Distinct Roles of Cdc42 in Thymopoiesis and Effector and
Memory T Cell Differentiation
Fukun Guo1*, Shuangmin Zhang1, Pulak Tripathi2, Jochen Mattner2,3, James Phelan2, Alyssa Sproles2,
Jun Mo4, Marsha Wills-Karp2, H. Leighton Grimes2, David Hildeman2, Yi Zheng1
1Division of Experimental Hematology and Cancer Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio, United States of America, 2Division of
Immunobiology, Children’s Hospital Research Foundation, Cincinnati, Ohio, United States of America, 3Microbiology Institute-Clinical Microbiology, Immunology and
Hygiene, Universita ¨tsklinikum Erlangen und Friedrich-Alexander Universita ¨t Erlangen-Nu ¨rnberg, Erlangen, Germany, 4Division of Pathology, Children’s Hospital Research
Foundation, Cincinnati, Ohio, United States of America
Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its
physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that
Cdc42 maintains naı ¨ve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have
further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc422/2
thymus, positive selection of CD4+CD8+double-positive thymocytes was defective, CD4+and CD8+single-positive
thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies,
and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-
stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization
and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1
and CD8+effector and memory cells in vitro and in vivo. Finally, Cdc422/2mice exhibited exacerbated liver damage in an
induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and
plays a restrictive role in effector and memory T cell differentiation and autoimmunity.
Citation: Guo F, Zhang S, Tripathi P, Mattner J, Phelan J, et al. (2011) Distinct Roles of Cdc42 in Thymopoiesis and Effector and Memory T Cell Differentiation. PLoS
ONE 6(3): e18002. doi:10.1371/journal.pone.0018002
Editor: Ralph Tripp, University of Georgia, United States of America
Received January 4, 2011; Accepted February 17, 2011; Published March 24, 2011
Copyright: ? 2011 Guo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grant R01 HL085362 (to Yi Zheng)(www.nih.gov) and Cincinnati Children’s Hospital Trustee
Grant (to Fukun Guo)(www.cchmc.org). No additional external funding was received for this study. The funders had no role in study design, data collection and
analysis, decision to publish, and preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
T cell development in thymus proceeds through a series of
differentiation stages. The most immature populations in thymus
comprise CD42CD82double-negative (DN) thymocytes. The
differentiation of DN thymocytes to CD4+CD8+double-positive
(DP) cells is dependent on the expression and rearrangement of
TCRb and TCRa. DP cells further undergo positive and negative
selection, and differentiate to CD4+or CD8+single-positive (SP) T
cells. CD4+or CD8+SP T cells migrate to peripheral tissues, e.g.
spleen and peripheral blood, where they are maintained as naı ¨ve T
Upon recognition of peptide-MHC complex on antigen-
presenting cells (APC), naı ¨ve T cells undergo actin cytoskeletal
rearrangement, TCR clustering, and formation of immunological
synapse (IS). These cellular events elicit a cascade of intracellular
signaling changes including activation of ZAP70 and LAT and
subsequent ERK, JNK and p38 MAP kinases, leading to naı ¨ve T
cell clonal expansion and differentiation into effector and memory
There are several types of CD4+effector cells, among which T
helper (Th) 1 and 2 are the best studied . Th1 and Th2 cells
exert their immune functions through secretion of distinct patterns
of cytokines: Th1 cells mediate clearance of intracellular
pathogens by producing IFN-c and TNF-a while Th2 cells are
involved in elimination of parasitic organisms by secreting IL-4,
IL-5, and IL-13 [3,4,5]. On the other hand, cytotoxic CD8+
effector cells play essential roles in the protection against
intracellular pathogens and tumor cells by generating IFN-c,
TNF-a, granzymes, perforin, and FAS ligand (FasL) . Aberrant
cytokine production is involved in the pathogenesis of a variety of
autoimmune diseases. For example, IFN-c contributes to the
development of experimental autoimmune myasthenia gravis and
liver damage in a liver-specific autoimmune disease model induced
by alphaproteobacterium Novosphingobium aromaticivorans (N.
aro) [7,8,9]. A small fraction of effector cells can further
differentiate into memory cells, which are major players in recall
immune responses . CD4+memory cells are generally thought
to maintain similar cytokine expression patterns of their
Cdc42 of the Rho GTPase family is an intracellular signal
transducer that cycles between an inactive GDP-bound form and
an active GTP-bound form under tight regulation . Mostly by
overexpression of dominant active or negative mutants, Cdc42 has
been shown to regulate actin cytoskeleton reorganization, cell
migration, proliferation, and survival . In T cells, overexpres-
sion of a dominant mutant suggests that Cdc42 plays a role in
actin and tubulin cytoskeleton polarization, migration, and in
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development [12,13,14,15]. However, this approach is hampered
by its nonspecific nature, as dominant mutants of Cdc42 may
affect other Rho GTPases . Indeed, distinct cell functions of
Cdc42 have been observed in studies of Cdc42 knockout mouse
models. For example, contrary to the prevailing view that Cdc42
promotes cell growth and survival, hematopoietic stem cells
(HSCs) and HSC-derived myeloid cells deficient in Cdc42 exhibit
hyperproliferative properties, and Cdc42-deficient HSCs do not
display survival defects, whereas Cdc42-deficient myeloid cells
show enhanced survival [17,18]. Further, Cdc42-deficient fibro-
blastoid cells and B lymphocytes do not show migratory defects,
whereas primary fibroblasts and neutrophils display a dependence
on Cdc42 for cell migration [19,20,21,22]. Thus, defining the
physiologic role of Cdc42 requires genetic and cell type-specific
To examine the physiological contribution of Cdc42 in T cells,
we have generated a Lck-cre driven T cell-specific Cdc42
conditional knockout mouse model. By characterizing this model,
we have recently reported that Cdc42 is required for coordinating
IL-7R-mediated T cell survival and TCR-mediated T cell
activation in maintaining naı ¨ve T cell homeostasis . To define
the role of Cdc42 in multiple stages of T cell development, in the
present study we have examined thymopoiesis, IS formation in
peripheral naı ¨ve T cell activation, and subsequent effector and
memory T cell differentiation, in Cdc422/2mice. We demon-
strate that Cdc42 is required for thymocyte development by
impact on positive selection, cell migration, and proliferation.
