Proline-rich tyrosine kinase-2 is critical for CD8 T-cell
short-lived effector fate
Sören Beinkea,b, Hyewon Pheea,b, Jonathan M. Clingana,c, Joseph Schlessingerd, Mehrdad Matloubiana,e,
and Arthur Weissa,b,e,1
aDepartment of Medicine, Division of Rheumatology,bThe Howard Hughes Medical Institute,eRosalind Russell Medical Research Center for Arthritis, and
cGraduate Program in Biomedical Sciences, University of California, San Francisco, CA 94143-0795; anddDepartment of Pharmacology, Yale University School
of Medicine, New Haven, CT 06510
Contributed by Arthur Weiss, August 6, 2010 (sent for review June 15, 2010)
T-cell interactions with antigen-presenting cells are important for
CD8 T-cell effector or memory fate determination. The integrin leu-
kocyte function-associated antigen-1 (LFA-1) mediates T-cell adhe-
sion but the contribution of LFA-1–induced signaling pathways to
T-cell responses is poorly understood. Here we demonstrate that
proline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cell
activation by synergistic LFA-1 and T-cell receptor stimulation. Fur-
thermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesion
and LFA-1 costimulation ofCD8 T-cell migration. During lymphocytic
choriomeningitis virus infection in vivo, PYK2 deficiency results in
a specific loss of short-lived effector CD8 T cells but does not affect
memory-precursor CD8 T-cell development. Similarly, lack of LFA-1
primarily impairs the generation of short-lived effector cells. Thus,
PYK2 facilitates LFA-1–dependent CD8 T-cell responses and pro-
motes CD8 T-cell short-lived effector fate, suggesting that PYK2
may be an interesting therapeutic target to suppress exacerbated
CD8 T-cell responses.
integrin|costimulation|chemotaxis|lymphocytic choriomeningitis virus|
cytokine expression, and acquire cytotoxic effector functions to
eliminate the target cells (1). Although the majority of cytotoxic
CD8effector T cellsare short-lived,a smallfraction oftheantigen-
survive long term and respond more vigorously to rechallenge with
the same antigen. The signaling pathways that promote terminal
CD8 T-cell differentiation are of great interest for intervention
of allograft rejection, prevention of tissue damage during overly
aggressive antiviral responses, and immunization against viruses
CD8 T-cell short-lived effector or memory fate is directed by the
cytokines IL-2, IL-7, IL-12, IL-15, and IFN-γ, and involves the tran-
scription factors T-bet, eomesodermin, Runx3, and Blimp-1 (2, 3).
Furthermore, signals that CD8 T cells receive during the priming by
antigen presenting cells (APCs) can influence their fate. Initial an-
programs in naive T cells (4–6). However, both the strength and the
duration of the antigenic stimulus have been shown to influence the
amplitude of the CD8 T-cell response or shift the ratio between ef-
fector and memory CD8 T-cell fate (7–11). Thus, prolonged CD8
T-cell interactions with APCs may facilitate the terminal differenti-
ation of effector cells by extending antigen-mediated signals, and
as a consequence of the early termination of signals (12). Further-
more, long-lived interactions of CD8 T cells with APCs throughout
the first cell division were recently suggested to control effector and
memory fate of CD8 Tcells byaffecting the asymmetricdistribution
of effector or memory fate determinants to the proximal or distal
depends on the ability of CD8 cells to migrate in response to che-
mokines, which is also important for appropriate CD8 T-cell differ-
uring the acute phase of immune responses to intracellular
entiation (14–16). Therefore, the spatial and temporal regulation of
versus memory fate decisions.
Both chemokine and antigen receptors induce signaling path-
ways that mediate the reorganization of the cellular cytoskeleton.
