Discordant responses to HAART, consisting of either incomplete CD4 cell count rescue despite HIV viraemia control, or maintenance of high CD4 cell count in the presence of sustained viral replication, immunologic non-responders (INR) and virologic non-responders (VNR), respectively, have been described in up to 30% of patients [1–3]. The lack of a proper quantitative immune recovery in INR could also be associated with impaired immunologic function [4–6], pointing to relevant clinical implications on the best therapeutic approach. Indeed, INR may be more susceptible to opportunistic infections  and display an increased long-term risk of disease progression and death [7,8]. Although several processes have been individually cited as relevant in the pathogenesis of INR, a comprehensive model of the immune pathways and T-cell homeostatic balances in discordant responses is still missing [3,9–14]. Similarly, the actual mechanism(s) responsible for the progressive and relentless CD4 cell count decline in untreated HIV infection still remain a matter of controversy . Cases of maintenance of CD4 cell depletion despite viral suppression, and others of continuous CD4 cell count recovery in patients who remain viraemic, have somewhat discredited the classical view that CD4 cell loss reflects solely direct virus cytopathogenicity. Instead, a more complex model which also includes alterations in immune activation, T-cell turnover and homeostatic regulation, is now favoured. The identification of specific immunologic pathways involved in the lack of CD4 cell count rescue in INR as compared to full responders (FR) and VNR, could provide a more thorough comprehension of the immunopathogenesis of discordant responses, and possibly HIV disease, with potential implications for alternative treatment strategies.
In the light of these premises, we performed a cross-sectional study, aimed at an in-depth analysis of immune reconstitution in a group of INR versus FR and VNR. In particular, T-cell dynamics were investigated studying thymopoiesis, peripheral T-cell activation, proliferation and apoptosis and were analysed together with T-lymphocyte homeostasis parameters, i.e., immunophenotypic subsets and interleukin (IL)-7/IL-7 receptor system. Moreover, we investigated whether T-cell dynamics and immune reconstitution patterns in INR could be associated with increased reservoir of infected CD4 cells in peripheral blood, as measured by the frequency of CD4 cells carrying intracellular HIV DNA.
Patients and methods
Between July 1998 and December 2002 we performed a cross-sectional, observational, institutional review board-approved study of all the HIV-infected HAART-treated patients attending the Institute of Infectious and Tropical Diseases, Milan University. Of 1200 HAART-treated patients, we selected patients who started HAART with a CD4 count nadir < 100 cells/μl and who were on stable HAART for at least 24 months. Of 201 patients found, 64 fulfilled the following clinical features for at least 6 months in three consecutive determinations, and were divided into three groups: (i) FR, HIV RNA < 50 copies/ml and CD4 count ≥ 500/μl; (ii) INR, HIV RNA < 50 copies/ml and CD4 count ≤ 200 cells/μl; and (iii) VNR, HIV RNA ≥ 10 000 copies/ml and CD4 count ≥ 350 cells/μl. Exclusion criteria were initiation of therapy during primary HIV infection, concurrent use of immunomodulants, reported non-adherence to antiretroviral therapy.
Plasma HIV-RNA levels were quantified by a nucleic acid signal-amplification assay (bDNA; Chiron, Emeryville, California, USA) with a detection limit of 50 copies/ml.
Lymphocyte surface phenotypes were evaluated by flow-cytometry on fresh peripheral blood (Coulter ESP; Beckman Coulter, Hialeah, Florida, USA) using directly labelled antibodies [fluorescein isothiocyanate (FITC), phycoerytrin (PE), and phycoerytrin-cyanin 5 (PCy5)] (CD45RA-FITC, CD62L-PE, CD38-PE, CD4-FITC, CD8-FITC, Becton Dickinson, San Josè, California, USA; CD127-PE, CD4-PCy5, CD8-PCy5, Coulter, Florida, USA). The following combinations were used: CD4/CD8/CD127, CD45RA/CD62L/CD4, CD4/CD38, CD8/CD38.
