ARTICLES

Calcineurin imposes unresponsiveness through targeted proteolysis of signaling proteins

Vigo 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 and the transcription factor NFAT. We show here that Ca2+-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related θ γ

http://www.nature.com/natureimmunology protein Nedd4, resulting in degradation of two key signaling proteins, PKC- and PLC- 1. T cells from Itch- and Cbl-b–deficient mice 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 containing 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 in anergic T cells.

In addition to activating signaling pathways that have a positive effect, In a relatively common scenario, a single second-messenger or sig- receptor stimulation induces negative feedback pathways that attenuate naling molecule simultaneously mediates both positive and negative or terminate positive signaling. One of the best-documented mecha- outcomes downstream of surface receptors. This point has been well nisms of countering productive responses involves removal of phos- illustrated in T cells, in which Ca2+ signaling is essential not only for phate groups or other activating modifications from proteins that proliferation and effector function but also for imposition of an aner- mediate signal transduction (receptors, kinases, adapter proteins and gic state in which positive signals cannot be initiated or are substan- transcription factors1). In another mechanism, positive signals increase tially delayed or attenuated6. Sustained signaling through Ca2+ and © 2004 Nature Publishing Group the amounts or activities of negative regulators or inhibitory proteins; calcineurin results in sustained activation of the transcription factor indeed, many genes that are rapidly induced by activation of signaling NFAT, which in turn induces many genes encoding effector , pathways encode proteins that have negative effects in the same path- chemokines and other products in the productive immune response7. way2. In a third mechanism, activated signal transducers are selectively However the same transcription factor, when preactivated in the targeted for degradation, terminating ongoing signals and also interfer- absence of its transcriptional partner AP-1 (Fos-Jun), induces a differ- ing with subsequent stimulation. Cytoplasmic signaling proteins and ent set of genes encoding known or presumed negative regulators of T nuclear transcription factors tend to be polyubiquitinated and targeted cell signaling, thus mediating an opposing program of T cell anergy or for proteasomal degradation3, whereas ligand-activated surface recep- tolerance8. Among the negative molecules induced in these conditions tors, including receptor tyrosine kinases, –coupled receptors are several tyrosine phosphatases that would be expected to downreg- and the T cell receptor (TCR) are more often degraded by endocytosis ulate TCR signaling by opposing the effects of tyrosine kinases such as and trafficking to the lysosome4,5. Induced endocytosis of activated Zap70, and Itk; diacylglycerol kinase-α, which metabolizes the receptors at the plasma membrane and sorting of the receptors into second messenger diacylglycerol; and genes encoding several proteases multivesicular bodies at the endosomal membrane is regulated through and E3 ubiquitin ligases, which might induce T cell anergy by promot- tagging of receptor or adaptor proteins with ubiquitin4. Together these ing degradation of downstream signaling molecules in T cells. mechanisms ensure a balanced response to extracellular signals and The interface between the T cell and the antigen-presenting cell protect cells from the deleterious effects of chronic activation. (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 in Molecular 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 Einstein College 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

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 255 ARTICLES

Figure 1 Decreased PLC-γ1 levels correlate with T cell anergy. (a) Changes in signaling proteins in a – + –––+ + + –+ anergic T cells. T cell anergy was induced by treatment of the T 1 cell clone D5 with (+) or CD3ζ LAT Vav-1 CD45 NFAT1 H without (–) 1 µM ionomycin for 16 h. The cells SHP-1 NFAT5 were washed to remove ionomycin and were Gads Wasp Zap70 incubated at higher cell density for 1–2 h at 37 p65 p85 SHP-2 °C. Whole-cell extracts were analyzed by Sos Lck PI3K p105 immunoblotting. (b–d) Two signals are required γ γ PTP-1B for maximal loss of PLC- 1. D5 cells were PLC- 1 Akt p50 µ Fyn anergized by treatment with 1 M ionomycin Calcineurin IKKγ PLC-γ2 (Iono) for 16 h, then were washed to remove MEKK-2 ionomycin and were incubated at higher cell IKKβ density for 1 h at 37 °C. Extracts were assayed for PLC-γ1 by immunoblotting. (b) Extracts were

) prepared either directly (lanes 1 and 2) or after α Unstimulated b Iono 16 h ––++c -CD3 – ++ – – d –3 resuspension at high cell density and incubation α-CD28 – – + – – α-CD3 and α-CD28 High density 1 h –– ++ 10

× for 1 h (lanes 3 and 4). (c) Cells were pretreated Iono – – – + + 50 Iono – + PMA – – – – + for 16 h with ionomycin and restimulated for γ PLC-γ1 PLC- 1 40 1 h with anti-CD3, anti-CD3 plus anti-CD28, 30 ionomycin, or PMA plus ionomycin. (d) The γ H]thymidine 20 PLC- 1 3 [ extent of anergy induction in a proliferation 1 2 3 4 10 assay and the extent of decrease in PLC-γ1

incorporation (c.p.m. Untreated Iono-pretreated after the step of incubation at high cell density 1 2 3 45 were evaluated in parallel in a single culture of untreated (–) and ionomycin-pretreated (+) D5 cells. α-, anti-. http://www.nature.com/natureimmunology

T cell–APC interface, adhesion molecules and T cell receptor signal and lysosomal degradation of key signaling proteins ‘downstream’ transduction machinery are organized into distinct supramolecular of the TCR. activation clusters in the mature immunological synapse9,10. T cell proliferation is strongly correlated with stability of the immunologi- RESULTS cal synapse over a period of many hours9–11, but paradoxically, the Sustained Ca2+ signals promote PLC-γ1 degradation time frame of formation of the mature immunological synapse (min- Sustained Ca2+ and calcineurin signaling induces a state of T cell utes) corresponds temporally to a period of T cell receptor degrada- unresponsiveness or anergy, characterized by upregulation of many tion and downregulation of signaling12. These anergy-associated genes6,8. Increased intracellular Ca2+ concentra- 2+ 2+ findings are not necessarily contradictory: the early and late phases of tions ([Ca ]i) in resting conditions, decreased Ca mobilization in signal transduction are well known to have different biological conse- response to receptor stimulation and upregulation of tolerance- quences13, and the immunological synapse is clearly a dynamic struc- associated genes have also been documented in tolerant B cells that ture that is capable of influencing T cell responses over a period of have been exposed continuously to antigen in vivo22,23. © 2004 Nature Publishing Group several hours by integrating signals from diverse cell surface receptors The transcriptional profile of anergic T cells included several genes in addition to the TCR14. encoding proteases and E3 ubiquitin ligases8, leading us to hypothe- Here we have investigated the mechanism of T cell unresponsive- size that T cell anergy was in part a consequence of proteolytic acti- ness (anergy) induced by Ca2+ and calcineurin, specifically testing vation in cells. To test this, we assessed the amounts of many the hypothesis that T cell anergy is in part a consequence of prote- signaling proteins in T cells anergized by sustained exposure to iono- olytic activation. We show that sustained Ca2+-calcineurin signaling mycin (Fig. 1a). We noted a limited number of changes, among them leads to transcriptional upregulation of at least three E3 ubiquitin a reproducible decrease in intensity of the PLC-γ1 band. This ligases, Itch15, Cbl-b16 and GRAIL17, and also induces increased decrease occurred mainly during a subsequent step of cell stimula- expression of Tsg101, the receptor involved in sorting monoubiqui- tion or homotypic cell adhesion, as PLC-γ1 decreased after incuba- tinated proteins to the lysosomal degradation pathway18. tion of the ionomycin-treated cells at high cell density for an hour, as Restimulation of these sensitized cells results in membrane localiza- well as after restimulation with antibody to CD3 (anti-CD3), anti- tion of Itch and the related E3 Nedd4, which target the key sig- CD3 plus anti-CD28, or phorbol 12-myristate 13-acetate (PMA) naling proteins -γ1 (PLC-γ1) and plus ionomycin (Fig. 1b,c). We did not find the decrease in PLC-γ1 C-θ (PKC-θ) for monoubiquitination and lysosomal degradation. after restimulation with ionomycin alone, ruling out the possibility Anergic T cells form immunological synapses normally, but consis- of a direct effect of Ca2+-calcineurin signaling (Fig. 1c). The decrease tent with downregulation of signaling proteins after restimulation, reflected lowered amounts of PLC-γ1 protein, and was not due to cell the synapses are unstable and abnormal structures accumulate death, post-translational modification of PLC-γ1, relocalization of rapidly. Mice lacking either Itch or Cbl-b develop autoimmune dis- PLC-γ1 to a different intracellular compartment or decreased PLC- ease15,19–21, and we show that T cells from these mice are resistant to γ1 gene transcription (data not shown). In experiments done with Ca2+-induced anergy, consistent with a function for both E3 ligases optimized conditions, there was a strong correlation between loss of in T cell anergy and tolerance. Our findings define a multistep pro- PLC-γ1 and extent of anergy induction in a parallel proliferation gram in which sustained signaling through Ca2+ and calcineurin assay (Fig. 1d). Thus, anergic T cells degrade PLC-γ1 in two separa- imposes T cell unresponsiveness by promoting enhanced expression ble stages. A period of sustained Ca2+-calcineurin signaling is and membrane localization of molecules involved in ubiquitination required to initiate the degradation program, but degradation is

