Transcriptional Regulation of IL-15

Expression during Hematopoiesis

This information is current as
of September 20, 2017.
Sara L. Colpitts, Spencer W. Stonier, Thomas A. Stoklasek,
Sierra H. Root, Hector Leonardo Aguila, Kimberly S.
Schluns and Leo Lefrançois
J Immunol 2013; 191:3017-3024; Prepublished online 21
August 2013;
doi: 10.4049/jimmunol.1301389
http://www.jimmunol.org/content/191/6/3017

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Supplementary http://www.jimmunol.org/content/suppl/2013/08/21/jimmunol.130138
Material 9.DC1
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 The Journal of Immunology

Transcriptional Regulation of IL-15 Expression during

Hematopoiesis

Sara L. Colpitts,* Spencer W. Stonier,† Thomas A. Stoklasek,* Sierra H. Root,*

Hector Leonardo Aguila,* Kimberly S. Schluns,† and Leo Lefrançois*,1
Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we
have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8+ and
CD82). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived
cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to
no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineage2Sca-1+c-Kit+) expressed high levels of IL-15,
suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was
Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate.

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Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory
activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15+CD8+ and IL-152/low
CD82 DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells. The
Journal of Immunology, 2013, 191: 3017–3024.

nterleukin-15 is a common g chain (CD132) cytokine that

I
functions through a unique mechanism of delivery termed trans-
presentation. IL-15 has a very high affinity (1.4 3 10211 M)
for its private receptor, IL-15Ra (1). The two proteins bind within
the cell and are shuttled to the surface as a complex (2). IL-15 is
then presented in trans to responding cells, such as NK cells, CD8
memory T cells, and intraepithelial lymphocytes (IELs), that ex-
press the other two shared chains of the receptor, CD132 and IL-2/
15Rb (CD122) (3). The generation of both IL-15 and IL-15Ra
knockout (KO) mice has greatly facilitated our ability to study the
dramatic downstream effects that exist in the absence of IL-15
signaling (4, 5). Importantly, transferring NK cells or CD8
T cells from IL-15Ra KO mice to wild-type (WT) recipients
demonstrated that IL-15–dependent cells need not express the IL-
15Ra chain to mediate the downstream effects of IL-15 signaling
(6–8). For this reason, trans-presentation is largely accepted as the
*Department of Immunology, University of Connecticut Health Center, Farmington,
CT 06030; and †Department of Immunology, University of Texas MD Anderson
Cancer Center, Houston, TX 77054
1
Dr. Lefrançois passed away during preparation of this article for print.
Received for publication May 24, 2013. Accepted for publication July 4, 2013.
S.L.C. is supported by a postdoctoral fellowship from the American Cancer Society
(Grant PF-11-152-01-LIB), and S.W.S. was supported by National Institutes of
Health Predoctoral Training Grant in Cancer Immunology CA009598. This work
was also supported by National Institutes of Health Grants 1RC1HL100569 (National
Heart, Lung and Blood Institute) (to H.L.A.), AI 070910 (to K.S.S.), and R01
AI76457 and U01 AI 095544 (to L.L.).
Address correspondence and reprint requests to Department of Immunology, c/o
Dr. Lynn Puddington, University of Connecticut Health Center, 263 Farmington Avenue,
Room L3092, Farmington, CT 06030. E-mail address: Leolefrancois2013@gmail.com
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; BMDC, bone marrow–derived
dendritic cell; cDC, conventional dendritic cell; DC, dendritic cell; DL1, Delta-like 1;
DN, double-negative; EmGFP, emerald GFP; ETP, early thymic progenitor; Flt3L,
Flt3 ligand; GITR, glucocorticoid-induced TNFR; HSC, hematopoietic stem cell;
IEL, intraepithelial lymphocyte; KO, knockout; Lin, lineage; LSK, lineage2Sca-1+
c-Kit+; MFI, mean fluorescence intensity; pDC, plasmacytoid DC; Tg+ or Tg2 ,
transgene positive or negative; WT, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
primary mechanism of IL-15 delivery. Whether cis-presentation of
IL-15 occurs in vivo is currently unclear but remains feasible (9).
First, CD8 T cells are known to upregulate IL-15Ra expression
following activation (10), and second, impaired development and
activation of IL-15–dependent cells is observed in transgenic mice
engineered to express a chimeric cytokine receptor composed of
the external domain of IL-15Ra fused to the intracellular portion
of IL-2Ra, suggesting that cell-intrinsic signaling through IL-
15Ra is required for the generation of optimal immune responses
(11).
Many elegant studies have addressed the cell–cell interactions
that drive IL-15–dependent responses, including the cellular
sources of IL-15 in vivo (reviewed in Ref. 12). Early studies used
the generation of bone marrow (BM) chimeras to distinguish IL-
15 derived from hematopoietic (radio-sensitive) versus non-
hematopoietic (radio-resistant) sources (6, 8, 13). In total, these
studies showed that individual populations of IL-15–dependent
cells are varied in their requirements for IL-15 from distinct
sources. For example, peripheral NK cells and memory CD8
T cells are partially restored when IL-15 production is limited to
hematopoietically derived cells whereas IL-15 trans-presentation
by radio-resistant intestinal epithelial cells is necessary and suf-
ficient for the generation and maintenance of CD8aa+ IELs (14).
Consistent with their association intracellularly, these studies have
also shown that the same cell must express both IL-15 and the IL-
15Ra to mediate activity (15, 16). Recent approaches have used
transgenic expression of IL-15Ra limited to certain subsets of
cells and conditional deletion of IL-15Ra using Cre-lox technol-
ogy to more closely examine the significance of IL-15 from se-
lected in vivo sources (17–21). These studies have implicated
a role for dendritic cells (DCs) in trans-presenting IL-15 to many
subsets of immune cells, yet they also suggest that other cellular
sources of IL-15 likely work in conjunction with DCs to maintain
overall peripheral homeostasis.
The overt lymphoproliferative disease observed when IL-15 is
produced in excess is evidence that IL-15 expression must be
tightly regulated (22). However, IL-15 itself has proven much
more difficult to study. This is likely the combined result of overall

