A septin requirement differentiates autonomous and contact-facilitated T cell proliferation

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1 A septin requirement differentites utonomous nd contct-fcilitted T cell prolifertion Adrin M Mujl 1, Juli K Gilden 1,3, Audrey Gérrd 1, Mkoto Kinoshit 2 & Mtthew F Krummel 1 T cell prolifertion is initited y T cell ntigen receptor (TCR) triggering, solule growth fctors or oth. In chrcterizing T cells lcking the septin cytoskeleton, we found tht successful cell division hs discrete septin-dependent nd septin-independent pthwys. Septin-deficient T cells filed to complete cytokinesis when prompted y phrmcologicl ctivtion or cytokines. In contrst, cell division ws not dependent on septins when cell-cell contcts, such s those with ntigen-presenting cells, provided niche. This septin-independent pthwy ws medited y phosphtidylinositol-3-oh kinse ctivtion through comintion of integrins nd costimultory signls. We were le to differentite etween cytokine- nd ntigen-driven expnsion in vivo nd thus show tht trgeting septins hs strong potentil to moderte detrimentl ystnder or homeosttic cytokinedriven prolifertion without influencing expnsion driven y conventionl ntigen-presenttion. T cell prolifertion rpidly expnds the numer of ntigen-specific cells, process tht is necessry to control infection. Typiclly, this type of cell division is initited y T cell interction with its cognte ntigen on n ntigen-presenting cell (APC), nd its mgnitude is determined y the strength of the TCR recognition event in tht cell-cell contct 1 3. Antigen-specific T cell clonl expnsion hs een reported to occur in the lymph node, where swrming T cells engge in cell-cell contcts with proximl APCs nd other ctivted T cells 4,5, nd this my represent niche for cell division. Yet cell division cn lso e driven y high locl cytokine concentrtions in the environment, in the possile sence of such cell-cell interction. This scenrio is considered possile hzrd for utoimmunity, s when non virus-specific ystnder cells encounter high concentrtions of cytokines produced y virl-specific T cells during n immune response in lymph node 2,6. Cytokine-driven cell division is lso importnt for homeosttic mintennce, wherein cytokines such s interleukin-7 (IL-7) or IL-15, in conjunction with trnsient low-ffinity peptide-mhc (p-mhc) TCR interctions, support turnover of clones 7. Asymmetric cell division hs een proposed to e pthwy tht cn influence the individulity of dughter cells 8, ut completion of cytokinesis hs een considered invrint. To our knowledge, it hs not een possile to clerly distinguish cytokine-driven cell division from TCR-driven cell division. The physicl event of cell division requires mny processes, including the functions of specific kinses 9, specific cytoskeleton proteins such s myosins nd, notly, septins Septins re GTP-inding proteins tht self-ssemle into tetrmeric, hexmeric or octmeric quternry structures nd then into lrge filments, rings nd guzes, in vitro nd in vivo, nd re ssemled on the cell cortex The mmmlin septins, of which there re 13, cn e divided into four groups; one from ech clss is required to form cnonicl complex. In mmmls, septin 7, the only one in its clss, ppers to e indispensle for the genertion of filments, nd its depletion leds to loss of other septin proteins 14,17 19, presumly s result of qulity-control processes. Septins were originlly identified s cell-division cycle muttions 2 nd re evolutionrily conserved in their crucil role in cytokinesis. Similrly to yest, septins in mmmls hve een found to e essentil for completion of cytokinesis 12,21. Septins re usully, ut not exclusively, found ssemled s ring t the clevge furrow 12,22. They hve een suggested to e essentil in coordinting myosin motor proteins during cell scission 1,23, remodeling the memrne s cytokinesis progresses 24 nd nchoring the midody ring structure to the memrne 25 s mother nd dughter cell seprtion is resolved. One exception to the requirement of septins for mmmlin cytokinesis is in T cells; septin 7 depletion in D1 cell lines leds to loss of the other septins ut results in ner-norml cell division in response to cues driven y APCs 17. T cell cytokinesis in the sence of septins hs lso een confirmed in Sept7 knockout mice 19. To investigte how T cells evde this highly conserved requirement, we generted mice with T cell specific depletion of septin 7 nd exmined CD8 + T cell ctivtion nd function under vriety of conditions. We found tht the requirements for septins in T cell division differ depending on whether T cells engge in cell contcts during cytokinesis. This finding led us to exmine how prolifertion occurs in septin-deficient (septin-null) CD8 + T cells in order to isolte the compenstory pthwys. Our results provide insight into the possiility of ttenuting cytokine-driven expnsion specificlly while leving ntigen-driven expnsion unffected. RESULTS Development of septin-deficient T cells is unimpired We engineered T cells to lck ll septins y crossing mice ering Cre recominse expressed from the promoter nd enhncer of Cd4 1 Deprtment of Pthology, University of Cliforni, Sn Frncisco, Sn Frncisco, Cliforni, USA. 2 Division of Biologicl Sciences, Ngoy University Grdute School of Science, Ngoy, Jpn. 3 Present ddress: Deprtment of Medicl Microiology nd Immunology, University of Wisconsin School of Medicine nd Pulic Helth, Mdison, Wisconsin, USA. Correspondence should e ddressed to M.F.K. (mtthew.krummel@ucsf.edu). Received 24 Mrch; ccepted 21 Octoer; pulished online 21 Decemer 215; doi:1.138/ni.333 nture immunology DVANCE ONLINE PUBLICATION

