il-7 determines the homeostatic fitness of t cells by distinct mechanisms at different signalling...

IL-7 determines the homeostatic fitness of T cells bydistinct mechanisms at different signalling thresholdsin vivo

Claire Pearson�, Ana Silva�, Manoj Saini and Benedict Seddon

Division of Immune Cell Biology, MRC National Institute for Medical Research, The Ridgeway,

Mill Hill, London, UK

The cytokine interleukin (IL)-7 is essential for Treg-cell homeostasis. It remains unclear,

however, whether IL-7 regulates the homeostatic fitness of T cells quantitatively and, if so,

by what mechanisms. We addressed this question by analysing T cells exposed to

different levels of IL-7 signalling in vivo. Using TCR transgenic mice that conditionally

express IL-7Ra, we show that T-cell longevity in the absence of survival cues is not a cell-

intrinsic property but rather a dynamic process of which IL-7 signalling is a key regulator.

Naı̈ve T cells deficient in IL-7Ra expression underwent rapid cell death within hours of in

vitro culture. In contrast, the same T cells from lymphopenic hosts, in which IL-7 is non-

limiting, were able to survive in culture independently of growth factors for many days.

Surprisingly, different levels of IL-7 signalling in vivo evoked distinct molecular mechan-

isms to regulate homeostatic fitness. When IL-7 was non-limiting, increased survival was

associated with up-regulation of anti-apoptotic Bcl2 family members. In contrast, in T-cell

replete conditions i.e. when IL-7 is limiting, we found evidence that IL-7 regulated T-cell

fitness by distinct non-transcriptional mechanisms. Together, these data demonstrate a

quantitative aspect to IL-7 signalling dependent on distinct molecular mechanisms.

Key words: Fitness . Homeostasis . IL-7 . T cells

Supporting Information available online

Introduction

A commonly evoked concept in studies of lymphocyte homeostasis is

that of cellular fitness. Whether a cell lives or dies in a particular

context, such as effector cell transition to a memory state, or survival

of recent thymic emigrants entering a replete peripheral compart-

ment, is a function of its relative fitness [1]. The cytokine IL-7 plays

a fundamental role in homeostasis of the peripheral T-cell

compartment [2–4]. IL-7 is limiting in replete conditions and is a

key determinant of the T-cell compartment size, since total T-cell

numbers in mice lacking one or other CD41 and CD81 subsets are

near-identical to those in mice with both subsets [5–7]. Conversely,

genetic over-expression of IL-7 [8, 9] or its administration in vivo

[10] increases T-cell numbers. It is likely, therefore, that IL-7 is a key

determinant of homeostatic fitness. Lymphocytes are unlikely to

have unfettered access to the multiple environmental cues required

for their survival, but rather receive such signals on a sporadic basis.

Consistent with this view, chemokines direct T cells to sites of IL-7

production within lymph nodes [11]. Thus, the homeostatic fitness

of a T cell and consequently whether it lives or dies in a given

homeostatic context may depend on how frequently it receives

survival signals such as IL-7 and how long the cell can persist in the

absence of such survival signalling.

�These authors contributed equally to this work.

Correspondence: Dr. Benedict Seddone-mail: [email protected].

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201141514 Eur. J. Immunol. 2011. 41: 3656–3666Claire Pearson et al.3656

The mechanisms by which IL-7 maintains T-cell survival, and

therefore regulate cellular fitness, have been the subject of

numerous studies. Many of these have focused on the transcrip-

tional control of key regulators of apoptosis such as anti-apoptotic

factors Bcl2 and Mcl1. Evidence from knockout mice illustrates the

importance played by the balance in expression of Bcl2 family

members. The defects in thymopoeisis in mice lacking IL-7 or

IL-7Ra can be substantially rescued by over-expression of Bcl2

[12, 13], or by compound deficiency with pro-apoptotic molecules

such as Bax [14] or Bim [15]. In vitro, it has long been recognized

that IL-7 stimulation of mutant T-cell lines or primary T cells

up-regulates Bcl2 [12, 13, 16–18], as well as Mcl1 [19]. Conver-

sely, there have been other reports suggesting that Bcl2 expression

is reduced in the absence of IL-7 signalling [3, 20–22]. However,

this is not observed in in vitro cultured T cells where particular

care was taken to isolate viable cells [23]. Therefore, although IL-7

can transcriptionally regulate Bcl2 expression, it remains unclear

whether this accounts for the full range of IL-7 activity in vivo.

While the identity of signals that regulate T-cell survival are

known, it remains unclear how such survival signals determine

homeostatic fitness in order to regulate T-cell homeostasis

in vivo. Are survival signals digital, permitting cell survival when

intact and resulting in cell death in their absence, or do

T cells indeed exhibit varying degrees of fitness depending on their

current exposure to such survival signals? In this study, we report

evidence for different mechanisms of IL-7 regulated T-cell survival

evoked at different levels of IL-7 signalling.

Results

IL-7 signalling strength in vivo determines naı̈ve CD81

T-cell fitness

To examine T-cell survival in the absence of IL-7 signalling, we

used a mouse model in which class I-restricted F5 TCR transgenic

mice conditionally express IL-7Ra using the tetracycline regula-

tory system (F5 TreIL-7R rtTAhuCD2 Il7r�/�, F5 TetIL-7R hereon,

see Materials and Methods) [24]. Induction of IL-7Ra expression,

by feeding mice doxycycline (dox) throughout life (F5 TetIL-

7RON), overcomes the block in thymic development that normally

occurs in Il7r�/� F5 mice and allows the generation of a normal

peripheral compartment of F5 T cells. In contrast to the high

levels of IL-7Ra found in the thymus, peripheral T cells from dox-

fed F5 TetIL-7R mice express much lower levels of IL-7Ra that are

not functional in vivo [24]. Nevertheless, we have previously

shown that withdrawal of dox food from F5 TetIL-7R mice for

three days (F5 TetIL-7ROFF) is sufficient to guarantee complete

loss of residual IL-7Ra expression (referred to as IL-7R– F5 T cells

hereon). Importantly, surface IL-7Ra protein is undetectable on

IL-7R– F5 T cells and cells fail to phosphorylate STAT5 in

response to IL-7 stimulation in vitro [2].

