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The Journal of Immunology, 2006, 177: 6685-6694.
Copyright © 2006 by The American Association of Immunologists, Inc.

Death of CD4+ T Cells from Lymph Nodes during Primary SIVmac251 Infection Predicts the Rate of AIDS Progression1

Laurence Viollet, Valérie Monceaux, Frédéric Petit, Raphaël Ho Tsong Fang, Marie-Christine Cumont, Bruno Hurtrel2 and Jérôme Estaquier3

Unité de Physiopathologie des Infections Lentivirales, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological and virological events that occur during the earliest stages of SIV infection are now considered to have a major impact on subsequent disease progression. In the present study, we demonstrate a clear correlation between progression to AIDS and the rate of in vitro CD4+ (but not CD8+) T cell death in lymph nodes. The dying CD4+ T cells were effector memory T cells, which are critical for the immune response to pathogens. However, there was no correlation between the rate of the viral replication within lymph nodes and the extent of Fas ligand-mediated death, despite the increased sensitivity of CD4+ T cells to death in response to recombinant human Fas ligand. CD4+ T cell death was caspase and apoptosis-inducing factor independent but was clearly associated with mitochondrion damage. Interestingly, higher expression levels of the active form of Bak, a proapoptotic molecule involved in mitochondrial membrane permeabilization, were observed in SIV-infected macaques progressing more rapidly to AIDS. Finally, we demonstrated that the strain of SIV we used requires CCR5 and BOB/GRP15 molecules as coreceptors and caused death of unstimulated noncycling primary CD4+ T cells. Altogether, these results demonstrate that CD4+ T cell death occurring early after SIV infection is a crucial determinant of progression to AIDS and that it is mediated by the intrinsic death pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The primary acute phase of HIV or SIV infection is characterized by an early burst in viral replication, the dissemination and seeding of the virus in all peripheral lymphoid organs, and the induction of the host immune response to the virus (1, 2, 3, 4, 5). Several reports demonstrated that the plasma viral load that is reached at the end of this primary phase (~2–6 months postinfection) predicts the rate of disease progression, from rapid development of AIDS to nonprogressive infection (6, 7, 8, 9, 10). Moreover, several reports have also found that activated memory CD4+ T cells are rapidly depleted in the blood and the lymphoid tissues during primary SIV infection (11, 12).

The excessive induction of apoptosis has been proposed as one of the main mechanisms responsible for the CD4+ T cell depletion in vivo during HIV and SIV infections (13). Studies performed in pathogenic and nonpathogenic primate models of HIV and SIV infections have further suggested a correlation between the pathology of those infections in vivo and the level of CD4+ T cell apoptosis ex vivo (14, 15). However, most studies that investigated the potential relationship between HIV or SIV infection and apoptosis have focused on the chronic asymptomatic phase and were mainly performed on cells from peripheral blood, rather than on cells from lymphoid organs, where viral replication and immune responses occur. We recently found that during primary infection of rhesus macaques with the pathogenic SIVmac251 strain, the number of dying cells in the lymph nodes (LNs)4 is significantly higher in rapid progressors than in the slow progressors (10).

However, several important questions remain unresolved, such as the phenotype of the T cells that undergo apoptosis during primary SIV infection, and the molecular mechanisms that explain the increased propensity of T cells to undergo apoptosis. Two major apoptotic pathways have been identified: 1) the extrinsic pathway mediated by death receptors, and 2) the intrinsic pathway which is death receptor independent but is induced by diverse stimuli such as growth factor deprival, UV exposure, and drugs (16). These pathways converge to a central sensor, the mitochondria, and induce the release of apoptogenic factors from the mitochondria to the cytosol, which leads to apoptosis. The release of those apoptogenic factors from the mitochondria is under the control of members of the B cell lymphoma (Bcl-2) family (16).

Here, we identify from LNs the phenotype of the apoptotic cells as CD4+ effector memory T cells (and not CD8+ cells), a critical population for the establishment of the immune response against pathogens. We also reveal that the death pathway of these CD4+ T cells is caspase and apoptosis-inducing factor (AIF) independent, but is associated with a loss of mitochondrial membrane potential. Interestingly, we demonstrate higher N-terminal epitope Bak staining in moderate progressors (MPs) than in slow progressors (SPs).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and viruses

Rhesus macaques (Macaca mulatta), purchased from China and housed and cared for in compliance with French regulations (<http://www.pasteur.fr/recherche/unites/animalerie/fichiers/Decret2001-486.pdf>), were inoculated i.v. with either the pathogenic SIVmac251 strain (ten 50% animal-infectious doses) or the live attenuated SIVmac251{Delta}nef isolate. The pathogenic SIVmac251 isolate was initially provided by R. Desrosiers (17), titrated in Chinese rhesus macaques (M. mulatta) by i.v. inoculation, and provided by Anne-Marie Aubertin (Institut National de la Santé et de la Recherche Médicale Unité 74, Strasbourg, France). A 1-ml volume of stock virus contained 4 x 104 50% animal-infectious doses as determined by the VACMAN program. The attenuated virus, SIVmac251{Delta}nef, was also provided by Dr. Aubertin. It was derived from the SIVmac251 BK28 clone by three modifications: 1) the premature stop codon at position 8785 in the env gene was mutated to restore a complete env open reading frame; 2) the nef initiator codon ATG was mutated to ACG at position 9059; and 3) nucleotides 9225–9401 in the nef region, which do not overlap either the 3' end of env or the U3 part of the long terminal repeat, were deleted as previously described (18).

Infections were performed with the same batch of virus titrated in vivo on rhesus macaques and stored in liquid nitrogen. The recipient animals were seronegative for simian T lymphocyte virus-1, simian retrovirus-1, and herpesvirus B. They were 8.50 ± 0.97 years old. The inguinal and axillary LNs were sequentially collected under general anesthesia at 0, 7, 14, and 60 days after infection. All procedures with animals were performed after general anesthesia with ketamine (Imalgène 1000; Mérial).

