Early correction of cell cycle perturbations predicts the immunological response to therapy in HIV-infected patients
Paiardini, Mirkoa,b; Cervasi, Barbaraa,b; Galati, Domenicoc; Dominici, Sabrinab; Albrecht, Helmutd; Sfacteria, Alessandrae; Magnani, Maurob; Silvestri, Guidoa; Piedimonte, Giuseppee
From the aDivision of Infectious Diseases and Vaccine Research Center, Emory University, Atlanta, Georgia USA, bIstituto di Chimica Biologica ‘G. Fornaini', Universita’ di Urbino, cDipartimento di Internistica Clinica e Sperimentale, Seconda Universita’ di Napoli, Italy, dDivision of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia, USA, eDipartimento di Sanita’ Pubblica Veterinaria, Universita’ degli Studi di Messina, Italy.
Requests for reprints to: Dr M. Paiardini, Instituto di Chimica Biologica ‘G. Fornaini', Universita’ delgi Studi di Urbino ‘Carlo Bo', Via Saffi2, Urbino 61029, Italy Email: firstname.lastname@example.org
Received: 25 February 2003; revised: 17 June 2003; accepted: 7 July 2003.
Objective: To determine whether changes in the indices of HIV-associated cell cycle dysregulation (i.e., increased expression of cyclin B1 and abnormal nucleolar structure) may predict the level of immunological reconstitution in HIV-infected patients treated with highly active antiretroviral therapy (HAART).
Methods: Cross-sectional and longitudinal analysis of viral load, CD4 T cell counts, cyclin B1 expression, and AgNOR number and area of distribution in 30 HIV-infected patients who were studied before and up to 6 months after initiation of HAART.
Results: In HIV-infected individuals, the level of cell cycle dysregulation correlated with the type of response to HAART. While low levels of dysregulation were present in patients with complete (both virological and immunological) response to HAART, high levels were present in HAART-treated patients with limited CD4 T cell increases despite persistent viral suppression (immunological non-responders). Importantly, the level of correction of cell cycle dysregulation after 60 days of therapy predicted the level of immune reconstitution after 6 months.
Conclusion: These observations suggest that correction of cell cycle dysregulation predicts a good immunological response to HAART and that sequential analysis of cell cycle dysregulation might help to identify patients that could benefit from alternative, immune-based interventions in addition to standard HAART.
The natural history of HIV infection is characterized by a progressive CD4 T cell depletion and development of AIDS. Although the ‘direct’ cytopathic effect of HIV on infected cells is a well-established pathogenic phenomenon, HIV infection is also associated with an ‘indirect’ effect through ‘bystander’ apoptotic cell death of large numbers of uninfected CD4 and CD8 T cells [1–7]. The high level of lymphocyte apoptosis observed during HIV infection is likely related to the hyperimmune activation that follows infection with a lymphotropic and lympholytic virus [1–7] and is maintained by the abundance of antigenic stimuli and pro-inflammatory/pro-apoptotic cytokines [8–12]. As a result, during HIV infection, the level of activation-induced cell death of T lymphocytes is higher than in other instances of immune activation, and this may contribute significantly to the progressive lymphocyte depletion. As such, the pathogenesis of AIDS is related to both the direct impact of HIV on the infected cells and the deleterious consequences of the host immune response, which result in high levels of bystander apoptosis (indirect mechanisms of immunodeficiency). It is important to note that it is still unclear to what extent the direct and indirect mechanisms of CD4 T cell depletion contribute to the pathogenesis of AIDS in different HIV-infected individuals, and that there is no reliable laboratory index to measure the impact of the indirect effect in the clinical setting [13–15].
