The p110? isoform of phosphatidylinositol 3-kinase regulates
migration of effector CD4 T lymphocytes into peripheral
Molly S. Thomas, Jason S. Mitchell, Christopher C. DeNucci, Amanda L. Martin,
and Yoji Shimizu1
Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota
Medical School, Minneapolis, Minnesota, USA
has been studied extensively. However, the specific
role of the p110? isoform of PI- 3K in CD4 T lym-
phocyte function has yet to be defined explicitly. In
this study, we report that although p110? does not
proliferation, it plays a crucial role in regulating CD4
effector T cell migration. Naı ¨ve p110??/?CD4 lym-
phocytes are phenotypically identical to their wild-
type (WT) counterparts and do not exhibit any de-
fects in TCR-mediated calcium mobilization or Erk
cells become activated and proliferate comparably
with WT cells in response to antigen in vivo. Inter-
estingly, however, antigen-experienced, p110?-defi-
cient CD4 OT.II lymphocytes exhibit dramatic de-
fects in their ability to traffic to peripheral inflamma-
tory sites in vivo. Although antigen-activated, p110?-
deficient CD4 T cells express P-selectin ligand, ?2
integrin, ?1 integrin, CCR4, CXCR5, and CCR7
comparably with WT cells, they exhibit impaired F-
actin polarization and migration in response to stim-
ulation ex vivo with the CCR4 ligand CCL22. These
findings suggest that p110? regulates the migration
of antigen-experienced effector CD4 T lymphocytes
into inflammatory sites during adaptive immune re-
sponses in vivo. J. Leukoc. Biol. 84: 814–823;
The role of PI-3K in leukocyte function
Key Words: inflammation ? chemokines ? leukocyte ? activation
The PI-3K family of enzymes has been studied extensively in
multiple cellular systems and participates in a variety of signal
transduction pathways that regulate multiple cellular func-
tions, including growth, metabolism, survival, proliferation,
differentiation, apoptosis, adhesion, and migration [1, 2]. Class
1A PI-3Ks consist of the p110?, p110?, and p110? catalytic
subunits that bind to the p85? regulatory subunit, which is
regulated by tyrosine phosphorylation and is commonly down-
stream of growth or antigen receptors. Class 1B PI-3K is
comprised solely of p110?, which binds to the p101 regulatory
subunit, regulated through its association with the ?? subunit
of heterotrimeric G-proteins and activated downstream of sev-
en-transmembrane G-protein-coupled receptors, including
chemokine receptors [1, 3].
The contribution of PI-3K activity to the function of several
leukocyte populations, such as neutrophils, mast cells, and
monocytes, has been well documented [4–9]. However, the
specific, individual contributions of the p110? and -? isoforms
of PI-3K to naı ¨ve and effector T lymphocyte function have not
been characterized completely. There are, at best, modest
impairments in thymocyte development and lymphocyte mat-
uration in p110??/?or p110??/?mice. In contrast, mice
lacking both of these isoforms (p110?/??/?or p110?knockout
?D910A) display dramatic reductions in thymocyte development
and reduced numbers of mature CD4 and CD8 T lymphocytes
in the periphery [10, 11]. Naı ¨ve CD4 lymphocytes isolated from
p110?/?-deficient mice have an activated phenotype and se-
crete increased levels of the Th2 cytokines IL-4 and IL-5
following TCR stimulation in vitro, suggesting that these two
isoforms of PI-3K may also function in regulating some aspects
of T cell activation [10, 11]. Studies using p110??/?and
p110??/?mice have demonstrated that the p110? isoform of
PI-3K is the main isoform responsible for generating phospha-
tidylinositol-3,4,5-trisphosphate (PIP3) downstream of TCR en-
gagement in mature lymphocytes . In addition, the p110?
isoform of PI-3K has been shown to regulate PIP3accumula-
tion and localization at the immune synapse following TCR
stimulation . Kinase-inactive p110? (p110?D910A) and
p110??/?mice exhibit defects in antigen-specific CD4 T cell
proliferation and differentiation in vitro and defective T-de-
pendent antibody production in vivo but exhibit normal Erk
activation and adhesion to integrin ligands in vitro [12–15].
Similarly, p110? has also been shown to regulate antigen-
dependent responses in mast cells . p110??/?mice have
been shown to exhibit decreased contact hypersensitivity and
delayed-type hypersensitivity (DTH) reactions, presumably as
1Correspondence: University of Minnesota Medical School, 6-112 Has-
selmo Hall, 312 Church St., SE, Minneapolis, MN 55455, USA. E-mail:
Received August 23, 2007; revised May 1, 2008; accepted May 9, 2008.
814 Journal of Leukocyte Biology
Volume 84, September 2008
0741-5400/08/0084-814 © Society for Leukocyte Biology
a result of impaired migratory responses of p110??/?dendritic
cells . Impaired generation of T-dependent antibodies fol-
lowing immunization with T-dependent haptens has also been
reported in p110??/?mice . Furthermore, CD4 T lympho-
cytes, isolated from p110??/?mice, are reported to exhibit
reduced proliferative responses following TCR stimulation in
vitro . However, the antigen-dependent responsiveness of
p110?-deficient CD4 T cells has yet to be described.
It has been demonstrated previously that p110? PI-3K is a
major regulator of neutrophil, monocyte, and mast cell migra-
tion, and p110? plays a more dominant role in regulating B cell
migration [4, 18–21]. Although p110? PI-3K regulates PIP3
accumulation at the membrane downstream of CXCR4 stimu-
lation, naive p110??/?T cells exhibit only modest reductions
in CCR7- and CXCR4-mediated migration in vitro that are
dependent in part on the chemokine being analyzed and the
dose of chemokine tested . Modest defects in p110??/?T
cell homing to secondary lymphoid tissue have also been
reported [19, 22]. The adaptor protein dedicator of cytokinesis
2 (DOCK2), which participates in downstream Rac-dependent
pathways involved in actin remodeling and cell migration [1,
22, 23], has been proposed to be a major regulator of naive T
cell migration via a mechanism that is independent of PI-3K.