While essential for TCR clustering and actin polarization in the
course of mature IS formation, Cdc42 suppresses CD4+Th1 (but
not Th2) and CD8+effector and memory T cell differentiation and
autoimmunity. These systematic studies suggest a stage-specific
role of Cdc42 in T cell developmental cascade.
Cdc42 deficiency causes defective positive selection in
To examine the role of Cdc42 in multiple stages of T cell
development, we first characterized thymopoiesis in Cdc422/2
mice. By flow cytometry analysis (FACS), we found that the
proportion and numbers of DP thymocytes were increased and SP
thymocytes were decreased in Cdc422/2mice (Figure 1A) .
Consistent with these changes, the cortex of Cdc42-deficient
thymus was prominent while the medulla of mutant thymus was
small and inconspicuous (Figure 1B). Because the cortex is a place
where DP thymocytes reside and undergo selection, we next
examined thymocyte positive selection in Cdc422/2mice. The
percentage of DP thymocytes expressing CD69, a marker
upregulated in positively-selected thymocytes, was significantly
reduced in Cdc42 null mice, compared to that in wild-type (WT)
mice (Figure 1C). To determine at which point thymocyte
development is blocked in Cdc422/2
expression of TCRb together with CD69 in total thymocytes, by
FACS. As shown in Figure 1D, the percentage of TCRloCD692
cells, which correspond to preselection DP thymocytes, was higher
TCRhiCD69+, and TCRhiCD692cells, which contain mainly
DP thymocytes at the initial stage of positive selection, immature
SP thymocytes, and mature SP thymocytes, respectively, was lower
in Cdc422/2mice. These results suggest that Cdc42-deficient
thymocytes were blocked at the earliest stage of positive selection.
To reaffirm the inhibitory effect of Cdc42 deficiency on
thymocyte positive selection, we crossed Cdc422/2mice with
SMARTA TCR transgenic mice. SMARTA TCR transgenic
mice, we analyzed
mice express TCRVa2Vb8 that specifically recognizes lymphocytic
choriomeningitis virus (LCMV) epitope gp61–80 presented by class
II major histocompatibility complex (MHC) molecule I-Ab.
Thus, the resultant Cdc422/2;SMARTAtg/+from Cdc422/2
crossbreeding with SMARTAmice aredominated with monoclonal
CD4+T cells. FACS analysis of thymocytes from Cdc422/
2;SMARTAtg/+mice revealed fewer CD4+SP thymocytes and
lower expression of Va2 TCR in DP thymocytes, compared to
WT;SMARTAtg/+mice (Figure 1E). Taken together, these results
show that Cdc42 is required for thymocyte positive selection and
Cdc42 knockout impairs migration, survival, and TCR
signaling of thymic T cells
Cell migration and adhesion are known to be critical for
thymopoiesis . We found that depletion of Cdc42 dampened
migratory activity of SP thymocytes toward MIP-3b (Figure 2A).
However, adhesion of Cdc42-deficient SP thymocytes to fibro-
nectin remained unchanged (Figure 2B). These data suggest that
Cdc42 is important for SP thymocyte migration but not adhesion
and that defective migration may underlie the developmental
block in Cdc422/2thymocytes.
Because Cdc42 can regulate cell survival , we next
examined the survival status of Cdc422/2thymocytes. To our
surprise, Annexin V staining of freshly isolated thymocytes did not
reveal a survival defect in Cdc422/2DP and SP thymocytes
(Figure 2C). However, we found that disruption of Cdc42 led to a
minor but statistically significant increase in apoptosis in SP
thymocytes that were stimulated in vitro by CD3 and CD28
antibodies (Figure 2D). Thus, Cdc42 may be involved in
protecting thymocytes from anti-CD3-induced apoptosis.
The defect in thymic T cell development in Cdc422/2mice
suggests a perturbation in thymocyte TCR signaling. Indeed, we
found that TCR ligation-induced thymocyte growth was abrogat-
ed in the absence of Cdc42 (Figure 2E). Moreover, TCR
engagement-triggered ERK and JNK MAPK activation was
enhanced in Cdc422/2
thymocytes (Figure 2F). However,
ablation of Cdc42 had no effect on TCR-induced p38 activation
(Figure 2F). These findings are contrary to some of the previous
reports of a positive role of Cdc42 in the regulation of ERK, JNK,
and/or p38 activities and reflect the cell type-specific signaling
function of Cdc42 .
Cdc42 is required for TCR clustering and actin
polarization during the formation of mature IS in
peripheral naive T cells
Mature SP thymocytes emigrate from thymus to peripheral
tissues where they are maintained as naı ¨ve T cells. Upon binding
of TCR with antigen-MHC complex, naı ¨ve T cells differentiate to
effector and memory cells to exert their immune functions [1,2].
