Interleukin-7 (IL-7) is a key regulator of T-cell homeostasis, with essential functions in thymopoiesis and in the survival of naive T cells, as indicated by the profound lymphopenia in IL-7 or IL-7Rα-deficient hosts [1,2]. IL-7 is also required for the long-term survival of memory T cells after the clonal expansion phase, especially within the CD4+ T-cell subset . In addition, IL-7 availability controls the homeostatic proliferation of CD4+ T cells, in normal as well as lymphopenic conditions . Thus, IL-7 regulates the CD4+ T-cell population at multiple levels, including development, survival, proliferation, and niche size.
IL-7 effects are mediated by signaling via the receptor IL-7R, comprising the common γc chain CD132 and the private chain IL-7Rα/CD127. IL-7 binding to its receptor activates the associated Janus kinases JAK1 and JAK3, which in turn activate the transcription factor STAT5 through phosphorylation at tyrosine Y694 [1,5]. Phosphorylated STAT5 then dimerizes, migrates to the nucleus, and acquires the capacity to bind its DNA targets and activate transcription. STAT5 is also regulated through serine phosphorylation, which controls its interactions with other transcription factors. In particular, phosphorylation of serine S726 located in a conserved proline-serine-proline (PSP) motif of the STAT5 transactivation domain can be induced by treatment with γc family cytokines in primary T cells . Phosphorylation of STAT5 at S726 appears to have a predominantly negative regulatory role on transcription, though its activity may depend on the promoter context [7–9]. In primary T cells, STAT5 acts directly as a transcription factor but also indirectly in triggering a second wave of signaling dependent on the phosphoinositide 3-kinase (PI3K) pathway [10,11]. These combined pathways promote T-cell survival and growth, inhibit apoptosis, and facilitate entry into the cell cycle [12,13].
Importantly, IL-7 signaling is perturbed in both CD4+ and CD8+ T cells of patients with progressive HIV infection, resulting in decreased proliferative responses, decreased induction of the survival factor Bcl-2, and increased apoptosis [14–20]. Given the key role of IL-7 in CD4+ T-cell homeostasis, defective IL-7 signaling may contribute significantly to the CD4+ T-cell loss characteristic of HIV disease. One mechanism for IL-7 response impairment is receptor downregulation. Expression of CD127 is markedly decreased in patient CD8+ T cells, especially in the more differentiated memory and effector subsets, resulting in a loss of IL-7 responses within these subsets [21,22]. The downregulation of CD127 is less marked in CD4+ T cells, but still significant within the memory subset, resulting in a decreased percentage of responding cells in this population [23,24]. The downregulation of CD127 in progressive HIV infection tightly correlates with the induction of immune activation markers [22,25–27] and may be explained by a chronic excess of cytokine or T-cell receptor (TCR) signals .
Several lines of evidence indicate that IL-7 signaling is also impaired at a postreceptor level in progressive HIV infection. We had previously shown that IL-7 functional responses, such as the induction Bcl-2 and CD25, correlated with CD127 expression in CD4+ T cells from healthy donors, but not in those from viremic patients . This was particularly striking in the naive CD4+ T-cell subset, in which CD127 receptor expression was preserved, while the decrease in Bcl-2 induction was significant . The early signaling response, measured by the phosphorylation of STAT5 at Y694, was unexpectedly efficient in naive CD4+ T cells from viremic patients, with an induction of pY694-STAT5 per cell that was even higher than that detected in healthy donors . Thus, neither receptor downregulation nor pY694-STAT5 phosphorylation could account for the loss of Bcl-2 induction in the naive subset. These findings pointed to a block downstream of the early STAT5 phosphorylation step, but above the late IL-7-dependent signaling responses leading to Bcl-2 induction. To investigate this issue, we set to identify the precise step that may be defective in the IL-7 signal transduction chain in patient CD4+ T cells.
