of July 26, 2010
This information is current as
online May 3, 2010;
2010;184;5949-5953; originally publishedJ. Immunol.
Mention, Odile Richard-Le Goff and James P. Di Santo
Nuno L. Alves, Nicholas D. Huntington, Jean-Jacques
IL-7 Expression In Vivo
Dynamically Regulates Intrathymic
Epithelial Cell Cross-Talk
Cutting Edge: A Thymocyte-Thymic
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Cutting Edge: A Thymocyte-Thymic Epithelial Cell
Cross-Talk Dynamically Regulates Intrathymic
IL-7 Expression In Vivo
Nuno L. Alves,*,†,‡Nicholas D. Huntington,*,†Jean-Jacques Mention,*,†
Odile Richard-Le Goff,*,†and James P. Di Santo*,†
Thymic epithelial cells (TECs) are the predominant
intrathymic source of the essential thymopoietin IL-7.
Whether thymocyte-TEC interactions have a role in
the regulation of IL-7 expression is not known. By
of IL-7 (Il7+TECs), we show that Il7+TECs segregate
from emerging medullary TECs during thymic organo-
genesis. Although Il7+TECs normally diminish with
age, we found that Il7+TECs are markedly retained in
alymphoid Rag22/2Il2rg2/2IL-7 reporter mice that
manifest a profound thymopoietic arrest. Transfer of
Tcra2/2or wild-type (but not Rag22/2) hematopoietic
progenitors to alymphoid IL-7 reporter recipients nor-
malizes the frequency of Il7+TECs and re-establishes
cortical TEC/medullary TEC segregation. Although
thymocyte-derived signals are often considered stimula-
feedback mechanism in which signals derived from
TCRb-selected thymocytes modulate TEC-dependent
The Journal of Immunology, 2010,
epithelial cells (TECs) represent a predominant stromal cell
component and have a paramount role in T cell development
and selection by providing essential thymopoietic signals,
including the Notch ligand D-like 4 (Dll4), IL-7, and self-
peptide–MHC complexes (1). The thymic epithelium is clas-
sically divided into two specialized and spatially distinct sub-
sets, cortical TECs (cTECs) and medullary TECs (mTECs),
that manifest different functional properties (2). The gen-
eration of a functionally competent and diversified TEC
hymocyte differentiation involves a complex in-
tegration of exogenous signals provided by a three-
dimensional thymic stromal cell network. Thymic
compartment is a prerequisite for normal thymopoiesis. Im-
portantly, functional maturation of TECs depends on in-
structive signals provided by thymocytes, thus defining the
symbiotic bidirectional thymic cross-talk (1, 2). Neverthe-
less, the molecular events underlying the functional segre-
gation of distinct TEC subsets are not fully elucidated.
Although compelling evidence points to IL-7 as a master
regulator of thymopoiesis in man and mice, we know surpris-
ingly little about the mechanisms that control IL-7 expression
in vivo. One possibility is that IL-7 is expressed at constant
and low levels in normal conditions such that the number of
IL-7–consuming cells in vivo would ultimately determine
IL-7 bioavailability. Alternatively, IL-7 expression might be
dynamically regulated and could thereby dictate lymphoid
homeostasis (3, 4). Although these different models have been
considered in the context of peripheral T cell homeostasis, they
understanding of the mechanism that controls IL-7 expression
remains speculative, in part due to the paucity of experimental
models that can monitor IL-7–expressing cells.
We have recently provided a temporal-spatial analysis of
IL-7–expressing TECs in vivo using bacterial artificial
chromosome transgenic reporter mice in which IL-7 pro-
moter elements control yellow fluorescent protein (YFP)
expression [B6.Cg-Tg(Il7-EYFP)5Pas mice] (5). YFP+(re-
ferred hereafter as Il7+) TECs expressed abundant Il7 tran-
scripts, localized to the corticomedullary junction in the
adult thymus and their frequency declined during postnatal
life (5). Based on these findings, we hypothesized that thy-
mocyte-TEC interactions might regulate TEC production of
thymopoietic factors (including Dll4 and IL-7) that are re-
quired for early T cell development while promoting func-
tional TEC diversification (6). In this regard, it has been
recently shown that thymocyte-specific signals downregulate
Dll4 expression in cTECs (7). In this study, we exploit
IL-7 reporter mice to dissect the intrathymic regulation of
IL-7 expression. Our results indicate that thymocyte-derived
*Cytokines and Lymphoid Development Unit, Institut Pasteur;†Institut National de la
Sante ´ et de la Recherche Me ´dicale U668, Paris, France; and‡Grupo de Activac ¸a ˜o Celular
e Expressa ˜o Gene ´tica, Instituto de Biologia Molecular e Celular, Porto, Portugal
Received for publication February 24, 2010. Accepted for publication April 7, 2010.