However, T-cell polarity additionally requires directed interac-
tions of T cells. The T-cell integrin, leukocyte function-associated
antigen-1 (LFA-1), facilitates T-cell adhesion by binding to its
ligand intercellular adhesion molecule-1 (ICAM-1), which is ex-
pressed on the surface of many cell types (17). In resting T cells,
the extracellular domain of LFA-1 is in an inactive folded con-
the absence of antigen or chemokines. T-cell receptor (TCR) or
chemokine receptor engagement triggers signaling pathways (in-
side-out signaling) that induce a conformation change and clus-
tering of LFA-1, allowing it to bind ICAM-1 with high affinity.
in signaling) are activated that induce cytoskeletal rearrange-
ments. Although some specific components of the inside-out
signaling pathway that regulates LFA-1 activity have been iden-
tified in recent years, the role of LFA-1 outside-in signaling in T
cells is less well-studied. Src family kinases, Syk kinases, the
adaptor protein SLP-76, and Vav have been shown to be involved
in integrin-mediated functions in neutrophils and platelets (18–
22). The role of these signaling proteins in regulating LFA-1
function in T cells has been difficult to study because proximal
TCR signaling also critically depends on them and their loss
results in impaired T-cell development. Interestingly, SLP-76
binding to adhesion and degranulation-promoting adapter pro-
tein (ADAP) has recently been identified to be critical for LFA-1
ever, the physiological importance of LFA-1 outside-in signaling
for overall T-cell responses is unclear.
Proline-rich tyrosine kinase-2 (PYK2) is closely related to the
nonreceptor tyrosine kinase focal adhesion kinase (FAK). Both
kinases have been implicated in the regulation of the actin cyto-
skeleton (25). PYK2 is highly expressed in immune cells and acti-
vated in response to LFA-1, antigen receptor, or chemokine re-
ceptor stimulation (26–33). In macrophages and B cells, PYK2 has
been shown to be important for chemokine-induced migration (34,
35). TCR-induced phosphorylation and activation of PYK2 is de-
pendent on the Src family kinase FYN, but does not require LCK,
which mediates ZAP-70 activation and the canonical antigen re-
ceptor signaling pathways leading to Ca2+/NFAT, NF-κB, and
MAP kinase activation (28, 29). Normal T-cell development in
Author contributions: S.B., M.M., and A.W. designed research; S.B., H.P., and J.M.C. per-
formed research; J.S. and M.M. contributed new reagents/analytic tools; S.B., H.P., M.M.,
and A.W. analyzed data; and S.B., M.M., and A.W. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 14, 2010
| vol. 107
| no. 37www.pnas.org/cgi/doi/10.1073/pnas.1011556107
PYK2-deficient mice suggested that PYK2 is not critical for TCR
signalingpathways that mediateT-cellselectioninthethymus (35).
LFA-1 stimulation of T cells induces PYK2 colocalization with the
a nonredundant function in T cells is not known.
Here we study PYK2-deficient T cells to determine whether
PYK2 is essential for T-cell responses in vitro or in vivo. Our
CD8 T-cell activation and migration by regulating T-cell polarity.
Furthermore, this signaling pathway is critically important for
the generation of short-lived effector but not memory-precursor
effector CD8 T cells during lymphocytic choriomeningitis virus
(LCMV) infection in vivo.
PYK2-Deficient CD8 T Cells Are Impaired in Activation by Synergistic
TCR and LFA-1 Stimulation. Because the tyrosine kinase PYK2 is
and B). Simultaneous TCR and integrin stimulation has been
reported to result in enhanced PYK2 activation, indicating that
PYK2 might integrate signaling pathways downstream of these
receptors (38). Therefore, we also stimulated PYK2-deficient T
cells simultaneously through both TCR and LFA-1 under con-
with TCR stimulation, we titrated the anti-CD3 antibody down to
limiting concentrations, which alone were insufficient to induce
T-cell proliferation, but additional LFA-1 stimulation by ICAM-1
synergistically facilitated proliferation of wild-type T cells. This
synergistic response was severely impaired in PYK2-deficient T
cells. Interestingly, and consistent with previous reports, only wild-
type CD8 but not wild-type CD4 T cells responded to LFA-1 cos-
CD8 T cells (Fig. 1C). These data indicate that PYK2 facilitates
synergy between LFA-1 and TCR signaling during CD8 T-cell ac-
tivation under limiting TCR-stimulation conditions.
