In addition to activating signaling pathways that have a positive effect,receptor stimulation induces negative feedback pathways that attenuateor terminate positive signaling. One of the best-documented mecha-nisms of countering productive responses involves removal of phos-phate groups or other activating modifications from proteins thatmediate signal transduction (receptors, kinases, adapter proteins andtranscription factors1). In another mechanism, positive signals increasethe amounts or activities of negative regulators or inhibitory proteins;indeed, many genes that are rapidly induced by activation of signalingpathways encode proteins that have negative effects in the same path-way2. In a third mechanism, activated signal transducers are selectivelytargeted for degradation, terminating ongoing signals and also interfer-ing with subsequent stimulation. Cytoplasmic signaling proteins andnuclear transcription factors tend to be polyubiquitinated and targetedfor proteasomal degradation3, whereas ligand-activated surface recep-tors, including receptor tyrosine kinases, G protein–coupled receptorsand the T cell receptor (TCR) are more often degraded by endocytosisand trafficking to the lysosome4,5. Induced endocytosis of activatedreceptors at the plasma membrane and sorting of the receptors intomultivesicular bodies at the endosomal membrane is regulated throughtagging of receptor or adaptor proteins with ubiquitin4. Together thesemechanisms ensure a balanced response to extracellular signals andprotect cells from the deleterious effects of chronic activation.
In a relatively common scenario, a single second-messenger or sig-naling molecule simultaneously mediates both positive and negativeoutcomes downstream of surface receptors. This point has been wellillustrated in T cells, in which Ca2+ signaling is essential not only forproliferation and effector function but also for imposition of an aner-gic state in which positive signals cannot be initiated or are substan-tially delayed or attenuated6. Sustained signaling through Ca2+ andcalcineurin results in sustained activation of the transcription factorNFAT, which in turn induces many genes encoding effector cytokines,chemokines and other products in the productive immune response7.However the same transcription factor, when preactivated in theabsence of its transcriptional partner AP-1 (Fos-Jun), induces a differ-ent set of genes encoding known or presumed negative regulators of Tcell signaling, thus mediating an opposing program of T cell anergy ortolerance8. Among the negative molecules induced in these conditionsare several tyrosine phosphatases that would be expected to downreg-ulate TCR signaling by opposing the effects of tyrosine kinases such asZap70, Lck and Itk; diacylglycerol kinase-α, which metabolizes thesecond messenger diacylglycerol; and genes encoding several proteasesand E3 ubiquitin ligases, which might induce T cell anergy by promot-ing degradation of downstream signaling molecules in T cells.
The interface between the T cell and the antigen-presenting cell(APC) is an important site for regulation of TCR signaling. In the
1Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA. 2Program inMolecular Pathogenesis and Department of Pathology, Skirball Institute of Molecular Medicine, New York University School of Medicine, New York, New York 10016,USA. 3Division of Cell Biology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA. 4Department of Microbiology, Columbia University,College of Physicians and Surgeons, 701 West 168th Street, HHSC, New York, New York 10032, USA. 5Present addresses: Department of Pathology, Albert EinsteinCollege of Medicine, Bronx, New York 10461, USA (F.M.) and Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-ku,Kwangju 500-712, Korea (S.-H.I.). Correspondence should be addressed to A.R. ([email protected]).
Published online 15 February 2004; doi:10.1038/ni1047
Calcineurin imposes T cell unresponsivenessthrough targeted proteolysis of signaling proteinsVigo Heissmeyer1, Fernando Macián1,5, Sin-Hyeog Im1,5, Rajat Varma2, Stefan Feske1, K Venuprasad3, Hua Gu4,Yun-Cai Liu3, Michael L Dustin2 & Anjana Rao1
Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin andthe transcription factor NFAT. We show here that Ca2+-induced anergy is a multistep program that is implemented at least partlythrough proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitinligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomalsorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the relatedprotein Nedd4, resulting in degradation of two key signaling proteins, PKC-θ and PLC-γ1. T cells from Itch- and Cbl-b–deficientmice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containinglymphocyte function–associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.
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T cell–APC interface, adhesion molecules and T cell receptor signaltransduction machinery are organized into distinct supramolecularactivation clusters in the mature immunological synapse9,10. T cellproliferation is strongly correlated with stability of the immunologi-cal synapse over a period of many hours9–11, but paradoxically, thetime frame of formation of the mature immunological synapse (min-utes) corresponds temporally to a period of T cell receptor degrada-tion and downregulation of tyrosine kinase signaling12. Thesefindings are not necessarily contradictory: the early and late phases ofsignal transduction are well known to have different biological conse-quences13, and the immunological synapse is clearly a dynamic struc-ture that is capable of influencing T cell responses over a period ofseveral hours by integrating signals from diverse cell surface receptorsin addition to the TCR14.
Here we have investigated the mechanism of T cell unresponsive-ness (anergy) induced by Ca2+ and calcineurin, specifically testingthe hypothesis that T cell anergy is in part a consequence of prote-olytic activation. We show that sustained Ca2+-calcineurin signalingleads to transcriptional upregulation of at least three E3 ubiquitinligases, Itch15, Cbl-b16 and GRAIL17, and also induces increasedexpression of Tsg101, the receptor involved in sorting monoubiqui-tinated proteins to the lysosomal degradation pathway18.Restimulation of these sensitized cells results in membrane localiza-tion of Itch and the related E3 ligase Nedd4, which target the key sig-naling proteins phospholipase C-γ1 (PLC-γ1) and protein kinaseC-θ (PKC-θ) for monoubiquitination and lysosomal degradation.Anergic T cells form immunological synapses normally, but consis-tent with downregulation of signaling proteins after restimulation,the synapses are unstable and abnormal structures accumulaterapidly. Mice lacking either Itch or Cbl-b develop autoimmune dis-ease15,19–21, and we show that T cells from these mice are resistant toCa2+-induced anergy, consistent with a function for both E3 ligasesin T cell anergy and tolerance. Our findings define a multistep pro-gram in which sustained signaling through Ca2+ and calcineurinimposes T cell unresponsiveness by promoting enhanced expressionand membrane localization of molecules involved in ubiquitination
and lysosomal degradation of key signaling proteins ‘downstream’of the TCR.
RESULTSSustained Ca2+ signals promote PLC-γ1 degradationSustained Ca2+ and calcineurin signaling induces a state of T cellunresponsiveness or anergy, characterized by upregulation of manyanergy-associated genes6,8. Increased intracellular Ca2+ concentra-tions ([Ca2+]i) in resting conditions, decreased Ca2+ mobilization inresponse to B cell receptor stimulation and upregulation of tolerance-associated genes have also been documented in tolerant B cells thathave been exposed continuously to antigen in vivo22,23.
