Successful control of HIV-1 replication following the initiation of highly active antiretroviral therapy (HAART) has drawn attention to the real extent of the improvement of immune integrity that may be achieved in treated patients. Recovery of peripheral blood CD4 T-cell numbers and function above threshold levels in patients on HAART has been shown recently to provide an accurate estimate of immune reconstitution allowing interruption of primary prophylaxis for several opportunistic pathogens, including Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium-intracellulare complex and cytomegalovirus (see [1,2] for a review). HAART is generally associated with a decrease in viral replication to undetectable levels, an increase in both memory and naive CD4 lymphocytes [3,4] and a variable recovery of T-cell responses to a number of recall antigens [5–8]. However, recovery of T-cell responses to HIV-1 antigens in vitro are not achieved , unless patients are treated during acute infection .
During HIV-1 infection, an increase in total, activated (HLA-DR, CD38) and proliferating (Ki67) CD8 T cells is commonly detected [3,4], with up to a sevenfold enhancement of their daily production [3,4,9]. This enhancement of T-cell proliferation is, however, paralleled by a progressive decrease in HIV-1-specific cytotoxic T lymphocyte (CTL) activity during the course of disease progression in untreated patients .
After initiation of HAART, a progressive decrease in total CD8 T cells towards normal levels is observed, with concomitant decrease of their activation state as determined by expression of HLA-DR and CD38 [3,4,11] and repertoire stabilization with reduced CD8 T-cell oligoclonality . However, even after 12 months of HAART in patients with primary HIV-1 infection and after 24 months of HAART in chronically infected patients, a substantial fraction of CD8 T cells with an activated phenotype (HLA-DR,CD38) still persists. Moreover, the mean percentage and total CD8 T-cell counts in lymph nodes and peripheral blood remain twofold increased [3,4].
Virus replication resumes early after interruption of HAART even after several years of apparently successful treatment [13,14], and after adjunctive immunotherapy with recombinant interleukin-2 that may lower significantly the burden of latently infected CD4 T cells [15,16]. In this regard, low-level residual HIV-1 replication persists in lymphoid tissue of successfully treated patients with undetectable plasma viraemia [17,18], virus being produced by residual cells at an unchanged rate .
These studies suggest that, although a satisfactory degree of CD4 immune reconstitution after several months from initiation of HAART may allow safe interruption of primary prophylaxis for opportunistic infections, an incomplete recovery of CD8 functions persists and may prevent clearance of small numbers of infected CD4 cells that still harbour actively replicating virus.
Mechanisms that underlie the inability of cytolytic effectors – both CD8 CTL and natural killer (NK) cells – to clear cells displaying residual virus replication are still poorly understood. They may include decreased antigen-driven T-cell stimulation [8,20,21], reduced expression of CD3zeta or CD28 [22,23] and functional inhibition of HIV-1-specific CTL activity as a consequence of an increased expression of HLA class I-specific inhibitory NK receptors (iNKR) [24,25]. In this context, we showed previously that HIV-specific CTL isolated from infected patients were inhibited in their cytolytic activity against HIV-1-expressing autologous target cells as a consequence of the surface expression of iNKR . This finding strongly suggested an involvement of iNKR in the down-regulation of HIV-specific CTL activity. This appears even more likely because of the frequent coexpression of multiple iNKR on CD8 T-cell clones derived from infected patients .
In the present report we analysed the expression of six HLA class-I-specific iNKR (p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2) on CD3 peripheral blood mononuclear cells (PBMC) of patients treated with HAART who experienced a persistent complete control of virus replication below the level of detection for at least 2 years, to determine the possible decrease in iNKR expression on peripheral blood CD8 T cells.
Patients and methods
Patients and cell samples
Participants included eight patients with advanced HIV-1 infection (CD4 cells < 120 × 106/l and previous virological failure to antiretroviral agents) who were enrolled at the Department of Infectious Diseases, University of Milan in a protocol of salvage HAART including a protease inhibitor and two nucleoside reverse transcriptase inhibitors. Peripheral venous blood was collected before HAART was commenced and then after 3, 6, and 24 months of treatment.
