IL-7-dependent STAT-5 activation and CD8 T cell proliferation are impaired in HIV infection

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1 Article IL-7-dependent STAT-5 activation and CD8 T cell proliferation are impaired in HIV infection Agatha Vranjkovic,* Angela M. Crawley,*, Andrea Patey,* and Jonathan B. Angel*,,,1 *Ottawa Hospital Research Institute and Division of Infectious Diseases, Ottawa Hospital-General Campus, Ottawa, Ontario, Canada; and Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ontario, Canada RECEIVED JULY 30, 2010; REVISED NOVEMBER 9, 2010; ACCEPTED NOVEMBER 12, DOI: /jlb ABSTRACT This study tests the hypothesis that IL-7 signaling and activity of CD8 T cells are impaired in HIV infection. IL-7 is necessary for optimal CTL activity and T cell survival and proliferation. Defects in IL-7R signaling may contribute to impaired activity of IL-7 observed in progressive HIV disease. A decreased proportion of CD8 T cells expressing the IL-7R chain (CD127) in progressive HIV disease would be expected to affect IL-7 activity. Alternatively, disease-associated defects of remaining CD8 CD127 T cells may influence IL-7 responsiveness. Therefore, the IL-7 responsiveness of CD8 CD127 T cells from HIV and untreated or treated HIV individuals was investigated. Blood was collected from HIV and untreated or effectively treated HIV ( 50 viral copies/ml for 1 year) individuals, and CD8 CD127 T cells were isolated and cultured with IL-7. Indicators of IL-7 signaling (P-STAT5) and activity (Bcl-2 and proliferation) were evaluated by flow cytometry. Isolated CD8 CD127 T cells from untreated HIV individuals expressed significantly less P-STAT5 in response to IL-7 compared with CD8 CD127 T cells from HIV individuals. In effectively treated HIV individuals, CD8 CD127 T cells also expressed significantly lower levels of P-STAT5 compared with HIV individuals. IL-7-dependent proliferation of CD8 CD127 T cells from untreated HIV individuals was similarly impaired. In contrast, IL-7-induced Bcl-2 expression was not impaired in CD8 CD127 T cells from HIV individuals. These data demonstrate that IL-7/IL-7R dysfunction in HIV infection may contribute to IL-7-specific signaling defects. Decreased, IL-7-dependent activation of STAT5 and impaired proliferation may negatively impact the maintenance of CD8 T cell responsiveness in HIV infection. J. Leukoc. Biol. 89: ; Introduction The elimination of acute viral infections such as influenza [1] and respiratory syncytial virus [2] and the containment of chronic infections such as CMV [3] and Herpes simplex virus [4] are mediated by the proliferation and maintenance of virus-specific CD8 CTLs. Infection with HIV also elicits strong virus-specific CTL responses [5], which initially appear to be effective in recognizing HIV antigens and suppressing viral replication [5, 6]. However, the initial suppression of viremia is insufficient for complete viral clearance, and HIV infection leads to a gradual decline in CTL function. Spiegel et al. [7] demonstrated the presence of HIV- and CMV-specific CD8 T cells in patients with advanced disease. Thus, it would appear that in the context of advanced HIV infection, virus-specific CD8 T cells remain present in the circulation but are unresponsive to their cognate antigens. Many mechanisms, including the down-regulation of the CD3- chain [8, 9], reduced perforin expression [10] and reduced IFN- expression [11], and maturational arrest of CD8 effector T cells [12] have been proposed and evaluated as a potential explanation for decreased CTL activity during HIV infection; however, none has been clearly shown to explain the impaired CTL activity. Identifying the reason for this impaired function may lead to ways of enhancing HIV-specific CTLs and provide significant benefits in the management of HIV disease. IL-7 signaling occurs via the IL-7R complex, which is composed of two subunits: the IL-7R chain (CD127) that binds IL-7 and the common IL-2R chain (CD132). By signaling through the JAK/STAT pathway, the PI3K pathway, and the MAPK pathway, as well as