Evaluation of spontaneous CD4 cell apoptosis
The percentage of apoptotic CD4 cells was measured on fresh patient peripheral blood mononuclear cells (PBMC) separated from blood plasma by Ficoll-Hypaque (Sigma-Aldrich, Milan, Italy) using flow cytometry Annexin V/7-aminoactinomycin D staining as described previously . Apoptotic cells were measured on live gated cells using morphological parameters (forward- and side-scatter).
Evaluation of thymic output
VαJα coding-joint T-cell receptor excision circles (TREC) were quantified with a previously described quantitative PCR-ELISA  in immunomagnetically purified CD4 and CD8 T cells (CD4 and CD8 Positive isolation Kit, Oxoid, Milan, Italy) (purity > 95%). Briefly, after Ficoll separation of PBMC, CD4 and CD8 cells were immediately immunomagnetically separated, pelleted, and frozen at −80°C. DNA was then extracted and soon after the TREC assay was performed. TREC frequency was measured as copies/μg. We also calculated total TREC content, i.e., TREC/μl blood: (TREC/CD4) × (absolute number of CD4 cells), assuming that 1 μg genomic DNA equals approximately 150 000 cells.
Ki67 expression analysis
T-cell proliferation was evaluated by flow cytometry Ki67 expression. Briefly, 1 × 106 Ficoll-separated PBMC were stained with PE-conjugated anti-CD4 or anti-CD8, and fixed in 300 μl 2% paraformaldehyde. The cells were resuspended in 50 μl phosphate-buffered saline and 15 μl Nonidet 1.5% and stained with 10 μl anti-Ki67 FITC (clone MIB-1, Immunotech, Westbrook, Maine, USA). Ki67 antigen expression was measured on live gated cells by means of flow cytometry.
Plasma IL-7 measurement
Plasma IL-7 was measured using an ELISA kit (Quantikine HS human IL-7; R&D, Minneapolis, Minnesota, USA) following the manufacturers' instructions.
Memory and naive CD4 purification
After PBMC separation by Ficoll, and immunomagnetic CD4 separation, CD4 cells were directly labelled with 20 μl/1 × 106 cells each of FITC-conjugated anti-CD45RA (Becton Dickinson) and PE-conjugated anti-CD45R0 (Becton Dickinson) for 30 s at 4°C, and sorted using an EPICS Elite ESP cell sorter (Coulter; Beckman Coulter) (purity > 95%). For each patient, four to six separate aliquots of sorted naive and memory CD4 cells were obtained, pelleted and frozen at −80°C until use.
Analysis of intracellular HIV-DNA content
Analysis of intracellular HIV-DNA content was performed on purified memory and naive CD4 cells. DNA was extracted from sorted memory and naive CD4 cells as described previously . DNA was separately isolated from each aliquot of naive and memory CD4 cells, and individually analysed by different PCR, with concordant results.
Intracellular HIV DNA was amplified with specific primers selected within the highly conserved p24 HIV gag gene region (A. Casabianca, unpublished data). The cloned 303-bp gag gene PCR product in the pGEM® vector (Promega Corporation, Madison, Wisconsin, USA) generated a pGAG plasmid, used to obtain a standard curve. Appropriate serial dilutions of the pGAG plasmid in 10 μl noninfected DNA were amplified in parallel with the unknown samples. In the same sample a similar assay has been developed to quantify the 18S rRNA gene to normalize sample-to-sample variations values of intracellular DNA . For 18S rRNA amplification, twofold dilutions for each DNA sample were tested to ensure the absence of inhibitor(s) during amplification and the mean of the results was used for data analysis. Both PCR for gag gene and 18S rRNA gene were performed in a 50 μl final volume. Ten microlitre DNA samples were added to 40 μl containing 1 × PCR reaction buffer (67 mM Tris/HCl pH 8.8, 16.6 mM (NH4)2SO4, 0.01% Tween 20), 3 mM MgCl2, 200 μM (each) dNTP, 400 nM each primer, bovine serum albumin 200 ng/μl, and 1.5 U of Hot-Rescue DNA Polymerase (DIATHEVA s.r.l, Fano, Italy). The following conditions were used: after one cycle at 95°C for 10 min, a three-step PCR procedure was used consisting of 15 s at 95°C, 15 s at 63°C and 30 s at 72°C for 60 cycles. Finally, 30 μl of the amplified product were resolved by electrophoresis on 2.5% agarose gel staining with ethidium bromide at 110 V for 1 h. The signal intensity associated with DNA bands was determined by fluorescence, using the Gel Doc™ 1000 Video Gel Documentation System with Molecular Analyst® software Version 2.1 (Bio-Rad, Hercules, California, USA).