We first tested the hypothesis that STAT5 phosphorylation may be defective at the regulatory serine S726. We found that S726 phosphorylation was efficient and even hyperactivated in naive CD4+ T cells of viremic patients. Increased S726 phosphorylation correlated positively with that of Y694, pointing to a general hyperphosphorylation of STAT5 in progressive HIV infection. We then asked whether activated STAT5 could efficiently migrate into the nuclear compartment. Quantitative image analysis revealed that this step was impaired in viremic patients, with both forms of phosphorylated STAT5 showing an abnormal accumulation within the cytoplasm. Thus, HIV infection perturbed IL-7 signal transduction by impairing the access of STAT5 to the nuclear compartment.
Patients and methods
The group of viremic patients (n = 23) consisted in patients with untreated chronic HIV-1 infection. Inclusion criteria were a viral load more than 10 000 HIV-1 RNA copies/ml plasma, a CD4+ T-cell count more than 100/μl, the absence of antiretroviral treatment or an interruption of treatment for at least 6 months, and no evidence for primary HIV infection. The patients had a median age of 37 years (range 23–64), a median viral load of 31 245 HIV RNA copies/ml (range 12 000–352 767), and a median CD4 cell count of 443/μl (range 317–1108). The control group (n = 23) consisted in anonymous healthy individuals who donated blood at the Etablissement Français du Sang (EFS, Paris, France). The flow cytometry experiments concerned subgroups of 13 viremic patients and 13 healthy donors, whose characteristics are reported in Table 1. The study, designated EP33–2, was promoted by the Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS). The study was approved by the Comité de Protection des Personnes de l’Ile de France VII under number 05–15. All participants gave written informed consent prior to blood sampling.
Intracellular phospho-specific labeling
STAT5 phosphorylation was detected by flow cytometry after intracellular labeling. Briefly, 400 μl aliquots of heparinized blood were stimulated with 2 nmol/l recombinant human glycosylated IL-7 (Cytheris, Issy-les-Moulineaux, France). Blood was stimulated for 15 min at 37°C, before fixation and red cell lysis with the Lyse/Fix Phosflow buffer (BD Biosciences, San Jose, California, USA) for 10 min at 37°C. After lysis, cells were permeabilized with methanol and stained according to our previously published protocol . For pY694-STAT5 analysis, cells were stained with the following antibody combination: CD45RA-V450 Horizon and pY694-STAT5-Alexa Fluor 647 (pY694-STAT5-AF647; BD Biosciences), CD3-allophycocyanin-eFluor 780 (eBioscience, San Diego, California, USA), CD25-PE (Dako, Glostrup, Denmark), CD4-ECD (Beckman Coulter, Fullerton, California, USA), and FoxP3-AF488 (eBioscience). For pS726-STAT5 analysis, a rabbit antibody specific for pS726 (sc-12893-R; Santa Cruz Biotechnology, Santa Cruz, California, USA) was used as a primary reagent in combination with the same cell surface markers and with a FoxP3-AF647 antibody (eBioscience). The secondary reagent consisted in a goat antirabbit IgG (H+L)-AF488 serum (Invitrogen, Carlsbad, California, USA). Each experiment included a ‘fluorescence minus one’ (FMO) control in which the pSTAT5-specific antibody was replaced by the corresponding isotypic control, either IgG1-AF647 (BD Biosciences) or purified rabbit IgG. Fluorescence was acquired on the same day on a CYAN flow cytometer (Coulter) using the SUMMIT V4.3.02 software. Flow cytometry data were analyzed with the FlowJo V8.8.4 software (Tree Star, Ashland, Oregon, USA).
As antibodies to IL-7 receptor chains CD127 and γc did not give detectable binding to methanol-permeabilized cells, a separate set of experiments was carried out to evaluate expression of these markers on unpermeabilized whole blood cells, as described previously . The following antibody combinations were used: CD25-FITC, CD132-PE, CD45RA-V450 and CD3-V500 (BD Biosciences); HLA-DR-PECy7 and CD127-APC-AF750 (eBioscience); and CD4-ECD.
