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Progenitor Cells
Ex Vivo Expansion of T Lymphoid
IL-18 Acts in Synergy with IL-7 To Promote
De Wiele, Jonathan D. Wren and T. Kent Teague VanA. Taylor, Christopher C. Pack, Joel Gaikwad, C. Justin
Siva K. Gandhapudi, Chibing Tan, Julie H. Marino, Ashlee
http://www.jimmunol.org/content/194/8/3820
doi: 10.4049/jimmunol.1301542
March 2015;
2015; 194:3820-3828; Prepublished online 16J Immunol
References http://www.jimmunol.org/content/194/8/3820.full#ref-list-1
, 20 of which you can access for free at: cites 54 articlesThis article
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Copyright © 2015 by The American Association of
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The Journal of Immunology
IL-18 Acts in Synergy with IL-7 To Promote Ex Vivo
Expansion of T Lymphoid Progenitor Cells
Siva K. Gandhapudi,* Chibing Tan,* Julie H. Marino,* Ashlee A. Taylor,*
Christopher C. Pack,* Joel Gaikwad,
†
C. Justin Van De Wiele,*
,‡
Jonathan D. Wren,
x,{
and
T. Kent Teague*
,‡,‖,#
Although IL-18 has not previously been shown to promote T lymphopoiesis, results obtained via a novel data mining algorithm
(global microarray meta-analysis) led us to explore a predicted role for this cytokine in T cell development. IL-18 is a member of the
IL-1 cytokine family that has been extensively characterized as a mediator of inflammatory immune responses. To assess a potential
role for IL-18 in T cell development, we sort-purified mouse bone marrow–derived common lymphoid progenitor cells, early thymic
progenitors (ETPs), and double-negative 2 thymocytes and cultured these populations on OP9–Delta-like 4 stromal layers in the
presence or absence of IL-18 and/or IL-7. After 1 wk of culture, IL-18 promoted proliferation and accelerated differentiation of
ETPs to the double-negative 3 stage, similar in efficiency to IL-7. IL-18 showed synergy with IL-7 and enhanced proliferation of
both the thymus-derived progenitor cells and the bone marrow–derived common lymphoid progenitor cells. The synergistic effect
on the ETP population was further characterized and found to correlate with increased surface expression of c-Kit and IL-7
receptors on the IL-18–treated cells. In summary, we successfully validated the global microarray meta-analysis prediction that
IL-18 affects T lymphopoiesis and demonstrated that IL-18 can positively impact bone marrow lymphopoiesis and T cell devel-
opment, presumably via interaction with the c-Kit and IL-7 signaling axis. The Journal of Immunology, 2015, 194: 3820–3828.
Using a bioinformatics approach (1), IL-18 was predicted
to be involved in T cell development and differentiation
by a program called global microarray meta-analysis
(GAMMA). GAMMA performs a meta-analysis of ∼16,600 pub-
licly available microarray experiments, across different techno-
logical platforms (two-color, one-color, and RNA sequencing),
to identify and rank gene pairs that are strongly correlated across
experimental conditions (1). The approach identifies sets of genes
that are frequently transcribed and repressed together, regardless of
the experimental condition being analyzed, implying that the pair is
required for the same biological purpose, whether such a relation-
ship is formally documented or not. To identify what these sets of
genes have in common, an automated literature-mining program,
implicit relationship identification by in silico construction of an
entity-based network from text (IRIDESCENT) (2–4), was used to
find published associations the gene set has with diseases, pheno-
types, and function. GAMMA performed well on computational
benchmarks using genes of known function and was also success-
fully used to predict function for several different poorly charac-
terized or uncharacterized genes that were subsequently validated
by laboratory experiments (5–9). Of the genes that were identified
using this predictive strategy when employed for discovery of novel
factors regulating thymopoiesis, IL-18 was of interest because it has
not been directly linked to early T cell development.
IL-18 was originally described as an IFN-g–inducing factor be-
cause it was able to augment the production of IFN-gfrom T cells
and NK cells (10). As a part of the IL-1 cytokine family, IL-18 is
a multifunctional component of both the innate and the adaptive
immune response. Under various conditions the IL-18R1 and IL-
18R accessory protein are expressed on a variety of immune cells,
including NK cells, macrophages, neutrophils, B cells, and fully
differentiated Th1 cells (reviewed in Ref. 11). IL-18 has been
shown to work in synergy with other cytokines, including IL-12 and
IL-4, and has been broadly implicated in autoimmune and inflam-
matory diseases as well as chronic allergic rhinitis and asthma (12).
In the periphery, IL-18 is known to exert an influence on numerous
and diverse T cell processes. It increases Fas ligand–mediated cy-
totoxicity on T cells (13) and stimulates the development of CD8
effector T cells (14). IL-18 also promotes chemotaxis of T cells
(15). Furthermore, IL-18 drives CD4 T cell effector responses, in-
ducing IFN-gproduction by Th1 cells and promoting production of
IL-4, IL-5, and IL-13 in Th2 cells (16–18). IL-18 can also enhance
Th2 responses (with IL-2) and IL-18R1 is indispensable for Th17
*Department of Surgery, University of Oklahoma School of Community Medicine,
Tulsa, OK 74104;
†
Department of Biological Sciences, Oral Roberts University,
Tulsa, OK 74171;
‡
Department of Pharmaceutical Sciences, University of Oklahoma
College of Pharmacy, Tulsa, OK 74135;
x
Department of Arthritis and Clinical Im-
munology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104;
{
Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104;
‖
Department of Psychiatry,
University of Oklahoma School of Community Medicine, Tulsa, OK 74104; and
#
Department of Biochemistry and Microbiology, Oklahoma State University Center
for the Health Sciences, Tulsa, OK 74107
ORCID: 0000-0002-4680-5440 (T.K.T.).
Received for publication August 8, 2013. Accepted for publication February 13,
2015.
This work was supported by seed funding from the Chapman Foundation, as well
as by National Institutes of Health Grants 5P20GM103636 and 8P20GM103456
(to J.D.W.), Oklahoma Center for the Advancement of Science and Technology
Grant HR07-095 (to T.K.T.), and by additional funding from the University of
Oklahoma School of Community Medicine, Integrative Immunology Center.
Address correspondence and reprint requests to Dr. T. Kent Teague, University of
Oklahoma College of Medicine, Schusterman Center, 4502 E. 41st Street, Tulsa, OK
74135. E-mail address: kent-teague@ouhsc.edu
Address correspondence regarding GAMMA or IRIDESCENT to Dr. Jonathan
D. Wren, Department of Arthritis and Clinical Immunology, Oklahoma Medical
Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104. E-mail
address: jdwren@gmail.com
Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; CLP, common ly m-
phoid progenitor cell; DL, Delta-like; DN, double-negative; DP, double-positive; ETP,
early thymic progenitor; GAMMA, global microarray meta-analysis; HSC, hematopoi-
etic stem cell; IRIDESCENT, implicit relationship identification by in silico construction
of an entity-based network from text; ISP, immature single-positive; SP, single-positive.