The transcriptional profile of anergic T cells included several genesencoding proteases and E3 ubiquitin ligases8, leading us to hypothe-size that T cell anergy was in part a consequence of proteolytic acti-vation in cells. To test this, we assessed the amounts of manysignaling proteins in T cells anergized by sustained exposure to iono-mycin (Fig. 1a). We noted a limited number of changes, among thema reproducible decrease in intensity of the PLC-γ1 band. Thisdecrease occurred mainly during a subsequent step of cell stimula-tion or homotypic cell adhesion, as PLC-γ1 decreased after incuba-tion of the ionomycin-treated cells at high cell density for an hour, aswell as after restimulation with antibody to CD3 (anti-CD3), anti-CD3 plus anti-CD28, or phorbol 12-myristate 13-acetate (PMA)plus ionomycin (Fig. 1b,c). We did not find the decrease in PLC-γ1after restimulation with ionomycin alone, ruling out the possibilityof a direct effect of Ca2+-calcineurin signaling (Fig. 1c). The decreasereflected lowered amounts of PLC-γ1 protein, and was not due to celldeath, post-translational modification of PLC-γ1, relocalization ofPLC-γ1 to a different intracellular compartment or decreased PLC-γ1 gene transcription (data not shown). In experiments done withoptimized conditions, there was a strong correlation between loss ofPLC-γ1 and extent of anergy induction in a parallel proliferationassay (Fig. 1d). Thus, anergic T cells degrade PLC-γ1 in two separa-ble stages. A period of sustained Ca2+-calcineurin signaling isrequired to initiate the degradation program, but degradation is
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Figure 1 Decreased PLC-γ1 levels correlate withT cell anergy. (a) Changes in signaling proteins inanergic T cells. T cell anergy was induced bytreatment of the TH1 cell clone D5 with (+) orwithout (–) 1 µM ionomycin for 16 h. The cellswere washed to remove ionomycin and wereincubated at higher cell density for 1–2 h at 37°C. Whole-cell extracts were analyzed byimmunoblotting. (b–d) Two signals are requiredfor maximal loss of PLC-γ1. D5 cells wereanergized by treatment with 1 µM ionomycin(Iono) for 16 h, then were washed to removeionomycin and were incubated at higher celldensity for 1 h at 37 °C. Extracts were assayedfor PLC-γ1 by immunoblotting. (b) Extracts wereprepared either directly (lanes 1 and 2) or afterresuspension at high cell density and incubationfor 1 h (lanes 3 and 4). (c) Cells were pretreatedfor 16 h with ionomycin and restimulated for 1 h with anti-CD3, anti-CD3 plus anti-CD28,ionomycin, or PMA plus ionomycin. (d) Theextent of anergy induction in a proliferation assay and the extent of decrease in PLC-γ1 after the step of incubation at high cell densitywere evaluated in parallel in a single culture ofuntreated (–) and ionomycin-pretreated (+) D5cells. α-, anti-.
actually implemented during a subsequent step of cell stimulation orhomotypic cell adhesion.
Calcineurin is required for PLC-γ1 and PKC-θ degradationWe used the calcineurin inhibitor cyclosporin A (CsA) to evaluate theinvolvement of calcineurin in PLC-γ1 degradation in anergic T cells(Fig. 2a). D5 T cells subjected to 16 h of ionomycin pretreatment fol-lowed by cell-cell contact showed a substantial decrease in PLC-γ1,PKC-θ and RasGTPase-activating protein (RasGAP), but no changesin several other signaling proteins, including RasGRP1, Lck, Zap70and PLC-γ2 (Fig. 2a, lanes 1 and 2). Degradation was completelyblocked by inclusion of CsA during the ionomycin treatment step(Fig. 2a, bottom right, lane 3). Ionomycin also induced an increase ofabout twofold in total protein ubiquitination (Fig. 2a, bottom right,lanes 1 and 2), which was blocked by CsA (Fig. 2a, lane 3). Primary Tcells anergized by stimulation with anti-CD3 alone showed decreasedPLC-γ1, PKC-θ, RasGAP and, to a lesser extent, Lck, but not PLC-γ2,compared with cells productively stimulated with both anti-CD3 andanti-CD28 (Fig. 2b). The data are consistent with the hypothesis thatcalcineurin activates ubiquitin-dependent proteolytic pathways thatpromote protein degradation.
We assessed the functional consequences of PLC-γ1 degradation inanergic T cells by examining Ca2+ mobilization (Fig. 2c). We ren-dered primary T helper type 1 (TH1) cells anergic by ionomycin pre-treatment, then labeled them with the Ca2+ indicator fura-2. Weinduced Ca2+ mobilization by TCR-CD3 crosslinking, after which westimulated the cells with ionomycin to identify healthy, responsivecells. Consistent with the idea of a central function for PLC-γ1 (but
not PLC-γ2) in Ca2+ mobilization and T cell activation24, anergic Tcells responded very poorly to TCR stimulation compared withuntreated T cells. As reported before25,26, we also noted a strongimpairment of Ca2+ mobilization in T cells from DO11.10 transgenicmice that were orally tolerized by ovalbumin feeding (data notshown). Thus, the PLC-γ1 degradation observed in anergic T cellscorrelates with a pronounced impairment of Ca2+ mobilization inresponse to TCR triggering. Because this effect occurs without obvi-ous delay, PLC-γ1 degradation may be initiated during the fura-2labeling step as a result of cell-cell contact. Alternatively, other mech-anisms such as dephosphorylation by tyrosine phosphatases upregu-lated during the step of sustained Ca2+-calcineurin signaling8 maycontribute to loss of PLC-γ1 activity in anergic T cells (these possibil-ities are not mutually exclusive).
PLC-γ1 is a substrate for Nedd4 and ItchAll three targets of the Ca2+-calcineurin–dependent degradationprogram, PLC-γ1, PKC-θ and RasGAP, have C2 domains(Supplementary Fig. 1 online). These domains can mediate Ca2+-dependent phospholipid binding or may serve as Ca2+-dependentor Ca2+-independent protein interaction domains27. C2 domainsare also found in the Itch and Nedd4 family of E3 ubiquitin ligases28
(Supplementary Fig. 1 online), leading us to test the hypothesis thatthese E3 ligases are involved in PLC-γ1 degradation. PLC-γ1 coim-munoprecipitated with both Nedd4 and Itch (Fig. 3a) and was asubstrate for ubiquitination and degradation by Itch and Nedd4(Fig. 3b, c). In 293 cells, ionomycin induced PLC-γ1 ubiquitination(Fig. 3b, lanes 4 and 5), and much of the ubiquitinated PLC-γ1
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Figure 2 Decreased PLC-γ1 and impaired Ca2+ mobilization correlate with T cell anergy. (a) Calcineurin-dependent degradation of target proteins in anergic T cells. D5 T cells were treated for 16 h with ionomycin (Iono), CsA or both, then were washed and incubated at increased cell density for 1 h. Cell extractswere prepared and analyzed by immunoblotting for proteins (margins) or for the extent of ubiquitin modification of total protein in the lysates (bottom rightblot). The faster-migrating band in the PKC-θ immunoblot (*) is the original Zap70 signal on the same blot, which was reprobed without prior stripping.Ionomycin treatment reproducibly upregulates PKC-θ protein in a manner insensitive to CsA; therefore, the appropriate comparison in this case is betweenlanes 3 and 2, not lanes 1 and 2. (b) Signaling proteins in primary TH1 cells activated by complete stimulation with anti-CD3 and anti-CD28 (left lanes) oranergized by incomplete stimulation with anti-CD3 alone (right lanes). Equal numbers of T cells were analyzed by immunoblotting for proteins (right margin).(c) Primary TH1 cells from 2B4 mice were left untreated (top) or were pretreated with ionomycin for 16 h (bottom) before fura-2 labeling, incubation withbiotinylated anti-CD3 and [Ca2+]i imaging. After an observation period of 100 s (left downward arrows), streptavidin was added to induce TCR crosslinking(TCR); at 600 s (right downward arrows), ionomycin (Iono) was added to identify responsive cells. Ca2+ mobilization was monitored by time-lapse videomicroscopy. Data represent individual (gray) and averaged (black) traces from ∼ 100 CD4+ and ionomycin-responsive single cells. α-, anti-.