The differentiation of effector and memory T cells requires naı ¨ve
T cell activation [1,2]. Previously we showed that Cdc42
deficiency in naı ¨ve T cells resulted in a hyperactive phenotype
. In support of this, we found that T cell activation marker
CD69 was upregulated in Cdc422/2naı ¨ve T cells (Figure 3A)
, in contrast to the downregulation of CD69 in Cdc422/2DP
thymocytes. This seemingly paradoxical observation could reflect
a distinct function of CD69 in peripheral T cells from that in
thymocytes where CD69 is a marker for positive selection. The
data suggest that Cdc42 plays a developmental stage-specific role
in regulating CD69 expression. Upon further examination of
the early events in the course of naı ¨ve T cell activation we found
that anti-TCR-induced actin polymerization was impaired in
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established role of Cdc42 in actin cytoskeleton reorganization.
TCR cap is an asymmetric membrane structure that is formed
through ligand-induced TCR clustering, depending on actin
cytoskeletal rearrangement . Anti-TCR stimulated TCR
capping/clustering activity was reduced by ,50% in Cdc42 null
naı ¨ve T cells (Figure 3C). To determine if Cdc42 deficiency also
causes defects in actin polymerization and TCR clustering during
immune synapse formation, we mixed Cdc422/2;SMARTAtg/+
or WT;SMARTAtg/+naı ¨ve T cells with APCs preloaded with
LCMV epitope gp61-80, incubated cell mixture for 15 or 30 min,
and stained F-actin and TCRVa2. Actin polymerization and
TCR clustering at the interface between Cdc422/2;SMARTAtg/+
T cells and APCs were impaired at 30 min after mixing T cells
with APCs, as reflected by a marked reduction of the size of
polarized F-actin and TCR clusters in Cdc422/2;SMARTAtg/+T
cells (Figure 3D). Interestingly, Cdc422/2;SMARTAtg/+T cells
appeared to undergo normal actin polymerization and TCR
clustering at 15 min after mixing T cells and APCs (Figure 3D).
Since TCR clusters initially form at the periphery of immature IS
and subsequently translocate to the center of IS to facility IS
maturation [26,27,28,29], our data suggest that Cdc42 is not
required for TCR microcluster formation but essential for its
centralization during the maturation of IS.
naı ¨ve T cells (Figure 3B), consistent with the
Cdc42 deficiency enhances effector and memory T cell
Cdc42 deficiency causes
(Figure 3A), raising a possibility that subsequent effector and
memory T cell differentiation may be altered in the absence of
Cdc42. To examine this, we first cultured CD4+naı ¨ve T cells from
Cdc422/2and WT mice in vitro under standard Th1- and Th2-
polarizing conditions, and analyzed IFN-c and IL-4/IL-5
production, respectively. Disruption of Cdc42 led to more IFN-
c-producing cells and IFN-c secretion (Figure 4A). However, the
frequency of IL-4-producing cells and production of IL-5 were not
altered in the absence of Cdc42 (Figure 4A).
We next examined effector cell differentiation in vivo. To this
end, Cdc422/2and WT mice were inoculated with LCMV and
sacrificed 9 days later and analyzed for LCMV-specific CD4+and
CD8+T cells, using MHC class II and class I tetramers containing
LCMV epitopes, I-Abgp61-80 and Dbgp33-41, respectively. We
found that compared to WT mice, Cdc422/2mice had higher
frequency of LCMV-specific (tetramer+) CD4+and CD8+effector
cells (Figure 4B). This increase persisted when the cells transited
into the memory compartment, as frequency of LCMV-specific
CD4+and CD8+T cells remained higher in mutant mice by 50
days after infection (Figure 4C). Moreover, while IL-4-producing
CD4+memory cells remained unchanged in the absence of Cdc42,
IFN-c- and IL-2-producing CD4+and CD8+memory cells were
increased in Cdc422/2mice. In vivo Brdu labeling experiment
revealed that LCMV-specific Cdc422/2memory T cells had a
significantly increased proliferative activity (Figure 4D). Surpris-
ingly, Cdc42 deficiency did not alter survival of LCMV-specific
naı ¨veT cell hyperactivation
memory cells (Figure 4D). These results suggest that Cdc42
restrains proliferation and differentiation of CD8+and CD4+
effector and memory cells.
Cdc42 deficiency causes an exacerbated liver damage in
Aberrant effector and memory T cell differentiation may lead to
autoimmune responses. We thus investigated if Cdc422/2mice
showed autoimmune phenotypes. The serum level of anti-nuclear
autoantibodies (ANA) in Cdc422/2mice was comparable to that
in WT mice (Figure 5A), suggesting that no spontaneous
autoimmunity is developed in mutant mice. This could attribute
to an increased frequency of regulatory T cells (Figure 5B).
We next examined the role of Cdc42 in an induced liver-specific
inflammatory disease model. Cdc422/2and WT mice were
inoculated with alphaproteobacterium N. aro that induces
antibodies against microbial Pyruvate Dehydrogenase Complex
E2 (PDC-E2) and its mitochondrial counterpart . The mice
were analyzed 6 weeks after infection. We found that livers from
Cdc422/2mice were moderately larger than that from WT mice
after N. aro challenge (Figure 5C). Strikingly, splenocytes from
N.aro-infected Cdc422/2mice produced 3-fold more IFN-c when
restimulated in vitro with bone marrow-derived dentritic cells
preloaded with N.aro (Figure 5D). Moreover, histological
examination of livers from Cdc422/2mice revealed exacerbated
portal inflammation with increased leukocytic and lymphocytic
infiltration (Figure 5E). These data suggest that Cdc42 suppresses
inflammation in this mouse model.