We reasoned that homeostatic fitness of CD81 T cells should

be manifest in their ability to persist in the absence of survival

signals. We therefore isolated F5 T cells and determined their rate

of death in vitro. T cells from control F5 donors ((F5

Rag1�/��C57Bl6/J.CD45.1)F1, IL-7R1 F5 hereon) underwent

progressive apoptosis over several days that was prevented by

addition of IL-7 (Fig. 1A). In the absence of continued

IL-7Ra expression in vivo, IL-7R– F5 T cells disappear relatively

fast, with a half life of �14 d (Supporting Information Fig. 1), a

phenotype that implies their reduced homeostatic fitness. Inter-

estingly, upon culture in vitro, IL-7R– F5 T cells underwent

apoptosis far more rapidly than controls, particularly at early

time points (Fig. 1A). As expected, in the absence of IL-7Raexpression, death of IL-7R– F5 T cells was not prevented by

addition of IL-7 (Fig. 1A).

We next examined the effect of non-limiting IL-7 in vivo on

T-cell fitness. Control F5 T cells were transferred into

T-cell-deficient Rag1�/� hosts in which there is consequently no

T-cell competition for IL-7. Although F5 T cells proliferate in

response to lymphopenia in Rag1�/� hosts, they retain a naı̈ve

Figure 1. IL-7 signalling in vivo determines F5 T-cell fitness. (A) IL-7R–

F5 T cells from F5 TetIL-7ROFF (circles) or IL-7R1 F5 T cells from F5Rag1�/� (squares) donors were enriched for CD81 T cells and culturedfor the indicated time points, either alone (empty symbols, dashedlines) or in the presence of 50 ng/mL of IL-7 (filled symbols, solid lines).Cells were stained with 7AAD and survival was determined by flowcytometry analysis. (B) F5 Rag1�/� splenocytes were transferred toRag1�/� (circles) or Il7�/�Rag1�/� (triangles) host mice for between 7and 14 days. Splenocytes from host mice and intact F5 Rag1�/�

(squares) mice were enriched for CD81 T cells and cultured for theindicated time points, either alone (empty symbols, dashed lines) orwith 50 ng/mL of IL-7 (filled symbols, solid lines). Percentage ofsurviving cells were normalized to the percentage of live cells on day0. Graphs are representative of at least three independent experiments.

Eur. J. Immunol. 2011. 41: 3656–3666 Molecular immunology 3657


phenotype [25, 26]. After 7–14 d, survival of transferred cells was

compared with T cells from intact F5 donors. Remarkably, F5

T cells recovered from Rag1�/� hosts survived in vitro in the

complete absence of any survival or growth factors for many days

(Fig. 1B), and exogenous IL-7 had little additional effect on their

survival. T-cell survival was Bcl2 dependent, since addition of

specific inhibitor ABT-737 caused death of all cells by 24 h (data

not shown). Although naı̈ve T cells proliferate in lymphopenic

hosts, persistence of F5 T cells in vitro was a function of survival

and not cell division, as F5 T cells did not continue to divide in

vitro, even in the presence of exogenous IL-7 (Supporting

Information Fig. 2A). While not further enhancing survival, IL-7

did maintain the increased cell size observed in F5 T cells

transferred to Rag1�/� hosts, suggesting that the trophic prop-

erties of IL-7 are more short lived and do require persistent IL-7

signalling (Supporting Information Fig. 2B) and also confirmed

that IL-7 signalling had ceased in IL-7 free cultures. In Rag1�/�

hosts, there is a lack of T-cell competition for other factors

important for CD81 T-cell survival, such as DCs expressing

self-peptide–MHC (spMHC) and IL-15, which could also

influence the fitness of F5 T cells. Additionally, IL-7Ra is also a

component of the heterodimeric thymic stromal lymphopoietin

(TSLP) receptor, that has also been implicated in maintenance of

naı̈ve CD41 T cells [27, 28], and loss of signalling through this

receptor could also be contributing to death of IL-7R– F5 T cells.

Therefore we directly addressed the role of IL-7 in enhancing T-

cell fitness by transferring the same cells to IL-7-deficient Rag1�/�

mice. Significantly, cells recovered from Il7�/� Rag1�/� hosts

died in vitro in the absence of IL-7 with similar kinetics to control

F5 T cells and their survival was enhanced by addition of

exogenous IL-7 to cultures (Fig. 1B). This demonstrated that the

enhanced fitness of F5 T cells transferred to Rag1�/� hosts was

indeed IL-7 dependent.