migrated as a doublet corresponding to mono- and diubiquitinatedforms (Fig. 3b, arrow, top). Coexpression of Itch strongly enhancedPLC-γ1 ubiquitination, increasing the amounts of mono-, di- andpolyubiquitinated forms, but the ionomycin dependence of ubiqui-tination was less notable in these conditions (Fig. 3b, lanes 2 and 3).When we cotransfected 293 cells with PLC-γ1 and Nedd4 or Itchexpression vectors and treated the cells with ionomycin, we noted aloss of PLC-γ1 (Fig. 3c, top, lanes 3, 4 and 7, 8). This decrease wasblocked by coexpression of a dominant negative Nedd4 proteinbearing an alanine substitution at the active site cysteine of theHECT domain (Fig. 3c, top, lanes 5 and 6). The subcellular localiza-tion of Itch and Nedd4 was altered in anergic T cells, as the combina-tion of ionomycin treatment and homotypic cell adhesion causedstrong translocation of both proteins to the detergent-insoluble
membrane fraction (Fig. 3d). In the same conditions, the membraneadapter LAT localized to both detergent-soluble and detergent-insoluble membrane fractions and was equally abundant in thesefractions in resting and anergized cells. Thus, the C2 domain–con-taining E3 ligases Itch and Nedd4 are strong candidates for mediat-ing PLC-γ1 degradation in T cells anergized by sustained Ca2+
signaling. Although C2 domains do not necessarily engage in Ca2+-dependent protein-protein interactions27, they are likely to promotecolocalization of target and effector proteins at the plasma mem-brane, possibly through interactions with specific phospholipids oradapter proteins such as the annexins29. Indeed, annexins I and VIwere among the proteins most highly induced in a proteomic analy-sis of tolerant T cells in vivo26, indicating that the mechanism forlocalizing E3 ligases to the membrane fraction in anergic T cells is
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Figure 3 E3 ubiquitin ligases of the HECT type induce ubiquitination and degradation of PLC-γ1. (a) Physical interaction of Nedd4 and Itch with PLC-γ1. AU.1epitope–tagged PLC-γ1 was coexpressed in HEK 293 cells with Myc-tagged Itch or Myc-tagged Nedd4. Anti-Myc immunoprecipitates (top two panels) orwhole-cell lysates (bottom two panels) were analyzed by immunoblotting (proteins, right margin). PLC-γ1 in immunoprecipitates was detected with a ‘cocktail’of monoclonal antibodies. (b) Itch induces mono-, di- and polyubiquitination of PLC-γ1. HEK 293 cells were transfected in duplicate with expression vectorsencoding hemagglutinin (HA)-tagged ubiquitin, AU.1-tagged PLC-γ1 and/or Myc-tagged Itch (above lanes), and one culture of each pair was stimulated with 3 µM ionomycin (Iono) for 30 min before cell extraction. Cell extracts were immunoprecipitated with anti-AU.1 and analyzed for ubiquitin-modified (Ub–) ortotal immunoprecipitated PLC-γ1 (top two panels) or were directly analyzed for PLC-γ1 and Itch expression by immunoblotting (bottom two panels). (c) Itch and Nedd4 promote PLC-γ1 degradation. HEK 293 cells were mock-transfected (Mock) or were transfected with Itch, Nedd4 or a catalytically inactive Nedd4mutant (DN Nedd4) and AU–PLC-γ1 and were stimulated with ionomycin (Iono +) or were left unstimulated (Iono –). Top, cell lysates were analyzed for PLC-γ1 expression by immunoblotting with antibodies to the AU tag. Bottom, immunoblotting of the lysates from unstimulated cells (lanes 1, 3, 5 and 7) withantibodies to endogenous Itch and Nedd4 shows that the amounts of ectopically expressed and endogenous Nedd4 were similar, whereas the expression ofintroduced Itch was about fourfold greater than that of endogenous Itch. (d) D5 cells were left untreated (–) or were stimulated with ionomycin (+) for 16 h,then were washed and incubated at higher cell density for 2 h. Cytoplasmic, detergent-soluble and detergent-insoluble membrane fractions were analyzed by immunoblotting. (e) The proteasome inhibitor MG132 promotes accumulation of a modified form of PKC-θ. D5 cells were left untreated (Iono –) or werepretreated with ionomycin (Iono +), then were washed and incubated in the absence (–) or presence (+) of MG132 (treatments above lanes). Extracts wereimmunoblotted for PLC-γ1 and PKC-θ. (f) PKC-θ becomes monoubiquitinated in cells subjected to sustained Ca2+ signaling. D5 cells were left untreated (–) orwere pretreated with ionomycin (+), and cell lysates were immunoprecipitated with anti-PKC-θ. Immunoprecipitates were analyzed for ubiquitin modification(Ub–) by immunoblotting. α-, anti-; IP, immunoprecipitation.
also induced and/or activated as part of the anergy program, eitherduring the first step of sustained Ca2+-calcineurin signaling or inresponse to T cell–APC contact.
PKC-θ is monoubiquitinated in anergic T cellsThe proteasome inhibitor MG132 did not prevent PLC-γ1 degrada-tion, nor did it inhibit the loss of PKC-θ protein noted in ionomycin-pretreated D5 T cells subjected to homotypic adhesion (Fig. 3e).Instead, MG132 slightly increased the accumulation, only in aner-gized T cells, of a modified form of PKC-θ visible in a long exposureof the imaging film to the immunoblot. This species migrated withan apparent molecular weight of about 10 kDa greater than that ofPKC-θ itself, indicating that it represented a monoubiquitinatedform. The monoubiquitin modification is transient and technicallydifficult to detect: sorting of monoubiquitinated proteins into theinternal vesicles of multivesicular bodies involves an obligatory deu-biquitination step18, and therefore the proteins reside in the ubiqui-tinated state for only a very short time. Moreover, in contrast topolyubiquitination, the monoubiquitin modification presents onlyone epitope for detection with the antibodies to ubiquitin comparedwith many more epitopes for polyubiquitination. Nevertheless, wewere able to demonstrate unambiguously that PKC-θ wasmonoubiquitinated in anergic T cells, by immunoprecipitating PKC-θ and immunoblotting for ubiquitin (Fig. 3f). Untreated T cellsshowed no ubiquitin modification, whereas ionomycin-pretreated T cells that were allowed to interact homotypically showed a distinctband at a molecular weight corresponding to that of monoubiquiti-nated PKC-θ, with no apparent signal at higher molecular weights.These results indicated that degradation of signaling proteins inanergic T cells was not accomplished through the proteasome, whichbinds with high affinity only to proteins tagged with four or moreubiquitin moieties3, but rather through the lysosomal pathway, inwhich monoubiquitination promotes sorting of proteins associatedwith the limiting membrane of endosomes into small internal vesi-cles that accumulate in the lumen as the endosomes mature18,30.