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301389
3018
low levels of IL-15 in the steady-state and possibly poor detection
reagents, which in some cases may be further hindered when IL-15
is bound to IL-15Ra. To overcome these hurdles in IL-15 detection,
we generated a bacterial artificial chromosome transgenic mouse
strain in which emerald GFP (EmGFP) is produced under control of
the IL-15 promoter and all upstream regulatory elements (EmGFP/
IL-15). We employed this tool to identify previously unappreciated
differences in the respective levels of IL-15 produced by individual
subsets of conventional DCs (cDCs) during homeostasis and suc-
cessfully measure changes in IL-15 expression in DCs following
virus infection (23). In this study, we use EmGFP/IL-15 reporter
mice to further identify novel subsets of cells capable of producing
IL-15 and examine developmental pathways that regulate IL-15
expression in immune cells, which largely result in limiting IL-
15 production to cells of the myeloid lineage.
Materials and Methods
Mice
C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME) and the National Cancer Institute–Charles River (Fredericksburg,
MD), and IL-152/2 mice were purchased from Taconic (Germantown,
NY). EmGFP/IL-15 reporter transgenics were bred and maintained at the
University of Connecticut Health Center. All mice were used between 6
and 20 wk of age and housed according to the guidelines at the University
of Connecticut Health Center and MD Anderson Cancer Center. All
experiments were performed with the approval of the respective Institu-
tional Animal Care and Use Committees.
Tissue isolation
Spleen and thymus were harvested in complete media and crushed through
a 70-mm filter. BM was isolated by rapid centrifugation directly into
complete media. Peritoneal exudate cells were collected by injecting ∼10
ml PBS into the peritoneal cavity and collecting the solution after 5 min.
RBC lysis was performed by incubating single-cell suspensions in Tris
ammonium chloride for 5 min at 37˚C.
Flow cytometry and cell sorting
All Abs and Live/Dead viability dye were purchased from BD Biosciences,
BioLegend, and eBioscience. Fc block (anti-CD16/32; clone 2.4G2) was
purchased from Bio X Cell (West Lebanon, NH). IL-15 was detected with
rabbit anti–IL-15 biotin (PeproTech, Rocky Hill, NJ) followed by
streptavidin-allophycocyanin (Jackson ImmunoResearch Labratories, West
Grove, PA). Background staining was determined by examining analogous
populations from IL-152/2 mice.
In vitro culture
For IL-15 detection, 107 whole splenocytes or BM cells were cultured
overnight with or without LPS (1 mg/ml; Sigma-Aldrich, St. Louis, MO) or
IFN-a (300 U/ml; PBL Interferon Source and provided by Willem Over-
wijk [MD Anderson]). For in vitro culture with OP9 stromal cells, ex-
periments were performed as previously described (24). Briefly, ∼4–5 3
104 sorted lineage (Lin)2Sca-1+c-Kit+ (LSK) cells (CD32CD192CD11b2
CD11c2Gr-12NK1.12Ter1192Sca-1+CD117+) were seeded on confluent
OP9 or OP9-Delta-like 1 (DL1) stromal cells in OP9 media (MEMa, 20%
FBS, penicillin/streptomycin) supplemented with 5 ng/ml Flt3 ligand
(Flt3L) and 1 ng/ml IL-7 (PeproTech). Cells were replenished with new
media and cytokines on day 5. To generate BM-derived DCs (BMDCs),
8 3 106 whole BM cells were plated at 2 3 106 cells/ml in six-well plates
with media (IMDM, 10% FBS, 2-ME, nonessential amino acids, sodium
pyruvate, penicillin/streptomycin) supplemented with 100 ng/ml Flt3L
(Celldex Therapeutics, Needham, MA) for 9 d.
Adoptive transfer
Approximately 1–5 3 104 purified pre-DCs (CD32CD192B2202Ter1192
NK1.12CD11c+MHC II2CD135+CD172a2) isolated from the BM were
transferred to congenic recipients, and the spleens of recipient mice were
harvested after 6 d, as previously described (25).
Statistics
Statistical analysis was performed using Prism software (GraphPad Soft-
ware, San Diego, CA) using a Student t test or ANOVA where appropriate.
DEVELOPMENTAL REGULATION OF IL-15
Results
IL-15 is expressed at the highest levels in myeloid lineages
To facilitate the detection of IL-15 in vivo, our laboratory has
recently generated a transgenic bacterial artificial chromosome
reporter mouse in which EmGFP is expressed under the control of
the promoter and upstream regulatory elements of the IL-15 gene
locus (23). Our analysis of these mice confirmed previously
published studies that show IL-15 production by DCs and mono-
cytes (26, 27). However, the ability to use flow cytometry and
EmGFP expression as a surrogate for IL-15 promoter activity has
revealed previously unappreciated differences in the relative levels
of EmGFP/IL-15 expression in individual subsets of cells, par-