Recent studies have demonstrated that DOCK2, not p110?,
controls egress of T lymphocytes out of thymus and secondary
lymphoid tissue, as well as T cell movement within peripheral
lymph nodes (LN) [22, 23]. Although p110? is not necessary
for the basal migration of lymphocytes through secondary lym-
phoid tissues, the question of whether p110? controls the
movement of effector T cells into inflammatory sites in periph-
eral tissues remains to be addressed.
The loss of p110? activity ameliorates the pathogenesis of
several autoimmune diseases, using established animal models
of collagen-induced arthritis and systemic lupus erythematosus
[2, 24, 25]. Although these autoimmune diseases are depen-
dent on the development of aberrant effector CD4 T cell
responses, the specific effect of the loss of p110? in CD4 T
cells to the pathogenesis of these two autoimmune diseases is
unclear. Moreover, the defects in T-dependent antibody pro-
duction and DTH reactions, previously observed in p110?-
deficient mice, are also dependent on the proper initiation and
execution of CD4 effector T cell functions [26–28]. Here, we
sought to investigate the hypothesis that p110? specifically
regulates the migration of antigen-activated effector CD4 T
cells into peripheral inflammatory sites. Our results demon-
strate that although p110? does not control initial antigen-
dependent CD4 T cell activation, this isoform of PI-3K regu-
lates the trafficking of antigen-experienced effector CD4 T
cells into peripheral inflammatory sites through its regulation
of F-actin polarization and migration downstream of inflamma-
tory chemokine receptors.
MATERIALS AND METHODS
p110??/?mice were obtained from Dr. Charles Abrams (University of Pennsyl-
vania, Philadelphia, PA, USA) . OT.II transgenic mice were obtained from Dr.
Marc Jenkins (University of Minnesota, Minneapolis, MN, USA). p110??/?were
back-crossed onto the C57BL/6 and OT.II transgenic background for more than 10
generations and then bred to generate p110??/?OT.II transgenic Thy1.1? mice.
Thy1.2 congenic recipients were obtained from the National Cancer Institute (NCI;
Bethesda, MD, USA) or Taconic (Hudson, NY, USA). All experiments involving
the use of animals were completed under the approval of the University of
Minnesota Institutional Animal Care and Use Committee.
Antibodies and reagents
The following antibodies and reagents were purchased from eBioscience (San
Diego, CA, USA): streptavidin-PE, CD4-FITC, CD8-FITC, B220-FITC,
Thy1.1-FITC, B220-PE-Cy5.5, mouse IgG-FITC, CD62L-allophycocyanin,
CD62L-PeCy7, Thy1.1-PE, Thy1.1-allophycocyanin, CD29-PE, CD49d-PE,
purified CD49d, biotinylated rat IgG, and the corresponding isotype control
antibodies. The following antibodies and reagents were purchased from BD
PharMingen (San Jose, CA, USA): purified CXCR5, recombinant murine
(rm)CXCL13, purified P-selectin-Fc chimera, I-Ad-FITC, CD4-PE-Cy7, V?2
TCR-PE, and CD4-Pacific Blue. CFSE, calcein-acetoxymethylester (AM), and
indo-1-AM were purchased from Invitrogen (Carlsbad, CA, USA). Ionomycin,
PKH-26 reference beads, IFA, CFA, probenecid, and LPS were purchased
from Sigma-Aldrich (St. Louis, MO, USA), and PMA was purchased from LC
Laboratories (Woburn, MA, USA). Anti-FITC microbeads, streptavidin mi-
crobeads, and LS columns were purchased from Miltenyi Biotec (Auburn, CA,
USA). The peptide OVA323–339(ISQAVHAAHAEINEAGR) has been reported
previously  and was synthesized by Invitrogen. Purified anti-CCR4 (clone
K14) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Goat anti-human IgG-PE and anti-goat IgG-PE were purchased from Jackson
ImmunoResearch (West Grove, PA, USA). Mouse/human CCL19-Fc, followed
by incubation with a biotinylated anti-human Fc secondary antibody and
streptavidin-PE (eBioscience), was used to detect CCR7 expression. rmCCL21,
rmCCL22, and rmCCL3 were purchased from Peprotech (Rocky Hill, NJ,
USA). The anti-phospho-Erk (anti-p-Erk; rabbit monoclonal 197G2), anti-total
ERK (rabbit polyclonal), anti-p-AKT (Ser 473, rabbit monoclonal 58711F),
and anti-total AKT (rabbit polyclonal) antibodies used in Western blotting
analysis were purchased from Cell Signaling (Danvers, MA, USA).
Phenotypic analysis of naı ¨ve lymphoyctes
Naı ¨ve lymphocytes were harvested from wild-type (WT) or p110??/?OT.II
transgenic animals and analyzed for GFP, TCR (V?2), CD62L, CD44, and
CCR7 expression on the CD4 lymphocytes using a FACSCalibur flow cytom-
eter (Becton Dickinson, San Jose, CA, USA) and FlowJo analysis software
(Tree Star Inc., Ashland, OR, USA).
For analysis of AKT activation by Western blotting, naı ¨ve WT or p110??/?T cells
were purified from peripheral LN by negative selection (purity, ?95%). The cells
were washed twice in PBS. Purified cells (3?106) were left unstimulated or
stimulated with 5 or 10 ?g/ml rmCCL21 for 1 min at 37°C. The cells were
immediately placed on ice and then pelleted. The cells were resuspended in 1?
Nonidet P-40 lysis buffer (1% Triton, 50 mM Tris, 150 mM NaCl, 4 mM EDTA,
1 mM PMSF, 1 ?g/ml aprotinin, 1 ?g/ml leupeptin) and incubated on ice for 30
min. Lysates were clarified by centrifugation, reduced, and resolved on a 4–12%
Bis-Tris acrylamide gel (Invitrogen). Proteins were transferred to a PVDF mem-
brane, and the membranes were subsequently blocked in PBS ? 0.2% casein and
then probed with anti-p-AKT (Ser473, rabbit mAb) or anti-total AKT (rabbit
polyclonal), followed by goat anti-rabbit IgG-Alexa 680 secondary antibody (In-
vitrogen). Proteins were visualized by scanning membranes on an infrared scanner
(Odyssey, LI-COR Biosciences, Lincoln, NE, USA).
