Opposing roles for RhoH GTPase during T-cell
migration and activation
Christina M. Bakera,1, William A. Comriea,1,2, Young-Min Hyuna, Hung-Li Chungb, Christine A. Fedorchuka, Kihong Lima,
Cord Brakebuschc, James L. McGrathb, Richard E. Waughb, Martin Meier-Schellersheimd, and Minsoo Kima,3
aDepartment of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, andbDepartment of Biomedical Engineering,
University of Rochester, Rochester, NY 14642;cDepartment of Molecular Pathology, University of Copenhagen, 2200 Copenhagen N, Denmark; and
dLaboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Timothy A. Springer, Immune Disease Institute, Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, MA, and approved
May 16, 2012 (received for review August 29, 2011)
T cells spend the majority of their time perusing lymphoid organs
in search of cognate antigen presented by antigen presenting cells
(APCs) and then quickly recirculate through the bloodstream to
another lymph node. Therefore, regulation of a T-cell response is
dependent upon the ability of cells to arrive in the correct location
following chemokine gradients (“go” signal) as well as to receive
appropriate T-cell receptor (TCR) activation signals upon cognate
antigen recognition (“stop” signal). However, the mechanisms by
which T cells regulate these go and stop signals remain unclear.
We found that overexpression of the hematopoietic-specific RhoH
protein in the presence of chemokine signals resulted in decreased
Rap1–GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell
chemotaxis; while in the presence of TCR signals, there were en-
hanced and sustained Rap1–GTP and LFA-1 activation as well as
prolonged T:APC conjugates. RT-PCR analyses of activated CD4+T
cells and live images of T-cell migration and immunological syn-
apse (IS) formation revealed that functions of RhoH took place
primarily at the levels of transcription and intracellular distribu-
tion. Thus, we conclude that RhoH expression provides a key mo-
lecular determinant that allows T cells to switch between sensing
chemokine-mediated go signals and TCR-dependent stop signals.
mokine signals as well as to form stable and prolonged T:antigen
presenting cell (APC) contacts to receive appropriate activation
signals upon cognate antigen recognition (1). Integrin-mediated
adhesion is needed for extravasation through high endothelial
venules (HEVs) and intranodal migration during which APC
scanning occurs (2). The fibroblastic reticular cell network serves
as a matrix upon which T cells migrate and expresses ICAM-1 as
well as CCL21, CCL19, and CXCL12 (3). Adhesive force gen-
erated by LFA-1 ligation is also essential for the maintenance of
the immunological synapse (IS) and the signal integration nec-
essary for complete T-cell activation (4).
Although enhanced LFA-1 adhesiveness is equally important
for both migration and T-cell activation, the biological outcomes
of chemokine and T-cell-receptor (TCR)–mediated LFA-1 ac-
tivation in T cells are quite different. Intracellular cues in re-
sponse to cognate interactions lead to cells deciding to stop
despite continued chemokine-driven migratory signals. Con-
versely, following a period of stable contact coincident with
T-cell activation, cells again become responsive to chemokine-
induced migratory signals and ignore the continued presence of
ligands for the TCR. Rap1, a member of the Ras family of small
GTPases, modulates the affinity and avidity of LFA-1 during
TCR and chemokine triggered LFA-1 adhesion to ICAM-1 (5).
Calcium and diacylglycerol-regulated guanine nucleotide ex-
change factor I (CalDAG–GEFI) and C3G are key GEFs that
activate Rap1 (6) and regulator of adhesion and polarization
enriched in lymphocytes (RAPL), through its association with
Rap1, is a crucial effector molecule involved in Rap1-mediated
LFA-1 activation during TCR and chemokine-triggered LFA-1
adhesion to ICAM-1 (7). Several negative regulators of LFA-1
egulation of a T-cell response is dependent upon the ability
of cells to migrate within a lymphoid organ following che-
activation have been described, including RhoH, a member of the
Rho family of small GTPases (8). Overexpression of RhoH is
inhibitory to chemokine-induced migration, whereas RhoH is in-
volved in proximal TCR signaling (9–13).