PYK2 Is Essential for LFA-1–Induced T-Cell Polarity. We then in-
vestigated how PYK2 might regulate LFA-1 costimulation of
T-cell responses. An important function of LFA-1 is to facilitate
T-cell adhesion by binding to its ligand ICAM-1 and to induce
the reorganization of the cellular cytoskeleton, thereby pro-
moting the ability of T cells to polarize. To test whether PYK2 is
important for LFA-1–mediated T-cell adhesion, the ability of
PYK2-deficient CD8 T cells to adhere to plate-bound ICAM-1
was tested (Fig. 2A). In these experiments, standard doses of
anti-CD3 antibody were able to induce normal binding of PYK2-
deficient CD8 T cells to ICAM-coated plastic plates, suggesting
that inside-out signaling leading to the induction of active LFA-1
is intact in these cells. However, basal adhesion and adhesion in
response to low-dose anti-CD3 antibody stimulation was signifi-
cantly decreased in PYK2-deficient CD8 T cells. Moreover, after
the addition of MnCl2, which bypasses inside-out signaling by
artificially inducing a constitutively active confirmation of LFA-1,
adhesion of PYK2-deficient CD8 T cells to ICAM-1 was also im-
paired. These data indicate that PYK2 contributes to the adhe-
sion of CD8 T cells to ICAM-1–coated surfaces, but this function
is at least partially downstream of LFA-1 and can be bypassed by
strong TCR stimulation.
In addition to LFA-1 affinity, the ability of the cells to re-
organize their cytoskeleton and spread on the surface contributes
to the overall capacity of the cell to bind plate-bound ligands. To
investigate whether LFA-1 induced T-cell spreading is affected
by PYK2 deficiency, CD8 T-cell blasts were adhered to plate-
bound ICAM-1 and imaged by immunofluorescence microcopy
after intracellular staining for polymerized f-actin. Whereas wild-
type CD8 T-cell blasts had the ability to acquire a polarized
morphology in response to LFA-1 ligation and displayed poly-
merized actin at the leading edge and the uropod of migrating
cells, PYK2-deficient CD8 T-cell blasts were markedly impaired
in this response (Fig. 2B). These data indicate that PYK2 reg-
ulates an LFA-1 signaling pathway that contributes to the ability
of CD8 T cells to spread and polarize.
PYK2 Facilitates LFA-1–Dependent CD8 T-Cell Migration. LFA-1–
mediated cell contacts and cytoskeletal polarity are also impor-
tant for T-cell chemotaxis. PYK2 is activated by chemokine
receptors and mediates chemokine-induced migration in mac-
rophages and B cells (27, 32, 34, 35). Therefore, we examined
whether PYK2 is essential for chemokine-induced transmigration
of CD8 T cells in vitro (Fig. 2C). Interestingly, transmigration
induced by CXCL12 or CCL21 alone was not impaired in PYK2-
deficient CD8 T cells. However, coating the transmigration bar-
rier with the LFA-1 ligand, ICAM-1, resulted in a synergistic in-
crease in transmigration efficiency in wild-type CD8 T cells, but
was significantly lower in PYK2-deficient CD8 T cells. These data
show that although PYK2 is not required for LFA-1–independent
chemotaxis of CD8 T cells, it is essential for LFA-1–dependent
CD8 T-cell chemotaxis. Together, these results indicate that
PYK2 plays a critical role in the synergistic induction of CD8
3H-thymidine (cpm x103)
α α-CD3 Ab
low α α-CD3 Ab
high α α-CD3 Ab
high α α-CD3 Ab
low α α-CD3 Ab
low α α-CD3 Ab
ergistic TCR and LFA-1 stimulation in vitro. WT or PYK2-deficient T cells were
stimulated with plate-bound anti-CD3 antibody at standard concentrations
(0.5 μg per well) or at limiting concentrations (0.1 μg per well) in the presence
or absence of 0.3 μg per well plate-bound ICAM-1. (A) Proliferation was ana-
lyzedby3H-thymidine uptake at 72 h.Graph shows averagesignal± SD: *0.01
< P < 0.05; **0.001 < P < 0.01 (unpaired two-tailed Student’s t test). (B) Pro-
liferation was analyzed by CFSE labeling and FACS analysis. (C) IFN-γ and IL-2
expressionin CD8T cellswas determinedat 40 h by intracellular FACS staining
after 5-h Brefeldin A treatment. (A–C) Data are representative of three or
Impaired activation of PYK2-deficient CD8 T cells in response to syn-
Beinke et al.PNAS
| September 14, 2010
| vol. 107
| no. 37
T-cell polarity in response to simultaneous LFA-1 and antigen or
chemokine receptor triggering, which allows full CD8 T-cell ac-
tivation and migration.