Calcineurin induces E3 ligases and Tsg101 expressionWe asked whether Ca2+-induced anergy was associated with upregu-lation of the protein machinery involved in PLC-γ1 and PKC-θ degra-dation. In yeast, endosomal sorting is accomplished by theendosome-associated endosomal sorting complex required for trans-port (ESCRT-1 complex), which binds monoubiquitin- and diubiq-
uitin-tagged transmembrane proteins and sorts them into the invagi-nating structures that form the internal vesicles31; the resulting multi-vesicular bodies fuse with lysosomes and deliver their contents fordegradation18,30. The critical ubiquitin-binding component of theyeast ESCRT-1 complex is Vps23p, the mammalian homolog ofwhich is Tsg101 (ref. 18). Tsg101 is essential for downregulation of theactivated EGF-receptor, which is ubiquitinated by the E3 ligase Cbl30.Cbl proteins are known to diminish proximal TCR transduction bydownregulating the TCR16 as well as by ubiquitinating and inducingdegradation of TCR-coupled tyrosine kinases32.
Based on these considerations, we sought to determine whether E3ligases of the Cbl and Nedd4 families and the ubiquitin receptorTsg101 were upregulated during the development of T cell anergy.Tsg101, Itch and Cbl-b (the main Cbl family member in mature Tcells16) increased in a Ca2+- and calcineurin-dependent way duringthe priming step of anergy (Fig. 4a). Itch and Tsg101 increased aboutthreefold in ionomycin-treated D5 cells, and the increase was blockedby CsA. Cbl-b was induced even more highly, and its induction waspartly blocked by CsA. There was no change in the amount of Nedd4protein under these conditions, despite the membrane relocalizationof Nedd4 shown in Figure 3d. Upregulation of the E3 ligases reflectedan anergy-associated transcriptional program: PLC-γ1 mRNAexpression remained constant, but the amounts of mRNA encodingItch, Cbl-b and GRAIL (an anergy-associated E3 ligase17 encoded byRnf128) increased by 8- to 11-fold in ionomycin-treated T cells, andthis increase was mostly blocked by CsA17 (Fig. 4b). Thus, sustainedCa2+ and calcineurin signaling is associated not only with PLC-γ1 andPKC-θ degradation but also with upregulation of several moleculeswith involvement in protein monoubiquitination, endosomal sortingand lysosomal degradation: the Itch E3 ligase, linked here to themonoubiquitination and downregulation of PLC-γ1 and PKC-θ; theCbl-b E3 ligase, linked together with Cbl to the monoubiquitinationand downregulation of the TCR16; the endosome-associated E3 ligaseGRAIL17; and Tsg101, the receptor component of the endosomalESCRT-1 complex, which mediates sorting of monoubiquitinatedproteins into multivesicular bodies targeted for lysosomal fusion anddegradation18.
Disintegration of immunological synapses in anergyBecause TCR signaling occurs at the interface (immunologicalsynapse) between the T cell and the APC9,10, we monitored the for-mation and subsequent activity of the immunological synapse in
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Figure 4 Upregulation of E3 ligases in T cells subjected to sustained Ca2+ signaling. (a) Upregulation of Itch, Cbl-b and Tsg101 in anergic T cells. D5 cells were left resting (–) or were stimulated (+) with ionomycin (Iono), CsA or both. Cell extracts were evaluated for Itch, Tsg101, Cbl-b and Nedd4 byimmunoblotting, and relative protein expression was quantified (below lanes). (b) D5 cells were left untreated or were stimulated with ionomycin (Iono) or ionomycin plus CsA for 10 h, and expression of Itch, Cblb, Rnf128 (GRAIL) and Plcg1 mRNA was evaluated by real-time RT-PCR, normalized to amountsof mRNA encoding the ribosomal protein L32. Data represent the average ± s.d. of the ratio of mRNA expression in ionomycin-treated or ionomycin andCsA–treated to that in untreated cells. Results are representative of at least two independent experiments.
untreated and anergic T cells. In both cases, the immature immu-nological synapse, characterized by peripheral TCR–major histo-compatibility complex (MHC)–peptide contacts and centrallymphocyte function-associated antigen-1 and intercellular adhe-sion molecule 1 (LFA-1–ICAM-1) contacts, developed quickly intothe mature structure with a core TCR-MHC-peptide contact regionand a peripheral LFA-1–ICAM-1 ring (Fig. 5a,b, 5- and 6-min timepoints). Thus, anergic cells show no impairment of the membraneand cytoskeletal interactions necessary for synapse maturation.However, the mature synapse, which persisted stably in theuntreated T cells for at least an hour after the initial contact10–12,was unstable in anergic T cells. Anergic T cells showed partial or,occasionally, complete breakdown of the outer LFA-1 ring within10–20 min after the mature synapse was established, and often alsoshowed aberrant morphology of the inner TCR core (Fig. 5a,b, 10 min and later). Parallel analysis of fluorescence and contact areapatterns showed that anergic T cells had a ‘migratory’ phenotype, inwhich the outer LFA-1–ICAM-1 ring became disorganized, pullingaway from and distorting the TCR-MHC clusters, which weredragged behind the moving T cells (Fig. 5a). In this respect, anergicT cells (at 10 min and later) act like cells that do not receive a TCR-mediated ‘stop’ signal10,33,34. Migration of anergized T cells would
make a strong contribution to unresponsiveness because the T cell–APC contact would not be stably maintained.
We sought to determine whether synapse instability could beattributed to the loss of PLC-γ1 function. We allowed T cells toestablish mature synapses for 60 minutes, after which we obtainedimages of fields containing stable immunological synapses with cen-tral MHC clusters and complete ICAM-1 rings and recorded thelocations of the synapses. We then treated the stable synapsessequentially with the weak and strong phospholipase inhibitorsU73343 and U73122, respectively, and evaluated the effects of thedrugs on the previously imaged synapses. Whereas the weakinhibitor only had a slight effect on synapse integrity, the stronginhibitor effectively abolished the LFA-1 contact ring (Fig. 5c), aneffect that resembled the phenotype of disintegration of the outerLFA-1 ring observed in anergic T cells (Fig. 5a,b). These dataemphasize the requirement for PLC-γ1 in maintenance of themature immunological synapse and confirm previous reports thatboth anergic T cells and T cells treated with phospholipaseinhibitors do not bind fibronectin efficiently25. PLC-γ1-dependentdiacylglycerol production is required for effective ‘inside-out’ sig-naling25,35, whereas PKC-θ activation, which is ‘downstream’ of dia-cylglycerol production by PLC-γ1, has been linked to efficient
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formation of the immunological synapse (T.N. Sims, T. Soos, D.R.Littman and M.L.D., unpublished observations). These results indi-cate strongly that the aberrant synapse morphology of anergic Tcells can be attributed to reduced signaling through PLC-γ1 andPKC-θ and consequent reduced LFA-1 activity.
