Krüppel-like factor 2 regulates trafficking and homeostasis of gammadelta T cells.
ABSTRACT gammadelta T cells are generated in the thymus and traffic to secondary lymphoid organs and epithelial surfaces, where they regulate immune responses. alphabeta T cells require sphingosine 1-phosphate receptor type 1 (S1P(1)) and CD62L for thymic emigration and circulation through secondary lymphoid organs. Both of these genes are regulated by the transcription factor Krüppel-like factor 2 (KLF2) in conventional alphabeta T cells. It is unclear if gammadelta T cells use similar mechanisms. In this study, we show that thymic gammadelta T cells express S1P(1) and that it is regulated by KLF2. Furthermore, KLF2 and S1P(1)-deficient gammadelta T cells accumulate in the thymus and fail to populate the secondary lymphoid organs or gut, in contrast to the expectation from published work. Interestingly, KLF2 but not S1P(1) deficiency led to the expansion of a usually rare population of CD4(+) promyelocytic leukemia zinc finger(+) "gammadelta NKT" cells. Thus, KLF2 is critically important for the homeostasis and trafficking of gammadelta T cells.
- SourceAvailable from: Rita Ostan[Show abstract] [Hide abstract]
ABSTRACT: Recent longitudinal studies in dietary daily intake in human centenarians have shown that a satisfactory content of some micronutrients within the cells maintain several immune functions, a low grade of inflammation and preserve antioxidant activity. Micronutrients (zinc, copper, selenium) play a pivotal role in maintaining and reinforcing the performances of the immune and antioxidant systems as well as in affecting the complex network of the genes (nutrigenomic) with anti- and pro-inflammatory tasks. Genes of pro- and anti-inflammatory cytokines and some key regulators of trace elements homeostasis, such as Metallothioneins (MT), are involved in the susceptibility to major geriatric disease/disorders. Moreover, the genetic inter-individual variability may affect the nutrients' absorption (nutrigenetic) with altered effects on inflammatory/immune response and antioxidant activity. The interaction between genetic factors and micronutrients (nutrigenomic and nutrigenetic approaches) may influence ageing and longevity because the micronutrients may become also toxic. This review reports the micronutrient-gene interactions in ageing and their impact on the healthy state with a focus on the method of protein-metal speciation analysis. The association between micronutrient-gene interactions and the protein-metal speciation analysis can give a complete picture for a personalized nutrient supplementation or chelation in order to reach healthy ageing and longevity.Mechanisms of ageing and development 01/2014; · 4.18 Impact Factor
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ABSTRACT: Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (TRM cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of TRM cells is unknown. We found that CD8(+) TRM cells lacked expression of the transcription factor KLF2 and its target gene S1pr1 (which encodes S1P1, a receptor for sphingosine 1-phosphate). Forced expression of S1P1 prevented the establishment of TRM cells. Cytokines that induced a TRM cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S1P1 provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.Nature Immunology 10/2013; · 24.97 Impact Factor
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ABSTRACT: Sphingosine-1-phosphate (S1P) is a central factor responsible for lymphocyte distribution in the body. S1P is able to control the integrity of various effector cell populations within many lymphoid tissues by directing lymphocyte egress. In this review, we give an overview of the generation and degradation of S1P in specific lymphoid microenvironments. Furthermore, we discuss, sometimes contradictory, the functions of the five S1P receptors on different cells in diverse tissues and give an idea of additional counteracting chemotactic signals for lymphocyte immigration and emigration. We focus special attention to recent discoveries of S1P-specific transporters, like spinster-homolog-2 and the active secretion of S1P by endothelial cells, erythrocytes and platelets. In addition, we describe the microanatomical structures as well as entry and egress routes into lymphoid organs which lymphocytes use for efficient trafficking. Finally, we give an overview of open questions regarding the regulation of lymphocyte homing from primary lymphoid organs to secondary lymphoid organs and back again.Archivum Immunologiae et Therapiae Experimentalis 11/2013; 62(2). · 2.38 Impact Factor
The Journal of Immunology
Kru ¨ppel-Like Factor 2 Regulates Trafficking and
Homeostasis of gd T Cells
Oludare A. Odumade, Michael A. Weinreich, Stephen C. Jameson, and
Kristin A. Hogquist
gd T cells are generated in the thymus and traffic to secondary lymphoid organs and epithelial surfaces, where they regulate
immune responses. ab T cells require sphingosine 1-phosphate receptor type 1 (S1P1) and CD62L for thymic emigration and
circulation through secondary lymphoid organs. Both of these genes are regulated by the transcription factor Kru ¨ppel-like factor
2 (KLF2) in conventional ab T cells. It is unclear if gd T cells use similar mechanisms. In this study, we show that thymic gd
T cells express S1P1and that it is regulated by KLF2. Furthermore, KLF2 and S1P1-deficient gd T cells accumulate in the thymus
and fail to populate the secondary lymphoid organs or gut, in contrast to the expectation from published work. Interestingly,
KLF2 but not S1P1deficiency led to the expansion of a usually rare population of CD4+promyelocytic leukemia zinc finger+“gd
NKT” cells. Thus, KLF2 is critically important for the homeostasis and trafficking of gd T cells.