PYK2 Is Critical for the Expansion of CD8 T Cells in Response to LCMV.
LFA-1 function is critical for the activation of T cells by APCs in
CD8 T cells by APCs, PYK2-deficient CD8 T cells expressing the
transgenic P14 TCR, which recognizes the gp33-41 epitope of
LCMV, were stimulated with syngeneic dendritic cells pulsed
with various doses of gp33-41 peptide in vitro. PYK2 deficiency
impaired P14 T-cell proliferation in response to stimulation, par-
ticularly at low doses of peptide; high doses of gp33-41 peptide
were able to bypass the requirement for PYK2 (Fig. 3A).
We next investigated the significance of PYK2 for CD8 T-cell
responses in vivo. To determine whether PYK2 is essential for the
expansion of CD8 T cells in response to LCMV infection, PYK2-
deficient P14 CD8 T cells were adoptively transferred together
with wild-type P14 CD8 T cells at a 1:1 ratio into host mice. Five
and 8 d after LCMV Armstrong challenge, the populations of
transferred PYK2-deficient and wild-type P14 CD8 T cells in
blood and spleen were determined by FACS analysis of congenic
markers (Fig. 3 B and C). At the peak of the response on day 8,
PYK2-deficient P14 CD8 T-cell numbers in the blood were ap-
proximately three times lower than wild-type P14 CD8 T cells.
PYK2-deficient P14 CD8 T-cell numbers in the spleen were
similarly reduced, suggesting that the defect is a consequence of
impaired expansion rather than altered distribution of the cells
because of a migration defect. A difference between PYK2-de-
ficient andwild-type P14 CD8 T-cellnumber was noted asearly as
day 5 after LCMV infection. CFSE-labeling of P14 CD8 T cells
before adoptive transfer as above demonstrated that although all
transferred PYK2-deficient P14 CD8 T cells entered cell division,
their numbers were reduced compared with wild-type P14 CD8 T
cells after four to five cell divisions (Fig. 3D). However, CD25 up-
regulation on P14 CD8 T cells 24 or 36 h after LCMV challenge
was not affected by PYK2-deficiency, further suggesting that the
impaired expansion of PYK2 knockout P14 CD8 T cells is not
a consequence of an impact on proximal TCR signaling or the
receipt of IL-2 signals (Fig. S1). Furthermore, the small pop-
ulation of PYK2-deficient P14 CD8 T cells that was present
during the acute expansion phase did not display any differences
from wild-type P14 CD8 T cells in expression of the activation
markers glycosylated CD43 (1B11), CD44, and CD62L, or IFN-γ,
and TNF up-regulation upon restimulation with gp33-41 peptide
in vitro (Fig. S2 A–C). This finding suggests the PYK2-deficient
CD8 T cells were activated normally but failed to fully expand
during the acute phase of their response to LCMV.
CD8 T-Cell Intrinsic PYK2 Deficiency Results in a Loss of Short-Lived
Effector Cells. Rapid expansion is an attribute that is specifically
associated with short-lived effector CD8 cells. To investigate
whether PYK2-deficient P14 CD8 T cells have specific defects in
the generation of short-lived effector or memory-precursor effec-
tor cellpopulations,expressionofthespecific markersIL-7Rα and
This finding revealed that in both blood and spleen, the frequency
of IL-7Rαlow/KLRG1highshort-lived effector cells was markedly
reduced and that of IL-7Rαhigh/KLRG1lowmemory-precursor ef-
fector cells increased in PYK2-deficient CD8 T cells (Fig. 4A).
control Mn2+ high
% CD8 T cell adhesion
to plate-bound ICAM-1
α α α-CD3 Ab
WWWT T T + + + I I IC C CA A AM M M1 1 1P P PY Y YK K K2 2 2 K K KO O O + + + I I IC C CA A AM M M1 1 1
sion, polarity, and transmigration. (A) WT or PYK2-deficient T cells were
stimulated as indicated in ICAM-1–coated microwell plates and adherent
CD8 T cells were quantified by FACS. (B) WT or PYK2-deficient CD8 T-cell
blasts were allowed to adhere to ICAM-1–coated cover slides. F-actin was
stained with Alexa488-phalloidin and analyzed by fluorescence microscopy.