Genetic evidence for involvement of Itch and Cbl-b in anergyMice deficient in either Itch or Cbl-b have autoimmune pheno-types15,19–21, indicating that these E3 ligases are important in sup-pressing immune responses to self antigens. To evaluate theparticipation of Itch and Cbl-b in Ca2+-induced T cell anergy, wetested T cells from Itch–/– (Itchy) and Cblb–/– mice (Fig. 6). Wetreated the cells with increasing doses of ionomycin, then stimulatedthem with anti-CD3 plus anti-CD28, after which we assayed prolifer-ation ([3H]thymidine incorporation). Itch–/– and Cblb–/– T cells wereresistant to anergy induction at low doses of ionomycin, and thiseffect was partially overcome at higher doses of ionomycin (Fig. 6a).We also assessed the ability of Itch–/– and Cblb–/– T cells to degradePLC-γ1 and PKC-θ in response to anergy-inducing conditions. Asexpected, PLC-γ1 protein decreased in wild-type T cells after thecells were anergized with anti-CD3 stimulation in the absence ofcostimulation, but Itch–/– and Cblb–/– T cells did not show thisdecrease (Fig. 6b). Likewise, wild-type T cells showed the expecteddecrease in PKC-θ protein after ionomycin pretreatment followedby restimulation with anti-CD3, but we did not find this effect in T cells from Itch–/– and Cblb–/– mice (Fig. 6c). Finally, we comparedthe kinetics of synapse disintegration in control and Cblb–/– T cellsthat had been anergized by pretreatment with ionomycin (Fig. 6d).As expected, control 5CC7 TCR transgenic T cells exposed to pep-tide-loaded MHC and LFA-1 molecules in lipid bilayers formed
synapses that were stable throughout the observation period of 50 min (Fig. 6d), whereas 5CC7 T cells that were pretreated withionomycin for 16 h formed the mature synapse quickly (<5 min) oncontact with the bilayer but then showed synapse disorganizationand developed the migratory phenotype. Synapses formed byuntreated Cblb–/– T cells were as stable as those formed by wild-typeT cells, but synapses formed by ionomycin-pretreated Cblb–/– T cellswere mostly protected from synapse disintegration, as judged bytheir stability for up to 35 min of observation. Thus, Cbl-b con-tributes substantially to the early disintegration of the immunologi-cal synapse in anergic T cells. However, the synapses break down atlater times in ionomycin-pretreated Cblb–/– T cells (50 min), indi-cating that other factors are also involved.
DISCUSSIONBased on our data, we propose that T cell anergy is initiated andimplemented through a complex multistep negative signalingprocess, at least one facet of which involves the coordinate actions ofseveral E3 ligases. The program is initiated by sustained Ca2+-calcineurin signaling and culminates in proteolytic degradation ofPLC-γ1 and PKC-θ, two central participants in the TCR signalingcascade24,36. The first step of the program involves calcineurin-mediated upregulation of three E3 ligases, Itch, Cbl-b and GRAIL,as well as Tsg101, the ubiquitin-binding component of the endoso-mal sorting complex. This process may be mediated through NFATin the absence of AP-1 cooperation8. Degradation of signaling pro-teins is implemented during a second step of T cell–APC contact,during which the immunological synapse forms normally and theE3 ligases Itch, Nedd4 and Cbl-b move to detergent-insoluble mem-brane fractions, where they may colocalize with activated substrate
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Figure 6 Cblb- and Itch-deficient T cells areresistant to anergy induction. (a) CD4 T cells fromC57BL/6 (WT), Cblb–/– and Itch–/– mice werestimulated with anti-CD3 and anti-CD28 for 2 dand were left resting for 5 d. Cells were then leftuntreated or were treated for 16 h with 25–100ng/ml of ionomycin (Iono), after whichproliferative responses to anti-CD3 and anti-CD28stimulation were measured by [3H]thymidineincorporation. (b) TH1 cells from C57BL/6 (WT),Cblb–/– and Itch–/– mice were allowed todifferentiate for 1 week, then were stimulatedwith plate-bound anti-CD3 in the presence ofCTLA4-Ig (Anergized) or with anti-CD3 and anti-CD28 (Activated) for 2 d, then were allowed to‘rest’ for 3 d in media without interleukin 2. Cellextracts were analyzed for PLC-γ1 and actin byimmunoblotting. (c) TH1 cells from C57BL/6(WT), Itch–/– and Cblb–/– mice were left untreated(–) or were treated for 16 h with ionomycin (+),were washed, then were restimulated (+) or not(–) with plate-bound anti-CD3 (α-CD3). Cellextracts were analyzed for PKC-θ and actin byimmunoblotting. (d) Formation of immunesynapses was evaluated as described in Figure 5,with TH1 cells from wild-type or Cblb–/– 5CC7TCR–transgenic mice and lipid bilayers displayingICAM-1 and I-Ek pigeon cytochrome C (PCC)molecules. Top, individual representative cells(genotypes, left margin) observed over a timecourse of 50 min. Bottom, quantification ofresults, showing the percentage of cells withstable synapses at 35 min after synapseformation was initiated.
proteins. The membrane fraction may include ‘raft’ membranes,endosomal membranes or both, consistent with previous findingsthat PLC-γ1, RasGAP, Tsg101 and GRAIL are all found associatedwith endosomes4,17. As a result, the active, membrane-proximalpool of signaling proteins becomes monoubiquitinated and capableof stable interaction with Tsg101. This interaction in turn results insorting of the monoubiquitinated proteins into multivesicular bod-ies and their targeting for lysosomal degradation. In the third step,degradation of active PLC-γ1 and PKC-θ leads to diminished TCRand LFA-1 signaling. Once this happens, the mature synapse cannotbe maintained11 and the inability to sustain stable APC contact fur-ther reduces the antigen responses of anergic T cells. Overall, there-fore, a stable difference in gene expression profile between normaland anergic T cells is transformed through ubiquitin modificationinto a transient increase in turnover of activated signaling proteins,thereby altering the migration activity of T cells and establishing apersistent unresponsive state.