Quantitative image analysis of STAT5 subcellular localization
Peripheral blood mononuclear cells (PBMCs) were resuspended at 2 × 106 cells/ml in RPMI 6140 medium supplemented with 2 mmol/l glutamine, antibiotics, and 0.5% human AB serum, and then cultured in 24-well plates at 1 × 106 cells/well in the presence or absence of 500 pmol/l recombinant human IL-7 for 30 min at 37°C. After adding 1 ml of ice-cold phosphate-buffered saline (PBS), cells were transferred onto precoated poly-L-lysine coverslips deposited in 24 well plates and centrifuged for 7 min at 1200 rpm at 4°C. After fixation with 4% paraformaldehyde (PFA) and permeabilization with 0.4% triton X-100 in PBS, cells were stained with primary antibodies to pY694-STAT5 (clone C11C5; Cell Signaling Technology, Danvers, Massachusetts, USA) or to pS726-STAT5 (sc-12893-R) and CD4 (BD Pharmingen, San Jose, California, USA) at 1 : 100 in staining buffer [PBS, 2% fetal bovine serum (FBS), 0.1% Triton X100] for 1 h at room temperature. Cells were washed once and then incubated with secondary antibodies goat antirabbit IgG (H+L)-AF594 and goat antimouse IgG (H+L)-AF488 (Invitrogen) for 1 h at room temperature. Coverslips were mounted onto glass slides in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, California, USA) to stain nuclei. Fluorescence was imaged on an upright Zeiss Axioplan 2 microscope using a Plan Apochromat 63x/1.4-oil immersion objective. Images were collected with a cooled CCD camera piloted by the Axiovision 4.6 imaging software (Zeiss, Oberkochen, Germany). Optical sectioning was performed according to the ‘structured illumination’ principle  using the ApoTome system (Zeiss). A script was implemented in the Acapella 2.0 software (PerkinElmer, Waltham, Massachusetts, USA) to measure the distribution of pY694-STAT5 or pS726-STAT5 in the nuclear and cytoplasmic compartments. The total cell area was defined as the area enclosed by CD4-FITC staining. The nucleus was defined as the closed area containing DAPI staining, and the cytoplasm as the total cell area minus the nucleus area. pSTAT5 accumulation in these compartments was defined as the sum of gray values corresponding to the pSTAT5-AF594 staining in each area. Analyses, including cell segmentation and total gray value computation, were carried out automatically on a minimum of 50 CD4+ T cells per slide. The ratio of pSTAT5 accumulation in the cytoplasm to that in the whole cell area (Rcyto/cell) was computed for each cell. The median Rcyto/cell ratio obtained for all CD4+ T cells analyzed per slide is reported.
Analyses were performed with the Prism 5.0 software (GraphPad Software, La Jolla, California, USA), using nonparametric statistical tests in all cases. Correlations were analyzed with Spearman's coefficient R. All significant differences between groups (P < 0.05) are reported on data plots.
Distinct regulation of STAT5 phosphorylation at S726 and Y694 in response to interleukin-7
IL-7-dependent signaling was investigated by flow cytometry in 13 healthy controls (HD group) and 13 patients with a median viral load of 40 204 HIV-1 RNA copies/ml (VIR group; Table 1). The phosphorylation status of STAT5 was evaluated with antibodies specific for pS726 and pY694. Intracellular labeling was carried out directly on whole blood samples, to achieve an accurate detection of STAT5 activation level, without perturbations due to Ficoll gradient separation. Unstimulated CD4+ T cells from healthy donors were found to harbor high levels of pS726-STAT5 at baseline, as shown for a representative individual (Fig. 1a; gray line). Stimulation with IL-7 led to a limited induction of pS726-STAT5 (Fig. 1a; black line). In the HD group, IL-7 stimulation resulted in a median increase in pS726-STAT5 mean fluorescence intensity (MFI) of 1.34x in naive (Fig. 1b, left) and 1.39x in memory CD4+ T cells (Fig. 1c, left), which was statistically significant (P < 0.05 in both cases). In contrast, pY694-STAT5 expression was minimal in unstimulated CD4+ T cells from healthy donors, but showed a marked induction after IL-7 treatment, with a median increase of 12.6x in naive CD4+ T cells (Fig. 1d, left) and of 16.0x in memory CD4+ T cells (Fig. 1e, left; P < 0.0001 in both cases). Thus, phosphorylation of the regulatory serine S726 appeared mostly constitutive and inducible only to a limited extent, whereas phosphorylation of the tyrosine Y694 proved to be highly sensitive to IL-7 stimulation, especially within the memory CD4+ T-cell subset.
