1 Department of Experimental Medicine and Biochemical Science, Tor Vergata University Hospital, and 5 Department of Neuroscience, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome; 2 Department of Infectious and Tropical Diseases, University of Rome La Sapienza, Viale del Policlinico 155, 00161 Rome; 3 Clinical Immunology Unit, S. Giovanni Hospital, Via Codirossoni, 00169 Rome; 4 Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome; 6 IRCCS, S. Lucia, Via Ardeatina 306, 00179 Rome; 7 Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone 31, 96168 Messina, Italy
Received 3 September 2003; returned 22 October 2003; revised 24 November 2003; accepted 8 December 2003
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Abstract |
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Patients and methods: Twenty-two HIV-1-infected patients undergoing PART were enrolled in a long-term, open longitudinal study. Data derived from 17 patients with successful response to therapy (TS; median time of follow-up 36 months, range 2436 months) were used for correlation studies. Apoptosis was evaluated after short-term culture of peripheral blood lymphocytes by flow cytometry analysis of isolated nuclei or of annexin V/CD4, annexin V/CD8 double-stained cells.
Results: Sustained, noticeable levels of apoptosis inhibition in peripheral blood mononuclear cells were measured, in the long-term, in 16 of the 17 TS patients. Levels of total cell apoptosis correlated with levels of CD8+ apoptotic cells more significantly than with levels of CD4+ apoptotic cells. In addition, CD4+ cell counts were correlated inversely with levels of CD8+ apoptotic cells in a highly significant fashion, but not with levels of CD4+ apoptotic cells.
Conclusions: Our data indicate that the increase of CD4+ lymphocytes in HIV patients, as a consequence of successful response to PART, may be related to changes in apoptosis level occurring in the CD8+, and not in the CD4+, cell compartment.
Keywords: HIV, antiviral, CD8+ cells
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Introduction |
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Materials and methods |
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This was a longitudinal, open study in a well-characterized cohort of HIV-1-infected patients starting PART. PART was defined as two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor or, in three patients, a non-NRTI. Patients were enrolled from the Department of Infectious and Tropical Disease, University of Rome La Sapienza, or from the AIDS Center, S. Giovanni Hospital, Rome. Enrolled patients were: (i) routinely followed up by medical visits; (ii) checked at baseline and monthly for plasma VL during the first 6 months and every 3 months thereafter; (iii) checked at baseline and monthly for CD4+ and CD8+ cell counts; and (iv) checked at baseline and monthly for apoptosis during the first 6 months and every 3 months thereafter.
Eligibility criteria at enrolment required that: (i) the patients were naive or that they had been off antiretroviral therapy for at least 4 months (one patient only); (ii) their age was 18 years; (iii) they had the ability to be compliant with the study; and (iv) they had not been suffering from opportunistic or any kind of severe infection, cancer, autoimmune disorders, or major vascular or neurological diseases. No patient lost eligibility for the last two criteria during the course of the study. Most of the patients remained on their assigned treatment, with the exception of four subjects who changed or briefly interrupted and reinitiated the therapy, without modification of the overall response. The study received ethics approval from the institutions participating in the study and informed consent was obtained from patients.
Cell cultures and evaluation of apoptosis
PBMCs were isolated from heparinized blood by Ficoll-Hypaque gradient (Lymphoprep; Nycomed, Oslo, Norway) according to standard methods. In order to evaluate spontaneous apoptosis of short-term cultured PBMCs, as previously described,12 the cells were cultured for 66 h in RPMI 1640 (Life Technologies, Paisley, UK) supplemented with 10% FCS (Life Technologies), 2 mM glutamine, 50 IU/mL penicillin and 50 IU/mL streptomycin (Hyclone, Cramlington, UK). At the end of incubation time, apoptosis of samples from all patients was evaluated by flow cytometry analysis of isolated nuclei following detergent treatment and propidium iodide staining, using a method that distinguishes nuclei from apoptotic, necrotic or viable cells, as previously described.19 Moreover, in order to selectively detect cell death in CD4+ and CD8+ cells, quantitation of apoptosis was also assessed, in additional samples from a subgroup of the patients, by evaluating the percentage of annexin V-positive CD4+ or CD8+ cells. This evaluation was performed by double-fluorescence flow cytometry analysis, following staining with fluorescein-conjugated annexin V (Annexin V-FITC Apoptosis Detection Kit; BD-PharMingen, San Diego, CA, USA) plus phycoerythrin-conjugated CD4 or, alternatively, CD8 monoclonal antibodies (PE-CD4 and PE-CD8; Becton Dickinson, Mountain View, CA, USA). Staining with relevant isotype-matched control monoclonal antibodies (Becton Dickinson) was also performed. Flow cytometry analysis was performed, immediately after staining, on a FACScan or a FACScalibur flow cytometer (Becton Dickinson). Events were gated on forward-scatter versus side-scatter in order to include viable and dead lymphocytes and to exclude debris, doublets and, where present, non-lymphoid cells. As a consequence, presence of CD14+ cells in analysed samples was null or negligible.