activation of various Src family kinases, IL-7R signaling has been demonstrated to play a major role at various stages of T cell development. IL-7R signaling is involved in thymopoiesis and the generation of mature T cells [13], the survival of naïve T cells via up-regulation of Bcl-2 expression [14], and the homeostatic expansion of naïve and memory T cells via proliferation. In well-characterized murine models, Kaech et al. [15] demonstrated the dependence of memory CD8 T cell genesis on IL-7 and that CD127 expression distinguishes effector cells (mostly CD127 ), which successfully develop from activated CD127 naïve cells into mem- Abbreviations: ART antiretroviral therapy, MFI mean fluorescence intensity, P-STAT5 phosphorylated STAT5, Tx treated with ART, untx untreated, VL viral load 1. Correspondence: Division of Infectious Diseases, Ottawa Hospital-General Campus, 501 Smyth Rd., Room G-12, Ottawa, Ontario, K1H 8L6, Canada. jangel@ohri.ca /11/ Society for Leukocyte Biology Volume 89, April 2011 Journal of Leukocyte Biology 499

2 ory cells (CD127 ) [16, 17]. IL-7 signaling has also been demonstrated to increase the cytotoxic potential of CD8 T cells [18, 19]. Dysregulation of the IL-7/IL-7R system has been identified in several human diseases [20 24], including HIV infection. In HIV individuals, CD127 is expressed on a lower proportion of CD4 and CD8 T cells than in their HIV counterparts, with significant reductions observed on naïve and memory subsets [25 27] during a time at which there are increased concentrations of circulating IL-7 [28, 29]. Despite this increase in IL-7, CTL activity becomes progressively impaired with advanced disease, suggesting an inability of CD8 T cells to respond to IL-7. Impaired CD4 and CD8 T cell function and decreased CD127 expression have recently been linked to abnormal activation of the immune system in HIV patients, contributing to the observed immunodeficiency [30, 31]. Colle et al. [32] demonstrated that in CD4 T cells, a significant, positive correlation between CD127 expression and IL-7-dependent Bcl-2 up-regulation in HIV individuals is not evident in untreated and treated HIV individuals, further suggesting that defects within the IL-7/IL-7R signaling pathway (downstream of the IL-7R) may contribute to a decrease in IL-7 responsiveness. CD127 down-regulation on CD8 T cells during HIV infection may contribute to the observed impairment in CTL activity. Enhanced IL-7 production reported in HIV individuals is thought to be the host s attempt to counteract lymphopenia [28] with the additional effect of down-regulating CD127 expression. An alternative explanation for CD8 T cell dysfunction is impairment in IL-7-mediated signaling caused by host or viral factors, which would impede the biological function of IL-7 on CD8 T cells in HIV infection. To address these questions directly, CD8 CD127 T cells from HIV, untreated HIV, and treated HIV individuals were isolated, and IL-7 responsiveness was assessed by examination of an aspect of IL-7 signaling as well as important IL-7-associated activities. MATERIALS AND METHODS Patient subjects and study design All research conducted using blood from human subjects was approved by the Ottawa Hospital Research Ethics Board (Ontario, Canada), and donors provided written, informed consent. The functional capacity of CD127 was studied in a cross-sectional study. Samples were collected from HIV individuals (n 11). Untreated HIV subjects (n 21) were naïvetoart (n 16) or off therapy (n 5) for at least 1 year. Treated patients (n 8) have been on effective ART, receiving two nucleoside RT inhibitors with a protease inhibitor or non-nucleoside RT inhibitor, and had an undetectable ( 50 copies/ml) plasma VL for at least 1 year. CD127 expression in whole blood Blood was collected into heparin-containing tubes, and 100 l blood was incubated with CD8-PC5 and CD127-PE mab (5 l each; Beckman Coulter, Fullerton, CA, USA) for 30 min at room temperature in the dark. Samples were then processed by the Q-Prep/ImmunoPrep lysing system (Beckman Coulter), followed by flow cytometric