Ghazawi FM et al, 2013

ORIGINAL ARTICLE

IL-7 downregulates IL-7Ra expression in human CD8T cells by two independent mechanisms

Feras M Ghazawi1,2,5, Elliott M Faller1,2,5, Scott M Sugden1,2, Juzer A Kakal1,2 and Paul A MacPherson1,2,3,4

Interleukin (IL)-7 is an essential nonredundant cytokine, and throughout the lifespan of a T-cell signaling via the IL-7 receptor

influences cell survival, proliferation and differentiation. It is therefore no surprise that expression of the IL-7 receptor

alpha-chain (CD127) is tightly regulated. We have previously shown that IL-7 downregulates expression of CD127 at the cell

surface and now elucidate the kinetics of that suppression and demonstrate that IL-7 downregulates CD127 transcripts and

surface protein in primary human CD8 T cells by two separate pathways. We show that IL-7 induces the initial reduction in cell-

surface CD127 protein independent of transcriptional suppression, which is delayed by 40–60 min. Although IL-7-mediated

downregulation of CD127 transcripts is dependent on Janus kinase (JAK)/STAT5, the early downregulation of surface CD127

protein is independent of JAK activity. The data further illustrate that low levels of IL-7 induce smaller and transient decreases

in CD127 transcripts and surface protein, whereas higher concentrations induce more profound and sustained suppression.

Such flexibility in receptor expression likely allows for fine-tuned immune responses in human CD8 T cells in different

microenvironments and in response to different immunological challenges.

Immunology and Cell Biology (2013) 91, 149–158; doi:10.1038/icb.2012.69; published online 4 December 2012

Keywords: interleukin-7; interleukin-7 receptor; CD127; cytotoxic T lymphocytes

Interleukin (IL)-7 has essential, nonredundant roles in the develop-ment, maintenance and function of T cells.1,2 It is a master regulatorof human and murine T-cell homeostasis and is required for survivalof naive and mature peripheral T cells3 and for the establishment andmaintenance of T-cell memory.4–7 IL-7 also has a pivotal role in theactivation and proliferation of cytotoxic CD8 T cells.8–10 IL-7enhances TCR-dependent T-cell activation and proliferationin vivo11 and significantly augments the cytotoxic activity of effectorT cells.12–16 IL-7 signaling is initiated upon binding of IL-7 to itshigh-affinity site on the IL-7 receptor alpha-chain (CD127), whichthen dimerizes with the second component of the IL-7 receptor, thecommon g-chain (CD132), which is shared with the receptors forIL-2, -4, -9, -15 and -21.1,17 The interaction of the two receptorsubunits brings the associated Janus kinases (JAKs)-1 and -3 intoproximity and allows their subsequent trans-phosphorylation andactivation. This leads to phosphorylation of CD127 at a criticaltyrosine residue (Y449) by JAK1, allowing docking and subsequentphosphorylation and activation of signal transducer and activatorof transcription (STAT) proteins and the p85 subunit ofphosphoinositide 3 (PI3) kinase, both of which subsequentlyregulate the expression and activity of a number of downstreamgenes and gene products.1,18 Despite the central importance of IL-7and its receptor in the development, maintenance and function of

T cells, the mechanisms regulating expression of the IL-7 receptor inresponse to IL-7 signaling have been only partially characterized.19–22

We and others have shown that IL-7 downregulates expression ofthe CD127 receptor subunit on the surface of T cells.19–25 Althoughsome groups have shown transcriptional suppression of the CD127gene in response to IL-7,19,22 others have found that IL-7 does notaffect CD127 mRNA levels in human T cells.21 Further, where IL-7has been shown to suppress CD127 gene transcription, it has not beenestablished whether the loss of CD127 protein from the cell surface isthe result of transcriptional downregulation and reduced proteinsynthesis or the result of a second mechanism acting at the cellmembrane.19,22 Indeed, it was recently reported IL-7 downregulatessurface CD127 protein in the T-leukemia cell lines HPB-ALL andTAIL7 through receptor internalization.24 Whether this applies toprimary human CD8 T cells has yet to be determined. In addition, thedetailed kinetics and roles that PI3K and JAK and downstreamphosphorylation of STAT proteins have in regulating the amount ofCD127 on the cell membrane or CD127 gene transcription or bothhave never been delineated.

In view of this, we set out to clearly and systematically define themechanisms by which IL-7 downregulates CD127 expression on thesurface of resting human CD8 T cells and to determine to what extentthe early downregulation of surface CD127 protein is dependent on

1Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa,Ontario, Canada; 3Department of Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada and 4Division of Infectious Diseases, Ottawa Hospital GeneralCampus, Ottawa, Ontario, Canada

Correspondence: Dr PA MacPherson, Division of Infectious Diseases, The Ottawa Hospital, General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6.E-mail: [email protected]

5These authors contributed equally to this study.

Received 20 August 2012; revised 16 September 2012; accepted 29 October 2012; published online 4 December 2012

Immunology and Cell Biology (2013) 91, 149–158& 2013 Australasian Society for Immunology Inc. All rights reserved 0818-9641/13

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http://dx.doi.org/10.1038/icb.2012.69

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transcriptional suppression. We demonstrate for the first time thatIL-7 suppresses expression of its own receptor in primary humanCD8 T cells by two independent mechanisms, one transcriptional andone at the level of surface protein. Through these pathways, IL-7provides negative feedback on its own signaling cascades, allowing forfine-tuned immune responses in human CD8 T cells in differentmicroenvironments and in response to different immunologicalchallenges.

