The loss of CD4 T lymphocytes is a central factor in the progression of HIV infection to AIDS. The key role of these cells in regulating and amplifying the immune response means that any decline in their number results in deficits in both humoral and cell-mediated immunity . Understanding how these CD4 T cells are eliminated is critical for the development of effective new therapies for AIDS. Many mechanisms, including syncytia formation, lysis by cytotoxic T lymphocytes (CTL), and direct cytopathic effects of HIV, have been suggested to explain the curtailed lifespan of HIV-infected CD4 cells. One of the most intriguing phenomena is that the number of dying CD4 T cells in HIV-infected patients greatly exceeds the number of HIV-infected cells . This suggests that HIV has several other detrimental bystander effects on uninfected CD4 T cells. Reports of in vitro experiments show that several HIV-1 proteins, including gp120, Tat and Nef, can use a variety of disparate death pathways to initiate apoptosis in uninfected cells, regardless of whether they are attached to the virion or released by infected cells [3,4]. An alternative proposal is that, in view of the high level of immune activation in HIV-infected individuals, uninfected CD4 T cells may be killed by activation-induced cell death (AICD) through Fas . The relative contribution of these phenomena and their relationship to the receptor tropism to the overall CD4 T cell decline in vivo remains unclear.
In earlier reports, we proposed an alternative hypothesis to explain, at least in part, the progressive CD4 T cell decline that occurs in the course of HIV disease. Specifically, we showed that after HIV infection a substantial fraction of the CD4 T cell population expresses natural killer (NK)p44L, an activator ligand of the natural cytotoxicity receptor NKp44. This expression renders them sensitive to autologous NK lysis . The expression of NKp44L is strongly correlated with both the decline in the CD4 cell counts and the increase in viral load . Ward et al. have recently confirmed the specific expression of NKp44 ligand on CD4 T cells after in vitro infection by HIV-1 . We also showed that NKp44L is induced by a highly conserved motif of gp41 peptide (called 3S), and that antibodies directed against this peptide inhibit in vitro NKp44L expression and CD4 T cell sensitivity to NK lysis . CD4 cell counts in infected individuals were correlated with antibody titres . Taken together, these data suggest that NK cell cytotoxicity may aggravate HIV infection and impair CD4 cell homeostasis. This conclusion is consistent with previous reports by several groups that NK cells may contribute to HIV disease progression [8,9].
To further elucidate the role of NK cells and their ligands in CD4 cell depletion in vivo, we used the SHIV model of HIV-1 disease, since the 3S motif of the gp41 protein is present exclusively in HIV-1 strains, and not in either HIV-2 or SIV . To compare the effect of coreceptor usage, cynomolgus macaques were challenged with one of two viruses, which present the 3S-gp41 motif in the SHIV envelope. SHIV89.6P is a highly pathogenic dual-tropic (CXCR4 and CCR5) virus that causes rapid elimination of CD4 T cells within the first 2 weeks of infection . SHIV162P3 is exclusively a CCR5-tropic virus. Its profile (viraemia and CD4 cell count) in cynomolgus macaques closely resembles naturally transmitted HIV strains . Our results show that CXCR4 and CCR5 as coreceptors might affect NK cell activity differently. NK lysis was correlated most highly with CD4 cell depletion in the macaques infected by the CCR5-tropic virus, in comparison with the CXCR4-tropic virus, which directly targeted CD4 T cells.
Ten adult cynomolgus macaques (Macaca fascicularis), each weighing 4–6 kg, were imported from Mauritius. They were housed in individual cages in level 3 biosafety facilities. All experimental procedures were conducted in compliance with European Community legislation for animal care (Official Journal of the European Communities, L358, December 18, 1986). Five animals were challenged intravenously with 1 ml containing 46.6 × 108 copies of SHIV162P3. This came from a stock prepared as a pool of plasma collected from cynomolgus macaques at peak of viraemia during primary infection (days 12–17 after intrarectal inoculation with SHIV162P3 stock obtained from the NIH AIDS Research & Reference Reagent Program, catalogue number 6526). The other five animals were intravenously infected with 10 AID50 doses of a cell-free stock of the SHIV89.6P kindly provided by A.M. Aubertin (ULP, Strasbourg, France) .
