Immunodiscordant responses to HAART are characterized by poor recovery of CD4 T cells despite complete virological suppression . Blunted immunological responses have been associated with a number of adverse immunological features that include high levels of CD4 and CD8 T-cell activation [2,3], poor thymic production of new CD4 T cells [4,5] and increased sensitivity to cell death of circulating cells . The general hallmark of immunodiscordance also includes higher levels of inflammatory markers  and bacterial translocation across intestinal mucosa . In general, these features seem to be associated with low CD4 T-cell nadir values , although causal relationships are not clearly understood.
The inflammatory environment and the high level of CD4 and CD8 T-cell activation seem to impact mortality and morbidity in HIV-infected individuals [9,10]. Therefore, immune activation has been the target of different clinical interventions using immunomodulatory drugs (hydroxychloriquine) or anti-cytomegalovirus (CMV) agents (valganciclovir) with some short-term beneficial effects [11,12]. A potential consequence of higher activation and proinflammatory signals is the increased risk for residual viral replication in immunodiscordant patients, and therefore these individuals might represent an appropriate target for treatment intensification. Intensification approaches have controversial effects on HIV-infected individuals; whereas short-term effects (24 weeks) are elusive [13,14] or observed in a limited percentage of patients , long-term effects (48 weeks) seem to be more consistent and led to sustained decreases of CD8 T-cell activation , suggesting that some level of residual HIV replication is occurring in apparently suppressed patients.
We have evaluated the potential contribution of the persistent de-novo infection to the immunological abnormalities described in immunodiscordant individuals. Immunological markers were analyzed in a randomized clinical trial assessing the effect of the addition of raltegravir to HAART standard regimens in stable virologically suppressed patients showing incomplete CD4 T-cell recovery (CD4 <350 cells/μl). Although clinical data show poor CD4 T-cell count benefit (Negredo & Massanella et al. unpublished observation), raltegravir intensification has significant immunological effects that differ among CD4 and CD8 T cells. Moreover, in CD8 T cells, the effects are also divergent among the activation makers analyzed.
Materials and methods
Study design and patients
A prospective, open-label, ranomized study (ClinicalTrials.gov number NCT00773708) to assess the effect of raltegravir addition to standard antiretroviral therapy for 48 weeks in immunodiscordant individuals (Discor-Ral Study) was carried out in the Hospital Universitari Germans Trias i Pujol (Badalona, Spain). The protocol was approved by the Institutional Ethics Review Committee. All participants provided written informed consent.
We enrolled HIV-1-infected patients on suppressive HAART (viral load <50 copies/ml) for at least 2 years (with >4 determinations) and CD4 T-cell counts always below 350 cells/μl. This cohort of immunodiscordant individuals has been followed for several years and its immunological features have been reported previously [3,6]. Patients were randomly assigned 2 : 1 to intensify their current antiretroviral therapy with raltegravir (400 mg twice daily) for 48 weeks (intensified arm, n = 30) or to continue their current therapy regimen (control arm, n = 14).
Blood samples were drawn at baseline and weeks 2, 4, 12, 24 and 48 and processed by standard protocols to obtain plasma and peripheral blood mononuclear cell (PBMC) samples . Prestudy values of viral load and CD4 T cells (up to 48 weeks) and epidemiological data were also recorded.
