The course of HIV infection has been profoundly changed by HAART, that prolongs both survival and the duration of time free from opportunistic infections . These positive effects are clear to clinicians and patients, but it is evident that many patients' everyday lives have been adversely affected by the administration of complex therapeutic regimens. Moreover, HAART is associated with several side effects, many of which can be ascribed to mitochondrial toxicity of the drugs [2–6].
In order to reduce drug-associated toxicity or delay the onset of side effects that may lead to poor adherence to antiretroviral therapy, several approaches have been explored. One of these is the maintenance of HAART, but with the substitution of certain drugs and the simplification of treatment. Another approach is the use of treatment interruptions, which are performed by using two main strategies. The first, defined ‘structured’ treatment interruption, consists of different periods of alternating on and off cycles of HAART, the length of the cycles either being fixed in advance or determined on viral load values [7–10]. The second is guided by the number of CD4+ T cells [11–15]: patients interrupt treatment when CD4+ T-cell count is above a certain level (in our experience > 500 cells/μl), and restart when the count falls below the predefined threshold (350 cells/μl in our studies).
For many years, our group has been conducting several studies to investigate the main features of the CD4 cell-guided interruptions. In a large multicentric study, we have observed that patients who started therapy with a CD4+ T-cell count between 250 and 350 cells/μl and who later interrupted therapy can remain off therapy with CD4 cell count > 350 cells/μl for a substantial period of time . Then, we observed that HAART-induced damages to peripheral blood lymphocytes' mitochondria can be repaired, but only after, at least, 6 months of treatment interruption . This suggests that interruptions of shorter periods are unlikely to allow restoration of mtDNA and thus decrease at least this type of HAART-related toxicity.
Treatment interruptions also represent a model to better understand the interactions between virus and host. Indeed, withdrawal of the therapy provokes an immediate rebound of viral activity, resembling what happens during primary infection. Few data exist on the fine characteristics of the immune responses during the re-exposure to the virus, as during treatment interruptions. Accordingly, the aim of the present study was to evaluate the impact of this strategy on the phenotype of peripheral T cells, paying a particular attention to markers of cell differentiation, activation, apoptosis and survival.
Patients and methods
Since January 2001, a longitudinal prospective study on CD4 cell monitored treatment interruption has been ongoing in the Clinic of Infectious and Tropical Diseases in Modena, Italy. All of the enrolled patients, currently more than 60, perform a clinical evaluation and a blood test at baseline and every 2 months, until the resumption of treatment. They interrupt HAART in agreement with their treating physician if they satisfy the following inclusion criteria: they must be older than 18 years, have received HAART for at least 12 months, and have a pre-interruption CD4+ T-lymphocyte count > 500 cells/μl. HIV plasma viral load at the time of discontinuation could have been < 50 or > 50 copies/ml. Patients treated with immuno-modulatory agents and hydroxyurea are excluded. The criteria for restarting treatment are: a CD4+ T-lymphocyte count < 350 cells/μl on two different occasions 1 month apart, a clinical manifestation of AIDS, or the patient's desire to resume HAART. Patients are advised of a possible higher risk of HIV transmission to sexual partners during the period of treatment discontinuation. Chiron branched-DNA is used for plasma HIV RNA, and a value < 50 copies/ml is considered undetectable. At each blood control, a sample is drawn to perform viro-immunological analyses.
The immunological study here described was performed on 17 patients, selected on the basis of the availability of biological material at all of the considered time points. Twelve (70.6%) were male, five were female; seven (41.2%) were former drug users, three (17.6%) men who have sex with men, seven heterosexuals; median age was 42 years (range, 34–57 years). Six were naive for antiretroviral therapy when they started HAART. Median time spent on treatment prior to discontinuation was 60 months (range, 9–103 months). At discontinuation, four patients (23.5%) were assuming a protease inhibitor-containing regimen, 13 patients (76.5%) a non-nucleoside reverse transcriptase inhibitor-containing regimen. Median CD4+ T-cell count at start of antiretroviral treatment was 380 cells/μl (range, 312–413 cells/μl), median nadir was 288 cells/μl (range, 156–392 cells/μl). At discontinuation, median CD4+ T-cell count was 858 cells/μl (range, 737–1194 cells/μl), and 13 patients had undetectable plasma viral load.