ticularly CD8+ and CD82 DCs (Ref. 23 and Fig. 1A), and these
results were recently corroborated using a second transgenic IL-15
reporter line (28). To further identify potentially novel cellular
sources of IL-15, we performed a large-scale analysis of both
innate and adaptive immune cell subsets in transgenic mice (Tg+)
and transgene-negative (Tg2) littermate controls using the mean
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fluorescence intensity (MFI) of EmGFP as an indicator of IL-15
transcription (Fig. 1A). The phenotypic definitions used to identify
each population of immune cells by flow cytometry are shown in
Supplemental Table I. We found that EmGFP/IL-15 was highly
expressed in splenic myeloid cells, including neutrophils, baso-
phils, and eosinophils, and mast cells isolated from the peritoneal
cavity (Fig. 1A, 1B). Alternatively, we found a lack of EmGFP/IL-
15 expression in lymphoid lineages, particularly CD4+ T cells and
follicular B cells (Fig. 1C, 1D). Interestingly, NK and NKT cells
along with marginal zone B cells, which are described as more
“innate-like” subsets of the immune system, maintained low but
detectable levels of EmGFP/IL-15 expression (Fig. 1D, 1E). How-
ever, unlike their cDC counterparts, plasmacytoid DCs (pDCs)
expressed EmGFP/IL-15 at very low levels (Fig. 1F). Thus, we
observed an apparent bifurcation regarding IL-15 promoter ac-
tivity that was largely associated with myeloid versus lymphoid
cell fate.
Initial attempts to measure cell-surface IL-15 expression using
a flow cytometry–based detection method have been largely un-
successful. However, two reports have detected IL-15 protein in
BMDCs and Gr-1+ polymorphonuclear cells (29, 30), and using
the same reagents we have also detected surface IL-15 on subsets
of DCs (17). Given the success of this particular Ab in detecting
trans-presented IL-15, we sought to measure IL-15 protein di-
rectly ex vivo on the populations of granulocytes that exhibited
high levels of IL-15 transcription based on EmGFP expression.
Unfortunately, we were unable to detect IL-15 expression directly
ex vivo on any of the cell populations identified in Fig. 1 isolated
from the spleen or BM (Supplemental Fig. 1A). However, we were
also unable to detect IL-15 directly ex vivo on CD11c+ spleno-
cytes (Supplemental Fig. 1B). In our previous studies, IL-15 was
only detectable when the cells were subjected to an overnight
in vitro culture or exposed to TLR ligands (17). Therefore, we
decided to take a similar approach to detect IL-15 protein on
granulocytic cells. When we cultured splenocytes overnight in
media alone, we detected low levels of IL-15 on neutrophils and
eosinophils, using CD11chigh DCs and IL-152/2 mice as a control
for IL-15 staining (Fig. 2A). Interestingly, when we performed
similar overnight cultures with BM cells, we were also able to
detect surface IL-15 on neutrophils, eosinophils, and macrophages
(Fig. 2B). To further explore the potential of these novel sources
of IL-15 to produce protein, we stimulated cells from the spleen
and BM with LPS or IFN-a to determine whether these known
inducers of IL-15 could augment IL-15 expression in the above
populations. Surprisingly, all subsets were able to upregulate IL-
The Journal of Immunology
FIGURE 1. IL-15 is broadly expressed in myeloid cells. (A) Individual
subsets of immune cells were identified in the spleens of EmGFP reporter
Tg+ and Tg2 littermate controls. The gating strategy for each subset is
described in Supplemental Table I. Data are presented graphically as the
MFI of EmGFP/IL-15 in each subset of immune cells (mean 6 SEM).
Representative histograms depicting relative levels of EmGFP/IL-15 ex-
pression in each population are shown in (B)–(F). Data are representative
of at least three experiments. Gray histograms show Tg2, black line
indicates Tg+.
15 expression to some degree compared with both unstimulated
controls and stimulated IL-15 KO cells (Fig. 2). Thus, in vitro
detection of IL-15 in neutrophils and eosinophils correlated with
the findings from our reporter mice indicating that these granu-
locytic populations have the capacity to produce IL-15 protein.
Hematopoietic progenitors express high levels of IL-15
All immune cells are derived from a small population of hemato-
poietic stem cells (HSCs) maintained in the adult BM. Because we
observed such dramatic variation in the levels of EmGFP/IL-15
expressed by mature cells of the hematopoietic system, we asked
to what degree EmGFP/IL-15 was expressed by hematopoietic pro-
genitors in the BM of transgenic reporter mice. We hypothesized that
HSCs would lack IL-15 expression and that IL-15 would be up-
