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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Jan. 2001, p. 74–78 1071-412X/01/$04.00ϩ0 DOI: 10.1128/CDLI.8.1.74–78.2001 Copyright 2001, American Society for Microbiology. All Rights Reserved.
Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo TIM NIEHUES,1,2 THOMAS W. MCCLOSKEY,1 JENNIFER NDAGIJIMANA,2 GERD HORNEFF,2 Department of Pediatrics, Division of Allergy and Immunology, North Shore University Hospital/New York University School of Medicine, Manhasset, New York, and Children’s Hospital, Heinrich Heine University, Du¨sseldorf, Germany2 Received 2 May 2000/Returned for modification 21 August 2000/Accepted 5 October 2000 Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing
apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V
assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for
apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed
significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating
stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of
apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that
in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4
depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8
compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a
condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences
between the CD4 and CD8 apoptotic responses to HIV.
While an increased rate of lymphocyte apoptosis has been idate the specificity of annexin binding for apoptosis, we documented for patients with human immunodeficiency virus examined whether PBMC which bound annexin V simulta- (HIV) infection (20), the precise mechanism(s) is still unclear.
neously demonstrated DNA strand breaks by the terminal Different pathways leading to apoptosis have been proposed; deoxynucleotidyltransferase-mediated dUTP nick end label- there is evidence for direct cytopathic effects by viral compo- ing (TUNEL) method. We found that the percentage of CD8 T nents as well as indirect effects on bystander cells (9, 11, 18, lymphocytes undergoing apoptosis was greater than that of 19). While both CD4 and CD8 T cells undergo apoptosis, the CD4 cells when measured immediately ex vivo or following induction and kinetics of cell death may be different for each overnight culture. Furthermore, the addition of activating subset. For example, telomeres have been observed to be sig- stimuli in vitro was able to increase the percentage of CD4 cells nificantly shorter in CD8 cells than in CD4 cells of HIV- which were dying, whereas the CD8 subset was unchanged.
infected adults, suggesting faster turnover in the CD8 popula- tion (7, 21, 24). In addition, in vitro addition of interleukin-2 MATERIALS AND METHODS
failed to rescue activated CD8 lymphocytes undergoing apo- ptosis (15), indicating that these cells may be committed to Study subjects. Peripheral blood samples were obtained from 67 children with
death in vivo. The deletion of activated responding CD8 T perinatal HIV infection. Children were grouped using Centers for Disease Con-trol and Prevention classification by the level of immune suppression: category 1, lymphocytes following an infection may be a homeostatic pro- none (n ϭ 23, age ϭ 8.2 Ϯ 3.7 years [mean Ϯ standard deviation]); category 2, cess serving to restore normal cell numbers, an event which moderate (n ϭ 24, age ϭ 7.5 Ϯ 4.0 years); category 3, severe (n ϭ 20, age ϭ may be amplified due to the chronic nature of HIV infection.
11.3 Ϯ 5.6 years). Treatments consisted of no antiretroviral therapy (n ϭ 6), Together, these data suggest that during HIV infection, CD8 reverse transcriptase inhibitor therapy (n ϭ 40), or combination therapy withreverse transcriptase inhibitors and protease inhibitors (n ϭ 21). In a subset of cells primarily undergo activation-induced cell death due to an patients for whom viral load measurements were available (n ϭ 46), the median environment of persistent inflammation.
number of RNA copies per ml was 2,050 (25th to 75th percentile, 400 to 11,000).
Cells undergoing apoptosis translocate phosphatidylserine Control blood samples were obtained from 10 HIV-negative healthy children (PS) to their outer cell membrane (23). Annexin V, which (age ϭ 3.5 Ϯ 2.9 years). These children were free of infection and were under- binds PS, was used to investigate lymphocyte subset apoptosis going elective surgery for nonmalignant disorders (inguinal hernia or phimosis).
In all cases, informed consent was obtained from the parents or guardians of the in a cohort of HIV-positive children and uninfected, healthy children per institutional review board-approved protocols.
pediatric controls. Importantly, the method of annexin V la- Cell isolation and culture. Following collection into heparinized tubes, sepa-
beling allowed us to quantitate apoptosis in peripheral blood ration of mononuclear cells was performed by conventional Ficoll-Hypaque mononuclear cells (PBMC) directly after isolation. To val- (Lymphoprep; Nycomed AS, Oslo, Norway) density gradient centrifugation.