Control samples were obtained from a group of 30 healthy adult volunteer blood donors from the local blood bank and from healthy laboratory workers.
In addition, PBMC were obtained from surplus peripheral blood at a scheduled follow-up visit from eight age/sex-matched patients with chronic asymptomatic hepatitis C virus (HCV) infection. These patients had negative HIV-1 serology and HCV plasma viraemia, normal or moderately increased (< 3 × normal limits) serum transaminase concentrations, absence of cryoglobulinaemia and no history of treatment for HCV during the previous 6 months.
Informed consent was obtained from patients, and human experimentation guidelines of the authors’ Institutions were followed in the conduct of clinical research.
PBMC were obtained from blood by density gradient centrifugation (Ficoll-Hypaque) and stored at −80°C until assayed. Cryopreserved cells were thawed immediately before analysis and controlled for cell viability by Trypan blue exclusion. Cell viability was ≥ 80% in all cases, with the exception of one sample from one HIV-1-infected patient who after 24 months of HAART had < 40% viable cells and was therefore not investigated further.
Antibodies and reagents
Monoclonal antibodies (MAb) specific for CD3 (JT3a), CD8 (B9.4), p58.1 (EB6), p58.2 (GL183), p70 (Z27), p140 (Q66), CD94 (XA185), NKG2A (Z270) and LIR1/ILT2 (F278) were used in this study [27–29]. Fluorescein isothyocyanate- and phycoerythrin-conjugated anti-isotype goat anti-mouse antibodies were purchased from Southern Biotechnology (Birmingham, Alabama, USA). The culture medium was RPMI 1640 (Bio-Whittaker, Europe, Verviers, Belgium) supplemented with 10% foetal calf serum (Boehringer, Mannheim, Germany), L-glutamine (2 mM/l) and 1% antibiotic mixture (penicillin 5 mg/ml, streptomycin 5 mg/ml, neomycin 10 mg/ml stock solution).
The reactivity of MAb with PBMC populations was assessed by indirect immunofluorescence and flow cytometry. Briefly, 1 × 105 cells were stained with the corresponding MAb, followed by an appropriate fluorescein isothyocyanate- or phycoerythrin-conjugated anti-isotype-specific goat anti-mouse antiserum (Southern Biotechnology, Birmingham, AL, USA) as second-step reagents. Negative control reagents were mouse MAb directed against irrelevant surface molecules. All samples were analysed on a flow cytofluorometer (FACScan, Beckton Dickinson, Mountainview, CA, USA). Results are expressed as logarithm of green fluorescence intensity (arbitrary units) versus logarithm of red fluorescence intensity (arbitrary units). For each analysis 10 000 events were counted. The proportion of CD3 cells staining with the MAb specific for each iNKR were expressed as percentage of the total CD3 PBMC in all instances. The proportion of CD8 PBMC displaying high-level fluorescence intensity (CD8-`bright') coexpressing all iNKR was also analysed and expressed as percentage of the total CD8-`bright’ PBMC. In all cases CD8-`bright', iNKR-positive PBMC represented ≥ 95% of the CD3 iNKR-positive cells.
