Soluble IL-7Rα (sCD127) Inhibits IL-7 Activity and Is Increased in HIV Infection Angela M. Crawley, Sylvie Faucher and Jonathan B. Angel This information is current as J Immunol 2010; 184:4679-4687; Prepublished online 19 of September 25, 2021. March 2010; doi: 10.4049/jimmunol.0903758 http://www.jimmunol.org/content/184/9/4679 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Soluble IL-7Ra (sCD127) Inhibits IL-7 Activity and Is Increased in HIV Infection

Angela M. Crawley,*,† Sylvie Faucher,‡ and Jonathan B. Angel*,†,x

Soluble CD127 (sCD127) appears to play an important role in the immunopathogenesis of several chronic infections, multiple scle- rosis, and various cancers. The function of sCD127 and whether it influences IL-7 bioavailability or activity is unknown. In this study, we demonstrated that recombinant and native sources of sCD127 significantly inhibited IL-7–mediated STAT5 and Akt phosphorylation in CD8+ T cells. IL-7–mediated proliferation and Bcl-2 expression were similarly reduced by sCD127. In each case, native sCD127 inhibited IL-7 activity to a greater degree than rsCD127. Anti–IL-7 activity was inherent to human plasma and could be reversed by depletion of CD127, revealing for the first time the biological activity of naturally occurring sCD127. Plasma sCD127 concentrations were increased in HIV+ individuals compared with HIV2 controls, correlated with IL-7 levels, and + remained unchanged in HIV individuals following 1 y of effective antiretroviral therapy. Determining the regulation and function Downloaded from of sCD127 may be critical for understanding both the pathogenesis of diseases in which IL-7 likely has a role (e.g., HIV infection, cancer) and its potential impact on IL-7 as a therapeutic approach. The Journal of Immunology, 2010, 184: 4679–4687.

t has recently been suggested that soluble CD127 (sCD127) modulating activity of their cytokine ligands, soluble cytokine plays a role in the immunopathogenesis of several diseases receptors have been shown to be of prognostic value in a number

I such as multiple sclerosis (MS), acute lymphoblastic leuke- of diseases and have also been developed as therapeutic agents in http://www.jimmunol.org/ mia, and HIV infection (1–3). Genetic evidence has revealed an the treatment of a number of inflammatory diseases (5). To date, association between soluble isoforms of CD127 and chronic a number of soluble cytokine receptors have been identified, and progressive MS, and increased levels of sCD127 transcripts have their mechanisms of release, unique functions, and putative been documented in children with acute lymphoblastic . pathophysiological roles have been described (4, 5). The first In HIV infection, individuals lacking HIV-specific cytotoxic ac- identified soluble was IL-2Ra (sIL-2Ra), and it tivity have increased concentrations of sCD127 (3). Although has been associated with leukemia disease progression and is increased levels of sCD127 have been detected in a number of a marker of poor prognosis in children with Hodgkin’s disease (6). disease states and have been associated with disease activity, the The IL-2Ra region, a known autoimmune susceptibility lo- biological function of sCD127 and its clinical relevance remains cus, possesses significant allelic heterogeneity related to either by guest on September 25, 2021 to be established. susceptibility and risk to MS and type 1 diabetes, and these allelic Soluble cytokine receptors have the ability to influence the variants independently correlate with plasma sIL-2Ra concen- extracellular bioavailability of their ligand and have been shown to trations (7). Antagonistic soluble receptors including TNFR I and play important roles in several aspects of the immune response II can inhibit TNF signaling and have been shown to prevent TNF- including inflammation, cellular proliferation, and apoptosis. In mediated tumor lysis (8). The IL-6R subunit gp130 has also been many cases, soluble cytokine receptors act as competitive inhib- shown to inhibit the proinflammatory activity of IL-6 (9). sIL- itors, or, alternatively, they can enhance the activity of the cytokine. 15Ra is an example of an agonistic cytokine receptor. Pre- The former effect has led to the idea that the release of soluble complexed IL-15–sIL-15Ra increases NK and CD8+ pro- cytokine receptors is a physiological mechanism for regulating the liferation and antitumor activity up to 50 times compared with IL- activity of cytokines, as production of the soluble receptor often 15 alone (10, 11). parallels synthesis of the ligand (4). In addition to their role in The biological role of sCD127 is not yet known, although it is plausible that sCD127 could bind circulating IL-7, thereby de- creasing IL-7 bioavailability. In HIV infection, there may be causal *Ottawa Hospital Research Institute; †Department of Biochemistry, Microbiology and Immunology, University of Ottawa; ‡National HIV Immunology Laboratory, National links between the increase in plasma IL-7 levels and higher con- HIV and Retrovirology Labs, Centre for Infectious Disease Prevention and Control centrations of sCD127. Therefore, we have investigated the impact x Health Canada; and Division of Infectious Diseases, Ottawa Hospital-General Campus, of sCD127 on IL-7 activity in human CD8+ T cells and quantified Ottawa, Ontario, Canada plasma sCD127 concentration in HIV infection to better un- Received for publication November 23, 2009. Accepted for publication February 20, 2010. derstand its role in immunopathogenesis. This work was supported by Grant ROGB131 from the Ontario HIV Treatment Network (OHTN), Grant HOP84649 from the Canadian Institutes of Health Re- Materials and Methods search, and Canadian Foundation for AIDS Research Grant 019014. A.M.C. is a re- Sample collection and cell isolation cipient of an OHTN fellowship and J.B.A. is an OHTN Career Scientist. Address correspondence and reprint requests to Dr. Jonathan B. Angel, Division of All research conducted using blood from human subjects was approved by Infectious Diseases, Ottawa Hospital-General Campus, 501 Smyth Road, Room the Ottawa Hospital and Ottawa Hospital Research Institute’s Research G-12, Ottawa, ON K1H 8L6, Canada. E-mail address: [email protected] Ethics Board (Ottawa, Ontario, Canada). Plasma was collected from Fi- coll-Paque gradients of heparinized blood from both HIV2 and HIV+ in- Abbreviations used in this paper: MFI, mean fluorescence intensity; MS, multiple 2 + sclerosis; pSTAT5, phosphorylated STAT5; sCD127, soluble CD127; WI-sup, super- dividuals and stored at 80˚C. The HIV individuals studied were natants of WI-26VA4 cell. recruited from the HIV clinic at the Ottawa Hospital. Blood was drawn, and plasma was isolated from individuals either naive to antiretroviral Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 therapy or off antiretroviral therapy for .6 mo at the time of sampling. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903758 4680 ROLE OF SOLUBLE IL-7Ra IN IL-7 ACTIVITY AND HIV

