Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes
Costa, Paolaa; Rusconi, Stefanob; Mavilio, Domenicoc; Fogli, Manuelaf; Murdaca, Giuseppec; Pende, Danielaa; Mingari, Maria Cristinad; Galli, Massimob; Moretta, Lorenzoe,f; De Maria, Andreac
From the aIstituto Nazionale per la Ricerca sul Cancro – IST-GE, Genova, the bClinica Malattie Infettive e Tropicali, University of Milan, the cDipartimento di Medicina Interna, the dDipartimento di Oncologia, Biologia e Genetica, the eDipartimento di Medicina Sperimentale, University of Genova, Italy, and the fIstituto Scientifico G. Gaslini, Genova, Italy.
Note: Paola Costa and Stefano Rusconi equally contributed to this work.
Requests for reprints to: A. De Maria, Dipartimento di Medicina Interna, University of Genova, 10 Largo R.Benzi, Genova 16132, Italy.
Received: 25 October 2000,
revised: 9 February 2001; accepted: 21 February 2001.
Sponsorship: Supported by M.U.R.S.T. (A.D.M.), Istituto Superiore di Sanità, Programma Nazionale AIDS 0B.67 (L.M.).
Background: Highly active antiretroviral therapy (HAART) is associated with a decrease in viral replication to undetectable levels and with an increase in CD4 T lymphocytes. Residual HIV-1 replication occurs together with incomplete recovery of cytotoxic CD8 T lymphocyte (CTL) numbers and function. We sought to determine whether expression of HLA class I-specific inhibitory natural killer receptors (iNKR) on the CTL of patients who had been treated successfully with HAART for 24 months could be involved, at least in part, in residual CTL functional inhibition.
Methods: Two-colour cytofluorometry was used to analyse the expression of six different iNKR including p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2 on the CD3, CD8 lymphocytes of eight patients with successful long-term suppression of viral replication before and after 3, 6 and 24 months of HAART. Healthy subjects were analysed as controls. HIV-1-specific cytotoxic activity was determined after 24 months of HAART in the presence and absence of iNKR-masking.
Results: No significant reduction of iNKR expression on CD8 T cells was observed by 6 months. Expression of p70 and p140 was inversely correlated with the increasing CD4 numbers. After 24 months CD8 T-lymphocytes expressing p58.1, p58.2, p70, p140 and CD94/NKG2A returned to levels indistinguishable from those of the healthy controls. A significantly increased proportion of CD8 CTL still expressed LIR1/ILT2, a receptor with broad HLA-class I specificity. Functional analysis of freshly separated cells revealed that the disruption of the interaction between LIR1/ILT2 and HLA-class I could partly restore HIV-1-specific lysis.
Conclusions: A decrease in CD3CD8iNKR cells is observed beyond 6 months of HAART. In some patients functional impairment due to LIR1/ILT2 expression may persist even after 24 months of successful HAART.
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.
1. Furrer H, Egger M, Opravil M. et al
. Discontinuation of primary prophylaxis against Pneumocystis carinii
pneumonia in HIV-1-infected adults treated with combination antiretroviral therapy. N Engl J Med 1999, 340: 1301 –1306.
2. Kovacs JA, Masur H. Prophylaxis against opportunistic infections in patients with human immunodeficiency virus infection. N Engl J Med 2000, 342: 1416 –1428.
3. Zaunders JJ, Cunningham PH, Kelleher AD. et al
. Potent antiretroviral therapy of primary human immunodeficiency virus type 1 (HIV-1): partial normalization of T lymphocyte subsets and limited reduction of HIV-1 DNA despite clearance of plasma viremia. J Infect Dis 1999, 180: 320 –329.
4. Fleury S, Rizzardi GP, Chapuis A. et al
. Long-term kinetics of T cell production in Hiv-infected subjects treated with highly active antiretroviral therapy. Proc Natl Acad Sci USA 2000, 97: 5393 –5398.
5. Autran B, Carcelain G, Li TS. et al
. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science 1997, 277: 112 –116.
6. Malhotra U, Berrey MM, Huang Y. et al
. Effect of combination antiretroviral therapy on T-cell immunity in acute human immunodeficiency virus type I. J Infect Dis 2000, 181: 121 –131.
7. Connick E, Lederman MM, Kotzin BL. et al
. Immune reconstitution in the first year of potent antiretroviral therapy and its relationship to virologic response. J Infect Dis 2000, 181: 358 –363.
8. Pitcher CJ, Quittner C, Peterson DM. et al
. HIV-1-specific CD4+ T cells are detectable in most individuals with active HIV-1 infection, but decline with prolonged viral suppression. Nature Med 1999, 5: 518 –525.
9. Hellerstein M, Hanley MB, Cesar D. et al
. Directly measured kinetics of circulating T lymphocytes in normal and HIV-1-infected humans. Nature Med 1999, 5: 83 –9.