Identical conditions were used for samples from HIV-infected children andhealthy uninfected controls. Each sample was processed within 1 h of collection.
In some experiments, PBMC were cultured overnight at a concentration of * Corresponding author. Mailing address: North Shore University 106/ml at 37°C and 5% CO2 in RPMI 1640 (Gibco Laboratories, Grand Island, Hospital, 350 Community Drive, Manhasset, NY 11030. Phone: (516) N.Y.) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 562-4641. Fax: (516) 562-2866. E-mail: spahwa@nshs.edu.
2 mmol of L-glutamine (Whittaker Bioproducts, Walkersville, Md.) per liter with EX VIVO APOPTOSIS DURING PEDIATRIC HIV INFECTION FIG. 1. Flow cytometric gating strategy for determination of total lymphocyte apoptosis and demonstration of simultaneous application of annexin V and TUNEL assays. Apoptotic lymphocytes (quadrant D2, 9%) which exhibited DNA strand breaks (TUNEL assay) also expressed PS on their cell membrane (annexin V assay). Monocytes bound annexin V despite the absence of strand breaks (quadrant E1). To accurately assess total lymphocyte apoptosis (quadrant F2, 17%), a two-tiered strategy of a large light scatter gate (gate B) combined with exclusion of CD14ϩ cells 100 U of penicillin G per ml and 100 ␮g of streptomycin per ml. For determi- those obtained with the large gate (22, 26, and 55%, respec- nation of the effect of activation on lymphocyte apoptosis, PBMC (106/ml) were tively). Worthy of note is our observation that all CD14ϩ cells incubated overnight with phytohemagglutinin (PHA; 1 ␮g/ml) or anti-CD3monoclonal antibody (0.1 mg/ml, clone HIT 3a; Pharmingen, San Diego, Calif.).
(monocytes) bound annexin V but most were TUNEL nega- Verification of annexin V assay. PBMC were cultured overnight and then
tive, a finding whose implications are at present unknown. One labeled with phycoerythrin (PE)-conjugated anti-CD14 monoclonal antibody possible explanation is that monocytes bind membrane frag- (Becton Dickinson, San Jose, Calif.), biotinylated annexin V (Pharmingen), and ments of apoptotic cells which contain PS. Such a situation streptavidin allophycocyanin (Molecular Probes, Eugene, Oreg.). Samples werefixed with Permeafix reagent (Ortho, Raritan, N.J.) for 40 min at room temper- would enable the monocytes to become labeled with annexin.
ature, followed by incubation with TUNEL solution as directed by the manufac- In contrast, the apoptotic lymphocytes which expressed PS as turer (Phoenix Flow Sytems, San Diego, Calif.). Positive- and negative-control determined by annexin V binding also exhibited DNA strand samples were prepared for each experiment. Samples were stored at 4°C until breakage by the TUNEL assay (Fig. 1). These preliminary flow cytometric analysis was performed.
findings allowed optimization of our gating for accurate mea- Quantification of apoptosis within defined lymphocyte populations. For de-
termination of apoptosis within specific cell populations, samples were labeled surement of lymphocyte apoptosis in subsequent experiments.
with PE-conjugated monoclonal antibodies directed against CD4 or CD8 (Bec- Apoptosis measured immediately ex vivo in T-cell subsets.
ton Dickinson). Background fluorescence was determined with isotype-matched Since our objective was to account for all apoptotic lympho- control antibodies. Immediately after isolation or following overnight culture, cytes within a sample, the large gate was used with monocytes PBMC were incubated at room temperature for 10 min with monoclonal anti-bodies and with annexin V conjugated to fluorescein (Boehringer Mannheim, excluded by gating on CD4bright or CD8bright cells, which limits Mannheim, Germany). Annexin binding buffer (Pharmingen) was used for all analysis to T lymphocytes. In order to determine the in vivo washes and incubation steps. Samples were analyzed by flow cytometry immedi- levels of cell death, we measured apoptosis immediately after ately after being washed. The time interval between blood draw and flow cyto- phlebotomy (Fig. 2) in CD4 and CD8 cells isolated from chil- metric analysis was less than 2 h in all experiments.