Abs and reagents

Fluorochrome-labeled mAbs used in this study were human specific and tested on primates, including anti-CD3-PerCP) (clone SP34-2; BD Biosciences), anti-CD4-FITC (clone M-T477; BD Biosciences) or -allophycocyanin, anti-CD8-PerCP (clone SK1; BD Biosciences) or -FITC, anti-CD45RA-PE-labeled (clone 2H4; Beckmann Coulter), anti-CD62L-FITC (clone SK11; BD Biosciences), anti-HLA-DR-PE (clone G46-6; BD Biosciences), anti-CD95-allophycocyanin (clone DX2; BD Biosciences), anti-CCR5-PE (clone 3A9; BD Biosciences) and anti-Ki67-FITC (clone Ki67; DAKO). Only anti-CD3-FITC (clone FN18; Biosource International) was macaque specific. The other Abs used were anti-BOB (BD Biosciences), anti-Bcl-2-PE (clone 83-8B; MBL), anti-Bcl-W (MBL), anti-phospho-p53 (Ser15; Cell Signaling), anti-Puma (bbc3 C-terminal; Sigma), anti-Bax (N-20; Santa Cruz), and anti-Bak (2-14; Calbiochem). These Abs recognized the active form of Bax and Bak proteins (conformational N-terminal epitope). We also used a recombinant human Fas ligand molecule (rhCD95L) (Alexis), a labeled goat anti-rabbit IgG Ab (Alexa Fluor 488; Molecular Probes), and a broad spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk; Bachem). The reagents used to assess dying cells were 3,3'-dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes), annexin V-FITC (Beckman Coulter), and annexin V-allophycocyanin (Bender Systems).

Cell culture and cell death quantification

Fresh LN cells were incubated for 24 h at 37°C with 5% CO2 in RPMI 1640 supplemented with 10% FCS (AbCys), penicillin (50 U/ml), streptomycin (50 U/ml), glutamine (2 mM), and sodium pyruvate (1 mM). Cells were incubated in the absence or presence of the rhCD95L (200 ng/ml) and were pretreated with zVAD-fmk (10 µM). Cell death was assessed by flow cytometry (19). Briefly, after staining with specific Abs (30 min at 4°C), cells were washed and then incubated with fluorescent labeled-annexin V (20 min at 4°C). However, technical constraints concerning CCR7 staining (indirect labeling in primates) and Ki67 staining for cycling T cell subpopulations (permeabilization procedure) preclude the quantitation of dying populations based on these markers. Mitochondrial membrane depolarization was assessed using the fluorescent probe DiOC6.

Fresh T cells of noninfected macaques were cultured in 24-well culture plates (BD Biosciences) at a concentration of 5 x 105/well. Abs to chemokine receptors (CCR5 and BOB/GPR15) (10 µg/well) were immobilized on the plate using 0.1 M Tris-HCl buffer (pH 9.6). Cells were cultured for 2 days, and the percentage of dying cells was quantified by flow cytometry using FITC-labeled annexin V (Beckman Coulter). Furthermore, cells were also incubated for 4 days in the absence (mock) or presence of SIV (multiplicity of infection (MOI), 0.01). Cells were incubated in the absence or presence of combination of the reverse transcriptase inhibitor didanosine (ddI; 1 µM), added at two time points (before SIV and 2 days later).

Lymphocyte immunophenotyping by flow cytometry

Cell surface staining was performed on axillary or inguinal fresh LN cells. Anti-CD3-PerCP, anti-CD4-FITC, or anti-CD8-FITC and anti-CD95-allophycocyanin Abs were added to 105 LN cells for a 15-min incubation at 4°C. After a washing, the cells were fixed before flow cytometric analysis (FACSCalibur; BD Biosciences). For intracellular staining, 105 fresh LN cells were incubated with anti-CD3-PerCP and anti-CD4-allophycocyanin for a 15-min incubation at 4°C and then fixed and permeabilized with 250 µl of Cytofix/Cytoperm (BD Biosciences). After washings with Perm&Wash (BD Biosciences), the cells were incubated for 45 min with anti-Ki67-FITC. After washings, the cells were fixed and analyzed by flow cytometry. A total of 10,000 events in the lymphocyte gate were analyzed using CellQuest software (BD Biosciences).

Expression of the members of the Bcl-2 family

Frozen cells from uninfected and infected macaques (both SPs and MP) were assessed the same day to preclude technical variabilty. CD8-PercP and CD4-allophycocyanin staining was first performed; then the cells were fixed and permeabilized (Perm&Fix; BD Pharmingen) before adding specific Abs to the Bcl-2 family. After a washing, FITC-labeled goat anti-rabbit IgG Ab was added (except for Bcl-2-PE) for 30 min at 4°C. Cells were finally washed, fixed, and analyzed by flow cytometry. A total of 10,000 events in the lymphocyte gate was analyzed using CellQuest software (BD Biosciences).

Western blotting

Fresh LN cells (16 x 106) of uninfected, MP, and SIVmac251{Delta}nef-infected animals, collected at day 60 postinfection, were incubated in the absence or presence of rhCD95L (500 ng/ml; Alexis Biochemical). After overnight culture, cells were washed and then lysed at 4°C in digitonin (2.5 µg/ml) buffer. After centrifugation, the cytosolic fraction (supernatant) was collected, whereas the pellet was lysed using Nonidet P-40 buffer. After centrifugation, the membrane fraction (supernatant) was retrieved. The extracts were quantified and then analyzed by electrophoresis on a 4/20% polyacrylamide gel (Bio-Rad). Proteins were then transferred to a nitrocellulose membrane (Hybond) and immunoblotted with a mouse monoclonal anti-AIF Ab (E-1; Santa Cruz Biotechnology), anti-Smac/Diablo (Alexis Biochemicals) and anti-heat shock protein 60 (clone 24; BD Biosciences). Western blots were visualized using a HRP-conjugated secondary Ab (Amersham Biosciences) followed by ECL (Amersham).