256 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY ARTICLES

a Iono –––++ ++– bcα-CD3 + – CsA ––++ ––++ α-CD28+ – 1,400 Untreated PLC-γ1 Iono PLC-γ1 PLC-γ2 1,200 1,000 PKC-θ 800 TCR 600 θ 400

Ras-GAP ]i, nM PKC- 200 * Lck 2+ 200 400 600 Lck [Ca 1,400 Iono-pretreated PLC-γ2 1,200 Iono RasGAP Zap70 1,000 800 600 TCR 400 200

0 200 400 600 Ubiquitin RasGRP1 Time (s)

123124 4 3

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 extracts http://www.nature.com/natureimmunology were prepared and analyzed by immunoblotting for proteins (margins) or for the extent of ubiquitin modification of total protein in the lysates (bottom right blot). 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 between lanes 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) or anergized 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 with 2+ biotinylated anti-CD3 and [Ca ]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 video microscopy. Data represent individual (gray) and averaged (black) traces from ∼100 CD4+ and ionomycin-responsive single cells. α-, anti-.

actually implemented during a subsequent step of cell stimulation or not PLC-γ2) in Ca2+ mobilization and T cell activation24, anergic T homotypic cell adhesion. cells responded very poorly to TCR stimulation compared with untreated T cells. As reported before25,26, we also noted a strong Calcineurin is required for PLC-γ1 and PKC-θ degradation impairment of Ca2+ mobilization in T cells from DO11.10 transgenic © 2004 Nature Publishing Group We used the calcineurin inhibitor cyclosporin A (CsA) to evaluate the mice that were orally tolerized by ovalbumin feeding (data not involvement of calcineurin in PLC-γ1 degradation in anergic T cells shown). Thus, the PLC-γ1 degradation observed in anergic T cells (Fig. 2a). D5 T cells subjected to 16 h of ionomycin pretreatment fol- correlates with a pronounced impairment of Ca2+ mobilization in lowed by cell-cell contact showed a substantial decrease in PLC-γ1, response to TCR triggering. Because this effect occurs without obvi- PKC-θ and RasGTPase-activating protein (RasGAP), but no changes ous delay, PLC-γ1 degradation may be initiated during the fura-2 in several other signaling proteins, including RasGRP1, Lck, Zap70 labeling step as a result of cell-cell contact. Alternatively, other mech- and PLC-γ2 (Fig. 2a, lanes 1 and 2). Degradation was completely anisms such as dephosphorylation by tyrosine phosphatases upregu- blocked by inclusion of CsA during the ionomycin treatment step lated during the step of sustained Ca2+-calcineurin signaling8 may (Fig. 2a, bottom right, lane 3). Ionomycin also induced an increase of contribute to loss of PLC-γ1 activity in anergic T cells (these possibil- about twofold in total protein ubiquitination (Fig. 2a, bottom right, ities are not mutually exclusive). lanes 1 and 2), which was blocked by CsA (Fig. 2a, lane 3). Primary T cells anergized by stimulation with anti-CD3 alone showed decreased PLC-γ1 is a substrate for Nedd4 and Itch PLC-γ1, PKC-θ, RasGAP and, to a lesser extent, Lck, but not PLC-γ2, All three targets of the Ca2+-calcineurin–dependent degradation compared with cells productively stimulated with both anti-CD3 and program, PLC-γ1, PKC-θ and RasGAP, have C2 domains anti-CD28 (Fig. 2b). The data are consistent with the hypothesis that (Supplementary Fig. 1 online). These domains can mediate Ca2+- calcineurin activates ubiquitin-dependent proteolytic pathways that dependent phospholipid binding or may serve as Ca2+-dependent promote protein degradation. or Ca2+-independent protein interaction domains27. C2 domains We assessed the functional consequences of PLC-γ1 degradation in are also found in the Itch and Nedd4 family of E3 ubiquitin ligases28 anergic T cells by examining Ca2+ mobilization (Fig. 2c). We ren- (Supplementary Fig. 1 online), leading us to test the hypothesis that γ γ dered primary T helper type 1 (TH1) cells anergic by ionomycin pre- these E3 ligases are involved in PLC- 1 degradation. PLC- 1 coim- treatment, then labeled them with the Ca2+ indicator fura-2. We munoprecipitated with both Nedd4 and Itch (Fig. 3a) and was a induced Ca2+ mobilization by TCR-CD3 crosslinking, after which we substrate for ubiquitination and degradation by Itch and Nedd4 stimulated the cells with ionomycin to identify healthy, responsive (Fig. 3b, c). In 293 cells, ionomycin induced PLC-γ1 ubiquitination cells. Consistent with the idea of a central function for PLC-γ1 (but (Fig. 3b, lanes 4 and 5), and much of the ubiquitinated PLC-γ1

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 257 ARTICLES

a beHA-ubiquitin + + + + + Iono– – – + + + γ PLC- 1 + + + Myc-Itch + + + – – High cell – + ++– + Myc-Nedd4 ––+ AU–PLC-γ1 – + + + + density Myc-Itch ––+ Iono + –+ – + MG132––+ –– +

IP α-Myc γ γ α Ub–PLC- 1 PLC-γ1 Blot α-PLC-γ1 PLC- 1 IP -AU Blot α-HA

IP α-Myc Nedd4 Blot α-Myc Itch α IP -AU PLC-γ1 Short PKC-θ Blot α-AU Lysate PLC-γ1 exposure Blot α-AU Lysate Lysate Nedd4 Blot α-Myc Itch Blot α-Myc Itch 123 Long Lysate PLC-γ1 exposure PKC-θ Blot α-AU