In this study, we report that Cdc42 deficiency blocks
thymopoiesis at DP stage, resulting in a decreased production of
SP thymocytes. We further show that disruption of Cdc42 causes a
dampened actin polymerization and TCR clustering in naı ¨ve T
cells during the formation of mature IS. Moreover, ablation of
Cdc42 induces enhanced naı ¨ve T cell differentiation to effector
and memory cells in vitro and in vivo. Lastly, we detected more
severe liver damage in Cdc42-deficient mice in a liver-specific
autoimmune disease model. Our findings suggest that while Cdc42
is required for thymopoiesis, it plays a restrictive role in effector
and memory T cell differentiation and autoimmunity.
Cdc42 regulates thymopoiesis through complex mechanisms.
First, Cdc422/2DP thymocytes expressed less CD69, suggesting
that positive selection is impaired in mutant mice. Given that
Cdc422/2mice have more preselection DP thymocytes, T cell
development in mutant mice appears to be impeded at early stage
of positive selection. Second, Cdc422/2SP thymocytes are
dampened in migration. Thirdly, Cdc422/2SP thymocytes were
more susceptible to anti-CD3-induced cell death. Finally,
thymocytes display a defective proliferation, in
response to anti-CD3/-CD28 stimulation, and aberrant TCR
signaling. Therefore, a combined effect of Cdc42 on positive
selection, migration, survival, growth, and TCR signaling
contributes to its regulation of thymic T cell development. It is
Figure 1. Defective positive selection in Cdc422 2/ /2 2thymocytes. (A) Flow cytometry of thymocytes from wild type (WT) and Cdc422/2mice.
Numbers in dot plots indicate percent cells in each corresponding quadrant; right, average frequency of thymocyte subsets. n=12. **P,0.01. Error
bars represent SD. (B). Thymus sections from WT and Cdc422/2mice, stained with hematoxylin and eosin. The medulla exhibits lighter staining. Data
are representative of 3 mice. (C) Flow cytometry of the expression of CD69 in WT and Cdc422/2DP thymocytes. Numbers above bracketed lines
indicate percent CD69+cells; right, average frequency of CD69+DP cells. n=5. **P,0.01. Error bars represent SD. (D) Flow cytometry of CD69 and
TCRb on total thymocytes from WT and Cdc422/2mice. Numbers adjacent to outlined areas indicate percent cells in each gate; right, average
frequency of thymocyte subsets gated at left. n=5. **P,0.01. Error bars represent SD. (E) Average frequency of CD4+SP thymocytes and TCR Va2hi
DP thymocytes from WT;SMARTAtg/+and Cdc422/2;SMARTAtg/+mice. n=5. **P,0.01. Error bars represent SD.
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Figure 2. Defective migration, survival, and TCR signaling in Cdc422 2/ /2 2thymocytes. (A) Migration to SDF-1a of DP thymocytes or to MIP3b
of SP thymocytes from wild type (WT) and Cdc422/2mice. Data were expressed as numbers of cells migrated to the exterior of transwell chambers
relative to numbers of cells initially seeded in the interior of transwell chambers (% of input)n=5. *P ,0.05; **P ,0.01. Error bars represent SD. (B)
Adhesion to fibronectin of thymocytes from WT and Cdc422/2mice. Data were expressed as numbers of adhered cells relative to numbers of cells
plated (% of input). n=6. Error bars represent SD. (C) Apoptosis of Ex vivo thymocytes from WT and Cdc422/2mice. Freshly isolated thymocytes were
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noted that contrary to a positive role of Cdc42 in the regulation of
ERK, JNK, and/or p38 activities reported in some of the previous
studies, ERK and JNK activities were enhanced while p38 activity
remained unchanged in response to TCR stimulation in Cdc422/2
thymocytes. Given that ERK is known to promote thymocyte
positive selection , the elevated ERK activity could be due to a
compensatory effect for the compromised positive selection in
Cdc422/2thymocytes. On the other hand, since JNK positively
regulates thymocyte negative selection , the increased JNK
activity could be suggestive of an enhanced negative selection in
Cdc422/2thymocytes. These results, in the context of diverse
signaling functions of Cdc42 , are consistent with the notion
that Cdc42 plays a cell type-specific role in regulating ERK, JNK,
and p38activities. The thymopoietic phenotypes of Cdc422/2mice
are reminiscent of and complementary to the previous observations
in mouse models deficient in Rac1, Rac2 or RhoH, other members
of Rho family GTPases. In Rac1 and Rac2 double knockout mice,
T cell development is severely impaired at checkpoints of b selection
and positive selection. The developmental block is associated with
defects in thymocyte proliferation, survival, adhesion, and migra-
tion. Further, loss of Rac1 and Rac2 suppresses TCR-mediated
interleukin-2 production and Akt activation and leads to hyper-
activation of Notch signaling [1,33]. Similarly, RhoH knockout
mice display a defective T cell maturation during transition from
DN3 to DN4 and during positive selection. And RhoH deficiency
leads to defective TCR signaling manifested by decreased activation
of CD3f, LAT, PLCc, Vav1, and Erk, and by reduced calcium
Cdc42 is well appreciated as a key regulator of actin
cytoskeleton rearrangement [36,37]. Consistent with this view,
Cdc42 deficiency diminishes anti-TCR-induced actin polymeriza-
tion of naı ¨ve T cells. Moreover, anti-TCR-induced TCR capping/
clustering is attenuated in Cdc422/2naı ¨ve T cells. Essentially, by
examining Cdc422/2transgenic monoclonal T cells, we found
that actin polymerization and TCR clustering were impaired at
later but not early stage of IS formation with APCs. In line with
recent studies showing that TCR microclusters initially emerge at
the periphery of IS and then move to the center of IS, resulting in
mature IS formation [26,27,28,29], it is logical to reason that
Cdc42 may promote centralization but not emergence of TCR
microclusters. Nonetheless, the hypothesis warrants further and
stringent examination in the future. It is not clear at this point why
Cdc42 deficiency has no effect on TCR microcluster formation.