2 % of mx (SL8) c nti-tcr/cd28 (Cd4-Cre) to mice ering loxp-flnked Sept7 (Sept7 flox/flox ) to generte Sept7 conditionl knockout () mice 19. These mice were susequently crossed to trnsgenic mice ering the OT-I TCR llele. Mice from oth non OT-I nd OT-I ckgrounds demonstrted nercomplete loss of septin 7 in peripherl T cells, with smll proportion (5 1%) of cells tht retined septin expression, s ssessed y flow cytometry (Supplementry Fig. 1). To remove this contminting popultion, we took cre in ll future nlyses to eliminte these cells escpees from our experimentl nlysis when possile either y detection of intrcellulr septin 7 or use of mtmg mice 26, in which Cre recominse ctivity converts mtomto + cells to mgfp + cells. As in smll hirpin RNA (shrna) studies in cell lines 17, genetic deletion of septin 7 led to coordinte loss of other key T cell expressed septin fmily memers in peripherl T cells s ssessed y immunolotting (Supplementry Fig. 1). The Cd4-Cre llele is expressed in pre doule-positive (pre- DP, for CD4 nd CD8 expression) phse of thymic development 27, nd we oserved initil onset of septin loss in doule-negtive (DN, CD4 CD8 ) thymocytes with mximl loss y the single-positive (SP) stge in mice (Supplementry Fig. 1c). We oserved similr proportions of DN, DP nd SP thymocyte popultions (Supplementry Fig. 1d), equivlent cellulrity in secondry lymph node orgns (Supplementry Fig. 1e) nd similr frequencies of CD4 + nd CD8 + T cells within those orgns (Supplementry Fig. 1f). Additionlly, nive resting septin-deficient CD8 + T cells mintined norml mounts of filmentous ctin (Supplementry Fig. 1g), nd septin-deficient OT-I T cells showed morphologicl defects tht phenocopied pulished findings with septin knockdown in T cell clones 17,22 (Supplementry Fig. 1h,i). These results demonstrte tht development of septindeficient T cells in this mouse model is lrgely intct. Selective prolifertion defects in the sence of septins We sought to exmine T cell prolifertion in the context of control (Cd4-Cre + Sept7 flox/+ or CD4-Cre Sept7 flox/flox ) or Figure 1 Septin-deficient (SL8) T cells exhiit selective cytokinetic defects upon APC-independent stimultion. (,) Prolifertion () nd cellulr DNA content DAPI () of live ctivted cells, ssessed y flow cytometry (), nd cell numer recovery () in nd control CD8 + OT-I T cells 72 h fter ctivtion in vitro through culture with SL8-pulsed (1 ng/ml), on plte-ound nti-tcr nd solule nti-cd28 (nti-tcr/cd28) or stimultion with PMA nd ionomycin (). (c) Confocl imges of fixed nd control CD8 + polyclonl T cell nuclei stined with DAPI 48 h fter ctivtion. Scle rs, 1 µm. (d) Kinetics of OT-I multinucletion formtion through comprison of nd content y flow cytometry 72 h fter ctivtion s in. (e,f) Intrcellulr expression of c-myc (e) nd cell surfce expression of CD71 (f) expressed in nive or ctivted live nd control CD8 + OT-I T cells 24 h fter ctivtion. (g) Cell surfce CD69 levels expressed y live nd control CD8 + OT-I T cells 24 h fter culture together with pulsed with given OT-I peptide nd concentrtion. n.s., not significnt; **P <.1; ****P <.1, unpired t-test (). Dt re represesnttive of two (c), three (e g) or six (,d) independent experiments or re pooled from the verge vlues of technicl triplictes from three independent experiments (, men ± s.e.m.). d nti-tcr/cd28 g Conc. (ng/ml): N4 T4 Q4H7 V4 Cell numer ( 1 3 ) % of mx CD69 (SL8) n.s. e % of mx f % of mx c-myc CD71 nti-tcr/cd ** **** 6 4 CD8 + T cells isolted from these respective mice. Although septins re required for cell division in vrious types of eukryotic cells, T cells cn proliferte proficiently in the sence of septin 7 nd ssocited septin fmily memers 17,22,19. Consistent with this, we found tht, when cultured in vitro with one mrrow derived dendritic cells () pulsed with the OT-I peptide ntigen SL8, CD8 + OT-I T cells divided (Fig. 1 nd Supplementry Fig. 2), progressed in the cell cycle nd expnded in numer t similr rte to wild-type cells (Fig. 1). Unexpectedly, however, when ctivted with plte-coted nti-tcr ntiody or solule phorol myristte cette (PMA) nd ionomycin, septin-deficient OT-I T cells underwent fewer cell divisions, s ssessed y dilution of the cytosolic dye (Fig. 1 nd Supplementry Fig. 2) nd cell recovery (Fig. 1) fter 72 h. Polyclonl CD8 + T cells exhiited these celldivision defects s well (dt not shown). Additionlly, wheres stimultion with generted lrgely conventionl G1 S G2/M cell-cycle profiles, stimultion in the sence of APCs resulted in i- nd multinucleted cells, s detected y flow cytometry (Fig. 1 nd Supplementry Fig. 2) nd confocl microscopy (Fig. 1c). This differentil lock in cell division ws lso ccompnied y n ccumultion of septin-deficient cells of incresed size (FSC-A hi ) s : nive : ctivted : ctivted nti-tcr/cd28 nti-tcr/cd28 DVANCE ONLINE PUBLICATION nture immunology

3 Figure 2 Septin-deficient T cells undergo cytokinetic filure fter cytokine exposure. () Prolifertion, s indicted y dilution, of live nive nd control CD8 + OT-I T cells fter in vitro culture with IL-7 (5 ng/ml) nd IL-15 (1 ng/ml) (top), or IL-2 (5, U/ml) (ottom) for 5 d. () Intrcellulr levels of phosphorylted STAT5 (p-stat5) in live control nd CD8 + OT-I T cells fter IL-2 exposure 24 h fter cells were stimulted with PMA nd ionomycin. (c) dilution nd levels in live nd control CD8 + OT-I T cells cultured in vitro with IL-7 nd IL-15 (top) or IL-2 (ottom). (d) Prolifertion of live nd control CD8 + OT-I T cells 72 h fter in vitro ctivtion, with or without IL-2 (1 2 U/ml) t initil plting. Dt re representtive of t lest three independent experiments. iono, ionomycin; nti-tcr/cd28, nti-tcr nd nti-cd28. c IL-7, IL-15 IL-7, IL-15 IL-2 [IL-2] (U/ml): % of mx p-stat d % of mx nti-tcr/cd28 compred to septin-competent cells from OT-I mice in T cell cultures stimulted with nti-tcr or PMA nd ionomycin IL-2 (Supplementry Fig. 2c). Together, these dt demonstrte tht T cells re not intrinsiclly unique in not requiring septins for cytokinesis, s hs een proposed 19 ; rther, only certin cytokinetic pthwys re septin independent. Moreover, in ssessing how the genertion of multinucleted cells reltes to cell division, we oserved tht septin-deficient cells were susceptile to cytokinesis filure with every division, not just the first one (Fig. 1d). This oservtion suggests tht filure to divide is stochstic event, with the limited expnsion of T cells to APC-independent stimuli resulting from rekthrough event with ech division. The division defect ws not n ovious result of differentil loss of filmentous ctin with some stimuli nd not others, s phlloidin stining t 24 h ws identicl etween nd control OT-I cells (Supplementry Fig. 2d). Additionlly, proximl signling in response to ll cues ws unffected y septin 7 depletion. This ws pprent in equivlent upregultion of c-myc nd CD71 fter stimultion (Fig. 1e,f). Further, OT-I cells stimulted y APC-dependent or APC-independent stimuli upregulted CD69 nd CD25 similrly (Supplementry Fig. 2e,f). Finlly, OT-I T cell clcium flux in response to nti-cd3 crosslinking or thpsigrgin lockde of srcoendoplsmic reticulum clcium trnsport ATPse (SERCA) uptke ws lso equivlent to tht of wild-type cells (Supplementry Fig. 2g). To determine whether the distinctions mong these stimuli relted to strength of signl, we cultured T cells from control nd OT-I mice with tht hd een pulsed with peptides differing in OT-I TCR-pMHC ffinity cross rnge of concentrtions nd mesured CD69 upregultion fter 24 h (Fig. 1g). Wek gonist peptides nd lower doses