In this study, we have addressed a longstanding question of how
T cells differentially regulate LFA-1 adhesiveness during chemo-
kine-mediated active migration (go) and TCR-mediated stable IS
formation (stop). Here, we provide evidence that RhoH performs
distinct functions by inhibiting chemokine-mediated migration
and enhancing TCR-induced adhesiveness, suggesting that RhoH
may serve as a rheostat that modulates the CD4 T-cell response.
Opposing Roles for RhoH in Chemokine-Receptor and TCR-Induced
Rap1 Activation. Chemokine and TCR signals induce the open
conformation of LFA-1, via inside-out signaling (2). A significant
body of primary literature supports the presence of common
intermediates between the inside-out signaling pathways down-
stream of chemokine and TCR-induced activation of LFA-1,
including CAS and Rap1 activation (Fig. S1). As CAS is reported
to impact Rap1 activation by modulation of C3G activity, we
focused on Rap1 activation in this study as a major point of
overlap between TCR and chemokine-receptor–induced LFA-
The conversion of LFA-1 from a bent to open conformation
is mediated by Rap1 and its GEFs. It has been speculated that
RhoH may interfere with Rap1 activation making this a prob-
able target for modulating both chemokine and TCR-induced
activation of LFA-1 (8). Importantly, RhoH, like Cbl-b, has a
negative impact on LFA-1–mediated adhesion (2). RhoH also
inhibits several effector functions of Rho family small GTPases,
such as NF-kB activation induced by Rac1 and cdc42 and RhoA
and p38MAPK activation by Rac1 and cdc42 (14), yet these
would not directly impact inside-out signaling to induce LFA-1
activation. Therefore, these observations had led us to hypoth-
esize that RhoH negatively regulates Rap1 activation, thus pre-
venting LFA-1 adhesion in T cells. To investigate the effects of
RhoH on Rap1 activation in T cells, we assessed the stimulation-
Author contributions: C.M.B., W.A.C., Y.-M.H., and M.K. designed research; C.M.B., W.A.C.,
Y.-M.H., C.F., K.L., and M.K. performed research; C.B., J.L.M., R.E.W., and M.M.-S. contrib-
uted new reagents/analytic tools; C.M.B., W.A.C., Y.-M.H., H.-L.C., C.F., M.M.-S., and M.K.
analyzed data; and C.M.B. and M.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1C.M.B. and W.A.C. contributed equally to this work.
2Present address: Department of Pathology and Laboratory Medicine, Children’s Hospital
of Philadelphia and Perelman School of Medicine at the Unniversity of Pennsylvania,
Philadelphia PA, 19104.
3To whom correspondence should be addressed. E-mail: Minsoo_Kim@urmc.rochester.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 26, 2012
| vol. 109
| no. 26www.pnas.org/cgi/doi/10.1073/pnas.1114214109
dependent activation of Rap1 in control and cells overexpressing
RhoH. To overexpress RhoH, we generated a fusion protein in
which monomeric red fluorescence protein (mRFP) was fused to
the C terminus of RhoH (RhoH–mRFP). The Jurkat cell line
was transiently transfected with control vector (mRFP) or
RhoH–mRFP, then stimulated with chemokine CXCL12 (Fig.
1A) or TCR cross-linking (Fig. 1B) for the indicated times. Ac-
tivation of Rap1 was rapid and transient upon chemokine-re-
ceptor or TCR stimulation, as seen in mRFP only expressing
cells (Fig. 1 A and B). CXCL12-induced Rap1 activation was
greatly reduced in cells that overexpressed RhoH (Fig. 1A). In
stark contrast, overexpression of RhoH resulted in enhanced and
prolonged activation of Rap1 upon TCR stimulation (Fig. 1B).
Transfection efficiency was verified by quantitation of tranfection
frequency and mRFP Western blots (Fig. 1A and Fig. S2) and
overexpression of RhoH–mRFP did not alter the surface ex-
pression of CXCR4 or TCR (Fig. S3). Furthermore, RhoH−/−T
cells revealed significantly greater constitutively active Rap1 than
WT cells (Fig. 1C). Previously RhoH−/−hematopoeitic stem cells
were also reported to have more active Rac1–GTP present in
basal state (15). Unlike in WT cells, TCR stimulation of RhoH−/−
T cells failed to increase, but slightly reduced Rap1 activation
(Fig. 1C). However, incubation of RhoH−/−T cells with CCL21
significantly enhanced Rap1 activation compared to TCR cross-
linking (Fig. 1C). The altered pattern of Rap1 activation in the
absence or overexpression of RhoH may not be explained by
changes in protein expression of C3G or CalDAG–GEFI (Fig.
