Proline-rich tyrosine kinase-2 is critical for CD8 T-cellshort-lived effector fateSören 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, andcGraduate Program in Biomedical Sciences, University of California, San Francisco, CA 94143-0795; and dDepartment of Pharmacology, Yale University Schoolof 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 forCD8 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 toT-cell responses is poorly understood. Here we demonstrate thatproline-rich tyrosine kinase-2 (PYK2) deficiency impairs CD8 T-cellactivation by synergistic LFA-1 and T-cell receptor stimulation. Fur-thermore, PYK2 is essential for LFA-1-mediated CD8 T-cell adhesionand LFA-1 costimulation of CD8 T-cell migration. During lymphocyticchoriomeningitis virus infection in vivo, PYK2 deficiency results ina specific loss of short-lived effector CD8 T cells but does not affectmemory-precursor CD8 T-cell development. Similarly, lack of LFA-1primarily 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 PYK2may be an interesting therapeutic target to suppress exacerbatedCD8 T-cell responses.

integrin | costimulation | chemotaxis | lymphocytic choriomeningitis virus |memory

During the acute phase of immune responses to intracellularpathogens or allografts, CD8T cells rapidly proliferate, induce

cytokine expression, and acquire cytotoxic effector functions toeliminate the target cells (1). Although the majority of cytotoxicCD8 effector T cells are short-lived, a small fraction of the antigen-specific CD8T cells, referred to asmemory-precursor effector cells,survive long term and respond more vigorously to rechallenge withthe same antigen. The signaling pathways that promote terminalCD8 T-cell differentiation are of great interest for interventionof allograft rejection, prevention of tissue damage during overlyaggressive antiviral responses, and immunization against virusesand cancer.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 byantigen presenting cells (APCs) can influence their fate. Initial an-tigen encounter can trigger both effector andmemorydifferentiationprograms in naive T cells (4–6). However, both the strength and theduration of the antigenic stimulus have been shown to influence theamplitude of the CD8 T-cell response or shift the ratio between ef-fector and memory CD8 T-cell fate (7–11). Thus, prolonged CD8T-cell interactions with APCs may facilitate the terminal differenti-ation of effector cells by extending antigen-mediated signals, andbrief interactionsmay result in themaintenance ofmemory potentialas a consequence of the early termination of signals (12). Further-more, long-lived interactions of CD8 T cells with APCs throughoutthe first cell division were recently suggested to control effector andmemory fate of CD8 T cells by affecting the asymmetric distributionof effector or memory fate determinants to the proximal or distaldaughter cell, respectively (13).Access toAPCsand cytokines largelydepends on the ability of CD8 cells to migrate in response to che-mokines, which is also important for appropriate CD8 T-cell differ-

entiation (14–16). Therefore, the spatial and temporal regulation ofT cell/APC interactions is critically important for short-lived effectorversus 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-associatedantigen-1 (LFA-1), facilitates T-cell adhesion by binding to itsligand 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-formation that prevents binding to ICAM-1 and T-cell adhesion inthe absence of antigen or chemokines. T-cell receptor (TCR) orchemokine 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.Consequently, signaling pathways downstream of LFA-1 (outside-in signaling) are activated that induce cytoskeletal rearrange-ments. Although some specific components of the inside-outsignaling pathway that regulates LFA-1 activity have been iden-tified in recent years, the role of LFA-1 outside-in signaling in Tcells is less well-studied. Src family kinases, Syk kinases, theadaptor protein SLP-76, and Vav have been shown to be involvedin integrin-mediated functions in neutrophils and platelets (18–22). The role of these signaling proteins in regulating LFA-1function in T cells has been difficult to study because proximalTCR signaling also critically depends on them and their lossresults in impaired T-cell development. Interestingly, SLP-76binding to adhesion and degranulation-promoting adapter pro-tein (ADAP) has recently been identified to be critical for LFA-1outside-in signaling in T cells usingmutant proteins (23, 24). How-ever, the physiological importance of LFA-1 outside-in signalingfor overall T-cell responses is unclear.Proline-rich tyrosine kinase-2 (PYK2) is closely related to the

nonreceptor tyrosine kinase focal adhesion kinase (FAK). Bothkinases 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 hasbeen 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, andMAP 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: aweiss@medicine.ucsf.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011556107/-/DCSupplemental.