While the indirect mechanisms of immunodeficiency likely act as significant negative prognostic factors during HIV disease, there are relatively few studies aimed at defining their molecular pathogenesis [16,17]. Interestingly, highly active antiretroviral therapy (HAART) appears to have a marked effect on both the direct (by suppressing HIV replication) and the indirect (by reducing the amount of antigenic load and, therefore, decreasing the level of ongoing immune activation) mechanisms of CD4 T cell depletion. It has been proposed that the indirect mechanisms of immunodeficiency may play a prominent immunopathogenic role in a subset of HIV-infected individuals, referred to as immunologically discordant or immunological non-responders (INR) to HAART because they maintain severe lymphocyte depletion despite sustained viral suppression, [18–21]. Understanding the pathogenic role of the indirect mechanisms of immunodeficiency will not only improve our knowledge of the immunopathogenesis of HIV but may also help to identify subsets of patients who would benefit from therapeutic interventions specifically aimed at reducing the indirect mechanisms of immunodeficiency in addition to standard HAART . Indeed, immune-based interventions using cytokines or other immune-modulating agents may be effective in reversing or counteracting the immune perturbations that are observed in HIV-infected individuals but are not caused directly by the virus [23–27].
In earlier studies, we showed that specific perturbations of cell cycle control are present in lymphocytes of both CD4 and CD8 T cell lineage isolated from HIV-infected patients with active viral replication and progressive immunodeficiency [28–31]. These perturbations consist mainly of increased activation of the cyclin B1/p34 cdc2 complex and the presence of multiple and enlarged argyrophilic nucleolar organizer regions (AgNOR) [28,29]. Although both findings are characteristic of cycling cells, the DNA content and metabolic profile of affected cells was consistent with a resting/G0 phase. This apparent paradox represents a perturbation of cell cycle control, which we termed cell cycle dysregulation (CCD). Importantly, CCD becomes more evident after in vitro treatment with mitogens and correlates with a fivefold increase in apoptosis [28–30]. Based on these studies, we proposed that CCD is related to the hyperimmune activation and T cell turnover that follows chronic HIV infection and that it may represent a molecular mechanism by which these events cause the increased sensitivity to apoptosis in uninfected T lymphocytes from HIV-infected patients [28–30]. Further, we hypothesized that the magnitude of CCD is an index of the pathogenic role played by the chronic aberrant immune activation and apoptosis of uninfected cells (i.e., indirect effects) on the progression of the immunodeficiency in individual HIV-infected patients.
To test the latter hypothesis, the present study involves a sequential quantitative analysis of CCD in a cohort of HIV-infected patients before and after HAART, with a specific focus on the correlation between the extent of CCD and the immunological and/or virological response to therapy.
This cross-sectional and longitudinal analysis of specific cell cycle abnormalities and their correlation with the immunological response to HAART (including at least two reverse transcriptase inhibitors and one protease inhibitor) was conducted on a group of 30 asymptomatic HIV-infected patients with plasma viremia > 10 000 copies/ml and CD4 counts < 500 × 106 cells/l. The control group comprised 20 HIV-uninfected healthy donors.
HIV viremia was measured by branched DNA technique (Quantiplex; Chiron, Emeryville, California, USA) according to standard procedures.
Lymphocyte isolation and flow cytometry
Peripheral blood mononuclear cells (PBMC) were isolated from acid citrate dextrose-treated blood by density gradient centrifugation and washed twice in phosphate-buffered saline (PBS) with 2% fetal bovine serum. Determination of CD4 T cell count was performed after direct staining with the anti-human CD4–fluorescein isothiocyanate (FITC) monoclonal antibody (Becton Dickinson, San Jose, California, USA), according to standard procedures. FITC-labelled anti-cyclin B1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) was used to detect the level of cyclin B1 expression. Phycoerythrin-labeled anti-Ki67 monoclonal antibody (Becton Dickinson) directed against the nuclear antigen Mib-1 was used to detect actively proliferating cells. Samples used for cyclin B1 and Ki67 staining were first surface stained for CD4 and CD8 (using anti-CD4–allophycocyanin and anti-CD8–peridinin–chlorophyll a complex antibodies; both from Becton Dickinson) then fixed and permeabilized using the CytoFix/Perm-Kit (Pharmingen, (San Diego, California, USA), and finally stained intracellularly with the proper monoclonal antibodies and controls. Flow cytometric acquisition and analysis of samples was performed on at least 100 000 events, gated according to forward- and side-scatter, on a FACScalibur flow cytometer driven by the CellQuest software package (Becton Dickinson).