2010, 184: 6060–6066.
positive (SP) ab T cell as they enter the thymic medulla. Post-
maturation, ab T cells emigrate from the thymus to seed peripheral
lymphoid organs. The sphingolipid receptor sphingosine 1-phos-
phate receptor type 1 (S1P1) is required for thymic emigration and
is only expressed at high levels by fully mature thymocytes (1).
Likewise, only mature thymocytes express CD62L (L-selectin),
which is required togain access toperipheral lymph nodes fromthe
blood (2, 3). We recently showed that the transcription factor
Kru ¨ppel-like factor 2 (KLF2, previously named LKLF) is required
for expression of S1P1 and CD62L in thymocytes (4). KLF2
transactivates both S1P1and CD62L promoters (4–6). Studies in
KLF2-deficient mice showed an accumulation of CD4+and CD8+
ab thymocytes in the thymus and a lack of ab T cells in secondary
lymphoid organs (4). These findings suggest that a critical role of
KLF2 in T cells is to induce expression of molecules required for
naive T cell trafficking.
gd T cells from embryonic day 17 fetal thymocytes have been
reported to express S1P1as determined by real-time PCR (7).
is inab T cells or ifitplays a functionalrole. Indeed,evidencewith
the S1P1analog FTY720 suggests that splenic gd T cells rely on
The Journal of Immunology,
cell progenitors with an MHC-restricted TCR undergo
positive selection in the thymus at the CD4+CD8+double-
positive (DP) stage and become a CD4+or CD8+single-
S1P1but that gut homing gd T cells do not (8). In this study, we
report that KLF2 (and S1P1) are expressed in gd thymocytes. In-
terestingly, we find that KLF2 deficiency in hematopoietic stem
cells leads to a reduced frequency of conventional gd T cells in the
peripheral lymphoid pool, but an increased incidence of promye-
locytic leukemia zinc finger (PLZF)+gd NKT cells (9, 10). Fur-
thermore, we show that both KLF2 and S1P1are required for
localization of gd T cells (and CD8aa+ab T cells) in the gut.
Overall, our findings suggest that KLF2 regulates lymphoid ho-
meostasis, affecting the composition and distribution of gd T cell
populations in steady state.
Materials and Methods
C57BL/6 (B6) and CD45.1+congenic B6.SJL-Ptprca(B6.SJL) mice were
purchased from The Jackson Laboratory (Bar Harbor, ME). Klf2GFP
knockin mice were previously described (11). Klf2fl/flmice were obtained
from Jerry Lingrel (University of Cincinnati, Cincinnati, OH). S1pr1fl/fl
mice were obtained from Richard Proia (National Institutes of Health,
National Institute of Diabetes and Digestive and Kidney Diseases, Be-
thesda, MD). VavCre mice (12) were obtained from Dimitris Kioussis
(National Institute for Medical Research, London, U.K.), through Bruce
Walcheck (University of Minnesota, Minneapolis, MN). All animal ex-
perimentation was approved by the University of Minnesota Institutional
Animal Care and Use Committee.
Mixed bone marrow chimera
Mixed bone marrow chimeras were generated by preparing a 1:1 mixture of
bone marrow from CD45.2+Klf2fl/flVavCre, S1p1rfl/flVavCre, or VavCre
together with marrow from C57BL/6.SJL (CD45.1+/CD45.2+) animals and
injecting it into lethally irradiated B6.SJL hosts. After 8 wk, thymus,
lymph node, spleen, and gut intraepithelial T lymphocytes (IELs) were
stained with FACS Abs and analyzed by flow cytometry.
Purification of IELs
IELs were purified as described in Ref. 13. The small and large intestine
were dissected from the mesentery and washed in RPMI 1640 supple-
mented with 10% FCS. Peyer’s patches were extracted, and then the in-
testine was cut longitudinally, rinsed out, and cut into ∼0.5-cm pieces. Gut
IELs were prepared via incubation of the small and large intestine in Ca2+
Mg2+-free HBSS with 1 mM DTT shaking for 20 min at 37˚C three times.
After each round, the supernatant was filtered with a 70-mm nylon filter.