However, in view that TCR microcluster formation is dependent
on actin polymerization and that Rac1, along with Cdc42, plays a
key role in actin polymerization [38,39], Rac1 may compensate
for the loss of Cdc42 to promote TCR microcluster formation
during initial phase of IS formation. It was originally proposed that
IS promoted TCR signaling transduction . However, most
recent studies suggest that IS plays a dual role in T cell activation:
TCR microclusters at the periphery of IS initiate TCR signaling
transduction, whereas centralized TCR microclusters are degrad-
ed, resulting in TCR signaling termination [26,27,28,29]. We thus
postulate that while being triggered normally, TCR signaling
could not be terminated in Cdc422/2cells. This hypothesis could
potentially explain sustained ERK activation and hyperactive
phenotypes observed in Cdc422/2T cells . The phenotypes of
TCR cluster dynamics caused by Cdc42 deficiency is reminiscent
of that resulted from inhibition of myosin IIA showing impaired
TCR microcluster translocation to the center of IS, with normal
TCR microcluster formation . Since we detected an impaired
activation of myosin light chain in Cdc422/2T cells (data not
shown), it is possible that Cdc42 functions upstream of myosin IIA
in the regulation of TCR microcluster kinetics.
In an in vitro CD4+effector T cell differentiation system, we
found that Th1 cell differentiation was enhanced in the absence of
Cdc42. However, we found no evidence of differences in Th2 cell
differentiation. Considering that Cdc422/2T cells bear increased
level of TCR signaling , our data are in support of the
literatures that strong TCR signals induce Th1 cell differentiation
[3,42,43]. The differentiation of Th1 effector cells are mainly
governed by JAK1/2/STAT1/3/4 signaling
transcriptional factor T-bet [3,4]. In this context, further work is
needed to determine if Cdc42 deficiency influences intracellular
signaling pathways that are discrete for Th1 cells. Of note, the role
of Cdc42 in Th cell differentiation are in sharp contrast to that of
Cdc42 immediate downstream effector WASP, disruption of
which leads to a diminished Th2 response while has no effect on
Th1 cells . This suggests that WASP is independent of Cdc42
in Th cell differentiation.
In an in vivo LCMV mouse model, we repeatedly detected an
increase in CD4+effector cells in Cdc422/2mice. CD8+effector
cells are also increased in mutant mice. Further, Cdc422/2mice
generate more LCMV-specific memory cells, compared to WT
mice. Consistent with the effects of Cdc42 deficiency on Th
effector cell differentiation, Cdc422/2CD4+memory cells
produce more Th1 signature cytokines IFN-c and IL-2, whereas
Th2 signature cytokine IL-4 remains comparable in Cdc422/2
and WT memory cells. The increased memory cells in Cdc422/2
mice are attributable to enhanced proliferative renewal. In
contrast to an increased apoptosis in Cdc422/2naı ¨ve T cells
, there is no survival defect in Cdc422/2memory cells. This is
an interesting observation as it assigns a developmental stage
specific role of Cdc42 in T cell survival regulation.
Cdc42 deficiency results in a constitutive activation of naı ¨ve T
cells, which may predispose Cdc422/2mice to autoimmune
diseases . However, serum anti-nuclear autoantibodies are
maintained at basal level in Cdc422/2mice. This self-tolerance
might be associated with increased regulatory T cells. It would be
interesting to assess how regulatory T cells act on memory
phenotype T cells to suppress the development of spontaneous
autoimmunity in Cdc422/2mice. One potential mechanism
could be that regulatory T cells kill memory phenotype T cells by
producing granzyme and/or perforin . Albeit Cdc422/2mice
do not appear to develop spontaneous autoimmunity in the
absence of infectious agents, they indeed mount more severe
stained with anti-CD4 and -CD8 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. n=4. *P ,0.05. Error bars
represent SD. (D) Apoptosis of cultured thymocytes from WT and Cdc422/2mice. Isolated thymocytes were cultured for 24 hours with anti-CD3/-
CD28 antibodies and stained with anti-CD4 and -CD8 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry.
Numbers outside the gates indicate percent cells in each gate and numbers inside the gates indicate absolute numbers of Annexin V+cells analyzed
in each gate; right, average frequency of Annexin V+thymocytes. Data are representative of three independent experiments. n=4. *P ,0.05. Error
bars represent SD. (E) Anti-CD3/-CD28-induced proliferation of thymocytes from wild type (WT) and Cdc422/2mice. TCRb+thymocytes were plated
on 96-well plates at 16106/well in 200 mL culture media in the presence or absence of plate-coated anti-CD3 (10 mg/mL) plus soluble anti-CD28
(2 mg/mL). The cells were cultured for 3 days and assayed for growth rate. n=6. **P ,0.01. Error bars represent SD. (F) MAP kinase activities in
thymocytes from WT and Cdc422/2mice. TCRb+thymocytes were stimulated with or without anti-CD3 (10 mg/mL) and -CD28 (2 mg/mL) for indicated
time. Western blotting was performed to assess the phosphorylation status of Erk, p38, and JNK. The result is a representative of three experiments.
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Figure 3. Defective TCR clustering and actin polymerization in Cdc422 2/ /2 2peripheral naive T cells during mature immunological
synapse formation. (A) Expression of T cell activation marker CD69 in naı ¨ve CD4+T cells from wild type (WT) and Cdc422/2mice. Splenocytes were
stained with anti-CD4, -CD62L, -CD44, and -CD69 antibodies. CD69 expression in CD4+CD62L+CD442naı ¨ve T cells was analyzed by flow cytometry.