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PYK2-deficient mice suggested that PYK2 is not critical for TCRsignaling pathways that mediate T-cell selection in the thymus (35).LFA-1 stimulation of T cells induces PYK2 colocalization with themicrotubuleorganizingcenterandPYK2relocalizes to the interfacebetweenTcells andAPCs (36, 37).Nevertheless,whetherPYK2hasa 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. Ourfindings demonstrate that PYK2 facilitates LFA-1 costimulation ofCD8 T-cell activation and migration by regulating T-cell polarity.Furthermore, this signaling pathway is critically important forthe generation of short-lived effector but not memory-precursoreffector CD8 T cells during lymphocytic choriomeningitis virus(LCMV) infection in vivo.

ResultsPYK2-Deficient CD8 T Cells Are Impaired in Activation by SynergisticTCR and LFA-1 Stimulation. Because the tyrosine kinase PYK2 isactivated in response toTCR stimulation, wefirst analyzedwhetherPYK2 deficiency affects T-cell activation in response to stimulationsolely with anti-CD3 antibody in vitro. However, only a small defectin the proliferation of PYK2-deficient T cells was observed (Fig. 1Aand B). Simultaneous TCR and integrin stimulation has beenreported to result in enhanced PYK2 activation, indicating thatPYK2 might integrate signaling pathways downstream of thesereceptors (38). Therefore, we also stimulated PYK2-deficient T

cells simultaneously through both TCR and LFA-1 under con-ditions inwhich these two stimuli synergize.Toachieve such synergywith TCR stimulation, we titrated the anti-CD3 antibody down tolimiting concentrations, which alone were insufficient to induceT-cell proliferation, but additional LFA-1 stimulation by ICAM-1synergistically facilitated proliferation of wild-type T cells. Thissynergistic response was severely impaired in PYK2-deficient Tcells. Interestingly, and consistent with previous reports, only wild-type CD8 but not wild-type CD4 T cells responded to LFA-1 cos-timulation (Fig. 1B) (39). IL-2 and IFN-γ production in response toTCR and LFA-1 costimulation was also reduced in PYK2 deficientCD8 T cells (Fig. 1C). These data indicate that PYK2 facilitatessynergy 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 ofT-cell responses. An important function of LFA-1 is to facilitateT-cell adhesion by binding to its ligand ICAM-1 and to inducethe reorganization of the cellular cytoskeleton, thereby pro-moting the ability of T cells to polarize. To test whether PYK2 isimportant for LFA-1–mediated T-cell adhesion, the ability ofPYK2-deficient CD8 T cells to adhere to plate-bound ICAM-1was tested (Fig. 2A). In these experiments, standard doses ofanti-CD3 antibody were able to induce normal binding of PYK2-deficient CD8 T cells to ICAM-coated plastic plates, suggestingthat inside-out signaling leading to the induction of active LFA-1is intact in these cells. However, basal adhesion and adhesion inresponse to low-dose anti-CD3 antibody stimulation was signifi-cantly decreased in PYK2-deficient CD8 T cells. Moreover, afterthe addition of MnCl2, which bypasses inside-out signaling byartificially 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 functionis at least partially downstream of LFA-1 and can be bypassed bystrong TCR stimulation.In addition to LFA-1 affinity, the ability of the cells to re-

organize their cytoskeleton and spread on the surface contributesto the overall capacity of the cell to bind plate-bound ligands. Toinvestigate whether LFA-1 induced T-cell spreading is affectedby PYK2 deficiency, CD8 T-cell blasts were adhered to plate-bound ICAM-1 and imaged by immunofluorescence microcopyafter intracellular staining for polymerized f-actin. Whereas wild-type CD8 T-cell blasts had the ability to acquire a polarizedmorphology in response to LFA-1 ligation and displayed poly-merized actin at the leading edge and the uropod of migratingcells, PYK2-deficient CD8 T-cell blasts were markedly impairedin this response (Fig. 2B). These data indicate that PYK2 reg-ulates an LFA-1 signaling pathway that contributes to the abilityof 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 chemokinereceptors and mediates chemokine-induced migration in mac-rophages and B cells (27, 32, 34, 35). Therefore, we examinedwhether PYK2 is essential for chemokine-induced transmigrationof CD8 T cells in vitro (Fig. 2C). Interestingly, transmigrationinduced 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, butwas significantly lower in PYK2-deficient CD8 T cells. These datashow that although PYK2 is not required for LFA-1–independentchemotaxis of CD8 T cells, it is essential for LFA-1–dependentCD8 T-cell chemotaxis. Together, these results indicate thatPYK2 plays a critical role in the synergistic induction of CD8