The resulting cell pellet was applied to a 703/403 isotonic Percoll gra-
dient. Postcentrifugation at 2200 rpm for 25 min at 25˚C, cells at the in-
terface were collected and washed.
Center for Immunology and Department of Laboratory Medicine and Pathology,
University of Minnesota, Minneapolis, MN 55414
Received for publication February 12, 2010. Accepted for publication March 30,
Training Grant CA009138 (to O.A.O).
Address correspondence and reprint requests to Dr. Kristin A. Hogquist, Center for
Immunology, University of Minnesota, 2-186 MBB, 2101 6th Street SE, Minneap-
olis, MN 55414. E-mail address: email@example.com
Abbreviations used in this paper: DN, double-negative; DP, double-positive; IEL, intra-
epithelialTlymphocyte;KLF2,Kru ¨ppel-likefactor2; L-IEL,largeintestine;LN,lymph
node; PLZF, promyelocytic leukemia zinc finger; S1P, sphingosine 1-phosphate; S1P1,
spleen; THY, thymus.
Abs were purchased from BD Biosciences (San Jose, CA) or eBioscience
57 monomers (provided by the National Institutes of Health Tetramer Fa-
cility) were incubated with streptavidin-PE or streptavidin-APC for 2 h at
Ab (D-9) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA),
and staining was performed according to Savage et al. (14) using the Foxp3
staining buffer set (eBioscience). After washing, cells were then stained
with anti-mouse IgG1-APC in 13 permeabilization buffer and rewashed.
All cells were analyzed on Becton Dickinson LSR II instruments (BD Bio-
sciences), and the data were processed using FlowJo (Tree Star, Ashland,
Cell sorting and real-time PCR
FACS was used to purify CD4+CD8+DP, dump-negative CD4+SP, and
double-negative (DN) GL3+NK1.12/CD1d2CD252gd T cells. Each
group was sorted in at least two independent experiments. For cell sorting,
CD8 T cells were first depleted with anti-CD8 FITC using MACS magnetic
beads (Miltenyi Biotec, Auburn, CA). Sorting was performed on an
FACSVantage (BD Biosciences) and was reliably .90% of target pop-
ulation. RNA was isolated from sorted populations using the RNeasy kit
(Qiagen, Valencia, CA), and cDNAwas produced using the SuperScriptIII
Platinum Two-Step qRT-PCR kit (Invitrogen, Carlsbad, CA). cDNA was
prepared at least twice from each sort. PCR products were amplified using
QuantiTect SYBR Green PCR kit from Qiagen and detected using an ABI
Prism 7000 Sequence Detection System (Applied Biosystems, Foster City,
CA). Hypoxanthine-guanine phosphoribosyltransferase was used to nor-
malize samples. Primers were as follows: hypoxanthine-guanine phosphor-
ibosyltransferase: 59-CTTCCTCCTCAGACCGCTTT-39 and 59-ACCTG-
GTTCATCATCGCTAA-39; S1P1: 59-GTGTAGACCCAGAGTCCTGCG-39
and 59-AGCTTTTCCTTGGCTGGAGAG-39; KLF2: 59-AGCCTATCTTG-
CCGTCCTTT-39 and 59-CGCCTCGGGTTCATTTC-39; CD62L: 59-GTG-
GAGCATCTGGAAACTGG-39 and 59-CGGCTACAGGAATGAAGAGG-
39; and b7integrin: 59-GGACGACTTGGAACGTGTG-39 and 59-CGTTT-
TGTCCACGAAGGAG-39. Fold changes were calculated using the ΔΔCt
method with DP values as baseline.
Statistical analysis using unpaired Student t tests was performed with Prism
4.0a for Macintosh (GraphPad, La Jolla, CA). A value of p # 0.05 was
considered to be statistically significant.
S1P1expression is dependent on KLF2 in gd T cells
Using multicolor cell sorting and quantitative real-time PCR, we
examined the expression of KLF2 and S1P1mRNA in gd T cells
from adult thymi. As shown in Fig. 1A, gd T cells in the thymus
express both KLF2 and S1P1mRNA. To further determine the
expression of KLF2 in individual cells, we used a knockin mouse,
which expresses a GFP–KLF2 fusion protein and allows for flow
cytometric detection of KLF2 (11). Expression of KLF2 in gd
T cells is heterogeneous, with ∼35% of thymic gd T cells ex-
pressing KLF2 (Fig. 1B). These are likely the most mature gd
T cells, by analogy to ab T cells, in which KLF2 is expressed only
in mature (Qa2hi, CD69low) CD4 SP (11).