regulated in myeloid cells, thus rendering them capable of trans-
presenting IL-15 and delivering a survival signal to responding NK
and T cells. Surprisingly, LSK cells, which constitute a heteroge-
neous population of both long-term and short-term HSCs, expressed
uniformly high levels of EmGFP/IL-15 (Fig. 3A). Thus, contrary to
our hypothesis, this result suggested that alternative mechanisms
selectively limited IL-15 expression in lymphoid lineages.
3019
Because T cells largely lacked EmGFP/IL-15 expression, and the
pathways that drive T cell commitment are well known, we ex-
amined the regulation of IL-15 during T cell development in the
thymus. We harvested thymocytes from Tg+ reporters and litter-
mate controls and determined the MFI of EmGFP/IL-15 expres-
sion in the CD32CD42CD82 double-negative (DN), CD32CD4+
CD8+ double-positive, and CD3+ single-positive CD4+ and CD8+
subsets. Similar to the LSK cells, early thymic progenitors (ETPs),
the c-Kit+ fraction of the CD44highCD252 DN1 subset (31),
expressed high levels of EmGFP/IL-15 (Fig. 3B, 3C). Such ele-
vated levels were maintained within the c-Kit+ fraction of CD25-
expressing DN2 cells as well. The downregulation of EmGFP/IL-
15 began as DN2 cells lost c-Kit expression (Fig. 3C), and
EmGFP/IL-15 expression was significantly reduced at the transi-
tion between DN2 and DN3 cells (Fig. 3B, 3C). By the DN4 stage,
EmGFP/IL-15 expression was completely lost in committed
T cells, and it remained absent from all downstream subsets in-
cluding double-positive and single-positive cells (Fig. 3B, 3C).
Thus, these data suggested that the signaling pathways controlling
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the progression of DN2 to DN3 cells could be responsible for
initiating the downregulation of IL-15.
Notch signaling mediates IL-15 downregulation during
thymopoiesis
The role of Notch in driving T cell development has been well
studied (32), and it is thought that Notch signaling not only ini-
tiates the necessary program for T cell development but also turns
off any residual expression of myeloid-specific factors (33). To
test whether Notch signaling was sufficient to drive IL-15 down-
regulation, we cultured purified LSK cells in vitro with OP9
stromal cells expressing the Notch ligand DL1. We sorted
EmGFP+ LSK cells from Tg+ mice and EmGFP2 LSKs from Tg2
littermate controls and cultured them in the presence of Flt3L and
IL-7 for 9 d (24). At that time, a large population of CD25+ DN2
and DN3 cells could easily be identified. Similar to our in vivo
analysis, we found that the DN2 to DN3 transition driven by
Notch signaling included a significant reduction in the MFI of
EmGFP/IL-15 (Fig. 4A). Interestingly, CD25+ DN2 cells grown
on OP9-DL1 cells had significantly reduced EmGFP/IL-15 ex-
pression compared with the small number of CD25+ DN2 cells
that emerge when LSKs are cultured on control OP9 stromal cells.
Thus, in OP9-DL1 cultures, the abundance of Notch ligands may
have initiated some degree of EmGFP/IL-15 downregulation in
DN2 cells that preceded the complete silencing of IL-15 expression
and additional phenotypic changes associated with the DN3 stage.
However, the presence of Notch ligands did not universally down-
regulate EmGFP/IL-15 expression because the small population of
Gr-1+ cells that escaped Notch-driven differentiation signals and
developed on the OP9-DL1 stromal cells expressed identical levels
of EmGFP/IL-15 as Gr-1+ cells that emerged after coculture with
control OP9 cells lacking DL1 expression (Fig. 4B). This finding
suggested that even in the presence of Notch signals, acquisition of
a myeloid fate includes the maintained expression of IL-15.
Coordinated regulation of IL-15 throughout DC development
Thus far, our results provided new evidence that IL-15 expression
was controlled as immune cells progress toward a mature lymphoid
fate. Considering the unique differences in EmGFP/IL-15 pro-
duction between CD8+ and CD82 DCs, we hypothesized that IL-15
would also be regulated throughout DC development, rather than
being controlled exclusively by extrinsic inflammatory mediators.
Therefore, we tracked EmGFP/IL-15 expression in BM precur-
sor cells downstream of LSKs but upstream of DC development.
Both macrophage/DC progenitors and common DC progenitors
3020
FIGURE 2. Detection of IL-15 pro-
tein from granulocytic myeloid cells.
(A and B) Cells (107) from the spleen
(A) or BM (B) of WT (black lines) and
IL-152/2 mice (gray filled histograms)
were incubated overnight in the ab-
sence or presence of 1 mg/ml LPS or
300 U/ml IFN-a. Numbers indicate the
MFI of IL-15 staining for WT (top)
and IL-152/2 samples (bottom). Plots
are representative of two to three ex-
