HIV infection leads to a progressive loss of CD4+ T cells as a consequence of different mechanisms induced by viral replication. HAART is able to reduce viral replication to undetectable levels measured by commercial assays. As a consequence, an increase in CD4+ T cells is generally seen in most treated patients, with at least partial restoration of immune responses. Differences have been noted in the capability of distinct antiretroviral regimens to recover CD4 cell counts. For instance, protease inhibitors seem to produce more pronounced CD4 gains than nonnucleoside reverse transcriptase inhibitors in certain scenarios .
Absolute CD4+ T-cell gains observed following viral suppression with antiretroviral treatment might derive from four different sources: increase in thymic production of new T cells; proliferation of pre-existing cells in the periphery; redistribution of cells from lymphoid organs to the circulation; and reduction in T-cell apoptosis [2,3]. Unveiling the origin of CD4+ T cells in a given patient is relevant because it may determine the functionality and antigen recognition capacity of these cells. Naive cells, especially those recently migrated from the thymus, hold a higher T-cell receptor diversity (TCR) and, therefore, are able to recognize a wide spectrum of foreign antigens. On the contrary, peripheral expansion of mature T cells will only multiply pre-existing clones of CD4+ T cells and, hence, their antigen recognition capacity will be limited.
Direct analysis of T-cell receptor excision circles (TRECs) may allow deducing the origin of circulating T cells . TRECs are stable DNA episoms formed during TCR rearrangement. They do not replicate upon cell division and, therefore, their presence is diluted within the cell population as it undergoes consecutive divisions. More recently, the surface marker CD31 has also been reported to identify naive CD4+ T cells recently migrated from the thymus [5–7]. The use of this marker offers some advantages compared with TREC quantitation, being the most important that CD31 detection by flow cytometry allows simultaneous staining with other surface markers, allowing recognition of the CD31 content in distinct T-cell populations.
HIV-induced apoptosis contributes to loss of both infected and uninfected CD4+ T cells during HIV-1 disease progression. The Fas/Fas ligand (FasL) apoptotic pathway has been widely examined as contributor of CD4+ T-cell loss in patients progressing to AIDS [8,9]. Other molecules, as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), have also been shown to reflect apoptosis of virus-infected cells . As HAART decreases the apoptosis of CD4+ and CD8+ T cells in lymphoid tissues , the restoration of CD4+ T cells may result in part from a reduction in cell death .
Raltegravir is the first integrase inhibitor approved for clinical use [12,13]. It has demonstrated potent antiviral activity in distinct patient populations [14,15]. Given its benign safety profile, raltegravir is currently often prescribed as part of simplification strategies in patients with undetectable viremia using other less convenient antiretroviral agents [16–19]. The aim of our study was to determine whether switching to raltegravir in a context of suppressed viremia under HAART may enhance CD4+ T-cell increases and if so, which are the main mechanisms involved.
Patients and methods
An exploratory, retrospective, case–control study was performed at out clinic. All consecutive HIV patients with undetectable plasma viremia (<50 HIV-RNA copies/ml) under HAART that switched to a raltegravir-based regimen during the second semester of year 2009 were identified. Only individuals for whom cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma specimens had been collected at the time of raltegravir switch and 6 months thereafter were included in the study. The control group consisted in HIV patients with undetectable plasma viremia that did not change its medication during the same period. Individuals in the control group were comparable with the raltegravir group in terms of age, time with suppressed viremia and baseline CD4 cell counts.
Immunophenotypic analyses were performed using flow cytometry on cryopreserved PBMCs. Cells were stained simultaneously with CD4-PC7 (Beckman Coulter, Fullerton, California, USA), CD45RA-ECD (Beckman Coulter), CD27-PE (BD Biosciences, San Diego, California, USA), CD38-PC5 (Beckman Coulter) and CD31-FITC (BD Biosciences). Cell populations were classified according to CD45RA and CD27 expression into four different maturation status: naive (CD45RA+CD27+), effector (CD45RA+CD27−), central memory (CD45RA−CD27+) and effector memory (CD45RA−CD27−) subsets.
CD38 was used to determine the level of activation, and CD31 was employed as an indicator of recent thymic emigrants. Both CD38 and CD31 expression were analyzed in each of the different T-cell subsets. Data acquisition was carried out with a five-color flow cytometer (Cytomics FC500; Beckman Coulter) and analysis was performed using CXP software (Beckman Coulter). Gating was done on lymphocytes based on forward and side-scatter characteristics and thereafter a minimum of 20 000 CD4+ events were acquired for further analysis of cell subsets.
Soluble markers of apoptosis
Apoptosis was evaluated through the soluble plasma markers FasL and TRAIL. They were measured using two commercial ELISAs (Diaclone, Besancon, France).