Plasma from an additional 14 HIV+ individuals was obtained in the context of Buckinghamshire, U.K.). Densitometry analysis of bands was a previously reported clinical trial of highly active antiretroviral therapy (12). performed using AlphaEase Fc Software 6.0 (a Innotech, San Leandro, For in vitro tissue culture experiments, PBMCs from healthy HIV seroneg- CA), and relative changes in protein expression of treated cells were cal- ative volunteers were isolated by Ficoll-Paque PLUS (Pharmacia Fine culated compared with unstimulated controls. Chemicals, Piscataway, NJ) gradient separation, washed twice in PBS, and resuspended in RPMI medium 1640 supplemented with penicillin-strepto- Bcl-2 expression mycin and 10% FCS at a concentration of 1 3 106 cells/ml. Isolation of CD8+ After 48 h of culture, intracellular Bcl-2 staining was performed using the T cells from PBMCs was conducted using a MACS CD8+ T Cell Isolation BD Biosciences FITC-conjugated Bcl-2 Ab reagent set, following the (Miltenyi Biotec, Auburn, CA) in accordance with the manufacturer’s in- manufacturer’s protocol (BD Biosciences). Cells were washed with PBS structions, achieving a purity of .98–99% as verified by flow cytometry. and resuspended in fixation buffer (100 ml/1 3 105 cells; eBioscience, San Purified CD8+ T cells were resuspended at 106 cells/ml in RPMI 1640 sup- Diego, CA) and incubated at room temperature for 20 min. Following plemented with penicillin-streptomycin and 20% FCS. incubation, cells were washed, resuspended in permeabilization buffer (100 ml/105 cells) with FITC-conjugated Bcl-2 Ab (2 ml/1 3 105 cells; BD IL-7 and IL-7R reagents Biosciences), and incubated at room temperature for 20 min. Samples were 5 Recombinant human IL-7 (Sigma-Aldrich, Oakville, Ontario, Canada), washed and then resuspended in flow staining buffer (0.5 ml/1 3 10 cells; a lyophilized product, was resuspended in PBS at a concentration of 10 mg/ eBioscience). Analysis was performed by flow cytometry, collecting ml and stored at 220˚C. Two sources of sCD127 were used: recombinant 10,000 events, with data represented as values of MFI. human CD127-Fc chimera (CD127-Fc, R&D Systems, Minneapolis, MN) and the culture supernatants of WI-26VA4 cells (WI-sup; American Type Cell proliferation Culture Collection, Manassas, VA), a cell line known to secrete CD127 Following isolation, cells were labeled with CFSE (CellTrace CFSE Cell (13). The use of these two protein sources enabled the consideration of Proliferation kit) using a method modified from the manufacturer’s spec- potential conformational differences affecting the activity of native and ifications (Invitrogen Canada, Burlington, Ontario, Canada). A stock so- Downloaded from recombinant forms of the receptor. Blocking mouse anti-human IL-7 lution (5 mM) of CFSE was prepared in DMSO followed by the (clone 7417.111, R&D Systems) and anti-CD127 (clone 40131, R&D preparation of a working solution (8 mM) of CFSE in PBS plus 0.1% BSA. Systems) mAbs were used as controls. For some experiments, aliquots of Cells were resuspended in CFSE working solution (1 3 107 cells/ml) and WI-sup were depleted of sCD127. Briefly, CD127 was removed from WI- incubated at 37˚C in the dark for 10 min. Cells were then incubated with sup by affinity chromatography using cyanogen-bromide activated Se- 15 volumes of cold complete RPMI 1640 on ice in the dark for 5 min and pharose beads (Sigma-Aldrich) coupled to mouse anti-human CD127 then washed and resuspended in complete RPMI 1640 (1 3 106 cells/ml). mAbs (R&D Systems). Similarly, sCD127 was depleted from human Cells were cultured with medium only, PHA (2.5 mg/ml; Sigma-Aldrich), http://www.jimmunol.org/ plasma samples. As a control, plasma samples were alternatively passed PHA plus IL-7 (10,000 pg/ml), or PHA plus IL-7 preincubated with over an unconjugated Sepharose column. CD127-Fc or WI-sup. The concentration of PHA used was the lowest tested concentration (i.e., submaximal dose) that induced at least two Cell culture rounds of cell division among activated cells. Cells were cultured for 4 d, Preincubation with CD8+ T cells, IL-7 (10–10,000 pg/ml) was pre- and then cell division was assessed by flow cytometry and analyzed using incubated with excess CD127-Fc (1000 ng/ml) or WI-sup (undiluted) or FCS Express 2 (De Novo Software, Los Angeles, CA). Individual cell undepleted or CD127-depleted plasma (1:10) for 1 h at 37˚C. Isolated divisions of the stimulated T cells were evaluated according to established CD8+ T cells (1 3 106 cells/ml) were subsequently cultured with IL-7 methods involving CFSE analysis of T cell division (14). Data are repre- alone or IL-7–CD127 complexes for various times, using a method adapted sented as the proportion of stimulated T cells that have undergone $3 from Stoklasek et al. (10) (see below). In experiments designed to block divisions. IL-7 activity, cells were preincubated with an excess of anti-CD127 Abs by guest on September 25, 2021 (15 ml/1 3 105 cells) (clone R.34.34, Beckman Coulter, Marseille, France) Quantification of plasma CD127 and IL-7 for 1 h prior to the addition of IL-7. Alternatively, IL-7 was preincubated Quantification of plasma CD127 was carried out using a CD127-specific with an excess of anti–IL-7 Abs (10 mg/ml) for 1 h before adding to cells. Ab-bead microfluorosphere assay that has been recently developed (15). no CD127 In addition, CD127-depleted WI supernatants (WI-sup ) were Goat anti-human CD127 polyclonal Abs (R&D Systems) were covalently preincubated with IL-7 and added to cells as above. coupled to carboxylated-modified fluorescent beads (Luminex, Austin, TX) were used to capture sCD127. The bound sCD127 was detected with IL-7 signaling pathways biotinylated mouse anti-human CD127 Ab (clone hIL-7-R-M21; BD Bi- + osciences) and streptavidin-PE (Invitrogen). Sample concentrations were The phosphorylation of STAT5 in CD8 T cells was analyzed by flow cytometry (Alexa Fluor 488 mouse anti-human STAT5 pY694, BD Bio- extrapolated from standard curves of recombinant human CD127-Fc chi- sciences, San Jose, CA) following 15 min of culture. Specific inhibition of mera (R&D Systems) generated using a five-parameter logistic curve fit- IL-7–induced phosphorylated STAT5 (pSTAT5) was confirmed by pre- ting equation (MasterPlex QT software, version 