Expression of cell cycle-dependent proteins
The expression of several cell cycle-dependent proteins (cyclin B1, cyclin D1, pRb, nucleolin) was measured by Western blot (all the monoclonal antibodies were from Santa Cruz Biotechnology) and the bands analyzed using SigmaGel (Handel Scientific, San Rafael, California, USA). The numerical values indicate the absolute area of the band (i.e., the total calibrated pixel intensity values of each band). Two to five replicates were performed for each sample. In all measurements, internal controls were always performed, including lysing equal cell numbers, loading into each lane equal volumes of equal protein concentrations (15 μg/lane), and, after the electrophoresis, by performing Coomassie brilliant blue staining. If different protein concentrations were observed at this time, the whole procedure was repeated and the initial loading protein concentration was adjusted based on the actin band.
AgNOR staining was performed as previously described . Briefly, lymphocytes were washed with PBS, suspended in 95% alcohol and transfered to a coverglass. After alcohol evaporation, coverglasses were stained (2 parts 50% AgNOR aqueous solution and 1 part 2% gelatine in 1% formic acid) for 12 min in the dark. AgNOR appear as black intranuclear dots, and the number per cell was evaluated in at least 500 cells. AgNOR area per cell was measured using Image-Pro Plus software (Media Cybernetics, Silver Spring, Colorado, USA). After definition of the grey threshold corresponding to the AgNOR alone, the AgNOR area was measured automatically.
PBMC were fixed on slides (Labtech, Idaho Falls, Idaho, USA) using 4% performic acid for 15 min. Cells were then permeabilized with 0.5% Triton X-100 and washed with PBS. Unconjugated mouse anti-nucleolin antibody (Santa Cruz Biotechnology) was added (1/100, 45 min at 37°C). After two washes, FITC-conjugated GAM-immunoglobulin (Sigma, St Louis, Missouri, USA) was added (1/200 dilution). After two washes, 5 μg/ml propidium iodide plus 200 μg/ml RNAase was added for 30 min. After washes, Moviol was added and slides were covered by a coverslip. Confocal microscope was from Leika, with a ×63–zoom 1.6 objective.
A two-tailed, two-sample Student's t test was used to calculate the P value for differences in means of AgNOR number, AgNOR area of distribution, and cyclin B1, cyclin D1, pRb, and nucleolin expression between HIV-infected patients and uninfected controls. A linear regression analysis was performed to evaluate the correlation between either AgNOR number or cyclin B1 expression and changes in CD4 T cell counts after 6 months of HAART in the same groups of patients.
Patient population and classification
Thirty untreated, asymptomatic HIV-infected patients with CD4 T cell count < 500 × 106 cells/l and viral load > 10 000 copies/ml were recruited to this study. The longitudinal analysis was performed at baseline (i.e., prior to initiation of therapy) and at 30, 60, 90, 120, 150, and 180 days after initiation of HAART. Viral load results and CD4 T cell counts for this cohort of patients are shown in Table 1. The virological and immunological response was evaluated after 6 months of therapy to determine the type of response to HAART. All patients (30/30) showed a virological response defined as a viral load decrease > 1 log10 copies/ml from baseline or to < 400 copies/ml of plasma (this latter result was achieved in 28/30 patients). However, 6 months after initiation of HAART, only 18/30 (60%) patients experienced a sustained (i.e., in at least two consecutive measurements) increase of their CD4 T cell count > 100 × 106 cells/l from baseline; these patients were defined throughout this study as complete responders (CR) to HAART. The remaining 12 patients (40%) did not experience a sustained increase in their CD4 T cell count and were defined as INR. (Trends of viremia and CD4 T cell counts in these two groups of patients are shown in Fig. 3a,b, below.)