Copyright Ó2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301542
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responses (19). Transgenic overexpression of IL-18 had dramatic
effects on the immune system; however, these studies did not focus
on the effects on early thymocytes, perhaps due to the important
role for this cytokine in Th1 and Th2 differentiation that has kept
the spotlight on peripheral immune cell mechanisms (20, 21). Al-
though the immunomodulatory functions of IL-18 are relatively
well defined, its potential role in T cell development, as predicted
by GAMMA, is not known. Previous studies have demonstrated
thymic expression of IL-18, and this cytokine has been shown to
promote the differentiation of fetal double-negative (DN) thymo-
cytes to thymic-derived dendritic cells (22, 23). Furthermore, thy-
mocyte stimulation with IL-18 can elicit production of Th1 and Th2
cytokines in the presence of IL-12 and IL-2, respectively (24).
These studies demonstrated the potential of IL-18 to signal within
the thymic microenvironment and indicated that IL-18 may indeed
be a factor capable of influencing early T cell development.
In an attempt to assess the potential role of IL-18 in T cell de-
velopment, we cocultured mouse bone marrow hematopoietic stem
cells (HSCs), common lymphoid progenitor cells (CLPs), thymus
early thymic progenitors (ETPs), and DN2 on OP9–Delta-like (DL)4
stromal cells, either with IL-18 alone or in conjunction with IL-7,
which is traditionally used to promote proliferation and survival of
thymocytes in this system (25). We found that IL-18 synergized
with IL-7 in promoting strong proliferation of CLP, ETP, and DN2
populations. The effect on the ETP cells was further characterized
and found to correlate with increased surface expression of CD127
and CD117 on these cells. Surprisingly, we found that IL-18 alone
was capable of promoting ETP proliferation to a magnitude similar
to that observed for IL-7. These findings demonstrate a novel role
for IL-18 in promoting in vitro T cell development from immature
precursors and further validate GAMMA as a method to predict
putative phenotype and function for genes.
Materials and Methods
Mice
C57BL/6 mice were bred and housed at the University of Oklahoma–Tulsa
Comparative Medicine satellite facility under the oversight of the Uni-
versity of Oklahoma Health Science Center Comparative Medicine Facility
(Oklahoma City, OK), an Association for Assessment and Accreditation of
Laboratory Animal Care–approved animal facility. Animal husbandry and all
experiments were performed in accordance with procedures outlined in the
Guide for the Care and Use of Laboratory Animals (National Research
Council). Protocols were reviewed and approved by the Institutional Animal
Care and Use Committee of the University of Oklahoma Health Science
Center. Mice used in this study were females ranging from 6 to 12 wk of
age. IL-18R1–deficient mice (strain B6.129P2-Il18r1
tm1AKI
/J) on a C57BL/6
background (26) were purchased from the The Jackson Laboratory (Bar
Harbor, ME).
Tissue harvest and cell staining
Thymuses were harvested and placed into complete tumor media as pre-
viously described (27). Thymuses were crushed through 70-mm nylon cell
strainers to produce single thymocyte suspensions. Cells were treated with
RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) and washed into complete
tumor media prior to counting. Thymocytes at a concentration of 1 310
8
cells/ml were incubated with mAb against mouse CD16/CD32 (Fc Block;
BD Biosciences, San Jose, CA) to block potential Fc-mediated binding and
then stained at a density of 1 310
8
cells/ml with primary mAbs for DN3a,
DN3b, and DN4a sorts, including CD4-bio, CD8-bio, TCRgd-bio, TCR-b–
bio, Lin-bio (28), CD25-PE, CD44-allophycocyanin-Cy7, and CD28-FITC,
for 45 min at 4˚C in the dark. After two washes, the cells were further
stained with SA-PE Texas Red for 30 min at 4˚C in the dark. Immature
single-positive (ISP; CD4
2
CD8
+
CD24
hi
TCR-b
2
) cells were sorted-purified
by staining with CD4-allophycocyanin, CD8-FITC, TCR-b–PE-Cy7, and
CD24-PE. ETP and DN2 cells were sort-purified, as shown in Fig. 1A, by
staining with the following fluorochrome- and biotin-coupled mAbs fol-
lowed by SA-PE Texas Red: CD4-bio, CD8-bio, TCRgd-bio, TCR–bio, Lin-
bio (28), CD25-PE, CD44-allophycocyanin-Cy7, c-Kit–FITC. To assess the
proliferation kinetics, ETPs were labeled with CFSE (Life Technologies,
Grand Island, NY) prior to placing in coculture. CFSE labeling was performed
by incubating cells at room temperature for 10 min in PBS solution containing
1% FBS and 5 mM CFSE and removing excess CFSE by washing cells with
culture media. One thousand cells from each population were cultured in
replicate wells in a 24-well plate containing confluent OP9–DL4 stromal cells
with/without cytokine(s). All cytokines were obtained from R&D Systems
(Minneapolis, MN). Unless otherwise indicated, cytokine concentrations were
as follows: IL-7 (5 ng/ml) and IL-18 (100 ng/ml).
For HSC (Lin
2
Kit
+
CD127
2
) and CLP (Lin
2
Kit
+
CD127
+
) isolation,
single-cell suspensions of bone marrow from wild-type mice were pro-
cessed to remove RBCs and block potential FC-mediated Ab binding by
treating cells with RBC lysis buffer followed by staining with anti-CD16/
CD32 Ab. Bone marrow cells (1 310
8
cells/ml) were then stained with
a biotin-conjugated lineage mixture (CD45RA [clone 14.8], Gr1 [clone
RB6-8C5], CD11b [clone M1/70], Ter119 [clone TER-119], CD45/B220
[clone RA3-6B2], CD2 [clone RM2-5], CD3 [clone 145-2C11], Cd8 [clone
53.6.7], CD49b [clone DX5[, and CD19 [clone 1D3]) and anti-mouse mAb
against c-Kit (c-Kit–allophycocyanin) and IL-7Ra (CD127-PE). This stain-
ing was followed by incubation with SA-PE Texas Red. Based on these
markers, HSCs and CLPs were discriminated and sort-purified.
After times indicated in Results, the cocultured cells were harvested by
aspirating and discarding half the culture volume and then resuspending
the cocultured cells by forceful pipetting in the remaining culture volume
followed by straining through a 30-mm mesh to remove any detached OP9–
DL4 monolayer cell aggregates from the plate. Any remaining OP9–DL4
cells were further discriminated from thymocytes by gating using forward
and side scatter; gating out the much larger OP9 cells. The cells were
stained with CD4-allophycocyanin, CD8–Pacific Blue, CD25-PE, CD44-
allophycocyanin-Cy7, CD28-FITC, TCR-b–PE-Cy7, TCRgd-bio, and Lin-
bio (28), followed by SA-PE Texas Red. To assess changes in IL-18R1,
CD117, and CD127 surface expression, cells were stained with FITC-
conjugated anti-IL–18R1 (R&D Systems; clone 112614), allophycocyanin-
conjugated anti-CD117 (BioLegend; clone 2B8), or PE-conjugated anti-
CD127 (eBioscience; clone A7R34) and the geometric mean fluorescence
intensity for IL-18R1, CD117, and CD127 staining on gated population
was compared with respective isotype control staining intensities.