Analysis of HIV-1 specific CTL activity in PBMC
To detect HIV-1 specific CTL activity, autologous Epstein–Barr virus transformed B-lymphoblastoid cell lines (B-LCL) were infected with recombinant vaccinia viruses expressing HIV-1 Env (vPE16), HIV-1 Gag and HIV-1 Pol (vVK2), and HIV-1 Nef (vTnef) (gift of P. Earl, Bethesda, Maryland, USA) and used as targets in triplicate wells in a short-term (4 h) 51Cr-release assay, as described previously . Autologous B-LCL infected with a control vaccinia virus (vSC8) expressing an irrelevant protein (lacZ) were used as a negative control. Briefly, freshly drawn PBMC were separated and plated in sterile 96-well V-bottom plates at different effector : target ratios. Autologous 51Cr-labelled B-LCL expressing appropriate antigens were used as targets. To analyse the effect of the disruption of LIR1/ILT2-HLA class I interaction, saturating amounts (10 μg/ml) of the F278 MAb was added to wells containing the appropriate dilution of PBMC effectors before the addition of labelled target cells. HIV-1-specific lytic units, expressed as number of units per 1 × 106 PBMC, were computed as described elsewhere .
To compare the results obtained for iNKR expression on CD3 lymphocytes, the Mann–Whitney U test for unpaired datasets was used. Results are expressed as box-plots showing median values and interquartile ranges to represent graphically the proportion of CD3 cells expressing any given iNKR in all the patient groups and in healthy controls. Speraman's rank correlation test for non-parametric data was used to analyse the relationship between the proportion of any given iNKR and total CD4 T-cells. Statistical analysis was performed using the StatView 4.2 program (Abacus Concepts, Berkeley, California, USA).
Patients, viral burden and CD4 T lymphocytes
Cryopreserved PBMC samples at baseline and then after 3, 6 and 24 months of HAART with two nucleoside reverse transcriptase inhibitors and one protease inhibitor were available for eight HIV-1-infected patients. The patients had virological failure to previous antiretroviral treatment and at the beginning of HAART had advanced immunodeficiency with mean CD4 T-cell counts 31 × 106/l (range, 3–115 × 106/l). A rapid decrease in viral load within the first 3 months and a sustained virological response throughout the observation period with undetectable plasma viraemia after 2 years were observed after initiation of HAART. After 6 months on HAART, all patients had undetectable viral load (< 200 copies/ml) with a mean increase in CD4 T-cell count of 123.9 × 106/l (SD ± 79.1 × 106/l; range, 62–305 × 106/l). Table 1 shows the demographic and treatment characteristics of the cohort, and the time-course of CD4 T-cell numbers and of plasma viraemia over the 24-month observation period.
Increased proportions of different iNKR on CD3CD8 lymphocytes in untreated HIV-infected patients and healthy controls
PBMC samples were obtained from a panel of 30 healthy adult blood donors from the local blood bank and from healthy laboratory workers. In most instances, healthy donors displayed only low levels of expression of iNKR on their CD8 peripheral blood T cells, with median expression ≤ 2% of CD3 PBMC for p58.1, p58.2, p70 and p140, < 4% for CD94/NKG2A and < 11% for LIR1/ILT2.
Analysis of CD3 PBMC in HIV-1-infected patients revealed a noticeable increase of cells expressing iNKR which reached statistical significance (Fig. 1). Moreover, most iNKR analysed were increased simultaneously in all patients in contrast with healthy donors (data not shown).
Coinfection with HCV was present in four out of the eight HIV-1-infected patients who underwent HAART in the present study. To define further whether and to what extent chronic HCV coinfection with persistent replication could contribute to the de novo expression of iNKR in vivo or to their delayed decrease during HAART, we analysed the proportion of CD3 PBMC expressing iNKR in eight HIV-1-seronegative patients with chronic HCV infection. Median expression of p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2 in these patients did not differ significantly from the healthy uninfected donor group, and was correspondingly significantly lower than in HIV-1-infected patients (data not shown). Thus, chronic HCV infection/replication alone does not result in a significant generalized up-regulation of iNKR expression on peripheral blood CD8 T cells in vivo and could reasonably be ruled out as a possible confounding parameter responsible for the observed increase in the expression of all iNKR molecules on CD8 T lymphocytes of the HIV-1-infected patients before HAART.