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Fig. 1. Impaired activation of PYK2-deficient CD8 T cells in response to syn-ergistic TCR and LFA-1 stimulation in vitro. WT or PYK2-deficient T cells werestimulated 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 presenceor absence of 0.3 μg per well plate-bound ICAM-1. (A) Proliferation was ana-lyzed by 3H-thymidine uptake at 72 h. Graph shows average signal± 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-2expression in CD8 T cells was determined at 40 h by intracellular FACS stainingafter 5-h Brefeldin A treatment. (A–C) Data are representative of three ormore experiments.

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T-cell polarity in response to simultaneous LFA-1 and antigen orchemokine 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 invitro. To investigate whether PYK2 is essential for the activation ofCD8 T cells by APCs, PYK2-deficient CD8 T cells expressing thetransgenic P14 TCR, which recognizes the gp33-41 epitope ofLCMV, were stimulated with syngeneic dendritic cells pulsedwith various doses of gp33-41 peptide in vitro. PYK2 deficiencyimpaired P14 T-cell proliferation in response to stimulation, par-ticularly at low doses of peptide; high doses of gp33-41 peptidewere 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 theexpansion of CD8 T cells in response to LCMV infection, PYK2-deficient P14 CD8 T cells were adoptively transferred togetherwith wild-type P14 CD8 T cells at a 1:1 ratio into host mice. Fiveand 8 d after LCMV Armstrong challenge, the populations oftransferred PYK2-deficient and wild-type P14 CD8 T cells inblood and spleen were determined by FACS analysis of congenicmarkers (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 weresimilarly reduced, suggesting that the defect is a consequence ofimpaired expansion rather than altered distribution of the cellsbecause of a migration defect. A difference between PYK2-de-ficient and wild-type P14 CD8 T-cell number was noted as early asday 5 after LCMV infection. CFSE-labeling of P14 CD8 T cellsbefore adoptive transfer as above demonstrated that although alltransferred 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 challengewas not affected by PYK2-deficiency, further suggesting that theimpaired expansion of PYK2 knockout P14 CD8 T cells is nota consequence of an impact on proximal TCR signaling or thereceipt of IL-2 signals (Fig. S1). Furthermore, the small pop-ulation of PYK2-deficient P14 CD8 T cells that was presentduring the acute expansion phase did not display any differencesfrom wild-type P14 CD8 T cells in expression of the activationmarkers glycosylated CD43 (1B11), CD44, and CD62L, or IFN-γ,and TNF up-regulation upon restimulation with gp33-41 peptidein vitro (Fig. S2 A–C). This finding suggests the PYK2-deficientCD8 T cells were activated normally but failed to fully expandduring the acute phase of their response to LCMV.

CD8 T-Cell Intrinsic PYK2 Deficiency Results in a Loss of Short-LivedEffector Cells. Rapid expansion is an attribute that is specificallyassociated with short-lived effector CD8 cells. To investigatewhether PYK2-deficient P14 CD8 T cells have specific defects inthe generation of short-lived effector or memory-precursor effec-tor cell populations, expression of the specific markers IL-7Rα andkiller cell lectin-like receptorG1 (KLRG1) was analyzed by FACS.This finding revealed that in both blood and spleen, the frequencyof IL-7Rαlow/KLRG1high short-lived effector cells was markedlyreduced and that of IL-7Rαhigh/KLRG1low memory-precursor ef-fector cells increased in PYK2-deficient CD8 T cells (Fig. 4A).

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Fig. 3. PYK2 deficiency impairs the expansion of CD8 T cells in response toLCMV. (A) WT or PYK2-deficient P14 CD8 T cells were stimulated with den-dritic cells and the indicated concentrations of LCMV gp33-41 peptide in vitro.Proliferation was analyzed by 3H-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 transferredintohostmice (CD45.1+/CD45.1+). Thenext day,micewere infectedwith LCMVArmstrong 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 fromblood at day 8; numbers shown represent frequencies of the indicated pop-ulation within CD8 T cells. (C) Graph shows average total P14 CD8 T-cellnumbers in blood and spleen from three or more experiments (day 5: n = 10mice; day 8: n = 20 mice; day 15: n = 6mice) ± SEM. (D) WT and PYK2-deficientP14 transgenic CD8 T cells that were CFSE-labeled before adoptive transferwere 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).