Flow cytometry
Freshly isolated thymocytes were stained as described above to discriminate
the DN, double-positive (DP), SP4, SP8, DN1–4, and the DN3/DN4 subsets
(29) to establish gating parameters for cells harvested from OP9–DL4
cocultures. A MoFlo XDP cell sorter with Summit v4.3 software (Beckman
Coulter, Fullerton, CA) was used for the experiments that included sorted
populations. Cells were analyzed using a BD LSR II four-laser flow
cytometer and FACSDiva (BD Biosciences) and FlowJo software (Tree
Star, Ashland, OR).
OP9–DL4 cocultures
The OP9–DL4 cell line was provided by Juan Carlos Zu
´n
˜iga-Pfl€
ucker and
maintained according to the protocols from his laboratory (30). For each
experiment a fresh vial was thawed and grown to 60–80% confluence on
treated plates; cells were then split and grown again to 60–80% confluence
before the final plating on experimental 24-well treated plates. Sorted bone
marrow and thymocyte subsets were cocultured in plates with the OP9–
DL4 stromal cells in aMEM (Invitrogen, Grand Island, NY) supplemented
with 16.5% FBS (Sigma-Aldrich) and penicillin-streptomycin (Sigma-
Aldrich) (culture media) and the cytokines indicated in the figures. Note
that no Flt3L was added to any of the cultures. After 7 d, the cells were
harvested from the wells, counted, and stained for flow cytometry. Viable cell
counts were obtained using 0.4% trypan blue (Lonza, Allendale, NY) staining
or annexin V and 7-aminoactinomycin D (7-AAD) staining technique.
Quantitative real-time RT-PCR
Total RNA from sort-purified thymocyte subsets (2 310
4
cells) and splenic
NK cells (1 310
5
) were isolated using MinElute columns from Qiagen
(Germantown, MD). Total RNA was reverse transcribed to cDNA using
a Qiagen Sensiscript reverse transcriptase kit. IL-18R transcript abundance
was measured by amplifying cDNA using IL-18Raand IL-18Rbprimers
from Quantitect (Qiagen) and quantified by the SYBR Green detection
method using a ViiA 7 real-time PCR system (Life Technologies).
GAMMA
The GAMMA analysis predicted thatIL-18 should play a role in thymopoiesis
and thymocyte differentiation based on the genes it is highly correlated with
across .16,600 human microarray experiments. After identifying gene sets
most specifically and consistently coexpressed with IL-18 across heteroge-
The Journal of Immunology 3821
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neous conditions, it used large-scale literature mining to identify what these
coexpressed genes have in common. These commonalities become the
inferred functions, roles, and phenotypes for IL-18. Known functions serve
as positive controls and, for IL-18, many of its major known roles were
correctly predicted on the basis of its coexpressed genes, such as its proin-
flammatory role in the immune response and its ability to influence cytokine
production, as well as genetic associations with other cytokines such as
IL-12, IL-10, and IL-1.
Data analysis
Flow cytometry data were analyzed using FACSDiva (BD Biosciences) and
FlowJo software (Tree Star). Statistical analysis was performed using
Graphpad Prism 6 software, and statistical significance between variables
was estimated by performing one-way ANOVA and a Fischer test for
multiple comparisons.
Results
IL-18 acts in synergy with IL-7 to induce expansion of ETPs on
OP9–DL4 stromal cells
An IL-18 dose response was performed to determine the effect of
IL-18 on immature thymocytes in culture. Sort-purified ETP cells
were cultured for 1 wk on OP9–DL4 stroma with IL-7 added at
a concentration of 5 ng/ml in conjunction with IL-18 at concen-
trations ranging from 0.1 to 100 ng/ml. Supplementing cocultures
with IL-18 significantly enhanced the expansion of ETP cells, as
determined by total cell yields on day 7, compared with control
treatments (Fig. 1B). We observed that the magnitude of ETP ex-
pansion in the presence of IL-18 alone was comparable to the ETP
expansion observed in the presence of IL-7 alone. Adding IL-7 and
IL-18 together to the cocultures greatly increased the cell yields
when compared with cultures containing either IL-7 or IL-18 alone.
The synergistic effects of IL-18 and IL-7 were evident only at higher
doses of IL-18 ($10 ng/ml). Requirement of a higher IL-18 dose for
cell response is not unusual, as relatively higher concentrations of
IL-18 are known to be required to activate cells in vitro (31). We
next tested whether IL-18 influenced expansion of other immature
thymocyte subsets, including DN1d/e, DN2, and DN3 populations.
We found that neither IL-7 nor IL-18 alone had an apparent effect
on the expansion of these thymocytes, although cocultures supple-
mented with both IL-7 and IL-18 showed a modest effect in pro-
moting expansion of the DN2 population (Fig 1C). These results
demonstrate that IL-18 can promote expansion of ETPs and can
synergize with IL-7 in a dose-dependent manner.
IL-7 and IL-18 stimulation enhances survival and proliferation
of ETPs in OP9–DL4 cocultures
To assess whether increased cell yields in IL-7– and IL-18–
stimulated ETP cocultures were due to enhanced survival or in-
creased proliferation, we measured cell viability in cocultures by
annexin V/7-AAD staining and monitored cell divisions using a
CFSE dilution assay. We observed that the percentage of live cells
in ETP cocultures stimulated with cytokines was significantly
higher than that observed in unstimulated cocultures (Fig. 2A). IL-7
and IL-18 were equally potent in increasing live cell percentages in
7-d cocultures. There was also no apparent synergistic effect in the
IL-7 plus IL-18 condition on cell survival, presumably because
there was minimal cell death observed in these cocultures stimu-
lated with IL-7 or IL-18 alone. Because the differences in cell
survival among the treatments are small, it is unlikely that enhanced
ETP expansion in stimulated cultures was entirely due to enhanced
survival. Hence, we compared the proliferation kinetics of unsti-
mulated and stimulated ETP cocultures. CFSE profiles demon-
strated that the ETPs had undergone more cell divisions than CFSE
staining can reliably detect by day 6, irrespective of the culture
conditions (Fig. 2B). However, CFSE profiles on day 4 clearly
showed that ETPs stimulated with either IL-7 or IL-18 alone or in
FIGURE 1. Enhanced ETP and DN2 thymocyte
expansion in the presence of IL-7 and IL-18. (A)
Gating strategy used for discriminating ETP and
DN2 thymocyte subsets. Sort-purified ETP or DN2
thymocytes were cocultured on OP9–DL4 stromal
cells in culture media supplemented with IL-18 or
IL-7 alone or in combination and cell yields on day
7 were measured using a hemocytometer. (B) Cell
number in ETP cultures stimulated with indicated
concentrations of IL-7 and IL-18. (C) Cell yields in
ETP and DN2 cultures stimulated with 5 ng/ml
IL-7 and 100 ng/ml IL-18. Data are presented as
means 6SEM of three replicate wells for each
experimental condition and are representative of
three experiments with similar results. **p,0.05,
***p,0.001.
3822 IL-18 ENHANCES T LYMPHOPOIESIS IN THE OP9–DL4 SYSTEM
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combination experienced more divisions compared with unstimu-
lated ETP cultures. Importantly, the synergistic action of IL-7/IL-18
coadministration was observed on day 4. Taken together, these
results support the contention that IL-18 promotes expansion of
ETPs by enhancing both survival and proliferation of ETPs.