1234 5 12346 5

cfd Iono –––+ ++Iono – + DN Nedd4Nedd4 Itch Mock Nedd4 IP α-PKC-θ Ub–PKC-θ Iono ––––+ +++ Blot α-Ub PLC-γ1 Itch IP α-PKC-θ PKC-θ http://www.nature.com/natureimmunology 13524687 Blot α-PKC-θ Nedd4 Itch LAT 151537 37 Cytoplasm Detergent Detergent soluble insoluble

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.1 epitope–tagged PLC-γ1 was coexpressed in HEK 293 cells with Myc-tagged Itch or Myc-tagged Nedd4. Anti-Myc immunoprecipitates (top two panels) or whole-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 vectors encoding 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–) or total 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 Nedd4

© 2004 Nature Publishing Group mutant (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) with antibodies to endogenous Itch and Nedd4 shows that the amounts of ectopically expressed and endogenous Nedd4 were similar, whereas the expression of introduced 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 were pretreated with ionomycin (Iono +), then were washed and incubated in the absence (–) or presence (+) of MG132 (treatments above lanes). Extracts were immunoblotted for PLC-γ1 and PKC-θ. (f) PKC-θ becomes monoubiquitinated in cells subjected to sustained Ca2+ signaling. D5 cells were left untreated (–) or were 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.

migrated as a doublet corresponding to mono- and diubiquitinated membrane fraction (Fig. 3d). In the same conditions, the membrane forms (Fig. 3b, arrow, top). Coexpression of Itch strongly enhanced adapter LAT localized to both detergent-soluble and detergent- PLC-γ1 ubiquitination, increasing the amounts of mono-, di- and insoluble membrane fractions and was equally abundant in these polyubiquitinated forms, but the ionomycin dependence of ubiqui- fractions in resting and anergized cells. Thus, the C2 domain–con- tination was less notable in these conditions (Fig. 3b, lanes 2 and 3). taining E3 ligases Itch and Nedd4 are strong candidates for mediat- When we cotransfected 293 cells with PLC-γ1 and Nedd4 or Itch ing PLC-γ1 degradation in T cells anergized by sustained Ca2+ expression vectors and treated the cells with ionomycin, we noted a signaling. Although C2 domains do not necessarily engage in Ca2+- loss of PLC-γ1 (Fig. 3c, top, lanes 3, 4 and 7, 8). This decrease was dependent protein-protein interactions27, they are likely to promote blocked by coexpression of a dominant negative Nedd4 protein colocalization of target and effector proteins at the plasma mem- bearing an alanine substitution at the cysteine of the brane, possibly through interactions with specific phospholipids or HECT domain (Fig. 3c, top, lanes 5 and 6). The subcellular localiza- adapter proteins such as the annexins29. Indeed, annexins I and VI tion of Itch and Nedd4 was altered in anergic T cells, as the combina- were among the proteins most highly induced in a proteomic analy- tion of ionomycin treatment and homotypic cell adhesion caused sis of tolerant T cells in vivo26, indicating that the mechanism for strong translocation of both proteins to the detergent-insoluble localizing E3 ligases to the membrane fraction in anergic T cells is

258 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY ARTICLES

a b 20 Resting Iono – ++– – ++– – ++– – ++– Iono CsA –++– –++– –++– –++– Iono + CsA

Cbl-b Nedd4 Itch Tsg101 10

Relative 1 1 1 1 1.2 3.48 0.75 0.99 3.45 0.75 0.99 7.11 3.03 3.51 1.08 0.92 intensity Relative mRNA expression

Plcg1 Itch Cblb Rnf128

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 by immunoblotting, 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 amounts of mRNA encoding the ribosomal protein L32. Data represent the average ± s.d. of the ratio of mRNA expression in ionomycin-treated or ionomycin and CsA–treated to that in untreated cells. Results are representative of at least two independent experiments.

also induced and/or activated as part of the anergy program, either uitin-tagged transmembrane proteins and sorts them into the invagi- during the first step of sustained Ca2+-calcineurin signaling or in nating structures that form the internal vesicles31; the resulting multi- response to T cell–APC contact. vesicular bodies fuse with lysosomes and deliver their contents for degradation18,30. The critical ubiquitin-binding component of the θ http://www.nature.com/natureimmunology PKC- is monoubiquitinated in anergic T cells yeast ESCRT-1 complex is Vps23p, the mammalian homolog of The proteasome inhibitor MG132 did not prevent PLC-γ1 degrada- which is Tsg101 (ref. 18). Tsg101 is essential for downregulation of the tion, nor did it inhibit the loss of PKC-θ protein noted in ionomycin- activated EGF-receptor, which is ubiquitinated by the E3 ligase Cbl30. pretreated D5 T cells subjected to homotypic adhesion (Fig. 3e). Cbl proteins are known to diminish proximal TCR transduction by Instead, MG132 slightly increased the accumulation, only in aner- downregulating the TCR16 as well as by ubiquitinating and inducing gized T cells, of a modified form of PKC-θ visible in a long exposure degradation of TCR-coupled tyrosine kinases32. of the imaging film to the immunoblot. This species migrated with Based on these considerations, we sought to determine whether E3 an apparent molecular weight of about 10 kDa greater than that of ligases of the Cbl and Nedd4 families and the ubiquitin receptor PKC-θ itself, indicating that it represented a monoubiquitinated Tsg101 were upregulated during the development of T cell anergy. form. The monoubiquitin modification is transient and technically Tsg101, Itch and Cbl-b (the main Cbl family member in mature T difficult to detect: sorting of monoubiquitinated proteins into the cells16) increased in a Ca2+- and calcineurin-dependent way during internal vesicles of multivesicular bodies involves an obligatory deu- the priming step of anergy (Fig. 4a). Itch and Tsg101 increased about biquitination step18, and therefore the proteins reside in the ubiqui- threefold in ionomycin-treated D5 cells, and the increase was blocked tinated state for only a very short time. Moreover, in contrast to by CsA. Cbl-b was induced even more highly, and its induction was © 2004 Nature Publishing Group polyubiquitination, the monoubiquitin modification presents only partly blocked by CsA. There was no change in the amount of Nedd4 one epitope for detection with the antibodies to ubiquitin compared protein under these conditions, despite the membrane relocalization with many more epitopes for polyubiquitination. Nevertheless, we of Nedd4 shown in Figure 3d. Upregulation of the E3 ligases reflected were able to demonstrate unambiguously that PKC-θ was an anergy-associated transcriptional program: PLC-γ1 mRNA monoubiquitinated in anergic T cells, by immunoprecipitating PKC- expression remained constant, but the amounts of mRNA encoding θ and immunoblotting for ubiquitin (Fig. 3f). Untreated T cells Itch, Cbl-b and GRAIL (an anergy-associated E3 ligase17 encoded by showed no ubiquitin modification, whereas ionomycin-pretreated Rnf128) increased by 8- to 11-fold in ionomycin-treated T cells, and T cells that were allowed to interact homotypically showed a distinct this increase was mostly blocked by CsA17 (Fig. 4b). Thus, sustained band at a molecular weight corresponding to that of monoubiquiti- Ca2+ and calcineurin signaling is associated not only with PLC-γ1 and nated PKC-θ, with no apparent signal at higher molecular weights. PKC-θ degradation but also with upregulation of several molecules These results indicated that degradation of signaling proteins in with involvement in protein monoubiquitination, endosomal sorting anergic T cells was not accomplished through the proteasome, which and lysosomal degradation: the Itch E3 ligase, linked here to the binds with high affinity only to proteins tagged with four or more monoubiquitination and downregulation of PLC-γ1 and PKC-θ; the ubiquitin moieties3, but rather through the lysosomal pathway, in Cbl-b E3 ligase, linked together with Cbl to the monoubiquitination which monoubiquitination promotes sorting of proteins associated and downregulation of the TCR16; the endosome-associated E3 ligase with the limiting membrane of endosomes into small internal vesi- GRAIL17; and Tsg101, the receptor component of the endosomal cles that accumulate in the lumen as the endosomes mature18,30. ESCRT-1 complex, which mediates sorting of monoubiquitinated proteins into multivesicular bodies targeted for lysosomal fusion and Calcineurin induces E3 ligases and Tsg101 expression degradation18. We asked whether Ca2+-induced anergy was associated with upregu- lation of the protein machinery involved in PLC-γ1 and PKC-θ degra- Disintegration of immunological synapses in anergy dation. In yeast, endosomal sorting is accomplished by the Because TCR signaling occurs at the interface (immunological endosome-associated endosomal sorting complex required for trans- synapse) between the T cell and the APC9,10, we monitored the for- port (ESCRT-1 complex), which binds monoubiquitin- and diubiq- mation and subsequent activity of the immunological synapse in