IL-7R– F5 T cells are highly susceptible to the inductionof apoptosis

We wished to examine the molecular mechanisms that were

responsible for the range of cellular fitness observed in F5 T cells

receiving different strengths of IL-7 signalling in vivo. First, we

asked whether IL-7R– F5 T cells had an increased susceptibility to

apoptosis. We examined caspase activity in IL-7R– F5 T cells at

the earliest stages of in vitro culture by assessing fluorescently-

labelled caspase inhibitor peptide (FLICA) binding to active

caspases. While little caspase activity was apparent in control F5

T cells, caspase activation was readily detectable in a significant

population of IL-7R� F5 T cells during the 1 h in vitro duration of

the assay (Fig. 2A). We also assessed onset of apoptosis by

measuring annexin V binding to phosphatidylserine, whose

translocation from inner to outer membrane leaf is an early

event during cell death. While few viable IL-7R1 or IL-7R– F5

T cells were annexin V1 ex vivo, 1 h culture of IL-7R– F5 T cells

was sufficient to induce a substantial population of high forward

scatter (FSChi) Annexin V1 cells not evident in control IL-7R1 F5

T cells (Fig. 2B). Finally, we also assessed specific activation of

caspase 3, one of the executioner caspases, in IL-7R– F5 T cells

directly ex vivo and following culture in vitro. Ex vivo, neither

IL-7R1 F5 control nor IL-7R– F5 T cells had elevated levels of

activated caspase 3, suggesting that there were not high levels of

detectable apoptosis in vivo. However, following culture for 24 h,

activated caspase 3 was readily detectable in both cell types but

was particularly elevated in IL-7R– F5 T cells in which viability

was also more reduced (Fig. 2C). Taken together, these data

indicate that the reduced fitness of IL-7R– F5 T cells is associated

with a very substantial elevation in their susceptibility to

induction of apoptosis.

IL-7 signalling regulates Bcl2 expression in lympho-penic but not replete conditions in vivo

It has long been recognized that T cells cultured in vitro with IL-7

up-regulate Bcl2 and this is thought to be a key mechanism

through which cell survival is promoted. We therefore investi-

gated whether modulation of Bcl2 expression in vivo by IL-7

Figure 2. Rapid induction of apoptosis in IL-7R� F5 T cells. Caspaseactivation and apoptosis induction was measured in the live popula-tions of F5 Rag1�/� (IL-7R1 F5) and F5 TetIL-7ROFF (IL-7R� F5) splenicCD81 T cells by flow cytometry. (A) Total caspase activity wasdetermined ex vivo by 1-h incubation with fluorescent inhibitor ofcaspase activation (FLICA) reagent (see Materials and Methods).(B) Contour plots are of FSc versus Annexin V staining of IL-7R� F5and IL-7R1 F5 T cells either ex vivo (0 h column) or following 1 h cultureat 371 (1 h column). (C) Contour plots are of 7aad versus FSC and SSCversus active caspase 3 (act-Casp3) staining on live-gated CD81 T cellsat 0 h, and at 24 h after in vitro culture in medium alone, plots are of7aad versus FSc and SSc versus active caspase 3 staining on totalCD81-gated cells. Values displayed indicate the percentage of cells inthe indicated gates. Data are representative of two (A, C) or four(B) independent experiments.

Eur. J. Immunol. 2011. 41: 3656–3666Claire Pearson et al.3658


signalling could account for the differential survival of IL-7R– F5

T cells and IL-7R1 F5 T cells from lymphopenic hosts.

Examination of F5 T cells transferred to Rag1�/� hosts revealed

a robust increase in Bcl2 expression levels (Fig. 3A and C),

consistent with the continued survival of these cells in vitro in the

absence of exogenous growth factors (Fig. 1B). The increase in

Bcl2 levels observed was similar to that previously reported in F5

T cells cultured in vitro with exogenous IL-7 [2]. Surprisingly, in

IL-7R� F5 T cells that were incapable of receiving IL-7 signalling

[2], Bcl2 levels were identical to those in control IL-7R1 F5 T cells

(Fig. 3B and C). IL-7R– F5 T cells were recovered from F5

TetIL-7R mice taken off dox no more than seven days previously

and at this relatively early stage of IL-7R loss (Supporting

Information Fig. 1), there was little change in splenic F5 T cell

numbers compared with dox-fed controls (data not shown).

Therefore, these data suggest that basal Bcl2 expression by naı̈ve

CD8 T cells in replete F5 hosts does not depend on IL-7 signalling.

To further examine whether or not basal Bcl2 expression

depends on IL-7 signalling, we examined Bcl2 levels in thymo-

cytes, since both IL-7Ra and Bcl2 expression are dynamically

regulated during development. IL-7Ra is expressed in DN

thymocytes, required for normal DN survival and expansion [29],

but is completely lost in DPs. Following successful positive

selection, CD4 and CD8 single positive (SP) thymocytes re-

express IL-7Ra (Fig. 4A). Correlating with IL-7Ra, Bcl2 levels

were high in WT DNs, greatly reduced in DPs and expression

restored in SPs of WT thymocytes (Fig. 4A), consistent with the

view that IL-7 signalling is regulating Bcl2 expression in vivo

during thymic development. To test whether Bcl2 expression in

this developmental context was directly dependent on IL-7

signalling, we examined thymic development of Il7r�/� and dox-

fed F5 TetIL-7R mice. In Il7r�/� mice, although thymus size is

approximately 100-fold less than WT [30], the gross thymic

phenotype is remarkably normal in terms of the four major

subsets defined by CD4 and CD8 expression. Interestingly, regu-

lation of Bcl2 expression during thymic development was

virtually identical to that of WT (Fig. 4A). In dox-fed F5 TetIL-7R

Figure 3. IL-7 regulates Bcl2 expression levels by F5 T cells only inlymphopenic conditions. Bcl2 expression was measured ex vivo insplenic CD81 T cells by flow cytometry. Levels of Bcl2 by IL-7R1 F5T cells from F5 Rag1�/� donors (solid line) were compared with (A)IL-7R1 F5 T cells transferred first to Rag1�/� hosts for between 7 and 14days (IL-7R1 F5 in Rag1�/�; dashed line) or (B) IL-7R– F5 T cells from F5TetIL-7ROFF donors (dashed line). Isotype controls are shown in greyfilled histogram. (C) Bar chart indicates the Bcl2 mean intensity offluorescence (MFI) ratio to IL-7R1 F5 T cells, by IL-7R1 F5 T cells inRag1�/� hosts (n 5 4) or IL-7R– F5 T cells (n 5 13). Error bars indicate SD.Data are representative of three independent experiments.