Plasma viral load measurement
Viral RNA was prepared with the High Pure Viral RNA Kit (Roche Diagnostics, Meylan, France) from 200 μL EDTA-treated cell-free plasma, in accordance with the manufacturer's instructions. RNA was eluted in 50 μl nuclease-free water and immediately frozen at −80°C until analysis. Ten-fold dilutions of SIVmac251, titrated by branched-chain DNA assay and diluted in EDTA-treated plasma from uninfected macaques, were used to plot a standard curve. Three titrated SIVmac251-infected EDTA-treated plasma samples and two EDTA-treated plasma samples from SIV-negative macaques were used, respectively, as positive and negative controls for RT–PCR. Standards, controls and viral RNA samples were extracted and tested in parallel, in the same conditions. The SIVmac251 gag cDNA sequence was used as a positive control for PCR. RT–PCR was performed in an iCycler real-time thermocycler (Bio-Rad, Marnes-la-Coquette, France). As described by Hofmann-Lehmann et al. , the probe and primers bind to the conserved SIV gag region. All amplifications were performed in duplicate, and the standard RNA template dilution yielded a correlation coefficient of up to 0.97 over seven orders of magnitude, with a sensitivity of 60 copies/ml.
MultiScreen 96-well filtration plates (Millipore, Guyancourt, France) were coated with monoclonal antibody against monkey interferon (IFN)-γ (clone GZ,4, Mabtech, Nacka, Sweden) at a concentration of 10 μg/ml in phosphate-buffered saline (PBS) at 4°C. Peripheral blood mononuclear cells (PBMC) were recovered by density gradient centrifugation and 2 × 105 cells were added to each well. SIV-Gag peptide pools were then added in triplicate to a final concentration of 10 μg/ml of each peptide in the culture medium.
Plates were incubated for 24 h, washed with 0.1% Tween 20 in PBS and then treated overnight with biotinylated anti-IFN-γ antibody (clone 7-B6-1, Mabtech). Spots were revealed with alkaline phosphatise–streptavidin conjugate (Sigma-Aldrich, St-Quentin Fallavier, France) and then developed by adding NBT/BCIP substrate (Sigma-Aldrich). The spots were counted with an Automated Elispot Reader System with KS software (Carl Zeiss, Le Pecq, France). The background was calculated as twice the mean number of IFN-γ spot-forming cells per 106 PBMC (IFN-γ SFC/million PBMC) in unstimulated samples. Samples yielding more than 50 IFN-γ SFC/million PBMC after removal of the background were scored as positive.
Flow cytometric analysis
Four-colour FACS analysis was performed on freshly harvested blood cells. Isotype-matched immunoglobulin served as the negative control (BD Biosciences Pharmingen, San Diego, California, USA). Briefly, 100 μl of peripheral blood was stained for 30 min, at room temperature under gentle agitation, with an appropriate antibody cocktail provided by BD Biosciences Pharmingen including: anti-CD45 (TÜ116), anti-CD3 (SP34), anti-CD4 (L200), and anti-CD8 (RPA-T8); NKp44L expression was determined using anti-NKp44L mAb (#7.1, IgM), as described previously . After staining, the cells were washed with PBS and erythrocytes were then lysed gently with 1 ml of the FACS lysing solution kit (BD Biosciences Pharmingen), for 10 min under slow agitation (250 rpm at RT). After extensive washing in PBS, and resupension with 300 μl PBS, at least 30 000 events were analysed on a FACScalibur (BD Biosciences Pharmingen). Results were analysed with CellQuest Pro software (BD Biosciences Pharmingen) and expressed as the percentage of all mAb-positive CD45 cells, without discrimination on FCS/SSC profile.