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 259 ARTICLES

a

b

c

100 http://www.nature.com/natureimmunology 75 50 25 0

Stable synapses (%) Stable DMSO Weak Strong inhibitor inhibitor

Figure 5 Ionomycin-anergized T cells show decreased stability of the immunological synapse. (a,b) Primary TH1 cells from 2B4 TCR transgenic mice were left untreated (top rows; control) or were pretreated with ionomycin (bottom rows), then were incubated for 40 min on planar phospholipid bilayers containing Oregon green–labeled I-EK–agonist MCC peptide complexes and indocarbocyanine-labeled ICAM-1. The distribution of ICAM-1 (red) and I-Ek–MCC (green) molecules in T cell–bilayer contact zones was captured at different times. The gray panels in a are interference reflection microscopy images in which cell- bilayer contacts appear as dark areas. We have obtained similar results in more than four independent experiments. (c) Involvement of PLC-γ1 in synapse stability. Mature T cell synapses were allowed to form, then weak (U73343) or strong (U73122) PLC-γ inhibitors were added. Right, percentage of cells with

© 2004 Nature Publishing Group mature synapses relative to the same cells before the addition of inhibitors, as shown for one representative experiment of three total.

untreated and anergic T cells. In both cases, the immature immu- make a strong contribution to unresponsiveness because the nological synapse, characterized by peripheral TCR–major histo- T cell–APC contact would not be stably maintained. compatibility complex (MHC)–peptide contacts and central We sought to determine whether synapse instability could be lymphocyte function-associated antigen-1 and intercellular adhe- attributed to the loss of PLC-γ1 function. We allowed T cells to sion molecule 1 (LFA-1–ICAM-1) contacts, developed quickly into establish mature synapses for 60 minutes, after which we obtained the mature structure with a core TCR-MHC-peptide contact region images of fields containing stable immunological synapses with cen- and a peripheral LFA-1–ICAM-1 ring (Fig. 5a,b, 5- and 6-min time tral MHC clusters and complete ICAM-1 rings and recorded the points). Thus, anergic cells show no impairment of the membrane locations of the synapses. We then treated the stable synapses and cytoskeletal interactions necessary for synapse maturation. sequentially with the weak and strong phospholipase inhibitors However, the mature synapse, which persisted stably in the U73343 and U73122, respectively, and evaluated the effects of the untreated T cells for at least an hour after the initial contact10–12, drugs on the previously imaged synapses. Whereas the weak was unstable in anergic T cells. Anergic T cells showed partial or, inhibitor only had a slight effect on synapse integrity, the strong occasionally, complete breakdown of the outer LFA-1 ring within inhibitor effectively abolished the LFA-1 contact ring (Fig. 5c), an 10–20 min after the mature synapse was established, and often also effect that resembled the phenotype of disintegration of the outer showed aberrant morphology of the inner TCR core (Fig. 5a,b, LFA-1 ring observed in anergic T cells (Fig. 5a,b). These data 10 min and later). Parallel analysis of fluorescence and contact area emphasize the requirement for PLC-γ1 in maintenance of the patterns showed that anergic T cells had a ‘migratory’ phenotype, in mature immunological synapse and confirm previous reports that which the outer LFA-1–ICAM-1 ring became disorganized, pulling both anergic T cells and T cells treated with phospholipase away from and distorting the TCR-MHC clusters, which were inhibitors do not bind fibronectin efficiently25. PLC-γ1-dependent dragged behind the moving T cells (Fig. 5a). In this respect, anergic diacylglycerol production is required for effective ‘inside-out’ sig- T cells (at 10 min and later) act like cells that do not receive a TCR- naling25,35, whereas PKC-θ activation, which is ‘downstream’ of dia- mediated ‘stop’ signal10,33,34. Migration of anergized T cells would cylglycerol production by PLC-γ1, has been linked to efficient

260 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY ARTICLES

Figure 6 Cblb- and Itch-deficient T cells are resistant to anergy induction. (a) CD4 T cells from C57BL/6 (WT), Cblb–/– and Itch–/– mice were stimulated with anti-CD3 and anti-CD28 for 2 d Iono a (ng/ml) d and were left resting for 5 d. Cells were then left 0 Time (min) 5 20 35 50 untreated or were treated for 16 h with 25–100 25 ng/ml of ionomycin (Iono), after which 40 50 ) 100 proliferative responses to anti-CD3 and anti-CD28

–3 5CC7 3 30 stimulation were measured by [ H]thymidine 10 × incorporation. (b) TH1 cells from C57BL/6 (WT), 20 Cblb–/– and Itch–/– mice were allowed to H incorporation (c.p.m. differentiate for 1 week, then were stimulated

3 5CC7 10 anergized with plate-bound anti-CD3 in the presence of CTLA4-Ig (Anergized) or with anti-CD3 and anti- WT Itch–/– Cblb–/– CD28 (Activated) for 2 d, then were allowed to ‘rest’ for 3 d in media without interleukin 2. Cell b Activated + ––++ – Anergized – + ––+ + Cblb –/– extracts were analyzed for PLC-γ1 and actin by PLC-γ1 immunoblotting. (c) TH1 cells from C57BL/6 Breakdown (WT), Itch–/– and Cblb–/– mice were left untreated product (–) or were treated for 16 h with ionomycin (+), Cblb –/– Actin anergized were washed, then were restimulated (+) or not (–) with plate-bound anti-CD3 (α-CD3). Cell WT Itch–/– Cblb–/– extracts were analyzed for PKC-θ and actin by n = 748 immunoblotting. (d) Formation of immune c Iono ––+ + ––+ + ––+ + 75 α-CD3 – + ––+ + – + – + – + synapses was evaluated as described in Figure 5, 50 n = 423 with T 1 cells from wild-type or Cblb–/– 5CC7 PKC-θ n = 501 H TCR–transgenic mice and lipid bilayers displaying 25 k http://www.nature.com/natureimmunology ICAM-1 and I-E pigeon cytochrome C (PCC) Actin n = 347 –/– –/– molecules. Top, individual representative cells Stable synapses (%) 5CC7 5CC7 Cblb Cblb WT Itch–/– Cblb–/– anergized anergized (genotypes, left margin) observed over a time course of 50 min. Bottom, quantification of results, showing the percentage of cells with stable synapses at 35 min after synapse formation was initiated.