Only the data obtained during the first 10 months off therapy are presented and discussed here. Among the population studied, six of 17 patients had restarted therapy within the 12 months of discontinuation (median, 8.5 months; range, 5–10 months) because their CD4 cell count decreased to a value < 350 cells/μl on two different occasions 1 month apart. In order to identify possible markers related to the immunological outcome, patients were thus divided in two groups: those who did not restart (group A), and those who restarted (group B) therapy during the observation time. The length of follow up after discontinuation was 12 months, but since samples at the 12-month timepoint were not available for all patients, analysis was performed for only for the first 10 months of interruption.
Immunophenotyping was performed in 15 patients, soluble CD95 (sFas) plasma level was measured in 11, and all of the other parameters, including the quantification of the cells expressing T-cell receptor rearrangement sj excision circles (sjTREC), are for all 17 individuals. To obtain reference values, six healthy subjects, aged 20–30 years, were included in the immunophenotype analysis. Plasma concentrations of interleukin-7 (IL-7) and sFas were determined in 23 and 42 healthy subjects (aged 20–60 years), respectively.
Isolation of peripheral blood mononuclear cells and phenotype analysis by flow cytometry
Blood was collected by venous sampling into EDTA. Plasma was separated from blood (130 × g, 25 min, room temperature) and then platelets were removed from plasma (1000 × g, 15 min, 4°C), which was stored at −80°C. Peripheral blood mononuclear cells (PBMC) were isolated and frozen using standard procedures. PBMC were thawed and stained (20 min in the dark, 4°C) with monoclonal antibodies recognizing the following surface antigens: CD3, CD4, CD5, CD8, CD16, CD19, CD38, CD45, CD45RA, CD57, CD95, CD127, CCR7, in different combinations. Cytofluorimetric analysis were performed by using a flow cytometer CyFlow Space (Partec GmbH, Münster, Germany), as described . A minimum of 10 000 events for each sample were acquired by using FloMax software (Partec); files were then analysed as described . Lymphocyte populations were selected on the basis of physical parameters and CD45 expression; CD4/CD3 or CD8/CD3 cell populations were then identified and, within these subsets, expression of other surface markers was evaluated. As far as CD38 is concerned, we could identify dim (+) or bright (++) cells, corresponding to activated or overactivated lymphocytes, respectively . The high expression of CD38 on monocytes was used to identify CD38bright cells within T lymphocytes in all of our analyses.
Quantification of percentage of sjTREC positive CD4 or CD8 T cells by real-time PCR
CD4 or CD8 T cells were positively isolated by magnetic sorting (MACS, Miltenyi GmbH, Bergisch Gladbach, Germany); the purity of the cell populations was always > 95%, as assessed by parallel cytofluorimetric analysis. The percentage of CD4 and CD8 T cells containing sjTREC was then measured by an original method that has been recently developed, that uses a real-time PCR approach, as described .
Quantification of plasma IL-7 and sFas levels
Plasmatic concentration of IL-7 and sFas were determined by using commercially available kits (Quantikine, R&D Systems, Minneapolis, MN), according to the manufacturer's instructions.
Anova for repeated measures and the Mann Whitney test were used to compare the data among the groups of subjects. Statistics were performed by using Stata 7.0 under Windows XP.
Changes in viro-immunological parameters after treatment interruption
As already described in our previous studies [11,16,17], in almost all patients we observed a viral rebound (Fig. 1a) that was significantly more marked in group B (Fig. 1b), along with a reduction in CD4 T cell absolute number and the inversion of the CD4/CD8 ratio (data not shown).
Plasma viral load was stable after the fourth month off therapy (Fig. 1a), while the decrease in CD4 T cells was arrested after the sixth month. The trend in the changes of CD4 cell count was similar in the two groups, even if group A had always higher values (not shown). After 6 months of interruption, group A also had a CD4 cell count value that was consistently higher than that observed before treatment (data not shown).