analysis. Isolation of CD8 CD127 T cells Blood from donors was collected into heparin-containing tubes, and PBMCs were isolated by Ficoll-Paque PLUS (Pharmacia Fine Chemicals, Piscataway, NJ, USA) gradient separation following the manufacturer s instructions. The isolation of CD8 CD127 T cells was performed as described previously [33]. First, CD8 T cells were isolated from PBMCs by negative selection using the T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA), yielding a 90% pure population (data not shown), with minimal numbers of CD4 T cells and no contaminating monocytes. Then, CD127 T cells were isolated from the CD8 T cells by positive selection, typically yielding an enriched CD127 population ( 95% CD127 ) comparable with similar methods used to enrich other CD8 T cell subsets [34]. Isolation of CD8 CD127 T cells does not induce cell proliferation or CD127-associated cell signaling (e.g., activation of STAT5) as described previously [33]. T cell proliferation assay Following cell isolation, CD8 CD127 T cells were labeled with CFSE (CellTrace TM CFSE cell proliferation kit, Invitrogen, Carlsbad, CA, USA) using a method modified from the manufacturer s specifications as described previously [33]. Labeled cells were cultured (10 5 cells/200 l) in 96-well microtiter plates with medium only, PHA (0.5 g/ml; Sigma-Aldrich, St. Louis, MO, USA), human ril-7 (10,000 pg/ml; BD Biosciences, San Jose, CA, USA), or PHA (0.5 g/ml) IL-7 (10,000 pg/ml). The concentration of PHA used was the lowest tested concentration (i.e., submaximal dose) that induced at least two rounds of cell division among activated cells, which were cultured for 5 days, and cell division was assessed by flow cytometry, analyzing cells undergoing greater than or equal to three cell divisions (% CFSE low ), as described previously [33]. Intracellular P-STAT5 expression Isolated CD8 CD127 T cells (10 5 cells/200 l) were incubated in 96-well microtiter plates with medium or IL-7 (1000 pg/ml) for 15 min and stained for P-STAT5. Analysis was performed by flow cytometry. Cells were labeled with the anti-human STAT5 antibody (Alexa Fluor 488 mouse antihuman STAT5 py694, BD Biosciences) using a fixing and permeabilization kit (Invitrogen), following a method modified from the manufacturer s instructions. The percentage of P-STAT5-positive cells was measured from a cut-off set using an unstained control. MFI was measured over the entire lymphocyte population. Intracellular Bcl-2 expression Isolated CD8 CD127 T cells (10 5 cells/200 l) were incubated in 96-well microtiter plates with medium or IL-7 (10,000 pg/ml) for 48 h. Cells were resuspended in Reagent A fixation buffer (100 l/10 5 cells; Invitrogen), incubated at room temperature for 15 min, and washed in PBS. Cells were then resuspended in Reagent B permeabilization buffer (100 l/10 5 cells; Invitrogen) with FITC-conjugated Bcl-2 antibody (2 l/10 5 cells; BD Biosciences) and incubated at room temperature for 20 min. Samples were then washed in PBS and resuspended in PBS, and analysis was performed by flow cytometry. The percentage of Bcl-2-positive cells was measured from a cut-off set using an isotype control. MFI was measured over the entire lymphocyte population. Statistical analysis Data were analyzed for statistical significance using the Student s unpaired or paired t tests as appropriate, and correlations were evaluated using Pearson s correlation test (GraphPad Prism software, Version 4.0, GraphPad, La Jolla, CA, USA), and P values 0.05 were considered significant. RESULTS Study population To determine the effect of antigen and IL-7 on the proliferation and function of memory CD8 T cells during HIV infection, CD8 T cells were isolated from HIV individuals. Untreated 500 Journal of Leukocyte Biology Volume 89, April