16236 | www.pnas.org/cgi/doi/10.1073/pnas.1011556107 Beinke et al.

However, this reflected a decrease in total numbers of PYK2-deficient short-lived effector cells, whereas PYK2-deficientmemory-precursor effector cells were generated at similar numbers as wildtype (Fig. 4B). In vivo BrdU labeling on day 5 after infectiondemonstrated that a smaller fraction KLRG1+ PYK2 P14 CD8T cells underwent cell divisions compared with KLRG1+ wild-type P14 CD8 T cells (Fig. 4C). PYK2 knockout and wild-type P14CD8 T cells remaining at day 50 after LCMV infection expressedequivalent 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 dataindicate that PYK2 is essential for the generation of short-livedeffector CD8 T cells, but memory-precursor T cells develop in-dependently of PYK2.

Short-Lived Effector CD8 T-Cell Generation Is LFA-1–Dependent. CD8T-cell responses against LCMVwere previously reported to occurindependently of LFA-1 (40, 41). However, if the defect in short-lived effector cell expansion in PYK2-deficient P14 CD8 T cellsis 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 cellsdeficient in the CD11α subunit of LFA-1 together with the same

number of wild-type P14 CD8 T cells before LCMV Armstronginfection of host mice. Analyses of congenic CD45 markersrevealed impaired expansion of CD11α-deficient P14 CD8 T cellsin the blood at day 8 after infection (Fig. 5 A and C). There wasalso a shift in the frequencies of IL-7Rαlow/KLRG1high short-livedeffector versus IL-7Rαhigh/KLRG1lowmemory-precursor in CD11α-deficient P14 CD8 T cells compared with wild type P14 CD8 T cells(Fig. 5B). CD11α deficiency primarily affected total numbers ofshort-lived effector P14 CD8 T cell butmemory-precursor P14 CD8T-cell numbers were also reduced to some degree (Fig. 5C). Thedecrease CD11α-deficient P14 CD8 T-cell numbers in spleens wascomparably 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-cellgeneration similar to PYK2, but has additional functions duringCD8 T-cell responses that are probably attributed to its more ex-tensive role in T-cell adhesion.

DiscussionThis study reveals that PYK2 plays a critical role in integratingLFA-1 and TCR or chemokine signaling pathways to facilitatethe synergistic induction of cell polarity during CD8 T-cell acti-vation and migration. Furthermore, we demonstrate that PYK2and LFA-1 are important for the generation of short-lived ef-fector CD8 T cells, suggesting that cell polarity may be a criticaldeterminant of CD8 T-cell fate.

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Fig. 4. PYK2deficiency results in a loss of short-lived effector cells but doesnotaffectmemoryprecursor cell generation. (A–C)WTandPYK2-deficient P14CD8T cells were adoptively transferred and mice were infected with LCMV Arm-strongas in Fig. 3. (A) Representative FACS analysis of IL-7Rαlow/KLRG1high short-lived effector versus IL-7Rαhigh/KLRG1low memory precursor cells at day 8 afterinfection. (B)Graphshowsaveragetotal cellnumbersof short-livedeffectorcellsor memory precursor cells from three or more experiments (day 5: n = 10mice;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 incorporationon day 5 after LCMV infection. Data are representative of three experiments.

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Fig. 5. CD11α deficiency primarily affects the generation of short-lived ef-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 andadoptively transferred into host mice (CD45.1+/CD45.1+). The next day, chi-meric mice were infected with LCMV Armstrong. (A) Representative FACSanalysis of congenic markers on blood cells at day 8 after infection. Numbersshown represent frequencies of the indicatedpopulationwithinCD8T cells. (B)Representative FACS analysis of IL-7Rαlow/KLRG1high short-lived effector versusIL-7Rαhigh/KLRG1low memory precursor cells in the blood at day 8 after in-fection. (C)Graph showsaverage total cell numbers fromtwoexperiments (day8: n = 10mice; day 15: n = 10mice) ± SEM: *0.01 < P < 0.05; **0.001 < P < 0.01;***P < 0.001 (unpaired two-tailed Student’s t test).