NK cytotoxicity assay and ELISA
Cytotoxicity of NK cells from PBMC samples was evaluated in a 4-h 51Cr release assay, against the MHC class I-deficient erythroleukaemia K562 cell line at several effector: target cell ratios, as described previously . Anti-3S antibodies were quantified with ELISA, as described previously . Anti-3S antibody quantities were expressed in arbitrary units (AU). This test has a detection limit of 10 AU/ml.
Statistical analysis used the Mann–Whitney or Wilcoxon tests, appropriate for small sample sizes, with Graphpad Prism 4 software.
Effect of R5 and X4 viruses on CD4 count and viral load
To allow comparison between the monocytotropic SHIV162P3 and dual-tropic SHIV89.6P infections, the study included 10 cynomolgus macaques. Plasma viraemia was very similar in SHIV162P3- and SHIV89.6P-infected animals. The mean peak of viral load during acute infection (2 weeks postchallenge) was 1.7 × 107 and 1.9 × 107 copies/ml, respectively. After 40 days, plasma viraemia levels were within the range previously recorded in both groups of chronically infected macaques (Fig. 1a) [11,14]. In addition, PBMC-based IFN-γ ELISPOT assays assessed cellular immune responses during SHIV infection. As expected, cynomolgus macaques infected with either SHIV162P3 or SHIV89.6P developed specific responses against Gag; these responses increased slightly 40 days following infection, but did not differ significantly between the two groups of animals (P = 0.812 at day 230) (Fig. 1b). Taken together, these results indicate that these two viruses, with their different co-receptor tropisms, behave similarly in their capacity to replicate and to induce a specific cellular immune response against SIV-Gag.
Next, we compared the T cell dynamics in the two groups. Infection with the acutely cytopathic SHIV89.6P induced a profound and sustained drop in the CD4 T cell count, which fell to 122 ± 57 cells/μl within 3 weeks. These numbers subsequently stabilized over time to a value that never exceeded 500 cells/μl, far below baseline (Fig. 1c). CD8 T cell levels behaved differently, returning to baseline after a slight diminution (data not shown). In contrast, all animals infected with SHIV162P3 showed an early transient but significant decline in CD4 T cells (P = 0.016), which then returned to preinfection values after 21 days and progressively decreased thereafter (Fig. 1c).
Effect of R5 and X4 viruses on NKp44L expression
In view of our recent findings that NKp44L is specifically induced during HIV-1 infection and that its expression is highly correlated with CD4 T cell depletion, we assessed the relationship between NKp44L expression and CD4 T cell counts in SHIV-infected animals throughout the course of this study . As Fig. 2 shows, NKp44L expression increased progressively in SHIV162P3-infected animals to obtain a steady-state level ranged from 175 and 203 CD4NKp44L T cells/μL, between days 58 and 174. The percentage of CD4 T cells expressing NKp44L was significantly and inversely correlated with the circulating CD4 cell count (P = 0.03; Fig. 2 and data not shown). In the SHIV89.6P-infected macaques, however, a transient strong increase of NKp44L was detected on CD4 T cells, 2 weeks after the challenge, similar to the maximal level observed in SHIV162P3-infected macaques, (around 220 CD4NKp44L T cells/μl), followed by a profound decline until a steady-state level ranged from 33 to 62 CD4NKp44L T cells/μl (Fig. 2). In both groups, strong NKp44L induction was accompanied by a profound decline in the CD4 cell counts during primary infection (Fig. 1b), and its continued expression was correlated with the CD4 cell counts during the disease course in SHIV162P3-infected macaques.