Changes in cell cycle abnormalities with therapy
Increased levels of cyclin B1, as measured by Western blot, are a consistent feature of the HIV-associated CCD [28,29]. To assess more directly the levels of cyclin B1 in specific T cell subsets of HIV-infected patients and controls, flow cytometric analysis was performed after intracellular staining with an anti-cyclin B1 antibody and surface staining with anti-CD4 and anti-CD8 antibodies (Fig. 1a,b). The results confirmed that cyclin B1 expression, measured either as number of positive cells or mean fluorescence intensity in positive cells, was significantly increased in both CD4 and CD8 T cells from the HIV-infected patients. In addition, the mean fluorescence intensity for cyclin B1 was higher in T cells from HIV-infected patients even when measured in the subset of cells that were defined as cyclin B1 negative (data not shown). Based on earlier studies in which cyclin B1 expression was studied in different phases of the cell cycle [28,29], we had hypothesized that these increased levels of cyclin B1 associated with HIV infection did not simply reflect an increased rate of cycling T cells but rather a more complex perturbation of cell cycle control. This was confirmed by performing flow cytometric analysis of cyclin B1 expression in conjunction with staining for the proliferation marker Ki67 [32,33]. As shown in Fig. 1c, the vast majority of cyclin B1-positive T cells in HIV-infected patients, both CD4 and CD8, did not express measurable levels of Ki67 (80.3% and 73%, respectively), suggesting that the overexpression of cyclin B1 is unrelated to the number of cycling cells. In contrast, the majority of cyclin B1-positive T cells isolated from healthy donors coexpressed the Ki67 proliferation marker, indicating their commitment to cell cycle (data not shown).
Several immunological abnormalities described in HIV-infected patients can be corrected by effective antiretroviral therapy [34–36]. Of these parameters, CD4 T cell count is considered to be the most important index of good immunological response to HAART. To determine whether correction of CCD correlates with virological (i.e., suppression of HIV plasma viremia) or immunological (i.e., increase in CD4 T cell count) parameters of response to HAART, the levels of cyclin B1 in our cohort of 30 HIV-infected patients undergoing HAART was measured sequentially. The patients included in this part of our study were divided into two groups: CR and INR. As shown in Fig. 2, the levels of cyclin B1 showed a marked decrease after 6 months of HAART in CR patients (Fig. 2c), but not in INR patients (Fig. 2b), in whom the levels of this protein remained significantly higher than those of normal controls (Fig. 2a). These results indicate that correction of the increased levels of cyclin B1 is observed only in patients with good immunological response to HAART.
The second consistent cell cycle abnormality of lymphocytes from HIV-infected patients with active viral replication is the presence of an abnormal nucleolar structure, as measured by both AgNOR and nucleolin staining [28–30]. To determine whether nucleolar abnormalities are also affected by HAART, AgNOR staining and subcellular localization of nucleolin was sequentially examined by confocal microscopy in our cohort of patients. After 6 months of HAART, the diffuse extranucleolar localization of nucleolin and appearance of multiple AgNOR dots, typical of a large proportion of lymphocytes from untreated HIV- infected patients [28,29], was re-established in CR patients (Fig. 2f,i), who showed a pattern of AgNOR and nucleolin staining similar to that observed in healthy controls (Fig. 2d,g), but not in INR patients (Fig. 2e,h).
Together with the results showing the correction of the cyclin B1 overexpression in CR but not in INR HIV-infected patients, these findings indicate that abnormalities of CCD are corrected only in patients with good immunological response to HAART.