Data are representative of an average view field from more than three
experiments. (C) Migration of WT or PYK2-deficient CD8 T cells in the ab-
sence or presence of the indicated chemokines was measured using control
or ICAM-1–coated transwell chambers. Migratory activity is presented as
a percentage of migrating CD8 T-cell number divided by input CD8 T-cell
number. (A and C) Graphs show average cell numbers from three in-
dependent experiments ± SEM: *0.01 < P < 0.05; **0.001 < P < 0.01; ***P <
0.001 (unpaired two-tailed Student’s t test).
PYK2-deficient CD8 T cells are defective in LFA-1–mediated adhe-
# cells (x106)
# cells / ml (x106)
3H-Thymidine (cpm x103)
LCMV. (A) WT or PYK2-deficient P14 CD8 T cells were stimulated with den-
dritic cellsand theindicated concentrationsof LCMV gp33-41peptide in vitro.
Proliferation was analyzed by3H-thymidine incorporation. Graph shows av-
erage counts ± SD. (B–D) WT (CD45.1+/CD45.2+) and PYK2-deficient (CD45.2+/
CD45.2+) P14 CD8 T cells were mixed at a 1:1 ratio and adoptively transferred
Armstrong and P14 cells were analyzed at the indicated times. (B) Represen-
tative FACS plot of the analysis of congenic markers of P14 CD8 T cells from
blood at day 8; numbers shown represent frequencies of the indicated pop-
ulation within CD8 T cells. (C) Graph shows average total P14 CD8 T-cell
numbers in blood and spleen from three or more experiments (day 5: n = 10
mice; day 8: n = 20 mice; day 15: n = 6 mice) ± SEM. (D) WT and PYK2-deficient
P14 transgenic CD8 T cells that were CFSE-labeled before adoptive transfer
were analyzed by FACS after LCMV infection. (A and D) Data are represen-
tative of three experiments. (A and C) *0.01 < P < 0.05; **0.001 < P < 0.01;
***P < 0.001 (unpaired two-tailed Student’s t test).
PYK2 deficiency impairs the expansion of CD8 T cells in response to
| www.pnas.org/cgi/doi/10.1073/pnas.1011556107Beinke et al.
However, this reflected a decrease in total numbers of PYK2-
precursor effector cells were generated at similar numbers as wild
type (Fig. 4B). In vivo BrdU labeling on day 5 after infection
demonstrated that a smaller fraction KLRG1+PYK2 P14 CD8
T cells underwent cell divisions compared with KLRG1+wild-
CD8 T cells remaining at day 50 after LCMV infection expressed
equivalent levels of TNF and IFN-γ upon restimulation, sug-
gesting that PYK2-deficient memory cells are functionally com-
petent in regard to cytokine expression (Fig. S2D). These data
indicate that PYK2 is essential for the generation of short-lived
effector CD8 T cells, but memory-precursor T cells develop in-
dependently of PYK2.
Short-Lived Effector CD8 T-Cell Generation Is LFA-1–Dependent. CD8
T-cellresponses against LCMV were previously reported to occur
independently of LFA-1 (40, 41). However, if the defect in short-
lived effector cell expansion in PYK2-deficient P14 CD8 T cells
is related to the role of PYK2 in regulating LFA-1 costimulation,
then a similar phenotype should be caused by LFA-1 deficiency.
To test this hypothesis, we adoptively transferred P14 CD8 T cells
deficient in the CD11α subunit of LFA-1 together with the same
number of wild-type P14 CD8 T cells before LCMV Armstrong
infection of host mice. Analyses of congenic CD45 markers
revealed impaired expansion of CD11α-deficient P14 CD8 T cells
in the blood at day 8 after infection (Fig. 5 A and C). There was
also a shift in the frequencies of IL-7Rαlow/KLRG1highshort-lived
deficient P14 CD8 T cells compared with wild typeP14 CD8 T cells
(Fig. 5B). CD11α deficiency primarily affected total numbers of
T-cell numbers were also reduced to some degree (Fig. 5C). The
decrease CD11α-deficient P14 CD8 T-cell numbers in spleens was
comparably smaller. This result could be because of the accumu-
lation of LFA-1–deficient P14 CD8 T cells in the spleen as a con-
sequence of impaired transmigration from the spleen to the blood.
