A hallmark of HIV infection is the depletion of CD4 T cells, leading to progressive immunodeficiency, opportunistic infections, and death.1-3 Combinational antiretroviral therapy (cART) stops this process, and long-term viral suppression leads to sustained recovery of CD4 cell counts with a rapid increase within the first 3-6 months and a second slower increase lasting over several years.4,5 However, there is considerable individual variation in this process. In 10%-30% of patients, CD4 cell counts remain critically low despite treatment-induced viral suppression.5,6 Several risk factors for insufficient CD4 T cell have been defined. These include older age, treatment interruptions, incomplete viral suppression, low CD4 T-cell count before initiation of antiretroviral therapy, and coinfections like viral hepatitis.6-8 Furthermore, certain components of cART like zidovudine, tenofovir, and didanosine have been associated with impaired immune reconstitution.9,10
Recently, it has become more and more evident that not only direct cytopathic effects of HIV but also the immune activation by chronic HIV infection leads to a profound impairment of the immune functions.11 Chronic immune activation in HIV-infected patients leads to exhaustion and premature senescence of the immune system.12 One parameter of this exhaustion is the activation-associated T-cell molecule programmed death-1 (PD-1), which by interaction with its 2 ligands, PD-L1 and PD-L2, conveys negative signals to T cells.13 Importantly, expression of PD-1 is highly correlated with impaired T-cell function and functional senescence of the immune system.14 High level of PD-1 expression is a general phenomenon in chronic viral infection and has been shown in mice and men.14,15 Blocking of PD-1/PD-L pathways restores T-cell function in vitro.16,17 In HIV-infected patients, PD-1 expression is directly correlated with individual viral loads, and effective cART has been shown to downregulate the expression of this receptor on CD4 and CD8 T cells.15,18 HIV-infected patients who can control viral replication (controller) and patients who do not progress due to very low viral loads (long-term nonprogressors) exhibit significant lower PD-1 expression as compared with patients with high levels of viral replication and progressive disease.19
Previous studies have focused on PD-1 expression in viremic patients, but no detailed analysis of PD-1 expression in successfully treated aviremic patients has been done up to date. In this study, we have analyzed PD-1 expression in patients with failing immunological recovery but successful control of viral replication on sustained antiretroviral treatment (nonimmune reconstituters, non-IR).
MATERIAL AND METHODS
Patients and Healthy Controls
Blood samples from 40 patients infected with HIV-1 (20 responder/20 poor responder) who were virologically suppressed on cART >12 months were obtained during routine checkups at the outpatient department of the HIV unit of the Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University of Vienna, Austria. Informed consent was obtained from all patients, and the study was approved by the local ethic committee (Ethic Committee of the General Hospital of Vienna, Austria). The basic characteristics of the patients are summarized in Table 1. For control purpose, a group of healthy elderly (age >80 years) and children (age <10 years) was chosen.
Flow Cytometry Analysis
Tlymphocytes were analyzed by 4-color flow cytometry. For fluorescence activated cell sorting (FACS) analysis of whole blood, the ADG lysis kit (An der Grub, Kaumberg, Austria) was used. Monoclonal antibodies directed against CD4, CD8, CD25, CD28, CD38, CD45RA, CD62L, CD69, CD95, CCR5, HLA-DR, and PD-1 were obtained from BD Bioscience (Palo Alto, CA). Anti-PD-L1 was obtained from R&D (Minneapolis, MN), and anti-PD-L2 antibody from eBioscience (San Diego, CA). Unlabeled antibodies were detected by allophycocyanin-labeled goat anti-mouse IgG (Fc-γ)-specific antibodies (Jackson ImmunoResearch, West Grove, PA).
FACS analyses were done on a FACS Calibur using the CellQuest software (BD Bioscience). Samples that showed less than 20% positive cells were scored negative.
Plasma samples were diluted 5-fold with endotoxin-free water and heated to 70°C for 10 minutes to inactivate plasma proteins before analysis. Endotoxin plasma levels were then quantified with a commercially available limulus test (QCL-1000 assay; Cambrex, Milan, Italy) according to the manufacturer's protocol.