CD4 T cell reconstitution and early changes in cyclin B1 expression and nucleolar structure during therapy
As shown in Fig. 2, HAART induced correction of CCD only in CR patients. To determine whether the extent of correction of CCD may predict the level of immune reconstitution after 6 months of HAART, a longitudinal analysis (days 0, 30, 60, 90, 120, 150, and 180 after initiation of HAART) of the following parameters was performed: viral load, CD4 T cell count, cyclin B1 expression, and AgNOR number per cell. HAART-treated patients were retrospectively divided into CR and INR groups as described above. Figure 3 shows the trend of viral loads and CD4 T cell counts in the CR (Fig. 3a) and the INR (Fig. 3b) groups, and the trend of cyclin B1 expression and AgNOR number in the CR (Fig. 3c) and the INR (Fig. 3d) groups. This sequential analysis confirmed that a significant increase in CD4 T cell count was only apparent in the CR group, although viral suppression was achieved in both CR and INR, and that correction of the cell cycle abnormalities was obtained in CR, but not in INR. To determine whether the early effect of HAART on the parameters of CCD correlated with the extent of immunological reconstitution measured as CD4 T cell count, a series of linear correlation analyses were performed between the amount of decline seen in cyclin B1 expression and in the average AgNOR number per cell after 30, 60, and 90 days of HAART, and the absolute increase of CD4 T cell count after 6 months of therapy. Interestingly, the analysis showed significant correlations between changes in both cyclin B1 levels (r2 = 0.90; P < 0.01) and AgNOR number (r2 = 0.92; P < 0.01) at day 60 post-initiation of HAART and the extent of CD4 T cell increase after 6 months of therapy (data not shown). In all, these findings indicate that measurement of the changes in cyclin B1 expression and AgNOR staining induced by 60 days of HAART may effectively predict the level of CD4 T cell reconstitution after 6 months of therapy.
Cell cycle dysregulation in HIV-infected patients involves cyclin D1 and pRb proteins
Since the extent of CCD predicts the immunological response to HAART, laboratory measurements of these indices could be useful to identify a subset of HIV-infected patients that may benefit from immune-based therapy in addition to standard HAART. To determine whether the expression of other cell cycle-related proteins may be changed during HIV infection and, therefore, possibly to increase the number of available laboratory markers for CCD, the intracellular levels of cyclin D1 and pRb were measured by Western blot in PBMC isolated from the cohort of 30 HIV-infected patients at baseline and 20 uninfected controls. As shown in Fig. 4 and Table 2 (and similar to previously described results [28,29]), the intracellular levels of cyclin B1 and nucleolin were consistently increased in PBMC from HIV-infected patients compared with controls. Interestingly, the intracellular levels of cyclin D1 and pRb, two proteins that are mainly expressed during the G1/S phase of cell cycle, were also significantly increased in lymphocytes from HIV- infected patients. Cyclin D1 levels were fivefold higher, while pRb levels were increased approximately threefold (Table 2). It is of note that in this group of patients the intracellular levels of cyclin B correlated directly with those of both cyclin D and pRb (data not shown). In all, these findings indicate that the HIV-associated cell cycle perturbation involves phase-dependent proteins other than cyclin B1 and nucleolin, thus suggesting more complex changes in the physiology of lymphocytes during HIV infection, and potentially providing additional laboratory indices to measure the in vivo extent of CCD.
The pathogenic mechanisms responsible for HIV- associated immunodeficiency can be divided into ‘directly’ HIV related (i.e., killing of infected cells) and ‘indirectly’ HIV related (i.e., apoptosis of uninfected T cells) [1–7]. The relative contribution of these direct and indirect mechanisms may differ significantly among HIV-infected individuals. Notably, the indirect mechanisms of immunodeficiency may play a critical role in maintaining low CD4 T cell counts in those patients where HAART induces persistent suppression of viremia without significant immune reconstitution (INR). While HIV viremia is a reliable index of the direct pathogenic effects of HIV [37–39], the contribution of indirect effects is hard to quantify because of a lack of established laboratory measures. Since the indirect mechanisms of HIV-associated immunodeficiency could conceivably be corrected by immune-based interventions, a reliable in vivo quantification of these indirect mechanisms may be clinically useful.
In earlier studies [28–31], we described the presence of a perturbation of the normal cell cycle control in lymphocytes from HIV-infected patients, and we proposed that this CCD contributes to AIDS-associated lymphocyte depletion by decreasing the threshold for apoptosis. According to this view, CCD is an index of the role played in vivo by the indirect mechanisms of lymphocyte depletion, and its measurement would provide a quantitative marker of these indirect mechanisms in individual patients. Consistent with this hypothesis is the finding that CCD is corrected by HAART in patients with a good virological and immunological response, but persists in treated INR. In addition, we found that the level of correction of CCD at 60 days after initiation of HAART correlated directly with the level of immunological response after 6 months.