IL-18 accelerates the differentiation of immature thymocytes
To determine whether IL-18 could influence the differentiation of
immature thymocytes, sort-purified ETPs and DN2s were cocultured
with OP9–DL4 stromal cells in the presence of IL-7 or IL-18 alone
or in combination. After 7 d, differentiation of thymocytes in the
cocultures was analyzed by discriminating thymocyte populations
using surface markers. Both ETP and DN2 cocultures supplemented
with IL-7 showed an increase in total cell number after 7 d with-
out any notable changes in the percentages of different thymocyte
populations in comparison with untreated cocultures (Fig. 3A, 3B).
Similarly, ETP and DN2 cocultures treated with either IL-18 alone
or in combination with IL-7 showed an increase in total cell number
FIGURE 2. Enhanced proliferation and survival of ETP in the presence of IL-7 and IL-18. Sort-purified ETPs were cocultured with OP9–DL4 stromal
cells in media supplemented with IL-18 or IL-7 alone or in combination. (A) Survival of cells after 7 d of culture was assessed using annexin V and 7-AAD
binding (percentages of cells in each quadrant are represented as the means 6SEM of three replicate wells). (B) CSFE dilution profiles for ETPs expanding
under indicated conditions on days 3, 4, or 6. Histograms are representative of duplicate samples from each experimental condition and are representative of
at least three experiments with similar results. **p,0.05.
FIGURE 3. IL-7 and IL-18 accelerate ETP differentiation without skewing to a particular subset. Sort-purified ETP or DN2 thymocytes were cocultured
with OP9–DL4 stromal cells in media supplemented with IL-18 or IL-7 alone or in combination. Differentiation of ETP and DN2 cells into more mature
thymocytes was assessed by discriminating cells in the cultures using standard phenotypic markers. Thymocyte subsets identified in ETP/OP9–DL4 (A)or
DN2/OP9–DL4 (B) cocultures on day 7 under indicated conditions are shown. (C) Thymocyte subsets identified in ETP/OP9–DL4 cocultures during 5-d
expansion of ETPs under indicated conditions. (D) DN3a thymocytes sorted from day 7 ETP or DN2 cocultures or thymus from C57BL/6 mice were further
cultured for 8 d on OP9–DL4 stromal cells and their differentiation into SP4, SP8, or DP cells was analyzed using phenotypic markers. Dot plots were
representative of two to three replicates in each treatment and each experiment was repeated at least three times with similar results.
The Journal of Immunology 3823
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without skewing the percentages of any particular thymocyte pop-
ulation as compared with IL-7 alone or untreated cultures. However,
when we evaluated differentiation of the ETPs from the CFSE di-
lution assays at earlier time points (Fig. 3C), we saw that the dif-
ferentiation of ETPs into DN2 and DN3 subsets occurred at a faster
rate in IL-7– and IL-18–stimulated cultures compared with unsti-
mulated controls. The CFSE profiles also showed no preferential
expansion or differentiation of a particular thymocyte population in
cocultures stimulated in the presence of IL-18 (data not shown).
To determine the capacity for further development along the T cell
lineage, we evaluated DN3a thymocyte subsets generated in vitro
from ETP/OP9–DL4 cocultures and fresh ex vivo DN3a thymocytes
sort-purified from the thymus for their potential to develop into DP
cells. We found that all in vitro–generated DN3a subsets, irrespective
of the source and treatment conditions, differentiated into DP subsets
whencoculturedonOP9–DL4stromalcellsfor8d(Fig.3D)inthe
absence of IL-7. However, in the presence of IL-7 there were sig-
nificantly fewer DP thymocytes, as evidenced by the percentage of
cells within the DP quadrants, from DN3a populations generated
from ETP cocultures as well as from DN3a populations from thy-
mus. This effect is not surprising, as IL-7 has been shown to inhibit
the transition of DN2/DN3 thymocytes to the DP stage (32). These
results collectively suggest that IL-18 increased the expansion of
immature thymocytes without interfering with their differentiation
into more mature thymocytes.
ETP and DN2 subsets express IL-18 receptor transcript but not
discernible levels of IL-18 receptor protein as assessed by flow
cytometry
To further characterize the mechanisms by which IL-18 exerts its
effect on developing T cells, surface expression of the IL-18R1
(CD218a) was assessed on the four main thymocyte populations,
DN, DP, SP4, and SP8 (Fig. 4A). Only a small percentage of the
DN population and the SP4 population stained positively for the
IL-18R1. Subdivision of the DN compartment revealed that only
the DN1 population contained IL-18R1–expressing cells. Further
parsing of the DN1 compartment by CD24 and c-Kit staining
showed IL-18R1 surface expression to be restricted to the DN1e
population. This is somewhat surprising given that the DN1a/b
population (ETPs) expanded in the presence of IL-18, as did
both ETPs and DN2 cells with the combination of both IL-7 and
IL-18 as shown in Fig. 1C.
Because surface expression of the IL-18R1 was not detectable
in the IL-18–responsive ETP and DN2 populations using flow
cytometry, we assessed IL-18 receptor expression on ETP, DN1e/d,
DN2, and DN3 thymocytes using real-time RT-PCR. Both ETPs
and DN2 expressed low levels of IL-18R1 and IL-18R accessory
protein mRNA compared with DN1e/d populations and positive
control splenic NK cells (Fig. 4B). The OP9–DL4 stromal cells did
not express IL-18 receptor either at transcript level or at surface
expression (data not shown).
The IL-18 effect on thymocyte expansion is absent in cells from
IL-18R1–deficient mice
Although we did not detect surface expression of IL-18 receptors on
ETPs or DN2 thymocytes, the presence of IL-18 receptor transcripts
in these cells suggest the possibility for very low levels of receptor
expression. To determine whether the IL-18 effects in the cultures
were the direct result of IL-18 receptor engagement, we repeated
the OP9–DL4 cocultures described above comparing thymocytes
from IL-18R1–null mice to wild-type thymocytes. ETPs from either
C57BL/6 mice or IL-18R1–null mice (Fig. 5) were plated with no
cytokine, IL-7, IL-18, or a combination of IL-7 and IL-18. As ex-
pected, the IL-18R1–null mice did not show expansion in response
to IL-18. The synergy between IL-18 and IL-7 was also absent in
the null mice. This indicated that the IL-18 effects were mediated
through the IL-18 receptor even though we were unable to detect
IL-18R1 on the surface of the ETPs via flow cytometry.
