Gene regulation mediated by calcium signals in T lymphocytes

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1 Gene regultion medited y clcium signls in T lymphocytes Stefn Feske 1, Jen Giltnne 2, Ricrdo Dolmetsch 3, Louis M. Studt 2 nd Anjn Ro 1 Modultion of mny signling pthwys in ntigen-stimulted T nd B cells results in glol chnges in gene expression. Here we investigte the contriution of clcium signling to gene expression in T cells using cell lines from two severe-comined immunodeficiency ptients with severl cytokine deficiencies nd diminished ctivtion of the trnscription fctor NFAT nucler fctor of ctivted T cells. These T cells show strong defect in trnsmemrne clcium influx tht is lso pprent in their B cells nd firolsts. DNA microrry nlysis of clcium entry deficient nd control T cells shows tht C 2+ signls oth ctivte nd repress gene expression nd re lrgely trnsduced through the phosphtse clcineurin. We demonstrte n elorte network of signling pthwys downstrem of the T cell receptor, explining the complexity of chnges in gene expression during T cell ctivtion. Clcium plys n essentil role in lymphocyte ctivtion nd mturtion. Cross-linking of ntigen or Fc receptors on T cells, B cells, mst cells nd nturl killer cells results in rpid increses in intrcellulr free clcium concentrtions [C 2+ ] 1 4 nd consequent ctivtion of mny trnscription fctors, including NFAT, NF-κB, JNK1, MEF2 nd CREB 5. In turn, these trnscription fctors regulte the expression of severl inducile genes tht medite diverse genetic progrms, including effector immune function, cell prolifertion, cell differentition nd cell deth 6. Two stges of clcium moiliztion hve een distinguished in lymphocytes nd other nonexcitle cells. The first stge involves ctivtion of phospholipse Cγ y tyrosine kinses coupled to ntigen nd Fc receptors. This enzyme hydrolyzes phosphtidylinositol isphosphte to relese the second messenger inositol 1,4,5-trisphosphte (IP 3), which inds to its receptor in the endoplsmic reticulum (ER) memrne to cuse rpid ut trnsient relese of C 2+ from ER stores 7. The second stge involves sustined influx of extrcellulr C 2+ cross the plsm memrne, in process termed cpcittive or store-operted C 2+ entry 8,9. Cpcittive clcium entry (CCE) occurs through specilized plsm memrne C 2+ chnnels, which re notle in tht depletion of intrcellulr C 2+ stores suffices for their ctivtion. Electrophysiologiclly, these depletion-ctivted chnnels interchngely termed store-operted C 2+ chnnels (SOC) or clcium relese ctivted C 2+ chnnels (CRAC) show high selectivity for C 2+, very low single chnnel conductnce nd inwrd rectifiction 10. However, their moleculr identities nd mode of ctivtion remin controversil 11,12. The two stges of clcium moiliztion re differentilly coupled to signling pthwys in T nd B cells. The smll trnsient spike of incresed [C 2+ ] i elicited y store depletion is sufficient to ctivte certin signling pthwys nd trnscription fctors such s NF-κB nd JNK 5. However, these rief clcium stimuli re insufficient for ctivtion of other trnscription fctors, notly NFAT. NFAT normlly resides in the cytoplsm of resting cells in phosphorylted form; upon cellulr stimultion it is dephosphorylted y the clcium-dependent phosphtse clcineurin, whereupon it trnsloctes to the nucleus nd shows incresed trnscriptionl function NFAT ctivtion is continuously regulted y the opposing ctions of clcineurin nd NFAT kinses, nd oth the nucler trnsloction of NFAT nd its ctivtion of gene trnscription re reversed y tretment of T cells with the clcineurin inhiitors cyclosporin A (CsA) nd FK506 16,17. As result, sustined NFAT ctivtion requires cpcittive clcium entry nd sustined increse in [C 2+ ] i 5,18,19. We previously descried two severe-comined immunodeficiency (SCID) ptients with principl defect in T cell ctivtion nd gene expression tht ws ttriutle to strongly impired dephosphoryltion nd nucler trnsloction of NFAT 20,21. We show here tht the impirment of NFAT ctivtion in these ptients is secondry to pronounced defect in trnsmemrne clcium influx (CI). We used the ptients T cells s tool to study clcium-dependent gene expression fter stimultion. We show tht C 2+ -dependent signling pthwys medite oth gene induction nd gene repression in ctivted T cells y integrtion of inputs from clcium store depletion, CI, clcineurin ctivtion nd other signling pthwys downstrem of the T cell receptor (TCR). Results Incresed [C 2+ ] ex compenstes for NFAT defect We previously showed tht the defect in NFAT ctivtion in the SCID ptients T cells could not e ttriuted to muttions or other functionl defects, either in NFAT itself or in its regulting phosphtse clcineurin 21,22. Therefore, we sked whether the defect in NFAT ctivtion could e meliorted in the presence of n incresed concentrtion of extrcellulr C 2+ ([C 2+ ] ex). Control nd ptient T cells were stimulted with ionomycin in the presence of 0.8 or 2.8 mm [C 2+ ] ex for up to 2 h (Fig. 1). In control T cells (Fig. 1, left pnels), complete nd sustined dephosphoryltion of NFAT1 (Fig. 1, A,B), NFAT2 (Fig. 1, C,D) nd NFAT4 (dt not shown) ws oserved t oth [C 2+ ] ex. In 1 Center for Blood Reserch nd Deprtment of Pthology, 3 Children s Hospitl, Hrvrd Medicl School, Boston, MA 02115, USA. 2 Metolism Brnch, Division of Clinicl Sciences, Ntionl Cncer Institute, Bethesd, MD 20892, USA. Correspondence should e ddressed to A. R. (ro@cr.med.hrvrd.edu). 316