Flow cytometry analysis of blood samples
We stained fresh blood samples with the following antibody combinations: first, CD95- fluorescein isothiocyanate (FITC), PD-1–phycoerythrin (PE), human leukocyte antigen (HLA)-DR–peridinin chlorophyll protein complex (PerCP), CD3–allophycocyanin (APC)–Cy7, CD4–APC and CD8–PE–Cy7; second, HLA-DR–FITC, PD-1–PE, CD38–PerCP-Cy5.5, CD45RO–APC, CD3–APC-Cy7, and CD8–PE-Cy7; and third, CD45RA–FITC, CD31–PE, CD38–PerCP, CD3–APC–Cy7, CD4–APC and CD8–PE–Cy7. First and second Combinations were designed to evaluate immune activation, whereas third combination was designed to evaluate CD4 T-cell production (thymic output). An unstained control and a control antibody combination containing anti-CD3–APC-Cy7, CD4–APC and CD8–PE-Cy7 antibodies were performed for all samples. Additionally, a subgroup of patients (10 from the control group and eight from the intensified group) was assayed for changes in main CD4 T-cell subsets (naive, N; central memory, TCM; transitional memory, TTM; effector memory, TEM) using the following antibodies: CD45RA-FITC, CD4-PerCP, CCR7-PE-Cy7, CD27-APC and CD3-APC-Cy7. All antibodies were from BD Bioscience (Franklin Lakes, New Jersey, USA). Briefly, 20 μl of blood was incubated 15 min at room temperature with the different antibody combinations in V-bottomed 96-well plates. After incubation, blood was lysed with 200 μl of FACS Lysing solution (BD Bioscience, Franklin Lakes, New Jersey, USA) for 30 min at room temperature, washed in PBS and resuspended in PBS containing 1% formaldehyde. Cells were acquired in a LSRII flow cytometer (BD Bioscience) coupled with a high-throughput screening (HTS) loader. At least 10 000 lymphocytes were collected for each sample. Analysis was carried out using FLowJo software (Tree Star, Inc., Ashland, Oregon, USA).
Analysis of cell death
Cell death was evaluated by culturing 200 000 freshly isolated PBMC per well in 96-well plates in 100 μl of RPMI medium (containing 10% fetal calf serum) for 0, 1, and 4 days in the absence or presence of the pan-caspase inhibitor Z-VAD-fmk (50 μmol/l, R&D Systems, Minneapolis, Minnesota, USA). Cells were analyzed in an LSRII flow cytometer after incubation with 40 nmol/l of the potentiometric mitochondrial probe DIOC6 (Invitrogen, Madrid, Spain), 5 μg/ml propidium iodide (PI, Sigma, Madrid, Spain), and CD3-APC-Cy7, CD4-APC and CD8-PE-Cy7 antibodies. Total cell death was calculated as the percentage of cells showing low DIOC6 staining in cultures performed in the absence of the caspase inhibitor . Specific mechanisms of cell death were analyzed as described previously .
Analysis of soluble CD14
Soluble CD14 (sCD14) levels, a surrogate marker of bacterial translocation , were quantified in all plasma samples using commercially available ELISA assay (Diaclone, San Diego, California, USA). Plasma samples were diluted (1/50 or 1/100) and tested in duplicate.
We used the Mann–Whitney U test to compare medians between arms and the signed rank test (paired test) to perform intragroup comparisons. Differences in proportions between groups were analyzed through the Pearson's χ2, considering the continuity correction or the Fisher's exact test, as appropriate. Statistical analyses were performed with SAS 9.1 and the R package. Graphs were generated using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, California, USA). The rate of CD4 T-cell change in both arms was calculated using linear mixed models as described . Slope coefficients were compared between groups (Negredo and Massanella et al. unpublished observation).
Immunodiscordant individuals recruited for the study showed similar demographic characteristics in both groups (Table 1), in particular regarding factors influencing immune responses to HAART, such as age, time on antiretroviral therapy and time from HIV diagnosis. Moreover, the mean time of viral load suppression (<50 copies/ml) was 6 years in both groups and baseline immunological characteristics of these patients were consistent with previously reported features of immunodiscordance in the CD4 T-cell compartment , such as low CD4 T-cell counts, low nadir CD4 T-cell values, low percentage of naive CD4 T cells (measured by CD45RA and CD31 expression), high level of activation (measured by the CD38, HLA-DR or CD95 markers), exhaustion (PD-1), sensitivity to cell death in CD4 T cells and plasma levels of sCD14 (Table 1). Again, no significant differences between groups were observed in these variables.
Analysis of CD4 T-cell compartment
Changes in absolute CD4 T-cell counts in the control group showed the slow increasing trend characteristic of discordant individuals, and reached intragroup statistical significance (P < 0.05) at weeks 24, 36 and 48 (Fig. 1). Conversely, intensified group showed a faster significant increase peaking at week 12, reaching a plateau afterwards (P < 0.02 for weeks 12, 24, 36 and 48), resulting in similar CD4 T-cell counts in both groups at the end of the study (Fig. 1). Remarkably, the use of linear mixed models revealed a biphasic behavior of CD4 T cells in intensified individuals, which was significant when analysis was broken down in 0–12 and 12–48 week periods (P < 0.001, Fig. 1a). Full analysis is reported elsewhere (Negredo, Massanella et al. unpublished observation) and confirms a significant, but limited to 12 weeks, effect of intensification in CD4 T-cell dynamics.