formation of the immunological synapse (T.N. Sims, T. Soos, D.R. synapses that were stable throughout the observation period of Littman and M.L.D., unpublished observations). These results indi- 50 min (Fig. 6d), whereas 5CC7 T cells that were pretreated with cate strongly that the aberrant synapse morphology of anergic T ionomycin for 16 h formed the mature synapse quickly (<5 min) on cells can be attributed to reduced signaling through PLC-γ1 and contact with the bilayer but then showed synapse disorganization PKC-θ and consequent reduced LFA-1 activity. and developed the migratory phenotype. Synapses formed by untreated Cblb–/– T cells were as stable as those formed by wild-type © 2004 Nature Publishing Group Genetic evidence for involvement of Itch and Cbl-b in anergy T cells, but synapses formed by ionomycin-pretreated Cblb–/– T cells Mice deficient in either Itch or Cbl-b have autoimmune pheno- were mostly protected from synapse disintegration, as judged by types15,19–21, indicating that these E3 ligases are important in sup- their stability for up to 35 min of observation. Thus, Cbl-b con- pressing immune responses to self antigens. To evaluate the tributes substantially to the early disintegration of the immunologi- participation of Itch and Cbl-b in Ca2+-induced T cell anergy, we cal synapse in anergic T cells. However, the synapses break down at tested T cells from Itch–/– (Itchy) and Cblb–/– mice (Fig. 6). We later times in ionomycin-pretreated Cblb–/– T cells (50 min), indi- treated the cells with increasing doses of ionomycin, then stimulated cating that other factors are also involved. them with anti-CD3 plus anti-CD28, after which we assayed prolifer- ation ([3H]thymidine incorporation). Itch–/– and Cblb–/– T cells were DISCUSSION resistant to anergy induction at low doses of ionomycin, and this Based on our data, we propose that T cell anergy is initiated and effect was partially overcome at higher doses of ionomycin (Fig. 6a). implemented through a complex multistep negative signaling We also assessed the ability of Itch–/– and Cblb–/– T cells to degrade process, at least one facet of which involves the coordinate actions of PLC-γ1 and PKC-θ in response to anergy-inducing conditions. As several E3 ligases. The program is initiated by sustained Ca2+- expected, PLC-γ1 protein decreased in wild-type T cells after the calcineurin signaling and culminates in proteolytic degradation of cells were anergized with anti-CD3 stimulation in the absence of PLC-γ1 and PKC-θ, two central participants in the TCR signaling costimulation, but Itch–/– and Cblb–/– T cells did not show this cascade24,36. The first step of the program involves calcineurin- decrease (Fig. 6b). Likewise, wild-type T cells showed the expected mediated upregulation of three E3 ligases, Itch, Cbl-b and GRAIL, decrease in PKC-θ protein after ionomycin pretreatment followed as well as Tsg101, the ubiquitin-binding component of the endoso- by restimulation with anti-CD3, but we did not find this effect in mal sorting complex. This process may be mediated through NFAT T cells from Itch–/– and Cblb–/– mice (Fig. 6c). Finally, we compared in the absence of AP-1 cooperation8. Degradation of signaling pro- the kinetics of synapse disintegration in control and Cblb–/– T cells teins is implemented during a second step of T cell–APC contact, that had been anergized by pretreatment with ionomycin (Fig. 6d). during which the immunological synapse forms normally and the As expected, control 5CC7 TCR transgenic T cells exposed to pep- E3 ligases Itch, Nedd4 and Cbl-b move to detergent-insoluble mem- tide-loaded MHC and LFA-1 molecules in lipid bilayers formed brane fractions, where they may colocalize with activated substrate

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 261 ARTICLES

proteins. The membrane fraction may include ‘raft’ membranes, (especially T cell clones) show an initial tendency to interact homotyp- endosomal membranes or both, consistent with previous findings ically, but implementation of T cell anergy results in reduced binding that PLC-γ1, RasGAP, Tsg101 and GRAIL are all found associated of LFA-1 to its ligand ICAM-1. Anergic T cells show increased tran- with endosomes4,17. As a result, the active, membrane-proximal scription of the gene encoding LFA-1α40; upregulation of the pool of signaling proteins becomes monoubiquitinated and capable GRP-1 (cytohesin 3), overexpression of which leads to increased activ- of stable interaction with Tsg101. This interaction in turn results in ity of LFA-1 (refs. 41,42); and increased GTP loading of Rap1 (ref. 43), sorting of the monoubiquitinated proteins into multivesicular bod- which increases integrin adhesiveness and is crucial for antigen- ies and their targeting for lysosomal degradation. In the third step, dependent synapse formation44–47. Anergic T cells also show upregu- degradation of active PLC-γ1 and PKC-θ leads to diminished TCR lation of CD98 (ref. 8), which induces LFA-1 adhesion through and LFA-1 signaling. Once this happens, the mature synapse cannot activation of the small GTPase Rap1 (ref. 48). Paradoxically, however, be maintained11 and the inability to sustain stable APC contact fur- T cells from mice transgenic for constitutively active Rap1 did not ther reduces the antigen responses of anergic T cells. Overall, there- show an unresponsive phenotype, but instead showed increased pro- fore, a stable difference in gene expression profile between normal liferation in response to APC-peptide stimulation that correlated with β β 45 and anergic T cells is transformed through ubiquitin modification increased avidity of 1 and 2 integrins . A recent report may resolve into a transient increase in turnover of activated signaling proteins, this paradox, because it showed that much higher amounts of acti- thereby altering the migration activity of T cells and establishing a vated (GTP-loaded) Rap1 than in the constitutively active Rap1 trans- persistent unresponsive state. genic model could be achieved by inactivation of the gene encoding The attractive feature of such a downregulatory program is that sig- SPA-1, the main GTPase-activating protein for Rap1 in peripheral T naling molecules would be targets for degradation only when they are cells49. T cells from young SPA-1-deficient mice that had been immu- activated. In a normally activated T cell, signaling through the TCR nized with antigen in adjuvants developed an unresponsive phenotype results in maintained phosphatidylinositol-3,4,5 triphosphate pro- resembling the phenotype that develops in wild-type mice injected duction, PLC-γ1-dependent production of second messengers and with soluble antigen49. We postulate that the increased amount of Ca2+-mobilization, continuing for several hours11. In an anergic T Rap1-GTP in anergic T cells leads to preactivation of integrins45,