Analysis of Erk activation by Western blotting was preformed as described for
AKT above. Lymphocytes were stimulated with 20 ?g/ml anti-CD3 ? 2 ?g/ml
anti-CD28, and Erk activation was detected using anti-p-Erk (Tyr/Thr rabbit
mAb) or anti-total Erk (rabbit polyclonal) and goat anti-rabbit IgG-Alexa 680.
Calcium flux assays
Peripheral lymphocytes from WT or p110??/?mice were resuspended at a
concentration of 75 ? 106cells/ml in PBS ? 2% calf serum (CS) and stained
Thomas et al.
p110? controls CD4 effector T cell migration815
for CD8 and CD4 T cells, which were then resuspended in RPMI ? 1% FBS
to a concentration of 10 ? 106cells/ml and were loaded with 3 ?g/ml 100 mM
indo-1-AM and 100 mM probenecid for 30 min at 37°C in the dark. After the
collection of a 30-s base line, the cells were stimulated with 1 ?M ionomycin
or 8.4 ?g biotinylated anti-CD3 antibody ? 8 ?g streptavidin (to cross-link
anti-CD3). Calcium flux was analyzed using an LSRII flow cytometer and
FlowJo analysis software.
In vivo functional assays
Peripheral lymphocytes from WT or p110??/?OT.II transgenic Thy1.1? mice
were resuspended to a final concentration of 10 ? 106cells/ml in RPMI
containing 2% FCS and labeled with CFSE. The percentage of CD4? OT.II
(V?2?) transgenic lymphocytes in the bulk suspension was determined by
flow cytometry prior to CFSE-labeling. CD4?OT.II Thy1.1? lymphocytes
(0.5–1.0?106) were adoptively transferred i.v. into Thy1.2 congenic recipi-
ents. Twenty-four hours later, recipient animals were challenged with 10 ?g
OVA323–339peptide with 25 ?g LPS i.v., emulsified 1:1 in 100 ?l IFA i.p.,
emulsified 1:1 in 10 ?l IFA in the ear pinna, or emulsified 1:1 in 100 ?l CFA
s.c. At various time-points following challenge, the draining LN (i.v.?all
peripheral LN; s.c.?axial and cervical LN; ear?cervical LN; i.p.?pancreatic
and mesenteric LN) and spleen were harvested and dispersed into HBSS
containing 0.2% sodium azide and 2% FCS. The phenotype of the
CD4?Thy1.1? cells was determined by analyzing CFSE dilution and CD62L,
CD44, ?4 integrin, and fucosylated PSGL-1 expression. In animals challenged
i.p., peritoneal cells were recovered by washing the peritoneum with 5 ml
PBS/2% CS. In animals challenged in the ear, excised ears were digested in
RPMI/2% FCS containing 400 U/ml collagenase D in a rocking 37°C incubator
for 1 h. The phenotype of the adoptively transferred CD4?Thy1.1? cells was
determined as described above.
Ex vivo transmigration assays
Peripheral lymphocytes harvested from WT or p110??/?OT.II transgenic
Thy1.1? mice were labeled with CFSE as described above. OT.II lymphocytes
(1.0?106) were adoptively transferred into Thy1.2? recipients. Twenty-four
hours later, recipient animals were challenged with 100 ?g OVA323–339
peptide, emulsified 1:1 in 100 ?l CFA s.c. At various time-points following
challenge, the draining LN were harvested, and chemokine receptor expression
on the adoptively transferred CD4?Thy1.1? cells was determined. Bulk
lymphocytes (4?106) from the draining LN of challenged mice were added to
the upper chamber of a 5-?M Transwell plate and were allowed to migrate
toward 10 nM rmCCL21, 10 nM rmCXCL13, or 10 nM rmCCL22 in the lower
chamber at 37°C, 5% CO2, for 3 h. Migrated cells in the lower chamber were
harvested and stained for CD4?Thy1.1? lymphocytes. Migration was quan-
tified using PKH reference beads during flow cytometry analysis.
Ex vivo adhesion assays
WT (1.5?106) or p110??/?CD4?OT.II Thy1.1? lymphocytes were adop-
tively transferred into Thy1.2? recipients. Twenty-four hours later, recipient
animals were challenged with 100 ?g OVA323–339peptide, emulsified 1:1 in
100 ?l CFA s.c. On Day 7 postchallenge, the draining LN were harvested. The
phenotype of the adoptively transferred CD4?Thy1.1? cells was determined
by analyzing CCR4, CCR7, PSGL-1, ?1 integrin, and ?2 integrin expression.
The adoptively transferred donors were purified from the bulk suspension via
negative selection. Total cells (0.8–5?105) were added to each well of a
96-well plate that had been coated previously with 0.6 ?g/ml rmVCAM-1-Fc
or 6 ?g/ml rmICAM-Fc (R&D Systems Inc., Minneapolis, MN, USA) overnight.
The plates were washed twice and coated with 1 ?g/ml rmCCL22 for 1 h at
37°C. The plates were washed and then blocked with 2.5% BSA for 1 h at
37°C. The plates were quick-spun to allow the cells to settle to the bottom of
the wells and were incubated at 37°C for 5 min. The plates were washed, and
the number of adherent CD4?Thy1.1? cells was analyzed by flow cytometry.