RESULTS

IL-7 downregulates CD127 mRNA transcripts and surface proteinin a time- and dose-dependent mannerWe first set out to elucidate the kinetics of IL-7-mediated suppressionof CD127 surface protein and mRNA transcripts in primary human

CD8 T cells. To do this, purified CD8 T cells were incubated withincreasing concentrations of IL-7 (100–10 000 pg ml�1), and levels ofCD127 mRNA transcripts were measured by quantitative PCR(qPCR) and compared with untreated cells, normalizing to RPS18expression. Surface CD127 protein expression was monitored by flowcytometry. As shown in Figure 1a, IL-7 suppresses the level of CD127mRNA transcripts in CD8 T cells in a dose-dependent manner. Atleast 100 pg ml�1 IL-7 was required to see a decrease in the level ofCD127 mRNA (34±4% reduction), with 10 000 pg ml�1 inducing a78±8% decline in transcripts. Maximal suppression at all concentra-tions of IL-7 occurred at 3 h. At lower concentrations of IL-7 (100–1000 pg ml�1), the reduction in CD127 transcripts was transient witha dose response in time to recovery, whereas 10 000 pg ml�1 IL-7maintain transcriptional suppression for over 72 h. Similarly, the

Figure 1 IL-7 downregulates CD127 mRNA transcripts and surface protein in CD8 T cells in a time- and dose-dependent manner. Purified CD8 T cells were

incubated in media alone or in the presence of increasing concentrations of IL-7 (100–10 000pgml�1) for up to 72h. (a) CD127 mRNA transcript levels

were measured by qPCR normalizing to RPS18. (b) Surface CD127 protein expression was analyzed by flow cytometry. Values represent relative CD127

mRNA or surface protein expression (percent positive cells) compared with media controls. Error bars represent s.e. of the mean (s.e.m.) of four

independent experiments. (c) Representative flow-cytometry histograms from one individual in (b) showing CD127 expression on CD8 T cells incubated in

media alone or with IL-7 (250 and 10000pg ml�1) for 3–12 h.

IL-7 downregulates IL-7Ra expressionFM Ghazawi et al

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Immunology and Cell Biology

effects of IL-7 on CD127 surface protein are also dose dependent bothin the extent of the downregulation and in the duration ofsuppression (Figure 1b). A transient (17±3%) decrease in surfaceCD127 protein could be detected on CD8 T cells with as little as100 pg ml�1 of IL-7. This effect became more marked with increasingconcentrations of IL-7, where 5000 pg ml�1 induced a 41±2%reduction in CD127 surface protein within 3 h, with a maximalsuppression of 90±2% at 12 h. Recovery of CD127 on the cell surfacetook progressively longer with increasing doses of IL-7, but concen-trations of 5000–10 000 pg ml�1 maintained suppression for over72 h. Thus, we show that the effects of IL-7 on suppressing CD127mRNA transcripts and surface protein are both dose- and time-dependent, with transient reductions in expression at lower IL-7concentrations (100–1000 pg ml�1) and sustained suppression athigher concentrations (10 000 pg ml�1). The maximal reductions inCD127 mRNA and surface protein occur at 3 and 12 h, respectively, atall concentrations of IL-7.

IL-7 downregulates CD127 protein at the cell surface independentof transcriptional suppressionAs shown in Figure 1, the maximal effect of IL-7 on surface CD127protein expression was observed several hours following maximaltranscriptional suppression. This could suggest that loss of CD127protein from the cell membrane is entirely dependent on transcrip-tional suppression of the CD127 gene and natural turnover of surfaceprotein. We felt this was unlikely, however, because IL-7 induced amaximal reduction in surface CD127 protein within 12 h, whereas thenatural half-life of CD127 at the cell surface is approximately 55 h.26

To determine to what extent the downregulation of surface CD127protein is dependent on transcriptional suppression, we treated CD8T cells with IL-7 (10 ng ml�1) and measured CD127 mRNAtranscripts and surface protein at 20 min intervals by qPCR andflow cytometry, respectively. Interestingly, the downregulation ofCD127 protein at the cell surface occurred within 20 min, followingaddition of IL-7, and leveled off at 54±4% pretreatment levels overthe next 2 h (Figure 2a). In contrast, IL-7-induced suppression ofCD127 mRNA transcripts was delayed by 40–60 min post treatment(Figure 2b). These data then demonstrate that the initial down-regulation of CD127 protein at the cell surface actually precedes and istherefore likely independent of transcriptional suppression.