4, MiraiBio, South San treating cells with a Jak inhibitor (Calbiochem, Gibbstown, NJ). Expres- Francisco, CA). sion of phospho-STAT5 was measured by flow cytometry. Cells were In addition, an sCD127 ELISA was designed to measure sCD127 in plas- ma. Briefly, 96-well plates were coated overnight with mouse anti-human labeled with the anti-human STAT5 Ab using a fixing and permeabilization kit (Invitrogen, Camarillo, CA) using a method modified from the manu- CD127 mAb (clone R34.34; Beckman Coulter). Plates were blocked with facturer’s instructions. The cells were washed and incubated with fixing BSAfor 1h, washed, andthensamples wereincubated for1 h. BoundsCD127 solution for 20 min at 37˚C. Cells were washed again and incubated with was detected following a 1-h incubation with goat anti-human CD127 poly- cold methanol (100%) for 10 min at 4˚C. Following another wash, cells clonal Abs (R&D Systems) and bovine anti-goat IgG-HRP. Reactions were were incubated with the permeabilization reagent and the anti-STAT5 Ab developed using the SureBlue Reserve TMB microwell peroxidase substrate + (KPL, Gaithersburg, MD) and visualized with a plate reader at 450 nm. for 20 min. The proportion of phosphorylated STAT5 cells was calculated Sample concentrations were also extrapolated from standard curves of re- as follows: (mean fluorescence intensity [MFI] of cells incubated with plasma and IL-7)/(MFI of cells incubated with IL-7 alone) 3 100%. combinant human CD127-Fc chimera (R&D Systems). Phosphorylated Akt in cell lysates was assessed by Western blot after 30, Plasma IL-7 concentrations were measured using the IL-7 Quantikine 60, or 120 min of culture. As a loading control, the expression of b-actin ELISA (R&D Systems) according to manufacturer instructions. The IL-7 was detected using a mouse anti-human mAb (Calbiochem). Cell pellets capture Ab was coupled to beads as described above. The conjugated beads were incubated with plasma samples (diluted 1/5 in PBS containing were collected by centrifugation and lysed by incubating in lysis buffer (50 0.05% Tween-20, 1% BSA, 0.05% NaN , and 0.9 mg/ml EDTA [Sigma- mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 3 7 Aldrich]) for 2 h at room temperature. The bound IL-7 was revealed with mM MgCl2, and 1 mM EDTA) (40 ml/1 3 10 cells) for 1 h. Lysates were then centrifuged for 20 min at 14,000 rpm, 4˚C, and supernatants were the biotinylated detection Ab for 1 h and streptavidin-PE. The bead collected and stored at 220˚C. Lysate concentrations were quantified using analysis was done as described above. The lower detection limit of the a BCA Protein Assay kit (Pierce, Rockford, IL). A total of 5 mg protein assay was 0.5 pg/ml. was resolved by 10% SDS-PAGE and blotted onto polyvinylidene fluoride Statistical analysis membrane. Membranes were blocked overnight in 5% skim milk in TBS, probed with a rabbit anti-human pAktser Ab (1:5000) followed by an anti- Data from in vitro assays were graphed and analyzed by a one-way ANOVA rabbit-IgG HRP Ab (1:10,000) (Cell Signaling Technology, Boston, MA). test and the simultaneous Dunnett’s test or paired or unpaired (as appro- were detected using the Lumigen TMA-6 kit (GE Healthcare, priate) Student t tests. Plasma sCD127 concentrations were analyzed by The Journal of Immunology 4681 linear correlation analyses (p # 0.05) to determine any correlations with 52.7% (mean MFI = 17.78 6 1.27) compared with the IL-7– CD4 counts, viral loads (log transformed), or plasma IL-7 concentrations. rCD127-Fc chimera complexes, which reduced this by 12.3% All statistical analyses were conducted using GraphPad Prism 5.0 Software (mean MFI = 31.46 6 1.59; p = 0.0001) (Fig. 1D). (GraphPad, San Diego, CA). Anti–IL-7 Abs completely abrogated the effects of IL-7 on STAT5 phosphorylation (Fig. 1E). Anti-CD127 Ab (clone Results R34.34), also reportedly able to block the binding of IL-7 to sCD127 decreases IL-7–induced phosphorylation of STAT5 CD127, inhibited IL-7 activity, although to a lesser extent and in the range of what was seen with sCD127 (Fig. 1F). This confirms The principal intracellular signaling pathway of the g cytokine C a previous report demonstrating that this Ab has less than com- receptors in T cells involves the activation of Jak-STAT signaling plete IL-7 inhibition properties (17). CD127-depleted WI-sup had molecules. After 15 min of culture, increasing concentrations of no effect on IL-7–induced pSTAT5, confirming that the effect IL-7 significantly induced the phosphorylation of STAT5 in CD8+ caused by WI-sups was CD127-specific (Fig. 1G). T cells, and this effect quickly plateaued (Fig. 1A), consistent with previous reports (16). This increase was evident at IL-7 concen- trations as low as 10 pg/ml and plateaued at concentrations $100 sCD127 decreases IL-7–induced phosphorylation of Akt pg/ml (p , 0.0001 by ANOVA) after 15 min of incubation and The PI3K pathway is also activated upon IL-7 ligation of mem- decreased significantly after further culture (data not shown). To brane-associated CD127 and has been associated with IL-7–in- determine if sCD127 had an impact on the activation of this pathway duced T cell proliferation (18). Once activated, a cascade ensues, by IL-7, isolated CD8+ T cells were cultured with medium alone, IL- ultimately resulting in phosphorylation of Akt, a known mediator Downloaded from 7, or IL-7 plus each of two sources of sCD127 (CD127-Fc chimera of cell cycling molecules. To determine if IL-7–induced activation + or WI-sup), and pSTAT5 was quantitated. Culturing CD8 T cells of the Akt pathway is affected by sCD127, purified CD8+ T cells with either IL-7–CD127 Fc (Fig. 1B) or IL-7–WI supernatant (Fig. were treated as above and pAkt was measured by Western blot. 1C) complexes resulted in decreased expression of pSTAT5 com- IL-7 increased pAkt expression in a dose- and time-dependent pared with that seen with IL-7 alone (p = 0.025 and p # 0.0001, manner. Responses peaked after 1 h of culture (Fig. 2A), consis- respectively). This effect was more significant with WI culture su- tent with published observations (16). Therefore, all further ex- http://www.jimmunol.org/ pernatant, which reduced IL-7–induced pSTAT5 expression by periments involved culturing cells for 1 h with or without 1000 pg/ by guest on September 25, 2021

