The CD8 cytotoxic T lymphocyte response plays a vital role in the control of HIV replication. However, its eventual inability to halt AIDS progression in most patients without highly active antiretroviral therapy (HAART) is not understood. Under conditions of diminishing T cell help and chronic antigenic stimulation, CD8 T cells are thought to degenerate into an anergic and pre-apoptotic state characterized by altered differentiation patterns and functionality [1–6]. Deficiencies within the interleukin-2 (IL-2) system, normally critical in CD8 T cell differentiation, may contribute to the failure of CD8 T cells leading to AIDS [7–9].
Previously , we have demonstrated that CD8 T cells from chronically infected patients with high HIV viral load failed to enter into the cell cycle upon IL-2 stimulation even though the IL-2 receptor (IL-2R) α, β, and γc chains were highly expressed. However, CD8 T cells from HAART-treated patients, expressing lower levels of IL-2R components, responded to IL-2. This suggested that defects in the IL-2R-mediated signalling pathway may be involved. The activation of the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) signalling pathway (involving Jak-1, Jak-3, and STAT5) plays a critical role in IL-2-induced T lymphocyte proliferation [10–14]. Furthermore, STAT5 defects have been observed by others in unstimulated CD3 and CD4 subsets of peripheral blood mononuclear cell (PBMC) from HIV-positive patients [15,16]. Therefore, the present study investigates whether this pathway is functionally defective in CD8 T lymphocytes before and after HAART and the mechanisms that could be involved.
Chronically HIV-infected patients were recruited from Assistance Publique-Hôpitaux de Paris. Group A included 11 untreated patients with high viral load [mean 1.09 × 105 copies/ml (range, 0.22–1.35)] and low CD4 cell counts [223 × 106 cells/l (range, 57–473)]. For these patients HAART was initiated. Samples were taken at the time of HAART initiation (D0) and following 6 months of therapy (M6). After M6, viral load was < 20 copies/ml and CD4 counts were 243 × 106 cells/l (range, 131–957). Group B included 10 HAART-treated patients with viral load < 20 copies/ml and CD4 cell count of 527 × 106 cells/l (range, 258–1402).
CD8 T lymphocyte purification
CD8 T cells were purified from PBMC by positive selection using anti-CD8 antibody-coated beads (Dynal; Oslo, Norway), as described elsewhere . Cells were > 90% pure by flow cytometry with < 4% natural killer cells (data not shown).
Immunoprecipitation and immunoblotting
Cytoplasmic protein extracts were prepared in lysis buffer [0.2% NP-40, 10% glycerol, 50 mmol/l Tris and NaF, 10 mmol/l KCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA) and sodium orthovanadate, 10 μg/ml phenylmethylsulphonyl fluoride and leupeptin] and immunoprecipitated using anti-STAT5a, anti-Jak-3 (Santa Cruz, California, USA) and anti-STAT5b (BD Pharmingen, San Diego, California, USA) antibodies. Immunoprecipitates were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidine difluoride membranes (Millipore; Bedford, Massachusetts, USA). The membranes were probed sequentially with anti-phosphotyrosine antibody 4G10 (Upstate Biotechnologies; Lake Placid, New York, USA) and anti-STAT or Jak antibody. Expression was revealed using ECL Western blotting (Amersham Bioscience, Amersham, UK).
Phosphorylation of STAT5a, STAT5b and Jak-3 was examined after IL-2 stimulation and was expressed as the relative phosphorylation ratio (RPR), given by 100 (P-tyrsample/P-tyrstandard)/(totalsample/totalstandard). Protein phosphorylation and total protein expression levels were measured using a Fuji IR Las 1000+ CCD image quantification system (Fuji Photo Film, Tokyo, Japan). To evaluate the RPR, signal intensities were normalized to those of a standard extract prepared from IL-2-stimulated phytohaemagglutinin-stimulated PBMC. Equal quantities of the standard were carried through the immunoprecipitation procedure, loaded on each gel and subjected to immunoblotting with the patient samples.