VL and T cell counts
HIV-1 RNA VL levels were determined by using the Roche Amplicor HIV-1 Monitor (Roche Molecular System, Branchburg, NJ, USA; limit of detection 400 copies/mL) or by a quantitative ultrasensitive RTPCR assay (limit of detection 50 HIV RNA copies/mL). An arbitrary value of 200 or 25 HIV RNA copies/mL was assigned to samples below the detection limit of the two assays, respectively, for statistical and graphical management of data. CD4+ and CD8+ cell counts were evaluated on whole blood using flow cytometer analysis according to routine procedures.
Statistical analysis
Preliminary analysis showed that some of the data were not normally distributed, and that no cluster corresponding to different therapy regimens or to gender could be identified within the group of patients with successful response to therapy. As a consequence, data were analysed by non-parametric methods, and therapy and gender subgroups were not considered. Attempts to identify relatively homogeneous subgroups of patients were carried out using k-means cluster analysis. Baseline and follow-up values were compared using non-parametric Wilcoxon matched pairs signed ranks test for related samples, and for a non-parametric correlation, Spearmans rho correlation coefficients and corresponding two-tailed significances were calculated. For statistical analysis, the numbers of HIV RNA copies/mL were transformed as decimal logarithms, while CD4+ and CD8+ cell counts were transformed as corresponding natural logarithms.12 Statistical analysis was carried out using SPSS software for Windows (SPSS, Chicago, IL, USA).
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Results |
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Twenty-two patients were enrolled in the study. One of them died of acute myocardial infarction 6 months after undergoing PART and was excluded from the study. All the remaining 21 HIV-infected patients were eligible for evaluation. The median time of follow-up was 36 months (range 2436 months). The Centers for Disease Control classification at enrolment was as follows: 12 patients in class A (three A1, four A2, five A3), four in class B (one B1, one B2, two B3) and five in class C (C3). Patients were classified on the basis of their response to therapy into three subgroups, defined as follows: therapy success (TS; 17 patients), VL <400 copies/mL at the last observation time and overall increasing trend of CD4+ cell count; discordant response (DR, two patients), VL >400 copies/mL at the last observation time with increasing trend of CD4+ cell count (>100 cells/mL with respect to pre-PART); and therapy failure (TF, two patients), VL >400 copies/mL at the last observation time with no increases or increases <100 cells/mL in CD4+ cell count with respect to pre-PART. VL promptly dropped to and remained at undetectable levels in all the patients in the TS groups, except for three patients who experienced transitory viral rebounds concomitant with treatment interruption due to periods of non-adherence or change of therapy. In patients belonging to the DR and TF groups, after a drop in VL during the first 612 months, plasma viraemia was not further controlled by PART or showed only transitory decrease in the case of change of therapy. CD4+ and CD8+ cell counts showed a general trend to increase, less pronounced in CD8+ cells, with recurrent fluctuations, both in TS and DR patients. Conversely, in TF patients, CD4+ cells, after an initial increase, slowly, but constantly, declined after virological failure. In particular, this decline was noticeably delayed in one of the two TF patients who had a high CD4+ count at baseline. Differences in the response to therapy and in the trend of apoptosis during PART in the three subgroups of patients we identified suggested the need for a separate analysis of data. However, only the TS subgroup had the numerical requirements for a plausible statistical analysis. As a consequence, hereafter only the data from the TS group are reported and discussed. Characteristics of the patients classified into the TS group as a whole, at baseline and at the last observation time, including VL levels, CD4+ and CD8+ cell counts together with apoptosis levels, are reported in Table 1.
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Apoptosis showed a prompt, initial inhibition in response to PART and remained at low levels in the long-term in 16 out of the 17 patients of the TS group, with most of the values overlapping those previously reported by us for healthy controls in identical experimental conditions (mean value 10%).12 Nevertheless, recurrent minor fluctuations were detected in most of the patients. In fact, major, transitory blips of apoptosis (about a three-fold increase with respect to PART-suppressed baseline levels) were observed concomitant with brief interruptions or changes of therapy with viral rebounds in three of the TS patients. However, no other cluster of data corresponding to the patients with apoptosis blips, except VL rebound, was identified by the k-means cluster analysis. In particular, patients with blips did not differ from the rest of TS patients for NRTI back-bone or clinical manifestations of the disease. Apoptosis levels, as well as VL and CD4+ cell counts, in two representative patients of TS group, one with sustained inhibition (Figure 1a) and one of the three patients who experienced transitory blips (Figure 1b) of apoptosis, during the course of PART, are reported in Figure 1.