FIGURE 1. SolubleCD127 decreasesIL-7–induced phosphorylationofSTAT5 in CD8+ T cells invitro. IsolatedCD8+ T cells were culturedwith IL-7for 15 min, and then the expression of phosphorylated STAT5 was analyzed by flow cytometry. A, Increasing concentrations of IL-7 (1–1000 pg/ml) induced the phosphor- ylation of STAT5. pp , 0.0001 by ANOVA and p , 0.05 by Dunnett’s test versus unstimulated cells (medium); n = 6. Representative histrograms are shown of STAT5 phosphorylation analysis of CD8+ T cells cultured with preincubated IL-7 plus CD127-Fc chimera (dark gray fill) (B) or IL-7 preincubated with WI-26VA4 supernatant (WI-sup, gray fill) (C). In both B and C, the phosphorylation of STAT5 by IL-7 was reduced compared with IL-7 alone (black fill). These results are summarized in D (pp = 0.025; ppp , 0.001; pppp = 0.0001; all by paired Student t test, n = 6). The downregulation of IL-7–mediated STAT5 phosphorylation by IL- 7–CD127 complexes was CD127-specific as anti–IL-7 (E) and anti-CD127 (F) prevented full induction of STAT5 phosphorylation by IL-7. G, CD127-depleted WI supernatants didnot reduce this IL-7 activity. The dottedand solid lines in the histograms represent unstained cells and cells cultured with medium only, respectively. 4682 ROLE OF SOLUBLE IL-7Ra IN IL-7 ACTIVITY AND HIV