S4). Our data suggest another small GTPase effected by changes
in RhoH expression levels.
Opposing Roles for RhoH in Chemokine-Receptor and TCR-Induced
LFA-1 Activation. RhoH expression suppresses chemokine-me-
diated Rap1 activation, whereas it enhances Rap1 activation
induced by TCR ligation, suggesting that RhoH overexpression
may play a disparate role in LFA-1 adhesion induced by these
signals. To assess the LFA-1 adhesiveness of RhoH-over-
expressing cells stimulated under each condition, we generated
stable Jurkat cell lines expressing control (mRFP) or RhoH
(RhoH–mRFP) vector (Fig. 2A). T-cell binding on ICAM-1–
coated plates revealed that overexpression of RhoH reduced the
basal adhesion of cells greater than fourfold as seen by a de-
crease in frequency of cells bound to ICAM-1 in the absence of
stimulation (Fig. 2 B and C), confirming previous findings (8).
Consistent with the results of the Rap1 assay, RhoH-over-
expressing cells stimulated with CXCL12 failed to increase cell
binding on ICAM-1, whereas TCR cross-linking successfully in-
creased the number of cells bound to ICAM-1 (Fig. 2 B and C).
Although the magnitude of cells adhering to ICAM-1 was similar
between control and RhoH-overexpressing cells in the presence
of TCR cross-linking, the increase in adhesion in the presence of
stimulation was four- to fivefold greater in RhoH-overexpressing
cells (Fig. 2C). These results were further corroborated using
sorted transiently transfected primary human T cells expressing
control or RhoH–mRFP (Fig. 2 D–F). The effects of differential
expression of RhoH on chemokine signals in T cells were not
related to different onset of activation as seen in Fig. 2D. Finally,
to assess whether this effect is due to altered Rap1 activation,
and positively regulates TCR-mediated Rap1 activation. (A–C) Jurkat cells
were transiently transfected with mRFP or RhoH–mRFP, then at various time
after stimulation with CXCL12 or CD3 cross-linking, cell lysates were assessed
for active Rap1, total Rap-1, and b-actin. Representative blots of Rap1 acti-
vation by CXCL12 (A) or CD3 cross-linking (B) are shown and bar graphs of
each pair of blots consist of compiled densitometry analysis (Right). (C)
Freshly isolated CD4+T cells from WT and RhoH−/−mice were unstimulated
(NS) or stimulated with CD3 cross-linking or CCL21 and Rap1–GTP assessed.
Data are means ± SEM, n = 3, and statistically significant, *P < 0.05 vs. WT
NS, statistically significant,#P < 0.03 vs. RhoH−/−CD3.
RhoH negatively regulates chemokine-mediated Rap1 activationFig. 2.
positively regulates TCR-mediated LFA-1 adhesion. (A–C) Stable Jurkat cell
lines expressing pcDNA, mRFP, or RhoH–mRFP were generated and verified
by anti-mRFP immunoblot. Cells were stimulated with CXCL12 (B) or OKT3
and secondary antibody (C) to induce TCR cross-linking, then assessed in an
ICAM-1 adhesion assay. (D–G) Primary human lymphocytes transiently
transfected with GFP or RhoH–mRFP were sorted. ICAM-1 adhesion of
CXCL12 stimulation (D) or TCR stimulation (E) over time, CXCL12 titration (F)
or ICAM-1 adhesion of CXCL12 stimulation of control, RhoH overexpressing,
or double positive RhoH–mRFP and Rap1V12–GFP (G). Data are means ±
SEM. For B, n = 4 and P < 0.02 and C–G n = 3 independent donors.