3 Vranjkovic et al. IL-7 signaling is impaired in HIV infection TABLE 1. Baseline Data for Enrolled HIV and Untreated and Treated HIV Subjects CD4 mean SD VL mean SD (log 10 ) Average age SD (years) Sex (M/F) HIV,n 11 na a na /7 untx HIV,n /7 Tx HIV,n /4 a na Not applicable. HIV individuals were naïve to therapy or off ART for at least 1 year and had a mean ( sd) CD4 T cell count of 339 ( 180) cells/ l and a mean VL of 4.54 ( 0.65) log 10 copies/ml. Treated HIV individuals were receiving highly active ART with a plasma VL of 50 copies/ml for at least 1 year and had a mean CD4 T cell count of 733 ( 317) cells/ l (Table 1). Decreased, IL-7-induced P-STAT5 in CD8 T cells from HIV individuals IL-7/IL-7R signaling has an essential role in the maintenance of the T cell population, as well as their proliferation and differentiation. IL-7/IL-7R interactions have been shown to activate various signaling pathways, including the JAK/STAT pathway with P-STAT5. IL-7-induced P-STAT5 in CD8 CD127 T cells from HIV and untreated HIV individuals was therefore investigated. Expression of P-STAT5 was not detected in unstimulated cells from HIV and untreated or treated HIV individuals (Fig. 1). When stimulated with IL-7, P-STAT5 expression in CD8 CD127 T cells from HIV individuals increased significantly. In CD8 CD127 T cells from HIV individuals, IL-7 was still capable of inducing P-STAT5 expression, however to a significantly less degree than that observed in CD8 CD127 T cells from HIV individuals. A significantly lower percentage of cells expressing P-STAT5 (Fig. 1B) and P-STAT5 MFI (Fig. 1C) in IL-7-induced cells was detected in untreated HIV individuals compared with HIV controls (P for percentage, and P for MFI). P-STAT5 was also decreased in CD8 T cells from effectively treated HIV individuals compared with HIV controls (P for percentage, and P for MFI). There was no significant difference in IL-7-induced P-STAT5 levels between untreated HIV and treated HIV individuals. To determine if certain host immune factors were associated with the degree of IL-7 signaling, correlation analyses were performed. Whether analyzing all HIV individuals or only untreated HIV individuals, there was no correlation found between P-STAT5 expression and CD4 T cell count or plasma HIV RNA levels. There were also no correlations found between CD127 expression on CD8 T cells and P-STAT5 expression (data not shown). IL-7-induced proliferation of CD8 CD127 T cells is impaired in HIV individuals As it has been well described that IL-7 promotes the survival and proliferation of CD8 T cells, the effect of IL-7 on cell division of mitogen-stimulated CD8 CD127 T cells was evaluated. Incubation of CD8 CD127 T cells with IL-7 alone did not result in an appreciable number of cells undergoing multiple cell divisions (i.e., percent CFSE low ; Fig. 2A). Stimulation with a low (submaximal) concentration of PHA (0.5 g/ml; Fig. 2B) enables mature CD8 T cells to respond to the proliferative effects of IL-7, as described previously [35, 36]. Addi- Figure 1. Decreased IL-7-induced phophorylation of STAT5 in HIV infection. IL-7-induced P- STAT5 expression in CD8 CD127 T cells, isolated from HIV and untreated or treated HIV individuals, was investigated. Cells were cultured with medium or IL-7 (1000 pg/ml) for 15 min and then stained with FITC-conjugated anti-p-stat5 antibodies. (A) Representative flow cytometry histograms demonstrate a decrease in IL-7-induced P-STAT5 MFI in the untreated (HIV untx, n 18) and treated (HIV Tx, n 8) patients compared with HIV individuals (n 11). Significant lower levels of (B) the percentage expression (% of control) and (C) MFI values (change in MFI compared with control) in IL-7-induced P-STAT5 were detected in untreated HIV individuals compared with HIV individuals (by Student s ttest). Patients on ART also had significantly lower levels of P-STAT5 compared with HIV individuals (by Student s t test). Volume 89, April 2011 Journal of Leukocyte Biology 501