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Although TCR and chemokine receptor stimulation inducesPYK2 phosphorylation, neither TCR-mediated activation norchemokine receptor-induced migration of T cells was criticallydependent on PYK2. However, LFA-1 coligation by ICAM-1revealed that CD8 T cells require PYK2 for LFA-1–dependentactivation and migration. In line with the observation that TCRand integrin costimulation induces synergistic PYK2 phosphor-ylation, LFA-1 could provide an additional signal for PYK2 ac-tivation (38). Therefore, PYK2 may integrate proximal signalingpathways downstream of LFA-1 and TCR or chemokine recep-tors to fully induce the reorganization of the cytoskeleton. Thissynergistic increase in cytoskeletal reorganization and spreadingmaycontribute to CD8 T-cell activation and migration in the presence ofphysiologic concentrations of antigen and chemokines. It may con-stitute an additional safeguard mechanism to allow full responses ofCD8 T cells only when both integrins and antigen or chemokinereceptors 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-all activation by surface-bound ligands thanCD4Tcells because theyrapidly reorient and spread. CD4 T cells may also be more depen-dent on additional costimulatory signals, such as CD28 (42).During the in vivo response to LCMV, ablation of PYK2 resulted

in a specific loss of short-lived effector CD8 T cells, but memory-precursor effector CD8 T-cell generation was normal. BecausePYK2 was important for LFA-1–induced spreading but not forLFA-1 activity, LFA-1–dependent interactions of PYK2-deficientCD8 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. CD11αdeficiency also primarily affected short-lived effector cell genera-tion, but there was some impact on memory-precursor effector cellnumbers. Therefore, we propose that LFA-1 activation of PYK2,rather than LFA-1–mediated T-cell contacts per se, sets a thresholdfor short-lived effector versus memory-precursor effector CD8T-cell differentiation.PYK2-mediated T-cell polarity could be important for CD8 T

cell short-lived effector fate by regulating the kinetics or qualityof the CD8 T cell APC interaction. This process may indirectlyimpact on antigen triggering and exposure to cytokines. In linewith the report by Chang et al., failure to polarize in the absenceof PYK2 could also result in the miss-localization of effector-fatedeterminants from the APC proximal daughter cell during CD8T-cell division (13). Furthermore, the loss of LFA-1 signaling viaPYK2 may impact on the formation or molecular makeup ofsignaling microclusters (43). The molecular mechanism behindthe role of PYK2 in short-lived effector fate is very exciting, butalso difficult to study. Because we have demonstrated that PYK2is only critical for CD8 T cells under physiological levels of TCRor chemokine receptor stimulation in the presence of appropri-ate LFA-1 costimulation, the molecular mechanisms would bebest studied in vivo, but this exceeds the scope of the presentstudy 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 mountnormal antiviral responses (40, 41, 44, 45). However, in the presentstudy we revealed a defect in the generation of LCMV-specificshort-lived effector cells as a consequence of both PYK2 andCD11α deficiency. This result was achieved using simultaneousadoptive transfer of wild-type and knockout P14 TCR transgenicCD8 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 ofshort-lived effector and memory-precursor effector cells is simi-lar (46). Short-lived effector and memory-precursor CD8 T-cellpopulations 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–deficient dendritic cells and subsequent immunization, the authorsalso observed that after initial normal onset of the CD8 T-cellresponse, CD8 T-cell numbers were decreased after 7 to 14 d.However, this finding was interpreted as a defect in the memoryphase of the CD8 T-cell response. Characterization of short-livedeffector versus memory-precursor CD8 T-cell populations in thissystem would shed light into whether the report is consistent withthe data presented here. Furthermore, Zehn et al. demonstratedcurtailed OT1 CD8 T-cell responses to low-affinity variants ofthe SIINFEKL peptide (11). Interestingly, the low-affinity TCRstimulation allowed early egress of CD8 T cells from lymph nodes,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 versusmemory-precursor effectorCD8T cells. This resultmay open a veryinteresting window of opportunity for therapeutic intervention bytargeting PYK2. PYK2 may be an interesting candidate for the re-duction of acute CD8 T-cell responses without completely ablatingCD8 T cell-mediated surveillance of acute viral infections anddormant retroviruses. Prevention of allograft rejection after trans-plantationby targetingPYK2maybeproblematic becausememory-precursor effector cells that develop independently of PYK2 maycause acute or chronic rejection. However, PYK2 inhibition maybe useful to reduce acute pathogenic CD8 T-cell responses in thecontext of viral infections, which cause severe tissue damage leadingto death (i.e., fulminant hepatitis or influenza), although main-taining the beneficial anti-viral response.