We have previously shown that S1P1expression is dependent on
KLF2 in ab T cells. Similarly, we have shown that the basis for
altered trafficking in KLF2-deficient T cells is due to reduced
expression of essential trafficking molecules such as S1P1and
CD62L. As such, we sought to determine the expression of S1P1
and CD62L in KLF2-deficient gd T cells. KLF2-deficient mice are
embryonic lethal at day 13 due to the loss of hemodynamic reg-
ulation in response to fluid shear stress in endothelial cells (15). In
the past, we and others have used fetal liver chimeras or RAG2
blastocyst chimeras to create a hematopoetic cell-specific defect in
KLF2 (4, 16). In this report, we used a VavCre system that re-
sulted in the conditional deletion of the floxed KLF2 allele in all
hematopoetic cells. Efficient deletion of KLF2 mRNA was con-
firmed on both sorted CD4+SP and gd T cells via real-time PCR
(data not shown). Subsequent real-time PCR analysis of sorted gd
T cells from Klf2fl/flVavCre thymi demonstrated a 4-fold reduction
of S1P1mRNA (Fig. 1C). Furthermore, Klf2fl/flVavCre gd T cells
showed reduced expression of CD62L mRNA (data not shown)
and protein (Fig. 1C). Thus, S1P1 and CD62L expression are
dependent on KLF2 in gd T cells, as in ab T cells.
S1P1deficiency causes peripheral gd T cell lymphopenia
To determine the role of S1P1in gd T cells, we used a strategy
similar to that used with KLF2 (VavCre transgenic mice crossed to
mice with a floxed S1P1allele). We observed a higher percentage
and absolute number of gd T cells in the thymus of S1pr1fl/fl
VavCre mice compared with control VavCre mice (Fig. 2A, 2B).
This accumulation is consistent with a block in thymic egress in
the absence of S1P1. Furthermore, gd T cells were severely re-
duced in percentage (Fig. 2A) and absolute numbers (Fig. 2B) in
peripheral tissues of S1pr1fl/flVavCre mice. Hence, these data
suggest that S1P1is required for thymic emigration of gd T cells.
VavCre animals. Similar to S1pr1fl/flVavCre mice, we observed
Quantitative real-time PCR analysis for KLF2 and S1P1was performed
using RNA isolated from the sorted thymocytes. DN gd T cells were sorted
using a dump strategy to exclude CD4, CD8, TCRb, NK1.1, and CD1d-
aGal-cer tetramer-binding cells. Graphs show the mean difference 6 SD,
log scale; n = 4. B, Flow cytometric analysis of thymi from homozygous
Klf2GFPmice showing the expression of a KLF2–GFP fusion protein in the
indicated subsets. C57BL/6 mice or negative littermate mice were used as
controls (shaded gray). Note that the Klf2GFPDP population is identical to
negative littermate controls. Data in B are representative of analysis from
at least three animals of each genotype and five independent experiments.
Average percent (6 SD) of gd T cells expressing Klf2 is indicated above
the histogram. C, Left panel, quantitative real-time PCR analysis for S1P1
was performed using RNA isolated from sorted DP thymocytes or DN gd+
thymocytes from Klf2fl/flVavCre or control VavCre mice. Graph show the
mean difference 6 SD, linear scale; n = 4. Right panel, The expression of
CD62L in gd+thymocytes from VavCre (shaded gray) and Klf2fl/flVavCre
(black line) mice. Data are representative of at least three animals per
genotype, four independent experiments. ppp , 0.02.
S1P1expression is dependent on KLF2 in gd T cells. A,
The Journal of Immunology 6061
Klf2fl/flVavCre mice (Fig. 2A), though the absolute number was not
significantlyincreased (Fig. 2B). Surprisingly,gd T cell numbers in
the spleen were not reduced in Klf2fl/flVavCre mice (Fig. 2B). In-
terestingly, the level of surface TCRgd was consistently lower on
peripheral gd T cells from Klf2fl/flVavCre animals (Fig. 2A).
Peripheral gd T cell homeostasis is perturbed by KLF2
deficiency, but not S1P1deficiency
The lack of an effect on peripheral gd T cell numbers in KLF2-
deficient mice was surprising because S1P1deficiency resulted in
reduced numbers,andKLF2-deficient cells havereducedS1P1.Itis
possible that KLF2-deficient gd T cells have sufficient residual
surface S1P1to mediate normal emigration. However, given that
others have reported defective trafficking of T cells in S1P1+/2
animals (in which S1P1mRNA expression is only ∼50% reduced)
(17), it seemed likely that the substantial (∼75%) reduction of S1P1
observed in the Klf2fl/flVavCre gd T cell pool would be functionally
relevant. Furthermore, we noticed that a variable, and sometimes
substantial, proportion of the gd T cells present in the spleen of
Klf2fl/flVavCre mice expressedCD4 (Fig.3A) andareduced surface
TCR level (Fig. 2B). On average, we observed a 37-fold increase in
compared with wild-type control (Fig. 3B). This increase was not
observed in S1P1-deficient mice (Fig. 3A, 3B). Interestingly, a large
fraction of these CD4+gd T cells expressed Vd6.3/2 (Fig. 3C),
characteristic of a recently described subset of gd NKT (9, 10, 18).