This work was supported in part by Institut Pasteur, Institut National de la Sante ´ et de la
Recherche Me ´dicale, the Fondation pour le Recherche Me ´dicale (SPF20081214865),
the Ligue Nationale Contre le Cancer, and the Foundation for Science and Technology
(SFRH/45932/2008 and Program Cie ˆncia 2008).
Address correspondence and reprint requests to Dr. James Di Santo, Cytokines and
Lymphoid Development Unit, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris
Cedex 15, France, or Dr. Nuno L. Alves, Grupo de Activac ¸a ˜o Celular e Expressa ˜o
Gene ´tica, Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823,
4150-180 Porto, Portugal. E-mail addresses: firstname.lastname@example.org and email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this paper: BM, bone marrow; cTEC, cortical thymic epithelial
cell; E, embryonic day; MHC-II, MHC class II; mTEC, medullary thymic epithelial cell;
TEC, thymic epithelial cell; WT, wild-type; YFP, yellow fluorescent protein.
on July 26, 2010
signals curb IL-7 expression by TECs while promoting their
Materials and Methods
IL-7 reporter mice (5) were backcrossed to the Rag22/2Il2rg2/2background
(8). Mice were housed under specific pathogen-free conditions and experi-
ments performed in accordance with institutional guidelines. For fetal studies,
embryonic day (E) 0.5 was the day of vaginal plug detection.
Bone marrow chimeras
A total of 107bone marrow (BM) cells from 6-wk-old wild-type (WT),
Tcra2/2(9), and Rag22/2C57BL/6 donors were injected i.v. in 4-wk-old
sublethally irradiated (0.4 Gy) Rag22/2Il2rg2/2IL-7 reporter mice (8).
Isolation and flow cytometric analysis of TECs
TECs were isolated as described (5). Cell suspensions were stained with anti-
CD4, anti-CD80, anti–I-A/I-E, anti-Ly51 (PE), anti-CD8, anti-CD80 (al-
lophycocyanin), anti-CD45.2 (PerCPCy5.5), anti-CD205, and anti-Ly51
(biotin) Abs, and streptavidin (PE-Cy7) (BD Biosciences, San Jose, CA);
anti–I-A/I-E (allophycocyanin-Cy7) and anti-EpCAM (allophycocyanin) Abs
(eBioscience, San Diego, CA).
Thymi were prepared as described (5). Briefly, samples were fixed in 4%
paraformaldehyde (Sigma-Aldrich, St. Louis, MO), and 8-mm sections were
stained with anti-GFP, Alexa 488–anti-rabbit, Alexa 555–anti-rat Abs, Alexa
647- or Alexa 555-streptavidins (Invitrogen, Carlsbad, CA), and rhodamine-
UEA 1 (Vector Laboratories, Burlingame, CA). CDR-1 and MTS-10 were
kindly provided by Dr. R. Boyd, Monash Immunology and Stem Cell Lab-
oratories, Monash University, Australia (5). Counterstaining and image
analysis were as described (5).
Results and Discussion
Ontogeny of thymic Il7+TECs
We first analyzed the developmental origins of Il7+TECs
during thymic organogenesis. TEC ontogeny commences
during embryonic life between day 9.5 and 11.5 of murine
gestation (E9.5–E11.5), with endodermal outbudding of
the third and the fourth pharyngeal pouches and subsequent
formation of the thymus anlagen at E12.5 (10). To char-
acterize the emergence of intrathymic Il7+TECs, we per-
formed a combinatory phenotypic-temporal analysis using
cTEC and mTEC markers CD205 and CD80, respectively
(10). The colonization of the thymic anlagen by hemato-
poietic cells (CD45+) was already visible by E12.5, and
hematopoietic representation increased steadily thereafter,
such that CD45+cells comprised the majority of cells at
later stages of gestation and in the neonatal thymus (Fig. 1).
At E12, YFP expression was not yet detected within the
anatomical region, including the third and fourth pharyn-
geal pouches (Supplemental Fig. 1). Analysis of TEC dy-
namics showed that YFP expression was first detected
around E12.5, suggesting that IL-7 expression in TECs
starts between E12 and E12.5 of gestation (Fig. 1Bi). In-
terestingly, the ratio between YFP+and YFP2TECs de-
clined concomitantly with increased gestational age and the
increased number of thymocytes (Fig. 1Bi). This obser-
vation suggested the existence of a putative thymocyte-
induced negative feedback mechanism on IL-7–expressing
TECs. Moreover, YFP expression preceded that of MHC
class II (MHC-II) and coincided with the initiation of ex-
pression of CD205 (Fig. 1Bi, Bii). From E14.5 onwards,
YFP+and YFP2TECs expressed MHC-II at the same level,
and Il7+TECs continuously segregated from CD80+
mTECs that were first detected at E17.5 (Fig. 1Bii). These
results suggest that the initiation of IL-7 expression is
a primitive molecular event associated with early stages of
reporter mice ere analyzed. A, The frequency of CD45+cells augments during organogenesis. Bi, The frequency of Il7+TECs (CD452gate) declines throughout
organogenesis. Bii, Il7+TECs segregate from CD80+mTECs. Comparative phenotypic analysis of color-coded YFP+(black dot plot) and YFP2(gray contour
plot) TECs in relation to the expression of CD205 and CD80. A and B, Numbers indicate the percentage of gated cells. B, Italic numbers correspond to the ratio
between YFP+and YFP2TECs. Data are representative of two to three experiments/time point.