Figure 4. Expression of Bcl2 does not require IL-7Ra expression in vivo.(A) WT C57BL/6 and Il7r�/� thymocytes were analysed by flowcytometry to discriminate thymic subpopulations (contour plots): DN(CD4�CD8�); CD41CD81; CD8 SP (CD4�CD81); and CD4 SP (CD41CD8�).IL-7Ra (histogram top row) and Bcl2 levels (histogram middle andbottom rows) were measured ex vivo by flow cytometry in WT C57BL/6(solid line) and Il7r�/� (dashed line) thymic subpopulations. Isotypecontrol for Bcl2 staining is displayed in grey filled histogram. Theresults are representative of three individual mice and two indepen-dent experiments. (B) Histograms show FSc of DP and SP thymocytesfrom dox-fed F5 TetIL-7R mice (solid line) compared with the samesubsets from control F5 mice (grey fill). (C) Histograms show IL-7Rexpression by DP (broken line) and SP (solid line) thymocytes from dox-fed F5 TetIL-7R mice, as compared with DP thymocytes from F5 controlmice (grey fill). Histograms of Bcl2 expression are of DP and SPthymocytes from the strain indicated. Data are representative of sixindividual mice and of two independent experiments.



mice, IL-7Ra is expressed ectopically on DP thymocytes as

previously described [24]. Analysing cell size of thymocytes from

F5 TetIL-7R mice revealed an increase in cell size in both DP and

SP subsets (Fig. 4B), confirming that IL-7R signalling was func-

tional in these cells. As is true in WT thymocytes, F5 thymocytes

upregulate Bcl2 expression as they mature from DP and SP

stages. Significantly, ectopic expression of IL-7Ra on DPs of dox-

fed F5 TetIL-7R mice did not result in ectopic expression of Bcl2.

Rather, Bcl2 expression between the DPs and SPs of these mice

was similar to that observed in F5 control thymocytes (Fig. 4C).

Taken together, these data suggest that basal Bcl2 expression in

vivo is not dependent on IL-7 signalling, and that in normal

homeostatic conditions, IL-7 must be promoting survival by a

mechanism other than simply inducing expression level of Bcl2.

Transcriptional control of apoptosis by high but notlow levels of IL-7 signalling

Since Bcl2 expression levels could not account for the accelerated

apoptosis of IL-7R– F5 T cells, we used microarray analysis to

identify IL-7-regulated genes that may be involved in regulating

survival of these cells. We compared gene expression between F5

T cells from control, dox-fed F5 TetIL-7R and dox free F5 TetIL-7R

mice. We reasoned that control F5 T cells from intact F5 donors

receive low level IL-7 signalling due to the competitive nature of

the replete T cell compartment, while peripheral F5 T cells from

dox-fed and dox-free F5 TetIL-7R mice would be receiving no IL-7

signalling in vivo. To identify gene targets induced by non-limiting

IL-7 signalling, we also examined gene expression by control F5

T cells stimulated overnight with IL-7 at a concentration sufficient

to induce Bcl2 upregulation similar to that observed in F5 T cells

transferred to lymphopenic Rag1�/� hosts (Fig. 3) [2]. Figure 5A

shows the Principle Components Analysis (PCA) of the array data.

Biological replicates of IL-7-stimulated, control ex vivo F5 T cells

and cells from F5 TetIL-7R mice all clustered in a distinct manner

to one another. Our previous studies have suggested that the very

low levels of IL-7Ra expression on peripheral T cells in dox-fed F5

TetIL-7R mice are not functionally significant in vivo [24], and

consistent with this, the PCA analysis of F5 TetIL-7R array

data all clustered together, regardless of dox feeding of donors.

Comparison of gene expression between samples from dox-fed and

dox-free mice revealed no statistically significant differences

between these two data sets, supporting the view that the low

levels of IL-7Ra expression by peripheral F5 T cells in dox-fed F5

TetIL-7R mice were not functionally significant. We therefore

pooled the six replicate arrays from both dox-fed and dox-free F5

TetIL-7R donors to compare gene expression with control ex vivo

samples.

We specifically analysed expression of genes involved in

regulating the mitochondrial pathways of apoptosis. Figure 5B

summarizes changes induced by IL-7 stimulation of control F5

T cells compared with ex vivo F5 T cells, whereas Fig. 5C

compares gene expression between IL-7R– F5 T cells with control

IL-7R1 F5 T cells. IL-7 stimulation resulted in statistically

significant induction in expression of anti-apoptotic factors Bcl2,

Bcl-xL and Mcl1 expression (Fig. 5B and S3A) and reduced

expression of the pro-apoptotic BH3-only family member Bid.