Human T cells were obtained through depletion of CD11b, CD14, CD16, CD19, CD33, and major histocompatibility complex-class II bearing cells with the respective monoclonal antibodies by magnetic activated cell sorting (MACS; Miltenyi, Mönchengladbach, Germany). T-cell stimulation assays were essentially performed as described previously.20-22 Briefly, human T cells were cocultured with irradiated (6000 rad) T-cell stimulator cells expressing a membrane-bound anti-CD3 antibody and PD-L1, PD-L2, or control stimulator cells expressing the membrane-bound anti-CD3 antibody only. T-cell stimulator cells (2 × 104/well) and human T cells (1 × 105/well) were cocultured for 3 days in 96-well tissue culture plates. All assays were done in triplicates.
To assess T-cell proliferation, methyl-3[H]-thymidine (MP Biomedicals, Heidelberg, Germany) was added for the last 18 hours, and incorporation was measured on a microplate scintillation counter (Packard; Topcount Instrument, Meriden, CT).
Mann-Whitney U test was used to assess significance. Differences were considered significant at P < 0.05.
Twenty patients with good (IR) and 20 patients with failing immune recovery (non-IR) after cART were studied. The basic characteristics of both groups are shown in Table 1. A discordant immune response was defined as CD4 cell recovery to less than 300 cells per cubic millimeter or an absolute increase of less than 250 CD4 cells after at least 12 months of complete viral suppression. The mean age of the patients was 45.9 (±11.4) years for the immune reconstitution group and 49.8 (±8.1) years for patients with discordant immune response (P > 0,05); mean treatment duration was 8.3 (±5) years and 10.1 (±3.9) years, respectively (P > 0,05). Although the mean CD4 nadir was lower in patients with discordant immune response (79.1 ± 65.3) than in patients with good immune reconstitution (148.2 ± 96.5), this difference was not statistically significant (P > 0.05). Coinfection with hepatitis C was equally distributed between both groups, as were different antiretroviral medications and cytomegalovirus seropositivity.
PD-1 Expression on T Cells Is Increased in Patients With Discordant Immune Response
PD-1 has been described as a marker of chronic immune activation and T-cell exhaustion.23 Its expression is enhanced on T cells of viremic HIV-infected patient and correlates with the viral load in these patients. To assess if PD-1 expression might also be associated with poor immune reconstitution under antiretroviral treatment, T cells of patients with good and with discordant immune response were analyzed by FACS for PD-1 expression. PD-1 expression (>20% positive cells) on CD4 and CD8 T cells was observed in the majority of patients with failing immune recovery, whereas most of the patients with good immune response showed no or minimal PD-1 expression on their CD4 and CD8 T cells. Forty-eight percent of CD4 T cells expressed PD-1 in non-IR, whereas 14% of the CD4 T cells were found positive in patients with good immune recovery (median expression, P < 0.05) (Fig. 1A). Similar results were obtained when analyzing PD-1 expression on CD8 T cells: the median percentage of PD-1 positive CD8 T cells was 37% in non-IR versus 15% in patients with good immune reconstitution (P < 0.05; Fig 1A). In line with previous reports, no too minimal PD-1 expression was found on either CD4 or CD8 T cells of healthy controls (Figs 1A, B).
PD-1 Is a Unique Marker for Poor Immune Reconstitution
In healthy individuals, PD-1 expression is only induced on T-cell activation. We thus analyzed the expression of other T-cell activation markers on CD4 and CD8 T cells in our patients with failing immune recovery and compared it with patients with good immune reconstitution. We found that the activation markers, CD69 or CD25, were not expressed in either patient group. Furthermore, we found that T cells of HIV-positive aviremic patients did not express CCR5 and only low levels of CD38, HLA-DR, or PD-L1 (Fig. 1B). Importantly, the expression levels of these activation-associated molecules did not differ in patients with good and those with poor immune reconstitution and were slightly higher than in healthy controls (Fig 1C and data not shown).
In addition, we found PD-1 expression not to be restricted to a certain T-cell subset. PD-1 was not only expressed on terminally differentiated CD45RA+/CCR7− CD8 T cells but also on central memory and effector memory CD4 and CD8 T cells (CD45RA−/CCR7+ and CD45RA−/CCR7−, respectively) and, although to a lesser extend, on naive CD45RA+/CCR7+ T cells (data not shown). Furthermore, PD-1 expression was not restricted to T cells expressing PD-L1 or HLA-DR (Fig 1D).