RhoH negatively regulates chemokine-mediated LFA-1 adhesion and
Baker et al.PNAS
| June 26, 2012
| vol. 109
| no. 26
RhoH–mRFP+T cells were cotransfected with constitutively
active Rap1. Chemokine-induced T-cell adhesion on ICAM-1
substrate was recovered by the expression of constitutively active
Rap1 (Fig. 2G), suggesting that the effect of RhoH on T-cell
adhesion is a result of impaired Rap1 activation.
Conjugation of RhoH-Overexpressing T Cells. Diminished Rap1 ac-
tivation and LFA-1 adhesiveness in the presence of RhoH
overexpression would impact chemokine-induced migration. Cell
migration plots are shown for CXCL12-induced migration of
GFP or RhoH–mRFP expressing T cells (Fig. 3A and Movie S1).
Quantitative analysis of the velocities of cell migration revealed
that overexpression of RhoH reduces the speed of migration
in response to chemokine stimulation (Fig. 3B). RhoH-over-
expressing cells displayed a polarized morphology with relatively
short tails compared with control cells (Fig. S5). Recent evidence
suggests that activated Rap1 recruits the Par polarity complex to
regulate actin remodeling required for T-cell polarization (16).
Therefore, it is possible that the establishment of polarity is
inhibited or enhanced depending upon the levels of Rap1 acti-
vation and hence RhoH expression.
RhoH overexpression enhanced Rap1 activation and LFA-1
adhesiveness in the presence of TCR signals (Figs. 1B and 2 C
and E), suggesting that RhoH-overexpressing T cells may pro-
duce more stable T:APC conjugates. A cell:cell conjugation as-
say revealed that RhoH overexpression leads to an increased
frequency of conjugates over time (Fig. 3C). This effect is Ag
dose sensitive (Fig. 3 C and D). Furthermore, RhoH over-
expression resulted in enhanced dilution of PKH compared to
control T cells (Fig. S6). The result suggests that RhoH-over-
expressing T cells form stable T-cell adhesion to ICAM-1 in the
presence of TCR cross-linking at the earlier time points (Fig. 2E)
and maintain prolonged conjugate formation over time through
active LFA-1 (Fig. 3C), enabling enhanced signal integration for
Distinct RhoH Distribution Patterns During Chemokine-Mediated
T-Cell Migration vs. T:APC Conjugate Formation. RhoH lacks in-
trinsic GTPase activity and has specific amino acid substitutions
that lead to RhoH remaining GTP bound, thus the activity of
RhoH is not regulated by the typical GTP/GDP exchange cycles
(14). Rapid alteration in RhoH localization may serve as an ef-
ficient mechanism to enable RhoH to act in opposing ways to
signals rapidly being sensed by a T cell. A Rap1–Raichu fluo-
rescence resonance energy transfer (FRET) sensor revealed that
rapid Rap1 activation was selectively localized at the leading
edge of migrating T cells on CXCL12/ICAM-1–coated substrate
(Fig. 4A and Movie S2) and minimal FRET signal is detected in
cells on ICAM-1–only coated substrate (Fig. S7 and Movie S3).
Due to limitations in the FRET system, we were able to image
the global distribution pattern of Rap1 activation but not to the
positively regulates sustained T:APC conjugates. (A) Human T-cell blasts were
transiently transfected with control vector (GFP) or RhoH–mRFP vector. Cell
migration on CXCL12/ICAM-1 was analyzed; corresponding cell tracking
plots are shown (A, Movie S1). Images include fluorescent signal to indicate
transfected cells overlaid with migration tracks as indicated by colored lines.
(B) Velocity (millimeters per minute) of cell migration induced by CXCL12 or
CCL21 on ICAM-1 was analyzed. Data are combined from three independent
donors and are statistically significant P < 0.001 by Mann–Whitney test. (C
and D) DO11.10 TCR transgenic T-cell blasts were transiently transfected
with control vector or RhoH–mRFP and assessed in a flow cytometry-based
conjugate assay. (C) Representative plot of conjugate formation over time at
one antigen dose. (D) Conjugates at 6 h with NoAg or two doses of Ag. Data
are means ± SEM, n = 3 and statistically significant P < 0.05.