Indeed, these cells express the PLZF transcription factor, similar to
gd and canonical NKT cells (Fig. 3D). Thus, KLF2 deficiency, but
not S1P1deficiency,results in perturbedperipheral gdhomeostasis,
with a marked expansion of gd NKT.
The expansion of a gd NKT population in the periphery of KLF2-
deficient mice is unlikely to be driven by or secondary to ab T cell
which are equally severely lymphopenic (1). Nonetheless, to
approach. This approach also allows comparison of thymic emi-
gration of wild-type and knockout cells directly within the same
animal, which controls for variability in the microenvironment. We
created mixed bone marrow chimeras in which KLF2-deficient
(Klf2fl/flVavCre) and wild-type progenitors were mixed and used to
reconstitute lethally irradiated recipients using a congenic marker
to distinguish wild-type progenitors from KLF2-deficient progen-
itors and host cells (Fig. 4A). Indeed, in mixed bone-marrow chi-
meras without lymphopenia, we still observed gd NKT expansion
inthe KLF2 knockout-derived gdTcell population andnotinwild-
type gd T cells within the same mouse (Fig. 4B), suggesting an
autonomous effect. Importantly, we enumerated the conventional
gdTcells inbothlymphoidand nonlymphoidorgansusinga gating
strategy to exclude CD4+, Vd6.3/2+, PLZF+cells (Fig. 4C). In this
setting, KLF2- and S1P1-deficient gd T cells accumulated in the
thymus (3-fold) and were underrepresented in the periphery (3–6-
fold) (Fig. 4D). Hence, these data suggest that KLF2 deficiency
impairs thymic emigration of conventional gd T cells, an obser-
vation that was masked by the expansion of nonconventional gd
KLF2 is not expressed by resident gd or CD8aa+ab T cells in
the gut yet is required for their gut localization
Recently, Kunisawa et al. (8) proposed that gd T cells and CD8aa+
ab T cells migrated in a sphingosine 1-phosphate (S1P)-in-
dependent manner to the gut, whereas conventional CD4+ab
T cells and CD8ab+ab T cells required S1P1for entry into in-
testinal epithelium. Hence, we wished to evaluate the role of KLF2
in regulation of gut trafficking of these populations.
First, we determined the expression of KLF2 in multiple IEL
subsets at the protein level via flow cytometry using the KLF2GFP
reporter mice. We focused on four major subsets in the gut: CD4+
Interestingly, KLF2 was not expressed on the majority of CD8aa+
(control), Klf2fl/flVavCre, or S1pr1fl/flVavCre mice were stained with Abs to
CD4, CD8a, TCRgd, and TCRb and examined by flow cytometry. A, gd+
analyzed per group, five independent experiments. ppp , 0.02.
S1P1, but not KLF2, deficiency causes peripheral gd T cell
deficient mice. Single-cell suspensions from the thymus and spleen of
VavCre, Klf2fl/flVavCre, or S1pr1fl/flVavCre mice were stained with Abs to
CD4, CD8a, TCRgd, TCRb, Vd6.3/2, and/or PLZF and examined by flow
cytometry. A, C, and D, Two-parameter dot plots are shown after gating on
gd T cells. Numbers indicate the percentage of cells within each gate. B,
Bar graph represents the absolute number (log scale, mean 6 SD) of CD4+
TCRd+splenocytes; three to nine mice were analyzed per group, five in-
dependent experiments. pp , 0.05.
CD4+gd NKT cells are expanded in KLF2- but not S1P1-
6062 THE ROLE OF KLF2 IN gd T CELLS
or gd+IEL in the intestine (Fig. 5A). This correlates with a previous
study that reported undetectable S1P1mRNA in CD8aa+IEL (8).
In contrast, the majority of CD4+and CD8ab+ab T cells in the
large intestine do express KLF2 (Fig. 5A). Interestingly, we ob-
served an inverse correlation between KLF2 and CD69. In fact, the
only IELs that expressed KLF2 were CD69 negative (Fig. 5B).