Ex vivo F-actin polarization in CD4 effector
WT (2?106) or p110??/?CD4?OT.II Thy1.1? lymphocytes were adoptively
transferred into Thy1.2? recipients. Twenty-four hours later, recipient animals
were challenged with 100 ?g OVA323–339peptide, emulsified 1:1 in 100 ?l
CFA s.c. On Day 7 postchallenge, the draining LN were harvested. CCR7 and
CCR4 expression on the adoptively transferred CD4?Thy1.1? cells was
analyzed as described above. The adoptively transferred Thy1.1? donors were
purified from the bulk suspension via sorting on a FACSAria cell sorter (purity,
?99% Thy1.1?). The purified Thy1.1? cells were then resuspended to a
concentration of 1 ? 106cells/ml in RPMI ? 0.5% BSA and allowed to adhere
to poly-L-lysine-coated slides for 2 h at 37°C. The media were aspirated, and
the cells were then stimulated with media alone, 1 ?g/ml rmCCL21, or 1 ?g/ml
rmCCL22 for 10 min at 37°C. The cells were washed with PBS, permeabilized,
and stained with phalloidin-Alexa 594 (Invitrogen). F-actin polarization (phal-
loidin staining) was analyzed by confocal microscopy on an Olympus FluoView
1000 confocal microscope using Laser Sharp 3.0 software (Bio-Rad, Hercules,
CA, USA). Confocal images were blinded and scored for polarization by three
The presented results are represented as the mean ? SEM. Statistical signifi-
cance between experimental conditions was determined using a Students’s
t-test. P ? 0.05 was considered significant; *, P ? 0.05; **, P ? 0.005.
Phenotypic analysis of naı ¨ve, p110?-deficient
CD4 T lymphocytes
The p110?-deficient mice used in these studies were derived
through the insertion of GFP in-frame in the p110? gene, such
that GFP is expressed in lieu of p110? . Therefore, the
expression of GFP in p110?-deficient cells was monitored in
all of the experiments reported in this study to ensure the
effective loss of p110? expression (Fig. 1A). In addition, the
phenotype of naı ¨ve peripheral lymphocytes isolated from these
p110??/?and control WT mice was analyzed by flow cytom-
etry. As has been previously reported in other strains of p110?-
deficient mice , we did not observe any difference in the
numbers of CD4 T cells in the peripheral LN (data not shown)
or in the phenotype of naı ¨ve, peripheral CD4 T cells as
assessed by CCR7, CD62L, CD44, or TCR expression (Fig. 1A,
and data not shown). Activation of AKT or protein kinase B is
a proximal event downstream of PI-3K activation . There-
fore, the loss of p110? function in these lymphocytes was also
confirmed through the analysis of AKT activation following
chemokine receptor stimulation. AKT phosphorylation, follow-
ing stimulation with the CCR7 ligand CCL21, was dramatically
impaired in naı ¨ve, p110?-deficient T cells (Fig. 1B). Further-
more, we did not observe any deficit in signal transduction
pathways downstream of TCR ligation, as calcium responses
(Fig. 2A) and Erk activation (Fig. 2B) progressed normally
following anti-CD3 stimulation in p110?-deficient lympho-
cytes. These observations further demonstrate that the loss of
the p110? isoform of PI-3K does not affect the phenotype of
naı ¨ve CD4 T cells nor does it impact TCR-dependent signaling
events in vitro.
Antigen-dependent CD4 lymphocyte activation is
unaffected by the loss of p110?
We next examined whether p110? regulated antigen-depen-
dent CD4 T cell activation and function during immune re-
sponses in vivo. To test the ability of p110?-deficient CD4 T
cells to respond to an immunogenic stimulus in vivo, we
generated p110??/?OT.II TCR transgenic mice in which the
CD4 T cells recognize a defined peptide fragment of chicken
816Journal of Leukocyte Biology
Volume 84, September 2008
OVA. CFSE-labeled WT or p110??/?OT.II transgenic lym-
phocytes (1?106per recipient) were adoptively transferred
into normal Thy1.2 congenic recipients and subsequently chal-
lenged i.v. with 10 ?g OVA in conjunction with 25 ?g LPS as
an adjuvant. Peripheral LN were harvested at various time-
points following antigen challenge, and the proliferation (CFSE
dilution) and activation status (CD62L and CD44 expression)
of the adoptively transferred donors were analyzed by flow
cytometry. Interestingly, we discovered that the loss of p110?
does not impair antigen-dependent CD4 T cell activation or
proliferation in vivo (Fig. 3). The p110?-deficient OT.II trans-
genic donors did not exhibit any defects in their ability to
down-regulate L-selectin (CD62L) or up-regulate CD44 ex-
pression throughout the time course of the response (Fig. 3,
A–C). In addition, the p110??/?OT.II donors expanded with
similar kinetics as their WT OT.II counterparts, in which the
response peaked on Day 3 and was resolved on Day 9 following
OVA challenge (Fig. 3D). The difference in the extent to which
the p110??/?donor cells dilute CFSE in comparison with WT
is attributed to the endogenous GFP expression in the
Fig. 1. Phenotypic analysis of naı ¨ve, p110?-deficient CD4 lymphocytes. (A) Naı ¨ve,
p110?-deficient lymphocytes were analyzed for GFP, CCR7, and CD62L expression by
flow cytometry. Shaded histogram indicates isotype control staining, black line indi-
cates WT, and gray line indicates p110??/?CD4 T cells. (B) AKT phosphorylation in
naive T cells following stimulation with 5 or 10 ?g/ml rmCCL21 for 1 min was analyzed
by Western blotting. Densitometric analysis was preformed digitally using the Odyssey
imaging system and is presented as the ratio of p-AKT to total AKT in each sample.
Fig. 2. TCR-mediated activation of p110?-deficient CD4 lymphocytes. (A)
Calcium flux in naı ¨ve, p110?-deficient CD4 T cells following stimulation
with anti-CD3 antibody or ionomycin (iono; as a positive control) was
analyzed by flow cytometry. Black lines represent WT, and gray lines
represent p110??/?CD4 T cells. Individual lines represent analysis of individual samples. Data are representative of at least three independent
experiments in which n ? 3. strep, Streptavidin. (B) Erk phosphorylation in naive T cells following stimulation with 50 ng PMA or 20 ?g/ml anti-CD3
? 2 ?g/ml anti-CD28 antibody for 5 min was analyzed by Western blotting. Densitometric analysis was preformed digitally using the Odyssey imaging
system and is presented as the ratio of p-Erk to total Erk in each sample. Similar results were observed when Erk activation was analyzed by flow cytometry
(data not shown).