2 Figure 1. Dephosphoryltion nd nucler trnsloction of NFAT in SCID ptients T cells is fcilitted y high [C 2+ ] ex. () Dephosphoryltion: T cell lines estlished from one ptient (Pt) nd one control individul (Co) were left unstimulted or stimulted with ionomycin (Iono) in the presence of 0.8 mm (A,C) or 2.8 mm (B,D) CCl 2. Where indicted, cells were preincuted with CsA efore stimultion. Whole-cell extrcts were prepred t the indicted time points nd nlyzed y immunolotting. Arrows indicte the phosphorylted (P) nd dephosphorylted (dep) forms of NFAT1 (A,B) nd three phosphorylted splice vrints of NFAT2 (C,D). Results re representtive of severl experiments with T cells from oth ptients nd three independent controls. () Nucler trnsloction: ptient nd control T cell lines were left unstimulted (A,B) or stimulted with ionomycin in the presence of 0.8 mm (C,D), 2.8 mm (E,F) or 10.8 mm (G,H) extrcellulr CCl 2, nd NFAT1 locliztion ws ssessed y immunocytochemistry. contrst, in the ptients T cells (Fig. 1, right pnels), NFAT dephosphoryltion ws trnsient nd incomplete t 0.8 mm [C 2+ ] ex ut prolonged nd lmost complete t 2.8 mm [C 2+ ] ex. Similrly, NFAT nucler trnsloction could e prtilly restored in the ptients T cells y incresing [C 2+ ] ex. Before stimultion, NFAT1 ws found in the cytoplsm in oth control nd ptients T cells (Fig. 1, A,B). When cells were stimulted with ionomycin in the presence of 0.8 mm [C 2+ ] ex, complete nucler trnsloction ws oserved in control T cells (Fig. 1, C), wheres NFAT1 in ptients T cells remined fully cytoplsmic (Fig. 1, D). Incresing [C 2+ ] ex to 2.8 or 10.8 mm led to grded increse in nucler NFAT1 in the ptients T cells fter ionomycin stimultion (Fig. 1, D,F,H) without further effect in the control T cells (Fig. 1, C,E,G). At 2.8 mm [C 2+ ] ex, roughly 30% of the ptients T cells showed some mount of nucler trnsloction, lthough most of the NFAT1 protein still resided in the cytoplsm (Fig. 1, F), wheres t 10.8 mm [C 2+ ] ex, out 80 90% of the ptients T cells showed complete nucler trnsloction of NFAT1 (Fig. 1, H). Elevtion of [C 2+ ] ex to 10.8 mm lso resulted in low mount of cytokine gene trnscription in the ptients T cells, especilly for interleukin 5 (IL-5) nd, to lesser degree, lso for IL-2, IL-4 nd IFN-γ 22. These results show tht efficient expression of cytokine genes in T cells depends on complete nd prolonged nucler trnsloction of NFAT. A C E G B D F H e c d Figure 2.The SCID ptients T cells hve primry defect in trnsmemrne clcium influx. T cell lines estlished from one SCID ptient (P2), the ptient s mother (Mo) nd fther (F) nd norml control (Co) were loded with Fur-2 nd monitored for chnges in intrcellulr free clcium. Dt represent the mens of > single cells. () For TCR-medited cellulr ctivtion,t cells were incuted with nti-cd3 (α-cd3) in clcium-free Ringer solution, followed y cross-linking with got nti mouse IgG (g-α-m). Susequently, cells were perfused with Ringer solution contining 2 mm CCl 2 (2 C 2+ ). (, c) For direct store depletion,t cells were stimulted either with () ionomycin (iono) in 2 C + contining PMA or (c) thpsigrgin (TG) in clcium-free Ringer solution (0 C 2+ ), followed y perfusion in 2 C 2+.(d) T cell lines were treted s in c ut in the presence of vlinomycin to hyperpolrize the cells. Ech experiment is representtive of severl similr experiments conducted with oth ptients nd two independent controls. (e) Comprison of microscopic single cell recordings of control (upper pnels) nd SCID ptient s (lower pnels) T cells (treted s in c). Digitl imges were tken immeditely efore ddition of TG (left), 100 s fter ddition of TG (middle) nd 5 min fter ddition of 2 C 2+ (right). Grded color scle indictes Fur-2 emission rtios (340:380 nm), with purple representing low [C 2+ ] i nd yellow/red representing high [C 2+ ] i. pril 2001 volume 2 no 4 nture immunology 317

3 Figure 3. The clcium entry defect ffects SCID ptients B cells nd firolsts. EBV-trnsformed B cells () nd untrnsformed firolsts () from ptient 2 (P2) nd control (Co) were perfused with TG in clcium-free Ringer solution (0 C 2+ ), followed y Ringer solution contining 2 mm CCl 2 (2 C 2+ ) lone. (c) EBV-trnsformed B cell lines from ptient 2 nd control were left unstimulted or stimulted with ionomycin in the presence of the indicted CCl 2 concentrtions. Numers in the pnels refer to the pproximte percentges of cells with cytoplsmic, prtilly nucler nd fully nucler NFAT stining, respectively. Clcium influx defect in SCID T cells The strong dependence of NFAT ctivtion on [C 2+ ] ex indicted potentil defect in clcium moiliztion in the ptients cells. We used the C 2+ -sensitive dye Fur-2 nd compred stimultion-induced chnges in [C 2+ ] i in single cells y digitl video fluorescence imging, mimicking TCR-dependent ctivtion y cross-linking CD3 (Fig. 2). In the sence of [C 2+ ] ex, we oserved trnsient increse in intrcellulr clcium concentrtions from 50 nm to 100 nm in control T cells tht ws ttriutle to depletion of intrcellulr clcium stores. Roughly 400 s fter cross-linking, [C 2+ ] i reched resting concentrtions gin. Store depletion ws lso pprent in the SCID T cells, ut to lesser extent. Rising [C 2+ ] ex to 2 mm resulted in steep increse in [C 2+ ] i in the control T cells (pek 500 nm, with sustined plteu t 200 nm) tht ws not oserved in the ptient s T cells (Fig. 2). To determine whether the lck of [C 2+ ] i increse in the ptient s T cells resulted from impired TCR-medited signling leding to insufficient store depletion or inefficient ctivtion of plsm memrne C 2+ chnnels, we stimulted T cells with the clcium ionophore ionomycin (Fig. 2). At low concentrtions, this gent directly ctivtes trnsmemrne CI y depleting internl C 2+ stores 23. Ionomycin stimultion of control T cells in the presence of 2 mm [C 2+ ] ex resulted in steep increse in [C 2+ ] i from resting concentrtion of 80 nm to 1.5 µm; [C 2+ ] i remined t this concentrtion for t lest 30 min (Fig. 2 nd dt not shown). However, in