induced less ctivtion y this mesure, ut cells ehved identiclly to controls, demonstrting tht T cell sensed the density nd identity of TCR signls similrly to wild-type cells. Thus, the differences we oserved in T cell prolifertive cpcity stimuli did not stem from defective TCR signling or cell-cycle entry ut suggest, rther, tht APCs contriute key cellulr fctors tht fcilitte division of T cells. division defects with APC-independent stimuli Solule cytokines lso sustntilly drive T cell expnsion, so we tested whether this stimulus would led to cell-division defects in : PMA + IL-2 : PMA : PMA + IL-2 septin-null T cells. We found tht nive septin-deficient CD8 + OT-I T cells did not divide in vitro fter exposure to homeosttic cytokines IL-7 nd IL-15 or to high concentrtions of IL-2 (ref. 28) (Fig. 2 nd Supplementry Fig. 3). Defects in in vitro prolifertion did not pper to result from dysfunctionl signling, s the extent of phosphoryltion of STAT5, trget of these cytokine receptors, ws similr etween CD8 + OT-I cells nd control T cells (Fig. 2). As with TCR stimultion in the sence of APCs, solule cytokines induced multinucleted septin-deficient CD8 + T cells (Fig. 2c). The comintion of APC-independent ctivtion (y PMA or nti-tcr) with ddition of IL-2 lso filed to rescue in vitro prolifertion, which suggests tht the defect we oserved did not result from indequte cytokine production (Fig. 2d nd Supplementry Fig. 3). We concluded insted tht, in contrst to stimuli from, cytokines lone do not support cytokinesis of septin-null T cells. Rescue of defective prolifertion through cell contcts We next sought to determine whether were providing dditionl signls tht overcme lock in prolifertion in cells. To do so, we gin ctivted T cells in vitro with PMA nd ionomycin s se stimulus. Addition of peptide-pulsed to these PMActivted OT-I T cell cultures s n dd-ck lrgely restored cell division, s ssessed y dilution (Fig. 3 nd Supplementry Fig. 4). This prtil rescue ws equivlent when lcking ntigenic peptide were dded, demonstrting tht medite rescue independently of their ility to generte strong TCR signls. However, superntnt from competent BMDC T cell cultures, dded t 2% of the totl culture volume, ws unle to restore division, which suggests tht cell-cell contct ws primrily responsile for this rescue effect. In ddition, we found tht resting B cells were unle to support cell division in CD8 + OT-I T cells, wheres lipopolyscchride (LPS)-ctivted B cells fcilitted enhnced prolifertion (Fig. 3 nd Supplementry Fig. 5), though not to the sme extent s (Fig. 1). Tht nd LPS-treted B cells supported OT-I T cell division suggested tht this rescue ws medited y cellulr properties unique to highly ctivted APCs. nture immunology DVANCE ONLINE PUBLICATION

4 Figure 3 APCs medite rescue of septinnull CD8 + T cell cytokinetic defect through costimultory PI(3)K signling. () dilution of live or control CD8 + OT-I T cells stimulted with PMA nd ionomycin nd cultured t the time of plting with n dd-ck of SL8-loded (left, 1 ng/ml SL8), ntigen-free (middle) or 2% superntnt generted from wild-type BMDC-T cell cultures (right). () dilution of live or control CD8 + OT-I T cells fter 72 h of culture with resting or LPS-treted, SL8-pulsed splenic B cells. (c,d) dilution of live or control CD8 + OT-I T cells stimulted with PMA nd ionomycin (), cultured with ntigen-free nd treted with nti-cd8 nd nti-cd86 (nti-cd8/86) nd/or nti LFA-1 (c) or PI(3)K inhiitors LY2942 (1 µm) or GDC-941 (1 µm) (d) t vrious times fter plting. (e) dilution of or control CD8 + OT-I live ctivted T cells cultured with SL8-pulsed (1 1 ng/ml) nd treted with LY2942 (1 µm) or GDC-941 (1 µm) 36 h fter plting. Dt re representtive of three (,e), four () or five (c,d) independent experiments. In ddition to TCR signls, APCs provide numerous ccessory cues for T cells, nd we investigted severl of these. We found tht signling from costimultory molecules to CD28 nd from ICAM dhesion molecules to integrin LFA-1 represented prominent portion of the rescue; ntigen-free restored cell division to OT-I cells, nd this effect ws prtilly inhiited with locking ntiodies to CD8 nd CD86 or ntiodies to LFA-1 (Fig. 3c nd Supplementry Fig. 4). This locking ws even more profound when nti- + Antigen-free + Antigen-free CD8 nd nti-cd86 were comined with nti LFA-1. Surprisingly, lockde ws nerly s effective when these ntiodies were dded 24 h fter the initil stimultion with PMA nd ionomycin plus. This finding suggests tht rescue of cell division ws medited y in cell-cell contcts tht tke plce well fter the initition of TCR signls. To test whether these interctions were purely dhesive or resulted from signling, we repeted the BMDC dd-ck experiments with severl inhiitors tht trget phosphtidylinositol-3-oh kinse (PI(3)K), key downstrem signl trnsduction molecule in CD28 nd LFA-1 pthwys. We found tht pn-pi(3)k inhiitor compounds LY2942 nd GDC-941 locked the BMDC-medited rescue of OT-I cell division, with modest reduction in control T cell prolifertion (Fig. 3d nd Supplementry Fig. 4c). Wild-type cell viility, however, ws not grossly ffected t the dose used (dt not shown). Notly, lockde of PI(3)K signling reduced septin-null OT-I T cell prolifertion, whether inhiitors were dded 24 h fter BMDC ddition or t the onset of culture. To extend these findings to more physiologicl setting, we inhiited PI(3)K signling 36 h fter culturing nd control OT-I T cells with SL8- pulsed nd found tht tretment reduced T cell prolifertion (Fig. 3e nd Supplementry Fig. 4d). Notly, control T cell prolifertion ws modestly inhiited, with more sustntil loss in prolifertion when PI(3)K ws inhiited 24 h fter initil % of mx c % of mx % of mx d % of mx % of mx e % of mx + SL8-loded Time (h): Time (h): (SL8) Time (h): + Antigen-free LY2942 (36) nti-cd8/86 LY2942 () GDC-941 (36) + BMDC-T superntnt nti LFA-1 LY2942 (24) : PMA : PMA + dd-ck : PMA + dd-ck % of mx nti-cd8/86 nti LFA-1 nti-cd8/86 24 Resting B cells nti LFA-1 24 GDC-941 () LPS-ctivted B cells nti-cd8/86 nti LFA-1 24 GDC-941 (24) culture with (Supplementry Fig. 4e). Although the mgnitude of this effect ws lrger for septin-null T cells, these results imply tht ongoing PI(3)K ctivity is required for mximl prolifertion even in wild-type T cells. Consistently with these results, the differing cpcities of resting nd LPS-treted B cells in fcilitting OT-I T cell division were not due to differences in proximl T cell ctivtion, s ssessed y CD69 upregultion (Supplementry Fig. 5). T cell division driven y LPS-treted B cells ws medited through prolonged signling vi CD28 nd LFA-1 nd dependent on PI(3)K (Supplementry Fig. 5,c). Together, these findings support model in which APCs estlish niche of cell-cell contct interctions tht is chrcterized y PI(3)K signling nd complements or compenstes for septin function in CD8 + T cell division. Polrized contcts support T cell division ering CD8, CD86 nd ICAM-1 re likely to represent polrized surfce during cell division, nd we sought to ddress whether tht feture is sufficient to complement septin deficiency. To ddress this, we cultured PMA-ctivted OT-I T cells in wells coted with dhesive molecules including ICAM-1, fironectin nd ntiodies ginst CD44 nd CD28. Of these, ICAM-1 nd nti-cd28 were uniquely cple of enhncing division of septin-null CD8 + T cells (Fig. 4 nd Supplementry Fig. 3c). These findings suggest tht % of mx LY2942 (36) GDC-941 (36) DVANCE ONLINE PUBLICATION nture immunology