Thomas et al.
p110? controls CD4 effector T cell migration817
p110??/?cells (see Fig. 1A). These results demonstrate that
p110? does not regulate the initial events downstream of
antigen-dependent CD4 T cell activation.
Loss of p110? impairs the migration of antigen-
activated CD4 lymphocytes back to sites of
initial antigen encounter
We hypothesized that p110? may specifically regulate the
trafficking of newly activated, effector CD4 T cells into periph-
eral inflammatory sites. To test our hypothesis, it was necessary
to deposit antigen in a site where it could not freely diffuse,
allowing us to accurately track the insinuation of the immune
response in the draining LN as well as the migration of those
newly activated lymphocytes back to the site of antigen depo-
sition. We chose to deposit 10 ?g OVA323–339antigen emul-
sified in IFA in the ear pinna of congenic recipients that
received 1 ? 106CFSE-labeled WT or p110??/?OT.II lym-
phocytes 24 h earlier. It is well established that lymphocytes
differentially regulate the expression of adhesion molecules,
such as L-selectin, PSGL-1, and integrins, to leave secondary
lymphoid tissue and gain access to peripheral inflammatory
sites [31–36]. Therefore, at various time-points following anti-
gen challenge, the draining LN and challenged ear were har-
vested, and the proliferation (CFSE dilution) and phenotype
[CD62L, PSGL-1, and ?4 integrin (CD49d)] of the adoptively
transferred donor cells were analyzed by flow cytometry.
In line with our previous observations, there was no differ-
ence in the activation status or proliferation of p110?
OT.II transgenic donors in the draining LN on Day 7 postan-
tigen challenge, when the response peaks compared with their
WT counterparts (Fig. 4A, and data not shown). Interestingly,
we observed a dramatic reduction in the number of p110??/?
OT.II transgenic donors that had migrated to the inflamed ear
on Day 10 following antigen challenge, the peak of effector
CD4 T cell accumulation in the ear (Fig. 4A). However, the
phenotype of the few p110??/?OT.II donor cells that were
able to migrate into challenged ears was similar to WT OT.II
transgenic donors in that they were CFSEdilute, PSGL-1hi,
CD49dlow, and CD62Llow(Fig. 4B, and data not shown). This
defect in migratory capacity of p110??/?OT.II lymphocytes
could not be attributed to differences in the endogenous in-
flammatory environment in the ears of the recipient mice, as
the number of endogenous CD4 lymphocytes (CD4?Thy1.1–)
isolated from the challenged ears throughout the time course
was equivalent between recipients that had received WT or
p110??/?OT.II donor lymphocytes (data not shown). In addi-
tion, these observations were not dependent on the particular
site of initial antigen challenge, as we also observed a signif-
icant reduction in the ability of p110??/?OT.II donor lym-
phocytes to migrate into the peritoneum on Day 10 following an
i.p. challenge with OVA/IFA (Fig. 4C). These results further
suggest that p110? may specifically regulate the migration of
effector CD4 T cells, rather than the initial events involved in
T cell activation and priming.
The p110? isoform of PI-3K controls chemokine
receptor-mediated migration of effector
For newly activated CD4 T cells to appropriately traffic to
peripheral inflammatory sites, they must be able to respond to
numerous inflammatory signals. First, the antigen-experienced
effector CD4 T cells must be able to appropriately interpret
signals from chemokines and integrin ligands presented on
inflamed endothelial venules that would allow them to adhere
and extravasate into the surrounding tissue. Second, the cell
must be able to migrate along a gradient of inflammatory
chemokines produced in the inflamed tissue site. Therefore, to
better understand the mechanism underlying the observed
defect in migration of p110??/?OT.II effector CD4 T cells
into peripheral inflammatory sites, we chose to further inves-
Fig. 3. Antigen-dependent CD4 T cell ac-
tivation is unaffected by the loss of p110?.
The phenotype and proliferation of WT and
p110??/?OT.II transgenic donor lympho-
cytes on Day 3 (A), Day 6 (B), and Day 9 (C)
following i.v. OVA/LPS challenge were de-
termined by flow cytometry. Light-gray his-
togram indicates isotype control staining,
dark-gray, shaded histogram indicates adop-
tively transferred, unchallenged donor cells,
and black line indicates adoptively trans-
ferred, OVA-challenged CD4?Thy1.1? do-
nor cells. (D) The total number of WT or
p110??/?OT.II donor cells (CD4?Thy1.1?)
in the peripheral LN of recipient mice was
quantified using flow cytometry. Data are rep-
resentative of three independent experiments
in which n ? 3. *P ? 0.05.
818 Journal of Leukocyte Biology
Volume 84, September 2008
tigate the ability of these effectors to respond to inflammatory
chemokines ex vivo.
It has been proposed that the interaction between CCR4?
effector T cells and CCR4 ligands (CCL17 and CCL22) on
inflamed, cutaneous venules signals the activation of ?1 and
?2 integrins on the T cell and is necessary for the migration of
activated lymphocytes into peripheral cutaneous sites [33, 34,
37, 38]. Given that we primarily used s.c. routes of antigen
delivery in our experimental system, we investigated whether
defects in CCR4-induced adhesion and migration contributed
to the migratory defects observed in p110??/?OT.II effector
CD4 T cells in vivo. WT or p110??/?OT.II lymphocytes were
adoptively transferred into WT congenic recipients and chal-
lenged s.c. 24 h later with 100 ?g OVA emulsified in CFA. The
phenotype and responsiveness of the adoptively transferred
WT or p110??/?OT.II donors in the draining LN were ana-
lyzed at various time-points following challenge. We empiri-
cally determined that proliferation and chemokine receptor
expression and responsiveness peaked on Day 7 following s.c.