For all samples, quantitative results were calculated by a linear regression analysis of the plot between the log of fluorescence and the log of the starting plasmid molecules . Intracellular HIV DNA values were normalized with 18S rRNA, and expressed per 1 × 104 cells.
Medians and interquartile ranges were used to describe continuous variables. Kruskall-Wallis/Anova non-parametric test were performed for continuous variables, χ2 test for categorical variables. Statistics were performed using EPI6 software and SPSS software.
Sixty-four HIV-positive patients were recruited: 27 INR, 15 VNR, 22 FR. No significant differences were observed among groups in demographic, clinical and HIV-related parameters (Table 1). As defined by the inclusion criteria, at time the of analysis CD4 cell counts differed significantly among groups, with INR displaying the lowest median CD4 cell counts, and FR the highest (Table 1; P < 0.01 in each pair-wise comparison). No significant differences among groups were observed in both highest pre-HAART HIV RNA and at HAART initiation (Table 1). Furthermore, there was no significant difference in the median time of plasma HIV-RNA reduction to below 50 copies/ml or in the median viral load reduction following HAART initiation in INR versus FR (P > 0.05). Conversely, VNR displayed significantly lower median viraemia decrease as compared to the other groups (P < 0.01 for each pair-wise comparison) (Table 1). At the time of study, HIV virus load was significantly higher in VNR as compared to the other groups (P < 0.01 in each pair-wise comparison), whereas no significant differences were observed between INR and FR (P > 0.05; Table 1). No significant differences were observed among groups in terms of drug regimen at HAART initiation, as more than 90% of the patients (58/64) started HAART with a protease inhibitor (PI)-based regimen (Table 1). However, at time of analysis INR were more likely to be on a PI-containing treatment as compared to FR who had been mainly switched to a non-nucleoside reverse transcriptase inhibitor (NNRTI) regimen, due to treatment simplification (P < 0.01) (Table 1).
Peripheral CD4 and CD8 cell activation, turnover and apoptosis
Immune activation was measured by circulating CD38CD4 and CD8 (Fig. 1a and b). Both FR and INR displayed a trend toward lower CD4CD38 versus VNR, albeit not reaching statistical significance (INR, 11.7%; FR, 12.8%; VNR, 15.8%; P > 0.05 in each pair-wise comparison, P = 0.06 for FR versus VNR) (Fig. 1a). A similar trend was observed in CD8+ in FRs, showing lowest CD8+CD38+ percentages (Fig. 1b). Surprisingly, INR displayed median CD8CD38 comparable to VNR, and significantly higher than FR (FR, 8.4%; INR, 23.5%; VNR, 24.7%; P < 0.05 for FR versus INR and VNR; P > 0.05 for INR versus VNR) (Fig. 1b). Our results show that, despite complete viral suppression, INR still maintain a significant immune T-cell hyper activation selectively within CD8, comparable to VNR.
T-cell proliferation was defined by the percentage of Ki67-expressing CD4 and CD8 cells [21–23] (Fig. 1c and d). INR displayed the highest median Ki67CD4 (INR, 2.0%; FR, 0.4; VNR, 0.3%; P < 0.05 for INR versus each other group), whereas FR and VNR displayed a similar CD4 cell proliferation (P > 0.05) (Fig. 1c). A similar yet not statistically significant pattern was seen in Ki67CD8 (INR, 1.0%; FR, 0.8%; VNR, 0.4%; P > 0.05 for each pair-wise comparison) (Fig. 1d).