4 Figure 2. IL-7-induced proliferation is impaired in CD8 CD127 T cells from HIV individuals. In HIV individuals, (A) IL-7 alone did not induce appreciable cell division. Incubation of CD8 CD127 T cells with a low dose (submaximal) of PHA (0.5 g/ml) was used to stimulate T cells in vitro to study the effects of IL-7. (B) A submaximal dose of PHA resulted in cells from HIV individuals undergoing two to three cell divisions, and (C) PHA IL-7 resulted in significantly more proliferation (n 7). (D) Similar to HIV individuals, CD8 CD127 T cells from untreated HIV individuals did not divide in response to IL-7 alone (n 14). (E) Submaximal doses of PHA induced less cell division in cells from HIV individuals compared with HIV individuals. (F) The combination of PHA IL-7 resulted in a smaller increase in the absolute number of CFSE-labeled cells undergoing greater than or equal to three divisions compared with that observed in HIV individuals. (G) When stimulated with IL-7 alone, CD8 CD127 T cells from treated HIV individuals did not divide (n 5). Similar to untreated HIV individuals, (H) PHA and (I) PHA IL-7 induced less cell division in cells from treated HIV individuals compared with HIV individuals. SSC, Side-scatter. (J) The data of cells undergoing greater than or equal to three divisions (% CFSE low ) from experiments in HIV and untreated or treated HIV individuals are summarized in a bar graph [n 7 (HIV ), n 14 (HIV untx), n 5 (HIV Tx)]. (K) The degree of IL-7-induced proliferation [i.e., (IL-7 PHAinduced % CFSE low cells) (PHA-induced % CFSE low cells)] for cells from HIV and untreated and treated HIV individuals is shown in a bar graph (P and 0.025, respectively, by Student s t test). tion of IL-7 significantly increased the proportion of CFSE low PHA-stimulated CD8 CD127 T cells from HIV individuals compared with cells cultured with PHA alone (Fig. 2C and J). Similar to cells from HIV individuals, culture with IL-7 alone did not induce proliferation in cells from untreated or treated HIV individuals (Fig. 2D and G). Submaximal dose of PHA induced slightly less cell division in untreated HIV individuals compared with HIV individuals, however this difference did not reach statistical significance (P 0.084; Fig. 2E and J). Similar findings were observed in cells from treated HIV individuals. Although PHA IL-7 induced proliferation of CD8 CD127 T cells from untreated and treated HIV individuals (Fig. 2F and I), the proportion of CFSE low cells was significantly lower than that observed in HIV individuals (P and 0.032, respectively; Fig. 2J). A significant difference is also observed when data are analyzed for the increase in proliferation induced by IL-7 (P 0.03 and 0.025, respectively; Fig. 2K). No differences between untreated and treated HIV individuals were observed in any of the parameters tested. IL-7-induced Bcl-2 production in HIV and untreated or treated HIV individuals It has been well described that IL-7 is required for T cell homeostasis and survival and that this survival may be attributed to the production of antiapoptotic molecules such as Bcl-2 [37]. Numerous groups have shown that IL-7-induced Bcl-2 production plays a critical role in lymphoid development and the regulation of immune responses [30]. In HIV infection, decreased CD127 expression has been associated with increased apoptosis of CD8 T cells [38], likely as a result of the increased proportion of CD8 CD127 T cell effector cells destined to die in the contraction phase. Whether other inherent defects in IL-7 signaling are also involved is not known. Therefore, IL-7-induced expression of Bcl-2 in isolated CD8 CD127 T cells from HIV and untreated or treated HIV individuals was investigated. Unstimulated CD8 CD127 T cells from HIV and untreated HIV individuals had similar levels of Bcl-2 expression (Fig. 3A). Interestingly, the data suggest that treated HIV individuals had higher basal levels of Bcl-2 compared with HIV individuals (P 0.06; Fig. 3A). Stimulation with IL-7 enhanced Bcl-2 production by CD8 CD127 T cells from HIV and untreated or treated HIV individuals; however, there was no significant difference among these groups in the level of IL-7-induced Bcl-2 production (Fig. 3B). Likely, as a result of baseline Bcl-2 expression being higher in treated HIV individuals, the absolute increase in Bcl-2 induced by IL-7 was lower in this group (i.e., IL-7 induced a greater proportional increase in Bcl-2 production in HIV individuals than in treated HIV individuals; Fig. 3C). 502 Journal of Leukocyte Biology Volume 89, April