All results are expressed as medians and interquartile ranges (IQR). Comparisons between raltegravir and control groups were performed using nonparametric tests (Mann–Whitney U-tests), and changes over the study period were evaluated using nonparametric tests for paired samples (Wilcoxon signed-rank test). All statistical analyses were carried out using the SPSS v15 software (SPSS Inc., Chicago, Illinois, USA).
A total of 37 HIV patients with undetectable plasma viremia during at least the prior 6 months and with stored cryopreserved specimens were identified. Of them, 19 had changed their antiretroviral combination for a raltegravir-based regimen and 18 had kept on the same medication. The switch to raltegravir had been mainly made in an attempt to improve metabolic abnormalities and improve upon other toxicities of antiretroviral agents included in the prior regimen. Exposure to the former medication had been for longer than 1 year in all instances, with a median of suppressed viremia of 26 [13–31] months and no significant differences between both groups of patients. The total time on antiretroviral therapy did not differ significantly comparing both study groups and was on average of 35 months. Most patients switched from a protease inhibitor-containing regimen, mainly atazanavir/r and lopinavir/r. The main baseline characteristics of the study population by treatment group are summarized in Table 1. No significant differences were found in baseline median CD4 cell counts neither in median CD4 nadir when comparing both groups of patients.
Absolute CD4+ T-cell count evolution
At baseline, median CD4+ T-cell count was 322 cells/μl (IQR 242–594) in the raltegravir group and 312 cells/μl (IQR 141–478) in controls (P = 0.485). After 6 months, a significant gain in CD4+ T cells was seen in patients switched to raltegravir [median increase to 448 cells/μl (288–575), P = 0.026], whereas it did not change significantly in the control arm [median increase to 330 cells/μl (176–425), P = 0.813] (Table 2). Of note, only seven out of 18 (38.9%) controls experienced any CD4 gain, whereas it was seen in 13 out of 19 (68.4%) raltegravir patients (P = 0.07).
Maturation status of CD4+ T cells
Surface markers CD27 and CD45RA were used to classify cell populations according to their maturation status. At baseline, CD4+ T cells were mostly central memory in both patient groups [64% (48–70) and 64% (55–69) for raltegravir and controls, respectively], followed by naive, effector memory and lastly effector T-cell subsets (Table 2). After 6 months, the distribution of CD4+ T cells changed significantly in the raltegravir group, with a proportionally larger increase in naive cells (P = 0.014), at expenses of the effector memory subset, which significantly decreased (P = 0.005). In contrast, control patients maintained stable the distribution of CD4+ T cells during the 6-month study period.
CD31 was used to identify recent thymic emigrants. In the whole population of CD4+ T cells, there was no significant change in the level of expression of this marker during the 6-month study period in any of the treatment arms (P = 0.277 and P = 0.679 for raltegravir and controls, respectively). However, raltegravir patients showed a trend toward an increased CD31 expression from 25.2% at baseline to 28.1% at month 6 (P = 0.17) in the naive subset. In controls, the increase was minimal, from 20.4% at baseline to 21.1% at 6 months (P = 0.811) (Fig. 1). Moreover, in 12 out of 19 (63.2%) raltegravir patients, the proportion of CD31+ T cells increased at 6 months [median increase of 5.31% (3.42–8.27)], whereas it increased in only nine of 18 (50%) control patients [median increase of 3.23% (1.41–7.50)], although these differences did not reach statistical significance.
During the 6-month study period there was no significant change in the activation level of the whole CD4+ T-cell population in any of the treatment groups (P = 0.059 and P = 0.231 in raltegravir and controls, respectively). However, the analysis of activation within the different T-cell subsets showed that there was a significant decline in activation of the effector subset in both treatment arms (P = 0.014 and P = 0.031 for raltegravir and controls, respectively). In contrast, the proportion of CD38+ naive cells increased in raltegravir patients (P = 0.036), whereas conversely it decreased in controls (P = 0.039) (Fig. 1).
Association of CD31 and CD38 markers in the naive T-cell subset
Given that there was a significant increase in CD38 expression and to a lesser extent in CD31 on CD4+ naive T cells in raltegravir patients, we analyzed whether there was a correlation between the expression of these markers. In the whole study population there was a significant (P < 0.001) and positive association between the two parameters in the naive CD4+ T-cell subset, both at baseline and at after 6 months (Fig. 2), suggesting that they might evolve together.
Soluble markers of apoptosis
Soluble FasL and TRAIL were quantified in plasma at baseline and after 6 months. No significant changes were recognized in any treatment arm, although there was a trend toward a decrease in the level of soluble FasL in raltegravir patients [from 563 pg/ml (459–831) at baseline to 500 pg/ml (385–673) at month 6; P = 0.071). Measurement of TRAIL levels did not reveal any significant change over time in any of the groups (Table 3). Finally, there was no significant association between soluble markers of apoptosis and CD4 cell counts or CD4+ T-cell activation.