To evaluate whether the increased T-cell activation and proliferation in INR were linked to heightened spontaneous cell death, ex vivo CD4 cell apoptosis was measured. INR displayed significantly higher median apoptotic CD4 cell percentages versus FR (INR, 5.9%; FR, 2.9%; VNR, 4.5%; P < 0.05 for INR versus FR; P > 0.05 for INR versus VNR), with no significant differences between FR and VNR (P > 0.05) (Fig. 1e).
Altogether, our data demonstrate a differential T-cell activation and proliferation in discordant patients. In particular, INR maintain a highly activated and proliferating peripheral T-cell pattern, which in turn may continue to drive CD4 cell apoptosis.
T-cell subsets analysis, thymic output, IL-7/IL-7 receptor system
We comparatively measured circulating memory-(CD45RA−) and naive-(CD45RA+CD62L+) CD4 and CD8 cells (Fig. 2a–d). INR displayed the highest median memory CD4 cell proportions, followed by VNR, and then FR (INR, 67.1%; VNR, 41.3%; FR, 23.0%; P < 0.01 in each pair-wise comparison) (Fig. 2a). A similar outgrowth of memory T cells in INR was observed in CD8 (INR, 66.0%; VNR, 48.3%; FR, 43.4%; P < 0.05 for INR versus FR; P > 0.05 for the other pair-wise comparisons) (Fig. 2b). Conversely, INR presented the lowest naive CD4 cell percentages, with no significant differences between FR and VNR (INR, 19.4%; VNR, 30.0%; FR, 37.9%; P < 0.05 for INR versus each of the other groups; P > 0.05 for FR versus VNR) (Fig. 2c), and a similar trend in CD8 cells (INR, 11.3%; VNR, 16.6%; FR, 30.7%; P < 0.05 for FR versus each of the other groups; P > 0.05 for INR versus VNR) (Fig. 2d).
Having shown a predominant memory/activated/apoptotic T-cell pattern in INR, we investigated the contribution of thymic output by comparing CD4 and CD8 TREC (Fig. 3a and b). INR displayed median TREC frequency comparable to FR within CD4 (INR, 11 777; FR, 5661 copies/μg) and CD8 cells (INR, 3696; FR, 3324 copies/μg) (P > 0.05). Although both INR and FR showed a tendency toward lower CD4 and CD8 TREC versus VNR, this did not reach statistical significance (VNR: CD4, 43 954 copies/μg, P = 0.140 for INR versus VNR; P = 0.07 for FR versus VNR. CD8: 37 537 copies/μg, P = 0.06 for INR versus VNR; P = 0.122 for FR versus VNR). When adjusted for total T-cell numbers, total TREC content/μl blood in INR appeared slightly and yet not significantly reduced as compared to FR in CD4 (INR, 11 copies/μl versus FR, 24 copies/μl; P > 0.05) and CD8 cells (INR, 18 copies/μl cells versus FR, 7 copies/μl; P > 0.05) (Fig. 3a and b). Total TREC content analysis also confirmed a clear trend toward highest levels in VNR versus each of the other group, now reaching significance in comparison to INR (CD4, 477 copies/μl; CD8, 299 copies/μl; P > 0.05 for VNR versus FR; P < 0.01 for VNR versus INR) (Fig. 3a and b).
To investigate specific alterations in T-cell homeostasis regulation, we analysed the IL-7/IL-7 receptor system (Fig. 3c–e). INR displayed the highest levels of free IL-7, with no significant differences between the other groups (INR, 8.8 pg/ml; FR, 3.2 pg/ml; VNR, 3.7 pg/ml; P < 0.05 for INR versus FR and VNR; P > 0.05 for FR versus VNR) (Fig. 3c), suggesting maintenance of the IL-7 compensatory feedback.