5 Vranjkovic et al. IL-7 signaling is impaired in HIV infection Figure 3. IL-7-induced Bcl-2 expression. IL-7-induced Bcl-2 expression in CD8 CD127 T cells from HIV (n 7) and untreated (n 15) or treated HIV (n 6) individuals was investigated. (A) Following 48 h in culture with medium alone, CD8 CD127 T cells from treated HIV individuals tended to have higher Bcl-2 expression compared with HIV or untreated HIV individuals (P 0.06). (B) Representative flow cytometry histograms demonstrate no difference in IL-7-induced Bcl-2 expression (MFI) in the untreated (HIV untx) and treated (HIV Tx) patients compared with HIV individuals. (C) The degree of Bcl-2 induced with IL-7 was significantly less in treated HIV individuals than in HIV individuals in the percent expression (left panel) and MFI values (right panel); however, there were no statistically significant differences between HIV and untreated HIV individuals. DISCUSSION Reduced CD127 expression on CD8 T cells of HIV patients may contribute to the observed decline in CTL activity associated with HIV disease by affecting the ability of these cells to respond to IL-7. Alternatively, disease-associated defects of T cells may also influence IL-7 responsiveness of cells that express CD127. As IL-7 is important for T cell homeostasis and the replacement of diminishing T cell pools, there is a rationale for investigating the responsiveness of CD8 CD127 T cells to IL-7 in HIV infection. In this cross-sectional study, we report that isolated CD8 CD127 T cells from untreated HIV individuals expressed significantly less P-STAT5 in response to IL-7 when compared with those from HIV individuals. In effectively treated HIV individuals, CD8 CD127 T cells also expressed significantly lower levels of P-STAT5 compared with that observed in HIV individuals. Furthermore, IL-7-dependent proliferation of CD8 CD127 T cells from untreated HIV individuals was also impaired. Isolation of CD8 CD127 T cells enabled this study to directly assess IL-7 dysfunction of CD8 T cells in HIV infection. The description by Benoit et al. [39] that IL-7 stimulation failed to activate STAT5 in a significant proportion of CD8 T cells from HIV individuals suggests that IL-7/IL-7R signaling is impaired in HIV infection. The cause of impaired, IL-7-induced STAT5 activation in HIV individuals and its impact on immunopathogenesis are as of yet unknown; however, it is reasonable to believe that this observation and that of others [30, 40] can be attributed in part to the well-documented downregulation of CD127 on CD8 T cells in HIV infection. The data presented here address the additional possibility that reduced IL-7 activity in HIV infection is not only a result of a decrease in CD127 expression but may also be a result of HIVinduced defects in CD127 signaling. Altered IL-7R signaling has been demonstrated in other disease states such as in breast cancer [24]. PBMC obtained from 13 of 19 patients with breast cancer did not respond to stimulation with IL-7, as defined by P-STAT5, despite normal levels of CD127 expression. Analogous to what we have observed with IL-7-induced STAT5 signaling in CD8 T cells, CD4 T cells isolated from HIV individuals have been shown to have similarly reduced IL-2 responsiveness [41]. Kryworuchko et al. [42] attributed these defects to dysfunction in the JAK/STAT signaling pathway in CD8 T cells. Although a different cell type with distinct responses to cytokines compared with CD8 T cells, CD4 T cells have also been shown to be less responsive to cytokine stimulation in HIV infection. In addition, when CD4 T cells from HIV donors are cultured with the HIV-1 env protein, IL-2-induced activation of JAK-1 and JAK-3, as well as their targets, STAT5a and STAT5b, were reduced [43], suggesting that viral factors may contribute to this impaired cytokine signaling. Furthermore, we found that IL-7 activation of STAT5 in CD8 T cells from HIV individuals did not correlate with CD4 counts or VLs (data not shown), suggesting that virologic control is not