http://www.nature.com/natureimmunology cell in which the Itch, Cbl-b, Nedd4 and GRAIL E3 ligases are upreg- which in turn co-opts TCR signaling by prematurely activating the E3 ulated and/or preactivated for membrane localization, PLC-γ1 and ligase-associated anergy program that we have defined here. However, PKC-θ activation would be rapidly followed by E3-mediated the resulting degradation of active PLC-γ1 and PKC-θ interferes with monoubiquitination at ‘raft’ or endosomal membranes, and this, LFA-1 activity, thus conferring the phenotype of synapse disorganiza- through Tsg101, would immediately sequester the active tion that we note in anergic T cells. within endosomes where they cannot be reactivated. Thus, imposi- There is controversy about the precise function of the immunolog- tion of T cell anergy would be a localized and efficient process in ical synapse in TCR signaling. The kinetics of formation of the mature which PLC-γ1 and PKC-θ would be eliminated only in the context of immunological synapse correspond to a period in which there is sub- active, membrane-localized signaling complexes, and massive deple- stantial T cell receptor internalization and degradation as well as a tion of the bulk of cellular PLC-γ1 would not be required (and is not decrease from the early peak of tyrosine kinase signaling12. However, found). Consistent with this hypothesis, anergic T cells show no a stable immunological synapse seems to be required for ongoing appreciable downregulation of PLC-γ2, which despite having the Ca2+ mobilization, phosphatidylinositol 3 kinase activation and same domain organization as PLC-γ1 is not part of the signaling path- interleukin 2 production9–11. Our findings offer a synthesis of these way downstream of the TCR24. two opposing views, showing that the synapse is a dynamic structure © 2004 Nature Publishing Group Our data indicate that lack of anergy induction is the molecular mech- whose stability requires ongoing signaling through the TCR and anism underlying the autoimmune phenotypes of Cbl-b-deficient and which in turn modulates the overall level of T cell responses by inte- Itch-deficient (Itchy) mice. Itchy mice show splenomegaly and lympho- grating positive and negative signals from a variety of surface recep- cyte infiltration in several tissues and chronic inflammation in the tors including the TCR, costimulatory receptors and integrins. skin15,19, whereas Cblb ablation is associated with spontaneous T cell In summary, we have shown that Ca2+-calcineurin signaling, which activation and autoantibody production21 and enhanced experimental is essential for the productive immune response, also promotes T cell autoimmune encephalomyelitis20. Furthermore, Cblb is a principal gene anergy through a program of ubiquitin modification and targeted linked to susceptibility to type I diabetes in rats37. Because Itch–/– and degradation of signaling proteins. Thus, our studies confirm that Cblb–/– T cells have very similar phenotypes of resistance to Ca2+- T cell anergy involves an active process that precedes unresponsive- induced anergy (impaired proliferation and impaired degradation of ness to antigen. It is notable that two main participants in this are E3 PLC-γ1 and PKC-θ), it is likely that these two proteins cooperate to ubiquitin ligases whose involvement in negative signaling has already evoke T cell anergy, either in the context of a multiprotein complex38 or been confirmed in mouse models of autoimmune disease. Further by acting sequentially in a pathway of anergy induction. Cbl family biochemical analysis of the Ca2+- and calcineurin-induced anergy members are essential for internalization and downregulation of the T program should uncover additional participants, potentially encoded cell receptor16, possibly through their ability to bind the CIN85 paralog by the many genetic loci that predispose to autoimmune disease. CD2AP39; thus, one likely scenario is that Cbl-b regulates TCR internal- ization through monoubiquitination and sorting into internal vesicles Note added in proof: The Itchy cells used in these studies show a TH2 bias of late endosomes, whereas Itch functions to monoubiquinate PLC-γ1 that has been attributed to increased JunB, a postulated Itch target50, and PKC-θ, routing them into the same lysosomal degradation pathway raising the possibility that the autoimmune phenotype of Itchy mice is as the TCR. The E3 ligase GRAIL, which resides in the endosomal mem- due to T cell hyperproliferation in response to increased interleukin 4. brane and is upregulated in anergic T cells17, could synergize with these However, this explanation is not compatible with the finding that back- effectors to further enhance protein ubiquitination and degradation. crossing Itchy mice to the B10 background eliminates their TH2 bias in Consistent with several previous studies, our findings point to a vivo, but the mice still develop autoimmune disease (V. Parravicini and complex function for integrins in T cell anergy. Anergic T cells R. Zamoyska, personal communication).

262 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY ARTICLES

METHODS firmed that anergized TH1 cells subjected to restimulation showed a reduction Mice. BALB/cJ mice and DO11.10, 2B4, AND and 5CC7 TCR-transgenic mice in PKC-θ protein compared with their counterparts that were not restimu- were obtained from Jackson laboratories and were maintained and bred in lated; however, a corresponding anti-CD3 stimulation of resting TH1 cells did pathogen-free conditions in a barrier facility. not result in such a decrease (data not shown).

Cell culture, cell stimulation and anergy induction ex vivo. The mouse D5 Expression plasmids. Nedd4 (KIAA0093) cDNA and Itch cDNA15 were 8 + (Ar-5) TH1 cell clone was grown as described . CD4 cells were isolated from inserted via SalI-NotI into pRK5 vectors containing an N-terminal sequence spleens and lymph nodes of mice with positive selection with anti-CD4 mag- encoding the Myc epitope. R. Abraham (Duke University, Durham, NC) and netic beads (Dynal) and were differentiated into TH1 cells for 2 weeks with J. Huibregtse (University of Texas at Austin, Austin, TX) provided expression standard protocols8. For most experiments, anergy was induced by treatment plasmids for PLC-γ1 and Nedd4, respectively. × 6 µ of primary TH1 cells or the D5 TH1 clone (1 10 cells/ ml) with 1 M iono- mycin for 16 h. This method has the advantage of providing a homogeneous Cell extracts, immunoprecipitations and immunoblots. D5 cells were × 6 µ population of anergic T cells that have synchronously experienced the same extracted at a concentration of 1 10 cells/10 l in radioimmunoprecipita- intensity of prior Ca2+ signals and so are suitable for biochemical investiga- tion assay (RIPA) buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1 mM DTT, tion. CsA was included in some experiments at a concentration of 2 µM. Cells 10 mM MgCl2, 1% Nonidet P-40, 0.1% SDS and 0.5% sodium deoxy- were washed to remove ionomycin and were incubated at higher cell density cholate) supplemented with protease and phosphatase inhibitors (1 mM µ µ (3 × 106 cells/ ml) for 1–2 h at 37 °C. In the experiment in Figure 1a, the high- phenylmethyl sulfonyl fluoride, 25 g/ml of aprotinin, 25 g/ml of leu- β density incubation step was included but had not been planned. For the exper- peptin, 10 mM NaF, 8 mM -glycerophosphate and 0.1 mM sodium ortho- iment in Figure 1c, D5 cells were restimulated with 1 µg/ml of anti-CD3 with vanadate). Re-extraction of the resulting DNA pellet with hot SDS sample γ or without 2.5 µg/ml of anti-CD28, or with 20 nM PMA, 1 µM ionomycin or buffer followed by immunoblotting did not show any residual PLC- 1 in the SDS fraction of either resting or ionomycin-anergized D5 cells, confirming both. For the experiment in Figure 2b, primary TH1 cells were stimulated with plate-bound anti-CD3 to induce anergy or with a combination of anti-CD3 the efficiency of extraction with RIPA buffer (data not shown). For assess- µ and anti-CD28 to induce productive activation. In both cases the cells go ment of proteins in cell extracts, 7.5–30 l of RIPA extracts were separated through a phase of active proliferation, but cells that received only anti-CD3 by 9–12% SDS-PAGE, and proteins were electrotransferred onto nitrocellu- µ stimulation responded much less well to subsequent restimulation than cells lose membranes. For immunoprecipitation, 500–1,000 l of RIPA cell previously stimulated with both anti-CD3 and anti-CD28. For the experi- extracts were used. For coimmunoprecipitation from lysates of transfected http://www.nature.com/natureimmunology –/– –/– HEK 293 cells, cells from one 10-cm dish were lysed in 50 mM HEPES, ments of Figure 6, TH1 cells from C57BL/6, Cblb or Itch mice that had been differentiated in vitro for 1 week were stimulated for 2 d with plate- pH 7.5, 100 mM NaCl, 1 mM EDTA, 0,5% Nonidet P-40 and 10% glycerol plus phosphatase and protease inhibitors. Lysates were precleared with bound anti-CD3 (3 µg/ml) in the presence of CTLA4-Ig (10 µg/ml) to induce either protein A– or protein G–Sepharose, immunoprecipitation proceeded anergy or with anti-CD3 (0.25 µg/ml) and anti-CD28 (2 µg/ml) to induce for 4 h and the resulting precipitates were washed three to four times with activation, then were left resting for 3 d in media without interleukin 2. The the buffer used for cell extraction. For detection of monoubiquitinated extent of anergy induction was evaluated by intracellular staining or PKC-θ, cell lysates from 1 × 108 untreated or ionomycin-pretreated D5 cells in standard proliferation assays8. HEK 293 cells were grown and transfected were immunoprecipitated with anti-PKC-θ. Immunoblots used antibody with Ca2+ phosphate with standard protocols. solutions in 5% milk and TBS (10 mM Tris-HCl, pH 8.0, and 150 mM Antibodies. Antibodies to Zap70 (Z24820), Lck (L15620), PKC-θ (610089), NaCl) and washes used TBS containing 0.05% Tween-20. Relative band Itch (I84520) and calcineurin (610259) were obtained from BD Transduction intensities were quantified by NIH IMAGE Quant and corrected for back- Labs. Antibodies to Fyn (06-133), RasGAP (05-178), SOS (06-246), Vav-1 (05- ground within each lane. 219) and Nedd4 (07-049) were purchased from Upstate Biotechnologies. 52 ζ Cell fractionation. Cell fractionation was done essentially as described Santa Cruz Biotechnology antibodies were used to detect CD3 (sc-1239), × 7 γ with 3 10 D5 cells. Cells were swollen for 15 min in hypotonic buffer E