10. Pantaleo G, De Maria A, Koenig S. et al
. CD8+ T lymphocytes of patients with AIDS maintain normal broasd cytolytic function despite the loss of human immunodeficiency virus-specific cytotoxicity. Proc Natl Acad Sci USA 1990, 87: 4818 –4822.
11. Bisset LR, Cone RW, Huber W. et al
. Highly active antiretroviral therapy during early HIV infection reverses T-cell activation and maturation abnormalities. Swiss HIV Cohort Study.
AIDS 1998, 12: 2115 –2123.
12. Soudeyns H, Campi G, Rizzardi P. et al
. Initiation of antiretroviral therapy during primary HIV-1 infection induces rapid stabilization of the T-cell receptor β chain repertoire and reduces the level of T-cell oligoclonality. Blood 2000, 95: 1743 –1751.
13. Harrigan PR, Whaley M, Montaner JS. Rate of HIV-1 RNA rebound upon stopping antiretroviral therapy. AIDS 1999, 13 (suppl 8): F59 –F62.
14. Garcia F, Plana M, Vidal C. et al
. Dynamics of viral load rebound and immunological changes after stopping effective antiretroviral therapy. AIDS 1999, 13 (suppl 8): F79 –F86.
15. Chun TW, Engel D, Mizell SB. et al
. Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active anti-retroviral therapy. Nature Med 1999, 5: 651 –655.
16. Davey RT Jr, Bhat N, Yoder C. et al
. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc Natl Acad Sci USA 1999, 96: 15109 –15114.
17. Finzi D, Blankson J, Siliciano JD. et al
. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nature Med 1999, 5: 512 –517.
18. Zhang L, Ramratnam B, Tenner-Racz K. et al
. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999, 340: 1605 –1613.
19. Hockett RD, Kilby JM, Derdeyn CA. et al
. Constant mean viral copy number per infected cell in tissues regardless of high, low, or undetectable plasma HIV RNA. J Exp Med 1999, 189: 1545 –1554.
20. Ogg GS, Jin X, Bonhoeffer S. et al
. Quantitation of HIV-1-specific Cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998, 279: 2103 –2106.
21. Gray CM, Lawrence J, Schapiro JM. et al
. Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). J Immunol 1999, 162: 1780 –1788.
22. Trimble LA, Shankar P, Patterson M, Daily JP, Lieberman J. Human immunodeficiency virus-specific circulating T lymphocytes have down-modulated CD3zeta and CD28, key signaling molecules ofr T-cell activation. J Virol 2000, 74: 7320 –7330.
23. Trimble LA, Lieberman J. Circulating CD8 Tlymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex. Blood 1998, 91: 585 –594.
24. De Maria A, Ferraris A, Guastella M. et al
. Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: impairment of specific cytolytic functions. Proc Natl Acad Sci USA 1997, 94: 10285 –10288.
25. De Maria A, Moretta L. HLA-class I-specific inhibitory receptors in HIV-1 infection. Hum Immunol 2000, 61: 74 –81.
26. De Maria A, Mavilio D, Costa P, Dignetti P, Fogli M, Mingari MC. Multiple HLA-class I-specific inhibitory NK receptor expression and IL-4/IL-5 production by CD8+ T-cell clones in HIV-1 infection. Immunol Lett 2000, 72: 179 –182.
27. Mingari MC, Ponte M, Bertone S. et al
. HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells. Proc Natl Acad Sci USA 1998, 95: 1172 –1177.
28. Pende D, Biassoni R, Cantoni C. et al
. The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J Exp Med 1996, 184: 505 –518.
29. Seventh Workshop and Conference on Human Leukocyte Differentiation Antigens
. Harrogate, June 2000.
30. Giavedoni LD, Velasquillo MC, Parodi LM, Hubbard GB, Hodara VL. Cytokine expression, natural killer cell activation, and phenotypic changes in lymphoid cells from rhesus macaques during acute infection with pathogenic simian immunodeficiency virus. J Virol 2000, 74: 1648 –1657.
31. Mingari MC, Schiavetti F, Ponte M. et al
. Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell population. Proc Natl Acad Sci USA 1996, 93: 12433. 12433.
32. Zinkernagel RM. Immunology taught by viruses. Science 1996, 271: 173 –178.
33. Alcami A, Koszinowski UH. Viral mechanisms of immune evasion. Immunol Today 2000, 21: 447 –464.
34. Orendi JM, Bloem AC, Borleffs JC. et al
. Activation and cell cycle antigens in CD4+ and CD8+ T cells correlate with plasma human immunodeficiency virus (HIV-1) RNA level in HIV-1 infection. J Infect Dis 1998, 178: 1279 –1287.
35. Bertone S, Schiavetti F, Bellomo R. et al
. Transforming growth factor-β-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes. Eur J Immunol 1999, 29: 23. 23.
36. Huard B, Karlsson L. KIR expression on self-reactive CD8+ T cells is controlled by T-cell receptor engagement. Nature 2000, 403: 325 –328.
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