Viral quantification

In situ hybridization. Viral replication in LNs was assessed by in situ hybridization using a 35S-labeled RNA probe derived from pBluescript encoding the SIVmac nef gene as previously described (10, 20). Infected cells, detected as spots, were counted in the paracortical zone on a minimum of three sections using a Nikon-FXA microscope. The number of positive cells (counted as spots) was then divided by the surface of the entire LN section, and results were expressed as the number of positive cells per 2 mm2 section. The mean count obtained for three slides was calculated.

Viral load. Quantification of serum SIV RNA levels was assessed by real time quantitative RT-PCR as previously described (10, 20). The fluorescence signals were detected with an ABI Prism 7700 sequence detector (PE Applied Biosystems) and were captured and analyzed using the Sequence Detector Software. The SIV RNA copy number was determined in reference to a standard curve prepared by RT-PCR amplification of serial dilutions of an in vitro-transcribed SIV gag RNA. The detection threshold of RT-PCR was 10 copies/ml.

Statistical analysis

Data were analyzed using the nonparametric Mann-Whitney U test and Student’s t test, and the confidence interval of the regression analysis was determined using the Spearman test (Prism software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Fas/CD95 expression during primary SIV infection

Macaques were infected either with the live attenuated SIVmac251nef-deleted isolate ({Delta}nef) or with the wild-type SIVmac251 strain (SIV+) and followed up for clinical progression to AIDS. We recently reported that {Delta}nef animals remain healthy for at least 6 years after infection (18). Infection of rhesus macaques with {Delta}nef resulted in a peak of viremia at day 14 postinfection (mean, 8.2 x 104 SIV RNA copies/ml; range, 4.8 x 104–1.3 x 105 copies/ml) (Fig. 1A). SIV+-infected macaques were sorted in two groups, MPs and SPs. MPs developed AIDS (a wasting syndrome with cachexia and opportunistic infections) and died 1–2 years after infection. whereas SPs died after 3–5 years. Both SPs and MPs displayed a peak of viremia at day 14 postinfection with a similar number of copies of SIV RNA (SPs: mean, 1.6 x 107 copies/ml; range, 5 x 106–2.9 x 107 copies/ml. MPs: mean, 1.7 x 107 SIV RNA copies/ml; range, 7 x 105 to 5.3 x 107 copies/ml) (Fig. 1A). This peak was followed by a decrease in viremia reaching a plateau at day 120 (Fig. 1A). The viremia plateau levels were lowest in the {Delta}nef-infected monkeys (mean, 58 SIV RNA copies/ml; range, 10–202 copies/ml), higher in SPs (mean, 564 SIV RNA copies/ml; range, 10 to 1904 copies/ml), and the highest in MPs (mean, 6.1 x 105 SIV RNA copies/ml; range, 9.9 x 103–2.1 x 106 copies/ml).


Figure 1
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  		FIGURE 1. Viral load and Fas/CD95 expression during primary SIV infection. A, Kinetics of plasma viremia in {Delta}nef-infected (n = 4), SIV+ SPs (n = 4), and SIV+ MPs (n = 8); B, proportion of LN CD4+ and CD8+ T cells expressing CD95. CD95 expression was assessed only for four MPs of eight. Each symbol represents one individual. Bar, mean. Statistical significance was assessed using the Mann-Whitney U test.

 
Because a higher CD95 expression was reported in HIV-infected individuals (21, 22, 23, 24), we determined the expression of Fas/CD95 in CD4+ and CD8+ T cells in the different monkey groups. CD95 expression was assessed only for four MPs of eight (94856, 94860, 94852, and 97087). Fresh lymphocytes were isolated from LNs retrieved at days 0, 7, 14, and 60 after infection and analyzed by flow cytometry. The fraction of CD4+ T cells expressing CD95 was unchanged during primary SIV infection. In MPs only, the percentage of CD8+ T cells expressing CD95 increased gradually with time. The increase in CD8+ T cells was significant between day 0 and day 7 (41.5 ± 3.7–55.7 ± 1.01%; p = 0.049) and between day 0 and day 60 (41.5–74 ± 10.4%; p = 0.034) (Fig. 1B). Interestingly, in one animal, there was a delay in the increase of CD95 expression on CD8+ T cells coinciding with a delay in viral replication. We found that the types of CD8+ T cell subsets that increased were of the CD45RA– CD8+ T cell subset (CD8+RA– at day 0 = 29 ± 4.8%; CD8+RA– at day 60 = 57 ± 13.8%; p = 0.034; data not shown).

Correlation between the extent of CD4+ T cell death during primary SIV infection and progression to AIDS

We recently found that extensive death induction in peripheral LNs during primary SIV infection was an early and predictive event of progression toward AIDS (10), but the phenotype of the cells that are more prone to die and the mechanisms involved in death were unknown. Thus, we determined the propensity of LN CD4+ and CD8+ T cells from SIV+ macaques to undergo death. Annexin V labeling performed on fresh cells showed that CD4+ T cell death increased between day 0 and day 14 (from 17.7 ± 4.3% to 33.7 ± 11.5%, p = 0.04) and then remained increased at day 60 (38.7 ± 12.4%; p = 0.03 between day 0 and day 60; Fig. 2A, left panel). A similar trend was observed in the kinetics of CD8+ T cell death, although only the increase between day 0 and day 60 (from 18.2% ± 5 to 40.2% ± 6.9) was significant (p = 0.03) (Fig. 2A, right panel).