Electrophoretic mobility shift assays
Nuclear extracts were prepared in a buffer containing 400 mmol/l NaCl, 10 mmol/l KCl, 20% glycerol, 20 mmol/l HEPES and 1 mmol/l EDTA and used for electrophoretic mobility retardation of double-stranded DNA probes containing a STAT5-binding consensus sequence (5′-AGATTTCTAGGAATTCAA TCC-3′) [18,19]. Binding reactions contained 1.5 μg nuclear extract, 1 ng 32P-labelled probe, 10 mmol/l Tris pH 8.0, 100 mmol/l KCl, 5 mmol/l MgCl2, 1 mmol/l dithiothreitol, 0.5 mg/ml bovine serum albumin, 5% glycerol, and 50 ng/μl poly(dI-dC). For supershift experiments, anti-STAT5 (STAT5a, STAT5b; Upstate Biotech.) or pan-STAT5 (Santa-Cruz) antibodies were also included at 50 ng/μl. DNA–protein complexes were resolved by SDS-PAGE and signal intensity quantified by a STORM 860 Phosphorimager (Molecular Dynamics, Amersham, UK).
Interleukin-2 receptor expression
PBMC were incubated with anti-IL-2Rα, anti-IL-2Rβ (Immunotech, Marseilles, France), anti-IL-2Rγ (Pharmingen) or isotype control antibody [mouse IgG1 (DAKO, Glostrup, Denmark), rat IgG2a and IgG2b (Caltag Laboratories, Burlingame, California, USA)], followed by the corresponding species-specific antibody conjugated with fluorescein isothiocyanate (Jackson Immunoresearch, West Grove, Pennsylvania, USA) and CD8-phycoerythrin antibody (DAKO). Analysis was performed on a FACScan flow cytometer (Becton-Dickinson, Mountain View, California, USA).
Failure of interleukin-2-induced STAT5 activation in CD8 T cells from a subset of untreated patients
IL-2-induced STAT5 activation was investigated by immunoblotting and electrophoretic mobility shift assay EMSA (Fig. 1). Strikingly, the results revealed the existence of a subset of group A patients at D0, here termed IL-2 low responders, whose CD8 T cells failed to activate STAT5 in response to IL-2.
Figure 1a shows a representative IL-2 low responder patient. At D0, it is clear that STAT5a phosphorylation was inhibited, but a restoration occurred after 6 months on HAART. Out of the 11 patients that could be evaluated for STAT5a phosphorylation, six were identified as IL-2 low responders, with a RPR < 4. All but one patient recovered the ability to respond after 6 months of HAART (P = 0.03). The remaining five patients (IL-2 responders) were clearly able to phosphorylate STAT5a at both D0 and M6 (Fig.1b). There was a statistically significant difference between IL-2 low responder and IL-2 responder patients at D0 (P = 0.03). Both group B and uninfected patients had the capacity to respond to IL-2 and phosphorylate STAT5a to levels indistinguishable from group A at M6 (Fig.1c). Patients identified as IL-2 low responders based on STAT5a activation also failed to phosphorylate STAT5b in response to IL-2 (data not shown).
EMSA experiments revealed that the patients classified as IL-2 low responders by STAT5 phosphorylation status also failed to activate STAT5 functionally (Fig. 1d–f). Indeed, STAT5 activation was not detectable in IL-2 low responders at D0 following 15 min of IL-2 stimulation but there was a similar activation to that in controls after 6 months of HAART (Fig. 1d). In contrast, STAT5 activation in the IL-2 responders at D0 or M6 was comparable to group B and HIV-negative donors. Moreover, out of the 10 patients analysed by EMSA from group A, five were classified as IL-2 low responders at D0 (Fig. 1f). All but one recovered responsiveness by M6 (P = 0.03). The other five patients from group A were responders and their response was significantly higher at D0 than that in IL-2 low responders (P = 0.001).
IL-2 low responder CD8 T cells fail to phosphorylate Jak-3 in response to IL-2
Investigations into the mechanism responsible for the inhibition of STAT5 activation in CD8 T cells from IL-2 low responders first involved testing for differences in IL-2R expression. As shown in Fig. 2a, there were no statistically significant differences between IL-2 responders and low responders at the level of IL-2R α, β or γc chain expression. Furthermore, comparing individual patients from the two subsets, IL-2R expression did not correlate with STAT5 activation status. This suggested that the failure in STAT5 activation was likely a signal transduction defect upstream of STAT5. Since Jak-3 is important in IL-2 responsiveness and mediates the phosphorylation of STAT5, IL-2-dependent Jak-3 phosphorylation was evaluated in the CD8 T cells of patients from each group (Fig. 2b). At D0, a clear failure of IL-2-induced Jak-3 activation was observed in the low responders, which was restored following 6 months of HAART. Similar to observations for STAT5, IL-2 responders from group A as well as controls (group B and HIV negative) retained the capacity to respond to IL-2 and phosphorylate Jak-3, irrespective of HAART.