5 Figure 4 Costimultory signling is sufficient to enhnce septin-null CD8 + T cell division. () dilution of live or control CD8 + OT-I T cells stimulted with PMA nd ionomycin nd cultured on uncoted surfces or plte-ound Fc-ICAM, fironectin, nti-cd44 or nti-cd28 for 72 h. () dilution of live or control CD8 + OT-I T cells stimulted with PMA nd ionomycin () for 48 h nd cultured on plte-ound Fc-ICAM during vrious intervls fter initil plting. Dt re representtive of four independent experiments. fcilitte septin-null division through specific signling, not merely through generl dhesion nd cellulr contct. Furthermore, y selectively plting OT-I T cells with ICAM-1 t vrious time points during stimultion, we determined tht rescue ws tking plce t lest 24 h fter initil ctivtion ut not t the time of initil stimultion (Fig. 4 nd Supplementry Fig. 3d). T cells egin dividing t lest 24 h fter ctivtion 4, so this temporl window coincided with the kinetics of T cell entry into the cell cycle nd ongoing division. Septin dependence seprtes prolifertion drivers in vivo Our in vitro findings suggest tht the requirement for septins in T cell division distinguishes whether cell division is driven y the presence or sence of specific contcts or niches. We therefore compred different ctivting stimuli in vivo. Using the Dec-OVA model of ntigen delivery, in which n ntiody to the dendritic cell (DC) mrker DEC-25 is conjugted to ovlumin (OVA), to lod ntigens onto lymph node resident DCs 29, we found tht doptively trnsferred septin-null CD8 + OT-I T cells expnded similrly to their cotrnsferred wild-type counterprts (Fig. 5). To test whether T cells continue to require the presence of ntigen-loded APCs fter initil ctivtion, we cultured leled nd control CD8 + OT-I T cells with SL8-pulsed for 36 h, t which point we isolted the T cells. We then replted nd control T cells or cotrnsferred them to ntigen-free mice. When the T cells were cultured in vitro, T cells showed notle cell division defects (Fig. 5). T cells, however, did not divide s proficiently s control T cells when trnsferred to ntigen-free host mice (Fig. 5). These findings suggest tht lthough T cells do not require ntigenering APCs during cell division, endogenous in vivo interctions tht re sent from the T cell only cultures re sufficient to support successful cell division. % of mx % of mx Fc-ICAM Fironectin Fc-ICAM ( 48 h) nti-cd44 Fc-ICAM ( 24 h) nti-cd28 Fc-ICAM (24 48 h) : PMA + dd-ck : PMA only : PMA + dd-ck : PMA + Fc-ICAM : PMA only : PMA + Fc-ICAM In contrst, when nti IL-2 complexes were delivered to generte cytokine-medited prolifertion in the sence of overt APC involvement, expnsion of doptively trnsferred septin-null OT-I T cells ws significntly reduced compred to tht of control cells (Fig. 5c). As n dditionl cytokine-medited in vivo chllenge, we trnsferred polyclonl wild-type nd cells into sulethlly irrdited mice. In these mice, T cells typiclly undergo slow form of lymphopeni-induced prolifertion, which is thought to mimic n cute form of homeosttic expnsion 3. Agin, the overll expnsion of septin-null CD8 + T cells ws impired (Fig. 5d). These findings support context-dependent requirement for septins in T cell division nd extend our model to criticl in vivo processes. Septin requirement for CD8 + T cell homeostsis We found evidence for defects in stedy-stte homeosttic mintennce of nive nd memory CD8 + T cells, which is in greement with findings tht septin 7 is required for cytokine-driven prolifertion. Although peripherl CD8 comprtments were comprle etween nd control OT-I mice t 6 8 weeks of ge, the frequency of CD8 + T cells declined fter 6 months (Fig. 6). In ddition, we found tht decline ws ccompnied y significnt loss of phenotypiclly nive CD44 lo int CD8 + T cells in ged OT-I mice. Of the CD8 + T cells tht remined in ged mice, most were CD44 hi, in contrst to control cells tht showed imodl expression (Fig. 6), which suggests lymphopenic environment in which surviving CD8 + T cells my e responding to ntigen 31,32. Given tht memory CD8 + T cell homeostsis lso relies on Figure 5 Septin deficiency differentites APC- nd cytokine-driven division in vivo. () Frequency of cotrnsferred control or CD8 + OT-I T cells expnded in drining inguinl lymph nodes 6 d fter sucutneous immuniztion of host mice with Dec-OVA nd nti-cd4. Ech symol represents n inguinl lymph node (left or right flnk) from host mouse. () Top, rtio of control to live T cells 48 h nd 72 h fter in vitro culture (left) nd representtive dilution profile 48 h fter T cell isoltion (right) of nive nd control CD8 + OT-I T cells cultured with SL8-pulsed (1 ng/ml) in vitro for 36 h, isolted nd cultured in vitro with low-dose IL-2 (1 U/ml). Bottom, rtio of control to T cells recovered from host-mouse spleen 48 h fter cotrnsfer (left) nd dilution profile (right) of nive nd control CD8 + OT-I T cells cultured with SL8-pulsed (1 ng/ml) in vitro for 36 h, isolted nd trnsferred to ntigenfree host mice. P =.351, one-smple t-test compring distriution to theoreticl men of 1. Ech symol represents n individul mouse (ottom) or in vitro culture smple (top). (c) Frequency of control or CD CD8 + OT-I T cells tht expnded in the spleen nd inguinl lymph nodes (LN) of CD host mice fter 7 d of dily CD8 + T cell rtio (control/) CD8 + T cell rtio (control/) % CD8 + cells h Spleen n.s. 72h c % CD8 + cells d % CD8 + cells **** **** Spleen **** **** Spleen LN LN intrperitonel delivery of IL-2 complex. (d) Frequency of cotrnsferred nd control polyclonl CD8 + T cells in the spleen nd skin-drining LN of sulethlly irrdited mice. Ech symol represents n individul mouse (c,d). n.s., not significnt; ****P <.1, with pired (,d) or unpired (c) t-test. Dt re representtive of three independent experiments with n = 3 5 mice (,d) or pooled from two (, top) or three experiments with n = 7 (, ottom) or 8 mice (c); error rs, men ± s.e.m. ( d). nture immunology DVANCE ONLINE PUBLICATION