Figure 2
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  		FIGURE 2. Correlation between the extent of CD4+ T cell death at day 60 postinfection and progression toward AIDS. A, Proportion of dying LN CD4+ and CD8+ T cells at days 0, 7, 14, and 60 after infection. Values are the mean ± SD of 8 SIVmac251+ animals at day 0, 6 at day 7 (3 SPs and 3 MPs) and 12 at days 14 and 60 (4 SPs and 8 MPs). B, Proportion of dying LN CD4+ and CD8+ T cells incubated in the presence or absence of rhCD95L, from SIV+ SPs (n = 4) and SIV+ MPs (n = 8) at day 14. C, Proportion of dying LN CD4+ and CD8+ T cells incubated in the presence or absence of rhCD95L, from {Delta}nef-infected animals (n = 3), SIV+ SPs (n = 4), and SIV+ MPs (n = 8) at day 60. Each symbol represents one animal. Bar, mean. Statistical significance was assessed using the Mann-Whitney U test. D, Correlation between spontaneous or rhCD95L-mediated CD4+ and CD8+ T cell death at 2 wk and viral replication, as measured by in situ hybridization at 2 wk postinfection. E, Correlation between spontaneous or rhCD95L-mediated CD4+ and CD8+ T cell death at 2 mo and viral replication, as measured by in situ hybridization at 2 mo postinfection. {square}, SPs; {blacksquare}, MPs. Statistical significance was assessed using the Spearman test.

 
We then determined the relationship between progression to disease (SPs vs MPs) and spontaneous or rhCD95L-mediated apoptosis of LN CD4+ and CD8+ T cells at 2 wk and 2 mo postinfection. The rate of spontaneous CD4+ T cell death at 2 wk was 21.5 ± 1.2% and 37.2 ± 11.3% in SPs and MPs, respectively (p = 0.006; Fig. 2B). The rate of spontaneous CD8+ T cell death was similar in MPs (28.1 ± 13.7%) and in SPs (21.7 ± 3.7%) (Fig. 2B). At 2 mo, we also assessed death in {Delta}nef macaques. The rates of spontaneous CD4+ T cell death were 12.8 ± 3.2, 27.2 ± 3.6, and 44.5 ± 11% in {Delta}nef, SPs, and MPs, respectively. The rate of spontaneous CD8+ T cell death was similar in both SPs (41.4 ± 3.5%) and MPs (39.7 ± 7.3%) but was lower in noninfected (18.2 ± 5%; Fig. 2A) and in {Delta}nef-infected monkeys (10.6 ± 2.9%; Fig. 2C).

Activation-induced cell death, involving members of the death receptor family, is one possible mechanism involved in silencing the immune response (16). Treatment with rhCD95L performed on fresh cells increased both CD4+ and CD8+ T cell death. At day 14, this increase was quite similar in CD4+ T cells (10.2% in SPs and 7% in MPs). In CD8+ T cells, a significant increase of cell death due to rhCD95L incubation was noticed in CD8+ T cells of SPs (33.8%) compared with MP (9.2%) (p = 0.02) (Fig. 2B). At day 60, this increase was equivalent in each group of monkeys, and was higher in CD8+ than in CD4+ T cells (12% cell death in CD8+ T cells vs 8% in CD4+ T cells; Fig. 2C). Thus, the impact of CD95L-mediated death seems minor. Although preincubation of the cells with zVAD-fmk, a broad caspase inhibitor, prevented rhFasL-mediated T cell death, zVAD-fmk had no effect on spontaneous T cell death (data not shown). We also found that the percentages of cycling CD4+ T cells in the LNs assessed by flow cytometry using Ki67 marker increased slightly on day 60, higher in MPs than in SPs (MPs, 7.7 ± 3.9% and SPs, 3.1 ± 1.7% vs 2.1 ± 0.9% before infection, p = 0.04 and p = NS, respectively) (Fig. 3). On day 14, the increased proportion of cycling cells was similar in SPs (4.3 ± 2.7%) and in MPs (5 ± 2.5%) despite the difference in the propensity to die. Therefore, the rates of CD4+ T cell death were consistently higher than the fractions of cycling CD4+ T cells.


Figure 3
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  		FIGURE 3. Dynamics of cycling CD4+ T cells during primary SIVmac251 infection. The percentage of CD4+ T cells expressing Ki67 was assessed by flow cytometry from fresh LN cells of SIV{Delta}nef-infected macaques (n = 3) (Figure 3) and 12 SIVmac251-infected macaques (4 were SPs; Figure 3), and 8 were MPs ({blacksquare}). Statistical significance was assessed using the Mann-Whitney U test.

 
Because the extent of viral replication in the LNs at 2 mo has a predictive value on the rate of progression to AIDS (8, 9, 10), we next measured whether the extent of CD4+ and CD8+ T cell death correlated with the extent of viral replication in LNs. Productively infected cells were quantitated by in situ hybridization. No correlation, but a trend, was observed between the rate of viral replication at day 14 and the extent of spontaneous or rhCD95L-induced CD4+ or CD8+ T cell death (Fig. 2D). On the contrary, at day 60, there was a positive correlation between the level of viral replication and the rate of CD4+ T cell death (p = 0.022), but not with the rate of CD8+ T cell death (p = 0.57; Fig. 2E, top panels). Furthermore, we did not observe any correlation between the extent of viral replication and the rate of rhCD95L-induced cell death in CD4+ (p = 0.112) or in CD8+ T cells (p = 0.21; Fig. 2E, bottom panels). We found that the frequency of SIV RNA+ CD4+ T cells at 2 mo postinfection never exceeded 1/100 (mean, 0.0031 ± 0.0025), supporting the idea that most of the dying CD4+ T cells are uninfected cells.