the ptient s T cells, the increse in [C 2+ ] i fter ionomycin tretment ws miniml (from resting concentrtion of 80 nm to 200 nm t its pek 30 s fter stimultion) nd trnsient (returning to seline 300 s fter stimultion initition) (Fig. 2). Thpsigrgin, n inhiitor of the SERCA (srcoplsmic endoplsmic reticulum clcium ATPse) which pumps clcium from the cytoplsm into the ER 24, ws used to deplete internl C 2+ stores independently of signls from surfce receptors. We treted SCID nd control T cells with thpsigrgin in C 2+ -free Ringer solution (Fig. 2c,e). Store depletion ws oserved s trnsient rise in [C 2+ ] i in oth SCID nd control T cells. Incresing [C 2+ ] ex to 2 mm led to steep increse of [C 2+ ] i in control T cells s well s in T cells from the ptient s mother nd fther; in contrst, the ptient s T cells showed n lmost complete lck of [C 2+ ] i increse (Fig. 2c,e). Similr results were otined with fluo-3 s clcium indictor in flow cytometry experiments (dt not shown). Thus, defective coupling of store depletion to opening of plsm memrne C 2+ chnnels is likely explntion for the lck of C 2+ moiliztion in the SCID T cells. Clcium influx through plsm memrne clcium chnnels is driven y the electrochemicl grdient cross the plsm memrne. To ddress the question of whether reduced memrne potentil in the SCID T cells ccounted for the defect in C 2+ influx, we ssessed the memrne potentil in ptient nd control T cells with pir of voltge sensitive dyes, CC2-DMPE nd DiSBAC 2(3), which undergo c chnges in fluorescence resonnce energy trnsfer when DiSBAC 2(3) reloctes from the outer to the inner leflet of the plsm memrne s result of ltertions in memrne potentil 25,26. Untreted T cells from SCID ptients nd controls perfused in stndrd Ringer solution showed no systemtic differences in resting memrne potentil s judged y 460:570 nm emission rtios. In ddition, oth control nd ptient T cells showed comprle increses of the 460:570 rtio in response to stepwise depolriztion with Ringer solution contining incresing KCl concentrtions in plce of NCl (dt not shown). Additionlly, the clcium entry defect ws not rescued y hyperpolriztion of the ptient s T cells with the potssium ionophore vlinomycin (Fig. 2d). Although we hve not yet fully ruled out the possiility of reduced driving force on clcium due to memrne polriztion defect, primry defect in CCE seems more plusile sed on the dt discussed ove. Forml proof of CCE defect wits ptchclmp mesurements on the SCID T cells. Clcium influx defect ffects B cells nd firolsts To determine whether other cell types showed the defect in C 2+ influx, we repeted the clcium imging experiments with Epstein-Brr virus (EBV)-trnsformed B cell lines from SCID ptient 2 nd controls (Fig. 3). Thpsigrgin tretment of control nd ptient B cells in C 2+ -free Ringer solution resulted in similr trnsient increses in [C 2+ ] i. Upon ddition of 2 mm CCl 2, only the controls showed high intrcellulr clcium concentrtions, with plteu t 1.5 µm [C 2+ ] i; in contrst, the SCID B cells showed much weker response, with [C 2+ ] i 600 nm. However, the defect ws not s striking s tht oserved in the ptient s T cells (compre with Fig. 2c). A similr impirment of C 2+ influx ws oserved in nonimmune cells from SCID ptients. Untrnsformed foreskin firolsts from one of the SCID ptients nd from helthy donor were grown in vitro nd stimulted with thpsigrgin in C 2+ -free Ringer solution. When perfused with 2 mm CCl 2, controls showed shrp spike of [C 2+ ] i to 500 nm tht lsted for 150 s efore reching plteu t 150 nm [C 2+ ] i. Conversely, the ptient s firolsts showed similrly trnsient, ut much weker, rise in [C 2+ ] i, which peked t 100 nm nd returned to seline in 4 min (Fig. 3). We investigted NFAT trnsloction in EBV-trnsformed B cell lines otined from SCID ptient 2 nd helthy controls (Fig. 3c). As expected, NFAT1 ws found in the cytoplsm in unstimulted control or ptient B cells independent of [C 2+ ] ex (Fig. 3c, A C). NFAT1 A B C D E F 318

4 A B C D E Figure 4. Clcium-dependent gene expression in T cells. () T cell lines of two ptients nd two norml controls were stimulted with PMA + ionomycin for 3 h nd gene expression nlyzed s descried in Methods. Genes re grouped into rod functionl groups. For ech gene, the rtio of mrna expression in stimulted versus unstimulted cells is listed in prentheses (control/ptient), with positive nd negtive vlues indicting fold gene induction nd repression, respectively.the rtio of these two vlues (induction/repression in control T cells divided y induction/repression in ptient T cells) is given outside the prentheses nd is plotted in the r grphs on se-2 logrithmic scle tht indictes the degree to which gene induction is ffected y the CI defect.a vlue of +2 indictes fourfold higher (tht is, 400% of ptient levels) induction of gene expression in control reltive to ptient T cells, wheres vlue of -2 indictes fourfold lower (tht is, 25% of control levels) induction in ptients reltive to controls. *Gene s identity ws independently verified y sequencing of DNA clones. Genes were incorported into this list if they showed t lest threefold induction or repression in one of the two cell popultions (ptient or control) nd t lest fourfold difference in induction or repression rtio in ptient reltive to control T cells. Cpitl letters ehind the vlues for ech gene refer to ctegories A E in. Cross-htching is used to emphsize rs in ctegories C nd E, which show opposing regultion y clcium versus other ctivtion-induced signling pthwys. A more extensive list of genes cn e found t () Ctegories A E indicte different ptterns of gene expression s descried in the text nd in the digrms to the right. trnsloction fter ionomycin tretment t 0.8 mm [C 2+ ] ex ws complete in control B cells (Fig. 3c, D), ut remined incomplete in the SCID B cells (Fig. 3c, E). Under the sme conditions, the ptient s T cells showed no NFAT nucler locliztion (Fig. 1, D). When we incresed [C 2+ ] ex to 10.8 mm, nucler trnsloction of NFAT1 ws complete s erly s 15 min fter stimultion in the control B cells ut pril 2001 volume 2 no 4 nture immunology 319