Analysis of lymphocyte activation markers
We investigated the expression of activation/cell death markers (such as CD38 and CD95/Fas), differentiation markers (CD45RA, CCR7) or survival molecules (CD127) among CD3/CD4 and CD3/CD8 T cells using the strategy indicated in Fig. 2. Treatment interruption resulted in small, even if significant, modifications of CD38 expression on CD4 T cells (Fig. 1c and e), while more consistent changes were observed among CD8 T lymphocytes (Fig. 1d and f). Indeed, in parallel with the viral rebound, we could observe a massive activation of CD8 T cells. In particular, after 2 months of therapy interruption, the percentage of CD8/CD38bright T lymphocytes remarkably increased, returning in the following months to levels similar to those at the moment of interruption (Fig. 1f). Nevertheless, a certain degree of CD8 activation was maintained throughout the period of therapy interruption (Fig. 1d), and it was correlated to the increased expression of CD95, especially on memory cells (data not shown). As far as CD38 expression is concerned (either total or bright), no differences were present between groups A and B (Fig. 1g–j).
The expression of CD95 was quite similar to that of CD38 (Fig. 3a and Fig. 4a). Also in this case, a significant increase was present among CD8 after the first 2 months of interruption, then CD95 showed negligible changes.
The analysis of soluble CD95 plasma levels showed that values were higher in patients than in healthy controls (7928 ± 908 pg/ml versus 5116 ± 397; P = 0.015), but that no significant changes occurred with time, and no differences were present between the groups (data not shown).
No significant changes were also observed with time or between the two groups as far as B lymphocytes (CD19) or natural killer cells (defined by the expression of CD16 and/or CD57) were concerned (data not shown).
Analysis of lymphocyte differentiation markers
By using CCR7 and CD45RA as differentiation markers, considering all patients, we could not observe any relevant modification of the naive and memory compartments among CD4 T lymphocytes (Fig. 3b–e). Nevertheless, the composition of CD4 subsets was different in the two groups of patients: in comparison with group A, group B showed and a higher amount of naive cells (Fig. 3f), and a lower percentage of effector memory cells (Fig. 3g).
As far as CD8 T lymphocytes are concerned, in all individuals we observed a significant decrease in naive cells and changes in the memory subsets, with the expansion of effector memory cells (Fig. 4b–e). In particular, at baseline group B presented the same amount of naive but significantly less terminally differentiated CD8 T cells than group A (Fig. 4f and g).
Quantification of TREC+ cells
The quantitative analysis of TREC+ cells revealed that at the moment of therapy discontinuation patients had a mean of 0.62% TREC+ cells among CD4 T lymphocytes, and a mean of 0.87% TREC+ cells among CD8 T lymphocytes. Such values did not change significantly over time, and no statistical differences were noted between groups A and B (data not shown).
Expression of IL-7 receptor and IL-7 plasma levels
Analysing all patients together, we found that IL-7 receptor was steadily expressed on CD4 T cells during treatment interruption, but that a significant decrease was observed among CD8 T lymphocytes after the second month of interruption (data not shown). As shown in Fig. 5a and b, the most relevant changes in CD127 expression were present among the subset of CD8/CD45RA T cells.
When we compared the two groups, we found that the amount of CD4 T cells expressing CD127 was different (Fig. 5c): group A expressed a higher amount of CD127 from the moment of interruption onwards. A similar trend was observed among CD8 T cells, in which a significant difference in IL-7 receptor expression was present between the two groups, even if both groups showed a decrease of CD127 expression during treatment interruption (Fig. 5d). Group B presented a higher amount of CD4/CD45RA+/CD127− cells (Fig. 5e), but a significantly lower amount of CD4/CD45RA−/CD127+ cells (P = 0.032, data not shown). No differences between the groups were present in terms of CD8/CD45RA+/CD127− (data not shown). In all patients we found an immediate increase in the percentage of CD8/CD45RA−/CD127− (in the first 2 months, P < 0.001, data not shown). Group A had a lower percentage of CD8/CD45RA−/CD127− (Fig. 5f), and a higher percentage of CD8/CD45RA/CD127 (P = 0.033, data not shown).