No significant differences were observed in CD4 and CD8CD127 fractions (CD4: INR, 60.7%; FR, 78.5%; VNR, 74.5%. CD8: INR, 47.8%; FR, 47.7%; VNR, 67.4%; P > 0.05 for each pair-wise comparison) (Fig. 3d and e). Interestingly, VNR displayed a consistent albeit non-significant trend toward highest levels of CD127CD8 (Fig. 3d and e), yet significantly lower than that of age-matched HIV-negative individuals (CD4CD127, 91.9%; CD8CD127, 86.1; P < 0.01 and < 0.05, respectively versus VNR; P < 0.01 versus each of the other groups), suggesting that the increased CD127CD8 in VNR may be interpreted as a tendency toward normalization.
Quantification of intracellular HIV-DNA in memory and naive CD4 cells
In a subgroup of six unselected INR, six VNR, and six FR, we investigated the possible association between immune reconstitution patterns and CD4 cell HIV reservoir through the comparative quantification of the frequency of total, memory and naive CD4 cells carrying intracellular HIV-DNA.
Compared to VNR, FR presented a trend toward lowest median CD4 cell HIV DNA, within both memory (FR, 90 copies/104 cells; VNR, 279 copies/104 cells) and naive CD4 cells (FR, 35 copies/104 cells; VNR, 128 copies/104 cells; P > 0.05 in each pair-wise comparison) (Fig. 4). Despite full HIV viraemia suppression, INR displayed a consistent trend toward highest median intracellular HIV DNA levels in total CD4 and, most importantly, within both memory and naive cells (441 copies/104 and 161 copies/104 cells, respectively) versus either FR or VNR, (P > 0.05 in each pair-wise comparison; P = 0.09 for INR versus FR) (Fig. 4). Despite the low number of patients, these data suggest an enhanced rate of HIV infection within the whole CD4 T-lymphocyte compartment in INR. Furthermore, HIV DNA content showed a constant trend toward higher median levels in memory versus naive CD4 cells, reaching statistical significance in INR (P < 0.05 for INR, P > 0.05 for VNR and FR) (Fig. 4), suggesting that the reconstituting peripheral T-cell pool may still display higher infectability by HIV of activated/memory versus naive CD4 cells.
Our study delineates specific T-cell homeostatic balances between antigen-driven activation, proliferation, cell death and thymopoiesis in HIV-infected patients with discordant immune-virological responses to HAART. Furthermore, by showing diverse HIV DNA levels in the different CD4 subpopulations according to viro-immunological recovery, our data suggest that HIV intracellular DNA content might indeed be involved in driving each specific T-lymphocyte immune phenotype.
While FR could be regarded as the prototype of broadest HAART-driven immune recovery, with contained T-cell activation and ‘preserved’ thymopoiesis, INR – despite full viral suppression–displayed heightened T-cell activation, specifically within CD8 cells, comparable to VNR and significantly higher than FR. This confirms the hypothesis that antigen-driven immune activation could significantly hinder CD4 cell count recovery. Moreover, our data indicate that CD4 and CD8 immune recovery is differentially regulated in discordant patients, with a selective outgrowth of activated CD8 cells, due to several possible mechanisms, including the strictest dependence of CD8 cells on antigen-driven immune activation [24–27], and higher rate of HIV infection and death in activated CD4 cells, rendering CD8 cell activation a more accurate marker of HIV-driven T-cell stimulation [13,24,28,29] and disease progression [30–32].