a factor in mediating IL-7-mediated P-STAT5 activation, although the sample size tested here was relatively small. This is in contrast to recent findings in CD4 T cells, in which IL-7 responsiveness was a good immune correlate of prevailing CD4 counts, although in CD4 T cells, this was found to be independent of factors influencing immune reconstitution and could be a result of increased CD127 expression with higher CD4 T cell counts [40]. It is possible that immune activation may influence IL-7 responsiveness, as it has been linked previously to this CD127 down-regulation [31]. Volume 89, April 2011 Journal of Leukocyte Biology 503

6 IL-7-dependent proliferation of PBMCs from untreated HIV individuals has been shown previously to be lower than in HIV individuals [30], and successful ART partially restored these IL-7-dependent responses. Here, we also demonstrate impaired IL-7-dependent proliferation of CD8 CD127 T cells, where CD8 CD127 T cells from untreated HIV individuals displayed a reduced degree of IL-7-dependent proliferation compared with those from HIV individuals, consistent with a recent report [44]. As IL-7 has been reported to drive T cell proliferation via STAT5 [45], the reduced proliferation of CD8 CD127 T cells from HIV individuals (Fig. 2) in response to IL-7 may be a result of a decreased ability to phosphorylate STAT5 and may be a mechanism contributing to the CD8 T cell impairment seen in HIV disease. Whether activation of STAT5 independently initiates cell cycle progression or whether additional pathways, such as the PI3K pathway [46], are involved remains to be examined. Restoration of IL-7-induced STAT5 signaling or proliferation with effective anti-hiv therapy was not observed in the present report, but this is possibly a result of the fact that a relatively small number of effectively treated patients were studied. Alternatively, HIV-induced impairment in IL-7 activity in CD8 T cells may not be fully restored or may even be irreversible, as it has been well documented that immune recovery is only partially restored with ART. Studies of greater size and duration of viral suppression should be able to address these issues. An additional mechanism contributing to the CD8 T cell impairment observed in HIV disease may be an altered ability to acquire sufficient nutrients to support cellular metabolism. Several groups have demonstrated that IL-7 maintains metabolic activity through the uptake of glucose from the extracellular milieu [45 47]. IL-7-mediated PI3K/AKT signaling results in the regulation of expression and activity of the glucose transporter GLU1 and the transferin receptor CD71, which are important in the control of cell growth [48]. As STAT5 is responsible for initiating the PI3K/AKT signaling cascade during IL-7/IL-7R signaling, it is possible that the decreased levels of P-STAT5 detected in untreated and treated HIV individuals also affect the cell s capacity to acquire nutrients, thereby impeding proliferation. In normal T cell development, IL-7 acts as an antiapoptotic factor, regulating members of the Bcl-2 family, including synthesis of Bcl-2, phosphorylation of Bad, and cytosolic retention of Bax [49]. It has been shown recently that the induction of Bcl-2 production in CD4 and CD8 T cells stimulated with IL-7 is reduced in HIV infection [50]. Similarly, CD8 T cells of viremic patients (in which many of the CD8 T cells are likely CD127 effector cells) expressed less Bcl-2 than HIV individuals [30]. It has been reported that low levels of Bcl-2 expression are observed in effector CD8 T cells [51]. This is consistent with what we have observed by excluding CD8 CD127 T cells that are Bcl-2 lo and may explain why CD8 CD127 T cells from HIV and untreated HIV individuals express similar levels of constitutive and IL-7-stimulated Bcl-2 expression (Fig. 3B). In treated HIV individuals, who tended to express higher baseline levels of Bcl-2 compared with HIV individuals, the degree of Bcl-2 induction with IL-7 was significantly less (Fig. 3C). Similar to Juffroy et al. [50], there were no differences in