The attractive feature of such a downregulatory program is that sig-naling molecules would be targets for degradation only when they areactivated. In a normally activated T cell, signaling through the TCRresults in maintained phosphatidylinositol-3,4,5 triphosphate pro-duction, PLC-γ1-dependent production of second messengers andCa2+-mobilization, continuing for several hours11. In an anergic Tcell in which the Itch, Cbl-b, Nedd4 and GRAIL E3 ligases are upreg-ulated and/or preactivated for membrane localization, PLC-γ1 andPKC-θ activation would be rapidly followed by E3-mediatedmonoubiquitination at ‘raft’ or endosomal membranes, and this,through Tsg101, would immediately sequester the active enzymeswithin endosomes where they cannot be reactivated. Thus, imposi-tion of T cell anergy would be a localized and efficient process inwhich PLC-γ1 and PKC-θ would be eliminated only in the context ofactive, membrane-localized signaling complexes, and massive deple-tion of the bulk of cellular PLC-γ1 would not be required (and is notfound). Consistent with this hypothesis, anergic T cells show noappreciable downregulation of PLC-γ2, which despite having thesame domain organization as PLC-γ1 is not part of the signaling path-way downstream of the TCR24.
Our data indicate that lack of anergy induction is the molecular mech-anism underlying the autoimmune phenotypes of Cbl-b-deficient andItch-deficient (Itchy) mice. Itchy mice show splenomegaly and lympho-cyte infiltration in several tissues and chronic inflammation in theskin15,19, whereas Cblb ablation is associated with spontaneous T cellactivation and autoantibody production21 and enhanced experimentalautoimmune encephalomyelitis20. Furthermore, Cblb is a principal genelinked to susceptibility to type I diabetes in rats37. Because Itch–/– andCblb–/– T cells have very similar phenotypes of resistance to Ca2+-induced anergy (impaired proliferation and impaired degradation ofPLC-γ1 and PKC-θ), it is likely that these two proteins cooperate toevoke T cell anergy, either in the context of a multiprotein complex38 orby acting sequentially in a pathway of anergy induction. Cbl familymembers are essential for internalization and downregulation of the Tcell receptor16, possibly through their ability to bind the CIN85 paralogCD2AP39; thus, one likely scenario is that Cbl-b regulates TCR internal-ization through monoubiquitination and sorting into internal vesiclesof late endosomes, whereas Itch functions to monoubiquinate PLC-γ1and PKC-θ, routing them into the same lysosomal degradation pathwayas the TCR. The E3 ligase GRAIL, which resides in the endosomal mem-brane and is upregulated in anergic T cells17, could synergize with theseeffectors to further enhance protein ubiquitination and degradation.
Consistent with several previous studies, our findings point to acomplex function for integrins in T cell anergy. Anergic T cells
(especially T cell clones) show an initial tendency to interact homotyp-ically, but implementation of T cell anergy results in reduced bindingof LFA-1 to its ligand ICAM-1. Anergic T cells show increased tran-scription of the gene encoding LFA-1α40; upregulation of the cofactorGRP-1 (cytohesin 3), overexpression of which leads to increased activ-ity of LFA-1 (refs. 41,42); and increased GTP loading of Rap1 (ref. 43),which increases integrin adhesiveness and is crucial for antigen-dependent synapse formation44–47. Anergic T cells also show upregu-lation of CD98 (ref. 8), which induces LFA-1 adhesion throughactivation of the small GTPase Rap1 (ref. 48). Paradoxically, however,T cells from mice transgenic for constitutively active Rap1 did notshow an unresponsive phenotype, but instead showed increased pro-liferation in response to APC-peptide stimulation that correlated withincreased avidity of β1 and β2 integrins45. A recent report may resolvethis paradox, because it showed that much higher amounts of acti-vated (GTP-loaded) Rap1 than in the constitutively active Rap1 trans-genic model could be achieved by inactivation of the gene encodingSPA-1, the main GTPase-activating protein for Rap1 in peripheral Tcells49. T cells from young SPA-1-deficient mice that had been immu-nized with antigen in adjuvants developed an unresponsive phenotyperesembling the phenotype that develops in wild-type mice injectedwith soluble antigen49. We postulate that the increased amount ofRap1-GTP in anergic T cells leads to preactivation of integrins45,which in turn co-opts TCR signaling by prematurely activating the E3ligase-associated anergy program that we have defined here. However,the resulting degradation of active PLC-γ1 and PKC-θ interferes withLFA-1 activity, thus conferring the phenotype of synapse disorganiza-tion that we note in anergic T cells.
There is controversy about the precise function of the immunolog-ical synapse in TCR signaling. The kinetics of formation of the matureimmunological synapse correspond to a period in which there is sub-stantial T cell receptor internalization and degradation as well as adecrease from the early peak of tyrosine kinase signaling12. However,a stable immunological synapse seems to be required for ongoingCa2+ mobilization, phosphatidylinositol 3 kinase activation andinterleukin 2 production9–11. Our findings offer a synthesis of thesetwo opposing views, showing that the synapse is a dynamic structurewhose stability requires ongoing signaling through the TCR andwhich in turn modulates the overall level of T cell responses by inte-grating positive and negative signals from a variety of surface recep-tors including the TCR, costimulatory receptors and integrins.
In summary, we have shown that Ca2+-calcineurin signaling, whichis essential for the productive immune response, also promotes T cellanergy through a program of ubiquitin modification and targeteddegradation of signaling proteins. Thus, our studies confirm that T cell anergy involves an active process that precedes unresponsive-ness to antigen. It is notable that two main participants in this are E3ubiquitin ligases whose involvement in negative signaling has alreadybeen confirmed in mouse models of autoimmune disease. Furtherbiochemical analysis of the Ca2+- and calcineurin-induced anergyprogram should uncover additional participants, potentially encodedby the many genetic loci that predispose to autoimmune disease.
Note added in proof: The Itchy cells used in these studies show a TH2 biasthat has been attributed to increased JunB, a postulated Itch target50,raising the possibility that the autoimmune phenotype of Itchy mice isdue to T cell hyperproliferation in response to increased interleukin 4.However, this explanation is not compatible with the finding that back-crossing Itchy mice to the B10 background eliminates their TH2 bias invivo, but the mice still develop autoimmune disease (V. Parravicini andR. Zamoyska, personal communication).
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METHODSMice. BALB/cJ mice and DO11.10, 2B4, AND and 5CC7 TCR-transgenic micewere obtained from Jackson laboratories and were maintained and bred inpathogen-free conditions in a barrier facility.