Restoration of normal levels of iNKR expression in T lymphocytes occurs after 24 months of sustained viral suppression on HAART
We next determined whether prolonged successful HAART could restore the proportions of iNKR-expressing CD3 T cells to normal values. The proportion of CD3 cells expressing each of the six iNKR monitored (i.e., p58.1, p58.2, p70, p140, CD94, LIR1/ILT2) was evaluated in all patients at baseline, after 3 months, 6 months and 24 months of treatment and are shown in Fig. 2. After 6 months of successful reduction of HIV-1 viral load which was accompanied by recovery of CD4 T-cell numbers, no noticeable decreases of iNKR expression toward normal percentages was detected and differences were still significant when compared with healthy controls (Fig. 2). Only after 24 months was a significant decrease of circulating CD3, iNKR-positive lymphocytes detectable for p58.1, p58.2, p70, p140 and CD94/NKG2A; mean values were similar to those of healthy controls (Fig. 2). Two-colour cytofluorometric analysis of iNKR expression on CD3 PBMC in a representative patient at baseline and after 24 months of HAART, as compared with a healthy control donor, is shown in Fig. 3a.
Overall, the expression of iNKR, as determined using double-immunofluorescence labelling with anti-CD3 or anti-CD8 MAb and a mixture of all the available MAb specific for the six different iNKR confirmed, in all patients, that iNKR were expressed predominantly (≥ 95%) on CD8 PBMC. We could not detect increased or significant iNKR expression on CD3CD4 PBMC in our patients (data not shown). This is in line with previous observations that report virtually no expression on CD4 T lymphocytes [24,31]. In addition, in selected patients up to 87% of the CD3CD8 PBMC (defined as CD8 ‘bright') expressed one or more iNKR in vivo before HAART (mean, 45.9%). The quantification of iNKR expression on CD3 T cells and on CD8 ‘bright’ T cells is shown in a representative patient in Fig. 3b, using a mixture of the individual MAb specific for the six iNKR. As shown, the proportion of iNKR-positive CD8 ‘bright’ cells is high before HAART, while less than one-third of the CD8 ‘bright’ PBMC still express iNKR after 24 months of HAART.
To exclude the possibility that the decrease in iNKR expression during HAART is a general consequence of the decrease in CD8 T cells, we further looked for a correlation between the proportion of CD8 iNKR-positive PBMC and the proportion of CD8 T cells in the cohort. No significant correlation could be found. In addition, the decrease in the median expression of iNKR on CD3 PBMC after 2 years of HAART was 48.2% (from 51% to 26.4% of CD3 T cells), while the proportion of CD8 T cells decreased only by 12% (from 58% to 51% of PBMC). Thus, it would seem unikely that the increase in CD3 CD8 iNKR-positive cells is the consequence of a general increase (or decrease on HAART) in CD8 T cells.
When further analysing the correlation between absolute CD4 counts and the proportion of iNKR expressed on CD3 CTL before and during HAART, a significant negative association was observed for p70 (P = 0.0015) and for p140 (P = 0.03), while this was not the case for the other iNKR. This observation suggests a staged, earlier recovery of CD3 CTL expressing these HLA B- and HLA A-specific iNKR, as compared with the other iNKR.
This pattern of recovery of normal populations of iNKR-positive CD8 T cells in HIV-1 patients on HAART regimens, is concordant with the current knowledge on CD8 PBMC activation and decrease of proliferation markers, including CD38, HLA-DR and Ki67 [7,11], during antiretroviral therapy and clearly lags behind the recovery of CD4 T cells and the decrease in plasma viraemia in these patients (Table 1).