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We investigated, using non-parametric bivariate Spearmans analysis, whether apoptosis levels detected by flow cytometry, following propidium iodide staining of hypodiploid nuclei, at baseline and during PART, correlated with immunological and virological parameters. Analysis was carried out on values of apoptosis obtained before starting treatment and after different time-points during PART (1, 2, 4, 6, 8, 12, 20, 24 and 36 months) in the group of responder patients, for a total of 160 observations. The data presented in Table 2 show that spontaneous apoptosis of PBMCs was directly and highly significantly correlated with the level of plasma VL (rho = 0.424, P < 0.0001). Moreover, apoptosis was inversely correlated with the CD4+ cell count (rho = 0.409, P < 0.0001) or, less significantly, with the CD8+ cell count (rho= 0.236, P = 0.002). These results extend our previous observation, which was limited to the first 6 months of therapy, to the long-term, confirming an extremely close correlation between proneness to apoptosis in short-term culture of PBMCs from patients responding to PART and established markers of virological and immunological response to therapy over a long period of time.
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In a subcohort of seven patients from the TS group, we investigated the relationships between susceptibility to apoptosis of the total lymphocyte population and selectively of the CD4+ and CD8+ PBMC from HIV patients before and during PART. For this investigation, cell death of lymphocytes present in the short-term culture of PBMCs, set up as described above, was assessed in samples stained for double fluorescence in order to selectively distinguish annexin V-positive cells and CD4+ and CD8+ lymphocytes. Figure 2 shows an example of how positivity for annexin V was detected separately in CD4+ and CD8+ cells (Figure 2b and d, respectively) following gating on red fluorescence for CD4 or CD8 positivity (Figure 2a and c, respectively). The death of both CD4+ and CD8+ cells contributed to the percentage of total cells positive for annexin. In fact, levels of total annexin V-positive cells were directly correlated with those of the single annexin-positive CD4+ and CD8+ subsets. However, the correlation of total annexin V-positive lymphocytes with annexin V-positive CD8+ cells was closer than that with annexin V-positive CD4+ cells (rho = 0.891 and 0.474, respectively). As expected, levels of total annexin V-positive cells were highly significantly correlated with levels of apoptosis detected in the parallel samples by means of flow cytometry analysis of hypodiploid nuclei following staining with propidium iodide. In the latter samples, no appreciable levels of nuclei from necrotic cells were detected, thus indicating that cell death by necrosis was negligible in annexin V-positive cells also in the absence of annexin V/propidium iodide double-stained samples.
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In order to assess the influence of apoptotic cell death of the distinct CD4+ and CD8+ subsets on the recovery of CD4+ cells in HIV-infected patients successfully responding to PART, analysis was undertaken to investigate the correlation between the absolute number of CD4+ cells and the percentage of annexin V-positive CD8+ and CD4+ cells during the course of the therapy. The results, shown in Figure 3(b) reveal that the log CD4+ cell count was not significantly correlated with the percentage of annexin V-positive CD4+ cells. Conversely, as shown in Figure 3(a), log CD4+ cell count was highly significantly inversely correlated with the percentage of annexin V-positive CD8+ cells (rho = 0.573, P <0.0001). Similarly, no significant correlation was found between changes in CD4+ cell count and the percentage of annexin V-positive CD4+ cells, whereas a highly significant correlation was found between changes in CD4+ cell count and the percentage of annexin V-positive CD8+ cells (rho = 0.471, P <0.0001; data not shown). Moreover, no significant correlation was found between the absolute number of CD8+ cells and the percentage of annexin V-positive CD8+ or CD4+ cells in the same group of patients undergoing therapy (data not shown). These results, together with those showing a highly significant correlation between total lymphocyte apoptosis and CD8+ cell apoptosis, indicate that the inverse relationship we found between CD4+ cell counts and total lymphocyte apoptosis is mainly attributable to the CD8+ subset.