RhoH negatively regulates chemokine-induced T-cell migration and
tion and localized toward the IS upon TCR activation. (A) Human T-cell blasts
were transiently transfected with Raichu–Rap1 FRET sensor, then allowed to
migrate on ICAM-1/CXCL12-coated coverslips. CFP and YFP are shown in gray
and intensity is pseudocolored below. FRET efficiency is shown in rainbow
colors, highest (red) to lowest (blue). (Right) Dynamic Rap1 FRET at select
time points; white arrows indicate leading edge of cell (A, Movie S2). (Lower
Left) Kymograph of the area selected by the white rectangle over time. (B)
Localization of RhoH–mRFP in transiently transfected human T-cell blast
migrating in response to CXCL12 is shown every minute. The distribution of
fluorescence intensity is shown in a pseudocolor scale, from low (black) to
high (red). (Lower panels, Movie S4). (C) Representative AL-57 (activation-
dependent LFA-1 mAb) staining (green) of RhoH–mRFP (red) transfected
cells. (D and E) Cell conjugates between transiently transfected Jurkat cells
and SEE-loaded Raji cells were tracked by contacts between APC and mRFP+
cells and then the localization of mRFP assessed, four representative images
each. Localization of mRFP was quantified by the frequency of conjugates
displaying a diffuse localization pattern of mRFP versus mRFP recruited to-
ward the APC (E). (F) Total LFA-1 (TS2/4, red) versus active LFA-1 (AL57,
green) in a representative T:APC conjugate reveals that active LFA-1 is also
found at the T:APC interface.
Rap1 and RhoH are segregated during chemokine-induced migra-
| www.pnas.org/cgi/doi/10.1073/pnas.1114214109Baker et al.
fine subcellular patterning of membrane proximity. In contrast,
the RhoH–mRFP signal was excluded from the leading edge of
the cell where active LFA-1 was detected, with the majority of
RhoH–mRFP localized to the uropod (Fig. 4 B and C and Movie
S4). Upon TCR activation, LFA-1, Rap1, and its interacting
partners are recruited to the IS (7) (Fig. 4F). To assess RhoH
localization during IS formation, conjugates were formed with
transiently transfected Jurkat cells. Unlike in migrating T cells,
RhoH–mRFP was localized toward the T:APC interface where
LFA-1 activation was also detected (9) (Fig. 4 D–F).
Enhanced Migration of RhoH-Deficient T Cells. To assess whether
there is a corresponding increase in migration in the absence of
RhoH, in vitro migration assays on ICAM-1– and CCL21-coated
surface were performed with sorted naïve T cells from WT or
RhoH−/−cells (Fig. 5 A and B and Fig. S8). A significant increase
in migration velocity and meandering index was detected among
RhoH−/−cells (Fig. 5 A and B and Movies S5 and S6). The
changes in migration do not track with altered levels of chemo-
kine receptors or integrins (Fig. S8). Thus, the absence of RhoH
leads to faster migration and less wandering of the cells.
Competitive homing assays were done to assess whether this
translates into altered homing in vivo. The ratio of RhoH−/−: WT
cells from various organs were assessed 1 d posttransfer (Fig.
S9). RhoH−/−cells have altered homing among lymphoid tissues,
which may be due to faster transit times such that RhoH−/−cells
enter and depart lymph nodes more quickly. To assess the rate at
which cells enter a lymph node, we used FTY720 as a blockade
for cell egress to detect the accumulation of cells in lymph nodes.