antigen challenge (data not shown). Therefore, we chose to
focus on this time-point for further analysis. Antigen-chal-
lenged donor CD4 T cells were purified from the draining LN
via positive selection, and their ability to adhere to the ?1
integrin ligand VCAM-1 and the ?2 integrin ligand ICAM-1
following CCL22 (CCR4 ligand) stimulation ex vivo was ana-
lyzed. WT and p110??/?OT.II effector T cells showed en-
hanced expression of the ?1 integrin subunit (CD29), the ?2
integrin subunit (CD11a), and the selectin ligand PSGL-1
when compared with unchallenged, donor T cells (Fig. 5A). In
addition, we did not observe any difference in CCR4 expres-
sion levels between WT and p110??/?OT.II effector T cells
(Fig. 5A). Antigen-experienced effector p110??/?OT.II effec-
tor T cells adhered to ICAM-1 and VCAM-1 comparably with
WT OT.II effectors (Fig. 5, B and C). Although stimulation with
CCL22 slightly enhanced the adhesion of WT and p110??/?
OT.II effectors to ICAM-1, this enhancement was not statisti-
cally significant (Fig. 5B). Stimulation with CCL22 did not
enhance the adhesion of WT or p110??/?OT.II effector T
cells to VCAM-1 (Fig. 5C). These results suggest that defects
in the migration of p110?-/ effector CD4 T cells into inflam-
matory sites are not associated with defects in integrin-depen-
Next, we directly tested the ability of p110??/?OT.II ef-
fectors to migrate in response to CCR4 ligands and other
inflammatory chemokines ex vivo. Antigen-experienced effec-
tor OT.II cells were generated as described above. We did not
detect any differences in the expression levels of the chemo-
kine receptors CCR7, CXCR5, or CCR4 on the antigen-expe-
rienced WT or p110??/?OT.II effector T cells on Day 7
following s.c. OVA challenge (Fig. 6A). Interestingly, how-
ever, we observed a significant reduction in the ability of
effector p110??/?OT.II donor lymphocytes to migrate in
response to the CCR4 ligand CCL22 as well as the CXCR5
ligand CXCL13 in ex vivo transmigration assays (Fig. 6B). We
also observed a reduction in the responsiveness of these
p110??/?effector CD4 lymphocytes to stimulation with the
CCR1 and CCR5 ligand CCL3, but this defect was not statis-
tically significant (data not shown). These results suggest that
an impaired migratory responsiveness to “inflammatory” che-
mokines may be responsible for the reduced migration of
p110?-deficient effector CD4 T cells into peripheral inflam-
matory sites in vivo.
Fig. 4. p110? controls the migration of effector CD4 lymphocytes into
peripheral inflammatory sites. (A) Proliferation of WT and p110??/?OT.II
(CD4?Thy1.1?) adoptively transferred donors in the draining LN and accu-
mulation of these donor lymphocytes in the ear at various time-points follow-
ing OVA challenge in the ear were quantified by flow cytometry. Data are
representative of three independent experiments in which n ? 3. *, P ? 0.05. (B) Phenotype of adoptively transferred WT and p110??/?OT.II donors in
challenged ears on Day 10. Light-gray histogram indicates isotype control staining, dark-gray, shaded histogram indicates expression levels on untransferred,
unchallenged donors for reference, and black line indicates adoptively transferred, OVA-challenged CD4?Thy1.1? cells. CFSE dilution is representative of
three independent experiments in which n ? 3, and phenotypic analysis of PSGL-1 and CD49d expression represent a single, independent experiment in which
n ? 4. (C) Proliferation of WT and p110??/?OT.II (CD4?Thy1.1?) adoptively transferred donors in the draining LN and accumulation of these donor
lymphocytes in the peritoneum at various time-points following i.p. OVA challenge were quantified by flow cytometry. Data are representative of at least four
independent experiments in which n ? 3. *, P ? 0.05.
Thomas et al.
p110? controls CD4 effector T cell migration819
The p110? isoform of PI-3K regulates F-actin
polarization downstream of inflammatory
Cells migrating along a chemotactic gradient must be able to
appropriately polarize their cytoskeletal machinery toward the
leading edge of the cell [39, 40]. Accumulation of the cytoskel-
etal protein F-actin at the leading edge of the cell is one
hallmark of cell polarization [39–41]. As the p110? isoform of
PI-3K has been shown to regulate F-actin polarization in
neutrophils , we next investigated whether these p110?-
deficient effectors would also display defects in their ability to
polarize F-actin effectively following CCR4 stimulation. Anti-
gen-experienced effector cells were generated in vivo as de-
scribed above. On Day 7 postantigen challenge, WT and
p110??/?OT.II effector CD4?Thy1.1? donors were purified
from the draining LN by FACS. These purified effectors
(?99% Thy1.1?) were analyzed for chemokine receptor ex-
pression and their ability to polarize F-actin following stimu-
lation with the CCR7 ligand CCL21 or the CCR4 ligand
CCL22. We observed a significant defect in the ability of
effector p110??/?OT.II cells to polarize F-actin following
CCR4 stimulation (Fig. 7, A and B). This observed defect is
Fig. 5. Adhesion of antigen-activated CD4 lymphocytes is not regulated by p110?. (A) The
phenotype of antigen-activated WT and p110??/?OT.II (CD4?Thy1.1?) donor lymphocytes was
analyzed by flow cytometry. Light-gray histogram indicates isotype control staining, dark-gray,
shaded histogram indicates adoptively transferred, unchallenged donors, and black line indicates
adoptively transferred, OVA-challenged donors. Data are representative of at least four independent
experiments for CCR4, CD29, and PSGL-1 expression and of a single independent experiment for
CD11a expression. (B) The adhesion of adoptively transferred, OVA-challenged WT and p110??/?