IL-18–stimulated ETPs significantly upregulate c-Kit and
IL-7Rareceptor expression
c-Kit signaling has been shown to play an important role in pro-
moting proliferation and differentiation of DN1 and DN2 thy-
mocyte populations (33). Additionally, Zhou et al. (34) showed
that rIL-18 can positively regulate the expression of c-Kit in hu-
man melanocytes. To evaluate a potential mechanistic concurrence
FIGURE 4. The IL-18 receptor is differentially expressed on thymocyte
subsets. (A) IL-18R1 protein expression evaluated by flow cytometry on
freshly isolated thymocytes from C57BL/6 mouse. (B) Real-time RT-PCR
assessment of IL-18R1 (filled bar) and IL-18R accessory protein (open
bar) transcript abundance relative to GAPDH in sort-purified thymocyte
subsets and NK cells from spleen. Data are representative of experiments
repeated at least twice with similar results.
3824 IL-18 ENHANCES T LYMPHOPOIESIS IN THE OP9–DL4 SYSTEM
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in our model, we tested the effects of IL-18 on regulation of c-Kit
and IL-7Raexpression on immature thymocytes differentiating in
the OP9–DL4 cocultures. ETPs cultured in the presence of IL-7 or
IL-18 alone for 7 d demonstrated significantly upregulated c-Kit
expression on the resulting DN2 and DN3 cells, although the ef-
fect was modest in comparison with the effect of adding both
cytokines simultaneously (Fig. 6). ETPs exposed to IL-7 plus IL-18
showed robust increases in surface c-Kit expression (∼13.5-fold
over control), which was significantly greater than what was ob-
served in ETP cultures exposed to IL-7 or IL-18 alone (Fig. 6A).
Because IL-18 had a synergistic effect with IL-7, we also assessed
the IL-7 receptor expression on these cells when treated with
IL-18. As expected, stimulating ETPs with IL-18 caused a signifi-
cant upregulation of surface IL-7Rain differentiating thymocytes
after 7 d of coculture (Fig. 6B). ETP cultures exposed to IL-7 with
or without IL-18 showed minimal IL-7Raexpression on the dif-
ferentiating thymocytes, presumably owing to activation-induced
receptor internalization.
IL-18 promotes the expansion of immature progenitor cells in
OP9–DL4 cocultures
Lack of IL-18–mediated proliferative effects in DN1e/d despite
IL-18 receptor expression by these cells suggested that IL-18 re-
ceptor expression alone is not sufficient for the proliferative effects.
Alternatively, we found that cells responding to IL-18 both express
c-Kit and IL-7R, as well as upregulate these receptors after stim-
ulation with IL-18. Hence, we hypothesized that the IL-18 prolif-
erative effects are not restricted to immature thymocytes and may
be present in other progenitor cells expressing c-Kit and IL-7
receptors. To test this hypothesis we further investigated the pro-
liferative effects of IL-18 on HSCs and CLPs isolated from mouse
bone marrow, which also express the c-Kit receptor. We observed
that supplementing cultures with IL-7 and IL-18 significantly en-
hanced the proliferation of both HSCs and CLPs cocultured on the
OP9–DL4 stromal cells (Fig. 7), although the proliferative effects of
IL-7 and IL-18 combination are modest in cocultures started with
HSCs compared with CLPs.
Discussion
Humans have ∼25,000 genes and, although their positions have
been known since the completion of the draft genome in 2000,
about a third of them still have yet to be characterized. Algo-
rithmic approaches to predicting function on the basis of tran-
scriptional network analysis, such as the GAMMA approach used
in the present study, enable function and phenotype to be pre-
dicted for genes (Fig. 8). In the case of IL-18, much was already
known; however, what we have shown in this study is that there
can still be unknown/unexplored functions for known genes that
FIGURE 5. The IL-18–induced increase in ETP expansion requires IL-
18 receptor expression. Sort purified ETPs from wild-type or IL-18R1
2/2
mouse thymocytes were cocultured with OP9–DL4 stromal cells in media
supplemented with IL-18 or IL-7 alone or in combination and cell yields
from day 7 cocultures were measured using a hemocytometer. Data are
presented as means 6SEM of three replicate wells from each experi-
mental condition and are representative of three experiments with similar
results. ***p,0.001.
FIGURE 6. IL-18 induced upregulation of c-Kit and IL-7Rasurface expression on ETP OP9–DL4 cocultures. Sort-purified ETPs from C57BL/6 mouse
thymocytes were cocultured for 7 d with OP9–DL4 stromal cells supplemented with IL-18 or IL-7 alone or in combination. DN2 and DN3 cell surface
expression of (A) c-Kit (CD117) and (B) IL-7Ra(CD127) was analyzed by flow cytometry. Histograms are representative of three replicates in each
treatment. Numbers in the histograms represent geometrical mean fluorescence intensity of CD117 or CD127 or their respective isotype control (ISO)
staining. Bars represent mean 6SEM of receptor expression, as determined by fluorescence intensity, from three replicate wells in each treatment. Data are
representative of three experiments with similar results. **p,0.05, ***p,0.001.
The Journal of Immunology 3825
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can be inferred on the basis of their close neighbors in the tran-
scriptional network.
As detailed in Results, IL-18 dramatically influenced T cell
development in our in vitro model. IL-18 augmented thymocyte
proliferation and accelerated differentiation capacity. Interest-
ingly, the proliferative effects of IL-18 are not restricted to T cell
progenitors in the thymus but also are seen in more immature
progenitor cells such as HSCs and in CLPs in bone marrow that
give rise to all lymphoid-derived cells, suggesting the possibility
that IL-18 can influence lymphopoiesis more broadly. Although
IL-18 has well-established roles in a number of other processes, its
involvement in early T cell development has not been well de-
fined. IL-18 has been reported to be expressed within the micro-
environment of the thymus (22) and therefore it is compelling to
think that it plays a functional role in T lymphopoiesis. IL-18
signaling-deficient mice demonstrate defects in peripheral Th1
responses, NK cell responses, and gd T cell homing to lymph
nodes (26, 35), although no gross defects in T cell development
were reported in either IL-18–deficient or IL-18R1–deficient mice.
The lack of a noticeable effect on T cell production (26, 35) suggests
that the low level of IL-18 expressed in the thymus is not essential
for T cell development under nonstressed conditions. However, the
effects of this cytokine on thymocyte development could be missed
unless the host system is stressed, as elevation of this cytokine would
only be predicted to occur as a result of infection. Injection of IL-18
cDNA in conjunction with cDNAs for IL-2 or IL-12 in mice resulted
in increased expression of cytokines (especially IFN-g) from early
thymocytes, giving at least indirect evidence that elevated levels of
IL-18 can alter the thymic environment (24).