FIGURE 2. IL-7–induced phosphorylation of Akt in CD8+ T cells is decreased by sCD127. Increasing concentrations of IL-7 (100, 1000, and 10,000 pg/ml) induced the phosphorylation of Akt in a dose- and time-dependent manner. A, A representative Western blot from four experiments is shown. B, Expression of phosphorylated Akt was assessed by Western blot analysis after CD8+ T cells were incubated for 1 h with medium (lane 1), IL-7 (1000 pg/ml, lane 2), IL-7 plus Downloaded from CD127-Fc chimera (lane 3), or IL-7 plus WI-26VA4 supernatants (WI-sup, lane 4). The results of three individuals are shown. C, The relative change in phosphorylated Akt expression in five individuals is summarized in the bar graphs. All data were calculated by densitometry analysis compared with unstimulated controls (Ctl). pp = 0.05; ppp = 0.05; pppp = 0.03 by http://www.jimmunol.org/ Student t test. by guest on September 25, 2021

ml of IL-7 in the presence or absence of sCD127. The addition of labeled and cultured for 5 d with submaximal concentrations of IL-7 preincubated with either rCD127-Fc or WI culture superna- the mitogen PHA. Such stimulation enables CD8+ T cells to re- tant significantly decreased IL-7–induced pAkt expression spond to the proliferative effects of IL-7 (Fig. 4A,4B), as reported (p = 0.047 and p = 0.028, respectively; Fig. 2B,2C). There ap- previously (19, 20). The proliferation of mitogen-stimulated CD8+ peared to be a greater inhibition of IL-7–mediated Akt phos- T cells increased significantly in the presence of IL-7 (p = 0.02). phorylation with WI supernatant compared with CD127-Fc, When IL-7 was preincubated with rCD127-Fc or WI-supernatant, although this difference did not reach statistical significance. the proportion of cells dividing three or more times reduced sig- nificantly compared with cells stimulated in the absence of these Downregulation of IL-7–induced Bcl-2 expression by sCD127 sources of sCD127 (p = 0.05 and p = 0.02, respectively; Fig. 4C– Complementing its role in T cell development and homeostasis, IL-7 E). Analysis of the proportion of cells dividing .1 division increases the expression of cell survival and antiapoptotic molecules yielded similar results (data not shown). Unlike the inhibition of including Bcl-2. After 48 h of culture with IL-7 (1000 pg/ml), in- STAT5 phosphorylation and Bcl-2 expression, there was no sig- tracellular Bcl-2 expression in isolated CD8+ T cells increased nificant difference in the proliferative effects of CD127-Fc com- significantly (p = 0.01; Fig. 3C). Preincubation of IL-7 with pared with WI-supernatant. rCD127-Fc did not significantly decrease Bcl-2 expression (Fig. 3A, 3C) compared with IL-7 alone. In contrast, WI-sups did decrease IL- Activity of sCD127 in human plasma 7–induced Bcl-2 expression (p = 0.03; Fig. 3B,3C). Inhibition of IL- Plasma samples were preincubated with IL-7 for 1 h, cultured with 7 activity was more evident in the presence of anti–IL-7 or anti- CD8+ T cells for 15 min, and then STAT5 phosphorylation was CD127 Abs (Fig. 3D,3E). This effect was further confirmed to be measured by flow cytometry. Thirteen independent plasma sam- CD127-specific because CD127-depleted WI-culture supernatant ples from eight different donors possessed anti–IL-7 activity, as its had no effect on IL-7–induced Bcl-2 expression (Fig. 3F). addition to isolated CD8+ T cells inhibited IL-7–induced phos- phorylated STAT5 levels. This provided the opportunity to de- IL-7–induced proliferation is reduced by sCD127 termine if sCD127 was responsible for this activity and therefore It has been well described that IL-7 induces T cell proliferation and may be able to block IL-7 activity in vivo. sCD127 was depleted hence is a potential IL-7 activity that could be inhibited by sCD127. from the plasma by CD127-Ab affinity chromatography, whereas To investigate this possibility, isolated CD8+ T cells were CFSE- duplicate samples of plasma were passed through an unconjugated The Journal of Immunology 4683 Downloaded from