Nature of dying CD4+ T cells during primary SIV infection

To precisely determine the nature of the dying CD4+ T cells, we performed quadruple color staining of LN T cells from SIV+ at 2 mo postinfection. During antigenic stimulation, T cell differentiation takes place according to the following sequence: naive T cells (CD45RA+ CD62L+) forming a population of central (central memory T cells) or early memory T cells that lose the CD45RA molecule. Further stimulation of central memory T cells leads to proliferation of intermediate memory cells that have lost CCR7 followed by the production of effector (effector memory T cells; TEM) which lack CD62L expression. These effector memory T cells may re-express the CD45RA molecule, in response to Ag stimulation, becoming terminal differentiated effector T cells (TDT) (25, 26). However, quantitation of CCR7 on dying cells was precluded by the need for indirect labeling. Therefore, we assessed dying subpopulations in CD4+ T cells, based on CD45RA and CD62L (Fig. 4A), from LNs retrieved before infection, and at day 60 in SPs and MPs macaques.


Figure 4
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  		FIGURE 4. Subsets of dying CD4+ T cells. A, Dot plots show the expression of the different subset markers on CD4+ T cells on day 0 and day 60 from one SP and one MP. B, Percentage of CD45RA+CD62L+ cells (45+62L+), CD45RA–CD62L– cells (45–62L+), CD45RA–CD62L– cells (45–62L–), and CD45RA+CD62L– (45+62L–) subset (top) and undergoing death (middle) from LNs of SIV– ({square}), SPs at day 60 (Figure 4), and MPs at day 60 ({blacksquare}). Bottom panel shows the proportion within each subset that undergoes death and was obtained by calculating the fraction of dying subset among T cell subset (e.g., % of dying 45–62L–/% of 45–62L– subset x 100). C, Death of effector memory T cells (45RA–62L–) expressing CD95 (45–95+) or not (45–95–) at day 60 in SPs (Figure 4) and MPs ({blacksquare}). D, Death of effector memory T cells (45RA–62L–) expressing HLA-DR (DR+) or not (DR–). Data are the mean ± SD of two noninfected macaques, three SP macaques, and three MP macaques. Statistical significance was assessed using the Mann-Whitney U test. {pi}, p < 0.05. E, Schematic model of dying CD4+ T cells during primary SIV infection. EM, Effector memory; CM, central memory; TCM, central memory T cells.

 
First, we found, on the basis of on these markers, that on day 60, LNs from MPs displayed a decrease in naive CD4+ T cells (28.2 ± 10.6%) and a trend toward decrease in percentage of naive CD4+ T cells in SPs (28.4 ± 15%) compared with before infection (50.7 ± 7.7%) (Fig. 4B, top panel). Conversely, CD4 subsets in LNs displayed a trend toward an increase in the percentage of central memory T cells in SPs (39.6 ± 20.1% vs 19.5% 5.6 before infection) and an increase in effector memory T cells from MPs (41.8 ± 11.2% vs 22.3 ± 6% before infection; Fig. 4B, top panel).

Secondly, the LN CD4+ T cells that are more prone to die were from the TEM and TDT subsets both in noninfected and SIV+ macaques (Fig. 4B, middle panel). However, our data demonstrated that TEM are more prone to die in SIV+ MPs (36.5 ± 13.7%) than in SPs (16.3 ± 5%) than in noninfected macaques (9.6 ± 3.2%) (Fig. 4B, middle panel). Thus, in noninfected macaques, the fraction of the dying TEM cells represents 42.4 ± 2.3%. In infected macaques, this percentage of dying cells within TEM subset rises to 67.1 ± 5% in SPs and 84.7 ± 12.7% in MPs. In contrast, although among the TDT subpopulations dying cells represent 73% in SIV–, 85% in SPs, and 74% in MPs of the TDT subset (Fig. 4B, bottom panel), the percentage of dead cells was not significantly increased in SIV+ macaques (Fig. 4B, middle panel).

Finally, we found that dying CD45RA– T cells were both CD95– and CD95+ (Fig. 4C) and that most of the dying LN CD4+ T cells were resting T cells (HLA-DR–; Fig. 4D). Given that percentages of dying cells at day 60 (27.2% in SPs and 44.5% in MPs; Fig. 2C) were much higher than the percentage of cycling cells (3.1% in SPs and 7.7% in MPs; Fig. 3), then it leads to the conclusion that most of dying cells were not cycling cells. We propose a schematic model of dying CD4+ T cell subsets during primary SIV infection (Fig. 4E). It will be interesting in future studies to identify more precisely the phenotype of these dying effector T cells using novel combinations of markers.

CD4+ T cell death is caspase and AIF independent but involves mitochondrial membrane depolarization

Mitochondria are central sensors for the regulation of the apoptotic cascade (27). We next determined whether dying cells (annexin V+) displayed mitochondrial membrane potential ({Delta}{psi}m) loss. Typical flow cytometric profiles shown are from one of three macaques from a noninfected, a {Delta}nef-infected, and a SIV-infected macaque (MP) at day 60. We found that cells displayed a membrane potential loss that was proportional to the rate of cell death. Both membrane potential loss and cell death increased in the presence of rhCD95L (Fig. 5A). Moreover, preincubation of the cells with zVAD-fmk did not prevent {Delta}{psi}m loss (data not shown). These findings indicate that in contrast to the essential role of caspase activation downstream of CD95 ligation, activation of caspases that are involved in producing certain features of the apoptotic phenotype (chromatin condensation and fragmentation) are dispensable for the spontaneous death of T cells from SIV+ animals.


Figure 5
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  		FIGURE 5. Association between CD4+ T cell death at day 60 postinfection and mitochondrial membrane potential loss. A, CD4+ T cell death after 24 h of culture, as assessed by flow cytometry using FITC-labeled annexin V; mitochondrial membrane depolarization was shown using DiOC6. Typical flow cytometric profiles shown are from one of three macaques from noninfected, {Delta}nef-infected, and SIV+ (MP) animals at day 60. B, AIF and Smac release during T cell death. Cytosolic (C) and membrane (M) fractions of LN T cells from one noninfected, one {Delta}nef-infected, and one SIV+ (MP) animal are shown. Heat shock protein 60 (Hsp60) was used as a control of cell fractionation. Cells were incubated for 24 h in the presence or absence of rhCD95L.