The association between CCD and INR phenotype supports the hypothesis that CCD represents an important indirect mechanism of immunodeficiency in HIV-infected patients and suggests that INR may benefit from immune-based interventions targeting the indirect mechanisms of immunodeficiency, such as cytokines that act by directly reconstituting the homeostasis of the immune system. Interleukin-2 is of particular interest in this context, given that it can revert CCD and decrease the level of apoptosis in vitro in lymphocytes from HIV-infected individuals . While interleukin-2 may increase CD4 T cell counts in HIV-infected patients [40–45], its use is costly and not devoid of side effects, so its role in the clinical management of HIV infection is still unclear. Results of our study suggest that measurement of CCD in vivo early after initiation of HAART may identify patients that will not show a sustained immunological response, and who may specifically benefit from the addition of interleukin-2 to their therapeutic regimen. To evaluate this latter possibility better, studies are now in progress to determine the in vivo effects of interleukin-2 therapy on the HIV-associated CCD.
The present study also provided some insights into the molecular features of CCD with respect to (i) the cause–effect relationship between CCD and HIV, (ii) the relationship between CCD and the stages of the cell cycle, and (iii) the molecular pathways that are affected by CCD. Although our hypothesis is that CCD reflects the chronic hyperimmune activation associated with HIV infection, an alternative hypothesis is that CCD is caused directly by HIV or by one of its gene products [45–47]. In our earlier studies, we concluded that this possibility is unlikely since no correlation was ever found between the level of HIV replication and the induction of CCD and apoptosis after in vitro activation of lymphocytes . The fact that the level of HAART-induced reversion of CCD does not correlate with the level of viral suppression further supports the idea that CCD is not directly induced by the virus. While our earlier work [28–31] had already suggested that CCD does not simply reflect the presence of increased numbers of cycling T cells, here we provide the first direct evidence for this hypothesis by using costaining for cyclin B1 and Ki67. We have shown that lymphocytes from HIV-infected patients, of both CD4 and CD8 T cell lineages, include an expanded population of proliferating cells (i.e., Ki67 positive) that may or may not express high levels of cyclin B1 (as predicted by the sequential expression of these two molecules), and an expanded population of non-proliferating (i.e., Ki67-negative), cyclin B1- positive T cells. This confirms that CCD is not merely a consequence of increased rates of T cell turnover but rather represents a complex dysregulation of cell cycle control that affects large numbers of cells not actively cycling at the time of analysis. Finally, the present study showed that the intracellular levels of cyclin D1 and pRb, two other phase-dependent proteins [48–50], were also increased during HIV infection. Although it is not clear to what extent the increased levels of cyclin D1 and pRb may reflect the higher rates of cycling T cells during HIV infection, it is also conceivable that CCD might involve molecular pathways other than those of cyclin B1/p34–cdc2 and nucleolin. More detailed experiments are required to test this hypothesis, including studies in which lymphocytes are activated in vitro with mitogens and a kinetic analysis of the expression of cyclin D1 and pRb over time.
In summary, our data suggest that changes in CCD may predict the level of immune reconstitution following HAART; therefore, measurements of CCD early after initiation of HAART may help to identify individual HIV-infected patients that may benefit from additional, immune-based interventions.
The authors wish to thank Drs Mark B. Feinberg, Maria Montroni, and Ann Chahroudi for the helpful discussion and critical review of the manuscript.
Sponsorship: This work was supported by NIH grant R01-AI052755-01A1 to G.S. and grants 30B.65 to G.P. and 35B.1 to M.M. from the Programma Nazionale di Ricerca sull'AIDS, Istituto Superiore di Sanita', Rome, Italy.
Note: Mirko Paiardini and Barbara Cervasi contributed equally to this work.
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