To further distinguish downregulation of surface CD127 proteinfrom transcriptional suppression, we treated CD8 T cells simulta-neously with IL-7 and dexamethasone (Dex), a glucocorticoidpreviously shown to upregulate CD127 transcripts in murine B andT cells.27,28 We reasoned if IL-7 is able to downregulate CD127 surfaceprotein independent of transcriptional suppression, we would see thisreduction at the cell surface, even when levels of CD127 transcripts areaugmented by the simultaneous addition of Dex. As expected(Figure 3a), treatment with Dex alone (20mM) increased CD127mRNA transcript levels in human CD8 T cells some 2.6-fold within1 h, whereas IL-7 alone (10 ng ml�1) reduced CD127 transcripts by64±5% at 4 h, reaching 80±4% suppression at 24 h. In contrast, thecombination of IL-7 plus Dex together maintained CD127 mRNAtranscripts at levels comparable to that in untreated cells. Interestingly,when added in combination with IL-7, Dex appeared to induce aninitial increase in CD127 transcripts within 1 h presumably due to therapid effect of Dex binding to the glucocorticoid receptor in thecytoplasm and its translocation to the cell nucleus. This was followedby an apparent decline in CD127 transcripts by 4 h likely due to thedelayed suppressive effects of IL-7. Although these variations wereconsistent, the changes were not statistically significant compared

with cells maintained in medium alone, and, ultimately, CD127mRNA leveled off to pretreatment levels at 24 h. Despite the initialincrease and overall lack of change in the level of CD127 transcripts,IL-7 still downregulated CD127 protein at the cell surface by 55±8%in the presence of Dex (Figure 3b). These data then confirm that IL-7downregulates surface CD127 protein independent of transcription.Notably, at 24 h, the level of surface CD127 protein was significantlyhigher in the presence of Dex plus IL-7 over IL-7 alone. This is mostlikely due to synthesis and replenishment of new CD127 protein inthe presence of normal transcript levels and highlights the contribu-tion of IL-7-induced transcriptional suppression in further limitingCD127 surface expression at later time points.

Finally, to fully separate IL-7-induced downregulation of CD127protein at the cell surface from transcriptional suppression, weutilized a Jurkat-CD127 cell line engineered to expresses the CD127cDNA from the cytomegalovirus promoter.22 Because IL-7 is notknown to attenuate transcription from the cytomegalovirus promoter,we expected CD127 transcripts would remain unchanged in thepresence of IL-7, whereas surface CD127 protein would bedownregulated. As shown in Figure 4, this is exactly what we found.Although treatment with IL-7 did not suppress CD127 mRNAtranscripts in the Jurkat-CD127 cell line (Figure 4a) over 6 h, IL-7did significantly downregulate CD127 protein at the cell surface

Figure 2 IL-7 induces the downregulation of surface CD127 protein within

20 min while suppression of CD127 mRNA is delayed by 40–60 min.

Purified CD8 T cells were incubated in media alone or in the presence of

IL-7 (10 ngml�1) and analyzed at 20 min intervals. (a) Surface CD127expression was measured by flow cytometry. (*Po0.05 compared with

media). (b) CD127 mRNA transcript levels were measured by qPCR

normalizing to RPS18 (*Po0.05 compared with media). Values represent

relative CD127 mRNA or surface protein (mean fluorescence intensities)

compared with media controls. Error bars represent s.e.m of at least four

independent experiments.


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(Figure 4b). This downregulation of surface CD127 protein in theabsence of changes in CD127 mRNA levels confirms that IL-7 inducesthe loss of CD127 protein from the cell surface independent of mRNAsuppression.

IL-7-mediated downregulation of CD127 mRNA transcripts isdependent on JAK/pSTAT5JAK/STAT and PI3K are considered the major IL-7-mediated signalingpathways in T cells,1,2 and we therefore set out to determine which, ifeither, of these pathways is involved in IL-7-induced downregulationof CD127 transcripts and surface protein. The role of IL-7 inactivating JAK3/STAT5 signaling is well established,20,22,23,29 and weconfirm here that IL-7 induces phosphorylation of STAT5 in primaryhuman CD8 T cells (Figure 5a). Phosphorylation of STAT3 inresponse to other g chain cytokines, such as IL-2,30 IL-4,30 IL-9,31

IL-1530 and IL-21,32 has been previously reported, but to ourknowledge, induction of STAT3 phosphorylation by IL-7 in humanCD8 T cells has not been investigated. To examine this, we treatedCD8 T cells with IL-7 (10 ng ml�1) or IL-10 (10 ng ml�1) as a positivecontrol33–35 and measured intracellular phospho-STAT3 by flowcytometry. As shown in Figure 5b, IL-10 induces STAT3 phosphor-ylation, whereas IL-7 does not. Finally, we used a synthetic STAT5

inhibitor that specifically targets the SH2 domain of STAT5 to blockits phosphorylation in the presence of IL-7. Figure 5c shows a dosetitration and complete block of STAT5 phosphorylation in thepresence of IL-7 plus 500mM inhibitor.

To examine the role of the JAK/STAT5 and PI3K signaling pathwaysin IL-7-induced downregulation of CD127 transcripts, we pretreatedCD8 T cells with inhibitors of either JAK or PI3K for 1 h followed byIL-7 (10 ng ml�1) for 80 min. The PI3K inhibitor (LY294002; 10mM)successfully blocked phosphorylation of Akt following IL-7 stimula-tion in primary human CD8 T cells, whereas the JAK inhibitor(1.5mM) blocked IL-7-induced STAT5 phosphorylation (data notshown). As shown in Figure 6a, preincubating cells with JAK inhibitorcompletely blocked IL-7-mediated downregulation of CD127 tran-scripts, whereas inhibition of PI3K signaling had no effect. Theseresults clearly indicate a requirement for JAK activity but not PI3K inthe downregulation of CD127 mRNA by IL-7. We next questionedwhether STAT5 phosphorylation had a role in the suppression ofCD127 transcripts by IL-7. To investigate this, CD8 T cells werepreincubated for 1 h with 500mM STAT5 inhibitor followed by IL-7(10 ng ml�1) for 60 min, and levels of CD127 mRNA transcripts weremeasured by qPCR. As shown in Figure 6b, inhibition of STAT5phosphorylation blocked the effect of IL-7, confirming that transcrip-tional suppression of CD127 by IL-7 is dependent on the JAK/STAT5signaling pathway.