The analysis of CD4 T-cell subsets contributing to the transient increase in CD4 T-cell counts revealed no significant changes in the frequency of recent thymic emigrants (CD45RA+CD31+), naive cells having proliferated in periphery (CD31− cells in the CD45RA+ subset) and memory cells (CD45RA−, Fig. 1b). Consistent with this observation, the analysis of absolute counts revealed transient increases in all populations in the intensified group. However, recent thymic emigrants increased in both groups, at week 24 in the control arm (P = 0.022, Fig. 2a) and at weeks 12–24 in the intensified arm (P = 0.003, P = 0.007, Fig. 2b). Peripheral proliferation of naive cells showed constant values in the control arm (Fig. 2a), although they appear to minimally contribute to CD4 T-cell increase in the intensified arm at week 24 (P = 0.027, Fig. 2b). Remarkably, we also observed a fast and significant increase of absolute memory cells in the intensified arm (weeks 12, 24 and 48, P = 0.033, P = 0.018 and P = 0.031, respectively, Fig. 2b), which was absent in the control arm (Fig. 2a). A detailed analysis of the evolution of central, transitional and effector memory cells in the intensified group did not show a specific contribution of any of these subsets to the transient effect of raltegravir (data not shown). Furthermore, at the end of the study the CD4 compartment showed similar composition in both groups (Fig. 1b).
In contrast to these transient changes, the activation of CD4 T cells, as measured by the frequency of HLA-DR+CD95+ or CD45RA−CD38+ in CD4 T cells (Fig. 3a and b, respectively) and the frequency of CD38+ in the memory (CD4+CD45RA−) compartment (Fig. 3c), remained unchanged over the study in both arms, suggesting that raltegravir intensification was unable to modify the exacerbated activation of CD4 T cells in immunodiscordant patients. Consistently, the sensitivity to ex vivo total cell death (Fig. 3d) or intrinsic apoptosis (data not shown), relevant markers for immunodiscordant responses [3,6], were unaffected over the study period in the intensified group. No differences were observed between groups at any time point, confirming that raltegravir intensification did not modify the main markers of immunodiscordant responses.
Analysis of CD8 T-cell compartment
Raltegravir intensification had also modest effects on absolute CD8 T-cell counts over the study (Negredo & Massanella et al. submitted); however, previous intensification treatments with raltegravir have shown significant but slow decays in some markers of CD8 T-cell activation . Thus, we have evaluated these markers in late time points of our study. Unexpectedly, the frequency of CD45RO+HLA-DR+ CD8 T cells remained unchanged over the study in both groups (Fig. 4a), whereas the intensified group showed a significant reduction in the frequency of CD45RO+CD38+ and HLA-DR+CD38+ cells at 24 and 48 weeks (P < 0.05 in both cases, Fig. 4b and c, respectively). We also analyzed the expression of CD38 and HLA-DR markers in gated memory cells, which seems to be the most sensitive surrogate of CD8 T-cell activation . Once again, the expression of HLA-DR and CD38 showed a divergent behavior. Whereas HLA-DR levels remained unchanged in control arm and showed only transient effects in intensified arm at week 24 (Fig. 4d), CD38 expression in memory CD8 T cells showed evident significant reductions in intensified individuals at weeks 24–48, with only minimal transient changes in the control group (Fig. 4e). As a whole, these data confirm a particular effect of raltegravir intensification in CD8 T cell activation in immunodiscordant individuals.
Analysis of plasma levels of sCD14
T-cell hyperactivation in immunodiscordant individuals has been associated with increased levels of microbial translocation . Although the pivotal role of this phenomenon in immunodiscordance is controversial [3,7], we have monitored the level of sCD14 in plasma at baseline, week 24–48 of the study (Fig. 4f). Consistent with previous reports on raltegravir intensification , no changes in plasma sCD14 were observed in intensified individuals, which maintained baseline values throughout the study period, confirming that antiretroviral intensification has no detectable effects on intestinal permeability to microbial products.