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Discussion |
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One of the key aspects of pathogenesis of HIV infection is whether CD4+ cell depletion is mainly due to direct destruction by the virus or rather to selective lymphopenia caused by other, virus-driven mechanisms. Recent reports sustain that the latter phenomenon is very probably involved in CD4+ cell depletion.20,21 It has been shown that PART inhibits both CD4+ and CD8+ cell decay rates in vivo.22 This implies that the death of T lymphocytes during HIV infection is reasonably, or at least partly, independent of direct killing by the virus, as suggested.23 Thus, inhibition of PBMC apoptosis during PART could be related to modifications occurring in either CD4+ or CD8+ cell subsets. These could involve indirect mechanisms such as the modulation of pro-inflammatory factors and other mediators, as hypothesized.18 However, from existing data it was not clear to what extent modulation of cell death of the two distinct T cell subsets contributes to generate overall PBMC apoptosis inhibition during successful PART. In this context, a major new finding of the present study is the direct evidence that the decrease in CD8+ cell death contributed more than the decrease in CD4+ cell death to determining the inhibition of total PBMC apoptosis in TS patients. Moreover, other evidence comes from our observation that levels of CD4+ cell counts during PART were inversely correlated with spontaneous apoptosis levels in CD8+, but not in CD4+, lymphocytes. This is in agreement with results of a recent cross-sectional observation.15 Correlation is not sufficient by itself to infer a causal relationship; in fact, we can simply hypothesize that both decrease in CD8+ cells apoptosis and increase in CD4+ cell counts are secondary phenomena to VL control during PART. In fact, it has been suggested that control of aberrant CD4 signalling as a consequence of reduction of virus burden may be one of the mechanisms of apoptosis decrease in CD8+ cells following antiretroviral therapy.24 Another mechanism for reduction of apoptosis in CD8+ cells following PART could be an altered level of cytokines acting as CD8+ cell pro-survival factors, such as interleukin-7 or -15. However, we can also hypothesize that the increase in CD4+ cells during PART directly depends on the inhibition of apoptosis in CD8+ cells. In fact, the rescue of CD8+ cells could play a key role in immune reconstitution during PART. Particularly, it could act on the specific response by developing cytotoxic T lymphocytes,25 or on the non-specific response by improving the release of factors exerting an antiviral action, such as the recently identified -defensins.26 Surprisingly, on the other hand, percentages of annexin V-positive CD4+ cells during PART were not correlated inversely with levels of CD4+ cell counts. This could be explained by the possibility that a portion of CD4+ cells rescued during PART were nevertheless prone to apoptosis. They could belong to the short-lived cells on the basis of the biphasic T-cell die-away kinetic, in which it has been hypothesized that HIV-infected patients exhibit different proportions of short-lived and long-lived subpopulations that are regulated by their rate of cell death.22 In any case, absence of correlation seems to exclude the possibility that increase in CD4+ lymphocytes in response to PART is the direct consequence of apoptosis inhibition in CD4+ cells. Moreover, it could be related to the delayed and usually incomplete restoration of CD4+ cell number, even after sustained undetectable VL, in PART-treated patients.27
Taken together our data show that CD4+ cell rescue during PART is not dependent on the inhibition of CD4+ cell apoptosis, but, rather, it is related to the decrease of apoptosis in the CD8+ subset. Further studies need to be performed in order to understand the mechanisms involved in this phenomenon, and its significance.
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Acknowledgements |
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Footnotes |
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B. Macchi and A. Mastino contributed equally to this work.
¶ B. Macchi and A. Mastino contributed equally to this work.
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References |
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2
.
Badley, A. D., Pilon, A. A., Landay, A. et al. (2000). Mechanism of HIV-associated lymphocyte apoptosis. Blood 96, 295164.
3 . Roeder, M. (1998). Getting to the HAART of T cell dynamics. Nature Medicine 4, 1456.[CrossRef][ISI][Medline]
4
.
Alimenti, J. B., Ball, T. B. & Fowke, K. R. (2003). Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS. Journal of General Virology 84, 164961.
5
.
Cannavo, G., Paiardini, M., Galati, D. et al. (2001). Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis. Blood 97, 175664.
6 . McCune, J. M., Hanley, M. B., Cesar, D. et al. (2000). Factors influencing T-cell turnover in HIV-1-seropositive patients. Journal of Clinical Investigation 105, R18.[ISI][Medline]
7
.
Mohri, H., Perelson, A. S., Tung, K. et al. (2001). Increased turnover of T lymphocytes in HIV-1 infection and its reduction by antiretroviral therapy. Journal of Experimental Medicine 194, 127787.