5 c Figure 5.T cells deficient in clcium influx fil to repress the expression of certin genes. () RNse protection ssys confirm down-regultion of five representtive clcium-repressed genes.t cell lines from ptients (P1, P2) nd controls (Co1, Co2) were left unstimulted ( ) or stimulted (+) with PMA + ionomycin nd cytokine concentrtions were ssessed y RPA. () Fold down-regultion of the five genes from the RPA experiment shown in.trnscript levels were quntified nd normlized to expression of the housekeeping gene L32 fter ckground sutrction nd n verge of the unstimulted:stimulted rtios from the two control nd the two ptient cell lines ws tken. Error rs indicte rnge of duplicte vlues for two ptients nd two controls.the higher the vlue, the stronger the down-regultion of gene expression fter stimultion. (c) Comprison of RPA nd microrry dt for the five genes shown in.the rtios of fold decreses in gene expression in control nd ptients T cells oserved in RPA nlyses (left) were compred with the sme rtios otined from the microrry experiments (right; nd see numer outside prentheses in Fig. 4). (d) Lck of induction of the CsA-sensitive NFAT2 splice vrint 50 in the ptients T cells fter prolonged stimultion.to confirm DNA rry dt showing tht NFAT2 is induced in control T cells ut not in ptient T cells (see Fig. 6),T cells were stimulted with PMA + ionomycin (P+I) nd NFAT2 ws detected y immunolotting using 7A6 ma. Filled nd open rrowheds indicte the phosphorylted nd unphosphorylted forms of the short inducile NFAT2 isoform, respectively. ecme complete only fter 30 or 60 min in the ptient s B cells (Fig. 3c, F nd dt not shown). Thus, unlike the ptient s T cells, the ptient s B cells show cler, ut prtil, defect in NFAT ctivtion. This is consistent with their prtil defect in C 2+ influx, which could result from low residul function of the depletion-ctivted C 2+ chnnels, from the ctivity of relted C 2+ -specific chnnel or from C 2+ lekge through nonselective ction chnnels t high [C 2+ ] ex. C 2+ signls medite gene ctivtion nd repression Studying the contriution of C 2+ signls to gene expression in T cells is difficult ecuse truly specific inhiitors of CCE re not known. Therefore, we used the CI-deficient T cells to identify C 2+ signling dependent genes using DNA microrrys, the preferred method for defining glol ptterns of gene expression, s it llows simultneous ssessment of the expression levels of thousnds of messenger RNAs 6, T cell lines of the two ptients nd two controls were left unstimulted or were stimulted with phorol myristte cette (PMA) + ionomycin for 3 h in RPMI medium in the presence of IL-2. cdna derived from unstimulted nd stimulted cells ws leled with Cy3 nd Cy5, respectively, nd incuted with specilized DNA Lymphochip microrrys contining genes relevnt to lymphocyte function 30. For ech gene, we clculted the verge fold increse in expression fter stimultion for oth control nd ptient T cell nd plotted the rtio of these two induction vlues on se-2 logrithmic scle (Fig. 4). Genes were chosen whose expression ws t lest threefold incresed (tht is, 300% of seline trnscription) or decresed (33% of seline trnscription) s compred to resting expression in either the control or ptient smples fter stimultion, nd lso showed fourfold or greter difference in expression etween these two popultions. Genes were then ssigned to severl groups ccording to their function. The list of genes is not comprehensive ut is ment to illustrte the different ptterns of gene expression; more complete list cn e found t Of the 111 genes represented 79 (71%) were ctivted in response to sustined CI: they were induced to t lest fourfold higher mounts in the control T cells thn in the ptients CI-deficient T cells (Fig. 4, positive vlues). Forty-eight of these (61%) showed little or no chnge d (less thn twofold, tht is, >50% nd <200% of seline trnscription) in expression in the ptients T cells (Fig. 4, ctegory B); 30 (38%) were t lest twofold (>200%) up-regulted in the ptients T cells fter stimultion, lthough the degree of up-regultion ws sustntilly less thn tht oserved in the controls (Fig. 4, ctegory A); nd one gene, oncosttin M, ws 9.5-fold (950%) up-regulted in control T cells ut 3.7-fold repressed (tht is, 27% of seline trnscription) in ptients T cells fter stimultion (Fig. 4, ctegory C, htched rs). These different ptterns indicte distinct underlying mechnisms for regulting gene expression (see lter). We found tht 32 of the 111 genes (29%) showed evidence of clcium-medited repression, which indicted t lest fourfold stronger induction in CI-deficient thn in control T cells (Fig. 4, negtive vlues for log se-2 induction rtio). Of these, 23 (72%) were expressed in sl mounts in resting T cells nd were repressed y clcium signls fter ctivtion: their expression ws strongly down-regulted in control T cells nd unchnged or less strongly down-regulted in the ptients T cells (Fig. 4, ctegory D). This ctegory includes the tyrosine kinse Lck; suunits of the serine nd threonine phosphtses PP1 nd PP2A; the trnscription fctors E2F-3, IRF-2 nd STAT1; nd the cell surfce receptors Fs, CD18, CD11A nd IL- 9Rα. The remining nine genes (28%) showed converse ehvior in tht gene expression ws ctully induced in the ptients T cells, ut either