 
AIF has been proposed as a major factor in caspase-independent cell death (28), although this remains controversial (29, 30). Thus, to assess the role of AIF in T cell death in SIV-infected macaques at day 60, we determined whether AIF was released from the mitochondria to the cytosolic fractions in LN cells incubated in the absence or presence of rhCD95L. The experiments were performed in one noninfected, one {Delta}nef, and one SIV+ monkey. We did not observe any difference in the pattern of spontaneous and rhCD95L-mediated AIF release between the three monkeys, suggesting that AIF did not play a decisive role and was not responsible for the increase in T cell death observed during SIV infection (Fig. 5B). However, Smac/Diablo was detected after release into the cytosolic fraction of T cells from SIV+ compared with the cytosolic fraction of T cells from {Delta}nef.

Increase in Bak expression in CD4+ T cells during primary SIV infection

Because we observed a {Delta}{psi}m loss during the LN T cell death (even in the presence of zVAD-fmk), we investigated the role of members of the Bcl-2 family on mitochondrial membrane depolarization. This family consists of both pro- and antiapoptotic members that elicit opposite effects on the mitochondria (14). The expression levels of members of the Bcl-2 family was analyzed in frozen LN CD4+ T cells before and 60 days after SIV infection in six macaques (three were SPs and three were MPs). Because the antiapoptotic molecule Bcl-2 is constitutively expressed and has been reported to be down-regulated in the blood during HIV infection (31), we first quantified the fraction of Bcl-2low. We found no significant increase in T cells expressing low Bcl2 (Bcl-2low) between day 0 and day 60. In contrast, we found a trend toward a decrease in the Bcl2low subpopulation in MPs (10.4 ± 3.5% at day 0 vs 3.8 ± 1.3% at day 60; Fig. 6A). We found no change in the expression of the antiapoptotic protein Bcl-W (Fig. 6B). We next assessed whether SIV infection was associated with a change in the pro-apoptotic Bax and Bak proteins, which are considered as main regulators of mitochondrial injury (32). There was no change in the fluorescence intensity associated with Bax between day 0 and day 60 in both SPs and MPs (Fig. 6C). However, expression of the active form of Bak (detected by measuring the N-terminal epitope) exhibited a trend toward an increase at day 60 in MPs (from 11.6 at day 0 to 44.9 mean of fluorescence intensity (MFI) at day 60; p = 0.049), but did not increase in SPs (Fig. 6D).


Figure 6
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  		FIGURE 6. Expression of pro- and antiapoptotic members of the Bcl-2 family in LN CD4+ T cells at days 0 and 60 postinfection. Left panel, Typical flow cytometric analyses at day 0 and day 60. Right panel, SPs ({circ}) and MPs (•). Each symbol represents one animal. A, Bcl-2 expression. The proportion of Bcl-2low was calculated as [Bcl-2low/(Bcl-2low + Bcl-2high)] x 100. B, Bcl-W expression. Data are expressed as the MFI of Bcl-W expression. C, Bax expression. Data are expressed as the MFI of Bax expression. D, Bak expression. Data are expressed as the MFI of Bak expression. E, Phosphorylated p53Ser15 expression. The proportion of phosphorylated p53 (p53P) was calculated as [p53+/(p53+ + p53–)] x 100. F, Puma expression. Data are expressed as the MFI of Puma expression. – – – –, Isotype Ab control. Statistical significance was assessed using the Mann-Whitney U test.

 
It has been proposed that Puma, which is induced by p53, could drive mitochondrial membrane depolarization during HIV infection (33). Thus, we analyzed in CD4+ T cells at days 0 and 60 the expression of the phosphorylated form of p53 on serine 15 (p53S15P), which is important in regulating the ability of p53 to induce apoptosis and Puma. We found a higher proportion of CD4+ T cells expressing phosphorylated p53 at day 60 only in one animal of six (Fig. 6E). Puma expression showed a trend to decrease in the CD4+ T cells from MPs (from 108 at day 0 to 36 MFI at day 60; p = 0.049; Fig. 6F) but not in those from SPs. Taken together, these results showed that LN CD4+ T cells from SIV+ monkeys that progress more rapidly toward AIDS display a higher staining of Bak, which may explain the loss of mitochondrial membrane depolarization in those cells.

SIV sensitizes CD4+ T cells of non-infected macaques to death

Because we and others (34, 35, 36, 37) have shown that most of the apoptotic cells are uninfected and that the simple binding and/or penetration of viruses (without integration) may be sufficient to prime T cell death, we assessed whether SIV primes CD4+ T cells from noninfected macaques to undergo cell death. In the absence of any additional stimulus, SIV (MOI 0.01) induced a process of CD4+ T cell death, which began to be detectable after 2 days and was maximal after 4 days (data not shown). Whereas supernatants from uninfected control-CEMx174 cultures caused <20% CD4+ T cell death after 4 days, incubation with SIV (MOI 0.01) led to the death of 40% of the CD4+ T cells, thus causing 20% CD4+ T cell death in excess of the control (Fig. 7C). At lower concentrations, we did not observe elevated CD4+ T cell death (Fig. 7D). Quiescent CD4+ T lymphocytes are not permissive to SIV infection in the absence of additional stimuli. p27 ELISA analysis indicated the absence of detectable virus production (data not shown). Pretreatment of CD4+ T cells with ddI (1 µM) did not prevent SIV-induced CD4+ T cell death, indicating that SIV proviral integration and expression were not required for the induction of T cell death. Consistent with previous reports (34, 35), suggesting that HIV-1 Env-mediated engagement of its coreceptor may by itself cause CD4+ T cell death, we found that plate-immobilized CCR5- and BOB/GPR15-specific Abs also induced CD4+ T cell death in the same proportion as the strain of SIV we used (Fig. 7, A and B). In contrast to CD4+ T cells, neither ligation of chemokine receptors using specific Abs nor SIV induced the death of CD8+ T cells (Fig. 7, B and C). Finally, our data demonstrated that incubation with SIV leads to a Bak conformational change (MFI 108 vs 131) in the same range as in SIV-infected macaques progressing more rapidly toward AIDS (Fig. 7E). In contrast, no changes in Bcl-2 and Bax expression or in the level of phosphorylation of p53 on serine 15 were observed.