The IL-18–induced proliferative effects on thymocytes that
we show in the present study builds on previous evidence for
a role for this cytokine in positive regulation of proliferation of
T lymphocytes. This role was demonstrated in an earlier study (36)
that showed IL-18 could induce proliferation of Ag-stimulated
memory T cells in the periphery. In vivo relevance of the IL-18
effect was more recently reported in a study that showed IL-18
drove homeostatic expansion of naive CD8 T cells in a lympho-
penic host (37). The results we report in the present study are in-
triguing because existing literature suggests that proinflammatory
cytokines and inflammation in general negatively impact T lym-
phopoiesis (22–24, 38, 39), which is in contrast with the IL-18
effects we have found on immature thymocytes and bone marrow
progenitors. For instance, thymic involution has been shown to
occur rapidly within 72 h of endotoxin challenge (39). Although
there is ample evidence suggesting that physiological stressors such
as infection and inflammation can cause thymic atrophy due to
apoptosis of DN and DP subsets (39, 40), the key players in re-
storing thymic homeostasis postinfection or inflammation remain
poorly defined. Because IL-18 showed effects similar to IL-7 in
promoting proliferation and survival of ETPs, one could predict that
IL-18 can act as a compensatory mechanism to boost thymic pro-
genitors during an infection or inflammation and to restore thymic
homeostasis. However intriguing, the complexities associated with
exploring this hypothesis are beyond the scope of the present study.
Previous studies have demonstrated dendritic cell potential of
DN1d and DN1e subsets in vivo (41) and the ability of IL-18 to
drive differentiation of fetal DN1 cells to dendritic cells (22).
However, in our OP9–DL4 cocultures, IL-18–induced prolifera-
tive effects appeared restricted to DN1a/b (ETP) and DN2 cells
that progressed toward DN3, as we observed no expansion of
the DN1d/e cells in this system (Fig. 4). In any case, the experi-
ments presented in the present study clearly demonstrate that under
controlled conditions, IL-18 can potentiate ETP proliferation and
differentiation toward the T lineage.
Notch signaling plays a crucial role in directing T cell de-
velopment by tightly regulating various developmental steps,
including commitment, selection, proliferation, and survival of
differentiating thymocytes (42). For instance, Notch can promote
the proliferation and survival of DN populations by positively
regulating the growth-promoting signal pathways mediated by
IL-7R and c-Kit (33, 43). There is ample evidence suggesting that
both IL-7R and c-Kit play important roles in promoting the pro-
liferation and survival of immature thymocytes (33, 44, 45).
Consistent with these studies, we observed higher levels of both
IL-7R and c-Kit on the surface of thymocytes expanded in the
presence of IL-18 and IL-7 (Fig. 6). These two receptor signaling
pathways reportedly directly interact with each other (46) and
positively regulate STAT5 signaling. Hence, it is plausible that the
IL-18 effects in augmenting thymocyte expansion are mediated
through positive regulation of a c-Kit and IL-7 signaling axis.
However, further studies are essential to confirm this contention and
to explore whether IL-18 modulates c-Kit and IL-7R expression
directly using transcriptional or posttranscriptional mechanisms
or indirectly by modulating notch signaling, which is known to
modulate the expression of these receptors on the surface.
Perhaps most importantly, we have demonstrated that IL-18 can
substitute for IL-7 in early thymic development processes and can
FIGURE 7. IL-18 promotes expansion of HSCs and CLPs in OP9–DL4
cocultures. Sort-purified HSCs (1 310
4
cells) and CLPs (500 cells) from
C57BL/6 mouse bone marrow were cocultured with OP9–DL4 stromal
cells in media supplemented with IL-18 or IL-7 alone or in combination
and cell yields from day 7 cocultures were measured using a hemocy-
tometer. Data are presented as means 6SEM of three replicate wells from
each experimental condition and are representative of three experiments
with similar results. **p,0.05 compared with controls,
##
p,0.05
compared with IL-7 or IL-18.
FIGURE 8. The GAMMA principle. By using a set of 20 coexpressed
genes with known functions as a proxy, we can infer what an unknown gene
does. Benchmark analyses using 5000 genes of known function confirm the
approach is accurate. Experimental validations so far have corroborated the
ability to predict function using this “guilt by association” approach.
3826 IL-18 ENHANCES T LYMPHOPOIESIS IN THE OP9–DL4 SYSTEM
by guest on April 23, 2019http://www.jimmunol.org/Downloaded from
synergize with IL-7 in promoting proliferation. This finding warrants
further investigation to determine whether coadministration of IL-18
and IL-7 will be valuable for therapeutic purposes. Administration
of rIL-7 has shown promise in clinical trials due to its ability to
promote lymphopoiesis under lymphopenic conditions (47, 48).
However, use of IL-7 has limitations such as its bias toward ho-
meostatic expansion of peripheral lymphocytes and its marginal
effects on enhancing progenitor cell populations that give rise
to lymphocyte diversity (49, 50). Furthermore, the duration and
dose of IL-7 necessary for effect pose a risk of graft-versus-host
disease due to its ability to enhance T cell functions (48, 51). IL-18
has also been put into the spotlight for its potential role in cancer
treatment and is showing promising results in preclinical and clin-
ical studies (52–54). Based on the robust synergistic effects ob-
served in our assays and the recently reported effects of IL-18 in
driving homeostatic expansion of naive CD8 T cells in lymphopenic
mice (37), it is possible that combining IL-18 and IL-7 as a com-
bination therapy in humans could overcome the limitations of IL-7
by enhancing the IL-7 effects on bone marrow and thymus pro-
genitor cells and decreasing the dose and duration of IL-7 needed
for lymphocyte reconstitution in clinical scenarios of lymphode-
pletion. Alternatively, this synergy could be exploited to expand
progenitor cells ex vivo for reconstitution into lymphopenic hosts.
As IL-7 has been characterized to be absolutely required for
“normal” T cell development, it will be interesting to further
characterize and compare the differences in phenotypically identical
cells that have been raised in IL-7 versus IL-18 environments. The
value of these studies lies in the ability to potentially co-opt these
signaling pathways for future potential therapeutic purposes by
enhancing our ability to elaborate T cells in vivo following lym-
phodepletion or in vitro for envisioned adoptive immunotherapy or
reconstitution efforts.
Acknowledgments
The BD LSR II flow cytometer was a gift from the Oxley Foundation.
Disclosures
A provisional patent disclosure has been filed for use of IL-18 and IL-7 for
treatment of lymphopenia. The following authors are listed on the provisional
patent: T.K.T., S.K.G., J.H.M., J.D.W., C.J.V.D.W., C.T., and A.A.T. The
remaining authors have no financial conflicts of interest.
References
1. Wren, J. D. 2009. A global meta-analysis of microarray expression data to
predict unknown gene functions and estimate the literature-data divide. Bio-
informatics 25: 1694–1701.
2. Wren, J. D. 2004. Extending the mutual information measure to rank inferred
literature relationships. BMC Bioinformatics 5: 145.
3. Wren, J. D., R. Bekeredjian, J. A. Stewart, R. V. Shohet, and H. R. Garner. 2004.
Knowledge discovery by automated identification and ranking of implicit rela-
tionships. Bioinformatics 20: 389–398.
4. Wren, J. D., and H. R. Garner. 2004. Shared relationship analysis: ranking set
cohesion and commonalities within a literature-derived relationship network.
Bioinformatics 20: 191–198.