repressed, induced or induced to significntly lesser extent (fourfold difference etween ptients nd controls) in control T cells fter stimultion (Fig. 4, ctegory E, htched rs). A striking rnge of induction:repression rtios ws oserved in this ctegory. For exmple, genes encoding the regultors of G-protein signling A28- RGS nd RGS-1 were 14- to 15-fold induced ( %) in the ptients T cells ut only 2- to 3-fold induced ( %) in control T cells; thioredoxin reductse (TXNRD1) nd IRF-1 were four- to fivefold ( %) up-regulted in the ptients T cells ut unchnged in control T cells; CXCR4 ws 2.8-fold up-regulted (280%) in the ptients T cells ut 2.3-fold repressed (43%) in control T cells; nd IL-16 (the only cytokine gene with this pttern of expression) ws 2.4-fold up-regulted (240%) in the ptients T cells ut 11- fold repressed (9%) in control T cells (Fig. 4). 320

6 Figure 6. Clcineurin is involved in expression of most of clciumregulted genes. Control nd ptient T cells were left unstimulted or stimulted with PMA + ionomycin for 3 h with or without preincution with CsA. Br sizes indicte rtios of gene expression in stimulted versus unstimulted cells for ech gene; control T cells, drk green; CsA-treted control T cells, light green; ptient T cells, yellow; positive vlues indicte fold induction; negtive vlues indicte fold repression. Inclusion into this list required t lest fourfold difference in inducile gene expression in the control T cells or fourfold difference etween control nd ptient T cells. Ctegories refer to the dependence of gene expression on CI nd clcineurin, s shown in Fig. 7. Ctegory 1, CI-dependent genes (greter thn twofold difference in induction:repression rtios etween control, CsA-treted control nd CI-deficient T cells). () Ctegory 1A, clcium signl lrgely chnneled through clcineurin (less thn twofold difference etween CsA-treted control nd CI-deficient T cells). () Ctegory 1B, clcineurin ccounts for only frction of the clcium effect on gene expression (greter thn twofold higher induction in CsA-treted control T cells compred with CI-deficient T cells). (c) Ctegory 1C, TNF-α gene induction is more sensitive to CsA thn to the CI defect (greter thn twofold lower induction in CsA-treted control T cells compred with CIdeficient T cells). For other detils see text nd Fig. 4. c To confirm the clcium-medited gene repression indicted y the DNA microrry nlysis, we used the independent method of RNse protection ssy (RPA) to ssess the trnscript levels of five genes tht showed more undnt expression in the CI-deficient T cells (STAT1, Fs, CXCR4, MAP3K5 nd CCR2; Fig. 5). RNA ws otined from cells tht were treted s for the microrry experiments (T cells of controls nd ptients were left unstimulted or stimulted with PMA + ionomycin for 3 h). Trnscript levels were quntified nd normlized to those of the housekeeping gene L32 nd chnges in expression levels were plotted s fold repression (Fig. 5). In control T cells, expression of these five genes ws strongly downregulted upon stimultion ( 5 to 130-fold), wheres in the CI-deficient T cells, trnscript levels were effectively unchnged (Fig. 5). The correltion etween the RPA nd microrry dt ws resonly good, when compring the rtio of inducile gene repression etween ptients nd controls (Fig. 5c). C 2+ -medited expression trnsduced y clcineurin To determine the extent to which C 2+ influx nd clcineurin re required for the overll gene expression progrm in ctivted T cells, we conducted microrry nlysis on cdnas from control nd CI-deficient ptient T cells left unstimulted or stimulted with PMA + ionomycin (to ssess CI-dependence), s well s from control T cells stimulted in the presence or sence of CsA (to ssess clcineurin dependence). Genes were chosen for further study if they showed fourfold or greter chnge in expression in either wild-type or ptient T cells fter stimultion or fourfold difference etween these two popultions (Fig. 6 & 7). Of 87 such induced or repressed genes, 22 (25%) were judged to e CI-independent, s they showed very similr induction or repression in control nd CI-deficient T cells (Fig. 7). In contrst, 65 of the 87 genes (75%) were judged to e CI-dependent, sed on fourfold or greter difference in degree of induction etween wild-type nd CI-deficient T cells (Fig. 6). We used the dt from this experiment to define CI-dependent genes tht re regulted y clcineurin, other clcium-dependent pthwys or oth. CsA influenced, to greter or lesser extent, induction of ll 66 CI-dependent genes in ctegory 1, which indicted tht principl function for clcineurin is trnsducing C 2+ signls during T cell ctivtion. pril 2001 volume 2 no 4 nture immunology 321

7 Figure 7. Clcineurin is involved in expression of most clcium-regulted genes. Control nd ptient T cells were treted s descried nd the dt is displyed s in Fig. 6. () Ctegory 2, CI-independent genes (less thn twofold difference etween control, CsA-treted control nd CI-deficient T cells). For other detils see text nd Fig. 4. () Ctegories refer to the dependence of gene expression on Cl nd clcineurin.the scheme is drwn under the ssumption tht TCR stimultion is mimicked y PMA + ionomycin. For ese of discussion, genes re sudivided into three reltively ritrry ctegories. The lrgest suctegory, 1A (55/65 genes), contins genes tht re strongly dependent on oth CI nd clcineurin, s their expression is s sensitive to clcineurin inhiition s to lck of CI. A much smller suctegory, 1B (9/66 genes), contins genes for which clcineurin trnsduces only prt of the C 2+ signl: induction of these genes ws less strongly suppressed y the clcineurin inhiitor CsA in control T cells thn it ws decresed in ptient T cells tht lck CI. A single