However, other BH3-only family members like Bim (Fig. 5B and

S3B) were slightly elevated or not affected. Similarly, expression

of the BH3-multidomain proteins Bax, Bak and Bok, was not

affected by IL-7 stimulation (Supporting Information Fig. 3B and

C). Thus, non-limiting IL-7 stimulation specifically upregulated

Figure 5. Regulation of gene expression by IL-7 signalling. Microarrayanalysis was performed to compare gene expression in purified CD81

populations of IL-7R1 F5 T cells from F5 Rag1�/� mice, either ex vivo(IL-7R1 F5; n 5 4) or following cultured with 50 ng/mL IL-7 for 24 h (F51

IL-7; n 5 4); and of F5 T cells from F5 TetIL-7RON (ON; n 5 3) or F5 TetIL-7ROFF (OFF; n 5 3) donors. (A) Principle components analysis (PCA) wasperformed in BioConductor software to assess clustering of microarraychip replicates. (B-C) Diagrams illustrate the relative expression ofgenes in the Bcl2 family. Genes with statistically significant alterationsin expression in (B) IL-7R1 F51IL-7 or (C) IL-7R- F5 compared with exvivo IL-7R1 F5 were highlighted in green (up-regulated relative tocontrol) and red (down-regulated relative to control). Gene expressionlevels unchanged between the different conditions are displayed inwhite. There were no significant differences in gene expressionbetween F5TetIL-7RON and F5TetIL-7ROFF microarray replicates andthese were therefore combined (n 5 6) for analysis in (B and C).



expression of anti-apoptotic molecules Bcl2, Bcl-xL and Mcl1 and

this is likely to be the primary mechanism of anti-apoptotic

activity by such IL-7 stimulation, consistent with previous reports.

In contrast, comparison of gene expression between IL-7R– F5

and control F5 T cells revealed surprisingly few differences in key

molecules regulating apoptosis (Fig. 5C). In addition to Bcl2,

levels of expression of other anti-apoptotic factors Bcl-xL and

Mcl1, BH3 only molecules Bad, Bik, Bim, Noxa, Puma and multi-

domain BH3 molecules Bax, Bak and Bok were all unaffected by

the absence of IL-7 signalling (Supporting Information Fig. 3).

Unexpectedly, expression of the pro-apoptotic BH3-only family

member Bnip3 was significantly reduced in the absence of IL-7Raexpression, while Bid also appeared reduced, but did not reach

statistical significance (Supporting Information Fig. 3B). There-

fore, there were no changes in the expression of Bcl2 family

members that could provide a simple explanation for the reduced

fitness of IL-7R� F5 T cells.

IL-7 signalling in vivo regulates mitochondrial homeo-stasis

Surprisingly, few Bcl2 family members were differentially

expressed between IL-7R- and IL-7R1 F5 T cells. However, it was

possible that IL-7 signalling in vivo was regulating survival by

influencing abundance of these key apoptosis regulators at a post

translational level, for instance by influencing protein stability

or turnover. We therefore assessed by Western blot the levels of

anti- and pro-apoptotic proteins in cell lysates from samples of

IL-7R� and IL-7R1 F5 T cells. As data in Fig. 6 show, abundance of

Bcl2, Bcl-xL, Mcl1, Bad and Puma were similar between IL-7R– and

IL-7R1 F5 T cells, consistent with prior transcript analysis

(Supporting Information Fig. 3A), and FACS analysis in the case

of Bcl2 (Fig. 3). Previous studies of cell lines have shown that IL-7

can promote cell survival by inactivating Bad through its Akt/PKB-

dependent phosphorylation [31]. However, detailed analysis of F5

transgenic mice that over-express Bad, consequently inducing

thymocyte apoptosis [32] (Supporting Information Fig. 4A),

revealed no evidence of defects in naı̈ve T-cell survival in vitro

(Supporting Information Fig. 4B) or in vivo (Supporting Informa-

tion Fig. S4C–S4E) and furthermore phosphorylation of Bad, and

thereby its inactivation, is even increased in IL-7R– F5 T cells

(Supporting Information Fig. 4F). Examining Bid and Bim-L levels

revealed small but significant reductions in protein abundance of

both in IL-7R– F5 T cells, which in the case of Bid, mirrored

differences observed transcriptionally (Supporting Information

Fig. 3B). Furthermore, the active cleaved form of Bid, tBid, was

not detected in either IL-7R1 or IL-7R– F5 T cells. Thus,

intriguingly, the only detected changes in abundance or activation

of anti-apoptotic and BH3-only molecules in IL-7R– F5 T cells

would rather be expected to inhibit their apoptosis.

Finally, we wished to examine whether there was any evidence

that mitochondrial homeostasis was perturbed in the absence of IL-7

signalling in T cells. We therefore examined mitochondrial integrity

of IL-7R– F5 T cells using the cationic dyes mitotracker red and

TMRE that are actively taken up by mitochondria and whose

Figure 6. Regulation by IL-7 of Bcl2, BH3-multi-domain and BH3 only family member proteinlevels in vivo. IL-7R1 F5 T cells from F5 Rag1�/�

donors and IL-7R� F5 T cells from F5 TetIL-7ROFF

donors were purified and cell lysates prepared.(A–C) Cell lysates were resolved with 12% SDS-PAGE and immunoblotted with the indicatedantibodies. Levels of the (A) pro-survival/anti-apoptotic proteins Bcl2, Bcl-xL, and Mcl1; (B) pro-apoptotic BH3-multidomain proteins Bax, Bak andBok; (C) BH3 only proteins Bad, Puma, Bid, Bim-ELand Bim-L were analysed with specific antibodies.Blots were reprobed with an anti–actin antibody(a-b indicate actin loading shared by differentprobes of the same gels) to confirm even proteinloading. (A–C) Values displayed refer to densito-metry quantification normalized to actin.(D) Densitometry quantifications showing ratio ofIL-7R� F5: IL-7R1 F5 for the analysed proteins.Results represent the mean1SD of between 2 and5 independent experiments. �po0.05 (Student’st-test), n 5 3 or more.



retention is dependent on the integrity of the mitochondrial

membrane. While total mitochondrial mass was similar between

IL-7R– and IL-7R1 F5 T cells (Fig. 7A), we found that both mito-

tracker red (Fig. 7B) and TMRE staining (Fig. 7C) of IL-7R– F5

T cells was reduced as compared with control IL-7R1 F5 T cells,

suggesting that the integrity of mitochondria in these cells is

compromised as compared with control F5 T cells. Such a finding is

consistent with the rapid induction of caspase activity and apoptosis

observed in IL-7R– F5 T cells (Fig. 2). So although we could not

identify any differences at the level of protein abundance of key

factors regulating mitochondrial homeostasis between IL-7R– and

IL-7R1 F5 T cells, these data strongly suggest that in replete hosts,

IL-7 still regulates T-cell survival through the mitochondrial

apoptosis pathway.