periments.
expressed high levels of EmGFP/IL-15, albeit at significantly re-
duced levels compared with the LSK cells (Fig. 5A, 5B). Thus,
there was a progressive downregulation of EmGFP/IL-15 through
each stage of DC development suggesting continuous regulatory
activity at the IL-15 locus. Pre-DCs, the final precursor population
prior to commitment to either the CD8+ or CD82 DC lineage (25),
expressed low but uniform levels of EmGFP/IL-15 (Fig. 5C, 5D).
Because we observed homogeneous expression of IL-15 in the
pre-DC precursor population, our results suggested that the overall
level of IL-15 expression by a given DC subset was linked to the
final developmental program for that particular subset. Therefore,
FIGURE 3. Murine HSCs express high levels of EmGFP/IL-15. (A) HSCs
were identified in the BM of reporter mice and littermate controls as LSK.
Data are representative of at least two independent experiments. (Lin2 =
CD32CD192CD11b2CD11c2Gr-12NK1.12Ter1192.) (B and C) Thymo-
cytes from reporter mice and littermate controls were examined for ex-
pression of EmGFP/IL-15. DN thymocyte populations were defined as
follows: CD32CD42CD82; ETP, c-Kit+CD252; DN2, CD44highCD25+,
both c-Kit+ and c-Kit2; DN3, CD442CD25+; DN4, CD442CD252. Double-
positive (DP) cells were CD32CD4+CD8+, and single positive (SP) cells
were either CD3+CD42CD8+ or CD3+CD4+CD82. The MFI of EmGFP/IL-
15 is shown graphically for each population in (B). Gray histograms show
Tg2, black line indicates Tg+. *p , 0.05. n.s., Not significant.
DEVELOPMENTAL REGULATION OF IL-15
to determine whether EmGFP/IL-15+ pre-DCs were progenitors of
both EmGFP/IL-15+ CD8+ DCs and EmGFP/IL-152/low CD82
DCs, sorted pre-DCs from the BM of Tg+ mice and Tg2 controls
were transferred to naive congenic recipients. After 6 d, EmGFP/
IL-15 expression by donor-derived DCs was measured. As pre-
viously shown (25), pre-DCs gave rise to both DC subsets with
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preferential generation of CD82 DCs. Nevertheless, we detected
two populations of DCs (CD8+ and CD82) expressing signifi-
cantly different levels of EmGFP/IL-15 (Fig. 5E, 5F) derived from
a population of pre-DCs expressing uniform levels of EmGFP/IL-
15 (Fig. 5C). Thus, although little is known with regard to the
precise transcriptional elements that regulate the development of
CD8+ versus CD82 DCs, these results indicated that regulation at
the IL-15 locus occurred during this cell fate decision.
Just as the OP9-DL1 system can be used to mimic T cell de-
velopment in vitro, culturing BM cells in vitro with Flt3L can be
used to generate a diverse mixture of cDCs and pDCs. Importantly,
unlike BMDCs generated by culture with GM-CSF, the CD103+
and SIRPa+ (CD172a+) subsets of cDCs produced in Flt3L-
containing cultures more accurately reflect the CD8+ and CD82
subsets observed in vivo (34). We hypothesized that if the level of
IL-15 expressed by individual subsets of DCs was linked to their
cellular fate, the Flt3L-derived BMDCs should exhibit a similar
pattern of EmGFP/IL-15 expression between CD103+ and SIRPa+
DCs as did CD8+ and CD82 DCs, respectively. Indeed, the in vitro
generation of BMDCs with Flt3L exactly mimicked the pattern of
IL-15 expression by DC subsets in vivo (Fig. 5G). pDCs lacked
expression of EmGFP/IL-15, and CD103+ DCs expressed signif-
icantly higher levels of EmGFP/IL-15 than SIRPa+ DCs when
compared with the average MFI of EmGFP/IL-15 in BMDCs
grown from Tg2 controls (Fig. 5G). Thus, our findings suggested
that Flt3 signaling initiates a developmental program that includes
the regulation of IL-15 expression in developing DCs.
Discussion
IL-15 is an essential cytokine required for the development and
maintenance of several populations of immune cells. However, the
unfortunate combination of low expression levels and, perhaps, poor
detection reagents has made studying certain elements of IL-15
biology extremely difficult. By using a transgenic IL-15 reporter
mouse, we showed that IL-15 expression can be detected in numerous
subsets of hematopoietically derived immune cells. Our findings in-
dicated that granulocytic cells such as neutrophils, eosinophils, and
basophils have the potential to make IL-15 in vivo. However, it is
important to note that proven reagents for detecting IL-15 protein
following in vitro culture failed to detect any measurable levels of
IL-15 on any myeloid cells, including DCs, directly ex vivo. Thus,
whereas the IL-15 reporter communicates transcriptional rather than
translational activity, it nevertheless remains an excellent tool for
dissecting the regulation of IL-15 expression in vivo.
The Journal of Immunology 3021