Hyperphosphorylation of STAT5 in naive CD4+ T cells of viremic patients
Analysis of pS726-STAT5 expression in the VIR group showed a limited induction after IL-7 stimulation, with a median MFI increase of 1.27x in naive CD4+ T cells (Fig. 1b, right; P = 0.007) and 1.34x in memory CD4+ T cells (Fig. 1c, right; P = 0.002), which was within the same range as the induction factor measured in the HD group. Interestingly, the absolute pS726-STAT5 MFI appeared higher in the VIR group than in the HD group, with a difference that was significant in the naive CD4+ T-cell compartment after IL-7 treatment (P < 0.05). Thus, S726 appeared hyperphosphorylated in naive CD4+ T cells of viremic patients, even though the expression of the IL-7 receptor was slightly decreased in this subset (Table 1).
Analysis of pY694-STAT5 expression in the VIR group showed a potent induction after IL-7 stimulation, with induction factors of 12.9x and 13.8x within the naive and memory CD4+ T-cell subsets, respectively (P < 0.0001 in both cases). Again, the absolute pY694 MFI proved to be higher in the VIR group than in the HD group, when considering naive IL-7-stimulated cells (P = 0.002). Basal pY694-STAT5 expression was also increased in naive CD4+ T cells from viremic patients (P = 0.018). These data suggested a chronic hyperphosphorylation of Y694-STAT5 in naive CD4+ T cells from viremic patients, consistent with our previous findings .
Because regulatory T cells (Tregs) respond less efficiently to IL-7 than conventional memory CD4+ T cells [31,32], a separate analysis was carried out to evaluate STAT5 phosphorylation in the Treg population. Tregs were defined by a CD3+ CD4+ FoxP3+ CD25hi phenotype and were subdivided into a CD45RA+ naive subset (NTreg) and a CD45RA- memory subset (MTreg). Viremic patients showed an increased percentage of NTregs as compared to healthy donors (P < 0.05; Table 1), whereas no differences were detected for the MTreg subset in this cohort. Interestingly, basal pY694-STAT5 levels were increased in both Treg subsets of viremic patients (basal pY694 in NTregs: MFIVIR = 27.9 vs. MFIHD = 17.9, P = 0.001; in MTregs: MFIVIR = 26.1 vs. MFIHD = 18.6, P = 0.006). In addition, the expression of pY694-STAT5 after IL-7 stimulation was also increased in the NTreg subset of viremic patients (stimulated pY694 in NTregs: MFIVIR = 235 vs. MFIHD = 178, P = 0.004; in stimulated MTregs: MFIVIR = 165 vs. MFIHD = 137, P = 0.12). A trend for increased basal phosphorylation of S726-STAT5 was also observed in NTregs and MTregs of viremic patients (P = 0.09 in both cases; data not shown), though it did not reach statistical significance. Taken together, these analyses suggested that the abnormal activation of STAT5 extended to the Treg compartment.