Figure 3 IL-7 downregulates the expression of surface CD127 protein, even

when CD127 mRNA levels are augmented by Dex. Purified CD8 T cells were

incubated in media alone or in the presence of IL-7 (10 ngml�1), Dex

(20mM) or IL-7 plus Dex for 1, 4 and 24h. (a) CD127 mRNA transcript

levels were measured by qPCR normalizing to RPS18. (b) Surface CD127

expression was analyzed by flow cytometry (*Po0.05 and #P40.05

compared with media). Values represent relative CD127 mRNA or surface

protein expression (percent positive cells) compared with media control.

Error bars represent s.e.m of four independent experiments.

Figure 4 IL-7 downregulates the expression of surface CD127 protein in theJurkat-CD127 cell line without affecting CD127 mRNA levels. Jurkat-

CD127 cells were incubated in media alone or with IL-7 (10 ngml�1) for 1,

3 and 6 h. (a) CD127 mRNA transcript levels were measured by qPCR

normalizing to RPS18 (P¼0.126 at 1 h, 0.821 at 3 h and 0.328 at 6 h

relative to media). (b) Surface CD127 expression was analyzed by flow

cytometry (*Po0.006 compared with media). Data are represented as

relative changes compared with media controls. Error bars represent s.e.m

of three independent experiments.


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Although IL-7-mediated downregulation of CD127 protein at thecell surface occurs independently of JAK/STAT5 and PI3K,IL-7-induced degradation of CD127 is JAK dependentTo examine the requirement of the JAK/STAT5 and PI3K signalingpathways in IL-7-stimulated downregulation of CD127 protein at thecell surface, CD8 T cells were we pretreated with inhibitors of eitherJAK, PI3K or STAT5 for 1 h followed by IL-7 (10 ng ml�1) for 80 min

in the case of JAK and PI3K inhibitors or for 60 min in the case of theSTAT5 inhibitor. Levels of surface CD127 protein were then measuredby flow cytometry. As shown in Figures 7a and b, inhibition of JAK,PI3K and STAT5 phosphorylation had no effect on IL-7-mediated

Figure 6 IL-7-mediated suppression of CD127 transcripts is dependent on

JAK activity and STAT5 phosphorylation but not on PI3K. Purified CD8

T cells were maintained in media alone or preincubated with inhibitors of

JAK (1.5mM), PI3K (10mM) or STAT5 phosphorylation (500mM) for 1 h

followed by treatment with IL-7 (10 ngml�1) for 80min in the case of the

JAK and PI3K inhibitors (a) and for 60min in the case of the STAT5

inhibitor (b). CD127 transcript levels were measured by qPCR normalizingto RPS18. Data are represented as relative changes compared with media

control. (a) *Po0.005 relative to media. (b) P¼0.02 IL-7 compared with

media, P¼0.37 STAT5iþ IL-7 compared with STAT5i and P¼0.01

STAT5iþ IL-7 compared with IL-7. Error bars represent s.e.m of four

independent experiments.

Figure 5 IL-7 induces the phosphorylation of STAT5 but not STAT3 in

human CD8 T cells. Purified CD8 T cells were maintained in media alone or

treated with IL-7 (10ng ml�1) or IL-10 (10 ngml�1) for 45min and

then fixed, permeabilized and stained with fluorescein isothiocyanate

(FITC)-labeled anti-phospho-STAT3 or anti-phospho-STAT5 antibodies.

(a) Representative flow-cytometry histogram showing phospho-STAT5 in

untreated cells (gray fill) and cells stimulated with IL-7 (black line).

(b) Representative flow-cytometry histogram showing phospho-STAT3 in

untreated cells (gray fill) and cells stimulated with IL-7 (black line) or IL-10

(dashed line). (c) Purified CD8 T cells were maintained in media alone,

treated with IL-7 (10ng ml�1) for 8 h or preincubated with increasing

concentrations of STAT5 inhibitor (100–500mM) for 1 h and then treatedwith IL-7 (10 ngml�1) for an additional 8 h. Cells were then fixed,

permeabilized and stained with FITC-labeled anti-phospho-STAT5 antibodies

and analyzed by flow cytometry. Data show percent positive cells for each

condition (*Po0.03 and #P¼0.49 compared with media). Error bars

represent s.e.m of four independent experiments.