The quantification of IL-7 plasma concentration revealed that plasma level of this cytokine was lower in HIV+ patients than in healthy subjects (mean ± SEM, 2.01 ± 0.29 pg/ml versus 4.01 ± 0.39; P = 0.005), and did not change during therapy interruption (Fig. 5g). No differences were present between group A and B (Fig. 5h).
The strategy of CD4 cell monitored treatment interruption allows the patient to remain off therapy for periods that are much longer than those of the structured therapy interruptions. Such strategy represents an ideal model to study not only the immediate reaction of the immune system to the reactivation of the virus, but also the evolution of the response with time. Accordingly, the aim of this study was to investigate the changes of several immunological markers during CD4 cell monitored therapy interruption.
In almost all patients, after treatment interruption several alterations in lymphocyte subpopulations took place, partially resembling what occurs during primary HIV infection . A high viremia was accompanied by a decrease in CD4 T-cell count and an overactivation of CD8 T lymphocytes. As previously described , after a period of 2–3 months the immune system starts to control viral replication, which becomes stable, as shown by the fact that viral load reaches a new plateau. Unfortunately we could not compare such plateau with the viral load before starting therapy for the first time, as in most cases patients started therapy when this parameter was not available. The decrease in CD4 T-lymphocyte count was consistent in the first months, and decreased thereafter, as already reported . The viral rebound was associated with a strong CD8 lymphocyte activation. It is known that overactivation of the immune system, as revealed by high levels of CD38, is an important pathogenetic and prognostic factor for the progression of the infection . As CD8 overactivation was under control from the fourth month off therapy, CD4-guided treatment interruption does not seem to favour the disease progression very much, at least from this point of view.
In CD8 lymphocytes, CD95 expression could be induced either by a physiologic immune response (lymphocyte activation) or by defence mechanisms (lymphocyte apoptosis) related to the presence of the virus [24,25]. During primary infection, a marked expression of CD95 is indeed accompanied by massive apoptosis of these cells [25,26]. In the case of treatment interruptions, the increased expression of CD95 on most CD8 T cells was prolonged, with a trend that was similar to that of CD38dim cells, and different from that of CD38bright T lymphocytes. Thus, it might be that the immune regulation was quite different in acute compared with chronic infection, probably because in the latter the system is already compromised by years of viral persistence. It can be hypothesized that the presence of CD95, along with the persistent high level of plasma sFas, is an indicator of CD8 T-cell activation (maybe incomplete, considering the trend of CD38dim cells) rather than T-cell death.
We did not observe any substantial change in CD4 T-lymphocyte activation during treatment interruptions, and no main alterations in CD95 expression either with time or between the two groups of patients. As far as the decrease in CD4 T cells with time is concerned, we observed that the relative percentages of naive and memory CD4 T cells did not change significantly. In other words, the loss of CD4 T cells was similar among naive, central memory, effector memory and terminally differentiated cells. According to these observations, it is possible to hypothesize that the loss of CD4 is due to a direct action of the virus on all of the CD4 subsets rather than to the dysregulation (or triggering) of endogenous mechanisms such as, for example, that of activation-induced cell death, that are much more effective in memory cells.
We found that the percentages of TREC+ CD4 and CD8 lymphocytes did not change with therapy interruption, indicating that thymic output was probably still preserved in these patients. Thus, the reactivation of the virus had apparently a little effect on thymic precursors and functionality.
We showed that patients who restarted treatment presented a higher percentage of CD4 naive lymphocytes and a lower percentage of effector memory CD4, and that the trend of such subpopulations did not change during therapy interruption. Considering that no differences were observed as far as TREC+ cells are concerned, and thus that thymic output was probably similar in the two groups, we can hypothesize that in these individuals a higher, selective deletion of activated CD4 cells with an effector phenotype took place at some point of the infection, prior to treatment interruption. This could suggest that such a cell population is capable of influencing the length of the interruption.