5. Daum, J. R., J. D. Wren, J. J. Daniel, S. Sivakumar, J. N. McAvoy,
T. A. Potapova, and G. J. Gorbsky. 2009. Ska3 is required for spindle checkpoint
silencing and the maintenance of chromosome cohesion in mitosis. Curr. Biol.
19: 1467–1472.
6. Towner, R. A., R. L. Jensen, H. Colman, B. Vaillant, N. Smith, R. Casteel ,
D. Saunders, D. L. Gillespie, R. Silasi-Mansat, F. Lupu, et al. 2013. ELTD1,
a potential new biomarker for gliomas. Neurosurgery 72: 77–90, discussion 91.
7. Clemmensen, S. N., C. T. Bohr, S. Rørvig, A. Glenthøj, H. Mora-Jensen,
E. P. Cramer, L. C. Jacobsen, M. T. Larsen, J. B. Cowland, J. T. Tanassi, et al. 2012.
Olfactomedin 4 defines a subset of human neutrophils. J. Leukoc. Biol. 91: 495–500.
8. Lupu, C., H. Zhu, N. I. Popescu, J. D. Wren, and F. Lupu. 2011. Novel protein
ADTRP regulates TFPI expression and function in human endothelial cells in
normal conditions and in response to androgen. Blood 118: 4463–4471.
9. Towner, R. A., R. L. Jensen, B. Vaillant, H. Colman, D. Saunders, C. B. Giles,
and J. D. Wren. 2013. Experimental validation of 5 in-silico predicted glioma
biomarkers. Neuro-oncol. 15: 1625–1634.
10. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto,
K. Torigoe, T. Okura, Y. Nukada, K. Hattori, et al. 1995. Cloning of a new
cytokine that induces IFN-gproduction by T cells. Nature 378: 88–91.
11. Gracie, J. A., S. E. Robertson, and I. B. McInnes. 2003. Interleukin-18.
J. Leukoc. Biol. 73: 213–224.
12. Arend, W. P., G. Palmer, and C. Gabay. 2008. IL-1, IL-18, and IL-33 families of
cytokines. Immunol. Rev. 223: 20–38.
13. Dao, T., K. Ohashi, T. Kayano, M. Kurimoto, and H. Okamura. 1996. Interferon-
g-inducing factor, a novel cytokine, enhances Fas ligand-mediated cytotoxicity
of murine T helper 1 cells. Cell. Immunol. 173: 230–235.
14. Okamoto, I., K. Kohno, T. Tanimoto, H. Ikegami, and M. Kurimoto. 1999.
Development of CD8
+
effector T cells is differentially regulated by IL-18 and
IL-12. J. Immunol. 162: 3202–3211.
15. Komai-Koma, M., J. A. Gracie, X. Q. Wei, D. Xu, N. Thomson, I. B. McInnes,
and F. Y. Liew. 2003. Chemoattraction of human T cells by IL-18. J. Immunol.
170: 1084–1090.
16. Hoshino, T., R. T. Winkler-Pickett, A. T. Mason, J. R. Ortaldo, and H. A. Young.
1999. IL-13 production by NK cells: IL-13-producing NK and T cells are present
in vivo in the absence of IFN-g.J. Immunol. 162: 51–59.
17. Nakanishi, K., T. Yoshimoto, H. Tsutsui, and H. Okamura. 2001. Interleukin-18
is a unique cytokine that stimulates both Th1 and Th2 responses depending on its
cytokine milieu. Cytokine Growth Factor Rev. 12: 53–72.
18. Yoshimoto, T., H. Mizutani, H. Tsutsui, N. Noben-Trauth, K. Yamanaka,
M. Tanaka, S. Izumi, H. Okamura, W. E. Paul, and K. Nakanishi. 2000. IL-18
induction of IgE: dependence on CD4
+
T cells, IL-4 and STAT6. Nat. Immunol.
1: 132–137.
19. Gutcher, I., E. Urich, K. Wolter, M. Prinz, and B. Becher. 2006. Interleukin 18-
independent engagement of interleukin 18 receptor-ais required for autoim-
mune inflammation. Nat. Immunol. 7: 946–953.
20. Finotto, S., J. Siebler, M. Hausding, M. Schipp, S. Wirtz, S. Klein, M. Protschka,
A. Doganci, H. A. Lehr, C. Trautwein, et al. 2004. Severe hepatic injury in in-
terleukin 18 (IL-18) transgenic mice: a key role for IL-18 in regulating hepa-
tocyte apoptosis in vivo. Gut 53: 392–400.
21. Hoshino, T., Y. Kawase, M. Okamoto, K. Yokota, K. Yoshino, K. Yamamura,
J. Miyazaki, H. A. Young, and K. Oizumi. 2001. Cutting edge: IL-18-transgenic
mice: in vivo evidence of a broad role for IL-18 in modulating immune function.
J. Immunol. 166: 7014–7018.
22. Ito, H., E. Esashi, T. Akiyama, J. Inoue, and A. Miyajima. 2006. IL-18 produced
by thymic epithelial cells induces development of dendritic cells with CD11b in
the fetal thymus. Int. Immunol. 18: 1253–1263.
23. Youm, Y. H., T. D. Kanneganti, B. Vandanmagsar, X. Zhu, A. Ravussin,
A. Adijiang, J. S. Owen, M. J. Thomas, J. Francis, J. S. Parks, and V. D. Dixit.
2012. The Nlrp3 inflammasome promotes age-related thymic demise and
immunosenescence. Cell Reports 1: 56–68.
24. Rodriguez-Gala
´n, M. C., J. H. Bream, A. Farr, and H. A. Young. 2005. Syner-
gistic effect of IL-2, IL-12, and IL-18 on thymocyte apoptosis and Th1/Th2
cytokine expression. J. Immunol. 174: 2796–2804.
25. Holmes, R., and J. C. Zu
´n
˜iga-Pfl€
ucker. 2009. The OP9-DL1 system: generation
of T-lymphocytes from embryonic or hematopoietic stem cells in vitro. Cold
Spring Harb. Protoc. 2009: pdb.prot5156. doi:10.1101/pdb.prot5156
26. Hoshino, K., H. Tsutsui, T. Kawai, K. Takeda, K. Nakanishi, Y. Takeda, and
S. Akira. 1999. Cutting edge: generation of IL-18 receptor-deficient mice: evi-
dence for IL-1 receptor-related protein as an essential IL-18 binding receptor.
J. Immunol. 162: 5041–5044.
27. Teague, T. K., B. C. Schaefer, D. Hildeman, J. Bender, T. Mitchell,
J. W. Kappler, and P. Marrack. 2000. Activation-induced inhibition of interleukin
6-mediated T cell survival and signal transducer and activator of transcription 1
signaling. J. Exp. Med. 191: 915–926.
28. Van de Wiele, C. J., J. H. Marino, C. Tan, H. A. Kneale, J. Weber, J. N. Morelli,
B. K. Davis, A. A. Taylor, and T. K. Teague. 2007. Impaired thymopoiesis in
interleukin-7 receptor transgenic mice is not corrected by Bcl-2. Cell. Immunol.