Association of immunological changes with virological parameters
Immunological changes were analyzed according to virological variables. As the level of 2-LTR circles was only detectable in three intensified individuals, we have evaluated the potential association with the levels of plasma viral load as measured using ultrasensitive assays. Thus, in a post-hoc analysis, changes in CD8 T-cell activation and CD4 T-cell counts were compared in the intensified group between the 23 individuals showing undetectable viral load (<0.4 copies/ml) and the seven individuals with detectable viral load levels of viremia (median 0.96 copies/ml, see Table 1). The results showed similar responses in both subgroups; the levels of HLA-DR+ memory CD8 T cells remained constant in both subgroups (Fig. 4g), whereas significant reductions were observed in the frequency of CD38+ memory CD8 T cells (Fig. 4h). Similarly, no differences were observed in absolute CD4 T-cell increases among subgroups at weeks 12–48 (not shown and Fig. 4i). Thus, ultrasensitive viral load does not seem to predict immunological changes induced by intensification strategies in immunodiscordant individuals.
Raltegravir intensification in immunodiscordant patients has shown limited effects on CD4 T-cell recovery and virological parameters measuring long-term or short-term HIV DNA dynamics, total HIV DNA and 2-LTR circles in PBMC, respectively (Negredo & Massanella et al. unpublished observation). This observation is consistent with other intensification studies in immunodiscordant individuals reported by Hatano et al. and Byakwaga et al.. A comparison of these intensification trials shows that all of them have several limitations, a similar reduced size, and in our case, the lack a blinded control arm. However, our study shows a longer intensification period and has recruited individuals with the longest period of virological suppression [14,20]. Despite this, our data show a limited virological impact of raltegravir, although two immunological parameters seem to be specifically modified by intensification, namely the short-term dynamics of CD4 T-cell counts and the expression of CD38 in CD8 T cells. Interestingly these changes could not be associated with the level of baseline viremia (measured by ultrasensitive methods) or with the detection of 2-LTR episomal circles, which were identified only in three intensified individuals (Negredo & Massanella et al. unpublished observation). Thus, no virological tools seem to be available to predict immunological responses to intensification, at least in immunodiscordant individuals, due to the unexpectedly low levels of virological markers of HIV persistence in these patients.
From an immunological point of view, immunodiscordant individuals show two main features: a partial CD4 T-cell immunodeficiency and an immunosenescent highly activated CD8 T-cell compartment [3,4,6,7,21]. Raltegravir failed to impact the former, whereas had some beneficial effect on the latter. Thus, considering that the immunological effects of drug intensification are a direct consequence of its action on persistent residual replication, our data seem to rule out this factor as a major contributor to inadequate CD4 T-cell recovery. A similar conclusion has been reported in shorter intensification strategies [14,20].
Trends or significant short-term increases in CD4 T cells have been described in different raltegravir intensification or switch studies [13,22,23] (Massanella et al. unpublished observation). Although the switch to raltegravir seems to be associated with increased naive T-cell production , our data suggest that transient CD4 T-cell rise cannot be explained by major changes in the frequency of recent thymic emigrants (CD45RA+CD31+ CD4 T cells) nor to changes in the composition of the memory compartment, as the frequency of central, transitional and effector cells did not show significant changes. Therefore, main differences between the control and the intensified group seem to be associated with the absolute numbers of circulating memory cells, as observed in a previous raltegravir intensification study (Massanella et al. unpublished observation). The most plausible explanation for this observation is a redistribution of CD4 T cells from tissues, in which raltegravir may exert an anti-inflammatory effect through side inhibitory effects on other viruses  or by directly affecting residual HIV replication. The latter possibility is fully consistent with the reported link between markers of HIV replication and the effects of raltegravir intensification in memory CD8 T-cell activation [15,16].