Therefore, LFA-1 is critical for short-lived effector CD8 T-cell
generation similar to PYK2, but has additional functions during
CD8 T-cell responses that are probably attributed to its more ex-
tensive role in T-cell adhesion.
This study reveals that PYK2 plays a critical role in integrating
LFA-1 and TCR or chemokine signaling pathways to facilitate
the synergistic induction of cell polarity during CD8 T-cell acti-
vation and migration. Furthermore, we demonstrate that PYK2
and LFA-1 are important for the generation of short-lived ef-
fector CD8 T cells, suggesting that cell polarity may be a critical
determinant of CD8 T-cell fate.
# cells / ml blood (x106)
# cells / spleen (x106)
day 5day 8day 15
T cells were adoptively transferred and mice were infected with LCMV Arm-
lived effector versus IL-7Rαhigh/KLRG1lowmemory precursor cells at day 8 after
or memory precursor cells from three or more experiments (day 5: n = 10 mice;
day 8: n = 20 mice; day 15: n = 6 mice) ± SEM: **0.001 < P < 0.01; ***P < 0.001
(unpaired two-tailed Student’s t test). (C) FACS analysis of BrdU incorporation
on day 5 after LCMV infection. Data are representative of three experiments.
CD11α α KO
# cells / ml blood (x106)
# cells / spleen (x106)
WTCD11α α KO
fector cells during LCMV infection. (A–C) WT (CD45.2+/CD45.2+) or CD11α-de-
ficient (CD45.1+/CD45.2+) P14 CD8 T cells were mixed at a 1:1 ratio and
adoptively transferred into host mice (CD45.1+/CD45.1+). The next day, chi-
meric mice were infected with LCMV Armstrong. (A) Representative FACS
analysis of congenic markers on blood cells at day 8 after infection. Numbers
IL-7Rαhigh/KLRG1lowmemory precursor cells in the blood at day 8 after in-
8: n = 10 mice; day 15: n = 10 mice) ± SEM: *0.01 < P < 0.05; **0.001 < P < 0.01;
***P < 0.001 (unpaired two-tailed Student’s t test).
CD11α deficiency primarily affects the generation of short-lived ef-
Beinke et al.PNAS
| September 14, 2010
| vol. 107
| no. 37
Although TCR and chemokine receptor stimulation induces
PYK2 phosphorylation, neither TCR-mediated activation nor
chemokine receptor-induced migration of T cells was critically
dependent on PYK2. However, LFA-1 coligation by ICAM-1
revealed that CD8 T cells require PYK2 for LFA-1–dependent
activation and migration. In line with the observation that TCR
and integrin costimulation induces synergistic PYK2 phosphor-
ylation, LFA-1 could provide an additional signal for PYK2 ac-
tivation (38). Therefore, PYK2 may integrate proximal signaling
pathways downstream of LFA-1 and TCR or chemokine recep-
tors to fully induce the reorganization of the cytoskeleton. This
contribute to CD8 T-cell activation and migration in the presence of
physiologic concentrations of antigen and chemokines. It may con-
stitute an additional safeguard mechanism to allow full responses of
CD8 T cells only when both integrins and antigen or chemokine
receptors are engaged.
Interestingly, PYK2 function only seemed to be important for
CD8 but not CD4 T-cell activation. CD8 T cells may be more de-
pendent on the contribution of cytoskeletal reorganization to over-
rapidly reorient and spread. CD4 T cells may also be more depen-
dent on additional costimulatory signals, such as CD28 (42).