Discussion

IL-7 is essential for normal lymphocyte homeostasis. Here, we

investigated the contribution of IL-7 signalling in vivo to

homeostatic fitness of T lymphocytes. Varying the level

of IL-7 signalling in vivo revealed a crucial quantitative aspect to

the activity of IL-7. A surprisingly broad range of homeostatic

fitness, in terms of ability to survive, was apparent in F5

T cells receiving differing levels of IL-7 signalling in vivo. F5

T cells that had lost IL-7R signalling in vivo did not survive for long

in vitro. In contrast, the F5 T cells from hosts with non-limiting

levels of IL-7 persisted in vitro in the complete absence of any

survival signalling for many days. Interestingly, we found evidence

that the mechanisms by which IL-7 signalling in vivo regulated

T-cell fitness varied, depending on the homeostatic context.

IL-7 is arguably the most important cytokine for T-cell survival.

In the present study, we found that F5 T cells have a half life of

only 14 days in vivo in the absence of continued IL-7Ra expression,

which is shorter than is observed in the absence of TCR signalling

[33–35]. Interestingly, we found that F5 TCR transgenic T cells

exhibited highly distinct survival profiles depending on the host

environment they came from. Remarkably, F5 T cells recovered

from IL-7 sufficient lymphopenic Rag1�/� hosts survived in vitro

for several days in the complete absence of exogenous IL-7.

Conversely, IL-7R– F5 T cells underwent the most rapid apoptosis

in vitro. These data suggest that the homeostatic fitness of T cells

can be defined in terms of their ability to persist in the absence of

further survival signalling, as revealed by culture in vitro. It is

unclear how frequently T cells receive specific survival signals in

vivo, but there is likely to be a stochastic element to when T cells

receive survival signalling. Therefore, homeostatic fitness in the

terms described here would determine how long a T cell could

persist in the absence of survival signals and therefore how likely a

cell is to successfully receive further signals to support its persis-

tence in the repertoire. Consistent with this, the broad range of

homeostatic fitness we observed in F5 T cells from differing hosts

closely matched the behaviour of the cells in their native envir-

onments. The ability of F5 T cells from lymphopenic hosts to

survive for so long, even in the absence of survival signalling,

implies that there should be little T-cell death in vivo. Previous

studies of lymphopenia-induced proliferation of F5 T cells find

exactly this [26]. Conversely, the reduced fitness of IL-7R� F5

T cells is consistent with their relatively rapid loss in vivo. In this

context, the 14 d half-life of IL-7R� F5 T cells, short in comparison

with other studies, in fact seems long given the poor fitness of

these T cells in vitro. This implies the activity of other survival

factors on IL-7R– F5 T cells in vivo and we have previously shown

that IL-15 contributes to survival of F5 T cells in the absence of IL-7

[2]. Therefore, the decline of IL-7R� F5 T cells in dox-free F5

TetIL-7R mice could represent a failure of these T cells to receive

sufficient survival signals in time to prevent their death. Thus,

Figure 7. Dysregulated mitochondrial homeostasis in IL-7R– F5 T cells.Total mitochondrial mass was determined by staining cells withMitotracker green. Mitochondrial membrane potential (DCm) wasassessed ex vivo by staining spleen cells from IL-7R1 F5 and IL-7R– F5mice with the potentiometric dyes Mitotracker red and TMRE andanalysing the live CD81 T cells by flow cytometry. Mitotracker red andTMRE intensity reflects the DCm. Histograms show staining with(A) Mitotracker green, (B) Mitotracker red and (C) TMRE intensity byCD81-gated IL-7R1 F5 T cells (broken lines) and IL-7R� F5 T cells (solidlines). Data are representative plots from one of three mice and arerepresentative of two independent experiments.



T-cell persistence in vivo is not simply regulated by the presence or

absence of survival signalling, but rather is a dynamic process in

which cell fitness is constantly tuned by specific environmental

cues, of which IL-7 is a key factor.

Transcriptional regulation of anti-apoptotic proteins, such as

Bcl2 and Mcl1, has long been evoked as a key mechanism of IL-7

activity. In the present study, such regulation of Bcl2 family

members was apparent in vivo, in T cells transferred to lympho-

penic hosts, which resulted in substantially upregulated Bcl2

protein levels, and in CD81 T cells stimulated in vitro with

IL-7. Microarray analysis of these T cells revealed a number of

transcriptional changes, in addition to Bcl2 upregulation, that

could account for their enhanced survival. In both these cases,

IL-7 stimulation was non-limiting due to the relatively high cyto-

kine dose employed in vitro and the lack of host competition in the

lymphopenic host environment. In replete F5 mice, where the

homeostatic balance has resulted in a peripheral compartment

populated with the maximal number of mature T cells possible for

that host, IL-7 is available in limiting quantities. Interestingly, our

data suggest that under such conditions IL-7 regulates T-cell fitness

by mechanisms distinct from those that occur during non-limiting

IL-7 signalling. Although the correlation between IL-7Ra and Bcl2

expression in WT thymus implies a regulatory relationship

between IL-7 signalling and Bcl2 expression in vivo, our data show

this is clearly not the case for naı̈ve T cells. In thymus, develop-

mental regulation of Bcl2 between DP and SP stages did not

depend on IL-7R expression. Conversely, ectopic expression of

IL-7Ra in DP thymocytes of dox-fed F5 TetIL-7R mice did not

induce Bcl2 expression. Similarly, in peripheral T cells we found

that continued IL-7R expression was not required for normal Bcl2

expression in IL-7R– F5 T cells. This was evident both at the protein

level, by FACS and Western blot, and at the level of mRNA.