Contrasting with previous data, immunodiscordant intensified individuals show a specific reduction in the level of CD38 expression in memory CD8 T cells but maintain the expression of HLA-DR. This observation further supports the different nature of both markers, whereas HLA-DR is a classical activation marker and probably reflects CD8 T-cell turnover, the combination CD38/HLA-DR seems to be uncoupled from CD8 T-cell proliferation . In fact, immunodiscordant patients show higher frequency of HLA-DR+CD38+ CD8 T cells than full immunological responders, although the levels of expression of the proliferation marker Ki67 are similar in both groups . Despite this fact, CD38 is known to be strongly associated to HIV infection  and progression to AIDS . The CD38 gene has an interferon (IFN)-responsive element in its promoter, and its expression is regulated by alpha IFN in vitro and in vivo, particularly in memory CD8 T cells . Thus, specific decrease of CD38 expression in these cells may indicate a reduction in Type I IFN responses. Interestingly, these responses are triggered by TLR7 innate sensing of HIV-infected cells  and are increased in rapid progressors . Consistently, the development of HLA-DR+CD38− CD8 T cells during HIV infection has been associated with subsequent stable CD4 T-cell levels and good prognosis .
Our data also confirm the slow decrease in CD8 T-cell activation in immunodiscordant individuals upon raltegravir intensification when compared with other strategies such as valganciclovir  or hydroxichoroquine , acting on CMV replication or as nonspecific immunomodulator, respectively, and showing faster actions on T-cell activation in immunodiscordant individuals. The different mechanisms of action of these drugs along with quantitative or phenotypic differences (lifespan, sensitivity to IFN signals or other) between CMV and HIV specific CD8 T cells may account for different kinetics of activation decay. CMV specific CD8 T cells may represent more than 10% of total cells and show a predominantly CCR7−CD27−CD28− phenotype during latent infection, whereas HIV-specific cells are lower in number and are usually positive for CD27 . Again, the wide CD38 expression in CD8 T cells from HIV-infected individuals hardly fits with a classical antigen driven proliferation, and CD38 expression might result from an inflammatory status leading to a general antigen-independent T-cell activation.
Irrespective of the mechanisms involved in the control of activation markers, it seems relevant to reduce the level of activation in HAART-treated individuals due to its strong correlation with markers of clinical risk [9,10]. In this regard, immunodiscordant individuals add to the increased CD8 T-cell activation a partial CD4 T-cell immunodeficiency that is clearly associated with activation and sensitivity to cell death of CD4 T cells [3,6], parameters that remain unchanged by raltegravir intensification of standard antiretroviral therapy. It is still unclear whether reduction in CD8 T-cell activation may have long-term benefits on HIV-associated comorbidities and CD4 T-cell recovery.
This study was supported by the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA’ (RD06/0006), an unrestricted grant from Merck Sharp & Dohme (MSD), ‘Gala contra la sida’ Barcelona 2011 and ‘Les Nostres Cançons contra la sida’ Barcelona 2012’. J Blanco is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government (Generalitat de Catalunya). M.M. and M.J.B. were supported by Agència de Gestió d’Ajuts Universitaris i de Recerca from Generalitat de Catalunya and European Social Fund. M.M. is appointed to the Pompeu Fabra University PhD program. J.C. is supported by a ‘Sara Borrell’ grant from the Spanish Health Institute ‘ISCIII’. We are grateful to all patients that have participated or have shown their support to this study.
We also thank all the investigators that have contributed to this work through the Discor-Ral Study Group:
Silvia Marfil, Elisabet García; Fundació irsiCaixa-HIVACAT, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain
Anna Bonjoch, José Moltó, Antoni Jou, Patricia Echeverría, Josep M. Llibre; Fundació Lluita contra la SIDA, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Badalona, Spain
Josué Pérez-Santiago; University of California, San Diego, California, USA.
Conflicts of interest
E.N. has received research funding, consultancy fees or lecture sponsorships from GlaxoSmithKline, ViiV, Merck and Roche. B.C. has served as a consultant on advisory boards or participated in speakers’ bureaus or conducted clinical trials with Boehringer-Ingelheim, GlaxoSmithKline, Gilead, Janssen, Merck, Pfizer and ViiV. J.M.-P. has received research funding, consultancy fees or lecture sponsorships from GlaxoSmithKline, Merck and Roche. J.B. has received research funding, consultancy fees or lecture sponsorships from GlaxoSmithKline, ViiV and Merck. All other authors declare no competing interests.
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