OT.II donor cells isolated from the draining LN of recipient mice 7 days post-s.c. OVA challenge to
6 ?g/ml-immobilized rmICAM-1 (unstimulated) in the presence of 50 ng/ml PMA (?PMA) or 1 ?g/ml rmCCL22 (?CCL22) was analyzed. Data represent the
average percent cell adhesion in a single independent experiment in which n ? 5. (C) The adhesion of adoptively transferred, OVA-challenged WT and
p110??/?OT.II donor cells isolated from the draining LN of recipient mice 7 days post-s.c. OVA challenge to 0.6 ?g/ml-immobilized rmVCAM-1
(unstimulated) in the presence or absence of rmCCL22 (?CCL22) was analyzed. The average background level of adhesion (adhesion to BSA alone) in the
adhesion assays depicted in B and C was ?5%. Data represent the average percent cell adhesion pooled between two independent experiments in which n ? 3.
Fig. 6. p110? controls chemokine-mediated migration of effector CD4 lymphocytes. (A) The phenotype of antigen-activated WT and p110??/?OT.II
(CD4?Thy1.1?) donor lymphocytes isolated from the draining LN of recipient mice 7 days post-s.c. OVA challenge was analyzed by flow cytometry. Light-gray
histogram indicates isotype-control staining, dark-gray, shaded histogram indicates adoptively transferred, unchallenged donors, and black line indicates adoptively
transferred, OVA-challenged donors. Data are representative of at least four independent experiments. (B) The migration of adoptively transferred, OVA-challenged
WT and p110??/?OT.II donor T cells isolated from the draining LN of recipient mice on Day 7 post-OVA challenge to 10 nM rmCCL21 (CCR7 ligand), CCL22
(CCR4 ligand), or CXCL13 (CXCL13 ligand) was determined using ex vivo transmigration assays and analyzed by flow cytometry. Data from four independent
experiments, in which n ? 2, were pooled together. **, P ? 0.005.
820 Journal of Leukocyte Biology
Volume 84, September 2008
not attributable to differences in CCR4 expression levels between
WT and p110??/?effectors (Fig. 6A). However, p110??/?OT.II
effectors did not display any defect in their ability to polarize
F-actin in response to stimulation with the CCR7 ligand CCL21
(Fig. 7, A and B). These results demonstrate that the p110?
isoform of PI-3K regulates F-actin polarization downstream of
inflammatory chemokine receptors in effector CD4 T cells.
In these studies, we demonstrate that p110??/?CD4 T cells do
not exhibit defects in their ability to respond to TCR stimulation
in vitro. p110?-deficient CD4 T cells were able to mobilize
calcium and activate Erk equivalently following TCR stimulation.
Although p110??/?T cells have been reported to have defects in
proliferation in response to anti-CD3 antibody stimulation in vitro
, we did not observe any defects in the activation phenotype or
proliferative capacity of p110??/?OT.II transgenic T cells fol-
lowing antigen challenge in vivo. In addition, the kinetics of the
response and phenotype of antigen-experienced p110??/?OT.II
lymphocytes following systemic antigen challenge were similar to
their WT counterparts. This further suggests that the newly acti-
vated, p110?-deficient CD4 lymphocytes do not have any reduc-
tion in their capacity to respond to antigenic signals in vivo. These
results support other studies suggesting that p110?, not p110?, is
TCR in T cells [12–14, 42]. One recent study reported a defect in
anti-CD3-induced proliferation and ERK activation in vitro in
p110?-deficient T cells that could not be rescued with exogenous
CD28 or IL-2 stimulation . The basis for the discrepancy
between this recent report and our own studies, as well as others
[7, 12–15, 42], is currently unclear. However, despite this
discrepancy in T cell responses in vitro, our studies have
shown that p110? does not play a major role in the antigen-
dependent activation and clonal expansion of CD4 or CD8
naı ¨ve T cells in vivo .
In the current paradigm of T cell activation and effector differ-
entiation, CD4 T cells increase CXCR5 expression early after
initial activation, allowing a subset of these T cells to migrate to
the B cell follicle to lend cytokine help to antigen-specific B cells
. Another subset of newly activated T cells increases their
expression of inflammatory chemokine receptors (e.g., CXCR3,
CCR4, and CCR5) and commiserates with the site and nature of
initial antigen encounter by the APC [28, 44–46]. To gain access
into inflamed peripheral tissue sites, activated T cells rely on
complex interactions among selectin ligands, integrins, and che-
mokine receptors with selectins, chemokines, and integrin ligands
expressed on the inflamed vascular endothelium. Skin-homing
effector CD4 T cells preferentially increase their expression of
CCR4 and PSGL-1 [33, 37, 38]. It has been suggested previously
that the interaction between PSGL-1 on the activated T cell and
P-selectin on the inflamed endothelium is necessary for the rolling
of effector T cells on inflamed cutaneous venules. This rolling
allows CCR4?effector T cells to bind CCR4 ligands (CCL22,
CCL17) expressed on the endothelial surface, leading to the
enhancement of ?1 and ?2 integrin-mediated adhesion to the
inflamed cutaneous venule. This allows the cells to subsequently
extravasate into the surrounding tissue, perform their effector
function, and help resolve the inflammatory response [34, 36].
Our studies have shown that p110? is not required for the
activation-dependent changes in expression of chemokine re-
ceptors (CCR7, CXCR5, CCR4), integrins (?1 and ?2), and
PSGL-1 following s.c. antigen challenge in vivo. It is also
important to note that changes in L-selectin expression follow-
ing antigen challenge in vivo were not altered by the loss of
p110?, as recent work has revealed a role for p110? in con-
trolling L-selectin and CCR7 expression following TCR stim-
ulation . This provides further support for our hypothesis
that p110? does not participate in the initial priming or dif-
ferentiation events necessary for effective antigen-dependent
CD4 T cell activation during immune responses in vivo.