To determine if KLF2 or S1P1are required for homing to the gut,
we examined IEL in competitive mixed bone marrow chimeras
(similar to Fig. 4). Klf2fl/flVavCre cells and S1pr1fl/flVavCre cells
were underrepresented among CD4+and CD8ab+ab T cells (data
S1P1(8) for thymic emigration. Surprisingly, Klf2fl/flVavCre cells
and S1pr1fl/flVavCre cells were also profoundly underrepresented
(10–100-fold) among CD8aa+ab and gd IELs in the gut (Fig. 6;
data not shown for large intestine). These data show that KLF2 and
S1P1are required for gd T cell and CD8aa+ab T cell localization
to the gut in adult animals.
KLF2 was originally reported as a regulator of T cell quiescence,
this conclusion arising from the apparent collapse of the peripheral
T cell pool in KLF2-deficient animals and the ability of
A. B, Two-parameter dot plots showing the distribution of PLZF+(gd NKT cells) are shown after gating on gd T cells. CD45.1-negative cells mark the
experimental group. Numbers indicate the percentage of cells within each gate. C, Gating strategy to define conventional gd T cells in mixed bone marrow
chimeras. D, Analysis of the ratio of CD45.2+experimental to CD45.1+control cells among conventional gd T cells from the THY, SPL, and LNs of mixed
bone marrow chimeras. To allow comparison between different experiments, we normalized all groups to the chimerism ratio of DN progenitors in the
thymus. Bar graph shows the average (log scale, mean 6 SD) of three independent sets of chimeras with at least one mouse per genotype in each. Klf2fl/fl
VavCre ratios were significantly different from controls in all three tissues using an unpaired Student t test. pppp , 0.005. LN, lymph node; SPL, spleen;
KLF2 is required for thymic emigration of conventional gd T cells. Mixed bone-marrow chimeras were created using the strategy shown in
gut. Single-cell suspensions from the thymus, spleen, S-IEL, and L-IEL of
Klf2GFPmice were stained with Abs to CD4, CD8a, CD8b, CD69, TCRd,
and TCRb and examined by flow cytometry. A, KLF2 expression in TCRb
CD4+, TCRb CD8a+, TCRb CD8b+, and TCRgd+T cells. Representative
histograms from one of five independent experiments (except L-IEL, for
which n = 2). B, Representative dot plots of CD69 and KLF2 expression in
TCRgd+T cells in the thymus, spleen, and S-IELs and on TCRb CD8aa+
small intestine IELs. Data are representative of five independent experi-
ments. L-IEL, large intestine; S-IEL, small intestine.
KLF2 is not expressed in gd or CD8aa+ab T cells in the
ab IEL to the gut Mixed bone-marrow chimeras were created with equal
ratios of wild-type competitor and VavCre (control), Klf2fl/flVavCre, or
S1pr1fl/flVavCre marrow. Cells were isolated from thymus and small in-
testine and stained for CD4, CD8a, CD8b, CD45.1, CD45.2, TCRd, and
TCRb and analyzed by flow cytometry. To allow comparison between
different experiments, we normalized the ratios relative to the ratio in DP
thymocytes. The effect of KLF2 and S1P1deficiency in gd T cells (top
panel) and CD8aa+ab T cells (bottom panel) are indicated. Error bars
indicate the SD. n = 3 chimeras per genotype and three independent ex-
KLF2 and S1P1are required for homing of gd and CD8aa+
The Journal of Immunology 6063
overexpressed KLF2 to restrain T cell proliferation and activation
(16, 19, 20). Our subsequent studies argued that KLF2 was not
required for maintenance of the naive T cell pool, but rather acted
to induce expression of key molecules, including S1P1and CD62L,
which are upregulated late in thymocyte maturation and thus to
permit thymic emigration and peripheral trafficking to secondary
lymphoid organs (4). The regulation of CD62L and S1P1by KLF2
has been subsequently confirmed in other studies (5, 6, 21, 22).
In contrast to ab T cells, gd T cells are generated from thymic
precursors in embryonic waves associated with trafficking to
distinct tissue compartments (skin, reproductive tract, and gut). It
was previously shown that embryonic thymic gd T cells express
S1P1, suggesting that they might use a similar emigration pathway
as ab T cells (7). However, subsequent data suggested that S1P1
was necessary for gd T cell egress to the spleen and lymph node
but that migration of gd T cells or CD8aa+ab T cells into
nonlymphoid sites (the gut specifically) was S1P-independent (8).