Data are representative of five mice (B) Anti-TCR-induced actin polymerization in naı ¨ve CD4+T cells from WT and Cdc422/2mice. Naive CD4+T cells
from spleen were incubated with anti-TCRb antibody on ice and stimulated with anti-hamster IgG at 37uC. Cells were fixed, permeabilized, and
incubated with FITC-phalloidin, and analyzed by flow cytometry. Data are representative of four mice. (C) Anti-TCR-induced TCR capping/clustering in
Cdc42 in T Cell Development
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autoimmune responses in a N.aro-induced and T cell-mediated
liver-specific autoimmune disease model. The mice have increased
incidence of liver portal inflammation and splenocytes from the
mice produce higher level of IFN-c upon restimulation with N.
aro. These phenotypes could result from pre-activated states of
Cdc422/2T cells that hold lower threshold for antigen-induced T
In conclusion, we demonstrate that Cdc42 positively regulates
thymocyte development but negatively regulates naı ¨ve T cell
activation and differentiation to effector and memory T cells.
Thereby, Cdc42 plays a stage-specific role in T cell development.
Our data provide a novel insight into the mechanisms of T cell
development and adaptive immunity.
Materials and Methods
This study involved using mice. The study was carried out in
strict accordance with the recommendations in the Guide for the
Care and Use of Laboratory Animals of the Cincinnati Children’s
Hospital Research Foundation. The protocol was approved by the
Committee on the Ethics of Animal Experiments of the Cincinnati
Children’s Hospital Research Foundation (permit Number:
8D06052). Mice were anesthetized when necessary, using
ketamine (80–100 mg/kg im), aceptromazine (4–6 mg/kg im)
and atropine (0.1 mg/kg im). Anesthesia was maintained using
ketamine (30 mg/kg im) as needed. During the course of
experiments, mice were isolated in microisolator cages and cared
for in the Laboratory Animal Resource Center by trained
technician and two veterinarians. Animals were checked daily by
qualified personnel in the lab. The method of euthanasia used was
CO2euthanasia. This method was approved by the Animal Care
and Use Committee of the Cincinnati Children’s Hospital
Research Foundation and consistent with the recommendations
of the Panel on Euthanasia of the American Veterinary medical
Generation of mice lacking Cdc42 in T lymphocytes
Conditional targeted Cdc42
described previously . The flox allele contains loxP sites
flanking exon 2 of Cdc42 alleles. To delete Cdc42 in vivo in T cell
lineage, Cdc42flox/floxmice were mated with mice expressing Cre
recombinase under the control of Lck proximal promoter (from
Jackson Laboratory). Cdc42
with SMARTA mice expressing transgenic TCR (Vab2Vb8) to
generate Cdc422/2;SMARTAtg/+compound mice . All mice
were housed under specific pathogen-free conditions in the Animal
Facility at Cincinnati Children’s Hospital Research Foundation.
All mice used were 4–8 weeks of age.
flox/floxmice were generated as
flox/flox;Lck-Cre mice were crossed
Flow cytometry analysis
Single cell suspensions were prepared from thymus. Cells were
incubated for 20 min at room temperature with various
combinations of the following cell-surface marker antibodies:
anti-CD4, anti-CD8, anti-CD69, anti-TCRb (H57-597), and anti-
TCR Va2 (BD Biosciences). Immunolabeled cells were analyzed
by flow cytometry on a FACSCanto system using FACSDiVa
software (BD Biosciences).
Thymuses and livers were fixed in 10% buffered formalin,
embedded in paraffin, sectioned (5 mM), and stained with
hematoxylin and eosin.
Cell migration assay
Cell migration was measured using a transwell chamber.
Dulbecco modified Eagle medium (DMEM) containing 500 ng/
mL SDF-1 (PEPROTECH, Rocky Hill, NJ) or MIP-3b (R&D
System, Minneapolis, MN) was added to the exterior of the
transwell chamber. Flow cytometry sorted CD4+CD8+DP, CD4+
or CD8+SP thymocytes were suspended in the DMEM and added
to the interior of the transwell chamber and incubated for 4 hours.
Cells migrated through polyester membrane were counted.
Cell adhesion assay
Flow cytometry sorted CD4+CD8+DP, CD4+or CD8+SP
thymocytes were plated onto 10 mg/mL fibronectin-coated 96-well
plates and incubated for 2 hours. The unattached cells were
washed away with Dulbecco modified phosphate-buffered saline.
The attached cells were trypsinized and counted.
Cell apoptosis analysis
Freshly isolated thymocytes or thymocytes treated for 24 hours
with anti-CD3/-CD28 antibodies (BD Biosciences) were incubat-
ed with anti-CD4 and anti-CD8 antibodies for 20 min. Cells were
washed, incubated with Annexin V (BD Biosciences) for 20 min
and then analyzed by flow cytometry.
In vitro Proliferation assay
TCRb+thymocytes were sorted by flow cytometry, plated on
96-well plates with or without anti-CD3/-CD28 antibodies, and
cultured for 3 days. Cell growth rates were assayed by a
nonradioactive cell proliferation assay kit (Promega).
Whole-cell lysates were prepared and separated by 10% SDS-
polyacrylamide gel electrophoresis. The expression or activation
(phosphorylation) of ERK, JNK, and p38 was probed by using
corresponding antibodies (Cell Signaling Technology).
