation. Altogether, the parallel expansion in total and naive CD4 cells, TRECs and IL-7 plasma levels makes it possible to envisage a model wherein IL-2 induced increased IL-7 production, which, in turn, orchestrated the cellular reconstitution by both neothymopoiesis and peripheral expansion (Figure 2). Furthermore, the recent observation that IL-2-non-responders display decreased IL-7R expression on both CD4 and CD8 cells also suggests that IL-7-mediated signalling is indeed critical in influencing the immunological response to IL-2 immunotherapy in HIV+ patients.48 By showing a bi-directional interaction between IL-2 and IL-7, these data confirm the existence of an important in vivo cross-talk between these two cytokines, both with central roles in T cell homeostasis regulation, with potential clinical implications.



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Figure 2. Effect of adjuvant interleukin-2 (IL-2) immunotherapy plus highly active antiretroviral therapy (HAART) on naive CD4 cells, IL-7, TRECs and Ki67 levels. The figure shows the interaction between the different homeostatic parameters following adjuvant IL-2 in patients who fail to restore CD4 cell compartment, despite complete control of HIV-viraemia. The increase in naive CD4 cells was associated with a progressive increase in IL-7 plasma levels throughout the study period, which reached statistical significance at week 3423 cp,

 
The overall analysis of T cell homeostatic parameters suggests that the mechanisms responsible for the immune repopulation following IL-2 therapy in INRs are likely to be different from that promoted by HAART alone. In particular, a two-step model could be envisaged for intermittent IL-2 immunotherapy action.43 During the cycles, IL-2 results in a broad peripheral proliferation of mature T cells, possibly associated with heightened apoptosis, ultimately resulting in the pronounced expansion in the size of the peripheral T cell pool. At the completion of IL-2 administration, a marked reduction in T cell turnover is observed, and yet an increase in the CD4 cell pool is still maintained. Combined, these findings allow us to postulate that the persistent CD4 cell increase in IL-2 follow-up results from a state of decreased turnover and cell death and prolonged survival in the periphery. Additionally, IL-2 might also stimulate neothymic synthesis possibly through the increased production of IL-7. The net result of these competing forces determines the indiscriminate expansion of different T cell subsets, including phenotypically naive CD4 cells. In contrast, the stability in TRECs, Ki67 CD4 cells, and IL-7 plasma levels together with the preferential expansion of the memory CD4 pool suggests that HAART-alone driven cellular recovery preferentially involves different immunological events (i.e. cell redistribution, reduced apoptosis), in a context of continuous thymic output and enhanced yet stable cell turnover.

Quality of IL-2-driven immune recovery

Role of IL-2 in functional immune enhancement
The issue regarding the role of IL-2 immunotherapy in functional immune enhancement in HIV infection is still quite controversial. In particular, in a randomized controlled trial in HAART-naive patients, Levy et al.62 demonstrated that the odds of being a responder to in vitro lymphocyte proliferative responses to recall antigens was significantly higher in IL-2 patients versus HAART-alone controls, allowing for the speculation that the CD4 cells expanded during IL-2 adjuvant therapy are indeed functional and able to support T cell function. Quite interestingly, Sullivan et al.63 demonstrated a significant rescue of lymphoproliferative responses to HIV-1-specific antigens during IL-2-associated viral breakthroughs. However, in contrast to these data, Valdez et al.64 were unable to demonstrate any immunization response (lymphoproliferative response, CD8 ELISPOT, in vivo antibody responses) to both recall and HIV-specific antigens following IL-2 administration.

Furthermore, the recent observation that IL-2 immunotherapy leads to the preferential expansion of naive and central memory CD4 cells, without affecting the effector pool44 has relevant potential functional implications.65 Indeed, whereas it is unlikely that the expanded memory pool is efficient in mounting a substantial immunity toward active infections, a future improvement of host defences against recall or even neoantigens might indeed be hypothesized, given the relative rarity of these challenges in adulthood. Moreover, the potential of IL-2 to directly stimulate neothymopoiesis further reinforces the role of IL-2 in selectively reconstituting neoantigen responses, given that TREC increases have indeed been correlated with improved neoantigen immune function.66

Combined, these data strongly indicate that the selective IL-2-driven expansion of specific T cell subsets does indeed also associate with a qualitative CD4 recovery, thus priming a tentative rescue of the HIV-related T-helper function imbalances. However, also under this perspective, clear cut answers on whether or not this will translate into clinical benefits as well as further speculation on the correct timing of IL-2 immunotherapy with respect to immunizations will hopefully be derived from ongoing Phase III trials.27,28