gene (tht which encoded TNF-α) is plced in suctegory 1C, sed on the fct tht it showed reproducily higher induction in the ptients CI-deficient T cells thn in CsA-treted control T cells. The sis for this ehvior is not cler ut my involve component of induction through NF-κB. This trnscription fctor is CsA-sensitive in T cells, lthough its induction does not require sustined clcium influx, s it cn e elicited y trnsient elevtion of [C 2+ ] i. Discussion We hve shown tht the primry defect in the SCID ptients is strong impirment of clcium moiliztion in response either to TCR stimultion or exposure to the phrmcologicl gents ionomycin nd thpsigrgin. Stimultion of the ptients T cells through their TCRs filed to ctivte sustined rise in intrcellulr free clcium concentrtions; in ddition, ionomycin nd thpsigrgin were unle to ctivte sustined C 2+ influx, despite successful relese of C 2+ from the ER stores. We conclude tht the defect is downstrem of store depletion nd does not involve kinses or dpter proteins known to couple TCR ligtion to phospholipse C ctivtion, IP 3 production nd C 2+ moiliztion in T cells (for exmple, Lck, Itk, ZAP-70, LAT, SLP-76 nd Vv, reviewed in 1 ). However, it is noteworthy tht the store depletion relted trnsient increse in [C 2+ ] i ws lower in the ptients cells thn in the control cells fter CD3 cross-linking. The most likely explntion is n inility to replenish ER stores in the sence of trnsmemrne C 2+ influx. However, minor or secondry defect in TCR signling cnnot e entirely ruled out. The defect in the SCID T cells is lso unlikely to e ttriutle to depolriztion of the ptients cells reltive to control cells: direct mesurements of memrne potentil with voltge-sensitive dyes did not show systemtic differences etween CI-deficient nd control T cells. Also, delierte hyperpolriztion of the ptients T cells did not result in mesurle reconstitution of CI. From these dt, primry defect in CCE is the most plusile explntion for the C 2+ influx defect, lthough forml proof wits ptch-clmp mesurements of the SCID T cells. Hence, it is likely tht the muttion resides either in the depletion-ctivted clcium chnnels themselves or in component of the generl pthwy tht connects them to store depletion (reviewed in 10,12 ). The seven mmmlin homologs of the Drosophil photoreceptor TRP (trnsient receptor potentil) genes re cndidte CRAC chnnel genes 32,33. Similr to severl other proteins, the IP 3 receptor, which is locted in the ER memrne nd intercts directly with mmmlin TRP , hs een implicted in CRAC ctivtion 12. In ddition, similr clinicl phenotype, with oth T cell ctivtion defect nd n impirment of trnsmemrne CI 37,38, hs een descried in nother SCID ptient. It would e useful to know whether these two immunodeficiencies nd the C 2+ influx deficient Jurkt cells generted in other studies 39,40 fll into the sme complementtion group. The defect in trnsmemrne C 2+ influx is lso oserved in the ptients B cells nd firolsts, nd so my e responsile for functionl deficits in diverse cell types nd tissues. Indeed, the nonimmunologicl symptoms of the ptients, which include nonprogressive musculr hypotoni nd mild psychomotor nd mentl retrdtion 20,21, indicte functionl involvement of the mutted gene in muscle cells nd neuronl cells. The defect is of lesser mgnitude in B cells thn in T cells, which is consistent with norml isotype switching nd high to norml immunogloulin production y the ptients B cells 31. Potentilly, B cells express oth the mutnt gene nd relted gene, the product of which cn compenste for the C 2+ influx defect. Alterntively, EBV trnsformtion my prtilly overcome the C 2+ entry defect, either y induction of endogenous genes positively regulting clcium influx or ecuse compensting proteins re encoded in the EBV genome. Our results provide insight into the complex interply etween C 2+ - dependent signling pthwys nd other ctivtion-induced signling mechnisms in T cells. Only 25% of ll ctivtion-regulted genes 322

8 showed no dependence on C 2+ influx. In the lrger C 2+ -dependent ctegory, genes could e distinguished y whether they were positively or negtively regulted y clcium, the extent to which the clcium signls were trnsduced through clcineurin nd the extent to which other (nonclcium-medited) signling pthwys prticipte in their regultion. Genes tht were positively regulted y clcium fell into three ctegories, of which two (Ctegories A nd B) re distinguished y their reltive dependence on CI. Ctegory A genes re discernily up-regulted in CI-deficient ptients T cells, leit to lesser extent thn in control T cells; thus, their expression must e medited in prt y CI nd in prt y other ctivtion-induced signling mechnisms. These mechnisms could e completely clcium-independent or could depend on trnsient [C 2+ ] i increses tht do not involve CI. In contrst, ctegory B genes re strongly dependent on CI: they re effectively silent in the ptients T cells, indicting n solute requirement for [C 2+ ] i increses tht re sustined for mny hours. Genes in ctegories A nd B re present in ll functionl groups, nd include cytokine, chemokine nd growth fctor genes, cell surfce receptors, trnscription fctors nd signling proteins known to e induced during productive immune response. A third ctegory (Ctegory C) of positively regulted genes, represented y oncosttin M in our studies, is defined y upregultion in control T cells ut down-regultion in ptients T cells. This ehvior points to dul mode of regultion in which gene expression is ctivted y clcium ut repressed y other signling pthwys emnting from the TCR. Clcium-repressed genes (tht is, genes tht were up-regulted in ptients T cells reltive to controls) were under-represented