Functional analysis of HIV-1-specific CTL activity at 24 months
The decrease in the proportion of CD3CD8 PBMC expressing LIR1/ILT2 followed the same co-ordinated pattern as far as time-course and range variability is concerned. Surprisingly, however, a slightly but significantly increased mean expression persisted in HIV-1-infected patients with undetectable viraemia even after 2 years of HAART (Figs 2 and 3). In fact, after 2 years of HAART, seven out of eight patients still had > 18% of CD3PBMC expressing LIR1/ILT2. We therefore tried to verify further whether the finding of persistently increased mean expression of LIR1/ILT2 in CD3CD8 PBMC, as compared with healthy controls, could represent a functional correlate of decreased HIV-1-specific CTL activity, at least in selected patients. To this purpose, PBMC freshly isolated from a patient after 24 months of HAART were assayed against autologous B-LCL infected with recombinant vaccinia virus expressing the HIV-1 Env, Gag, Pol, and Nef antigens. The specific cytolytic assay was performed either in the presence or in the absence of anti-LIR1/ILT2 MAb to disrupt the functional interaction between this HLA class I-specific iNKR and HLA class I molecules on the target cells. At the time of the analysis the patient had suppressed viraemia (< 80 copies/ml) and 31% of his CD3 PBMC expressed LIR1/ILT2 receptors. Only low-level HIV-1-specific CTL activity (< 10% at 200 : 1 effector : target ratio) could be detected in PBMC. Addition of LIR1/ILT2-specific MAb resulted in a substantial increase of HIV-1-specific lysis (Fig. 4). This increase in CTL activity corresponds to a threefold increase in HIV-1-specific lytic units, from 2.34/1 × 106 PBMC to 6.79/1 × 106 PBMC. Similar results were obtained in another patient with > 18% LIR1/ILT2CD3 PBMC, while this was not observed in the only patient with < 18% LIR1/ILT2CD3 PBMC (data not shown).
Recent advances in antiretroviral therapy have proven to be very effective in reducing viral load and in increasing CD4 T-cell numbers in HIV-1-infected patients. Complete control of viral replication is, however, not achieved during successful HAART [17–19].
Actively replicating viruses have evolved multiple strategies to evade the immune response [32,33] including establishment of viral latency, inhibition of viral antigen processing and/or presentation, antigenic shift and drift with development of mutants and clonal exhaustion/deletion of the initially expanded virus-specific CD8 CTL. In the presence of HAART and therefore of a highly decreased rate of replication, however, other mechanisms may be involved in the failure of cytolytic effector cells (i.e., antigen-specific CD8 CTL and NK cells) to clear residual HIV-1 replication [20,23,25]. Among these mechanisms, the expression of iNKR by HIV-1-specific CTL may impair substantially their ability to lyse HIV-1-infected target cells .
In the present study we have characterized the pattern of expression and time course of the main inhibitory NK receptors belonging to both the immunoglobulin (KIR) superfamily (i.e., p58.1, p58.2, p70, p140, LIT1/ILT2) or to the C-type lectin family (i.e., CD94/NKG2A) on peripheral blood CD8 T lymphocytes in a cohort of HIV-1-infected patients who underwent a successful HAART regimen for 24 months.
Evidence is provided of a dramatic increase in the expression of all iNKR on CD8 CTL in patients with HIV-1 infection. This finding confirms, and further extends to all the presently identified iNKR, our previous observations . In addition, although coinfection with HCV was present in four of the eight HIV-1-infected patients who underwent HAART, chronic HCV coinfection could be reasonably ruled out as a possible confounding parameter responsible for the observed increase in the expression of all iNKR molecules on CD8 T lymphocytes in HIV-1-infected patients and for their delayed decrease during HAART.
Further work is required on a larger patient series to extend and define the possible involvement of CD94/NKG2A expression on CD3 T cells and also at the site of HCV replication in chronically infected patients.