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removal of CD127 from the cell surface. In all cases, IL-7 stimulated areduction in surface CD127 protein equally in the presence andabsence of inhibitors. As expected, IL-7 induced the phosphorylationof tyrosine 449 (Y449) in the cytoplasmic tail of CD127 within15 min, and inhibition of JAK prevented this from occurring(Figure 7c). Taken together, these data indicate that phosphorylationof Y449 by JAK is not required to mark CD127 for removal from thecell membrane. Interestingly, inhibition of JAK did prevent subse-quent degradation of CD127 protein. When examined by western blotof whole-cell extracts, CD127 was markedly reduced in CD8 T cellstreated with IL-7 for 6 h, but in the presence of JAK inhibitor, CD127remained at levels equivalent to media control following IL-7stimulation. Thus, although IL-7-mediated removal of CD127 from

the cell surface is independent of JAK, JAK activity is required totarget CD127 for degradation likely through phosphorylation ofY449. Finally, when CD8 T cells were stimulated with IL-7(10 ng ml�1) in the presence of JAK inhibitor, CD127 protein wasrapidly removed from the cell surface but then recovered over severalhours (Figure 7e). Although we cannot entirely exclude the absence oftranscriptional suppression as the cause of recovery, this is not thepattern seen when CD8 T cells were treated with IL-7 and Dex andCD127 transcripts were maintained at pretreatment levels. Here,inhibition of JAK allowed reaccumulation of CD127 on the cellmembrane. These data taken together suggest that IL-7 binding toCD127 directly triggers receptor internalization, perhaps, by inducinga conformational shift in the protein or by dimerization with CD132.

Figure 7 Although IL-7-induced downregulation of CD127 protein at the cell surface is independent of JAK activity and phosphorylation of tyrosine 449,

subsequent degradation of CD127 is dependent on JAK. Purified CD8 T cells were preincubated with inhibitors of JAK and PI3K (a) or STAT5

phosphorylation (b) followed by IL-7 (10 ngml�1) as described in Figure 6, and surface CD127 expression was analyzed by flow cytometry. Data are

represented as relative changes compared with media control (*Po0.05 compared with media). Error bars represent s.e.m of at least four independent

experiments. (c) Western blot showing phosphorylation of tyrosine 449 on the cytoplasmic tail of CD127 is induced by IL-7 and requires JAK. Purified CD8T cells were maintained in media alone, treated with IL-7 (10ng ml�1) for 15 min, JAK inhibitor (1.5mM) for 1 h and 15 min or JAK inhibitor for 1 h

followed by IL-7 (10ngml�1) for 15 min. Whole-cell lysates were then analyzed by western blot probing for phospho-Y449 using a polyclonal rabbit anti-

human CD127 phospho-Tyr449 antibody. Blots were then stripped and reprobed for total CD127 as control using a polyclonal goat anti-human CD127

antibody. (d) Western blot showing IL-7-induced degradation of CD127 is JAK dependent. Purified CD8 T cells were maintained in media, treated with IL-7

(10ngml�1) for 6 h, JAK inhibitor (1.5mM) for 6 h or JAK inhibitor for 1 h followed by the addition of IL-7 (10 ngml�1) for 6 h. Whole-cell lysates were

then analyzed by western blot probing for total CD127 with a polyclonal goat anti-human CD127 antibody. Blots were then stripped and reprobed for

b-actin as loading control. (e) CD127 reaccumulates on the cell surface in the presence of IL-7 and JAK inhibitor. Purified CD8 T cells were maintained in

media, pretreated with JAK inhibitor (1.5mM) for 1 h followed by the addition of IL-7 (10ngml�1) or treated with IL-7 alone, and CD127 surface expression

was monitored by flow cytometry at the times indicated. Data are represented as relative changes compared with media controls. Error bars represent s.e.m.

of at least four independent experiments.


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Once in the early endosome, CD127 may then be sorted, such thatCD127 phosphorylated at Y449 by JAK is targeted for degradation,whereas unphosphorylated CD127 is recycled back to the cell surface.

DISCUSSION

Although IL-7 signaling has pivotal roles in the development andfunction of T cells, the mechanisms regulating expression of the IL-7receptor have only been partially characterized. Although severalstudies have already reported that IL-7 downregulates the expressionof CD127 mRNA transcripts and surface protein in human andmurine T cells,19,22 conflicting data have been provided suggestingthat IL-7 does not affect CD127 gene transcription.21 Whether thesediscordant results are due to the source of T cells used in each study(for example, umbilical cord versus adult peripheral blood), cultureconditions or the single time point of analysis is unclear. Further, ithas not been determined whether IL-7-mediated downregulation ofCD127 protein at the cell surface is the result of transcriptionalsuppression and reduced protein synthesis or due to a secondpathway acting at the cell surface as has been reported forT-leukemic cell lines.24 Here we address a number of thesequestions, delineating the detailed kinetics and separatecontributions of IL-7-mediated suppression of CD127 genetranscription and the initial downregulation of CD127 surfaceprotein in primary human CD8 T cells, and provide evidence withrespect to the mechanisms and pathways by which these occur.