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FIGURE 4. Notch-driven regulation of IL-15 gene expression during thymopoiesis. (A and B) LSK cells were sorted from Tg+ and Tg2 mice and
cultured with OP9 or OP9-DL1 stromal cells in the presence of Flt3L and IL-7 for 9 d, as previously described (24). In (A), contour plots (left) indicate the
level of CD44 and CD25 expression on the population of Gr-12 cells. The relative level of EmGFP/IL-15 in CD25+ DN2 (CD44high) or DN3 (CD44low)
cells grown on either OP9 or OP9-DL1 cells is shown in the histogram (center) and depicted graphically (right). *p , 0.05 using ANOVA followed by
a Tukey post test. In (B), cells were harvested and analyzed for expression of CD45 and Gr-1 to identify hematopoietically derived granulocytes (density
plots, left). The relative level of EmGFP/IL-15 expression in each population is shown in the histogram (center) and graphically (right). Results are
representative of two individual experiments. n.s., Not significant using Student t test.

Studies have shown that certain subsets of IL-15–dependent cells
require IL-15 to be trans-presented by a specific subset of cells.
For example, limiting IL-15 trans-presentation to CD11c+ cells
was largely sufficient to restore memory CD8 T cells in the pe-
riphery but failed to reconstitute the IEL compartment of the in-
testine (17). Alternatively, IELs require IL-15 to be delivered by
intestinal epithelial cells (18, 21). Because many of the progenitor
and granulocytic populations that we have shown in this study
to express IL-15 are present at extremely low frequencies, it will
likely be difficult to examine their individual contributions in
maintaining IL-15–dependent homeostasis in vivo. Additionally, it
is highly likely that some degree of compensatory mechanisms
exists when manipulation of IL-15/IL-15Ra expression occurs.
For example, when IL-15Ra is conditionally deleted from both
CD11c+ and LysM+ cells, the defect in NK cell homeostasis is not
exacerbated above deletion in either subset alone, suggesting that
CD11c+ cells and LysM+ cells provide similar functions in the
absence of the other (21).
The BM has been suggested as a site of long-term memory CD8
and CD4 T cell maintenance (35–38), and this is perhaps the result
of IL-15 reservoirs present in that site. Snell et al. (39) identified
a subset of memory CD8 T cells in the BM that expressed high
levels of glucocorticoid-induced TNFR (GITR) but were absent
from secondary lymphoid tissues in the periphery and also from
the BM of IL-15–deficient mice. They further showed that
memory CD8 T cells upregulated GITR in response to IL-15
signaling in vitro and upregulated GITR upon migration into the
BM, suggesting that IL-15 signals were delivered specifically
within the BM. Similarly, studies in humans have demonstrated
the close proximity of CD8 and CD4 memory T cells to IL-15–
producing cells in the BM (40). Whereas others have shown that
IL-15 can be expressed by BM stromal cells (26, 41), to our
knowledge ours is the first study describing IL-15 expression by
hematopoietic progenitor cells residing in the BM. Interestingly,
HSCs have been shown to reside in a specific BM niche using
a CXCL12/CXCR4-dependent mechanism (42), and memory
T cell behavior in the BM can also be influenced by CXCL12 (36).
Furthermore, IL-15 signaling can induce CXCR4 expression on
in vitro–generated CD4 memory T cells, which perhaps results
in the juxtaposition of memory T cells with IL-15–producing
hematopoietic progentiors (43). Regardless, evidence clearly sug-
gests that some IL-15 is derived from hematopoietic sources in
the BM, enough to significantly increase the presence of NK
cells in the BM of IL-15 KO mice reconstituted with WT BM
(14). Interestingly, however, deletion of IL-15Ra from DCs had
no effect on the presence of memory CD8 T cells specifically
within the BM (21). This may not be surprising considering the
relatively low numbers of DCs present in this location and the
expression of IL-15 by a number of other cell types that likely act
in conjunction with stromal cells to drive IL-15–dependent re-
sponses in the BM.
Our studies show that steady-state IL-15 expression is linked to
a myeloid cell fate. There are numerous transcriptional networks at
play in the myeloid versus lymphoid cell fate decision, and it is
highly unlikely that a single transcription factor will be identified
that regulates all instances of IL-15 expression in hematopoietic
cells. However, one potential candidate is the ets family member
PU.1 (encoded by Sfpi1), which is essential for myeloid cell de-
velopment, including macrophages and DCs (44–48). Also im-
portant for B cell development, gradations of PU.1 expression
in progenitor cells drive the generation of B lymphocytes versus
macrophages (49). Alternatively, concurrent with commitment to
the T cell lineage is a loss of expression of myeloid-specific
transcription factors (33, 50). In fact, constitutive expression of
PU.1 is sufficient to block T cell development at the DN to double-
positive transition (51). However, recent studies have shown that
ETPs maintain a significant level of PU.1 expression and retain
myeloid potential, whereas DN3 cells, which are committed to
T cell development, lack PU.1 expression (33). We observed
a similar pattern of IL-15 expression in developing T cells where
IL-15 was expressed at high levels by ETPs, but Notch signaling
led to the downregulation of IL-15 in DN3 and DN4 cells, perhaps
3022 DEVELOPMENTAL REGULATION OF IL-15