IL-7 induction of Bcl-2 between untreated and treated HIV individuals. This is not surprising, as HIV protease increases apoptosis by cleaving Bcl-2; hence, HIV individuals treated with protease inhibitors may be expected to express more Bcl-2 [52]. Vingerhoets et al. [53] demonstrated decreased T cell sensitivity to IL-7 in HIV infection, where the inhibitory effect of IL-7 on spontaneous apoptosis in total CD8 T cells was less pronounced after 6 days in HIV individuals with decreased CD127 expression than in HIV individuals. The present study directly addresses the capacity of CD8 CD127 T cells to respond to IL-7, thereby avoiding the confounding factor of differing degrees of CD127 expression when comparing cellular responses of HIV and HIV individuals. The activation of STAT5 through JAK3 has been implicated in IL-7-dependent synthesis of Bcl-2 [45]. In the untreated HIV individuals, decreased P-STAT5 following IL-7 stimulation was detected; however, there was no difference in the amount of Bcl-2 synthesized following IL-7 stimulation. The production of Bcl-2 in response to IL-7 has been shown recently to depend on the Jak/STAT5 pathway [48], and the present data suggest that the defect observed in HIV individuals may be limited to this pathway. As a degree of STAT5 activation does occur in CD8 CD127 T cells from HIV individuals, a threshold level of P-STAT5 required to initiate Bcl-2 production may have been reached and may explain this observation. In addition, this observation may be explained by findings by Barata et al. [48], where PI3K activation was shown to be required for IL-7-mediated Bcl-2 up-regulation in a leukemic T cell line, suggesting that molecules, in addition to STAT5, are required and that phosphorylation of STAT5 may not be the limiting process. These data demonstrate a degree of dysfunction in the IL-7/ IL-7R system in HIV infection, which may be explained by IL-7- specific signal transduction defects. In sorted CD8 CD127 T cells, where CD127 expression is preserved, T cells from untreated or treated HIV individuals are less capable of responding to IL-7 compared with those from HIV individuals. Collectively, these results provide further insights into the mechanisms by which CD8 T cell activity is impaired in HIV infection. When considering IL-7 as a therapeutic agent, there is therefore likely a limit to which CTL activity could be enhanced, particularly in the setting of ongoing viral replication. However, enhancing IL-7 signaling by increasing the expression or function of CD127 may result in improved CD8 T cell function and immunologic control of viral replication. As the IL-7/IL-7R system and its disruption during HIV infection continue to be studied intensively, a better understanding of HIV immunopathogenesis will be gained and enable the development of novel, immune-based therapies. AUTHORSHIP A.V. designed and performed the research, analyzed data, and wrote the manuscript. A.P. assisted with flow cytometry methods. A.M.C and J.B.A. assisted in the design of the research and data analysis and aided in the writing of the manuscript. 504 Journal of Leukocyte Biology Volume 89, April

7 Vranjkovic et al. IL-7 signaling is impaired in HIV infection ACKNOWLEDGMENTS This research was supported by the Ontario HIV Treatment Network (OHTN) grant #ROGB131, the Canadian Institutes of Health Research grant #HOP84649, and the Canadian Federation of AIDS Research grant # A.M.C. is a recipient of an OHTN fellowship, and J.B.A. is an OHTN Career Scientist. The authors thank Drs. Paul MacPherson and Ashok Kumar for helpful discussion and critical review of the manuscript. DISCLOSURE The authors have no competing financial interests to declare. REFERENCES 1. Ada, G. L., Jones, P. D. (1986) The immune response to influenza infection. Curr. Top. Microbiol. Immunol. 128, Cannon, M. J., Openshaw, P. J., Askonas, B. A. (1988) Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus. J. Exp. Med. 168, Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D., Riddell, S. R. 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