Correlation analyses revealed a positive association between pS726-STAT5 and pY694-STAT5 expression, both within the naive subset (Fig. 1f; RVIR = 0.66, P = 0.013) and the memory subset (Fig. 1g; RVIR = 0.73, P = 0.005) of stimulated CD4+ T cells in viremic patients, whereas analyses in the HD group did not yield significant correlations (not shown). Similarly, analyses restricted to the Treg population showed a positive correlation between pS726-STAT5 and pY694-STAT5 expression, both in the NTreg subset (RVIR = 0.97, P < 0.001) and in the MTreg subset (RVIR = 0.75; P = 0.02) of viremic patients (data not shown). Thus, progressive HIV infection caused the hyperphosphorylation of both the regulatory serine S726 and the key tyrosine Y694, with these changes correlating together. These findings suggested that an abnormal activation of intracellular signaling contributed to the deregulation of IL-7 responses in HIV infection.
Abnormal cytoplasmic accumulation of pSTAT5 in viremic patients
We next focused on the step involving the nuclear translocation of STAT5, which occurs after its phosphorylation at Y694 by JAK kinases . STAT5 subcellular localization in CD4+ T cells stimulated or not with IL-7 was analyzed by immunofluorescence, using a microscopy system with optical sectioning that allowed colocalization measurements. pS726-STAT5 was found to concentrate predominantly within the nucleus of unstimulated CD4+ T cells from healthy donors (Fig. 2a, left). The localization of pS726-STAT5 remained similar 30 min after IL-7 stimulation (Fig. 2a, right). Intriguingly, some viremic patients showed a distinct distribution, with a significant fraction of pS726-STAT5 present within the cytoplasm, both before and after IL-7 stimulation (Fig. 2b). Consistent with the flow cytometry data, pY694-STAT5 was barely detectable in unstimulated CD4+ T cells (Fig. 2c, left). IL-7 addition to healthy donor CD4+ T cells resulted in pY694-STAT5 induction, which accumulated predominantly within the nucleus, but could also be detected within the cytoplasm (Fig. 2c, right). Analysis of CD4+ T cells from viremic patients showed a similar pattern, with a trend for a higher fraction of pY694-STAT5 remaining within the cytoplasm after IL-7 stimulation (Fig. 2d).
For quantitative analysis, the ratio of pSTAT5 fluorescence in the cytoplasm to that in the whole cell area (Rcyto/cell) was computed on a minimum of 50 CD4+ T cells per sample. This analysis confirmed an abnormal accumulation of pS726 STAT5 in the cytoplasm of CD4+ T cells from viremic patients, both in unstimulated cells (Fig. 3a; P = 0.009) and in IL-7-treated cells (Fig. 3b, P = 0.003). Within each group, the Rcyto/cell ratio did not change significantly before and after IL-7 stimulation (P ≥0.05), suggesting that IL-7 did not induce a major relocalization of pS726-STAT5. Subcellular localization of pY694-STAT5 was not quantified in unstimulated CD4+ T cells, as the immunofluorescence signal did not differ significantly from background staining. After IL-7 stimulation, pY694-STAT5 localized predominantly to the nucleus in the HD group (Fig. 3c, left). Viremic patients showed a distinct distribution, with a subset of patients having an abnormal accumulation of pY694-STAT5 within the cytoplasm (Fig. 3c, right, P = 0.0039). Of note, the Rcyto/cell ratios measured for pY694-STAT5 and pS726-STAT5 showed a strong positive correlation in the VIR group (RVIR = 0.81, P = 0.002; Fig. 3d), indicating that HIV infection perturbed the subcellular localization of both forms of pSTAT5. These findings implied that, in viremic patients, pSTAT5 did not fully relocalize to the nucleus in response to IL-7, and thus could not function efficiently as a transcription factor.
The subcellular localization of pSTA5 was also evaluated in samples from six treated patients with efficiently controlled viral load (viral load < 40 HIV RNA copies/ml in five patients; viral load = 62 HIV RNA copies/ml in the 6th patient). The median Rcyto/cell ratios were of 0.14 (range 0.12–0.15) for pS726-STAT5 in unstimulated CD4+ T cells, of 0.13 (0.11–0.16) for pS726-STAT5 in IL-7-stimulated cells, and of 0.22 (0.18–0.30) for pY694-STAT5 in IL-7-stimulated cells. These ratios were comparable to those measured for healthy donors (P ≥0.05 in the three cases) and were significantly lower than those measured for viremic patients (P < 0.05 in the three cases). Thus, efficient antiretroviral therapy was associated with a normalization of pSTAT5 localization.