FIGURE 3. Increased expression of intracelluar Bcl-2 by IL-7 is reduced by a native source of sCD127. A and B, Representative histograms depict the http://www.jimmunol.org/ intracellular Bcl-2 expression of isolated CD8+ T cells cultured with medium (black line), IL-7 (black fill), and IL-7 plus rCD127-Fc chimera (dark gray fill) or IL-7 plus WI-26VA4 supernatants (light gray fill) for 48 h of culture. The dotted lines represent unstained cells. C, The expression of Bcl-2, shown as MFI, is summarized in the bar graphs (n = 6). pp = 0.01; ppp = 0.03 by Student t test. This downregulation was CD127-specific as anti–IL-7 (D) and anti- CD127 (E) decreased IL-7–induced Bcl-2 expression, whereas CD127-depleted WI-26VA4 supernatants had no effect (F). affinity column (undepleted plasma). The depletion of sCD127 These results indicate that sCD127 present in human plasma is was confirmed using an sCD127-specific ELISA (data not shown). capable of inhibiting IL-7 activity, revealing for the first time its Depletion of sCD127 from 10 of these 13 samples reversed the biological activity.

anti–IL-7 activity, restoring pSTAT5 levels to near those seen in by guest on September 25, 2021 the absence of plasma (Fig. 5A,5B). In plasma samples that did Plasma CD127 concentrations are increased in HIV infection not possess anti–IL-7 activity, depletion of sCD127 had no effect The expression of plasma sCD127 can be detected in both HIV2 and on IL-7–induced STAT5 phosphorylation (data not shown, n = 8). HIV+ individuals, as shown previously by Western blot analysis

FIGURE 4. IL-7–induced proliferation of CD8+ T cells is reduced by sCD127. Representative scatter plots illustrate the cell division of CFSE-labeled CD8+ T cells stimulated for 5 d with medium only (gray dots) or PHA (black dots) (A) and PHA plus IL-7 (10,000 pg/ml) (B), PHA plus IL-7 preincubated with rCD127- Fc (C), or PHA plus IL-7 preincubated with WI-sup (D). Cells in divisions $3 divisions are shown in hatched boxes, and the proportion (%) of cells are indicated. E, The proportions of CD8+ T cells undergoing $3 divisions are summarized in the bar graph. pp = 0.02; ppp = 0.05; pppp = 0.02 by Student t test; n =5. 4684 ROLE OF SOLUBLE IL-7Ra IN IL-7 ACTIVITY AND HIV

FIGURE 5. Plasma sCD127 re- duces IL-7 activity. Undepleted or CD127-depleted plasma samples were preincubated with IL-7 (1000 pg/ml) for 1 h and then added to isolated CD8+ T cells and cultured for 15 min. The expression of pSTAT5 was then analyzed by flow cytometry. A,Two representative histrograms of pSTAT5 expression by CD8+ T cells are shown. Cells were cultured with IL-7 (1000 pg/ml) preincubated with me- dium only (black line), undepleted plasma (dark gray), or CD127-de- pleted plasma (light gray). B, De- pletion of sCD127 reversed the anti– Downloaded from IL-7 activity of plasma in 10 of 13 samples. The data are summarized in C. pp = 0.006 by paired Student t test. http://www.jimmunol.org/

(21). In this study, the concentration of sCD127 was quantified in the data with exclusion of a single very high value in the HIV+ group plasma of 55 HIV+ individuals not receiving antiretroviral therapy (sCD127 = 859 ng/ml) (p = 0.05). To determine if the concen- and compared with 58 HIV2 controls, using a recently described tration of sCD127 was associated with measures of HIV disease CD127-specific Ab-microfluorosphere assay (15). progression known to correlate with CD127 expression on CD8+ The mean concentration of plasma sCD127 was significantly T cells (e.g., CD4 count, viral load, plasma IL-7), potential cor- higher in HIV+ individuals (228.3 6 110.7 ng/ml) compared with relates of sCD127 concentration in HIV+ individuals were ex- healthy HIV2 controls (175.7 6 161.7 ng/ml) (p = 0.024) (Fig. amined. The mean 6 SD CD4 count of the HIV+ individuals was by guest on September 25, 2021 6A). Statistical significance remained following analysis of the 369.35 6 195.20 cells/ml. The mean 6 SD viral load (plasma HIV

FIGURE 6. Plasma sCD127 concentrations are increased in HIV infection. A, Plasma sCD127 was measured in HIV2 (n = 58) and HIV+ (n = 55) individuals using a CD127-specific Luminex assay. Plasma from HIV+ individuals had significantly more sCD127 than HIV2 individuals. pp = 0.024 by Student t test. The lower detection limit of this assay was 50 pg/ml (dotted line). There were no correlations between plasma CD127 concentrations and CD4 counts (B) or viral loads (C) (log transformed). D, Plasma sCD127 correlated positively with plasma IL-7 concentrations in HIV+ individuals (p = 0.024, r = 0.305). The lower detection limit of the IL-7 assay was 0.5 pg/ml. The Journal of Immunology 4685