Analysis of iNKR expression on CD8 T lymphocytes over an extended period of time encompassing 24 months showed that no substantial decrease in the expression of iNKR, including p58.1, p58.2, p70, p140 and CD94/NKG2A, was observed during the first 6 months of successful HAART. However, complete recovery to normal levels of iNKR expression on CD8 T cells takes place between 6 months and 2 years of antiretroviral therapy. In the present analysis only p70 and p140 were negatively correlated to the peripheral blood CD4 T-cell count, and showed a clear trend towards decreased expression on CD3CD8 PBMC during the first 6 months of HAART. This co-ordinated decrease in the expression of iNKR during HAART in HIV-1-infected patients considerably lags behind the reduction of virus replication to below detection limits, which was achieved by 6 months in all cases. In addition, the reduction to normal levels of CD3CD8 iNKR-positive cells in peripheral blood has a different and more protracted time course when compared with that of activation markers (e.g., CD38, HLA-DR). In fact, the expression of these surface antigens on CD3CD8 lymphocytes has been described to decrease significantly within the first 6 months of therapy [6,11] and is known to correlate with plasma RNA levels and viral replication . Moreover, the reduction in iNKR expression on CD3CD8 lymphocytes seems to be independent of the decrease in CD8 T cells that takes place during HAART in HIV-1-infected patients. Thus, the expression of iNKR on CD8 T cells of HIV-1-infected patients, with the possible exception of p70 and p140, appears to be associated with other factors, possibly including local production of cytokines [27,35] or low-level virus replication [17–19].
The persistence of a significantly elevated proportion of circulating CD8 T cells expressing LIR1/ILT2 after 24 months of virologically successful therapy in some HIV-1-infected patients on HAART may contribute, at least in part, to the inability of CD8 CTL to completely clear residual HIV-1-infected cells [18,19]. According to the present study, the expression of LIR1/ILT2 on a relevant proportion of CD8 CTL prevented lysis of autologous target cells bearing HIV-1 antigens in the patients that could be studied. On the other hand, in the only patient that after 24 months of HAART showed low proportions of CD8LIR1/ILT2 T cells (< 18%), HIV-1-specific lysis could be observed in the absence of LIR1/ILT2-mediated functional inhibition. Thus, in patients treated successfully with HAART, delayed decrease and persistent expression of iNKR could be involved, at least in part, in the failure to clear CD4 cells which bear residual HIV-1 replication. In addition, inhibitory signals delivered by HLA class I-specific iNKR expressed on HIV-1-specific CD8 CTL could help, at least in part, to explain the lack of detectable fresh CTL activity displayed by peptide/MHC tetramer-positive CD3 CD8 cells . The contribution of residual expression of LIR1/ILT2 or of any other iNKR on CD8 CTL after HAART to the residual impairment of HIV-1-specific lysis needs to be addressed further. In fact, although we found that HIV-1 specificities are contained in LIR1/ILT2 CD8 PBMC, only single-cell analysis could provide a definitive estimate of the extent of this observation. In this respect, the role played by residual iNKR expression on CD8 CTL could be particularly relevant if their proportion in lymph nodes would parallel the increased proportion of total CD8 and the proportion of cycling (Ki67) CD8 T cells that are reported at these sites .
Finally, the present findings raise questions on the possible involvement of continuous low-level HIV-1 expression in the maintenance of increased numbers of CD8 T cells expressing inhibitory NK receptors. In this respect, T-cell receptor engagement has been shown to sustain iNKR expression, indicating that iNKR expression on CD8 T cells in vivo may be maintained through continuous encounters with antigen . In principle, continuous treatment with HAART resulting in low-level ongoing HIV-1 replication could, therefore, mediate T-cell unresponsiveness to HIV-1 antigens, in a manner similar to that proposed for self-antigens . In this regard, expression of inhibitory NK receptors could therefore be viewed as one of the possible mechanisms. As iNKR-mediated inhibition of T-cell activation can be bypassed at high antigen concentrations , strategies based on structured treatment interruptions leading to intermittent and controlled HIV-1 antigen increases, will result in increased rates of T-cell receptor engagement and possibly in recovery of HIV-1-specific CTL function, provided that iNKR are not rapidly up-regulated during treatment interruption.
The authors thank. F. Torre and A. Picciotto (Di.M.I., University of Genova, Italy) for caring for some of the HCV-infected patients.
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