Role of IL-2 in immune tolerance
Aside from its role as a potent T cell growth factor, the actual functional activity of the CD4 cells expanded following IL-2 immunotherapy still remains to be clarified. Furthermore, both in vitro and in vivo animal models have shown that IL-2 is a master regulator of immune tolerance, via both the induction of Fas-mediated cell death and, most importantly, by promoting the development and peripheral expansion of CD4 CD25 regulatory T cells (Treg) which suppress in vivo excessive T cell responses.67 By bringing about a whole new perspective into the physiological function of IL-2, these observations also raise some concerns on the actual immunological effect of IL-2 immunotherapy, making it difficult to predict how it might affect in vivo interactions between proliferation and the suppressive phenotype of Tregs.68 The role and function of Tregs in the context of HIV infection have recently been investigated, yet remain quite controversial, with some preliminary data providing evidence that they may exert a detrimental effect by diminishing the HIV-specific T cell immune responses in vivo, possibly even hastening HIV disease progression.69 In contrast, an intriguing hypothesis proposes a possible positive effect of HIV-specific Treg cell-mediated suppressor function in the containment of several detrimental processes behind HIV pathogenesis, including activation-induced cell death and anergy, immune-mediated T cell destruction, and CD4 susceptibility to productive HIV infection, thus possibly delaying HIV disease progression.69 The enlightened understanding of the effect of IL-2 in specifically expanding and stimulating Treg cells in vivo combined with the most thorough investigation of Treg activity in HIV infection will allow for the most rational clinical manipulation of IL-2 signalling in HIV-infected patients.


    Conclusions and future directions
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 Abstract
 Rationale for immunotherapy in...
 Open issues: assessing the...
 Conclusions and future...
 References
 
Although HAART can efficiently control viral replication, it fails to induce major immune reconstitution. The adjuvant use of IL-2 offers the appealing perspective of filling the immunological gap left by HAART, including the potential for improved immunological control over HIV replication. Results from Phase I/II studies have indeed clarified several critical immunological issues on the actual role of IL-2 in HIV infection, allowing for clinical speculation.1724 In particular, intermittent schedules of IL-2 immunotherapy might indeed represent a valid and safe therapeutic option for HIV-infected patients with no immunological response to long-term HAART, insofar as its efficacy in rapidly reconstituting the CD4 cell pool to levels above the safety threshold values largely outweigh the side effects that it brings about.

From a more mechanistic standpoint, IL-2-associated immune reconstitution results from protean interactions with T cell dynamics and homeostasis, consisting of a preferential effect on CD4 cells, through the sequential stimulation of peripheral T cell turnover, apoptosis, survival and possibly a boost of neothymopoiesis, with a significant effect also on IL-7 production. The potential of IL-2 to interact with thymic output and to reverse the HIV-mediated T cell homeostasis imbalances by modulating the in vivo dynamics of T-lymphocytes and regulatory cytokines, without substantially affecting HIV viral load, offers the appealing prospect of obtaining major immune reconstitution in the treatment of HIV disease, with possible relevant implications in terms of functional immune enhancement, as also suggested by several in vitro and ex vivo studies.6264,68 Furthermore, a deeper understanding of the tolerogenic effect of IL-2 immunotherapy in HIV infection with respect to its role in sustaining the pool and function of Treg cells will be of great help in the re-evaluation of how best to clinically manipulate IL-2 signalling strength.

Notwithstanding these encouraging data, any definitive conclusions on whether or not the IL-2-mediated immunological effects could indeed be exploited in the clinic, translating into a clear clinical benefit, as well as the clinical setting that could mostly benefit from IL-2, possibly even with a specific indication, inevitably depend on the results of the large Phase III studies that are currently under way.27,28 Indeed, the completion of these on-going clinical studies will certainly provide a more direct confrontation of areas of controversy vis-à-vis IL-2-driven immune expansion and clinical benefit.

Despite the intrinsic barriers to validating clinical benefit in large-scale clinical end point trials, the need for new and effective surrogate markers of immune function is now becoming a priority. Indeed, while CD4 cell count and plasma HIV-RNA levels can reasonably be considered as valid markers to evaluate response to HAART, other putative markers for immune recognition have thus far failed to show any meaningful relationship with future clinical outcome.70 This is particularly true when it comes to IL-2 immunotherapy, as the immunological benefits that it confers are indeed complex, and not necessarily reflected in a direct and unequivocal effect on CD4 cell count and HIV viraemia control.


    Acknowledgements
 
We thank Professor Mauro Moroni for enlightened advice and continuous support, Anna Cristina Sacchetta for critical reading of the manuscript prior to submission and Chiron Italia for valuable advice. This work is supported by grants from Istituto Superiore di Sanità, Italy, the ‘National research program on AIDS’, from the ‘Ermenegildo Zegna’ Foundation, and from the AHSI Company.


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