RNA) was 4.05 6 0.9 log copies/ml. The mean 6 SD plasma IL-7 sCD127, demonstrating that natural inhibition of IL-7 activity can concentration was 12.28 pg/ml (SD 6 10.29, range 1.05–39.56). be enhanced by artificial means. Phosphorylation of Akt, a PI3K There was no significant correlation between plasma sCD127 pathway molecule common to gC cytokines, is important for concentration and CD4 count or plasma HIV RNA level (Fig. 6B, promoting T cell proliferation (18). Indeed, we demonstrated that 6C). Linear regression analysis indicated that there was a signifi- preincubation of IL-7 with sCD127 resulted in decreased pro- cant positive correlation between plasma sCD127 and plasma IL-7 liferation of CD8+ T cells in vitro (Fig. 4). The limited reduction concentration (undetectable values of IL-7 were assigned a value in IL-7–induced Bcl-2 expression by sCD127 suggests that there of 0.5 pg/ml for the analysis) (p = 0.027, r = 0.305; Fig. 6D). In is a low threshold of IL-7 signaling required to produce Bcl-2. HIV2 individuals, plasma IL-7 concentrations are consistently This would not be surprising given that in homeostasis, limited very low or undetectable (,8 pg/ml) (22, 23), and thus similar physiological concentrations of IL-7 in the circulation (22, 31) are analyses were not conducted in the HIV2 population. able to maintain the survival of a vast T cell population. Collec- As cell membrane-bound CD127 expression on CD8+ T cells tively, inhibition of early IL-7 signaling pathways by sCD127 can returns to near normal levels in HIV+ individuals following highly result in measurable decreases of IL-7–associated activities active antiretroviral therapy (24, 25), it is possible that sCD127 in vitro, suggesting the sCD127 is an IL-7 antagonist in this setting concentrations may decrease during antiretroviral therapy-induced and does not exhibit pleiotropic effects in vitro. Opposing effects immune reconstitution. Therefore, plasma sCD127 was quantified of soluble cytokine receptors have not been demonstrated in vitro; in samples from 14 antiretroviral naive HIV+ individuals receiving however, there have been discrepancies such as with sIL-15Ra, highly active antiretroviral therapy (12). Plasma sCD127 con- which inhibited IL-15 activity in vitro (32) yet enhanced this ac- centrations did not change significantly over 48 wk of effective tivity in vivo (10, 11, 33). The depletion of sCD127 from plasma Downloaded from therapy (Fig. 7). suggests that the inhibitory properties of sCD127 observed in vitro may be of biological relevance to the activity of IL-7 in vivo. It is Discussion not clear if this would occur in all individuals, as decreased ac- tivity was observed in 13 of 21 experiments (62%), and in two Despite the discovery of a soluble form of CD127 capable of cases where plasma decreased IL-7 signaling (Fig. 5B), depletion binding IL-7 almost 20 y ago (13), a functional role for the soluble