In this study, we observed KLF2 (and S1P1) mRNA in normal
adult thymic gd T cells. We showed that S1P1mRNA levels were
substantially reduced by KLF2 deficiency. In accordance with the
role of KLF2 in ab T cells, CD62L was also reduced in KLF2-
deficient gd T cells. CD62L expression was not completely absent,
consistent with its control via multiple other transcriptional regu-
lators (6). Because reduction of S1P1expression by as little as 50%
impairs T cell trafficking in vivo (17), it seemed likely that KLF2
deficiency would result in reduced thymic emigration of gd T cells.
However, despite having a phenotype suggestive of retention of gd
T cells in the thymus, a reduction of total gd T cell numbers in the
spleen was not observed. At least three factors confounded the
interpretation of this result. First, KLF2-deficient mice are highly
ab lymphopenic, and it was possible that this lymphopenia caused
the small number of gd T cells in the periphery to expand. Second,
KLF2-deficient mice display a cell-nonautonomous (bystander)
effect in which the overproduction of IL-4 causes multiple effects
on wild-type bystander CD8 T cells, including the upregulation of
CXCR3 and elevated levels of IgE (11). This overproduction of
cytokine could also contribute to gd T cell expansion. Finally, we
observed an expanded population of gd NKT cells (discussed be-
low) in KLF2-deficient mice. For these reasons, we sought to test
the trafficking of KLF2-deficient T cells in mixed bone marrow
chimeras in which wild-type competitor cells fill the peripheral
niche and dilute bystander effects to an undetectable level (11).
Using this approach, and applying a gating strategy that excluded
gd NKT cells, we report that KLF2-deficient conventional gd
T cells exhibit a thymic emigration defect, accumulating 4-fold in
the thymus and being reduced 3–6-fold in secondary lymphoid
organs. A similar approach was employed with S1P1-deficient
mixed bone marrow chimeras to show that S1P1, like KLF2, is
required for thymic emigration of gd T cells, which is in agreement
with the requirement of KLF2 for optimal expression of S1P1
mRNA in gd T cells. It is unclear at this point if S1P1is the ex-
clusive target of KLF2 as it relates to thymic emigration. We also
showed that KLF2 controls CD62L expression in gd T cells, so
presumably, entry into lymph nodes is coordinately regulated with
thymic emigration in gd T cells.
gd T cells. Typically, CD4 is only expressed on a small percentage
of gd T cells (23). This expression appears to be enriched among
a population of gd T cells with a skewed TCR repertoire, showing
an overrepresentation of TCRg1.1 and TCRd6.3 receptors (24).
Such cells rapidly produce IFN-g and IL-4 cytokines and express
NK1.1 (25) and the transcription factor PLZF (26). Indeed, such
cells require PLZF for their expansion (10). Because of these fea-
tures, CD4+gd T cells have been called gd NKT (18), although we
note that, like CD1d-tetramer binding NKT cells, not all gd NKT
express CD4 and NK1.1 (data not shown). Rather, PLZF itself
expansion has been observed now in several mutant mouse models:
TCRa-deficient mice (27), inducible T cell kinase-deficient mice
(26, 28) and mice lacking the Id3 transcription factor (29, 30).
an expanded gd NKT population (discussed above), including
KLF2-deficientmice, alldisplayperipherallymphopenia tovarious
alter the composition and differentiation state of T cells (31, 32).
However, because CD4+gd T cells were not expanded in S1P1-
deficient mice, it is unlikely that gd NKTexpansion was due solely
to lymphopenia in KLF2-deficient mice. A second observation was
that in mixed bone marrow chimeras with control and KLF2-
deficient cells, in which the emigration of control cells prevented
overt lymphopenia, we still observed an expansion of KLF2-
deficient gd NKTs. Thus, although many strains that display an
expanded gd NKT population also display lymphopenia, such
lymphopenia was not a primary driver of gd NKT expansion. It
remains to be identified what factors all of these strains have in
common that allow gd NKT expansion.
The fact that gd NKT cells are expanded in the periphery of
KLF2-deficient mice may imply that they have different require-
because the emigration defect is not 100%, even for conventional
T cells, it is unclear if gd NKT cells expand from a normal or re-
duced peripheral population in KLF2-deficient mice. Unfortu-
nately,this populationis numerically small innormalmice, making
it difficult to study via an intrathymic labeling approach. Current
studiesare underway todefinethe mechanisms controllinggdNKT
cell development in KLF2-deficient mice.
Compared to secondary lymphoid organs, the trafficking re-
35). Because we were studying gd T cells, we focused on the small
and large intestine, which are highly populated by gd T cells in
adult mice (36, 37). We first used Klf2GFPmice to determine the
expression of KLF2 in T cell subsets in the gut. In accordance with
data that sorted CD8ab+and CD4+cells from the large intestine
expressed S1P1but CD8aa+IEL did not (8), we observed a 5–10-
fold higher expression of KLF2 in CD8ab+and CD4+ab IEL in
Kunisawa et al. (8) did not measure S1P1mRNA in gd T cells, its
absence was inferred from the lack of an effect with FTY720
treatment (which did influence CD8ab+and CD4+IEL).