© 2004 Nature Publishing Group Mekk-2 (sc-1088), RasGRP (sc-8430), ubiquitin (sc-8017), PLC- 2 (sc-407), (10 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl and 1 mM DTT supple- Cbl-b (sc-1705), NF-κB p65 (sc-109), NF-κB p50 (sc-1192), IKKγ (sc-8330) 2 mented with protease and phosphatase inhibitors) and lysed by Dounce and Myc-tagged (sc-789) and hemagglutinin-tagged (sc-805) proteins. homogenization. Lysates were centrifuged at 100,000g for 30 min, yielding a Antibody to the AU.1 (MMS-130P) epitope tag was purchased from Covance; supernatant (cytosol) and a pellet that was resuspended in buffer E contain- anti-Akt (9272), from Cell Signaling; anti-Tsg101 (ab83), from Genetex; and ing 1% Nonidet P-40 and was recentrifuged at 100,000g for 30 min to anti-IKKβ (AHO0362), from Biosource. Antibodies to NFAT1 and NFAT5 separate the detergent-soluble fraction in the supernatant from the deter- were produced in the Rao lab. Antibodies to Gads and LAT were gifts from E. gent-insoluble fraction (pellet). The pellet was resuspended by sonication Clark (University of Washington, Seattle, WA) and L. Samelson (National in RIPA buffer and cleared by centrifugation. One fourth of the supernatant Cancer Institute, Bethesda, MD), respectively. Antibodies to p85 phos- from each centrifugation step (cytoplasm, detergent soluble and detergent phatidylinositol 3 kinase were obtained from the laboratory of L. Cantley; and insoluble fractions) was analyzed for by immunoblotting for Nedd4, Itch antibodies to SHP-1, SHP-2 and PTP-1B were provided by B. Neel (both from and LAT. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA). Endogenous PLC-γ1 was detected with a polyclonal antiserum provided by A. 2+ [Ca ]i imaging and immunocytochemistry. Intracellular calcium was Toker (Beth Israel Deaconess Medical Center, Harvard Medical School, µ measured in primary TH1 cells from 2B4 mice. Cells were loaded with 1 M Boston, MA), which was raised against the epitope APRRTRVNGDNR, repre- fura-2 aceto-methyl ester (Molecular Probes) for 30 min at 20 °C, washed senting the final C-terminal amino acids of the protein. The reactivity of this and resuspended in loading medium (RPMI + 10% FCS), incubated with antiserum with PLC-γ1 is unlikely to be affected by phosphorylation, as pro- 2.5 µg/ml of biotinylated anti-CD3 (2C11, Pharmingen) for 15 min at 20 °C posed earlier51, as the epitope does not contain any serine or tyrosine residues and attached to poly-L-lysine–coated coverslips mounted in a RC-20 closed and its single threonine residue is not part of any predictable phosphorylation bath chamber (Warner Instrument). The fura-2-loaded cells were perfused motif, as judged by the Scansite computer program. Furthermore, a commer- with Ringer’s solution containing 2 mM calcium (155 mM NaCl, 4.5 mM cial antibody source, comprising a pool of four different monoclonal antibod- KCl, 10 mM D-glucose, 5 mM HEPES, pH 7.4, 1 mM MgCl2 and 2 mM ies (05-163; Upstate Biotechnologies), also allowed us to visualize the CaCl2) and were stimulated by crosslinking of the surface-bound biotiny- differences in PLC-γ1 protein amounts in untreated and anergic T cells when lated anti-CD3 with 2.5 µg/ml of streptavidin (Pierce), after which healthy the antibody was used at a fivefold higher dilution than recommended. We cells were identified by their responsiveness to 1 µM ionomycin have also used a second monoclonal antibody to PKC-θ (E-7; Santa Cruz (Calbiochem). Single-cell video images were obtained on a Zeiss Axiovert Biotechnology) to immunoprecipitate PKC-θ in partially denaturing condi- S200 epifluorescence microscope with OpenLab imaging software tions from resting and anergized primary T cells. These experiments con- (Improvision). Fura-2 emission was detected at 510 nm after excitation at