6 Figure 6 Septins re required for homeosttic mintennce of nive nd memory CD8 + T cells in vivo. () Frequency of CD8 + T cells (left) nd CD8 + T cell CD44 surfce expression (middle nd right) in pooled lymph nodes from 6- to 8-weekold (top) or 6-month-old (ottom) OT-I mice. () Rtio of control to cells sorted from spleens of mice 14 d fter intrvenous immuniztion with Dec-OVA (top) or detected in spleens of ntigen-free host mice 8 weeks fter trnsfer of sorted OT-I T cells from immunized mice (ottom). n.s., not significnt; *P <.5. unpired t-test (); P >.5 (, top) 6 8 wk >3 wk CD4 CD % of CD8 + % of CD8 + 8 n.s * % of mx % of mx CD44 CD8 + T cell rtio (control/) CD8 + T cell rtio (control/) Dy 14 fter Dec-OVA immuniztion Spleen Week 8 fter trnsfer nd P =.243 (, ottom), one-smple t-test compring distriution to theoreticl men of 1. Ech symol represents n individul mouse ( nd, ottom) or n independent experiment (, top). Dt re pooled from 4 (, ottom), 5 (, ottom;, top) or 6 (, top) independent experiments with totl n = 6 (, top), 8 (, ottom), 5 (, top), or 6 (, ottom) mice (men ± s.e.m.). CD44 Spleen cytokines, we exmined mintennce of memory CD8 + T cells. Nive nd wild-type CD8 + OT-I T cells were cotrnsferred to host mice tht were immunized intrvenously (i.v.) with Dec- OVA. T cells were detected t similr frequencies to tht of wild-type T cells in the spleen 14 d lter (Fig. 6) ut were slightly lower in numer in lymph nodes (Supplementry Fig. 6) nd showed high expression of memory precursor cell mrkers CD44 nd CD62L (Supplementry Fig. 6). nd wild-type OT-I T cells were then sorted from the spleen nd lymph nodes nd cotrnsferred to ntigen-free mice. The frequency of nd control T cells in these mice ws determined 8 weeks lter, s studies hve demonstrted tht mny rounds of homeosttic turnover occurs over this period of time 33. Indeed, the frequency of T cells ws significntly lower thn tht of control T cells in ntigen-free host spleens fter 8 weeks (Fig. 6), which indictes tht septins hve role in fcilitting memory T cell homeostsis. In contrst, lthough we noted trend towrd preferentil wild-type T cell mintennce in the lymph node, it ws not s severe s in the spleen (Supplementry Fig. 6c). This oservtion my indicte different milieus etween the two orgns tht support homeosttic division ut remins n re of future investigtion. Together, these dt demonstrte tht lck of septins impedes T cell division in stimulus-dependent mnner in vivo. DISCUSSION We hve identified sitution nd mechnism tht differentites APC- nd niche-driven prolifertion from niche-independent division. This suggests tht it might e possile to trget specific types of T cell division with, for exmple, cytoskeletl inhiitors. This is of prticulr importnce, s homeosttic expnsion rising from lymphopenic conditions cn result in rejection of orgn trnsplnts nd, possily, in utoimmunity nd is not responsive to costimultory lockde A prepondernce of clinicl immunosuppressive gents re directed ginst ntigen-specific T cell expnsion, typiclly trgeting pthwys downstrem of T cell ctivtion, such s those involving mtor or clcineurin 35. Drugs tht re ville to restrict homeosttic division, such s mycophenolic cid, trget nonspecific processes, such s DNA synthesis 39, tht re chrcteristic of ll forms of T cell division nd thus leve ptients ill-equipped to fce pthogen chllenge. Our work suggests tht trgeting septins nd the pthwys they use might yield etter therpeutic strtegies in such cses. Pulished studies hve found tht T cells re mong select few eukryotic cells tht do not pper to require septins to complete cell division 17,19. We now understnd tht the T cell exception reflects role for septins in cell division tht is not cell-type dependent ut rther context dependent. In the cse of T cells, the context tht rescues cell division is the presence of highly dhesive nd ctivted surfce or cell type. In vivo lymph node imging studies of proliferting T cells hve shown tht ntigen-specific T cells divide independently of contct with leled APCs 5, though division fter DC contct hs lso een noted 4. Our findings tht APCs or surfces high in specific costimultory molecules nd integrins rescue septin-null T cell cytokinetic defects in vitro nd in vivo provide support tht niche in which T cells engge in contct interctions during the temporl window of division serves functionl role. We oserved spectrum of septin-null T cell prolifertive competence cross conditions of ctivtion (PMA nd ionomycin; nti-tcr nd nti-cd28; nd ntigen-pulsed APCs), n effect tht corresponds to the degree of PI(3)K signling generted y these stimuli. It is thus possile tht threshold of totl PI(3)K signling must e reched for T cells to divide in the sence of septins; however, cytokines such s IL-7 nd IL-2 generte PI(3)K ctivity through their receptors, nd we did not find prtil or full rescue of the cytokinesis defect in the presence of these cytokines. Distinctions in how nd when PI(3)K is ctive proly underlie this oservtion, nd it is lso possile tht the ctivting PI(3)K cue is counterlnced y dditionl cell-cell cues. On the sis of the dt, we fvor model in which APCs medite septin-null cell division y providing highly polrized source of PI(3)K signling nd/or stilized cell cortex t or ner the time of cell division. As septins hve een descried to estlish polrity in yest nd reinforce locl memrne comprtmentliztion y serving s diffusion rrier 41 43, it is tempting to speculte tht septins nd PI(3)K cooperte or ct redundntly to mintin polrity while T cells undergo cell division. Notly, comprtmentliztion of phospholipids hs een descried s importnt during cytokinesis, s the cytokinetic furrow is enriched for phosphtidylinositol-(4,5)- isphosphte 44,45. Although studies hve found PI(3)K signling within the first 8 h of T cell ctivtion to hve the most crucil impct on eventul prolifertion 46, our dt using PI(3)K inhiitors suggest tht PI(3)K signling even 24 h fter T cell ctivtion continues to regulte wild-type T cell prolifertive potentil nd kinetics. Indeed, dul PI(3)K septin phrmcologicl inhiition might prove selective in locking ntigen-independent prolifertion of leukemic cells while spring the prolifertion of ntigen-dependent host T cells. One notle result of our work is tht T cell signling, even t synpse, is independent of septins. Our originl impetus for studying these proteins ws the oservtion tht they ssemled densely s ring round the immune synpse (J.K.G. nd M.F.K., unpulished oservtions). To this extent, our collective dt re t odds with studies tht use shrna pproches to link septins to efficient colocliztion of the clcium sensor STIM1 nd the clcium chnnel DVANCE ONLINE PUBLICATION nture immunology