The effects of IL-7 on reducing CD127 transcripts and surfaceprotein are dose dependent both in the extent of the downregulationand in the duration of suppression with maximal effects of IL-7 onCD127 mRNA and surface protein at 3 and 12 h, respectively. Lowconcentrations of IL-7 (100–1000 pg ml�1) induced transient declinesin CD127 mRNA and protein, whereas higher concentrations(10 000 pg ml�1) sustained suppression for over 72 h. Although thismay be due to either a bona fide concentration effect or sustainedsignaling at higher IL-7 concentrations, either way, this allowsflexibility in regulating CD127 expression in different microenviron-ments with different levels of IL-7. Although decreased transcriptionof the CD127 gene was viewed as the primary mechanism by whichIL-7 downregulates surface CD127 protein,19,20,22,36 we now showthat transcriptional suppression contributes only at later time pointsand serves to maintain low CD127 levels after the receptor has beenremoved from the cell membrane by IL-7. We demonstrate by severalmethods that IL-7 reduces CD127 transcripts and surface proteinthrough separate pathways. In both the Jurkat-CD127 cell line andwhen added to primary human CD8 T cells in combination with Dex,IL-7 removed CD127 protein from the cell surface in the absence ofchanges in CD127 mRNA levels. We also show that IL-7-mediateddownregulation of CD127 protein at the cell surface is rapid andoccurs within 20 min before there is any reduction in CD127transcripts. This results in an initial new equilibrium in proteinturnover, maintaining lower but relatively constant CD127 expressionon the cell surface over the next 2 h until CD127 transcriptionalsuppression kicks in to prevent protein replenishment and furtherdownregulate CD127 expression. The contribution of transcriptionalsuppression in reducing CD127 expression at later time points isevident in CD8 T cells treated with IL-7 and Dex, where CD127mRNA and surface protein are higher at 24 h compared with cellstreated with IL-7 alone. This effect is less evident in the Jurkat-CD127cells because protein turnover on the surface of this cell line is muchmore rapid compared with primary cells (data not shown). Thesedata together suggest that brief exposure to IL-7 may allow rapidtransient decreases in surface CD127 protein, whereas sustained

exposure to IL-7 results in transcriptional suppression and furtherreductions in receptor expression over time. This would provide afinely tuned mechanism by which CD8 T cells could respond to smallchanges in IL-7 concentrations in the microenvironment and todifferent immunological challenges.

Suppression of CD127 mRNA transcripts by IL-7 is dependent onthe JAK/STAT5 signaling pathway. It is already established that IL-7stimulation leads to phosphorylation of CD127 at Y449 by JAK3,1

allowing docking via their SH2 domains and subsequentphosphorylation of STAT5 proteins.1,18,37 Phospho-STAT5 thendimerizes and translocates to the nucleus where it may eitherdirectly or indirectly suppress CD127 transcription. Given the delayin transcriptional suppression by 40–60 min following stimulationwith IL-7, we hypothesize that IL-7 signaling via STAT5 results in theupregulation of a transcriptional repressor or an epigenetic modifierprotein that in turn suppresses CD127 gene expression. Consistentwith this hypothesis, the translational inhibitor cycloheximide blocksthe ability of IL-7 to downregulate CD127 transcripts in human CD8T cells (Ghazawi et al., manuscript in preparation), confirming thatnew protein synthesis is in fact required for the transcriptionalsuppression. Park et al.19 reported a similar effect with cycloheximideand implicated the transcriptional repressor Gfi-1 in thedownregulation of CD127 gene expression in CD8 T cells in mice.However, to date, the repressor induced by IL-7 and responsible forsuppressing CD127 gene transcription in human CD8 T cells has yetto be identified.

The IL-7-mediated downregulation of CD127 protein on thesurface of human CD8 T cells is rapid and occurs within 20 min.Similarly, Swainson et al.20 reported that surface CD127 protein isreduced on human CD4 T cells within 30 min of IL-7 stimulation(10 ng ml�1), and Henriques et al.24 documented the rapidinternalization of CD127 protein from the surface of the leukemiccell line HPB-ALL following addition of IL-7 (50 ng ml�1). We showhere that, as expected, IL-7 induces the phosphorylation of tyrosine449 within the cytoplasmic tail of CD127 by JAK and that thisphosphorylation is not required for the initial removal of CD127protein from the cell membrane. Indeed, IL-7 still downregulatessurface CD127 protein in the presence of JAK inhibitor. However,inhibition of JAK does prevent subsequent degradation of CD127 andallows the reaccumulation of receptor on the cell surface over 12–24 h.Taken together, the data suggest that IL-7 binding to CD127 directlytriggers receptor internalization, perhaps, through dimerization withCD132 or by inducing a conformational shift in the protein. Once inthe early endosome, CD127 may then be sorted, such that CD127phosphorylated at Y449 by JAK is targeted for degradation, whereasunphosphorylated CD127 is recycled back to the cell surface. Thesepathways are currently being further delineated in our laboratory.