in a specific loss of short-lived effector CD8 T cells, but memory-
precursor effector CD8 T-cell generation was normal. Because
PYK2 was important for LFA-1–induced spreading but not for
LFA-1 activity, LFA-1–dependent interactions of PYK2-deficient
CD8 T cells with APCs may be sufficient to facilitate memory-
precursor cell development. Alternatively, memory-precursor ef-
fector cell development may be independent of LFA-1. CD11α
deficiency also primarily affected short-lived effector cell genera-
tion, but there was some impact on memory-precursor effector cell
numbers. Therefore, we propose that LFA-1 activation of PYK2,
for short-lived effector versus memory-precursor effector CD8
PYK2-mediated T-cell polarity could be important for CD8 T
cell short-lived effector fate by regulating the kinetics or quality
of the CD8 T cell APC interaction. This process may indirectly
impact on antigen triggering and exposure to cytokines. In line
with the report by Chang et al., failure to polarize in the absence
of PYK2 could also result in the miss-localization of effector-fate
determinants from the APC proximal daughter cell during CD8
T-cell division (13). Furthermore, the loss of LFA-1 signaling via
PYK2 may impact on the formation or molecular makeup of
signaling microclusters (43). The molecular mechanism behind
the role of PYK2 in short-lived effector fate is very exciting, but
also difficult to study. Because we have demonstrated that PYK2
is only critical for CD8 T cells under physiological levels of TCR
or chemokine receptor stimulation in the presence of appropri-
ate LFA-1 costimulation, the molecular mechanisms would be
best studied in vivo, but this exceeds the scope of the present
study and will be subject to further investigation.
Mice deficient in the LFA-1–specific CD11α subunit fail to re-
ject immunogenic tumors but were surprisingly reported to mount
study we revealed a defect in the generation of LCMV-specific
short-lived effector cells as a consequence of both PYK2 and
CD11α deficiency. This result was achieved using simultaneous
adoptive transfer of wild-type and knockout P14 TCR transgenic
CD8 T cells, which directly compared their competitive fitness.
Previous studies showed that after LCMV priming, CD11α-
deficient cytotoxic T lymphocytes lysed target cells normally.
However, this result is expected because the cytolytic activity of
short-lived effector and memory-precursor effector cells is simi-
lar (46). Short-lived effector and memory-precursor CD8 T-cell
populations were not specifically analyzed previously.
Interestingly, it has also been reported that ICAM-1 is dis-
pensable for short-lived, but essential for long-lasting, T cell/
dendritic cell interactions (47). Using in vivo transfer of ICAM-1–
also observed that after initial normal onset of the CD8 T-cell
response, CD8 T-cell numbers were decreased after 7 to 14 d.
However, this finding was interpreted as a defect in the memory
phase of the CD8 T-cell response. Characterization of short-lived
effector versus memory-precursor CD8 T-cell populations in this
system would shed light into whether the report is consistent with
the data presented here. Furthermore, Zehn et al. demonstrated
curtailed OT1 CD8 T-cell responses to low-affinity variants of
the SIINFEKL peptide (11). Interestingly, the low-affinity TCR
indicating that altered T-cell interactions with APCs could con-
tribute to this phenomenon.
Our studies reveal that PYK2 has not only a quantitative contri-
bution to the overall CD8 T-cell response, but a very specific qual-
itative impact on the differentiation of short-lived effector versus
interesting window of opportunity for therapeutic intervention by
targeting PYK2. PYK2 may be an interesting candidate for the re-
duction of acute CD8 T-cell responses without completely ablating
CD8 T cell-mediated surveillance of acute viral infections and
dormant retroviruses. Prevention of allograft rejection after trans-
precursor effector cells that develop independently of PYK2 may
cause acute or chronic rejection. However, PYK2 inhibition may
be useful to reduce acute pathogenic CD8 T-cell responses in the
to death (i.e., fulminant hepatitis or influenza), although main-
taining the beneficial anti-viral response.
Materials and Methods
Mice and Reagents. PYK2 and CD11α knockout mice (34, 48), BoyJ (CD45.1+),
and P14 TCR transgenic mice bearing the DbGP33-specific TCR were fully
backcrossed to C57BL/6. All animals were housed in specific pathogen-free
facility at the University of California, San Francisco, according to University
and National Institutes of Health guidelines. Antibodies were purchased
from BD Biosciences [CD3 (2C11), CD8, CD4, IFN-γ, IL-2, IL-7Rα, TNF, CD44,
CD62L] or Biolegend (CD45.1, CD45.2, KLRG1). Recombinant mouse ICAM-1-
FC, CXCL12, and CCL21 was purchased from R&D Biosciences.