Cytoplasmic localization of pSTAT5 correlates with immune activation
We then explored the characteristics of viremic patients with an abnormal cytoplasmic localization of pSTAT5. The increase in Rcyto/cell ratio did not show significant correlations with the CD4+ T-cell count nor with the viral load (not shown). However, the ratio measured for pS726-STAT5 in unstimulated CD4+ T cells correlated positively with expression of the activation marker HLA-DR (RVIR = 0.57, P = 0.039; Fig. 4a). The correlation did not remain significant after IL-7 stimulation (RVIR = 0.08, NS; not shown). Importantly, the Rcyto/cell ratio in unstimulated cells also correlated negatively with CD127 expression (RVIR = −0.63, P = 0.0086; Fig. 4b). CD127 downregulation was a hallmark of activated T cells, as indicated by the negative correlation between CD127 and HLA-DR expression in the viremic group (RVIR = −0.48, P < 0.05) and in the whole cohort studied (RVIR+HD = −0.56, P < 0.0005). These findings suggested that abnormal cytoplasmic localization of pS726-STAT5 depended on chronic immune activation, but was not a direct consequence of HIV replication or CD4+ T-cell depletion.
Analyses of the Rcyto/cell ratio for pY694-STAT5 in IL-7-stimulated cells further supported the notion that chronic immune activation perturbed STAT5 function. Indeed, the pY694 Rcyto/cell ratio correlated positively with HLA-DR expression (RVIR = 0.45, P = 0.04; Fig. 4c) and negatively with CD127 expression (RVIR = −0.65, P = 0.029; Fig. 4d). Thus, immune activation was associated with abnormal cytoplasmic localization of phosphorylated STAT5, both at baseline, as indicated by the pS726 ratio, and in response to IL-7, as indicated by the pY694 ratio. Taken together, our findings identify STAT5 nuclear relocalization as a key signaling step perturbed by HIV infection and suggest an underlying role for chronic immune activation in STAT5 dysfunction.
This work identifies a defect in a distal step of the IL-7 signal transduction chain in progressive HIV infection. The early signaling steps, as measured by phosphorylation of STAT5 at Y694 and S726, were increased rather than decreased in CD4+ T cells from viremic patients. Increased phosphorylation did not result in more efficient STAT5 nuclear translocation, but rather in the accumulation of phosphorylated STAT5 into the cytoplasm. This finding implies that STAT5 cannot efficiently function as a transcription factor and helps explain the defects in late IL-7-dependent functions, such as the induction of CD4+ T-cell survival and proliferation [29,33]. It was relevant that the impairment of STAT5 nuclear translocation correlated with signs of immune activation in CD4+ T cells. Chronic immune activation is thought to drive HIV pathogenesis, through the generation of exhausted, terminally differentiated T cells, the deleterious effects of inflammatory mediators, and the collapse of the regenerative capacity of the immune system [34,35]. This study pinpoints one of the mechanisms by which immune activation may contribute to CD4+ T-cell loss and dysfunction. By targeting the IL-7 system, HIV-driven immune activation perturbs the core mechanism that ensures CD4+ T-cell homeostatic proliferation and survival.