Correlation between the level of anti-3S antibodies and the CD4 cell count in SHIV162P3-infected macaques
We also reported that the expression of NKp44L on CD4 T cells during HIV-1 infection is controlled by anti-3S antibodies . To determine whether this process depends on coreceptor usage, we conducted a longitudinal kinetic study in both groups of macaques to determine anti-3S antibody levels. As a control, we also monitored anti-T20 antibody levels after infection in all 10 animals: irrespective of the SHIV strain used, anti-T20 antibody levels remained high throughout the period of observation (Fig. 3a), as they do in HIV-infected patients . Anti-3S antibody production appeared to be transient in macaques infected with SHIV162P3, increasing progressively through day 58 and then declining to baseline (Fig. 3b). Interestingly, in these macaques, the decline in CD4 cell counts was directly correlated with the decrease in the anti-3S antibody level (Fig. 3b). In addition, the presence of anti-3S antibodies in this group was inversely associated with NKp44L expression on CD4 T cells (data not shown). By contrast, in SHIV89.6P-infected animals, anti-3S antibodies level, nearly undetectable before the third week of infection, was minuscule; indeed it never exceeded 50 AU/ml. As compared to SHIV162P3-infected macaques, the level of anti-3S antibody remained significantly lower during the course of the disease (P = 0.008 at 58 days) (Fig. 3b).
High cytolytic activities in SHIV162P3-infected macaques
To determine whether the modulation of NKp44L was associated with increased NK lysis capacity, we compared both after SHIV162P3 and SHIV89.6P infection. The absolute number of NK (CD3−CD8+) cells began to decrease 10 days post-infection (Fig. 4a). However, the decline was significantly greater in the SHIV89.6P group, reaching a nadir at 14 days post-infection, together with the CD4 cell counts. In both groups, the NK cells count reached pre-infection values after 35 days and remained stable throughout the rest of the observation period (Fig. 4a). Assessment of the cytolytic capacities of these NK cells showed an early transient increase of specific NK lysis in both groups, peaking at day 80 for SHIV162P3-infected macaques and day 40 for SHIV89.6P-infected animals (Fig. 4b). Cytotoxicity remained high in the former, but returned to baseline in the latter. We then assessed the extent of NK cytotoxicity involved in the CD4 T cell depletion in both groups of animals. In the SHIV162P3-infected macaques, the progressive decline in CD4 cell counts was strongly associated with a high level of NK activity, which remained twice as high as in uninfected macaques (Fig. 4b). In contrast, after SHIV89.6P infection NK lysis was not associated at all with CD4 cell counts (Fig. 4b).
Our previous findings indicated that NKp44L is induced during HIV infection by the interaction of CD4 T cells with a highly conserved motif of the gp41 protein and suggested that the decrease in these cells may be due, at least partially, to NK cytolysis [5,7]. The experiments described here were performed in macaques to investigate in vivo whether such an effect could be detected in non-human primates and to compare the effect of infection with two SHIV strains using either CXCR4 or CCR5 as co-receptors. Our results indicate that the kinetics of CD4 cells decline differs in macaques infected with either SHIV162P3 or SHIV89.6P, while viral load and anti-SIV specific immune responses are similar in animals with both types of infection, as described previously [11,14]. Our results further demonstrate that during SHIV infection, the relationship between NKp44L expression on CD4 T cells, anti-3S antibody production and NK lysis was preserved during SHIV infection but differed substantially according to coreceptor usage.
In SHIV162P3-infected macaques, the pattern of CD4 cell counts was similar close to those in natural HIV infection, both during primary infection and in the chronic phase, in terms of CCR5 utilization, sustained viral replication, and gradual CD4T cell loss . However, the loss of CD4 T cells is much more rapid in macaques, as compared to humans. During SHIV162P3 infection, NKp44L expression was closely correlated with anti-3S antibodies and NK cell activity, which in turn were correlated with CD4 cell depletion. NKp44L expression may thus, at least in part explain CD4 cell depletion during the chronic phase of the disease.
CD4 cell depletion followed a different pattern in macaques challenged with SHIV89.6P. As previously described, SHIV89.6P induced a rapid and sustained decline in CD4 cells , similar to that observed in rapid progressors infected with CXCR4-tropic viruses, such as the rare infection of Δ32 homozygous individuals [15,16]. The association of these viruses with rapid disease progression may be due to its faster rate of viral production, increased cytopathogenicity or its ability to infect and deplete CD4 T cells [17,18]. These CXCR4-tropic viruses often arise in late infection and are associated with progression to AIDS [19,20]. These characteristics may explain why the NK system contributes so little to CD4 cell decline. Moreover, the transient and low production of anti-3S antibodies may be due to the rapid destruction of the 3S-specific CD4 helper T cells, as previously observed in HIV-1 infection .