Il7+TECs comprise a primitive thymic epithelium subset that emerges during early thymic organogenesis. Fetal and neonatal thymus from IL-7
5950CUTTING EDGE: THYMOCYTE-TEC CROSS-TALK REGULATES IL-7 EXPRESSION
on July 26, 2010
Frequency of Il7+TECs is augmented under severe lymphopenic
The results above indicated that thymic IL-7 expression might
by developing thymocytes. If true, ablation of thymocyte
differentiation might result in sustained IL-7 expression by
TECs. We therefore crossed IL-7 reporter mice onto the
severely lymphopenic Rag22/2Il2rg2/2background (8). As
expected, the number of CD45+hematopoietic cells was 1000-
fold reduced in Rag22/2Il2rg2/2IL-7 reporter mice compared
with age-matched immune-competent counterparts (Supple-
mental Fig. 2A). Strikingly, the proportion of Il7+TECs was
markedly increased in lymphopenic mice compared with
immune-competent mice, with most TECs expressing YFP
(Fig. 2A, 2B).
Thymic epithelial architecture is altered in lymphopenic
conditions as result of lack of instructive maturation signals
derived from developing thymocytes (11, 12). Under these
conditions, TECS retain an immature cortical-like phenotype
and do not segregate into mature cTECs and mTECs (11–
13). Our analyses of thymi from Rag22/2Il2rg2/2IL-7 re-
porter mice showed a predominance of cortical-like TECs
(CD205+, Ly51+, and CDR-1+) with a virtual absence of
CD80+, UEA+, and MTS10+mTECs. Il7+TECs displayed
a scattered distribution throughout the thymus, with the
majority expressing cortical (CD205+, Ly51+, and CDR-1+)
traits (Fig. 2A, 2C, Supplemental Fig. 2B, 2C). In contrast,
Il7+TECs localized predominantly to the corticomedullary
region in the immunocompetent thymus (Fig. 2C, Supple-
mental Fig. 2) (5). These results indicate the existence of an
intrathymic negative feedback mechanism involving thymo-
cyte-derived signals that diminish IL-7 expression by TECs.
Thymocyte-derived signals negatively impact on TEC-driven IL-7
We next assessed the influence of thymocyte subsets on TEC
IL-7 expression by reconstituting Rag22/2Il2rg2/2IL-7 re-
porter mice with BM precursors from Rag22/2, Tcra2/2, or
from WT mice. The thymi of recipient mice were analyzed
4 wk later. As expected, the number of CD45+cells was
progressively increased in the thymus of Rag22/2Il2rg2/2
IL-7 reporter mice reconstituted with Rag22/2or Tcra2/2and
WT BM precursors, the latter restoring normal T cell de-
velopment (Supplemental Fig. 3A). The conditioning irradia-
because their frequency was unchanged in nonirradiated and
nonreconstituted mice (Supplemental Fig. 3B). Strikingly,
although the ratio of Il7+TECs compared with YFP2TECs
remained largely unaffected in thymi reconstituted with
was markedly diminished upon reconstitution with either
Tcra2/2or WT BM precursors and coincided with the emer-
gence of CD80+mTECs (Fig. 3A, 3B).
Although cTEC differentiation is suggested to be dependent
on signals derived from early T cell progenitors, proper mTEC
maturation depends on signals provided by positively selected
and mature thymocytes (10, 14, 15). Accordingly, mTEC
differentiation process was stalled in nonreconstituted mice
(Fig. 3A, middle panels). However, the seeding of the thymus
of Rag22/2Il2rg2/2IL-7 reporter mice by Tcra2/2or WT
(but not Rag22/2) T cell precursors and the ensuing
phopenia. A, The expression of YFP is compared in TECs (CD452MHC-II+)
of Rag22/2Il2rg2/2control (WT), Rag22/2Il2rg2/2(Tg), and immuno-
competent (Tg) IL-7 reporter mice (top panels). The expression of CD205
and CD80 is shown in total TEC gate (middle panels) and colored gated for
Il7+(green) and total YFP2TECs (gray) (bottom panels). Numbers represent
the same as in Fig. 1. B, Frequency (i) and ratio (ii) of Il7+and YFP2TECs in
immunocompetent and Rag22/2Il2rg2/2IL-7 reporter mice at 4–6 wk of
age. Data represent average of more than six experiments (n = 10). C, Im-
munohistochemical analysis (5) of Rag22/2Il2rg2/2and immunocompetent
IL-7 reporter thymi (upper panels, original magnification 3100; lower panels,
original magnification 3200).