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FIGURE 5. IL-15 expression is tightly regulated throughout DC development. BM cells were harvested from Tg+ mice (black line) and Tg2 littermate
controls (gray histograms) to determine EmGFP/IL-15 expression by (A, B) Lin2Sca-12CD135+ macrophage-DC progenitors (MDP), common DC
progenitors (CDP), and (C, D) Lin22CD11c+MHC class II2CD135+CD172a2 pre-DC cells. Gating and histograms of EmGFP/IL-15 expression are shown
in (A) and (C) and depicted graphically in (B) and (D). The level of EmGFP/IL-15 expressed by LSK cells (see Fig. 3) is shown for statistical comparison in
(B). Lin22 = CD32CD192B2202Ter1192NK1.12. (E and F) Pre-DCs were purified by FACS from Tg+ and Tg2 controls and transferred to naive CD45.1
recipients between 3 and 4 wk of age. After 6 d, the spleens of recipient mice were harvested to determine the phenotype of donor-derived cells. Spleens
were first stained with anti–CD45.2-allophycocyanin followed by incubation with anti-allophycocyanin microbeads to enrich for the donor-derived pop-
ulation by MACS prior to flow cytometric analysis. One mouse that received no donor cells is shown as a control. Data in (E) are concatenated from one of
two independent experiments with three to four recipient mice per group per experiment. The data in (F) are pooled from the two independent experiments.
(G) BM cells from Tg+ and Tg2 mice were cultured in vitro with Flt3L, and after 9 d, cells were harvested and analyzed by flow cytometry. pDCs were
gated as B220+CD11cint, and cDC populations (either CD103+ or SIRPa+) were first gated as B2202CD11c+. The data are presented as the fold increase in
MFI of EmGFP over the average MFI of EmGFP from Tg2 controls for each population (F, G). *p , 0.05.