In all patients who interrupted therapy, we observed an expansion of effector memory CD8 lymphocytes along with a decrease in naive and central memory cells, but no changes in the percentage of terminally differentiated cells. Thus, according to previous data describing a skewed maturation of HIV-specific CD8 T cells , it can be argued that treatment interruption further exacerbates this phenomenon.
IL-7 is a master cytokine that regulates T-cell homeostasis and indeed the IL-7/IL-7 receptor system is profoundly affected in HIV infection, as in other persistent infections [28–30]. An increase in IL-7 plasma levels can occur during HIV-associated severe lymphopenia . Surprisingly, we found that in our patients IL-7 plasma concentration was lower than in healthy subjects, and did not increase significantly with time, even during the loss of CD4 cells. This could indicate that in these subjects a substantial loss of stromal cells that are able to produce IL-7 could had occurred, or that such a cytokine was more utilized than in healthy donors. A well preserved response to survival and proliferation signals such as those given by IL-7 or IL-2 can support the homeostatic maintenance of lymphocyte subsets and counter apoptosis . Patients who restarted therapy presented a lower expression of CD127, both on naive and memory CD4 or CD8 T lymphocytes. As far as CD4 T cells are concerned, group B presented a lower percentage of memory cells expressing CD127, and fewer effector cells. Since CD127 expression did not change when viral load increased, it could be possible that IL-7 acts as an important molecule for the antigen-independent proliferation of effectors .
The reduced expression of CD127 on CD8 lymphocytes could be explained by a down regulation of this molecule, related to cell activation and homeostasis . It has been reported that HIV infection per se is associated with decreased CD127 expression, even if the mechanism has still to be clarified . The loss of CD127 defines the expansion of an effector-like CD8 subpopulation, and has been considered a typical marker of the progression of infection . Our results show an expansion of memory CD8+/CD127− in all patients, along with a significantly higher amount of this subpopulation in patients who restart therapy. Our data strengthen the interest in better defining the functionality of these cells, whose presence not only correlates with several markers of disease progression, but seems to be important for the length of treatment interruptions. Thus, the expression of CD127 could be a meaningful immune marker also for monitoring therapy interruptions. However, we are well aware that the relatively small size of our study is not sufficient to reach the statistical power necessary to identify predictive factors of therapy interruption length, and further studies are needed to clarify this aspect.
In conclusion, several studies have shown that from a clinical point of view CD4-guided treatment interruption can be considered a safe strategy in the presence of an accurate selection procedure and strict control of patients [10,15–17,37,38] whose immune system has to be in a better condition than that of the patients of the SMART trial . For the length of the follow-up (up to 12 months), this study contributes to a better understanding of the immune response after treatment withdrawal. Indeed, treatment discontinuation induces immune modifications that are similar but not equal to a primary HIV infection, as the viral rebound meets an immune system already damaged by years of infection.
A prominent role in the maintenance of an immune response that could allow prolonged periods off treatment is probably played by the IL-7/IL-7 receptor system. Indeed, our results strongly reinforce the interest in this field of investigation, as the capability to respond to IL-7, by expressing and maintaining the expression of CD127, seems to be strongly associated with a favourable outcome of CD4 guided therapy interruptions.
The present study was conducted on patients undergoing a single treatment interruption. We do not know would happen to the immune system of patients who experience several treatment discontinuations on the basis of their CD4 cell count. Further immunological studies are thus needed before considering this strategy as a possible standard of care.
GeneMoRe Italy SRL (Modena, Italy) is kindly acknowledged for help in TREC analysis; Partec GmbH (Muenster, Germany) and Space srl (Milan, Italy) are acknowledged for technical support. Drs. L. Troiano, E. Roat, C. Giovenzana, L. Gibellini, G. Rocco and N. Prada are kindly acknowledged for precious help and discussions.
Sponsorship: This study was supported by Istituto Superiore di Sanità, Rome (Italy), Progetto Patologia, Clinica e Terapia dell'AIDS (grants 30D.56 to C. Mussini and 30F.15 to A. Cossarizza).
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
HAART; IL-7 receptor system; lymphocyte activation; therapy interruption; viral load