of sCD127 further decreased IL-7 activity, suggesting that other http://www.jimmunol.org/ receptor has not been described. Because there is an indication factors in the plasma may be involved. that sCD127 plays a role in the immunopathogenesis of a number The concentration of sCD127 (1000 ng/ml) required in this study of diseases (24, 26–29), and there are ongoing clinical inves- to reduce IL-7–induced activity of CD8+ T cells in vitro was near tigations of IL-7 as a potential HIV or cancer therapeutic, we the range of plasma IL-7 concentrations in healthy and HIV+ in- sought to investigate whether sCD127 was capable of influencing dividuals (175.7 ng/ml and 228.3 ng/ml, respectively; Fig. 5), and IL-7 activity. Both signaling and functional outcomes of IL-7 in tissue concentrations may be significantly higher (34). As such, CD8+ T cells were inhibited by sCD127. In addition, depletion of the physiological consequences of this may be more pronounced CD127 from plasma revealed for the first time the biological ac- than those observed in vitro. Other soluble cytokine receptors tivity of naturally occurring human sCD127. known to be produced at similar concentrations in vivo include by guest on September 25, 2021 sCD127 reduced the activation of IL-7 signaling pathways, as sTNFR with concentrations approaching 2000 ng/ml (35), and up indicated by the decreased phosphorylation of STAT5 and Akt to 75 ng/ml of IL-6R has been detected in the sera of HIV+ in- (Figs. 1, 2). Activation of STAT5 by IL-7 has been associated with dividuals (36). However, comparably lower concentrations of sIL- increased glucose uptake and activation of Akt (16), and murine 2Ra (6 ng/ml) were measured in the serum of patients with MS, models suggest that STAT5, although not independently, may yet these concentrations were able to inhibit IL-2 activity in vitro promote long-term survival of T cells by inducing Bcl-2 pro- (37). The primary source(s) of sCD127 in plasma has not been duction (30). In the case of the phosphorylation of STAT5, and to determined, but may be the result of alternative splicing of mRNA a lesser degree Akt phosphorylation, native CD127 (WI super- transcripts encoding the membrane-bound form of CD127 (13), natant) inhibited IL-7 signaling to a greater degree than its re- receptor shedding (21), receptor cleavage (38), or a combination combinant counterpart. This suggests that possible conformational thereof. differences between native and recombinant sources of sCD127 Using a quantitative CD127 assay, it was determined that plasma affect the nature of their interactions with IL-7, such as receptor- sCD127 concentrations were higher in HIV+ individuals compared ligand binding affinities. In a separate issue, anti–IL-7 and anti- with HIV2 individuals, consistent with our previous report and CD127 Abs decreased IL-7 activity more than either source of confirming the initial report by Carini et al. (3, 21). Recent reports describe the detection of sCD127 in human plasma, but either decreased levels (39) or similar levels (40) in HIV-infected in- dividuals compared with healthy individuals. In these reports, two different ELISA methods with different Abs were used. In the first report, a polyclonal anti-CD127 Ab for plate coating and a monoclonal Ab (41031 clone, R&D Systems) for detection of plate-bound sCD127 was used, whereas the second report de- scribes what appears to be a similar assay, but uses mAbs for coating (M21 clone, BD Biosciences, or 40131, R&D Systems) and a polyclonal Ab for detection. It is unclear if IL-7 in plasma interferes with CD127 detection by either of these assays, and it is possible that elevated levels of IL-7 in HIV+ individuals lead to FIGURE 7. Plasma sCD127 does not change over 48 wk in HIV+ in- decreased detection of sCD127 in this setting. Furthermore, the dividuals treated with effective antiretroviral therapy. The concentration of concentrations of sCD127 detected in these reports are consis- sCD127 was measured in the plasma of HIV+ individuals prior to and at tently ,50 ng/ml, which is the lower threshold of the Luminex 4 and 48 wk postinitiation of highly active antiretroviral therapy (n = 14). assay and considerably lower than the levels measured by this 4686 ROLE OF SOLUBLE IL-7Ra IN IL-7 ACTIVITY AND HIV sensitive assay. It is also possible that the narrow detection range Carini et al. (3), who detected more sCD127 in HIV+ individuals of the ELISA prevented the detection of a correlation between with undetectable HIV-specific CTL activity compared with those sCD127 concentration and IL-7 concentration in HIV+ individuals with detectable CTL activity. It has been suggested that, in health, as was observed in this study (Fig. 6D). the concentration of IL-7 is limited (49). Our results suggest that The biological role that sCD127 has in vivo remains to be the presence of increased amounts of sCD127 in disease would established, although these data suggest that the inhibition of IL-7 counter this otherwise altruistic model of IL-7 bioavailability activity by its soluble receptor may be of clinical relevance (Fig. 5). given its ability to inhibit the activity of IL-7. Furthermore, these effects may be more pronounced in primary Exogenous IL-7 has been shown to preferentially promote the and secondary lymphoid organs, where IL-7 concentrations would homeostatic proliferation of human naive T cells in vivo (50), be expected to be much higher than in the plasma (34). The a desirable clinical outcome for lymphopenic patients. Initial concentration of sCD127 in HIV+ individuals did not correlate clinical trial results indicate that IL-7 therapy improves T cell with other prognostic markers such as viral load or CD4+ T cell survival and proliferation of nonhuman primates and in humans in counts; however, there was a positive correlation with plasma IL- the context of HIV infection and in cancer (51–53). The influence 7, which is known to be increased in HIV disease (41) (Fig. 6). of sCD127 on IL-7 activity may be relevant to IL-7 therapy in Despite the immunorestorative effects of highly effective anti- which high concentrations of IL-7 are achieved (up to 10 mg/kg) retroviral therapy, which include increased CD4 counts and the re- (51), and this may become quite pronounced if the exogenous IL-7 expression of membrane-bound CD127 to near normal levels (24, induces additional release of sCD127. This resulting increase in 25), sCD127 concentrations remained stable over time (Fig. 7). the concentration of plasma sCD127 may potentially reduce the

Similarly, plasma IL-7 concentrations have been shown to remain efficacy of the exogenous IL-7. Elucidation of the exact role of Downloaded from elevated with effective antiretroviral therapy (26, 27, 42, 43). sCD127 on IL-7 physiology and function in vivo, both under Sustained concentrations of sCD127 despite the favorable re- normal conditions and in disease, is needed to more fully un- sponse to antiretroviral treatment is analogous to what has been derstand its role in the regulation of the immune system, its po- observed with sIL-2Ra, which is increased in severe MS yet is not tential role in disease pathogenesis, and the impact it may have on influenced by effective treatment (37). Stable concentrations of IL-7 therapies in HIV+ individuals or in cancer patients.

sCD127 in HIV infection may prevent or diminish any potential http://www.jimmunol.org/ immunorestorative effects of elevated concentrations of IL-7. Acknowledgments The present data, derived from in vitro experiments, suggest that We thank Drs. Paul MacPherson and Ashok Kumar for helpful discussion sCD127 inhibits the activity of IL-7. The fact that IL-7 activity was and critical review of the manuscript. not completely abrogated in the presence of excess amounts of sCD127 suggests low affinity between sCD127 and IL-7. It has been shown that IL-7 activities are mediated through high-affinity Disclosures interactions between IL-7 and membrane-bound CD127 (44). The The authors have no financial conflicts of interest. soluble form of a cytokine receptor may be expected to have lower affinity for its ligand compared with its membrane-bound form, as References by guest on September 25, 2021 is the case with soluble IL-2Ra (5). It is however possible that 1. Booth, D. R., A. T. Arthur, S. 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