The frequency of Il7+TECs is augmented under severe lym-
The Journal of Immunology5951
on July 26, 2010
thymocyte differentiation up to or beyond DP stage, re-
spectively (Supplemental Fig. 3A), allowed CD80+mTECs to
emerge (Fig. 3A, middle panels). Interestingly, Il7+TECs re-
tained a cortical-like phenotype (CD205+) under all con-
ditions and clearly segregated from the emerging CD80+
mTEC subset (Fig. 3A, bottom panels), recapitulating the
phenotype observed in immunocompetent thymus. These
results suggest that signals provided by DP thymocytes are
sufficient to decrease IL-7 expression by TECs. cTEC and
mTEC progenitors can be discriminated by the expression of
CD205 and claudin-3 and -4, respectively, at early phases of
thymic organogenesis (16, 17). Although Il7+TECs are
predominantly CD205low/+(Figs. 1–3), it is not known
whether mTECs (CD80+YFP2) may derive from a putative
precursor included in Il7+TECs (CD205+YFP+) or from an
alternatively (CD205+YFP2) TEC progenitor.
Collectively, our findings indicate that Il7+TECs comprise
a primitive TEC population and that signals delivered by
thymocytes beyond the stage of TCRb selection can nega-
tively influence IL-7 expression by TECs. Although thymo-
cyte-derived signals are often considered stimulatory for
proper TEC differentiation (1, 2, 14, 15), recent evidence
suggests that developing thymocytes may also negatively im-
pact functional properties of TECs, including Dll4 (7) and
IL-7 expression (described in this study), required to foster
early stages of thymopoiesis. Whether Dll4 and IL-7 expres-
sion are coordinately regulated in TECs is not known. Nev-
ertheless, this notion may be taken in consideration for future
clinical applications aimed at boosting T lymphopoiesis. In-
sufficient T cell reconstitution has been shown in adult pa-
tients suffering from conditioned lymphocyte depletion (18).
One may envisage that in this scenario, despite the transitory
lymphopenia, TECs had previously become silenced for
thymopoeitic factors required at early of thymopoiesis (e.g.,
IL-7). This functional insufficiency could be due to preceding
and continuous thymocyte-derived signals. As such, further
studies are warranted to elucidate the molecular events in-
volved in the initiation of IL-7 expression as well as to identify
the molecular players responsible for the downregulation of
IL-7 expression by TEC, which can include the concerted
action of multiple thymocyte-derived cell surface or soluble
factors. Understanding the mechanisms that allow the func-
tional rejuvenation of TECs may open the door to new
strategies that can enhance thymus reconstitution and func-
tioning in clinical settings in which more robust T cell re-
sponses are required, including the induced (e.g., post-BM
were reconstituted with Rag22/2, Tcra2/2, and WT BM precursors or were left nonreconstituted. A, The expression of YFP in TECs is compared in non-
reconstituted and reconstituted thymi (top panels). WT Rag22/2Il2rg2/2(6 BM) mice were used to set the YFP2gate (data not shown). Plots in middle and
bottom panels represent the same as in Fig. 2A: YFP+(black dot plot) and YFP2(gray contour plot) TECs. Numbers represent the same as in Fig. 1. B, Frequency
of CD80+TECs (squared), Il7+(gray), and total YFP2(black) TECs isolated/thymus from unreconstituted and reconstituted thymi (i). Ratios between Il7+and
YFP2(left panel) and between total CD80+and Il7+TECs (right panel) are represented (ii). Data represent average of four to five independent experiments (n =
Thymocyte-derived signals negatively impinge on IL-7 expression by TECs. Both Rag22/2Il2rg2/2IL-7 reporter mice (Tg) and control (WT) mice
5952CUTTING EDGE: THYMOCYTE-TEC CROSS-TALK REGULATES IL-7 EXPRESSION
on July 26, 2010
transplantation settings) or acquired (e.g., HIV infection)
We thank Drs. B. Rocha and L. Peaudecerf (Institut Necker, Paris, France) for
Tcra2/2mice, Dr. G. Eberl and S. Dularoy for technical help, Drs. A. M.
Carmo and A. Moreira (Instituto de Biologia Molecular e Celular, Porto,
Portugal) for scientific support, and Dr. P. Vieira (Institut Pasteur, Paris,
France) for critical reading of the manuscript.
The authors have no financial conflicts of interest.
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