7 ORAI nd susequent clcium flux in HeL, Jurkt nd humn emryonic kidney (HEK) cells 47,48. We, in contrst, did not find tht differences in septin-deficient T cell division cpcity stemmed from defects in TCR signling processes. One possile reson for this difference would e some form of compenstion in our cells for this specific septin function. The presence of cell division defects rgues ginst uiquitous compenstion, nd the ide of signlingspecific compenstion is not supported y dt in which norml clcium flux ws oserved despite Cre trnsfection of Sept7 flox/flox T cell lsts (dt not shown). An lterntive explntion for the pprent septin defect reported in cell lines my e tht these cells re experiencing cytokinetic filure nd, thus, tht defective signling is reltively distl to septin deficiency. At present, lthough we cnnot confirm tht there is requirement of septins for T cell clcium signling, further work my e needed to determine how nd when our findings lign with pulished signling studies. Why were we le to isolte T cells from the periphery of cko mice if cell division is possily compromised? One nswer is tht lthough the Cd4-Cre llele depletes septins in the pre-dp window, T cells in the post-dp stge typiclly do not divide gin unless clled upon to do so for homeosttic expnsion. Additionlly, with respect to homeosttic processes, lthough septin-null cells were clerly defective in expnding in vivo when trnsferred to sulethlly irrdited mice, the effect ws not s profound s the division defect to pure cytokines in vitro. It my e tht some mintennce, if not expnsion, of T cells in developing mice cn involve synptic cell-cell encounters tht re septin independent. For exmple, IL-15 is trns-presented to CD8 + T cells y nother cell ering the α-chin 49. Additionlly, T cells t stedy stte engge in cellulr contct with APCs presenting selfpeptide MHC nd/or firolstic reticulr cells tht produce homeosttic cytokines such s IL-7 (ref. 7). Wht is the redth of this septin requirement in homeosttic processes? Septin-null T cells ppered to selectively lose CD44 CD8 + popultions in lymph nodes 31,32, nd we lso oserved defective homeostsis of memory CD8 + OT-I T cells in the spleen. In light of the trend in septin requirement, these findings suggest tht endogenous cell-cell contcts in these settings do not medite T cell division s do endogenous cell-cell contcts for ctivted T cells. Although we found no specific defects in septindeficient T cells for effector genertion, s ssessed y surfce mrkers or interferon-γ t dy 6 fter immuniztion (dt not shown), lte-stge cellulr contcts such s those during symmetric cell division or in lte-stge T T interctions 8,5 my supply n dditionl, higher degree of complexity in T cell effector nd memory development s well s clonl urst size nd sustined survivl. In sum, the oserved distinction etween septin-dependent nd septinindependent immune cell cytokinesis indictes possiilities for enhnced selective trgeting of prolifertive processes. Methods Methods nd ny ssocited references re ville in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We thnk the Biologicl Imging Development Center personnel (UCSF) for technicl ssistnce with imging, J. Roose nd O. Ksiond (UCSF) for regents nd helpful discussion, E. Plmer (University Hospitl Bsel nd University of Bsel) nd D. Zehn (Swiss Vccine Reserch Institute nd Lusnne University Hospitl) for regents, nd M. Kuhns (University of Arizon College of Medicine) for criticl reding of the mnuscript. Supported y the US Ntionl Institutes of Helth (R1AI52116 to (M.F.K). AUTHOR CONTRIBUTIONS A.M.M. nd M.F.K. designed the experiments for ll primry text figures; A.M.M. did experiments; J.K.G. performed nd helped with experiments relted to initil chrcteriztion of mice; A.G. contriuted to experimentl design nd nlysis; M.K. provided the Sept7 flox/flox mice nd consulted on experiments; A.M.M. nd M.F.K. wrote nd revised the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t reprints/index.html. 1. Hogquist, K.A. & Jmeson, S.C. The self-osession of T cells: how TCR signling thresholds ffect fte decisions nd effector function. Nt. Immunol. 15, (214). 2. Murli-Krishn, K. et l. In vivo dynmics of nti-virl CD8 T cell responses to different epitopes. An evlution of ystnder ctivtion in primry nd secondry responses to virl infection. Adv. Exp. Med. Biol. 452, (1998). 3. Zehn, D., Lee, S.Y. & Bevn, M.J. Complete ut curtiled T-cell response to very low-ffinity ntigen. Nture 458, (29). 4. Mempel, T.R., Henrickson, S.E. & von Andrin, U.H. T-cell priming y dendritic cells in lymph nodes occurs in three distinct phses. Nture 427, (24). 5. Miller, M.J., Sfrin, O., Prker, I. & Chln, M.D. Imging the single cell dynmics of CD4 + T cell ctivtion y dendritic cells in lymph nodes. J. Exp. Med. 2, (24). 6. Ehl, S., Homch, J., Aichele, P., Hengrtner, H. & Zinkerngel, R.M. Bystnder ctivtion of cytotoxic T cells: studies on the mechnism nd evlution of in vivo significnce in trnsgenic mouse model. J. Exp. Med. 185, (1997). 7. Tkd, K. & Jmeson, S.C. Nive T cell homeostsis: from wreness of spce to sense of plce. Nt. Rev. Immunol. 9, (29). 8. Chng, J.T. et l. Asymmetric T lymphocyte division in the initition of dptive immune responses. Science 315, (27). 9. Nigg, E.A. Mitotic kinses s regultors of cell division nd its checkpoints. Nt. Rev. Mol. Cell Biol. 2, (21). 1. Oh, Y. & Bi, E. Septin structure nd function in yest nd eyond. Trends Cell Biol. 21, (211). 11. Surk, M.C., Tsng, C.W. & Trimle, W.S. The mmmlin septin MSF loclizes with microtuules nd is required for completion of cytokinesis. Mol. Biol. Cell 13, (22). 12. Estey, M.P., Di Cino-Oliveir, C., Froese, C.D., Bejide, M.T. & Trimle, W.S. Distinct roles of septins in cytokinesis: SEPT9 medites midody scission. J. Cell Biol. 191, (21). 13. Estey, M.P. et l. Mitotic regultion of SEPT9 protein y cyclin-dependent kinse 1 (Cdk1) nd Pin1 protein is importnt for the completion of cytokinesis. J. Biol. Chem. 288, (213). 14. Kinoshit, M., Field, C.M., Coughlin, M.L., Stright, A.F. & Mitchison, T.J. Self- nd ctin-templted ssemly of mmmlin septins. Dev. Cell 3, (22). 15. Kinoshit, M. Assemly of mmmlin septins. J. Biochem. 134, (23). 