It is well established that chronic infections lead to loss of CD28 expression on CD8 T cells. Although we found an increased number of CD28-negative CD8 T cells in the HIV-infected patients studied, PD-1 expression was equally distributed on CD28 positive and negative CD8 T cells (Fig 1E). This indicates that PD-1 expressing T cells are distinct from senescent T cells characterized by the loss of the CD28 receptor.
Plasma Endotoxin Levels Are Not Increased in Patients With Poor Immune Reconstitution
Increased endotoxin plasma levels in HIV-infected patients due to an impaired mucosal barrier function in the gut have been described and were shown to correlate with immune activation.24,25 In accordance with previous studies, we measured elevated endotoxin levels in viremic patients. Aviremic patients showed lower lipopolysaccharide levels than viremic patients, and we found that endotoxin levels did not differ between patients with good and those with poor immune reconstitution (Fig. 2).
T Cells in HIV-Infected Patients With Discordant Immune Response Differ From Senescent T Cells in Elderly
We hypothesized that persistent immune activation in chronic HIV infection may lead to premature aging of the immune system. We therefore compared T cells of patients with poor immune reconstitution with T cells of the elderly healthy individuals (older than 80 years). Although the aged immune system is particularly characterized by loss of CD28 expression on the CD8 T-cell subset,26 patients with failing IR did not have higher numbers of CD28-negative CD8 T cells than patients with IR. Furthermore, we found PD-1 expression to be a poor marker to define the aged immune system because its expression levels do not differ significantly between elderly and young healthy individuals (Figs. 3A, B).
PD-1 Expressing T Cells Are Inhibited by PD-L Ligation
In our patients, we found a strong negative correlation between the percentage of T cells expressing PD-1 and the CD4 T-cell counts, indicating that PD-1 might have a functional role in poor immune reconstitution (Fig. 4A). One explanation could be that PD-1 expressing T cells are more strongly inhibited by interaction with its ligands PD-L1 and PD-L2. To test this, we stimulated T cells from HIV-infected patients expressing high levels of PD-1 and T cells of healthy donors with stimulator cells expressing either a membrane-bound anti-CD3 antibody or with cells that coexpressed anti-CD3 with PD-L2. As shown in Figure 4B, the presence of PD-L2 led to a 20% inhibition of the proliferation of T cells in healthy donors. In contrast, T-cell proliferation in HIV-infected patients with failing immune recovery was reduced to about 50%, demonstrating that PD-1 overexpressing T cells are indeed more responsive to PD-1-mediated inhibition. When comparing in vitro T-cell responses of IR and non-IR, we observed a trend toward a stronger inhibitory effect of PD-1 ligands in non-IR, but these differences did not reach statistical significance. These results were obtained with both, PD-L1 and PD-L2.
Here, we show that PD-1 is a unique marker for failing immune reconstitution in HIV-infected patients on long-term suppressive cART. To our knowledge, this is the first report on a single marker to be specifically associated with a poor CD4 increase on HIV treatment initiation. Up to 30% of successfully treated patients-as defined by viral load suppression below the detection limit-does not reach normal CD4 cell counts and therefore remains at higher risk for clinical events.5,6 Only recently, it has been shown in large cohort studies that low CD4 cell counts dispose patients not only for classical HIV-associated diseases but also for pathologies currently not classified as AIDS-related malignancies and complications.27,28 Several risk factors for poor immune recovery, like low CD4 nadir, older age, coinfection, or certain components of cART, have been found to explain in a large portion of patients but not in all cases the lack of CD4 cell increase.5-8 However, no immunological pathway associated with the lack of immune recovery has been described so far.