The fold change in the homing index was greater in the presence
of FTY720 consistent with RhoH−/−T cells entering in larger
numbers than WT during this time frame (Fig. 5C). As retention
signals by CCR7 versus S1P sensing determine the homeostatic
retention time of T cells within the lymph node (17), we also
assessed the importance of RhoH expression for egress from
lymph nodes. There were no differences in cell egress between
WT and RhoH−/−mice (Fig. 5D) at a 20-h time point, although
this visualizes the sum of cell departure within the time frame
rather than the kinetics by which those cells departed. Overall,
these data suggest that RhoH expression effects chemokine re-
sponsiveness for entry or movement within lymph node rather
Altered RhoH Expression During T-Cell Activation. T-cell motility to
distinct locations within the lymph node at various times sub-
sequent to antigen exposure are necessary for effector functions
to be performed, including help for CD8 and B-cell responses, as
well as for efficient egress. Although maintaining RhoH ex-
pression is necessary for stable T:APC conjugation and proximal
TCR signaling, at the same time, our data showed that high
RhoH expression level inhibits chemokine-induced T-cell mi-
gration. Therefore, spatial regulation of RhoH may enable initial
T-cell activation, yet RhoH function must then be limited to
allow T cells to leave APC and migrate following chemokine
signals. To assess the expression levels of RhoH during T-cell
activation, the levels of mRNA were measured (14). RhoH
mRNA levels were rapidly reduced following TCR triggering and
before the first cell division (Fig. 6 A and C), whereas IL-2, an
indicator of activation, was up-regulated (Fig. 6 B and D). In
addition, there was a further significant reduction in RhoH
mRNA as cells went through later divisions (Fig. 6A). Also, T-
cell activation induces degradation of RhoH protein (18). Lim-
ited synthesis of more protein alongside degradation of the
existing protein would combine to result in an overall reduction
in RhoH expression. This may serve as a mechanism to turn off
activation and enable cells to sense and respond to the chemo-
kine milieu to adopt a migratory phenotype once more.
The crucial role of Rap1 in LFA-1 activation during T-cell mi-
gration and priming is well established (2, 5, 19). It was, however,
unexpected to find disparate downstream effects of RhoH
overexpression on chemokine and TCR-mediated Rap1 activa-
tion. In this study, we have demonstrated that RhoH impacts the
outcome of signals received by a T cell as a negative regulator of
have altered homing to lymphoid organs. (A and B) Migration of naïve
T cells isolated from wild type (WT) or RhoH-deficient (RhoH−/−) mice were
assessed on dishes coated with ICAM-1 and CCL21 (Movies S5 and S6). Shown
are velocity and meandering index from one representative experiment of
three (A). Cell migration plots of cell tracks overlaid with origins from center
are shown (B). (C and D) Adoptive transfer of cells 1 d before treatment with
FTY720 (C) or anti-CD62L (D) for 12 or 20 h, respectively. The fold change in
the homing index, as determined by [carboxy fluorescein succinimidyl ester
(CFSE)+sample/PKH+sample]/(CFSE+input/PKH+input) at 12 h over 0 h. These data
are means ± SEM, n = 4 experiments with 13 mice/time point for FTY720 and
n = 3 with 12 mice/time point for anti-CD62L, peripheral lymph node (pLN),
and mesenteric lymph node (mLN). Statistical significance P < 0.05.
RhoH-deficient T cells migrate in a more directional manner and
vation progresses. CFSE+T cells were stimulated with plate bound CD3/CD28
and assayed for RhoH (A) and IL-2 (B) transcripts normalized to GAPDH.
Unstimulated cells (T0), undivided (0), one to two divisions (1/2), three to four
divisions (3/4), and five divisions onward (5+) are shown as a fold change
relative to unstimulated. (C and D) OT-II TCR transgenic CD4 T cells were
stimulated with OVA323–339presented by irradiated RhoH−/−splenocytes and
RNA was isolated at indicated times. RNA analysis by the comparative CT
method was normalized to CD3d, a T-cell–specific gene. Data are means ±
SEM, n = 3 and considered statistically significant by Student’s t test, P < 0.05.
RhoH expression decreases at the transcriptional level as T-cell acti-
Baker et al.PNAS
| June 26, 2012
| vol. 109
| no. 26
chemokine-mediated migration while promoting TCR-signal–
induced conjugate formation with APCs. Our data establish
a previously unappreciated role for RhoH in the activation of
Rap1 and provide insight into the dynamic regulation of LFA-1
adhesion in the stop and go movement of lymphocytes. Thus, ex-
pression of RhoH may serve as a key functional determinant that
dictates T-cell migration, yet may maintain a naïve T cell in a state
whereby it may rapidly respond upon cognate antigen recognition.