in the ctegory of cytokine, chemokine nd growth fctor genes ut were strongly represented in ll other functionl groups. Agin, two different ctegories, D nd E, could e distinguished. Ctegory D genes show the converse ehvior of ctegories A nd B comined: store depletion nd CI repress sl gene expression to greter or lesser extents in oth control nd ptient T cells. Ctegory E genes re the converse of ctegory C: they re induced y nonclcium-dependent signling pthwys, nd this induction is repressed y store depletion nd/or CI. This ehvior indictes dul regultion wherey TCR signls other thn clcium induce gene expression, lthough this induction is normlly countered y CI-derived signls. Depending on the reltive degree of TCR-medited induction nd clcium-medited repression, wide rnge of induction:repression rtios is possile nd ws, in fct, oserved. Wheres the mechnisms y which C 2+ induces gene expression re well studied, less is known out how increses in [C 2+ ] i led to gene repression. Clcium ctivtes diverse trnscription fctors, including NFAT, NF-κB, Elk-1, Nur77, AP-1, ATF-2 nd CREB, y clmodulindependent protein kinses nd phosphtses Severl mechnisms of C 2+ -medited gene ctivtion hve een descried, mny of which involve relese of trnscriptionl repressor complexes from gene regultory elements 44,45. Prticulrly interesting re the diverse wys in which C 2+ promotes dissocition of histone decetylse complexes (HDAC) from the vicinity of the trnscription fctor MEF2D 46,47. By nlogy, it is conceivle tht C 2+ signls repress gene trnscription y recruiting or ctivting HDACs or relesing or inhiiting histone cetyltrnsferses in n ctive trnscriptionl complex. In the sence of truly specific inhiitors of C 2+ influx nd CCE, T cells deficient in trnsmemrne CI will e vlule not only for potentilly elucidting the mechnism of store-operted clcium entry ut lso for defining signling pthwys y which C 2+ positively nd negtively regultes gene expression. Methods Cse report. Detiled cse reports of the two SCID ptients investigted in this study hve een descried 20,21. Cell the culture conditions nd regents. Continuously growing T cell lines were derived from the peripherl lood lymphocytes (PBLs) of two ptients efore one mrrow trnsplnttion, their prents nd helthy donors, s descried 20. Cells were grown in the presence of 50 U/ml of recominnt humn IL-2 (Hofmnn LRoche, Nutley, NJ). B cell lines were generted y isoltion of CD19 + cells from PBLs with dyneds (Dynl, Lke Success, NY), trnsformed with EBV-contining superntnt derived from the mrmoset cell line B95-8 nd propgted in RPMI + 10% fetl ovine serum (FBS) (HyClone, Logn, UT) t 37 C, 5% CO2. Foreskin firolsts of the neworn Ptient 2 were provided y C. Niemeyer; Hs27 control firolsts from helthy neworn were from Americn Type Culture Collection (Mnsss, VA). Cells were grown in RPMI % FBS nd split 1:4 every 4 5 dys. For stimultion, cells were treted with 1 µm ionomycin (Cliochem, Sn Diego, CA) lone or in comintion with 16.2 nm PMA (Cliochem, L Joll, CA) in RPMI + 10% FBS (contining totl estimted 0.8 mm CCl2). Where indicted, cells were preincuted for 15 min with 1 µm CsA (Cliochem). Immunolotting nd immunocytochemistry. Immunolotting ws done s descried 21. Antiodies used were nti-t2b1 to COOH-terminl peptide of NFAT1c 48, ma 7A6 to NFAT2 (Alexis Biochemicls, Sn Diego, CA), nd polyclonl ntiserum to NFAT4 (gift of T. Hoey, Tulrik Inc., South Sn Frncisco, CA). Immunocytochemistry ws done s descried 21. Where indicted, cells were stimulted with 1 µm ionomycin or 1 µm thpsigrgin in RPMI + 10% FBS contining 0.8 mm CCl2. Anti-T2B1 for NFAT1 detection ws used t 1:1000, followed y Cy-3 conjugted sheep nti-rit IgG (Sigm). RNse protection ssy. RNse protection ssys were done ccording to mnufcturers protocol (Biotecx, Houston, TX), s descried 21. Briefly, cells were stimulted with 16.2 nm PMA nd 1 µm ionomycin in RPMI + 10% FBS for 3 h nd totl cellulr RNA ws extrcted with Ultrspec ccording to mnufcturers protocol. Cytokine RNA levels were nlyzed y RNse protection ssy using the RioQunt multiproe kits hck-3, hck-4 nd custom-mde templte set contining proes for STAT1, Fs, CXCR4, MAP3K5, CCR2 nd L32 (PhrMingen, Sn Diego, CA). Trnscript levels were quntified y utordiogrphy nd densitometric scnning of utordiogrms using Storm 860 phosphoimger nd imgequnt softwre (oth Moleculr Dynmics, Sunnyvle, CA). RNA loding ws estimted y mesuring the intensities of the protected frgments of the housekeeping gene L32. Video imging for clcium nd voltge rtios. Cells were loded t cells/ml with 1 µm Fur-2 AM ester (Moleculr Proes, Eugene, OR) in loding medium (RPMI + 10% FBS) for 30 min t room temperture, wshed nd resuspended in loding medium. Cells were ttched to poly(l)lysine coted coverslips for 15 min, mounted in RC-20 closed th flow chmer (Wrner Instrument Corp., Hmden, CT) nd nlyzed on TE-300 inverted epifluorescence microscope (Nikon, Melville, NY) with OpenL imging softwre (Improvision, Lexington, MA). Firolsts were grown directly on UV-treted sterile coverslips nd loded with fur-2. For recordings of cytoplsmic [C 2+ ]i, cells were perfused in Ringer solution (155 mm NCl, 4.5 mm KCl, 10 mm D-glucose, 5 mm Hepes t ph 7.4, 1 mm MgCl2, 2 mm CCl2) nd stimulted with either 1 µm ionomycin, 1 µm thpsigrgin or 5 µg/ml of nti-cd3 (HIT 3A) cross-linked with 5 µg/ml of rt nti mouse IgG2 (oth from PhrMingen). Where indicted, cells were first perfused in C 2+ -free Ringer (in which C 2+ ws sustituted y Mg 2+ ), followed y stndrd Ringer solution. Vlinomycin ws used t 1 µm where indicted. Fur-2 emission ws detected t 510 nm fter excittion of the dye t 340 nd 380 nm, respectively, nd rtios of 340:380 clculted for ech 5-s