The phosphorylation of STAT5 at S726 had not been previously explored in the context of HIV infection. As this posttranslational modification is thought to negatively regulate STAT5 transcriptional activity [7,9], it is possible that the excess of pS726-STAT5 detected in the naive CD4+ T cells of viremic patients contributes to decreased STAT5 function. However, we do not favor this hypothesis, as the phosphorylation of STAT5 at S726 increased in parallel with that at Y694, suggesting that inhibitory and activating modifications counterbalanced each other (Fig. 2f and g). Rather, we propose that the general increase in STAT5 phosphorylation reflects the activated status of patients CD4+ T cells. We  and others  have shown that baseline Y694 phosphorylation is consistently increased in CD4+ T cells of viremic patients, suggesting that these cells are chronically stimulated by cytokines and/or growth factors that activate STAT5, such as γc cytokines, thymic stromal lymphopoietin (TSLP), IL-3, IL-5, leukaemia inhibitory factor (LIF), or growth hormone . A candidate is IL-7 itself, which is known to be raised in the plasma of HIV-infected patients with depleted CD4+ T cells [4,38]. However, we did not detect a correlation between basal pY694-STAT5 levels and circulating IL-7 in the present study (not shown), suggesting the involvement of other cytokines or growth factors.
The accumulation of pSTAT5 in the cytoplasm of patient CD4+ T cells may result either from a defect in nuclear import or from a shuttling back from the nucleus. STAT5 nuclear import is mediated by a complex formed of importins α and β, the small G protein Rac1, and the small G protein regulator MgcRacGAP . A general block in the importin system is unlikely, as it would drastically compromise cellular viability. However, it is possible that chronic immune activation perturbs the function of the accessory factors MgcRacGAP, which would then specifically impact the import of STAT5. Alternatively, pSTAT5 may exit the nucleus because it cannot bind efficiently to its DNA targets . We have shown in a previous study that the induction of several STAT5 target genes, such as Bcl-2, FoxP3, and CD25, are defective in CD4+ T cells of viremic patients after IL-7 stimulation . Even though we did not evaluate these functions in the present study, we speculate that these functional defects may be direct consequences of decreased STAT5 DNA binding and/or transcriptional activity. It will be important in future studies to determine whether progressive HIV infection can alter the accessibility of STAT5 DNA targets within the chromatin.
The presence of phosphorylated STAT5 in the cytoplasm of patient CD4+ T cells may well have direct functional consequences, in addition to reducing the pool of nuclear STAT5. Indeed, recent evidence indicates that STAT5 does not only function as a transcription factor, but can also activate signaling proteins in the cytoplasm . In particular, constitutively active mutants of STAT5 have a predominantly cytoplasmic localization and mediate their effects through an interaction with the p85 subunit of PI3K, which results in Akt activation . It is also relevant that pY694-STAT5 has a predominantly cytoplasmic localization in cells from patients with myeloid leukemia . Phosphorylation of STAT5 at S726 may also have a cytoplasmic function, and not only an inhibitory effect on transcription, as it is required for the transforming capacity of STAT5 in mouse models of leukemia . Thus, the presence of cytoplasmic pSTAT5 in CD4+ T cells of viremic patients may lead to the abnormal activation of signaling pathways, which could be a driving factor in the chronic immune activation characteristic of HIV infection.
In conclusion, this study provides evidence for hyperphosphorylation and abnormal cytoplasmic localization of STAT5 in CD4+ T cells of viremic patients. These findings help understand the mechanism by which HIV infection impairs IL-7 responses and thus undermines CD4+ T-cell homeostasis.
The authors thank Huguette Berthé and Thomas L’Hyavanc for their help in recruiting patients, Michel Morre for the gift of recombinant human IL-7, and Alain Cosson for advice and reagents. They are especially grateful to the patients who participated in the study.
L.C., I.L., and J.T. conceived and designed the experiments. I.L., F.B., and A.D. performed the experiments. O.L., P.d.T., L.S., and J.F.D. were the clinical referents of the study and contributed to the analysis of the data. L.C. wrote the article. All the authors have read and approved the text as submitted to AIDS.
I.L. is the recipient of a fellowship from SENACYT-IFARHU (Panama). This work was supported by Agence Nationale de Recherche sur le SIDA et les hépatites virales (study ANRS EP33–2) and the Pasteur Institute, Paris, France.
Conflicts of interest
The authors declare no competing financial interests.
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