We studied the IL-2-dependent signal transduction pathway leading to STAT5 induction in purified CD8 T lymphocytes from a group of HIV-infected patients before and after approximately 6 months of HAART. CD8 T lymphocytes derived from 6 of 11 of the untreated HIV-positive patients studied (termed IL-2 low responders) failed to activate the STAT5 pathway in response to ex vivo stimulation with IL-2. Signal transduction via this pathway was restored following 6 months of HAART in all but one patient. Specifically, in response to IL-2 the tyrosine phosphorylation of STAT5a/b was inhibited in the CD8 T cells of IL-2 low responders. Attenuation of STAT5 phosphorylation translated into a reduced capacity for nuclear translocation and DNA-binding activity, as detected by EMSA. Since IL-2 has been reported to drive T cell proliferation partially via STAT5,, the defective STAT activation observed here is in keeping with our previous results  and further highlights a potential mechanism contributing to the CD8 T cell insufficiency observed in HIV infection.
It has been shown that activation of STAT5 is important in cell cycle progression and protection from apoptosis [10,13,20,21]. Therefore, failure of CD8 T cells to enter into cell cycle in response to IL-2 observed previously in patients with high virus load  may be partially because of faulty IL-2-induced STAT signalling. The lack of IL-2-dependent STAT activation could, therefore, have a significant negative impact on the maintenance of CD8 T cell responsiveness in HIV infection. This is supported by observations that CD8 T cells from HIV-positive patients are anergic and more prone to spontaneous apoptosis than uninfected controls and that culture with IL-2 in some cases was unable to deliver a protective effect .
We investigated why the CD8 T cells from certain group A HIV-positive patients were fully capable of mobilizing STAT5 while others were not. This effect could not be attributed to differences in CD4 T cell counts, virus load, IL-2R expression, IL-2 binding or other cell surface markers studied (HLA-DR, CD38, CD28, CD45RA, CD45RO) but did correlate with an impaired activation of the upstream kinase in this pathway, Jak-3 (Fig. 2 and data not shown). In the absence of Jak-3 activation, STAT5 would not be phosphorylated and the pathway would be blocked. Despite our efforts, reliable measurement of Jak-1 phosphorylation was not possible (data not shown). It may be that negative regulators of cytokine signalling from the CIS/SOCS family or certain phosphatases, including the Jak-1 and Jak-3 selective phosphatase TCPTP, may be involved in maintaining Jak-3 in a dephosphorylated state [23–27]. Preliminary results excluded a role for CIS and TCPTP. Furthermore, whether IL-2-induced Ras/MAP kinase and PI3-kinase pathways are involved remains to be investigated.
This is the first study identifying specific activation defects of the Jak/STAT pathway in response to IL-2 in CD8 T lymphocytes of HIV-infected individuals. It has been demonstrated that STAT5a/b expression was reduced in unstimulated CD3 T cells purified from HIV-positive patients; however, their functional status was not addressed . Recent work has also shown that a potentially dominant negative mutant of STAT5 was constitutively activated in the PBMC of HIV-positive patients . The absence of truncated versions of STAT5 in CD8 T cells was not unexpected as they have been mainly associated with CD4 subsets of HIV-infected individuals .
The restoration of IL-2 responses at the level of Jak/STAT function identifies an important benefit of HAART. It is possible that, through the control of virus replication, HAART interrupts the chronic antigenic activation of the immune system and thereby allows recovery of IL-2 responsiveness. The relevance of our data can also be extended to the benefit of IL-2 immunotherapy when given in combination with HAART. In this setting, IL-2 would potentially have beneficial effects on the cytotoxic function of T cells in HIV-seropositive patients. In conclusion, the restored responsiveness to a cytokine critical in cytotoxic T lymphocyte differentiation and proliferation may, in turn, help to maintain or restore the capacity of patient CD8 T cells to respond to HIV and other opportunistic pathogens.
The authors wish to thank Y. Percherancier, T. Planchenault, F. Bachelerie and J-L. Virelizier for the use of the CCD camera as well as V. Di Bartolo and O. Acuto for their advice and help with the phosphorimager. A. Kumar and K. Gee are acknowledged for critical reading of the manuscript. We also thank M.-T. Rannou, A. Cross, I. Jacquin and J. Desmares for their help in the recruitment of patients from the different hospital centres.
Sponsorship: This work was supported by grants to JT from the Agence Nationale de Recherches sur le SIDA (ANRS) and SIDACTION. MK was a recipient of a postdoctoral fellowship from the Canadian Institutes of Health Research and VP is supported by a studentship from the ANRS.
Note: MK and VP contributed equally to this work.
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