intervl fter sutrction of ckground. Clirtion vlues (Rmin, Rmx, Sf) were derived from cuvette mesurements s descried 49. For ech experiment, cells were nlyzed. For mesurements of the memrne potentil, T cells were loded with 1 µm coumrin lipid dye CC2-DMPE (Auror Biosciences, Sn Diego, CA) for 30 min t room temperture in 1 ml of serum-free RPMI 1640 in the presence of 2 µl of 10% w/v Pluronic- 127/DMSO solution (Auror Biosciences). The oxonol dye DiSBAC2(3) (Auror Biosciences) ws loded t 6 µm finl concentrtion for 45 min t room temperture in RPMI Cells were loded onto poly(l)lysine coted coverslips nd nlyzed y video imging t 405 nm (excittion) nd 460 nd 570 nm (emission), respectively. Depolriztion ws chieved y perfusing the cells in Ringer solution contining 20, 40, 60, 80, 100 nd 155 mm KCl, respectively, with susequent hyperpolriztion using stndrd 4.5 mm KCl Ringer solution. Messenger RNA smples nd microrry procedures. T cell lines from helthy controls nd the two SCID ptients were left unstimulted or stimulted with 16.2 nm PMA nd 1 µm ionomycin for 3 h with or without preincution with 1 µm CsA for 15 min. For Fig. 6, T cell lines were enriched for CD8 + cells to >90% purity using mgnetic ed seprtion (Dynl, Lke Success, NY). Cells were collected t the indicted time points nd poly(a) + mrna isolted with the FstTrck Kit (Invitrogen, Crlsd, CA). For ech experiment, fluorescent cdna proes were prepred y reverse trnscription of mrnas with Cy3- dutp nd Cy5-dUTP (NEN Life Sciences, Boston, MA) for unstimulted nd stimulted smples, respectively. Leled cdnas were incuted overnight onto Lymphochip microrrys (L. Studt, Bethesd, MD). Fluorescent imges of hyridized microrrys were otined using GenePix 4000A microrry scnner (Axon Instruments, Foster City, CA). pril 2001 volume 2 no 4 nture immunology 323

9 Imges were nlyzed with GenePixPro3.0 (Axon Instruments) nd single spots or res of the rry with ovious lemishes were flgged nd excluded from susequent nlyses. Fluorescence rtios were stored in custom dtse, nd normlized dt were extrcted from this dtse for further nlysis 28. Arry dt were filtered y selecting genes tht presented dt on t lest 75% of the rrys, hd spot dimeter of 25 µm nd signl of 200 in ech chnnel, or 1000 in one chnnel nd some seline signl in the other chnnel. In mny cses, the given vlues in Figs. 4 nd 6 re verges of severl representtions of one gene on the rrys. Genes re listed y their most commonly used nmes in the literture or, where miguous, sed on HUGO-pproved gene symols. The cdna clones on the Lymphochip microrry re ville from Reserch Genetics. For Fig. 4, the smples were hyridized on Mini-Lymphochip version 4.0, which contined 7396 cdna clones. Of these, 5335 (72.1%) pssed the spot qulity filters. For Fig. 6 the smples were hyridized on Mini-Lymphochip version 8.1, which contined 7392 cdna clones, of which 3177 (42.9%) pssed the spot qulity filters. Genes included in our nlysis were extrcted from this pool. Results for genes with uncler function, unchrcterized genes nd expressed sequence tgs re not represented in Figs. 4 nd 6 ut will e ville t Acknowledgements We thnk C. Niemeyer for providing us with the ptient dt. Supported in prt y grnts from the Deutsche Forschungsgemeinschft (Fe496/1-1) nd in prt y NIH grnts CA42471 nd AI Received 29 Novemer 2000; ccepted 12 Ferury vn Leeuwen, J. E. & Smelson, L. E.T cell ntigen-receptor signl trnsduction. Curr. Opin. Immunol. 11, (1999). 2. Brun, J., Sh fi, R. I. & Unnue, E. R. Crosslinking y lignds to surfce immunogloulin triggers moiliztion of intrcellulr 45C 2+ in B lymphocytes. J. Cell Biol. 82, (1979). 3. Hoth, M. & Penner, R. Depletion of intrcellulr clcium stores ctivtes clcium current in mst cells. Nture 355, (1992). 4. Turner, H. & Kinet, J. P. Signlling through the high-ffinity IgE receptor Fc epsilonri. Nture 402, (1999). 5. Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C. & Hely, J. I. Differentil ctivtion of trnscription fctors induced y C 2+ response mplitude nd durtion. Nture 386, (1997). 6. Tegue,T. K. et l. Activtion chnges the spectrum ut not the diversity of genes expressed y T cells. Proc. Ntl Acd. Sci. USA 96, (1999). 7. Berridge, M. J. Inositol trisphosphte nd clcium signlling. Nture 361, (1993). 8. Putney, J.W., Jr.A model for receptor-regulted clcium entry. Cell Clcium 7, 1 12 (1986). 9. Clphm, D. E. Intrcellulr clcium. Replenishing the stores. Nture 375, (1995). 10. Prekh,A. B. & Penner, R. Store depletion nd clcium influx. Physiol. Rev. 77, (1997). 11. Putney, J.W. & Rieiro, C. M. P. Signling pthwys etween the plsm memrne nd endoplsmic reticulum clcium stores. Cell Mol. Life Sci. 57, (2000). 12. Putney, J.W. Kissin cousins : intimte plsm memrne-er interctions underlie cpcittive clcium entry. Cell 99, 5 8 (1999). 13. Okmur, H. & Ro,A.Trnscriptionl regultion in lymphocytes. Curr. Opin. Cell. Biol., in press (2001). 14. Crtree, G. R. Generic signls nd specific outcomes: signling through C 2+, clcineurin, nd NF-AT. Cell 96, (1999). 15. Kini,A., Ro,A. & Armuru, J. Mnipulting immune responses with immunosuppressive gents tht trget NFAT. Immunity 12, (2000). 16. Timmermn, L.A., Clipstone, N.A., Ho, S. N., Northrop, J. P. & Crtree, G. R. Rpid shuttling of NF- AT in discrimintion of C 2+ signls nd immunosuppression. Nture 383, (1996). 17. Loh, C. et l. Clcineurin inds the trnscription fctor NFAT1 nd reversily regultes its ctivity. J. Biol. Chem. 271, (1996). 18. Dolmetsch, R. E., Xu, K. & Lewis, R. S. Clcium oscilltions increse the efficiency nd specificity of gene expression. Nture 392, (1998). 19. Li,W., Llopis, J.,Whitney, M., Zlokrnik, G. & Tsien, R.Y. 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