Statistical analysis. Differences between groups were determined in a paired
dren of immune categories 1, 2, and 3 as well as from healthy manner using the paired Student t test or the Wilcoxon signed rank test as uninfected control children. In HIV-positive children from all appropriate, depending on the normality of the data distribution, with Excel 5.0 immune categories, apoptosis was significantly higher in CD8 software (Microsoft, Redmond, Wash.) or Sigmastat software (Jandel Scientific, cells than in CD4 cells (Fig. 3). The percentage of apoptotic San Rafael, Calif.). P values below 0.05 were considered statistically significant.
CD4 cells was significantly higher than that of controls only inchildren with severe disease, while the percentage of apoptotic CD8 cells was significantly increased in all infected children.
Validation of annexin V assay. Our initial experiments were
As the majority of the patients were receiving antiretroviral designed to verify results obtained with the annexin assay in therapy at the time of this cross-sectional study, it was not our system. Additionally, we investigated appropriate flow cy- possible to evaluate the effects of treatment on apoptosis.
tometric gating schemes, taking into account the effect of in- However, elevated levels of CD8 T-lymphocyte death existed clusion of monocytes on the measurement of total lymphocyte despite effective control of viremia.
apoptosis. Cells that undergo apoptosis acquire morphological Apoptosis in T-cell subsets after in vitro activation. To fur-
alterations which are evident upon flow cytometric analysis as ther investigate the observed differences in cell death levels changes in light scatter patterns. As lymphocytes become apo- between CD4 and CD8 lymphocytes, apoptosis was examined ptotic, they no longer fall within a typical lymphocyte cluster in T-cell subsets of HIV-infected children after overnight cul- but can be differentiated from monocytes by lack of expression ture in the absence or presence of activation stimuli (Fig. 4).
of CD14 antigen (Fig. 1). We found that a large light scatter Upon overnight incubation with PHA, CD4 cells from HIV- gate contained significantly more apoptotic lymphocytes than a infected children showed an increase in the percentage of small gate, as values obtained for the percentages of apoptosis apoptotic cells, whereas the CD8 population did not change using the small gate for cells from children of categories 1, 2 (Fig. 5). In cells from uninfected children, the percentage of and 3 (9, 10, and 14%, respectively) were always lower than apoptotic CD4 and CD8 cells did not change after overnight FIG. 2. Representative histograms demonstrating apoptosis measured immediately ex vivo in T-cell subsets. Samples from healthy children (CONT) or HIV-infected children (HIVϩ) were labeled with anti-CD4 (A) or anti-CD8 (B) PE and annexin V fluorescein isothiocyanate immediately upon isolation. Flow cytometric analysis consisted of a combination of a large light scatter gate and a gate on CD4bright or CD8bright cells (not shown). The percentages of apoptotic CD4 T cells (control, 8%; HIV positive, 18%) and apoptotic CD8 T cells (control, 11%; HIV positive, 53%) were then determined.
stimulation with PHA. The addition of anti-CD3 monoclonal antibody also induced an increased percentage of apoptosis in CD4 T cells from HIV-infected children, whereas apoptosis in CD8 lymphocytes did not change (data not shown).
DISCUSSION
While the ability of HIV to induce lymphocyte cell death is well established, the causative mechanism(s) remains elusive.