Cell culture, cell stimulation and anergy induction ex vivo. The mouse D5(Ar-5) TH1 cell clone was grown as described8. CD4+ cells were isolated fromspleens and lymph nodes of mice with positive selection with anti-CD4 mag-netic beads (Dynal) and were differentiated into TH1 cells for 2 weeks withstandard protocols8. For most experiments, anergy was induced by treatmentof primary TH1 cells or the D5 TH1 clone (1 × 106 cells/ ml) with 1 µM iono-mycin for 16 h. This method has the advantage of providing a homogeneouspopulation of anergic T cells that have synchronously experienced the sameintensity of prior Ca2+ signals and so are suitable for biochemical investiga-tion. CsA was included in some experiments at a concentration of 2 µM. Cellswere washed to remove ionomycin and were incubated at higher cell density (3 × 106 cells/ ml) for 1–2 h at 37 °C. In the experiment in Figure 1a, the high-density incubation step was included but had not been planned. For the exper-iment in Figure 1c, D5 cells were restimulated with 1 µg/ml of anti-CD3 withor without 2.5 µg/ml of anti-CD28, or with 20 nM PMA, 1 µM ionomycin orboth. For the experiment in Figure 2b, primary TH1 cells were stimulated withplate-bound anti-CD3 to induce anergy or with a combination of anti-CD3and anti-CD28 to induce productive activation. In both cases the cells gothrough a phase of active proliferation, but cells that received only anti-CD3stimulation responded much less well to subsequent restimulation than cellspreviously stimulated with both anti-CD3 and anti-CD28. For the experi-ments of Figure 6, TH1 cells from C57BL/6, Cblb–/– or Itch–/– mice that hadbeen differentiated in vitro for 1 week were stimulated for 2 d with plate-bound anti-CD3 (3 µg/ml) in the presence of CTLA4-Ig (10 µg/ml) to induceanergy or with anti-CD3 (0.25 µg/ml) and anti-CD28 (2 µg/ml) to induceactivation, then were left resting for 3 d in media without interleukin 2. Theextent of anergy induction was evaluated by intracellular cytokine staining orin standard proliferation assays8. HEK 293 cells were grown and transfectedwith Ca2+ phosphate with standard protocols.
Antibodies. Antibodies to Zap70 (Z24820), Lck (L15620), PKC-θ (610089),Itch (I84520) and calcineurin (610259) were obtained from BD TransductionLabs. Antibodies to Fyn (06-133), RasGAP (05-178), SOS (06-246), Vav-1 (05-219) and Nedd4 (07-049) were purchased from Upstate Biotechnologies.Santa Cruz Biotechnology antibodies were used to detect CD3ζ (sc-1239),Mekk-2 (sc-1088), RasGRP (sc-8430), ubiquitin (sc-8017), PLC-γ2 (sc-407),Cbl-b (sc-1705), NF-κB p65 (sc-109), NF-κB p50 (sc-1192), IKKγ (sc-8330)and Myc-tagged (sc-789) and hemagglutinin-tagged (sc-805) proteins.Antibody to the AU.1 (MMS-130P) epitope tag was purchased from Covance;anti-Akt (9272), from Cell Signaling; anti-Tsg101 (ab83), from Genetex; andanti-IKKβ (AHO0362), from Biosource. Antibodies to NFAT1 and NFAT5were produced in the Rao lab. Antibodies to Gads and LAT were gifts from E.Clark (University of Washington, Seattle, WA) and L. Samelson (NationalCancer Institute, Bethesda, MD), respectively. Antibodies to p85 phos-phatidylinositol 3 kinase were obtained from the laboratory of L. Cantley; andantibodies to SHP-1, SHP-2 and PTP-1B were provided by B. Neel (both fromBeth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA).Endogenous PLC-γ1 was detected with a polyclonal antiserum provided by A.Toker (Beth Israel Deaconess Medical Center, Harvard Medical School,Boston, MA), which was raised against the epitope APRRTRVNGDNR, repre-senting the final C-terminal amino acids of the protein. The reactivity of thisantiserum with PLC-γ1 is unlikely to be affected by phosphorylation, as pro-posed earlier51, as the epitope does not contain any serine or tyrosine residuesand its single threonine residue is not part of any predictable phosphorylationmotif, as judged by the Scansite computer program. Furthermore, a commer-cial antibody source, comprising a pool of four different monoclonal antibod-ies (05-163; Upstate Biotechnologies), also allowed us to visualize thedifferences in PLC-γ1 protein amounts in untreated and anergic T cells whenthe antibody was used at a fivefold higher dilution than recommended. Wehave also used a second monoclonal antibody to PKC-θ (E-7; Santa CruzBiotechnology) to immunoprecipitate PKC-θ in partially denaturing condi-tions from resting and anergized primary T cells. These experiments con-
firmed that anergized TH1 cells subjected to restimulation showed a reductionin PKC-θ protein compared with their counterparts that were not restimu-lated; however, a corresponding anti-CD3 stimulation of resting TH1 cells didnot result in such a decrease (data not shown).
Expression plasmids. Nedd4 (KIAA0093) cDNA and Itch cDNA15 wereinserted via SalI-NotI into pRK5 vectors containing an N-terminal sequenceencoding the Myc epitope. R. Abraham (Duke University, Durham, NC) and J. Huibregtse (University of Texas at Austin, Austin, TX) provided expressionplasmids for PLC-γ1 and Nedd4, respectively.
Cell extracts, immunoprecipitations and immunoblots. D5 cells wereextracted at a concentration of 1 × 106 cells/10 µl in radioimmunoprecipita-tion assay (RIPA) buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM DTT, 10 mM MgCl2, 1% Nonidet P-40, 0.1% SDS and 0.5% sodium deoxy-cholate) supplemented with protease and phosphatase inhibitors (1 mMphenylmethyl sulfonyl fluoride, 25 µg/ml of aprotinin, 25 µg/ml of leu-peptin, 10 mM NaF, 8 mM β-glycerophosphate and 0.1 mM sodium ortho-vanadate). Re-extraction of the resulting DNA pellet with hot SDS samplebuffer followed by immunoblotting did not show any residual PLC-γ1 in theSDS fraction of either resting or ionomycin-anergized D5 cells, confirmingthe efficiency of extraction with RIPA buffer (data not shown). For assess-ment of proteins in cell extracts, 7.5–30 µl of RIPA extracts were separatedby 9–12% SDS-PAGE, and proteins were electrotransferred onto nitrocellu-lose membranes. For immunoprecipitation, 500–1,000 µl of RIPA cellextracts were used. For coimmunoprecipitation from lysates of transfectedHEK 293 cells, cells from one 10-cm dish were lysed in 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0,5% Nonidet P-40 and 10% glycerolplus phosphatase and protease inhibitors. Lysates were precleared witheither protein A– or protein G–Sepharose, immunoprecipitation proceededfor 4 h and the resulting precipitates were washed three to four times withthe buffer used for cell extraction. For detection of monoubiquitinatedPKC-θ, cell lysates from 1 × 108 untreated or ionomycin-pretreated D5 cellswere immunoprecipitated with anti-PKC-θ. Immunoblots used antibodysolutions in 5% milk and TBS (10 mM Tris-HCl, pH 8.0, and 150 mMNaCl) and washes used TBS containing 0.05% Tween-20. Relative bandintensities were quantified by NIH IMAGE Quant and corrected for back-ground within each lane.
Cell fractionation. Cell fractionation was done essentially as described52
with 3 × 107 D5 cells. Cells were swollen for 15 min in hypotonic buffer E(10 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl2 and 1 mM DTT supple-mented with protease and phosphatase inhibitors) and lysed by Douncehomogenization. Lysates were centrifuged at 100,000g for 30 min, yielding asupernatant (cytosol) and a pellet that was resuspended in buffer E contain-ing 1% Nonidet P-40 and was recentrifuged at 100,000g for 30 min to separate the detergent-soluble fraction in the supernatant from the deter-gent-insoluble fraction (pellet). The pellet was resuspended by sonicationin RIPA buffer and cleared by centrifugation. One fourth of the supernatantfrom each centrifugation step (cytoplasm, detergent soluble and detergentinsoluble fractions) was analyzed for by immunoblotting for Nedd4, Itchand LAT.