The rapid downregulation of surface CD127 protein by IL-7 beforeCD127 transcriptional suppression and the dose response in extentand duration of suppression highlights the importance of a fine-tunedresponse to IL-7 in CD8 T cells. IL-7 has several pivotal roles in CD8T-cell survival and function including but not limited to theupregulation of Bcl-2,23 induction of perforin expression,15

enhanced glucose metabolism38 as well as the establishment andmaintenance of memory.6,39,40 The fine-tuned modulation of surfaceCD127 protein may allow CD8 T cells to regulate these functionsin response to varying IL-7 concentrations in differentmicroenvironments and in response to different immunologicalchallenges. A disruption in this dynamicity could also lead to pooror inappropriate CD8 T-cell responses. Indeed, dysregulation of IL-7signaling has been implicated in a number of diseases, such as


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multiple sclerosis,41,42 rheumatoid arthritis and juvenile idiopathicarthritis, as well as in breast cancer.43,44 The expression of CD127 isalso dysregulated in Epstein–Barr virus,45–47 cytomegalovirus45,47 andhepatitis C virus48 infections. HIV infection in particular is associatedwith decreased expression of CD127 on CD4 and CD8 T cells.25,49–55

In fact, we have previously shown that soluble HIV Tat proteindownregulates CD127 on the surface of CD8 T cells isolated fromhealthy donors and targets the receptor for degradation by theproteasome.15,26 Understanding the detailed mechanisms by whichIL-7 regulates expression of its own receptor may well lead to noveltherapeutic strategies aimed at improving T-cell number and functionin lymphopenic conditions, as well as in chronic viral infections andseveral autoimmune diseases.

MATERIALS AND METHODS

Jurkat-CD127 cell line and primary human CD8 T cellsThe Jurkat-CD127 cell line was a kind gift from Dr Rene van Lier (Academic

Medical Centre, Amsterdam, The Netherlands). These cells contain the

retrovirally transduced CD127-IRES-GFP sequence and express the human

CD127 cDNA from the cytomegalovirus promoter.22

Primary human CD8 T cells were isolated as previously described.15,23,26

Peripheral blood mononuclear cells were isolated from the blood of healthy

volunteers by Ficoll-Paque density centrifugation followed by CD8 T-cell

purification using magnetic-activated cell sorting human CD8 microbeads and

the AutoMACS Isolation System (Miltenyi Biotech, Auburn, CA, USA).

Purified CD8 T cells and Jurkat-CD127 cells were cultured at a density of

1� 106 cells per ml at 37 1C in media comprised of RPMI 1640 (Hyclone,

Logan, UT, USA) supplemented with 100 U ml�1 penicillin, 100mg ml�1

streptomycin and 0.2 M L-glutamine (Gibco, Grand Island, NY, USA) and

either 20% fetal calf serum (Cansera, Rexdale, Ontario, Canada) for primary

CD8 T cells or 10% fetal calf serum for the Jurkat-CD127 cells. All research

conducted using human blood was reviewed and approved by the Ottawa

Hospital Research Ethics Board.

ReagentsIL-7 and IL-10, obtained from Invitrogen Biosource (Carlsbad, CA, USA), were

resuspended in phosphate-buffered saline plus 0.1% bovine serum albumin at

a stock concentration of 10 ngml�1 and stored at �80 1C. Dex was obtained

from BioVision (San Francisco, CA, USA) and was resuspended in dimethyl

sulfoxide at a stock concentration of 10 mM and stored at �20 1C. STAT5

inhibitor (573108; 0.1 M), JAK inhibitor 1 (420097; 10 mM) and PI3K inhibitor

(LY294002; 10 mM) were purchased from EMD Biosciences (San Diego, CA,

USA) and were dissolved in dimethyl sulfoxide at the indicated stock

concentrations and stored at �20 1C. CD8 T cells were incubated with these

inhibitors at the indicated concentrations for 1 h before the addition of IL-7.

Quantitative real-time PCRTotal RNA was harvested from 5� 105 Jurkat-CD127 or CD8 T cells using the

RNAqueous-4PCR kit for isolation of DNA-free RNA (Ambion, Austin, TX,

USA) according to the manufacturer’s instructions. RNA was quantified using

a Nanodrop ND-1000 instrument (Nanodrop, Wilmington, DE, USA) and

reverse transcribed using the iScript cDNA Synthesis Kit from Bio-Rad

(Hercules, CA, USA). Equal volumes of cDNA from each sample were then

mixed with IQ SYBR Green Supermix (Bio-Rad) and used to quantify CD127

transcripts relative to the RPS18 reference gene. Both reverse transcription and

qPCR were carried out in the iCycler thermal cycler from Bio-Rad. Samples

were incubated at 95 1C for 3 min and then amplified over 40 cycles as follows:

95 1C for 30 s, 59 1C for 20 s and 72 1C for 20 s. At the end of each cycle,

samples were held at 85 1C for 5 s for fluorescence acquisition. Following

amplification, samples were denatured for 1 min at 95 1C followed by final

extension at 72 1C for 1 min. CD127 transcripts were amplified using a forward

primer spanning the exon 4/5 boundary (50-ATGGACGCATGTGAATTTA

TC-30) and a reverse primer in exon 5 (50-GGGAGATGGATCCTATC-30). The

forward primer for RPS18 spanned the exon 2/3 boundary (50-CTGCCATTAA

GGGTGTGG-30) with the reverse primer in exon 3 (50-TCCATCCTTT

ACATCCTTCTG-30). Data analysis was carried out using the 2�DDCt principle

with the Bio-Rad Gene expression analysis macro program. The resulting

expression values were plotted using GraphPad Prism software (La Jolla, CA,

USA), and statistical analysis was done using a two-tailed, paired Student’s

t-test with 95% confidence intervals.