Putative pathways include induction via Fas ligand (2) or tu- mor necrosis factor-related apoptosis-inducing ligand (14) and loss of protective molecules such as Bcl-2 (3). Results of the analysis of T-cell apoptosis in freshly isolated blood samples from HIV-infected persons most likely reflect the rate of cell death in vivo, thus providing valuable insight into the patho- genesis of this disease. Previous reports of immediate ex vivo apoptosis in HIV-infected adults (12, 13, 16) and children (6), including a recent study which utilized the annexin V assay (16), indicated low levels of cell death. In contrast, a study utilizing the dye Apostain, which is reported to detect early apoptotic cells, found that over half of freshly isolated lympho- FIG. 3. Summary of immediate ex vivo apoptosis percentages in cytes underwent apoptosis in HIV-infected adults (1). These T-cell subsets. Mean percentages Ϯ standard deviations of annexin differences may be explained by the patient cohorts studied but binding CD4 and CD8 cells from HIV-infected children in immune are more likely due to the apoptosis assays used and the anal- categories 1 (n ϭ 10), 2 (n ϭ 10), and 3 (n ϭ 11) and from healthy ysis schemes employed. Our experiments were designed to controls (n ϭ 10) directly after isolation are shown. Asterisks indicate statistically significant differences between the percentages of apopto- account for all apoptotic lymphocytes while omitting poten- tic CD4 and CD8 cells within each immune category: ء, P Ͻ 0.05; ءء, tially confounding monocytes from the analysis, and thus our P Ͻ 0.01; ءءء, P Ͻ 0.001. Differences between groups were determined results demonstrate higher levels of cell death than many pre- in a paired manner using the paired Student t test or the Wilcoxon vious reports. Indeed, levels of cell death observed in a con- signed rank test. In addition, both CD4 and CD8 apoptosis levels in ventional lymphocyte gate were of a magnitude similar to those patients from category 3 were significantly higher than those from category 1 or 2 (for CD4, P was 0.004 for categories 1 and 3 and 0.01 previously reported, indicating that a basic variable such as for categories 2 and 3; for CD8, P was 0.004 for categories 1 and 3 and gating can have tremendous influence on the level of apoptosis EX VIVO APOPTOSIS DURING PEDIATRIC HIV INFECTION FIG. 4. Representative histograms demonstrating apoptosis in T-cell subsets after activation. Samples from a healthy child (A) and an HIV-infected child (B) were cultured overnight in the presence or absence of PHA. Cells were then labeled with anti-CD4 or anti-CD8 PE and annexin V fluorescein isothiocyanate. Flow cytometric analysis consisted of gating on CD4bright or CD8bright events and quantifying the percentages of CD4 control apoptotic cells (resting, 8%; activated, 10%), CD8 control apoptotic cells (resting, 21%; activated, 21%), CD4 HIV-positive apoptotic cells (resting, 18%; activated, 38%), and CD8 HIV-positive apoptotic cells, (resting, 46%; activated, 59%).
observed. We observed that in the majority of samples from HIV-infected children, CD8 apoptosis was significantly higher than in controls at all disease stages and apoptosis was present at higher levels in the CD8 population than in the CD4 pop- ulation. Because an earlier report of lymphocyte apoptosis in children (6) indicated that levels of immediate ex vivo CD4 and CD8 cell death were similar, we suggest that our effort to include all apoptotic lymphocytes in our analysis may explain The percentages of peripheral blood CD4 apoptosis were elevated only in children in immune category 3. Since most children were on antiretroviral therapies with moderate to complete virus suppression, relatively low CD4 apoptosis levels may reflect the adequacy of virus control. Alternatively, CD4 lymphocyte death may predominantly occur in locations other than the peripheral circulation. However, in agreement with our findings, in experiments conducted with tonsillar tissues from HIV-infected adults, Rosok and coworkers found higher levels of cell death in the CD8 population than in the CD4 population (22). CD8 lymphocyte death during HIV infection has been associated with cellular activation resulting in in- creased sensitivity to apoptosis (3, 5). The physiologic process FIG. 5. Summary of percentages of apoptosis in T-cell subsets fol- lowing activation. Shown are mean percentages Ϯ standard deviations of response to a pathogen, i.e., activation, may lead to a greater of annexin binding CD4 and CD8 cells of HIV-infected children (n ϭ propensity for CD8 death, a pathway which may be augmented 31) (A) and uninfected controls (n ϭ 10) (B) after overnight culture by the chronic nature of HIV. CD8 T lymphocytes from pa- with PHA or medium. For HIV-infected CD4 cells, the difference tients with acute Epstein-Barr and varicella-zoster virus infec- between the percentage of apoptosis with overnight culture in medium tions have been demonstrated to be highly sensitive to apopto- and that with overnight culture in PHA was statistically significant (P ϭ 0.007). Differences between groups were determined in a paired man- sis (4). Elevated levels of CD8 cell death may thus be a ner using the paired Student t test or the Wilcoxon signed rank test.
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verting enzyme-like protease involvement in Fas-induced and activation- induced peripheral blood T cell apoptosis in HIV infection: TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in ACKNOWLEDGMENTS
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