Furthermore, wider analysis of many other Bcl2 family members

revealed a similar scenario, that mRNA and protein levels were

normal in IL-7R– F5 T cells. Although there is evidence that IL-7

regulates Bcl2 expression in activated T cells [3] and early thymic

progenitors [18], our data suggest that late developmental and

steady state control of Bcl2 expression in naı̈ve T cells is not

dependent on IL-7 signalling. This view is consistent with studies

of mice expressing a conditional IL-7R transgene in T cells in which

loss of IL-7R expression was associated with reduced T cell fitness

but not a change in Bcl2 expression [36]. Thus, IL-7 must be

controlling naı̈ve T-cell survival by mechanisms other than simply

regulating expression level of Bcl2 family members.

Taken together, our data strongly suggest that IL-7 controls

homeostatic fitness of T cells in replete hosts by non-transcrip-

tional mechanisms. IL-7 can activate PI3K [23, 37] and down-

stream Akt/PKB whose kinase activity can potentially modulate

multiple pathways and that could constitute such non-transcrip-

tional mechanisms. Consistent with this view, IL-7 has been

reported to prevent apoptosis in IL-7 responsive cell lines by

inhibiting Bad activity following Akt/PKB phosphorylation of Bad

[31]. However, using F5 T cells over-expressing Bad, we could find

no evidence that Bad was regulating naı̈ve T-cell fitness in vitro, or

in vivo in a range of homeostatic environments or in the absence of

IL-7 signalling altogether. This is also consistent with experiments

showing that inhibition of PI3K does not block the pro-survival

properties of IL-7 [23] in vitro. However, in vitro, any potential

pro-apoptotic consequence of PI3K blockade may be masked by

the effects of upregulation of Bcl2 expression by IL-7. Furthermore,

it is unclear whether or to what extent IL-7 activates PI3K in naı̈ve

T cells in vivo. Thus, it is not possible to exclude a potential pro-

survival role for IL-7-dependent PI3K activation in vivo. The non-

transcriptional mechanisms by which IL-7 promotes T-cell survival

in vivo remain obscure. However, since we observed no differences

in abundance of key Bcl2 family members in IL-7R� F5 T cells, it

seems likely that regulation at the level of sub-cellular localization

of pro- or anti-apoptotic proteins and/or their interaction with one

another may rather account for the perturbed mitochondrial

homeostasis we observed in IL-7R� F5 T cells. Furthermore,

another study suggests that posttranslational regulation of glucose

transporters may be involved [36].

In conclusion, we show for the first time that homeostatic

fitness of T cells is dynamically regulated by IL-7, involving

multiple mechanisms that differ between lymphoreplete and

lymphopenic conditions. The view that T-cell fitness is not a

digital state of either survival or death but rather dynamic state is

consistent with concepts of competition for survival resources.

Such a view is also consistent with the recent insights into the

high mobility of lymphocytes within the 3-dimensional structure

of the lymph node [38, 39], and that the source of IL-7, and likely

other survival factors within these structures, is not homo-

geneously distributed, but rather focal and from specific cell types

[11]. In such a context, a dynamic fitness model of T-cell survival

would permit integration and interpretation of multiple and

likely sporadic survival cues. Distinct mechanisms of regulating

T-cell fitness also permit appropriate homeostatic responses.

Post-translational regulation of T-cell fitness, as occurs in

lymphoreplete conditions, allows for the most rapid response to

changing homeostatic conditions, while transcriptional changes

as occur in lymphopenia permit more sustained and robust

homeostatic responses by T cells. We identified a key role for IL-7

in regulating T-cell fitness. It will be interesting in future studies

to determine whether other signals known to be important for

T-cell homeostasis, such as TCR signalling induced by spMHC,

also influences T-cell fitness and by what mechanism.

Materials and methods

Mice

F5Il7r�/� TreIL-7R rtTAhuCD2 tetracycline-inducible mice (TetIL-

7R) have been described previously [24]. Breeders and weaned

pups were fed doxycycline (dox) in food (3 mg/g) to induce

IL-7Ra expression. (F5Rag1�/��C57BL/6J Ly5.1)F1 mice were

used as controls throughout. These strains and F5 Rag1�/�

BadhuCD2 [32], Rag1�/�, Il7r�/� and F5 Rag1�/� mice were bred

in a conventional colony free of pathogens at the NIMR, London.



All lines used were of the H-2b haplotype. Animal experiments

were performed according to the institutional guidelines and

Home Office regulations under project license 80/2092.