Western blot analysisCD8 T cells (1� 106 cells per condition) were collected by centrifugation at

6000 g for 10 min, and whole-cell extracts were prepared by resuspending the

cell pellets in 50ml RIPA buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM

EDTA, 1% NP-40 and 0.1% SDS with 1� Halt Protease Inhibitor Cocktail

(Thermo Scientific, Rockford, IL, USA)) and incubating at 4 1C for 45 min.

Protein concentration was determined by the Bradford protein assay. Equal

amounts of total cell protein were then resolved by SDS–PAGE and transferred

to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) using

a semi-dry transfer apparatus (Bio-Rad) for 30 min at 15 V. Following transfer,

membranes were blocked overnight in Tris-buffered saline with Tween-20

(TBST, 50 mM Tris–HCl pH7.6, 150 mM NaCl and 0.1% Tween-20), containing

5% nonfat milk. Total CD127 protein was detected using a polyclonal goat

anti-human CD127 antibody (R&D Systems, Minneapolis, MN, USA),

whereas CD127 phosphorylated at Tyr449 was detected using a polyclonal

rabbit anti-human CD127 phospho-Tyr449 antibody (Rockland, Biomol,

Hamburg, Germany) at a concentration of 1mg ml�1 in TBST blocking

solution. Horseradish peroxidase-conjugated Donkey anti-goat (1:5000) and

Donkey anti-rabbit (1:5000) from R&D Systems were used as secondary

antibodies, and proteins were visualized by chemiluminescence using the ECL

Advance Western Blotting Detection Kit (GE Healthcare, Chalfont St Giles,

UK) according to the manufacturer’s instructions. For reprobing for loading

controls, membranes were stripped for 30 min at 50 1C in stripping buffer (2%

SDS, 62.5 mM Tris–HCl pH 6.8 and 100 mM b-mercaptoethanol), washed five

times with TBST, blocked with 5% nonfat milk in TBST for 1 h and then

reprobed with a monoclonal mouse anti-human b-actin antibody (C4; Santa

Cruz Biotechnology, Santa Cruz, CA, USA) diluted (1:200, 1mg ml�1) in TBST

blocking solution followed by an horseradish peroxidase-conjugated Donkey

anti-mouse secondary antibody (R&D Systems) at 1:5000 dilution.

Flow cytometryAnti-CD8-phycoerythrin-Cy5 (PC5) (B9.11) and anti-CD127-phycoerythrin

(PE) (R34.34) fluorochrome-labeled monoclonal antibodies were purchased

from Immunotech Beckman Coulter (Marseille, France). Monoclonal anti-

phospho-STAT5-fluorescein isothiocyanate (FITC) (Y694) and anti-phospho-

STAT3-(Y705) antibodies were purchased from BD Biosciences (San Jose, CA,

USA). All fluorochrome-labeled antibodies used for flow cytometry were

titrated and used at saturating concentrations.

Surface CD127 protein expression was analyzed by incubating cells with

anti-CD127-phycoerythrin antibodies for 30 min in the dark at room

temperature followed by analysis on a Coulter Epics ALTRA flow cytometer

(Fullerton, CA, USA). The protocol was modified slightly in Figure 2a. To

prevent further internalization of CD127 while staining with the antibody,

CD8 T cells were treated with IL-7 for the times indicated and then incubated

with anti-CD127-phycoerythrin antibodies for 30 min on ice. Intracellular

phospho-STAT3 and phospho-STAT5 staining was performed as follows:

isolated CD8 T cells were fixed in 2% paraformaldehyde for 10 min at room

temperature and then permeabilized by incubating in cold 100% methanol for

an additional 10 min at 4 1C. After washing in phosphate-buffered saline, cells

were incubated with anti-phospho-STAT3-fluorescein isothiocyanate or anti-

phospho-STAT5-fluorescein isothiocyanate antibodies in the dark for 30 min

and then analyzed by flow cytometry. Live cells were gated on the basis of side

and forward scatter, and at least 10 000 events were recorded for each sample.

Isotype controls were performed for each fluorochrome-conjugated antibody.

Resulting profiles were analyzed with FCS Express 2 software (De Novo, Los

Angeles, CA, USA), and statistical analysis was done using a two-tailed, paired

Student’s t-test with 95% confidence intervals.

CONFLICT OF INTERESTThe authors declare no conflict of interest.


156


ACKNOWLEDGEMENTSThis work was supported by a grant from the Canadian Institutes of Health

Research (CIHR). PAM is the principal recipient of the research grant. FMG

and SMS are recipients of CIHR doctoral scholarships and EMF is a recipient

of a postdoctoral fellowship from the Ontario HIV Treatment Network. PAM

is supported in part by a mid-career salary award from the Department of

Medicine, University of Ottawa. We thank the volunteers who donated blood

samples for this study.

Author contributions: FMG carried out the qPCR in CD8 T cells and Jurkat-

CD127 cells and flow cytometry in CD8 T cells (Figures 1a, 2b, 3a, 4a, 6, 7b

and e), wrote and revised the manuscript. EMF carried out flow cytometry in

CD8 T cells (Figures 1b and c, 2a, 3b and 5). SMS performed flow cytometry

in Jurkat-CD127 cells and CD8 T cells and carried out western blot analysis

(Figures 4b and 7a, c–e). JAK participated in the ground work of the project.

PAM designed the research, analyzed and interpreted data, and co-wrote the

manuscript.

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Ghazawi FM et al, 2013