In addition to virus neutralization, a major challenge for immunotherapy is the prevention of CD4 cell depletion. The strategy for such an approach may well differ according to whether one seeks to prevent the death of infected T cells or an indirect effect on uninfected T cells, or both. Different approaches may be needed according to receptor specificity of the viral strain. Several factors in addition to their co-receptor usage distinguished the CXCR4- and CCR5-tropic viruses including rapid early infection and CD4 cell depletion, as well as a peak viral load that was predictive of disease outcome. This contrasts with what has been observed for CCR5-specific viruses, here and in other studies [14,21,22].
Both types of virus may induce CD4 T cell death either directly or by a bystander effect. Although numerous studies indicate that receptor-specific CD8 T cells may affect lysis of infected CD4 T cells [23,24], the role of the NK cell cytolytic functions in controlling disease development remains elusive. Reports of increased NK activity in some HIV-1-exposed but uninfected patients suggest that NK cells may help to protect against HIV-1 infection . Moreover, several studies show a diminution in the number and function of NK cells during HIV infection and progression to AIDS [8,9], which can have detrimental effects. One study reported a small percentage and sparse number of NK cells in a group of patients with rapid progression to AIDS ; another group reported that the initial un-stimulated NK cell cytotoxicity level does not predict subsequent disease course and observed that NK activity was relatively well preserved in healthy AIDS patients with low CD4 cell counts . On the other hand, various distinct allelic combinations of the KIR3DL1 and HLA-B loci significantly and strongly influence both AIDS progression and HIV RNA abundance . None of these studies mentioned any notable correlation between NK activity level and NK ligand expression in infected versus uninfected cells. HIV-infected cells are relatively resistant to NK lysis, and it has been suggested that this is due to a decrease in MHC class-1 expression . Interestingly, we and others have recently observed that HIV-infected CD4 T cells do not express NKp44L , and are therefore resistant to NK lysis (Vieillard V, Fausther Bovenclo H, Debré P. personal communication). This phenomenon, in contrast to the sensitivity of CD4 cells to NK lysis of cells expressing NKp44L, suggests that the differences observed in CD4 cell homeostasis for CCR5- and CXCR4-tropic SHIV may depend on a balance between the frequency of infected cells, which determines, at a minimum, NKp44L expression, and NK lysis activity. Our data strongly suggested that the NKp44L effect on CD4 cell depletion acted principally during the chronic phase of the infection, when the viraemia is low to undetectable. The number of infected CD4 T cells is low in CCR5-tropic virus infection. This suggests that a large proportion of the CD4 T cells at that point express NKp44L and are thus subject to indirect NK lysis. In contrast, the T-cell tropism of the CXCR4 virus, that is, its ability to infect CD4 T cells, accelerates disease course, which is characterized by early and profound CD4 T cell depletion, and strongly suggests that the virus is directly cytopathic and that these virus-infected cells, which do not express NKp44L, are relatively insensitive to NK cell activity.
Overall, our results provide new pathophysiological insight into HIV disease and into the various pathogenic roles the virus may play depending upon tropism, and could have a consequent effect on the NK cytotoxicity of CD4 T cells. These results suggest that new strategies for preventive and/or therapeutic immunization to stimulate anti-3S antibodies should be discussed with respect to the coreceptor tropism of the virus.
We thank Christophe Jouvert, the veterinarian in charge of animal facilities at CEA and Christophe Jouy, Patrick Flammant and Hélène Juin for excellent technical assistance.
AT2-SIV was provided by Dr Jeff Lifson through the EU Program EVA, Centre for AIDS Reagents, NIBSC, UK (contract QLKZ-CT-1999-00609).
Sponsorship: Supported by Sanofi-Pasteur and the Agence Nationale de Recherche sur le SIDA (ANRS).
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