Figure 7
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  		FIGURE 7. SIV mediates priming of resting CD4+ T cells for death. A and B, Engagement of chemokine receptor-mediated T cell death. Cells were incubated for 2 days in the absence (None: Ig control) or presence of plate-bound specific Abs (CCR5 or BOB/GRP15). A, Histograms show the expression of annexin V on CD4+ T cells by flow cytometry. B, Percentages of dying CD4+ and CD8+ T cells in response to chemokine receptor engagement. Data are the mean ± SD of three independent experiments. Significant differences (*, p < 0.05) were identified using a paired Student’s t test in comparison with isotype control (none). C, Cells were incubated for 4 days in the absence (Medium: Mock) or presence of SIV (MOI 0.01) (SIV) and in the absence or presence of ddI (1 µM). At day 4, the percentage of dying CD4+ and CD8+ T cells was determined. Results are the mean ± SD of two independent experiments. D, Dose response of SIV using different MOI. The percentage of dying CD4+ T cells was determined at day 4. E, Flow cytometric analyses at day 4 of Bcl-2 (FL2), phosphorylated p53(Ser15) (p53P) (FL1), Bak (FL1) and Bax (FL1) on CD4+ T cells (FL4). Percentages in the dot plots and MFIs in the histograms are shown. FL, Fluorescence.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results reveal that the rate of spontaneous CD4+ T cell apoptosis early after SIV infection postinfection is predictive of progression toward AIDS and that this apoptotic process follows the intrinsic (rather than the extrinsic) cell death pathway. This inference is based on several lines of evidence. First, dying LN T cells displayed signs of mitochondrial membrane potential loss despite the inhibition of caspase activation. Second, CD95 expression in CD4+ T cells remained unchanged during primary SIV infection, and most dying LN T cells were CD95– CD4+ T cells. Third, LN CD4+ T cells from MPs displayed higher N-terminal epitope availability of Bak than those from SPs, whereas levels of Bcl-2 and Bax proteins remained unchanged. Finally, exposure to SIV in vitro led to the death of the CD4+ T cells from noninfected donors concomitant with an active form of Bak.

Most studies that have investigated the potential in vivo relationship between HIV and SIV infections and apoptosis have focused on peripheral blood and on the chronic phase of infection (14). Previously, we reported that T cells from peripheral blood of SIV-infected macaques were more prone to die during primary infection (38). Moreover, two studies have indicated that cell death in tissues was higher in animals infected with pathogenic SIV than in those infected with the attenuated strain SIV{Delta}nef (39, 40). Recently, we also found that the extent of T cell death in LNs during primary infection predicts disease progression (10) and increase of apoptosis was also seen in lamina propria (41). To the best of our knowledge, this is the first report demonstrating the relationships between the extent of expression of activated Bak, CD4+ T cell death and further progression to AIDS during primary SIV infection. Thus, the extent of CD4+ (but not CD8+) T cell apoptosis at the peak of viral replication and at the set point is an early predictive marker of disease outcome.

It has been reported that overexpression of the proapoptotic proteins Bax and Bak triggers rapid cell death even in the presence of caspase inhibitors, and that this non-apoptotic cell death occurs through the generation of reactive oxygen species (ROS) (42, 43). Because Bak affects the mitochondria electron transport (following cytochrome c release and loss of ATP), which results in the disruption of the mitochondrial membrane potential (44), this may provide at least one mechanism leading to the death of the CD4+ T cells during primary infection. An increased production of ROS has been observed in PBMC from HIV-infected individuals (45). Thus, the involvement of ROS during primary SIV infection merits further analysis.

AIF has been proposed as a major effector released from the mitochondria into the cytosol in a caspase-independent pathway (28). However, this observation remains controversial (29, 30). Herein, we observed an equivalent expression level of the cytosolic form of AIF in both infected and uninfected macaques. Furthermore, the broad caspase inhibitor zVAD-fmk had no preventive effect on T cell death. Altogether, these observations suggest the existence of an alternative pathway independent of AIF and the caspase cascade in inducing the death of CD4+ T cells from SIV-infected macaques. Moreover, it has been reported that p53 phosphorylation on serine 15 is correlated with plasma viral load (33) and that its phosphorylation leads to an up-regulation of Puma in the LNs of HIV-infected individuals (46). Here, we found a decrease in Puma expression in MPs at 2 mo postinfection compared with SPs. Interestingly, the extent of Puma expression before infection was higher in MPs than in SPs. Therefore, the expression of Puma before infection might be predictive of the rapidity of the rate of progression toward AIDS, possibly reflecting a polymorphism in normal individuals. This remains to be addressed. Moreover, the results presented herein indicate that only minor changes in the phosphorylated p53 (p53S15P) expression occur in CD4+ T cells during primary SIV infection (one of the six animals displayed positive staining for phosphorylated p53). A possible explanation could be that our study focused on the early phase of infection, whereas in HIV-infected individuals, only the end-terminal phase of infection was addressed (33, 46). Despite the role of p53 in the regulation of pro-apoptotic genes, our results support the idea that the increase in Bak expression is independent of p53 signaling during primary SIV infection. Moreover, our data indicated that exposure to SIV in vitro-induced death of resting CD4+ T cells from noninfected macaques that was associated with expression of activated Bak independently of p53 phosphorylation on serine 15. In this context, SIV mediates death of nonactivated and nonproductively infected cells. Consistent with these observations, we found that ex vivo dying cells from SIV-infected macaques were at least in part CD95– and HLA-DR–. Given the percentage of cycling CD4+ T cells at the peak (4.6%) and at the set point (3.1% in SPs and 7.7% in MPs) compared before infection (2.1%), the propensity of T cells to die is much greater, supporting the idea that most of dying cells were nonactivated and potentially noncycling cells. Thus, the interaction between the envelope and the CD4-coreceptor complex may constitute a major potential candidate for SIV-mediated bystander killing of CD4+ T cells during acute viremia.