16. Rodl, A.A., Kozuowski, L., Goode, B.L., Druin, D.G. & Hrtwig, J.H. Actin nd septin ultrstructures t the udding yest cell cortex. Mol. Biol. Cell 16, (25). 17. Tooley, A.J. et l. Amoeoid T lymphocytes require the septin cytoskeleton for corticl integrity nd persistent motility. Nt. Cell Biol. 11, (29). 18. Aget-Ishihr, N. et l. Septins promote dendrite nd xon development y negtively regulting microtuule stility vi HDAC6-medited decetyltion. Nt. Commun. 4, 2532 (213). 19. Menon, M.B. et l. Genetic deletion of SEPT7 revels cell type-specific role of septins in microtuule destiliztion for the completion of cytokinesis. PLoS Genet. 1, e14558 (214). 2. Hrtwell, L.H. Genetic control of the cell division cycle in yest. IV. Genes controlling ud emergence nd cytokinesis. Exp. Cell Res. 69, (1971). 21. Mostowy, S. & Cossrt, P. Septins: the fourth component of the cytoskeleton. Nt. Rev. Mol. Cell Biol. 13, (212). 22. Gilden, J.K., Peck, S., Chen, M.Y.C. & Krummel, M.F. The septin cytoskeleton fcilittes memrne retrction during motility nd leing. J. Cell Biol. 196, (212). 23. Joo, E., Surk, M.C. & Trimle, W.S. Mmmlin sept2 is required for scffolding nonmuscle myosin II nd its kinses. Dev. Cell 13, (27). 24. El Amine, N., Kechd, A., Jnnji, S. & Hickson, G.R.X. Opposing ctions of septins nd Sticky on Anillin promote the trnsition from contrctile to midody ring. J. Cell Biol. 23, (213). 25. Kechd, A., Jnnji, S., Ruell, Y. & Hickson, G.R.X. Anillin cts s ifunctionl linker coordinting midody ring iogenesis during cytokinesis. Curr. Biol. 22, (212). 26. Muzumdr, M.D., Tsic, B., Miymichi, K., Li, L. & Luo, L. A glol doulefluorescent Cre reporter mouse. Genesis 45, (27). nture immunology DVANCE ONLINE PUBLICATION

8 27. Lee, P.P. et l. A criticl role for Dnmt1 nd DNA methyltion in T cell development, function, nd survivl. Immunity 15, (21). 28. Cho, J.-H. et l. Unique fetures of nive CD8 + T cell ctivtion y IL-2. J. Immunol. 191, (213). 29. Bonifz, L.C. et l. In vivo trgeting of ntigens to mturing dendritic cells vi the DEC-25 receptor improves T cell vccintion. J. Exp. Med. 199, (24). 3. Surh, C.D. & Sprent, J. Homeostsis of nive nd memory T cells. Immunity 29, (28). 31. Kieper, W.C. & Jmeson, S.C. Homeosttic expnsion nd phenotypic conversion of nïve T cells in response to self peptide MHC lignds. Proc. Ntl. Acd. Sci. USA 96, (1999). 32. Sprent, J. & Surh, C.D. Norml T cell homeostsis: the conversion of nive cells into memory-phenotype cells. Nt. Immunol. 12, (211). 33. Choo, D.K., Murli-Krishn, K., Anit, R. & Ahmed, R. Homeosttic turnover of virus-specific memory CD8 T cells occurs stochsticlly nd is independent of CD4 T cell help. J. Immunol. 185, (21). 34. Wu, Z. et l. Homeosttic prolifertion is rrier to trnsplnttion tolernce. Nt. Med. 1, (24). 35. Monti, P. & Piemonti, L. Homeosttic T cell prolifertion fter islet trnsplnttion. Clin. Dev. Immunol. 213, (213). 36. Khoruts, A. & Frser, J.M. A cusl link etween lymphopeni nd utoimmunity. Immunol. Lett. 98, (25). 37. Gttinoni, L. et l. Removl of homeosttic cytokine sinks y lymphodepletion enhnces the efficcy of doptively trnsferred tumor-specific CD8 + T cells. J. Exp. Med. 22, (25). 38. Tcho, N.K. & Turk, L.A. Lymphodepletion nd homeosttic prolifertion: implictions for trnsplnttion. Am. J. Trnsplnt. 12, (212). 39. Kitchin, J.E., Pomernz, M.K., Pk, G., Wshenik, K. & Shupck, J.L. Rediscovering mycophenolic cid: review of its mechnism, side effects, nd potentil uses. J. Am. Acd. Dermtol. 37, (1997). 4. Stoll, S., Delon, J., Brotz, T.M. & Germin, R.N. Dynmic imging of T cell-dendritic cell interctions in lymph nodes. Science 296, (22). 41. Brrl, Y., Mermll, V., Mooseker, M.S. & Snyder, M. Comprtmentliztion of the cell cortex y septins is required for mintennce of cell polrity in yest. Mol. Cell 5, (2). 42. Cudron, F. & Brrl, Y. Septins nd the lterl comprtmentliztion of eukryotic memrnes. Dev. Cell 16, (29). 43. Srikngs, J. & Brrl, Y. The emerging functions of septins in metzons. EMBO Rep. 12, (211). 44. Field, S.J. et l. PtdIns(4,5)P2 functions t the clevge furrow during cytokinesis. Curr. Biol. 15, (25). 45. Jnetopoulos, C. & Devreotes, P. Phosphoinositide signling plys key role in cytokinesis. J. Cell Biol. 174, (26). 46. Costello, P.S., Gllgher, M. & Cntrell, D.A. Sustined nd dynmic inositol lipid metolism inside nd outside the immunologicl synpse. Nt. Immunol. 3, (22). 47. Shrm, S. et l. An sirna screen for NFAT ctivtion identifies septins s coordintors of store-operted C2 + entry. Nture 499, (214). 48. Mléth, J., Choi, S., Mullem, S. & Ahuj, M. Trnsloction etween PI(4,5)P2-poor nd PI(4,5)P2-rich microdomins during store depletion determines STIM1 conformtion nd Ori1 gting. Nt. Commun. 5, 5843 (214). 49. Duois, S., Mriner, J., Wldmnn, T.A. & Tgy, Y. IL-15Pα recycles nd presents IL-15 in trns to neighoring cells. Immunity 17, (22). 5. Gérrd, A. et l. Secondry T cell-t cell synptic interctions drive the differentition of protective CD8 + T cells. Nt. Immunol. 14, (213). DVANCE ONLINE PUBLICATION nture immunology

9 ONLINE METHODS Mice. Sept7 flox/flox mice 19 were crossed to Cd4-Cre trnsgenic mice 27 to deplete septin 7 in T cells. These mice were red to ovlumin (OVA)-specific TCR trnsgenic OT-I mice 51 nd/or mtmg mice 26 (Jckson Lortory). Cells designted s experimentl controls were generted from Sept7 flox/flox ;Cd4-Cre or Sept7 flox/ ;Cd4-Cre + mice. These mice, long with C57BL/6 (The Jckson Lortory nd Simonsen) nd CD mice, were housed nd red under specific pthogen-free conditions t the University of Cliforni Animl Brrier Fcility. All experiments with mice were pproved y the Institutionl Animl Cre nd Use Committee of the University of Cliforni. Cell isoltion. CD8 + polyclonl or OT-I T cells were isolted from lymph nodes of 6- to 8-week-old mice using EsySep CD8 negtive-selection kits (STEMCELL Technologies). B cells were isolted from spleens of 6- to 8-week-old mice using EsySep B cell negtive selection kits (STEMCELL Technologies). In specified experiments, B cells were treted with 1 µg/ml LPS (Sigm-Aldrich) for 5 h efore use. were generted from treting cultured one mrrow cells with grnulocyte mcrophge colony-stimulting fctor (GM-CSF) for 7 11 d. IL-4 ws dded for the lst 2 d, with 1 µg/ml LPS stimultion 6 24 h efore use. nd B cells were incuted with 1 1 ng/ml SL8 OVA peptide (SIINFEKL) (Anspec) or vrint peptides N4, T4, Q4H7, V4 (gift from E. Plmer nd D. Zehn) for t lest 3 min t 37 C nd wshed three times. Immunolotting. Nive CD8 + T cells were isolted from 6-week-old nd littermte control OT-I mice. 1 6 cells per smple were