Flow cytometric analysis

Flow cytometry was carried out using thymus, spleen cells, or

peripheral blood lymphocytes (PBLs). Cell concentrations were

determined using a Scharfe Instruments Casy Counter (Scharfe

System, Reutlingen, Germany). Cells were incubated with

saturating concentrations of antibodies in 200 mL PBS-bovine

serum albumin (0.1%)-azide (1 mM) for 30 mins at 41C followed

by two washes in PBS-bovine serum albumin-azide. Monoclonal

antibodies used in this study were as follows: Pacific blue-CD4

(RM4-5; eBioscience, San Diego, CA, USA), PE-CD8a (53-6.7, BD

Biosciences, PharMingen), FITC, PE Cy5, allophycocyanin-CD8a(eBioscience), PE, PE Cy5, allophycocyanin-CD127 (A7R34,

eBioscience), allophycocyanin-TCRb (H57-597; eBioscience),

FITC-TCRb (BD Biosciences), FITC, AF-780-CD44 (IM7;

eBioscience), PE-Ly5.1 (BD Biosciences). Cell viability was

determined by 7-AAD (Sigma, St. Louis, MO, USA) exclusion

and labelling at 10 mg/mL. Four- and six-colour cytometric

staining was analysed on a FACSCalibur (Becton Dickinson,

San Jose, CA, USA) and a Cyan (Dako Cytomation), respectively.

Data were analysed using the Flowjo software v8.1 (Tree Star,

Ashland, OR, USA). Cells were labelled with 2mM carboxyfluor-

escein diacetate succinimidyl ester (CFSE; Molecular Probes) in

Dulbecco PBS (Invitrogen) for 10 min at 371C and washed twice.

Analysis of total active caspases was performed by adding

1� carboxyfluorescein-labelled VAD-fluoromethylketone (FMK)

FLICA (Chemicon) reagent to surface-stained cells and incubated

for 60 min at 371C with 5% CO2 in the dark prior to acquisition.

PE-Bcl2 (BD Biosciences) and active PE-caspase 3 (BD Bio-

sciences) staining of IC fix buffer (eBioscience) fixed samples was

carried out according to manufacturer’s instructions. Annexin

V-FITC binding was performed using calcium-containing annexin

binding buffer. Cells were stained with TMRE (Sigma-Aldrich,

St Louis, MO, USA) in PBS to a final concentration of 125 nM, and

incubated for 30 min at 371C with 5% CO2 to assess mitochon-

drial membrane potential (DCm). Total mitochondrial mass and

membrane potential were also determined using mitotracker

green and red dyes (Invitrogen), respectively, according to

manufacturers’ instructions.

Cell isolation and culture

For in vitro culture experiments, CD81 T cells were purified

490% by magnetic-activated cell sorting (MACS) using anti-

CD8a microbeads and LS columns (Miltenyi Biotec, Bergisch

Gladbach, Germany) following manufacturer’s instructions. For

microarray and Western blot analysis, CD81 T cells were purified

498% using the Easysep PE selection kit (StemCell Technolo-

gies) using PE-CD8a (eBioscience). Primary naı̈ve CD81 T cells

were cultured in 24-well plates at 1�106 cells/mL at 371C with

5% CO2 in RPMI-1640 medium (Sigma-Aldrich) supplemented

with glutamine, 2-mercaptoethanol and antibiotics (all Sigma-

Aldrich). Where used, IL-7 (Peprotech, Rocky Hill, NJ, USA) was

supplemented at 50 ng/mL.

Microarray preparation and gene expression analysis

CD81 T cells were sorted, and total RNA was prepared using the

RNEasy mini kit (Qiagen). RNA was quality and quantity

controlled for degradation on a BioAnalyzer 2100 (Agilent).

Since the starting quantity of RNA for each sample did not

exceed mg, two cycle amplification was performed, as recom-

mended by the manufacturer (Affymetrix). The GC-RMA (Robust

Multiarray Analysis) algorithm was applied to the probe level

data (CEL files). Quality control and data processing was

performed at the Bloomsbury Centre for Bioinformatics, Univer-

sity College London, using the limma, gcrma, simpleaffy,

annotate, annaffy and affycoretools R packages in Bioconductor.

Multiple testing correction was applied for the data using the

Benjamini and Hochberg False Discovery Rate. Annotation of

each probe set was derived using the NetAffx site (Affymetrix).

Microarray data were deposited in ArrayExpress (accession

number E-TABM-991).

Western blotting

Cell pellets containing 1� 106 cells were lysed at 41C in 1 mL 1%

NP40 lysis buffer. Protein lysates were run on 12% SDS-PAGE

acrylamide gels and protein content analysed on nitrocellulose

membrane with the following antibodies: Mcl1 (Rockland), Bcl2,

BclXL, Bok, Bax, total Bad, Bim, Bid, Bak, Puma, pBad (Ser112)

(all from Cell Signaling Technology), and Actin (Santa Cruz).

Densitometry calculations of proteins were calculated in the

ImageJ v1.43 (NIH, Public Domain).

Acknowledgements: The authors thank Biological Services for

animal husbandry and technical support, Hugh Brady for

providing Bad transgenic mice. This work was supported by the

Medical Research Council UK under programme code

U117573801.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: dox: doxycycline � DP: double positive � FLICA:

fluorescent labeled inhibitors of caspases � FSC: forward scatter �PCA: Principle Components Analysis � SP: single positive � SSC: Side

Scatter � TMRE: tetramethylrhodamine, ethyl ester, perchlorate

Current addresses: Dr. Claire Pearson, Translational Gastroenterology

Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK.

Dr. Manoj Saini, Centre for Molecular and Cellular Biology of

Inflammation, Kings College London, London, UK

Full correspondence: Dr. Benedict Seddon, Division of Immune Cell

Biology, MRC National Institute for Medical Research, The Ridgeway,

London NW7 1AA, UK

Fax: 144-20-8913-8531

e-mail: [email protected].

Received: 16/2/2011

Revised: 15/8/2011

Accepted: 14/9/2011

Accepted article online: 19/9/2011



il-7 determines the homeostatic fitness of t cells by distinct mechanisms at different signalling...

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