8 . Badley, A. D., Parato, K., Cameron, D. W. et al. (1999). Dynamic correlation of apoptosis and immune activation during treatment of HIV infection. Cell Death and Differentiation 6, 42032.[CrossRef][ISI][Medline]
9 . Gougeon, M. L., Lecoeur, H. & Sasaki, Y. (1999). Apoptosis and the CD95 system in HIV desease: impact of highly active antiretroviral therapy (HAART). Immunology Letters 66, 93103.[CrossRef]
10 . Ensoli, F., Fiorelli, V., Alario, C. et al. (2000). Decreased T cell apoptosis and T cell recovery during highly active antiretroviral therapy (HAART). Clinical Immunology 97, 920.[CrossRef][ISI][Medline]
11 . Chavan, S. J., Tamma, S. L., Kaplan, M. et al. (1999). Reduction in T cell apoptosis in patients with HIV disease following antiretroviral therapy. Clinical Immunology 98, 2432.[CrossRef]
12 . Grelli, S., Campagna, S., Lichtner, M. et al. (2000). Spontaneous and anti-Fas-induced apoptosis in lymphocytes from HIV-infected patients undergoing highly active anti-retroviral therapy. AIDS 14, 93949.[CrossRef][ISI][Medline]
13 . Roger, P. M., Breittmayer, J. P., Arlotto, C. et al. (1999). Highly active anti-retroviral therapy (HAART) is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients. Clinical and Experimental Immunology 118, 4126.[CrossRef][ISI][Medline]
14 . Roger, P. M., Breittmayer, J. P., Durant, J. et al. (2002). CD4+ T cell recovery in human immunodeficiency virus-infected patients receiving effective therapy is related to a down-regulation of apoptosis and not to proliferation. Journal of Infectious Diseases 185, 46370.[CrossRef][ISI][Medline]
15 . De OliveiraPinto, L. M., Lecoeur, H., Ledru, E. et al. (2002). Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro. AIDS 16, 32939.[CrossRef][ISI][Medline]
16
.
Sloand, E. M., Kumar, P. N., Kim, S. et al. (1999). Human immunodeficiency virus type I protease inhibitor modulates activation of peripheral blood CD4 T cells and decreases their susceptibility to apoptosis in vitro and in vivo. Blood 94, 10217.
17
.
Estaquier, J., Lelievre, J. D., Petit, F. et al. (2002). Effects of antiretroviral drugs on human immunodeficiency virus type 1-induced CD4+ T-cell death. Journal of Virology 76, 596673.
18 . Grossman, Z. & Herberman, R. B. (1997). T-cell homeostasis in HIV infection is neither failing nor blind: modified cell counts reflect an adaptive response of the host. Nature Medicine 3, 48690.[ISI][Medline]
19 . Matteucci, C., Grelli, S., De Smaele, E. et al. (1999). Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry. Cytometry 35, 14553.[CrossRef][ISI][Medline]
20 . Grossman, Z., Meier-Schellersheim, M., Sousa, A. E. et al. (2002). CD4+ T-cell depletion in HIV infection: are we closer to understanding the cause? Nature Medicine 8, 31923.[CrossRef][ISI][Medline]
21 . Feinberg, M. B., McCune, J. M., Miedema, F. et al. (2002). HIV tropism and CD4+ T-cell depletion. Nature Medicine 8, 5378.
22 . Grossman, Z. & Paul, W. E. (2000). The impact of HIV on naive T-cell homeostasis. Nature Medicine 6, 9767.[CrossRef][ISI][Medline]
23
.
Kovacs, J. A, Lempicki, R. A, Sidorov, I. A. et al. (2001). Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV. Journal of Experimental Medicine 194, 173141.
24
.
Tateyama, M., Oyaizu, N., McCloskey, T. et al. (2000). CD4 T lymphocytes are primed to express Fas ligand by CD4 cross-linking and to contribute to CD8 T-cell apoptosis via Fas/FasL death signaling pathway. Blood 96, 195202.
25 . Oxenius, A., Gunthard, H. F., Hirschel, B. et al. (2001). Direct ex vivo analysis reveals distinct phenotypic patterns of HIV-specific CD8+ T lymphocyte activation in response to therapeutic manipulation of virus load. European Journal of Immunology 31, 111521.[CrossRef][ISI][Medline]
26
.
Zhang, L., Yu, W., He, T. et al. (2002). Contribution of human alpha-defensin 1, 2 and 3 to the anti-HIV-1 activity of CD8 antiviral factor. Science 298, 9951000.
27
.
Lange, C. G. & Lederman, M. M. (2003). Immune reconstitution with antiretroviral therapies in chronic HIV-1 infection. Journal of Antimicrobial Chemotherapy 51, 14.
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