lysed in PBS contining 1% Triton X-1 in the presence of cocktil of protese nd phosphtse inhiitors (2 µg/ml protinin, 2 µg/ml leupeptin, 2 mm PMSF, 1 mm sodium fluoride, 1 mm iodocetmide nd 1 mm sodium orthovndte). After 15 min lysis on ice, lystes were centrifuged t 21, g for 1 min to remove insolule mteril, nd protein concentrtion in the superntnt ws quntified y the Bio-Rd detergent-comptile protein ssy to ensure equl loding. Smples were resolved y SDS-PAGE, nd immunolot nlysis ws performed using rit polyclonl primry ntiodies to Sept1, Sept6C, Sept9 (ref. 52), Sept7 (IBL-Americ, Inc.), nd HRP-conjugted got ntirit secondry ntiody (Jckson ImmunoReserch) Reltive protein undnce ws quntified using ImgeJ softwre (NIH). In vitro T cell ctivtion ssys. Isolted T cells were leled with.5 5 µm (Invitrogen) or CellTrce Prolifertion Dye (BD Biosciences) for 15 min t 37 C, cultured in 96-well pltes t density of per ml in complete RPMI nd hrvested for nlysis 72 h lter, unless noted otherwise. CD8 + T cells were cultured on plte-ound nti-cd3 (2C11; UCSF Hyridom Core) or nti-tcrβ (H57-597; produced in M.F.K. s l) with ddition of solule nti-cd28 t 2 µg/ml (PV-1; UCSF Hyridom Core). Alterntively, T cells were stimulted with 1 1, ng/ml PMA nd 125 ng/ml ionomycin. In specified experiments 1 2 U/ml of recominnt humn IL-2 (NIH AIDS Regent Progrm) or 2% superntnt hrvested from previous wild-type BMDC-T cell cultures ws dded t time of plting. For other indicted experiments, 96-well pltes were coted with the following ntiodies or proteins: 1 µg/ml nti-cd44 (IM7, ebiosciences), 1 µg/ml nti-cd28 (PV-1; UCSF Hyridom Core), 5 µg/ml recominnt mouse ICAM-1 Fc chimer protein (R&D Systems), or 1 µg/ml fironectin, ovine plsm (EMD Millipore). In some experiments, cells were moved to or removed from pltes contining ICAM-1 Fc Chimer protein, prepred s detiled ove. In coculture experiments, OT-I T cells were plted with ctivted t 1:1 rtio in flt-ottom wells or with isolted B cells t 1:1 rtio in roundottom wells. In specified experiments, 1 µm of PI(3)K inhiitors LY-2942 (Sigm-Aldrich) or GDC-941 (gift from J. Roose) ws dded to culture 24 h or 36 h fter plting. For BMDC rescue ssys, T cells were stimulted with PMA nd ionomycin, nd mture ctivted were dded t time of plting. To lock costimultory signling, 1 µg/ml nti-cd8 (16-1A1; UCSF Hyridom Core) nd 1 µg/ml nti-cd86 (GL-1; UCSF Hyridom Core) nd/or nti-cd11 (M17/4; UCSF Hyridom Core) ws dded to culture or 24 h fter plting. Alterntively, 1 µm of LY-2942 or 1 µm GDC-941 ws dded or 24 h fter plting. In other experiments, T cells were plted with ctivted t 5:1 rtio, isolted with CD8 negtive selection kit, nd re-plted with 1 U/ml of humn recominnt IL-2. Cytokine exposure. Isolted CD8 + OT-I T cells were cultured in 96-well pltes t density of cells per ml. 5, U/ml humn recominnt IL-2 or murine recominnt 5 ng/ml IL-7 (Peprotech) nd 1 ng/ml IL-15 (Peprotech) were dded to medium t time of plting, nd cells were hrvested for nlysis 5 d lter. Surfce nd intrcellulr flow cytometry stining. Cells were hrvested from lymph nodes, spleens or in vitro culture, wshed with PBS nd nonspecific inding locked with flow cytometry uffer (PBS, 2% FCS) nd nti-cd16/32 (2.4G2; UCSF Hyridom Core). Surfce proteins on cells were stined with the following ntiodies for 25 min t 4 C: nti-cd69 (H1.2F3; ebiosciences), nti-cd25 (PC61.5; ebiosciences), nti-cd71 (C2, BD Phrmingen), nti- CD8α (53-6.7; ebiosciences), nti-cd4 (RM4-5; BioLegend), nti-cd44 (IM7; ebiosciences), nti-cd45.1(a2; ebiosciences), or nti-cd45.2 (14; BioLegend). Cells were gin wshed nd resuspended with flow cytometry uffer efore dt collection, with ddition of lck ltex eds to smples to quntify cellulr numer. For intrcellulr stins, cells were wshed with PBS nd incuted with Zomie NIR fixle viility dye (BioLegend) t 4 C for 3 min to dye ded cells. Cells were next wshed nd fixed with 4% PFA for 15 min t 2 C. Lst, cells were wshed nd stined in flow cytometry uffer with.2% sponin long with nti septin 7 (ref. 52) or phlloidin proe (Invitrogen) for 3 min efore finl wsh nd resuspension in flow cytometry uffer. Alterntively, fixed cells were treted with cold ( 2 C) methnol for 3 min t 4 C. Cells were then stined for 1 h t 2 C in stining uffer (PBS, 1% BSA) contining nti c-myc (D84C12; Cell Signling Technology), wshed, nd stined with fluorescenceleled donkey F( )2 nti-rit (Acm) for 3 min t 2 C. For ssessing cellulr DNA content with dye, hrvested cells were wshed with PBS, fixed in 7% ethnol for 3 min on ice nd wshed twice with PBS. Cells were then incuted with PBS contining.1% Triton X-1,.1 mm EDTA, 1 µg/ml RNAse A (Thermo Scientific), nd 5 µg/ml dye (Thermo Scientific). Cells were gin wshed nd resuspended in flow cytometry uffer. STAT5 phosphoryltion ssy. Isolted CD8 + OT-I T cells were cultured in 96-well pltes nd ctivted through PMA nd ionomycin stimultion or culture together with SL8-pulsed ctivted. Anti-mouse IL-2 (15 µg/ml, JES6-1A12) ws dded to cultures t time of plting to restrict IL-2 delivery. After 24 h, recominnt humn IL-2 (.1 1 U/ml) ws dded to wells for 2 min t 37 C. Cells were wshed, fixed with 4% PFA for 15 min t 2 C nd wshed. Cells were resuspended with cold ( 2 C) methnol nd wshed with flow cytometry uffer four times. To stin, cells were incuted in PBS contining 2% BSA nd nti phospho-stat5 (C71E5; Cell Signling Technology) efore finl wsh nd resuspension in flow cytometry uffer. Clcium flux signling. Isolted CD8 + OT-I cells were leled with 1 µm rtiomeric clcium-inding dye Indo-1, AM (Life Technologies) in PBS for 15 min t 37 C. Cells were wshed twice with complete RPMI nd incuted t 37 C for 15 min to llow for complete de-esterifiction. Cells were coted with 5 µg/ml nti-cd3 (2C11; UCSF Hyridom Core) on ice, wshed nd trnsferred to 37 C 1 min efore dt collection. To induce CD3 crosslinking, 1 µg/ml of nti-armenin hmster (BioLegend) ws dded to CD3-coted cells fter 1 min of smple collection. Alterntively, 1 µm of thpsigrgin (Sigm-Aldrich) ws dded to uncoted cells. Cell smples were kept in heting chmer (37 C) during dt collection y flow cytometry. In vivo cell trnsfer nd Dec-OVA immuniztion. Isolted CD8 + T cells were resuspended in PBS nd doptively trnsferred y retro-oritl or til-vein injection to recipient mice leled wild-type nd polyclonl T cells were cotrnsferred to host mice tht hd een sulethlly irrdited. Inguinl lymph nodes nd spleen were hrvested nd nlyzed 14 d fter trnsfer leled wild-type nd CD8 + OT-I T cells tht hd een cultured together in vitro with SL8-pulsed were doi:1.138/ni.333 nture immunology