It was reported that the transcription level of RhoH, given the
lack of typical GTP exchange mechanisms (9, 14), controls reg-
ulation of its cellular function. Although maintenance of RhoH
expression in naïve T cells is essential for proximal TCR signaling
(12), down-regulation of RhoH mRNA is shown in Jurkat cells
treated with phorbol myristate acetate (PMA) and Th1 cells
stimulated through the TCR (14). Consistent with the study, our
data show rapid down-regulation of RhoH mRNA upon antigen
stimulation of naïve T cells and further reduction as cells proceed
through division. This may provide a mechanism for T cells to
become more responsive to chemokine-induced migration (by
removing an inhibitory signal for chemotaxis) as well as a tuning
mechanism to control T-cell activation (by removing a positive
signal for conjugate formation).
In the presence of chemokine stimulation, RhoH inhibits
LFA-1 adhesion, whereas Rap1 activation enhances LFA-1 af-
finity. Therefore, recruitment of activated Rap1 (a positive reg-
ulator for LFA-1 activation) to the front and redistribution of
RhoH (a negative regulator) to the rear of cells may be essential
for the spatial regulation of LFA-1 activation and deactivation
during T-cell migration. In the presence of TCR stimulation,
however, RhoH promotes Rap1 activation, thus RhoH, Rap1,
and active LFA-1 are located toward the APC. The combination
of reduced RhoH expression and differential localization de-
termine the outcome when T cells receive migratory signals or
reencounter activating signals.
The mechanisms by which RhoH differentially regulates Rap1
activation remain unclear. RhoH displays inhibitory effects on
many of the pathways induced by other Rho GTPases, including
NF-κB, MAP kinase p38 (14), and Zap70 (9), as well as LFA-1–
mediated adhesion (8). One way in which RhoH may perform dual
functions in T-cell migration and activation is by altering levels of
other Rho GTPases (10, 14, 15). A role for RhoH downstream of
CXCR4 signaling is identified by a failure to activate PAK1 in
response to strong stimulation via CXCR4 in the absence of RhoH
(13), further linking RhoH with regulation of the cytoskeleton by
Rac GTPases. However, constitutive activation of Rac1 increases
proliferation of thymocytes in DN3 and an accelerated transition
from DN3 to DN4 (20), whereas RhoH-deficient cells reveal di-
minished signaling and a block at this stage even in the presence of
activated Rac1 (21), suggesting that regulation by RhoH is more
complicated than only cytoskeletal effects. Defects in thymic se-
lection, alongside studies of proximal TCR signals implicate RhoH
as an important player in conjunction with Zap70 (9, 21). Zap70 is
also reported to be involved in outside-in signaling for high-affinity
LFA-1–mediated adhesion (22). Zap70 binds to immunoreceptor
tyrosine-based activation motifs (ITAMs) of the CD3 chains by its
tandem SH2 domains. Interestingly, RhoH contains an ITAM-like
motif (9), raising the possibility that, given appropriate circum-
stances, Zap70 may interact with RhoH. Whether any of these
mechanisms are a component of the effects of RhoH on Rap1
activation remain to be determined.
Our data suggest that RhoH expression serves as a sensing
mechanism for T cells to stop and go in response to the signals
they have received. Intravital imaging studies describe several
distinct phases of T-cell motility upon antigen challenge (23).