Concordantly, INR displayed the highest peripheral T-cell proliferation, further indicating the strict correlation with T-cell hyperactivation . Attempting to define the actual impact of increased activation and proliferation in peripheral T-cell homeostasis, we found a predominant memory pattern in INR, with lowest naive cells, and highest CD4 cell apoptosis, thus stressing the pathogenetic role of enhanced T-cell activation and proliferation in CD4 T-lymphopenia. The persistent activation and differentiation of naive T cells seems to be the ultimate responsible for the erosion of the naive pool in favour of a highly activated, short-lived memory pattern. To weigh peripheral T-cell dynamics against thymic-dependant pathways in the reconstituting T-lymphocyte immunophenotype, thymopoiesis was measured. Despite the predominant memory phenotype, total TREC content in INR, though more contained, was not substantially lower than in FR, supporting the hypothesis that thymic impairment could also be involved in the mechanisms underlying the lack of CD4 cell reconstitution [12,33]. Furthermore, our data also show highest plasma IL-7 in INR, consistent with the persistence of T-lymphopenia-driven compensatory loop [34,35], possibly implicated both in the tentative support of thymic output and in driving peripheral T-cell turnover. We cannot exclude, however, that high levels of IL-7 may merely reflect less consumption due to highly depleted T-cell pool rather than a direct consequence of increased production.
Aiming to further investigate the pathogenesis of different immune profiles in INR versus FR and VNR, we hypothesized the possible interference of intracellular reservoirs and residual viraemia, as reflected by intracellular HIV DNA content. Quite interestingly, a tendency toward heightened intracellular DNA in CD4 cells was shown in INR as compared with FR. This finding strongly supports the hypothesis of the persistence in INR of ongoing residual low-level viraemia, not detectable by standard assays, and probably associated with very little virus production either in peripheral blood, lymphoid organs or HIV reservoirs [3,10,11,36]. Although the small number of the patients assessed allowed for only descriptive considerations, limiting their statistical strength, our data show a parallel trend between the hyper-activated/hyper-proliferating T-cell pool in INR and the size of CD4 cell HIV reservoir. Furthermore, even though purified CD4CD45RA cells are likely to contain CD4CD45RACD27- effector cells, a proportion of these cells are truly naive, thus suggesting an altered viral burden within both memory and naive CD4 cell pool [37,38]. Combined, the expanded reservoir of infected CD4 cells, enhanced T-cell activation, and consistent naive T-cell depletion, on the one hand suggest that residual viraemia may be implied in the substantial increase in the rate of CD4 cell HIV infection, and further reinforce the role of increased antigen-driven T-cell activation and turnover as the ultimate driving force of continuous CD4 loss in INR .
By drawing specific immunopathogenetic models, our study may substantially contribute to a more rational clinical management of discordant patients with important therapeutic considerations, that include HAART intensification or modification [39,40], or immunoadjuvant approaches, the actual clinical efficacy of which still remains to be evaluated for future clinical trials [41–43].
The key role of T-cell activation in CD4 cell gain or loss, and the possible major contribution of an increased reservoir of HIV infected cells in dictating long-term immune recovery, would seem to strongly indicate the need for exhaustive investigations as to the actual viro-immunological equilibrium of discordant patients, in order to serve as possible guidance in therapeutic indications such as drug interruptions, changes or immune-based interventions.
We thank Alan Michael Rosen Camilla Tincati and Mark E. Jones for a critical reading of the manuscript and valuable grammatical advice, and Fulvio Adorni (CNR-ITBA, Milan, Italy) for assistance with statistical analysis. We thank Andrea Cossarizza for helpful discussion and suggestions. We are also grateful to Daniela Angioni for her continuous help with patients' records overview, and to Bianca Ghisi for excellent typing assistance; we particularly thank all the patients participating in the study, and the staff of the Institute of Infectious Diseases and Tropical Medicine, ‘Luigi Sacco’ Hospital who cared for the patients.
Sponsorship: Supported by grants from Istituto Superiore di Sanità, Italy ‘National research program on AIDS’ (50D.06; 30D.34; 30D.39), from the ‘Ermenegildo Zegna’ Foundation, from the AHSI Company, and from Progetto AIDS e Ricerca Corrente of the Ministry of Health, Italy.
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Study Group for Immunologic-non-Responders
Chiara Molteni, Greta Taskaris, Mauro Moroni, Ada Bertoli, Caterina Gori.