Materials and MethodsMice and Reagents. PYK2 and CD11α knockout mice (34, 48), BoyJ (CD45.1+),and P14 TCR transgenic mice bearing the DbGP33-specific TCR were fullybackcrossed to C57BL/6. All animals were housed in specific pathogen-freefacility at the University of California, San Francisco, according to Universityand National Institutes of Health guidelines. Antibodies were purchasedfrom 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 spleensor lymph nodes by MACS (MiltenYi Biotec) according to the manufacturersprotocol (purity >95%) and cultured in DMEM containing 10% FCS, 10 mMHepes, penicillin, streptomycin, 2 nM glutamate, 1 mM sodium pyruvate, 1×nonessential amino acids, and 50 mM 2-mercaptoethanol at 37 °C in thepresence of 5% CO2. Next, 2 × 105 T cells were stimulated in a 96-well platewith 0.5 or 0.1 μg/100 μL plate-bound anti-CD3 antibody per well in thepresence or absence of 0.3 μg /100 μL plate-bound ICAM-1-FC per well for 72h. P14 TCR transgenic CD8 T cells were stimulated in vitro with 1 μM to 40nM LCMV gp33-41 peptide and 2 × 104 CD11c MACS enriched syngeneicsplenic dendritic cells for 48 h. Proliferation was assessed by liquid scintilla-tion of 3H-thymidine uptake during the last 6 h of the culture or by FACSanalysis of CFSE labelled cells 72 h after stimulation. Cytokine expression wasdetermined 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 × 106 T cells were plated in 96-wellplates coated with 0.3 μg/100 μL plate-bound ICAM-1-FC per well on ice andstimulated with 0.5 μg/mL or 5 μg/mL soluble anti-CD3 antibody cross-linkedwith secondary antibody for 10 min at 37 °C. Nonadherent cells werewashed off before adherent cells were eluted and counted by FACS. CD8T-cell blast were generated by stimulating MACS-enriched CD8 T cells with10 ng/mL PMA and 200 ng/mL Ionomycin for 18 h and culturing them in thepresence of 50 U/mL IL-2 for 5 to 7 d. For immunofluorescent imaging of CD8T-cell blasts, cells were plated on ICAM-1-FC–coated glass coverslips for 10min at 37 °C and, after washing, F-actin was stained using Alexa Fluor 488phalloidin (Invitrogen) according to the manufacture’s protocol. Imageswere acquired using a Zeiss microscope.

16238 | www.pnas.org/cgi/doi/10.1073/pnas.1011556107 Beinke et al.

Transwell Migration Assay. Themigratory ability of T cells was measured using5-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 induplicates for each different chemokine (CXCL12, 400 ng/mL and CCL21, 1μg/mL). For transwell migration assays using filters coated with ICAM-1, 5-μmpolycarbonate 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 timeswith PBS and blocked with 2% BSA for 1 h at 37 °C. Filters were rinsed withPBS and dried. Coated filters were checked for leakage, then used fortranswell 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

cell/mice by i.v. tail-vein injection. Then, 24 to 48 h latermicewere infected i.p.

with 2 × 105 PFU 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 transferring1 × 106 cells per mice. FACS analysis of CFSE dilution was performed 66 h afterLCMVArmstrong infection. BrdU incorporation analysis was performed by i.p.injection of 10 μg BrdU per mouse at day 5 after LCMV Armstrong infectionand 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 withanimal maintenance and Andre Limnander and Byron Au-Yeung for in-spiring discussions, support, and critically reading the manuscript. This studywas supported in part by a long-term fellowship from the Human FrontierScience Foundation (to S.B.) and a Leukemia and Lymphoma Society SpecialFellow award (to H.P.); J.M.C. is a recipient of a National Science Foundationscholarship.

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