The exact dynamics of these phases may differ depending upon
the cell numbers, route of antigen challenge, antigen quantity
and quality, and presence of costimulatory signals, but the ability
of T cells to switch between periods of minimal motility and
highly migratory states is thought essential for full T-cell acti-
vation and effector function (24, 25). RhoH expression levels
may remain high during early phases after cell entry into a lymph
node to enhance TCR-mediated adhesion by LFA-1. The dis-
tribution of RhoH–mRFP is altered as a cell switches from mi-
gration to a phase of activation. A consequence of T-cell
activation is a decrease in the level of RhoH expression of which
a sufficient reduction leads to an enhanced ability of the cell to
respond to chemokine-driven migratory signals. Thus, activated
cells regain a high level of motility, undergo division, perform
effector functions in other locations within a lymph node, as well
as access sites of egress to depart and perform effector functions
at the site of injury (24). Furthermore, the efficiency of down-
modulation of RhoH expression could impact T:APC dwell
times thus strong signals lead to rapid loss of mRNA, whereas
weaker TCR engagements or low antigen dosage may lead to
slower kinetics of mRNA reduction. The quality of the TCR in-
teraction would then change the RhoH/Rap1 balance. Rapid
regulation of RhoH would enable a cell to counterbalance over-
stimulation or alter the threshold of activation. An inappropriate
T:APC dwell time may lead to death by overactivation, whereas
lack of RhoH may result in such a short contact time as to prevent
a primary response. A possible drawback in the current study,
however, is that the majority of Rap1 assay reported here has
been performed either after overexpressing RhoH protein or by
using RhoH-deficient T cells. Therefore, we cannot completely
exclude the possibility that some of the outcomes of our experi-
ment may have resulted from cellular reactions caused by the
nonphysiological protein expression level. Suppression of en-
dogenous RhoH with small interfering RNA (siRNA) would be
an excellent alternative approach. Unfortunately, we were unable
Therefore, without a proper assay to confirm the protein knock-
down, siRNA data may not directly support our conclusion be-
cause we cannot prove whether the experimental data resulted
entirely from the knockdown of RhoH or were due to nonspecific
reactions by the siRNA treatment. Another constraint in the
we can isolate from the RhoH−/−mouse. As shown in the current
study and by other investigators (9), RhoH regulates key signals in
T-cell functions and development. Thus, RhoH−/−mice manifest
striking defects at two important T-cell development transitions,
DN3 to DN4 and DP toSP (9). Because ofthese defects, RhoH−/−
mice suffer severe T-cell lymphopenia. Due to this phenotype,
comprehensive analysis of RhoH-deficient T cells for their Rap1
activation in various conditions has not been possible.
The mechanisms of stop and go signals are alluded to in many
studies, including the balance between the strength of TCR and
migratory signals received. In vitro some chemokine signals over-
ride ongoing TCR signals, whereas for others, the TCR signal is
dominant (26). Furthermore, chemokine-receptor internalization
and desensitization may allow TCR ligation to occur. The affinity
of TCR is 1–100 mM (27), whereas chemokine-receptor affinity,
such as CCR7 and CXCR4, is 3–15 nM (28). Some chemokine
receptors also serve in a costimulatory capacity (29). Chemokine-
induced integrin adhesiveness may destabilize the IS if this signal
occurs distal from the site of TCR engagement or it may synergize
with TCR to enhance activation if ligated within the IS. Up-reg-
ulation of negative regulators of activation such as CTLA4 may
also alter this balance. Indeed CTLA4 expression has a negative
impact on the TCR stop signal and coligation of CD3 and CTLA4
provides a positive migratory cue in vitro (30). The development of
autoimmunity in CTLA4-deficient mice may, in part, be due to the
sustained dwell time of T:APC interactions leading to activation by
self-antigens. One intracellular mechanism to regulate stop and go
signals is the counterplay between PKCθ and Wiskott–Aldrich
syndrome protein (WASP) (31). PKCθ causes periodic disruptions
| www.pnas.org/cgi/doi/10.1073/pnas.1114214109Baker et al.
of the IS in vitro and PKCθ−/−T cells make more stable contacts Download full-text
with APC in vivo. Cytoskeletal-associated WASP protein is nec-
essary to counteract PKCθ and stabilize the IS (31). The mecha-
nisms of stop and go signaling, including RhoH, will be key
determinants in the functional outcome of a CD4 response in the
face of antigenic challenge.
Materials and Methods
Primary T cells were isolated from whole blood by the polymorph layering
technique and cultured (32). Mouse CD4 T cells were isolated by negative
selection, and T-cell blasts from DO11.10 or OT-II were generated (33, 34).
Transient transfection was performed per manufacturer protocol (Lonza).
Detailed methods may be found in SI Materials and Methods. The Human
Research Studies Review Board and University of Rochester Committee on
Animal Care approved these studies.
ACKNOWLEDGMENTS. We thank Rachel Spoonhower, Pranita Sarangi,
and Nicole Morin for technical assistance; Dr. Jim Miller for reagents and
manuscript comments; Dr. Andrea Sant for the Mel-14 hybridoma; and
Nathan Laniewski for cell sorting. This project was supported by National
Institutes of Health Grants HL087088 and HL018208 (to M.K.).
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