Actin polymerization assay
Naı ¨ve CD4+T cells from spleen were purified by FACS cell
sorting. The cells were incubated with anti-TCRb antibody 1 mg/
ml on ice for 30 min. After washing, cells were stimulated with
anti-hamster IgG 3 mg/ml at 37uC for 30 min. Cells were fixed
with 4% paraformaldehyde, and permeabilized with 0.1% Triton
X-100. Cells were then incubated with fluorescein isothiocyanate
naı ¨ve CD4+T cells from WT and Cdc422/2mice. Naı ¨ve CD4+T cells from spleen were seeded on poly-L-lysine-coated slides, incubated with anti-TCRb
antibody on ice, and stimulated with biotin-conjugated anti-hamster IgG at 37uC. After fixation, cells were stained with Avdin-Texas Red and
visualized with a Zeiss fluorescence microscope Average percentage of capped cells was obtained by counting cells from 6 random fields. Data are
representative of 3 mice. (D) Antigen-presenting cells (APCs)-induced actin polymerization and TCR clustering in naı ¨ve T cells from WT;SMARTAtg/+
and Cdc422/2;SMARTAtg/+mice. Splenic naı ¨ve CD4+T cells bearing SMARTA transgenic TCRVa2Vb8 were incubated with gp61-80-loaded APCs (CHB.2
B cells) for 15 min or 30 min, fixed, permeabilized, stained with phalloidin (red) and anti-TCRVa2 (green), and visualized with a Leica
immunofluorescence microscopy. Differential interference contrast (DIC) show APC-T cell conjugates. Arrowheads point to F-actin and TCR clusters at
interface between APC and T cells. Data are representative of 20 to 30 APC-T cell conjugates. The size (area) of F-actin and TCR clusters was quantified
by Image J.
Cdc42 in T Cell Development
PLoS ONE | www.plosone.org8 March 2011 | Volume 6 | Issue 3 | e18002
(FITC)-phalloidin (Sigma) for 60 min, washed, and analyzed by
Purified naı ¨ve CD4+T cells were seeded on poly-L-lysine-
coated 2 chamber slides and incubated with anti-TCRb antibody
3 mg/ml on ice for 30 min. Cells were washed and then incubated
with biotin-conjugated anti-hamster IgG 3 mg/ml at 37uC for
30 min. After fixation, cells were stained with Avidin-Texas Red
(BD PharMingen) for 30 min and visualized with a Zeiss
fluorescence microscope and percentage of capped cells was
Figure 4. Enhanced effector and memory T cell differentiation in the absence of Cdc42. (A) Th1 and Th2 effector cell differentiation in vitro
from wild type (WT) and Cdc422/2naı ¨ve T cells. Splenic CD4+naı ¨ve T cells were cultured under Th1 or Th2 polarized conditions. At day 3, levels of Th2-
produced IL-5 and Th1-produced IFN-c in culture supernatants were quantified by ELISA (right). At day 6, the cells were restimulated as described in the
Methods. IL-4-secreting Th2 cells and IFN-c-secreting Th1 cells were analyzed 6 hours after restimulation, by flow cytometry (left). n=4. **P ,0.01. Error
bars represent SD. (B) Effector cell differentiation in vivo in WT and Cdc422/2mice. WT and Cdc422/2mice were administrated with lymphocytic
choriomeningitis virus (LCMV). At day 9, frequency of LCMV-specific CD4+and CD8+effector cells were analyzed by flow cytometry, using respective
tetramer staining reagents. n=6. *P,0.05, **P,0.01. Error bars represent SD. (C) Memory cell differentiation in vivo in WT and Cdc422/2mice. WT and
Cdc422/2mice were administrated with LCMV. At day 50, frequency of LCMV-specific CD4+and CD8+memory cells were analyzed as described in (B).
Frequency of cytokine-secreting LCMV-specific CD4+and CD8+memory cells were analyzed after in vitro restimulation with gp61-80 and gp33-41,
respectively. n=6. *P,0.05, **P,0.01. Error bars represent SD. (D) Memory cell proliferation and survival in WT and Cdc422/2mice. WT and Cdc422/2
mice were injected with Brdu beforebeing sacrificedatday50postLCMV infection. Brdu+LCMV-specific CD4+andCD8+memory cells were analyzedby
flow cytometry. Survival status of LCMV-specific CD4+and CD8+memory cells were analyzed by Annexin V staining.
Cdc42 in T Cell Development
PLoS ONE | www.plosone.org 9 March 2011 | Volume 6 | Issue 3 | e18002
T cell-APC conjugation and immunofluorescence staining
CHB.2 B cells, a B cell lymphoma line used as APCs, were
preloaded for 12 h with 10 mg/ml of gp61-80 peptide. The cells
were then mixed, by brief spinning, with wild type (WT) or
Cdc422/2splenic naı ¨ve CD4+T cells expressing SMARTA
transgenic TCRVa2Vb8. The cell mixtures were incubated for 15
min or 30 min, plated on poly-L-lysine-coated coverslips, fixed,
permeabilized with cytofix and cytoperm buffer (BD Biosciences),
and stained for F-actin with Rhodamine phalloidin (Sigma) and
TCRVa2 with FITC-anti-TCRVa2 antibody. Stained cells were
imaged with a fluorescence microscope equipped with a 40 x
objective lens and a deconvolution system (Leica) ‘driven’ by
Openlab software (Improvision) . The size (area) of F-actin
and TCRVa2 at the interface of 20 to 30 T cell-CHB.2 B cell
couples was quantified by using Image J (NIH).