250: 31–39.
29. Tan, C., A. A. Taylor, M. Z. Coburn, J. H. Marino, C. J. Van De Wiele, and
T. K. Teague. 2011. Ten-color flow cytometry reveals distinct patterns of expres-
sion of CD124 and CD126 by developing thymocytes. BMC Immunol. 12: 36.
30. Schmitt, T. M., and J. C. Zu
´n
˜iga-Pfl€
ucker. 2002. Induction of T cell development
from hematopoietic progenitor cells by Delta-like-1 in vitro. Immunity 17: 749–756.
31. Dinarello, C. A., D. Novick, S. Kim, and G. Kaplanski. 2013. Interleukin-18 and
IL-18 binding protein. Front. Immunol. 4: 289.
32.Huang,J.,K.P.Garrett,R.Pelayo,J.C.Zu
´n
˜iga-Pfl€
ucker, H. T. Petrie, and
P. W. Kincade. 2005. Propensity of adult lymphoid progenitors to progress to DN2/
3 stage thymocytes with Notch receptor ligation. J. Immunol. 175: 4858–4865.
33. Massa, S., G. Balciunaite, R. Ceredig, and A. G. Rolink. 2006. Critical role for
c-kit (CD117) in T cell lineage commitment and early thymocyte development
in vitro. Eur. J. Immunol. 36: 526–532.
34. Zhou, J., J. Shang, J. Song, and F. Ping. 2013. Interleukin-18 augments growth
ability of primary human melanocytes by PTEN inactivation through the AKT/
NF-kB pathway. Int. J. Biochem. Cell Biol. 45: 308–316.
35. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto,
H. Okamura, K. Nakanishi, and S. Akira. 1998. Defective NK cell activity and
Th1 response in IL-18-deficient mice. Immunity 8: 383–390.
36. Iwai, Y., H. Hemmi, O. Mizenina, S. Kuroda, K. Suda, and R. M. Steinman.
2008. An IFN-g-IL-18 signaling loop accelerates memory CD8
+
T cell prolif-
eration. PLoS ONE 3: e2404.
37. Walsh, M. C., E. L. Pearce, P. J. Cejas, J. Lee, L. S. Wang, and Y. Choi. 2014. IL-
18 synergizes with IL-7 to drive slow proliferation of naive CD8 T cells by
costimulating self-peptide-mediated TCR signals. J. Immunol. 193: 3992–4001.
The Journal of Immunology 3827
by guest on April 23, 2019http://www.jimmunol.org/Downloaded from
38. Billard, M. J., A. L. Gruver, and G. D. Sempowski. 2011. Acute endotoxin-
induced thymic atrophy is characterized by intrathymic inflammatory and
wound healing responses. PLoS ONE 6: e17940.
39. Wang, S. D., K. J. Huang, Y. S. Lin, and H. Y. Lei. 1994. Sepsis-induced apo-
ptosis of the thymocytes in mice. J. Immunol. 152: 5014–5021.
40. Gruver, A. L., and G. D. Sempowski. 2008. Cytokines, leptin, and stress-induced
thymic atrophy. J. Leukoc. Biol. 84: 915–923.
41. Moore, A. J., J. Sarmiento, M. Mohtashami, M. Braunstein, J. C. Zu
´n
˜iga-
Pfl€
ucker, and M. K. Anderson. 2012. Transcriptional priming of intrathymic
precursors for dendritic cell development. Development 139: 373–384.
42. Shah, D. K., and J. C. Zu
´n
˜iga-Pfl€
ucker. 2012. Notch receptor-ligand interactions
during T cell development, a ligand endocytosis-driven mechanism. Curr. Top.
Microbiol. Immunol. 360: 19–46.
43. Gonza
´lez-Garcı
´a, S., M. Garcı
´a-Peydro
´, J. Alcain, and M. L. Toribio. 2012.
Notch1 and IL-7 receptor signalling in early T-cell development and leukaemia.
Curr. Top. Microbiol. Immunol. 360: 47–73.
44. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, and
R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies
IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519–1526.
45. Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama,
K. Yokomuro, J. I. Miyazaki, and K. Ikuta. 1996. Interleukin 7 receptor-deficient
mice lack gd T cells. Proc. Natl. Acad. Sci. USA 93: 7172–7177.
46. Jahn, T., S. Sindhu, S. Gooch, P. Seipel, P. Lavori, E. Leifheit, and K. Weinberg.
2007. Direct interaction between Kit and the interleukin-7 receptor. Blood 110:
1840–1847.
47. Capitini, C. M., A. A. Chisti, and C. L. Mackall. 2009. Modulating T-cell ho-
meostasis with IL-7: preclinical and clinical studies. J. Intern. Med. 266: 141–153.
48. Sasson, S. C., J. J. Zaunders, and A. D. Kelleher. 2006. The IL-7/IL-7 receptor
axis: understanding its central role in T-cell homeostasis and the challenges
facing its utilization as a novel therapy. Curr. Drug Targets 7: 1571–1582.
49. Chu,Y.W.,S.A.Memon,S.O.Sharrow,F.T.Hakim,M.Eckhaus,P.J.Lucas,and
R. E. Gress. 2004. Exogenous IL-7 increases recent thymic emigrants in peripheral
lymphoid tissue without enhanced thymic function. Blood 104: 1110–1119.
50. Broers, A. E., S. J. Posthumus-van Sluijs, H. Spits, B. van der Holt, B. Lo
¨wenberg,
E. Braakman, and J. J. Cornelissen. 2003. Interleukin-7 improves T-cell recovery
after experimental T-cell-depleted bone marrow transplantation in T-cell-deficient
mice by strong expansion of recent thymic emigrants. Blood 102: 1534–1540.
51. Goldberg, G. L., J. L. Zakrzewski, M. A. Perales, and M. R. van den Brink. 2007.
Clinical strategies to enhance T cell reconstitution. Semin. Immunol. 19: 289–296.
52. Srivastava, S., N. Salim, and M. J. Robertson. 2010. Interleukin-18: biology and
role in the immunotherapy of cancer. Curr. Med. Chem. 17: 3353–3357.
53. Tarhini, A. A., M. Millward, P. Mainwaring, R. Kefford, T. Logan, A. Pavlick,
S. J. Kathman, K. H. Laubscher, M. M. Dar, and J. M. Kirkwood. 2009. A phase
2, randomized study of SB-485232, rhIL-18, in patients with previously un-
treated metastatic melanoma. Cancer 115: 859–868.
54. Simpkins, F., A. Flores, C. Chu, J. S. Berek, J. Lucci, III, S. Murray, J. Bauman,
H. Struemper, F. Germaschewski, Z. Jonak, et al. 2013. Chemoimmunotherapy
using pegylated liposomal doxorubicin and interleukin-18 in recurrent ovarian
cancer: a phase I dose-escalation study. Cancer Immunol Res 1: 168–178.
3828 IL-18 ENHANCES T LYMPHOPOIESIS IN THE OP9–DL4 SYSTEM
by guest on April 23, 2019http://www.jimmunol.org/Downloaded from