by overriding PU.1-initiated lineage commitment (52–54). Thus,
silencing of IL-15 in the lymphoid lineages occurred as progeni-
tors were directed toward a lymphoid fate. Because preliminary
studies have suggested that PU.1 can bind to a site in the first
intron of the IL-15 gene locus (E.V. Rothenberg, personal com-
munication), the possibility exists that PU.1 can modulate IL-15
expression in vivo. Interestingly, retroviral transduction of DN3 or
DN4 cells with PU.1 enforces a myeloid cell fate when cultured
on OP9 stromal cells (50). Whether or not this transcriptional re-
programming would include the upregulation of IL-15 could re-
veal a direct role for PU.1 in driving IL-15 transcription.
We further found that IL-15 expression was continuously modu-
lated during myelopoiesis, particularly in the development of DC
subsets. The Flt3/Flt3L signaling axis plays a dominant role in
controlling DC development (55). This conclusion has been
established in vivo (56, 57) but is also apparent using Flt3L (as
opposed to GM-CSF) to generate DCs from BM progenitors
in vitro (34). Considering these two commonly used approaches
for generating BMDCs, it is interesting that previous work shows
that CD8+ memory T cells are better maintained by Flt3L-derived
DCs than those grown in GM-CSF (58), which could suggest
a preferential role for Flt3 signaling in driving optimal IL-15
expression by terminally differentiated DCs. Flt3/Flt3L is also
critical for the generation of pDCs, which we have shown lack IL-
15 expression. pDCs are unique from their conventional coun-
terparts because they share similarities with developing lympho-
The Journal of Immunology
cytes, including the expression of RAG genes (59). Because we
have shown that IL-15 expression is turned off in lymphoid lin-
eages during their development, perhaps the transcriptional net-
work that is responsible for communicating lymphoidesque
characteristics on to this population of innate cells could also be
responsible for IL-15 downregulation during their development.
Finally, which signals and transcription factors collaborate to
control the differentiation of pre-DCs into CD8+ versus CD82
cDCs is currently unknown. However, our data, both in vivo and
in vitro, indicated that CD8+ DCs express higher levels of IL-15.
Id2, IRF8, and Batf3 have all been shown to be required for CD8+
DC development, but whether these specific transcription factors
act at the IL-15 locus remains to be examined. The differential
expression of IL-15 by cDC subsets has consequences in vivo, as
both CD8+ DCs and IL-15Ra were required to maintain a unique
population of CD122+ Ag–inexperienced CD8 T cells that exhibit
a memory phenotype (28). Importantly, our previous studies have
demonstrated that both subsets of cDCs have the capacity to up-
regulate IL-15 expression following virus-induced inflammation
in an IFN-a receptor–dependent fashion, suggesting that the
downregulation of IL-15 by immature CD82 DCs does not in-
volve permanent modifications to the gene locus (23).
In conclusion, the use of an IL-15 reporter transgenic system has
facilitated the identification of immune cells with the potential to
produce IL-15 in vivo. We have shown a large-scale bifurcation in
the two lineages of hematopoietically derived cells whereby IL-15
expression is primarily limited to myeloid cells and down-
modulated during lymphoid cell development. Surprisingly, IL-15
was expressed at high levels by HSCs in the BM, and IL-15
regulation occurred during both thymopoiesis and DC develop-
ment. In total, our findings suggested that molecular mechanisms
responsible for the development of cells in both myeloid and
lymphoid lineages were also able to exert regulatory control at the
IL-15 gene locus.
Acknowledgments
We thank Quynh-Mai Pham and the University of Connecticut Health Cen-
ter Flow Cytometry Core for technical assistance and Juan Carlos Zuniga-
Pflücker for OP9 and OP9-DL1 stromal cells.
Disclosures
T.A.S. and L.L. have a patent related to the IL-15 complex and have re-
ceived royalties from Marine Polymers Technology, Inc. The other authors
have no financial conflicts of interest.
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