We used both intact mice (data not shown) and mixed bone
marrow chimeras to determine if KLF2 or S1P1deficiency resulted
in alterations in the intestinal compartment. Contrary to the S1P-
independent model previously proposed (8), we detected a pro-
found reduction of KLF2 and S1P1-deficient gd T cells and
CD8aa+IELs in the gut, suggesting that KLF2 and S1P1are im-
portant in the emigration and/or gut homing of these populations in
the adult. Kunisawa et al. (8) concluded that gd+and CD8aa+ab+
IEL homingwere S1P-independentbasedon thelack ofaneffectof
long-term FTY720 treatment on the percent (or numbers) of gd and
CD8aa+ab IELs in the gut. It is possible, however, that T cells are
retained in the gut environment in a KLF2/S1P1-independent
fashion after initial entry in a KLF2/S1P1-dependent fashion. We
previously reported that b7 integrin is expressed at lower levels on
KLF2-deficient CD8 SP thymocytes (4), and this could contribute
to reduced IEL homing in knockout mice, because b7 integrin is
critical for this process (35). However, we found no differences in
b7 integrin mRNA levels on thymic gd T cells in KLF2-deficient
animals (data not shown).
6064 THE ROLE OF KLF2 IN gd T CELLS
Alternatively, it is possible that the low numbers of KLF2/S1P1-
deficient gd and CD8aa+ab IELs reflect a role for these mole-
cules in the thymic emigration of gd and CD8aa+precursors into
the circulation. This result is consistent with the requirement for
of CD4+and CD8ab+cells to the gut. Again, Kunisawa et al. (8)
was S1P-independent because short-term FTY720 treatment did
not affect the appearance of FITC-positive gd and CD8aa+recent
We do not have an explanation for this discrepancy at this time,
except to note that the highly pleiotropic effects of FTY720 on
immune and nonimmune cells, including on endothelial cells (38),
may contribute to their findings. Clearly, T cell intrinsic deficiency
of S1P1caused a profound reduction in the accumulation of gd
and CD8aa+ab T cells in the gut in the adult bone marrow chi-
meras. In this regard, our data support a thymic origin for gut IEL
progenitors in adult animals (39).
Despite the requirement for KLF2 and S1P1in localization or
homing of gd and CD8aa+ab T cells to the gut, once in that en-
vironment, such cells express very low levels of both molecules (8)
(Fig. 5). These populations are noted for their CD69+phenotype.
CD69 is upregulated in T cells activated through TCR ligation, al-
though it is unclear if this expression in IEL reflects recent Ag
stimulation (40). Alternatively, CD69 induction on IEL may more
generally reflect the T cell perception of microenvironmental cues,
independent of TCR specificity (41). Interestingly, our data showed
a clear inverse correlation between CD69 and KLF2 expression in
gut T cells. This same relationship is seen in T cells from all tissues
deficient (4) and S1P1-deficient (1) T cells, it is possible that the
initiating microenvironmental cues that T cells perceive in the gut
result inKLF2 downregulation. This inturn results in S1P1loss and
CD69 expression (42). Such a process might be predicted to con-
tribute to the long-term retention of T cells in the gut either by
the known role of S1P1for egress into the circulation (1) or the
proposed role of CD69 in cellular retention (43, 44).
Our findings are of relevance to understanding normal and
pathologic immune responses. In humans, gd T cells have been
(49). In fact, gd subset studies in mice suggest that Vg1+cells (i.e.,
gdNKT) enhance airway hyperresponsiveness,whereas Vg4+cells
suppress airway hyperresponsiveness(48). Wedemonstratedinthis
study that KLF2 and S1P1are important for thymic emigration/
trafficking of both conventional gd T cells and gut homing gd and
CD8aa+ab T cells, therefore suggesting potential strategies to
control specific subsets of gd T cell migration to lymphoid and
nonlymphoid organs. Additionally, our results indicate the need for
future studies aimed at understanding the mechanisms controlling
gd NKT expansion.
We thank Xiao-Jie Ding, Jason Vevea, and Carolina Mora-Solano for tech-
nical support. We also thank the members of the University of Minnesota
Medical Scientist Training Program and FlowCore facilities and Dr. David
Masopust and Dr. Kensuke Takada for reading the manuscript.
The authors have no financial conflicts of interest.
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6066 THE ROLE OF KLF2 IN gd T CELLS