Cell Isolation and in Vitro T-Cell Activation. T cells were purified from spleens
or lymph nodes by MACS (MiltenYi Biotec) according to the manufacturers
protocol (purity >95%) and cultured in DMEM containing 10% FCS, 10 mM
Hepes, penicillin, streptomycin, 2 nM glutamate, 1 mM sodium pyruvate, 1×
nonessential amino acids, and 50 mM 2-mercaptoethanol at 37 °C in the
presence of 5% CO2. Next, 2 × 105T cells were stimulated in a 96-well plate
with 0.5 or 0.1 μg/100 μL plate-bound anti-CD3 antibody per well in the
presence or absence of 0.3 μg /100 μL plate-bound ICAM-1-FC per well for 72
h. P14 TCR transgenic CD8 T cells were stimulated in vitro with 1 μM to 40
nM LCMV gp33-41 peptide and 2 × 104CD11c MACS enriched syngeneic
splenic dendritic cells for 48 h. Proliferation was assessed by liquid scintilla-
tion of3H-thymidine uptake during the last 6 h of the culture or by FACS
analysis of CFSE labelled cells 72 h after stimulation. Cytokine expression was
determined at 40 h after stimulation by treating cells with 10 μg/mL Bre-
feldin A for 5 h, followed by intracellular staining and FACS analysis.
T-Cell Adhesion. For T-cell adhesion, 1 × 106T cells were plated in 96-well
plates coated with 0.3 μg/100 μL plate-bound ICAM-1-FC per well on ice and
stimulated with 0.5 μg/mL or 5 μg/mL soluble anti-CD3 antibody cross-linked
with secondary antibody for 10 min at 37 °C. Nonadherent cells were
washed off before adherent cells were eluted and counted by FACS. CD8
T-cell blast were generated by stimulating MACS-enriched CD8 T cells with
10 ng/mL PMA and 200 ng/mL Ionomycin for 18 h and culturing them in the
presence of 50 U/mL IL-2 for 5 to 7 d. For immunofluorescent imaging of CD8
T-cell blasts, cells were plated on ICAM-1-FC–coated glass coverslips for 10
min at 37 °C and, after washing, F-actin was stained using Alexa Fluor 488
phalloidin (Invitrogen) according to the manufacture’s protocol. Images
were acquired using a Zeiss microscope.
| www.pnas.org/cgi/doi/10.1073/pnas.1011556107Beinke et al.
Transwell Migration Assay. The migratory ability of T cells was measured using
5-μm pore size Transwell plates (Corning Costar Corp.), as described pre-
viously (49). Cells were collected, stained with anti-CD4, -CD8, or -CD3 mAb,
and quantified using flow cytometry. Transwell assays were performed in
duplicates for each different chemokine (CXCL12, 400 ng/mL and CCL21, 1
μg/mL). For transwell migration assays using filters coated with ICAM-1, 5-μm
polycarbonate transwell filters were coated in 100 μL of PBS with ICAM-1
(R&D Systems, 3 μg/mL) overnight at 4 °C. All filters were washed three times
with PBS and blocked with 2% BSA for 1 h at 37 °C. Filters were rinsed with
PBS and dried. Coated filters were checked for leakage, then used for
transwell migration assays.
T-Cell Adoptive Transfer and in Vivo Analyses. Wild-type and PYK2- or CD11α-
deficient P14 CD8 T cells were mixed at a 1:1 ratio and transferred at 2 × 105
with 2 × 105PFU per mouse LCMV Armstrong and P14 CD8 T cell were char-
acterized by FACS 5, 8, or 15 d after infection. Proliferation in vivo was in-
vestigated by CFSE labeling of P14 CD8 T cells before adoptively transferring
1 × 106cells per mice. FACS analysis of CFSE dilution was performed 66 h after
injection of 10 μg BrdU per mouse at day 5 after LCMV Armstrong infection
and FACS analysis of splenic P14 CD8 T cells 1.5 h after the BrdU pulse.
ACKNOWLEDGMENTS. We thank Al Roque and Kristin Doan for help with
animal maintenance and Andre Limnander and Byron Au-Yeung for in-
spiring discussions, support, and critically reading the manuscript. This study
was supported in part by a long-term fellowship from the Human Frontier
Science Foundation (to S.B.) and a Leukemia and Lymphoma Society Special
Fellow award (to H.P.); J.M.C. is a recipient of a National Science Foundation
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| September 14, 2010
| vol. 107
| no. 37