Antiretroviral treatment drastically reduces plasma HIV-RNA in most adherent patients. As a consequence of viral suppression, CD4 gains and immune recovery occurs to some extent in most patients . Different mechanisms contribute to this CD4+ T-cell increase, beginning with memory T-cell redistribution from secondary lymphoid compartments  and followed by a rise in naive T cells rich in TRECs .
Not all antiretroviral drugs perform equally well in terms of immune recovery. For instance, maraviroc has proven to cause CD4+ T-cell increases beyond its effect on viral suppression , which could be the result of blocking the traffic of CCR5+ T cells from the circulation to peripheral tissues or, alternatively, due to some attenuation of T-cell activation . On the contrary, azidothymidine might be toxic for hematopoietic progenitor cells and, therefore, could partially disrupt CD4+ T-cell reconstitution . In our study, we assessed whether switching to a raltegravir-containing regimen in a context of suppressed viremia on HAART might lead to an enhanced CD4 gain and if so, which are the mechanisms behind it.
A higher CD4+ T-cell gain was found in patients switched to raltegravir than in controls maintaining unmodified their prior antiretroviral regimen in the context of undetectable viremia. In the STARTMRK trial, CD4 increases were superior at week 48 in antiretroviral-naive patients who were treated with raltegravir compared with efavirenz despite similar rates of viral suppression . To a lesser extent, these trends persisted for another 3 years . A faster and more pronounced effect on residual viremia by raltegravir could explain this finding, leading to stronger decreases in T-cell activation and enabling a better immune response. However, this hypothesis has not been confirmed in intensification studies that have assessed residual viremia  or T-cell activation , although one study found a decrease in T-cell activation followed by a transient increase in episomal DNA . Clearly, further studies are warranted to clarify this issue.
Cellular activation was not affected by raltegravir treatment in a different degree than using other antiretroviral drugs, discarding the hypothesis of enhanced CD4 gains on raltegravir as result of reductions in immune activation. In contrast, we observed an enlargement in the proportion of naive T-cells in patients switched to raltegravir, which was not seen in controls. Moreover, a trend toward greater CD31 expression, which reflects recent thymic emigrants, was recognized in naive CD4+ T cells in patients treated with raltegravir, which was not seen in controls. Thus, at least part of the CD4+ T-cell gain in these patients might result from a contribution of recent thymic emigrants. Accordingly, there was a significant increase in the proportion of naive CD4+ T cells expressing CD38 in raltegravir patients but not in controls. Although CD38 is generally used to assess T-cell activation, it is also constitutively expressed on naive T-cells, indicating immaturity regardless of activation status [28–31]. Interestingly, CD38 expression on CD45RA+ T cells has also been suggested to be associated with recent thymic emigrants , and we found a significant correlation in the naive CD4+ T-cell subset between CD38 and CD31 expression, supporting that these two markers evolve in parallel in naive T cells. Indeed, CD31 acts as a ligand for CD38 . Altogether, our results suggest that raltegravir therapy might increase the release of new CD4+ T cells from the thymus to a greater extent than other antiretroviral drugs. Further studies testing larger populations of treated patients are warranted to answer this question.
Finally, we examined whether CD4+ T-cell gains could be due to a decrease in T-cell death. Several factors favor apoptosis in HIV patients, including residual viral replication, older age, CCR5-induced activation, co-infections, microbial translocation, regulatory T-cell activity and some host genetic determinants . Antiretroviral treatment is generally able to reduce T-cell death to some extent and we found a trend toward a further decline in FasL levels in patients switched to raltegravir, which did not occur in controls, suggesting that raltegravir might intensify viral suppression in this setting, halting T-cell death. In this regard, ultrasensitive viral load testing could have provided further insights, but could not be performed in our specimens. A further limitation of our study is that the soluble markers we measured only indirectly correlate with T-cell apoptosis. On the contrary, a functional characterization of immune responses should allow an appreciation of the degree of qualitative immune recovery seen in these patients, besides the numerical changes in CD4 cell counts we saw. Finally, our conclusions are limited by the relative small size of the study population and the relative short follow-up examination. Clearly larger studies and with extended follow-up are warranted.
In summary, our results show that switching to a raltegravir-containing regimen in HIV patients with prolonged suppressed viremia on HAART induces a significant CD4+ T-cell gain in the short term. This increase mainly results from a raise in naive CD4+ T cells derived from the thymus.
C.G., C.d.M. and J.M.B. designed the study. C.G., N.R., M.L., N.Z. and J.M.B. performed the laboratory immunological studies. C.G., N.R. and V.S. revised the clinical charts and recorded the information from the study population. C.G., J.M.B. and V.S. did the statistical analysis. C.G., J.M.B. and V.S. wrote the manuscript.
This work was supported in part by grants from Fundación Investigación y Educación en SIDA (IES), Red de Investigación en SIDA (RIS, FIS-RD06/0006) and the European Union 6th Framework Programme (NEAT, LSHP-CT-2006-037570).
Conflicts of interest
There are no conflicts of interest.
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