Although the initial phase of T cell activation was unaffected,
there was a significant reduction in the number of antigen-expe-
rienced p110??/?OT.II donor T cells in the inflamed ear. This
defect in CD4 effector T cell migration is not site-specific, as we
also observed a significant reduction in the ability of antigen-
activated, p110??/?deficient effectors to traffic into an inflamed
peritoneum following an i.p. antigen challenge. Adhesion of ef-
fector CD4 T cells to the inflamed vascular endothelium is an
important step in the migration of activated T cells into peripheral
tissues sites. We observed that expression of the ?1 and ?2
integrin subunits increased on WT and p110?-deficient CD4
effector T cells on Day 7 following s.c. antigen challenge. How-
ever, p110?-deficient CD4 effector T cells were not impaired in
their ability to bind to VCAM-1 or ICAM-1 in ex vivo adhesion
Fig. 7. p110? PI-3K regulates CCR4-dependent F-actin polymerization. (A) Con-
focal images of purified WT (upper panel) or p110??/?(lower panel) effector CD4
T cells stimulated with media alone, 1 ?g/ml rmCCL21, or 1 ?g/ml rmCCL22 for 20
min and then stained with phalloidin-Alexa 594. Images are representative of more than 45 images analyzed per treatment group in two independent
experiments. (B) Blinded analysis of F-actin polarization in confocal images in A. Over 45 images per treatment group per experiment were blinded and scored
for polarization by three independent investigators. The results from the blinded analysis were pooled and graphed as percent polarization ? SEM. **, P ? 0.005.
Thomas et al.
p110? controls CD4 effector T cell migration821
assays. In vitro studies using naı ¨ve p110?- or p110?-deficient
leukocytes have also not implicated these isoforms of PI-3K in
regulating adhesion to integrin ligands [7, 14]. In addition, naı ¨ve
p65PI-3Ktransgenic p110??/?T cells, which express a constitu-
tively active form of Class 1A PI-3K and display an activated
phenotype, do not rely on p110? expression to migrate into
noninflamed peripheral tissues . Therefore, it is perhaps not
surprising that effector p110??/?OT.II lymphocytes did not
exhibit defects in their basal adhesion to VCAM-1 or ICAM-1 ex
vivo. We also did not observe changes in adhesion of these
antigen-experienced effector CD4 T cells to VAM-1 with the
addition of CCL22. However, we did observe a small increase in
the adhesion of effector T cells to ICAM-1 in the presence of
CCL22. The concentration of immobilized integrin ligand used in
on chemokine-mediated, inside-out integrin activation in these
effector T cells. In addition, activated effector CD4 T cells may
exhibit a higher basal state of integrin functional activity that is
not appreciably augmented by chemokine stimulation. It is also
possible that the other CCR4 ligand CCL17 may be a more potent
agonist for adhesion than CCL22.
Although adhesion of CD4 effector T cells to integrin ligands
was not affected by the loss of p110?, we observed significant
defects in the migration of antigen-experienced p110??/?
OT.II T cells to the CCR4 ligand CCL22. In addition, we have
demonstrated that p110? PI-3K regulates F-actin polarization
downstream of CCR4 stimulation. These results suggest that
p110? is critical for generating a polarized cell morphology
critical for effector T cell migration in response to chemokines.
Importantly, the loss of p110? in effector CD4 T cells did not
impact CCR7-dependent, F-actin polarization or migration,
demonstrating that these observed defects are a result of the
loss of p110? PI-3K and not a result of a global, functional
defect in p110?-deficient cells. We have also demonstrated
that p110??/?effector T cells exhibit severe impairments in
their ability to access the inflamed ear as well as the inflamed
peritoneum in vivo. These observations suggest that p110?
may not be acting solely downstream of CCR4, as CCR9-
dependent interactions with its ligand CCL25 are thought to
control effector T cell homing to intestinal sites [31, 32].
Moreover, we also observed a significant reduction in the
ability of p110??/?CD4 effector T cells to migrate in response
to the CXCR5 ligand CXCL13 and the CCR1/CCR5 ligand
CCL3 (data not shown). The observed defects in CXCR5-
dependent migration in p110?-deficient effectors suggest that
these effectors may be limited in their ability to migrate toward
the B cell follicle and may provide further insights into the
defects previously reported in T-dependent antibody produc-
tion in p110??/?mice . Therefore, we propose that p110?
is likely functioning downstream of multiple inflammatory che-
mokine receptors that are up-regulated following antigen-de-
pendent CD4 T cell activation and functions to fine-tune the
migratory capacity of effector CD4 T cells.
The findings presented here demonstrate that p110? PI-3K
regulates chemokine-dependent migration of antigen-experienced
effector CD4 T cells into peripheral inflammatory sites via regu-
lation of F-actin polarization downstream of inflammatory chemo-
kine receptors. In addition, these results provide further insights
into the previously reported deficiencies in T-dependent antibody
production, as well as the reduced ability to mount DTH reactions
p110? to specifically regulate effector CD4 T cell migration,
without impacting initial T cell activation or priming, makes it an
attractive target for therapeutic intervention in multiple allergic
and autoimmune diseases [2, 4, 20, 24, 25]. Careful manipulation
of p110? activity may allow for the inhibition of migration of
effector T cells into inflammatory sites, which could ameliorate
disease pathogenesis without drastically immunocompromising
This study was supported by National Institutes of Health
(NIH) grant AI064271 and the Harry Kay Chair in Biomedical
Research at the University of Minnesota (Y. S.), NIH Training
Grants T32DE007288 (M. S. T.), T32CA009138 (J. S. M.), and
T32AI07313 (C. C. D.), and an American Heart Association
predoctoral fellowship (A. L. M.). We thank S. Highfill, M.
Schwartz, and R. Srivastava for their valuable technical assistance
and the assistance of the Flow Cytometry Core of the Masonic
Cancer Center at the University of Minnesota, a comprehensive
cancer center designated by the NCI (supported in part by NIH
grant P30 CA77598). M. S. T. developed, performed, and ana-
lyzed the majority of the experiments presented in this manuscript
and wrote the manuscript. C. C. D. performed and analyzed ex
vivo adhesion assay experiments. J. S. M. performed the confocal
analysis of F-actin polarization in p110?-deficient effector lym-
phocytes. A. L. M. performed the CCR7 phenotyping of naı ¨ve
the experiments, analyzed and interpreted data, and edited the
manuscript. The authors have no competing financial interests to
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Thomas et al.
p110? controls CD4 effector T cell migration823