In Vitro Th1 and Th2 differentiation
Naı ¨ve CD4+T cells were sorted from spleen and cultured with
plate-bound anti-TCRb (30 mg/mL) and soluble anti-CD28
(1 mg/mL) antibodies under Th1- or Th2-polarized condition:
Th1: recombinant murine (rm) IL-12 (10 ng/mL), and anti-IL-4
antibody (10 mg/mL); Th2: rhIL-2, rmIL-4 (10 ng/mL), and anti-
IFN-c antibody (10 mg/mL). Cytokines secreted to culture
supernatant were collected on day 3 and analyzed using standard
enzyme-linked immunosorbent assay (ELISA). The cells were
restimulated for 6 hours on day 6 with plate-bound anti-TCRb
antibody (10 mg/mL) in the presence of monesin (Sigma). The
cells were then subjected to intracellular cytokine staining
following fixation and permeabilization with cytofix and cytoperm
Detection of antigen-specific T cells
Mice were injected i.p. with 0.25 mL of 26105pfu LCMV and
administrated with Brdu (500 mg/mouse) once a day for 3 days
before sacrifice. Spleens were harvested and single cell suspension
was prepared 9 days or 50 days after LCMV injection. LCMV-
specific T cells were detected as described previously. Briefly,
LCMV-specific CD4+T cells were detected by staining 26106
Figure 5. Exacerbated liver damage in Cdc422 2/ /2 2mice. (A) Level of anti-nuclear autoantibody (ANA) in serum from wild type (WT) andCdc422/2
mice. Peripheral blood was collected by tail vein bleeding and serum was prepared and analyzed for ANA by ELISA. n=5. Error bars represent SD. (B)
Regulatory T cells in WT andCdc422/2mice. Splenocytes were stainedfor CD4followed by intracellular stainingof Foxp3.CD4+Foxp3+regulatoryT cells
injected with Novosphingobium aromaticivorans (N. aro). Six weeks after N.aro infection, mice were sacrificed and livers were weighed. n=6. *P,0.05.
Error bars represent SD. (D) IFN-c production from WT and Cdc422/2splenocytes. WT and Cdc422/2mice were injected with N. aro. Six weeks post
infection, splenocytes were harvestedfrom WT or Cdc422/2mice andcocultured with bone marrow-deriveddentriticcells (DC) prepulsed with N.aro (N.
arotoDC:5:1).Cell cultures wereassayed72 hours laterfortherelease ofIFN-cbyELISA. n=6.**P,0.01.Error bars representSD.(E) Portalinflammation
in livers from WT and Cdc422/2mice. Six weeks post N.aro infection, livers from WT and Cdc422/2mice were sectioned, stained by hematoxylin and
eosin, andevaluated microscopically (left) for leukocytic andlymphocytic infiltration. Liver lesions were scored (right) by examining 5 sections separated
by 25 mM for portal inflammation using the following scale: 0= no inflammation, 1= sparse mononuclear cell infiltrates, 2= moderate inflammation,
3= intense inflammation, 4= intense inflammation and spillover into the periportal parenchyma. The score was based on the most severe infiltration
observed in the majority of portal fields. Statistical significance was calculated using a Mann-Whitney test based on exact p-value computations to
account for ties. n=6. *P,0.05. Error bars represent SE.
Cdc42 in T Cell Development
PLoS ONE | www.plosone.org10 March 2011 | Volume 6 | Issue 3 | e18002
splenocytes with I-Abgp61-80 tetrameric staining reagents for 2 h
at 37uC. During the last 45 min of incubation, cells were stained
with antibodies against CD4, CD44, and CD16/32 followed by
Annexin V staining or by fixation, permeabilization, and Brdu
CD4+CD44+CD16/322tetramer+cells and their survival status
and Brdu incorporation, by flow cytometry. LCMV-specific CD8+
T cells were detected by staining 2 x 106splenocytes with Dbgp33-
41 tetrameric staining reagents for 90 min at 4uC. During the last
45 min of incubation, cells were stained with antibodies against
CD8 and CD44 followed by Annexin V staining or by fixation,
permeabilization, and Brdu staining. Cells were analyzed for
CD8+CD44+tetramer+populations and their survival status and
Brdu incorporation, by flow cytometry. Splenocytes collected 50
days after LCMV infection were also cultured with peptides gp61-
80 or gp33-41 for 1 hour. Brefeldin A (Sigma) was added to the
culture and the cells were further incubated for 3 hours followed
by intracellular cytokine staining of CD4+and CD8+cells .
Cells were analyzedfor
Measurement of anti-nuclear autoantibodies
Blood was collected and serum was prepared and assayed for
anti-nuclear autoantibodies (ANA) using an ELISA kit (Alpha
Induction of liver-specific autoimmune disease
Bacterial N. aro (56107) (ATCC) was injected intravenously
into 4–7 week old mice on day 0 and day 14. Mice were sacrificed
6 weeks later and analyzed for spleen and liver weight and for liver
portal inflammation by hematoxylin and eosin (HE) staining of
paraffin-embedded liver sections. Portal inflammation was evalu-
ated microscopically for leukocytic and lymphocytic infiltration.
Splenocytes (105) from N.aro-infected mice were also co-cultured
with bone marrow-derived dentritic cells (105) prepulsed with
N.aro, for 3 days. Cell culture supernatant was collected and
assayed for cytokine release by ELISA (R&D Systems, Minneap-
olis, MN) .
Conceived and designed the experiments: FG PT J. Mattner DH YZ.
Performed the experiments: FG SZ PT J. Mattner JP AS J. Muo. Analyzed
the data: FG J. Mattner J. Muo DH YZ. Contributed reagents/materials/
analysis tools: FG J. Mattner J. Muo MW HLG DH YZ. Wrote the paper:
FG DH YZ.
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