[Ca2+]i imaging and immunocytochemistry. Intracellular calcium wasmeasured in primary TH1 cells from 2B4 mice. Cells were loaded with 1 µMfura-2 aceto-methyl ester (Molecular Probes) for 30 min at 20 °C, washedand resuspended in loading medium (RPMI + 10% FCS), incubated with2.5 µg/ml of biotinylated anti-CD3 (2C11, Pharmingen) for 15 min at 20 °Cand attached to poly-L-lysine–coated coverslips mounted in a RC-20 closedbath chamber (Warner Instrument). The fura-2-loaded cells were perfusedwith Ringer’s solution containing 2 mM calcium (155 mM NaCl, 4.5 mMKCl, 10 mM D-glucose, 5 mM HEPES, pH 7.4, 1 mM MgCl2 and 2 mMCaCl2) and were stimulated by crosslinking of the surface-bound biotiny-lated anti-CD3 with 2.5 µg/ml of streptavidin (Pierce), after which healthycells were identified by their responsiveness to 1 µM ionomycin(Calbiochem). Single-cell video images were obtained on a Zeiss AxiovertS200 epifluorescence microscope with OpenLab imaging software(Improvision). Fura-2 emission was detected at 510 nm after excitation at
NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 263
340 and 380 nm. The 340/380 ratio images were acquired every 5 s afterbackground subtraction. Calibration values (Rmin, Rmax and Sf) werederived from cuvette measurements with a calcium calibration buffer kit(Molecular Probes) and as described53.
Real-time PCR analysis. Total RNA was prepared from untreated or iono-mycin-pretreated D5 cells with Ultraspec reagent (Biotecx). cDNA was synthe-sized from 2 µg of total RNA as template, with a cDNA synthesis kit(Invitrogen). Quantitative real-time PCR was done in an I-Cycler (BioRad) witha SYBR Green PCR kit (Applied Biosystems). The sequences of the primer pairswere: L32 sense, 5′-CGTCTCAGGCCTTCAGTGAG-3′, and L32 antisense, 5′-CAAGAGGGAGAGCAAGCCTA-3′; PLC-γ1 sense, 5′-AAGCCTTTGACCCCTTTGAT-3′, and PLC-γ1 antisense, 5′-GGTTCAGTCCGTTGTCCACT-3′; Itchsense, 5′-GTGTGGAGTCACCAGACCCT-3′, and Itch antisense, 5′-GCTTCTACTTGCAGCCCATC-3′; Cblb sense, 5′-CTTAAATGGGAGGCACAGTAGAAT-3′, and Cblb antisense, 5′-CAGTACACTTTATGCTTGGGAGAA-3′;Rnf128 sense, 5′-GTAACCCGCACACCAATTTC-3′, and Rnf128 antisense, 5′-GTGAGACATGGGGATGACCT-3′. Thermal cycling conditions were 95 °C for5 min, then 40 cycles of 95 °C, 65 °C, and 72 °C for 30 s each, terminating with asingle cycle at 72 °C for 5 min. Signals were captured during the polymerizationstep (72 °C). A threshold was set in the linear part of the amplification curve,and the number of cycles needed to reach it was calculated for each gene.Melting curve analysis and agarose gel electrophoresis were done to test thepurity of the amplified bands. Normalization was done by using amounts ofmRNA for the ribosomal protein L32 as an internal control for each sample.
Formation of immunological synapses in lipid bilayers. Planar bilayers wereprepared essentially as described10, except that the moth cytochrome C(MCC) 88-103 peptide was loaded on glycosylphosphatidylinositol (GPI)–I-Ek for 24 h. Bilayers were prepared with Oregon green–labeled GPI–I-Ek andindodicarbocyanine-labeled GPI–ICAM-1 in parallel plate flow cells(Bioptechs). Control and ionomycin-pretreated T cells from TCR-transgenicmice were injected into the flow cell at a density of 1 × 106 cells/ml. Areas ofbilayers where cells were forming synapses were imaged with fluorescein isoth-iocyanate and indodicarbocyanine optics on an Olympus IX-70 invertedmicroscope equipped with a Hamamtsu ORCA-ER digital camera and a xenonarc lamp as a light source for fluorescence microscopy. The filter wheels, shut-ters and the camera were controlled with IPLAB software on a Macintosh plat-form. Bright field, interference reflection and fluorescence images werecollected and processed with Metamorph software. The background from thefluorescence images was subtracted with the produce-background correctionimage function, which is based on median filtering to subtract backgroundthat is nonuniform. The percentage of cells adhering was evaluated by com-paring bright field and interference reflection microscopy images.
Experiments with phospholipase inhibitors were done with AND T cellblasts (day 8). Cells were allowed to form immunological synapses on bilayerscontaining 80 molecules/µm2 of Oregon green I-Ek–MCC 88-103 and 200 molecules/µm2 of indodicarbocyanine–ICAM-1 in the presence of 0.01%dimethylsulfoxide (the carrier concentration for 1 µM U73122 and U73343).After 60 min, fields containing stable immunological synapses with centralMHC clusters (green) and complete ICAM-1 rings (red) were imaged and thelocations were recorded with an automated stage and IPLab software. The sta-ble synapses were then treated sequentially with 1 µM U73343 and 1 µMU73122 (weak and strong PLC-γ inhibitors, respectively). After each drugtreatment, the same fields were imaged within 10 min so that the effects of thedrugs on many individual synapses could be determined. The quantitativedata reflect the percentage of intact LFA-1–ICAM-1 rings after carrier or drugtreatment on 103 contact areas. In separate experiments, the effects of U73343and U73122 were stable for up to 1 h and U73122-dependent destruction ofthe LFA-1 adhesion ring was not dependent on prior treatment with U73343.We found these effects in three independent experiments with U73122 con-centrations from 0.1 to 1 µM (data not shown).
Note: Supplementary information is available on the Nature Immunology website.
ACKNOWLEDGMENTSWe thank members of the Rao and Dustin laboratories for discussions, and T. Starrfor the preparation of ICAM-1 and I-Ek for planar bilayer experiments. We also
thank A. Altman, H. Band, J. Brugge, C. Joazeiro, M. Katan for advice and reagents.Supported by National Institutes of Health grants RO1-AI48213, RO1-AI40127and RO3-HD39685 (to A.R.), RO1-AI50280 and R21-AI48542 (to Y.-C.L.) and AI-43542; an Irene Diamond Foundation grant (to M.L.D.); EMBO (V.H.); andthe Cancer Research Institute (S.-H.I. and S.F.).
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Received 29 September 2003; accepted 22 January 2004Published online at http://www.nature.com/natureimmunology/
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