We also demonstrated herein that TEMs were more prone to death during primary infection (Fig. 3E), explaining, at least in part, how HIV and SIV infections impair the immune response very early. Indeed, we have previously shown that the response to recall-Ags (e.g., bacillus Calmette-Guérin) is lost at an early stage following SIV infection (10). In humans, it has been shown that CD4+ T cell differentiation was abortive, that HIV-specific CD4+ T cells died during the asymptomatic phase (47, 48), and that early highly active antiretroviral therapy preserved CD4+ T cell immune response (49, 50). Given that the quality of the CD4+ T cells affects the cytotoxic CD8+ T cell response (51, 52, 53, 54, 55, 56, 57), it is likely that the death of CD4+ T cells early after infection impairs the control of infection and facilitates the progression toward AIDS. Finally, one consequence of such abnormal T cell apoptosis during primary infection could be to facilitate the dissemination of HIV/SIV in vivo by modulating immune responses. Engulfment of apoptotic cells have been shown to inhibit the production of proinflammatory mediators by macrophages/dendritic cells, by secretion of anti-inflammatory cytokines such as TGF-β (58). Such anti-inflammatory events can inhibit Ag presentation and promote microbial growth within macrophages such as HIV replication (59).

Interestingly, variable rates of T cell death were observed among the individual animals in this study, despite the fact that they received the same batch of virus and the same dose via the same route. These results suggest the existence of individual host factors that predispose to AIDS. It would be interesting to investigate how the host’s genetic polymorphism and/or individual life history may condition the extent of CD4+ T cell death. There are several possible explanations for the individual variations in CD4+ T cell death. First, differences in the signaling pathways that participate in apoptosis may lead to diverse individual CD4+ T cell sensitivity to death. Although we cannot formally rule out that the cells have already received a lethal hit via death receptor ligands in vivo (60), we did not inhibit death using a broad caspase inhibitor, suggesting a minor role of the death receptors and their ligands. Interestingly, a recent genomic analysis of human CD95 and CD95L polymorphisms also indicates no significant association between CD95/CD95L with AIDS (61). Second, because the sensitivity of cells to death also depends on the degree and duration of immune activation (activation-induced cell death), individual variations may be related to life history and prior infections. Third, individual variations in chemokine receptor expression may determine CD4+ T cell death because the sensitivity of CD4+ T cells to die in vitro depends on the levels and on the nature of the chemokine receptors expressed (34, 36, 62, 63). Herein, we found that a strain of SIV that uses CCR5 and BOB/GPR15 molecules as coreceptors causes death of noncycling primary CD4+ T cells, and that ligation of those coreceptors induced CD4+ T cell death. Given that SIV mediates death to a similar extent as its coreceptor, we cannot exclude the possibility that a nef-deleted virus could induce death through coreceptor engagement as well. The absence of priming for cell death in vivo in macaques infected with SIV{Delta}nef might be probably related to the lower rate of viral SIV{Delta}nef replication. This work emphasizes the critical role played by coreceptors in cell death induction. Thus, the extent of in vivo expression of BOB/GRP15 and of CCR5 Ags could impact on disease outcome. Strategies involving the use of molecules that block viral binding to CCR5 have been assessed in the past few years in the macaque model for their ability to prevent SIV infection or to induce a decrease in viral load but their efficacy is incomplete, suggesting either that the levels of CCR5 differ between individuals or that an alternative coreceptor is involved in cell death induction (64). Given that the engagement of coreceptors may also induce the release of interleukins that have been reported to exert proapoptotic activities, such as IL-10 (21, 65) and more recently type 1 IFN through a TRAIL-dependent pathway (66), the role played by such cytokines in response to ligation of CCR5 or BOB/GRP15 remains to be assessed.

These data lead us to suggest that strategies targeting cell death could slow down the immunopathogenesis of HIV/SIV infection, and particularly those targeting the intrinsic cell death pathway. Identification of host factors leading to increased CD4+ T cell death and inhibiting the mechanisms involved is an essential step to improve our arsenal against HIV infection.


   Acknowledgments
 
We thank Audrey Brussel and Jean-François Zagury for helpful discussions and Nathalie Arhel, John Zaunders, and Nuala Mooney for critical reading of the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Agence Nationale de Recherches sur le Sida (ANRS) (to J.E.); a doctoral fellowship from ANRS (to L.V.); and a postdoctoral fellowship from Sidaction (to F.P.). Back

2 This work is dedicated to Bruno Hurtrel. Back

3 Address correspondence and reprint requests to Dr. J. Estaquier, Unité de Physiopathologie des Infections Lentivirales, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris cedex 15, France. E-mail address: jestaqui{at}pasteur.fr Back

4 Abbreviations used in this paper: LN, lymph node; AIF, apoptosis-inducing factor; Bcl-2, B cell lymphoma; MP, moderate progressor; SP, slow progressor; rhCD95L, recombinant human Fas ligand molecule; zVAD-fmk, Z-Val-Ala-Asp-fluoromethylketone; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; MOI, multiplicity of infection; ddI, didanosine; TEM, effector memory T cells; TDT, terminal differentiated effector T cells; MFI, mean of fluorescence intensity; ROS, reactive oxygen species. Back

Received for publication December 14, 2005. Accepted for publication August 18, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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