PD-1-originally defined as an apoptosis-associated molecule on hybridoma cells-is an activation-induced inhibitory T-cell marker.29 In line with this, several studies demonstrated that PD-1 upregulation in human and murine T cells were chronically stimulated due to hepatitis, HI-viremia and lymphocytic choriomeningitis-viremia.14-16,30 Furthermore, it has been shown that suppression of viral load after the initiation of HIV therapy is also associated with a downregulation of PD-1 expression in T cells of patients with HIV.15
Persistent PD-1 expression in patients with poor immune reconstitution may be a consequence of chronic immune activation due to subclinical pathogen invasion. Reduced regain of CD4 T cells due to enhanced immune activation-as indicated by, for example, CD38 expression-has been reported previously.31 However, in our study, we could not observe the upregulation of CD38 or other activation-associated markers on T cells, indicating that PD-1 is a unique marker of poor immune recovery and not associated with general activation of the immune system. Furthermore, serum endotoxin levels, a surrogate marker for impaired mucosal barrier to bacterial invasion, did not differ in aviremic patients, irrespective of good or poor immune reconstitution. Thus, in this study, we do not find any direct of indirect evidence that persistence of a higher level of immune activation distinguishes between patients with adequate CD4 increase and patients without immune reconstitution. Based on these observations, we conclude that enhanced PD-1 expression in patients with poor immune recovery is not the result of enhanced immune activation due to persistent pathogen invasion. We also tried to address whether the observed differences in PD-1 expression might be the result of different distribution in the memory subsets in non-IR. However, in these patients, we found PD-1 expressed on T cells in central and in effector memory subsets, and in addition, we found no leads for a different memory distribution.
Reduced turnover of T cells could be another reason for exhaustion of T cells and therefore enhanced PD-1 expression. However, when comparing T cells of healthy elderly and young persons, we found that PD-1 was not differentially expressed in these groups, indicating that PD-1 is not a suitable marker for age-associated immune exhaustion. T cells of elderly were, however, characterized by the loss of CD28 expression on CD8 T cells, whereas CD28 expression although higher in infected than in HIV-negative healthy individuals did not differ between patients with good and poor immune reconstitution.
It is well established that PD-1 is an inhibitory T-cell molecule that on interaction with its ligands conveys negative signals to T cells.13,21,32-34 Furthermore, in vitro studies with T cells of viremic patients and murine in vivo studies have provided evidence that blocking the PD-1/PD-ligand pathway could restore the immune function in an exhausted immune system.17,23,35-39 Thus, persistence of PD-1 expression may not only be an epiphenomenon of poor immune recovery but may directly inhibit immune function. Indeed, we show, in our study, that PD-1 overexpressing T cells are more responsive to PD-L1-mediated and PD-L2-mediated inhibition of T-cell proliferation in vitro. To us, this seems to be an important aspect because there are several reports indicating that not only PD-1 but also its ligands are upregulated in chronic infections. Although there is clear evidence that PD-1 expression is normally reduced on initiation of highly active antiretroviral therapy, expression of its ligands seems to be affected by antiretroviral therapy to a much lesser extent.40,41 Although it did not reach statistical significance, a trend toward a stronger inhibition of T cells derived from non-IR compared with IR by PD-1 ligands was observed. It is possible that these small differences obtained in our short-term experiments results in a compromised expansion in the T cells of non-IR over time. Thus, PD-ligand-mediated impaired T-cell proliferation/expansion could also be an important mechanism underlying poor CD4 recovery. Furthermore, recent data suggest that not only T cells may be inhibited via the PD-1/PD-ligand pathway because it has been shown that also myeloid DC function can be inhibited by PD-1/PD-L1 inhibition via a phenomenon termed reverse signaling. This fact could thus further contribute to immune dysfunction in HIV-infected patients.40 In addition, a recent study reports that PD-1 expression correlates with increased susceptibility to spontaneous apoptosis in T cells, and thus, increased cell death could also further persistent low CD4 cell counts.42
In this study, we provide first evidence that PD-1 also plays a role in compromised immune function in aviremic patients under antiretroviral treatment. Thus, PD-1 function might be not only important in viremic patients but also play a role in other settings with CD4 cell reduction (eg, in patients successfully treated with cART).
There is growing evidence that blocking PD-1 might represent a novel therapeutic avenue to enhance immune responses.43-46 Our data indicate that patients with poor immune recovery might also benefit from therapeutic interventions that aim at blocking PD-1/PD-L interactions.
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