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 263 ARTICLES

340 and 380 nm. The 340/380 ratio images were acquired every 5 s after thank A. Altman, H. Band, J. Brugge, C. Joazeiro, M. Katan for advice and reagents. background subtraction. Calibration values (Rmin, Rmax and Sf) were Supported by National Institutes of Health grants RO1-AI48213, RO1-AI40127 derived from cuvette measurements with a calcium calibration buffer and RO3-HD39685 (to A.R.), RO1-AI50280 and R21-AI48542 (to Y.-C.L.) and (Molecular Probes) and as described53. AI-43542; an Irene Diamond Foundation grant (to M.L.D.); EMBO (V.H.); and the Cancer Research Institute (S.-H.I. and S.F.). Real-time PCR analysis. Total RNA was prepared from untreated or iono- COMPETING INTERESTS STATEMENT mycin-pretreated D5 cells with Ultraspec reagent (Biotecx). cDNA was synthe- The authors declare that they have no competing financial interests. 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) with Received 29 September 2003; accepted 22 January 2004 a SYBR Green PCR kit (Applied Biosystems). The sequences of the primer pairs Published online at http://www.nature.com/natureimmunology/ were: L32 sense, 5′-CGTCTCAGGCCTTCAGTGAG-3′, and L32 antisense, 5′- CAAGAGGGAGAGCAAGCCTA-3′; PLC-γ1 sense, 5′-AAGCCTTTGACCCC 1. Hunter, T. Protein kinases and phosphatases: the yin and yang of protein phosphory- TTTGAT-3′, and PLC-γ1 antisense, 5′-GGTTCAGTCCGTTGTCCACT-3′; Itch lation and signaling. Cell 80, 225–236 (1995). ′ ′ ′ 2. Sun, H., Charles, C.H., Lau, L.F. & Tonks, N.K. MKP-1 (3CH134), an immediate sense, 5 -GTGTGGAGTCACCAGACCCT-3 , and Itch antisense, 5 -GCT early gene product, is a dual specificity phosphatase that dephosphorylates MAP TCTACTTGCAGCCCATC-3′; Cblb sense, 5′-CTTAAATGGGAGGCACAGT kinase in vivo. Cell 75, 487–493 (1993). AGAAT-3′, and Cblb antisense, 5′-CAGTACACTTTATGCTTGGGAGAA-3′; 3. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, Rnf128 sense, 5′-GTAACCCGCACACCAATTTC-3′, and Rnf128 antisense, 5′- 425–479 (1998). ′ ° 4. Sorkin, A. & Von Zastrow, M. Signal transduction and endocytosis: close encounters GTGAGACATGGGGATGACCT-3 . Thermal cycling conditions were 95 C for of many kinds. Nat. Rev. Mol. Cell. Biol. 3, 600–614 (2002). 5 min, then 40 cycles of 95 °C, 65 °C, and 72 °C for 30 s each, terminating with a 5. Valitutti, S., Muller, S., Salio, M. & Lanzavecchia, A. Degradation of T cell receptor single cycle at 72 °C for 5 min. Signals were captured during the polymerization (TCR)-CD3-zeta complexes after antigenic stimulation. J. Exp. Med. 185, step (72 °C). A threshold was set in the linear part of the amplification curve, 1859–1864 (1997). 6. Schwartz, R.H. T cell anergy. Annu. Rev. Immunol. 21, 305–334 (2003). and the number of cycles needed to reach it was calculated for each gene. 7. Macian, F., Lopez-Rodriguez, C. & Rao, A. Partners in transcription: NFAT and AP-1. Melting curve analysis and agarose gel electrophoresis were done to test the Oncogene 20, 2476–2489 (2001). purity of the amplified bands. Normalization was done by using amounts of 8. Macian, F. et al. Transcriptional mechanisms underlying lymphocyte tolerance. Cell mRNA for the ribosomal protein L32 as an internal control for each sample. 109, 719–731 (2002). 9. Monks, C.R., Freiberg, B.A., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional seg- regation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998). http://www.nature.com/natureimmunology Formation of immunological synapses in lipid bilayers. Planar bilayers were 10. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T 10 prepared essentially as described , except that the moth cytochrome C cell activation. Science 285, 221–227 (1999). (MCC) 88-103 peptide was loaded on glycosylphosphatidylinositol (GPI)–I- 11. Huppa, J.B., Gleimer, M., Sumen, C. & Davis, M.M. Continuous T cell receptor sig- Ek for 24 h. Bilayers were prepared with Oregon green–labeled GPI–I-Ek and naling required for synapse maintenance and full effector potential. Nat. Immunol. 4, 749–755 (2003). indodicarbocyanine-labeled GPI–ICAM-1 in parallel plate flow cells 12. Lee, K.H. et al. T cell receptor signaling precedes immunological synapse formation. (Bioptechs). Control and ionomycin-pretreated T cells from TCR-transgenic Science 295, 1539–1542 (2002). mice were injected into the flow cell at a density of 1 × 106 cells/ml. Areas of 13. Murphy, L.O., Smith, S., Chen, R.H., Fingar, D.C. & Blenis, J. Molecular interpreta- bilayers where cells were forming synapses were imaged with fluorescein isoth- tion of ERK signal duration by immediate early gene products. Nat. Cell. Biol. 4, 556–564 (2002). iocyanate and indodicarbocyanine optics on an Olympus IX-70 inverted 14. Lee, K.H. et al. The immunological synapse balances T cell receptor signaling and microscope equipped with a Hamamtsu ORCA-ER digital camera and a xenon degradation. Science 302, 1218–1222 (2003). arc lamp as a light source for fluorescence microscopy. The filter wheels, shut- 15. Fang, D. et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in ters and the camera were controlled with IPLAB software on a Macintosh plat- TH2 differentiation. Nat. Immunol. 3, 281–287 (2002). 16. Naramura, M. et al. c-Cbl and Cbl-b regulate T cell responsiveness by promoting lig- form. Bright field, interference reflection and fluorescence images were and- induced TCR down-modulation. Nat. Immunol. 3, 1192–1199 (2002). collected and processed with Metamorph software. The background from the 17. Anandasabapathy, N. et al. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene fluorescence images was subtracted with the produce-background correction transcription is expressed in anergic CD4+ T cells. Immunity 18, 535–547 (2003). © 2004 Nature Publishing Group image function, which is based on median filtering to subtract background 18. Katzmann, D.J., Odorizzi, G. & Emr, S.D. Receptor downregulation and multivesicu- lar-body sorting. Nat. Rev. Mol. Cell. Biol. 3, 893–905 (2002). that is nonuniform. The percentage of cells adhering was evaluated by com- 19. Perry, W.L. et al. The itchy locus encodes a novel ubiquitin protein ligase that is dis- paring bright field and interference reflection microscopy images. rupted in a18H mice. Nat. Genet. 18, 143–146 (1998). Experiments with phospholipase inhibitors were done with AND T cell 20. Chiang, Y.J. et al. Cbl-b regulates the CD28 dependence of T-cell activation. Nature blasts (day 8). Cells were allowed to form immunological synapses on bilayers 403, 216–220 (2000). µ 2 k 21. Bachmaier, K. et al. Negative regulation of lymphocyte activation and containing 80 molecules/ m of Oregon green I-E –MCC 88-103 and by the molecular adaptor Cbl-b. Nature 403, 211–216 (2000). 2 200 molecules/µm of indodicarbocyanine–ICAM-1 in the presence of 0.01% 22. Healy, J.I. et al. Different nuclear signals are activated by the B cell receptor during dimethylsulfoxide (the carrier concentration for 1 µM U73122 and U73343). positive versus negative signaling. Immunity 6, 419–428 (1997). After 60 min, fields containing stable immunological synapses with central 23. Glynne, R. et al. How self-tolerance and the immunosuppressive drug FK506 pre- vent B-cell mitogenesis. Nature 403, 672–676 (2000). MHC clusters (green) and complete ICAM-1 rings (red) were imaged and the 24. Irvin, B.J., Williams, B.L., Nilson, A.E., Maynor, H.O. & Abraham, R.T. Pleiotropic locations were recorded with an automated stage and IPLab software. The sta- contributions of phospholipase C-γ1 (PLC-γ1) to T- cell antigen receptor-mediated ble synapses were then treated sequentially with 1 µM U73343 and 1 µM signaling: reconstitution studies of a PLC-γ1-deficient Jurkat T-cell line. Mol. Cell. U73122 (weak and strong PLC-γ inhibitors, respectively). After each drug Biol. 20, 9149–9161. (2000). 25. Wells, A.D. et al. Regulation of T cell activation and tolerance by phospholipase treatment, the same fields were imaged within 10 min so that the effects of the Cγ-1-dependent integrin avidity modulation. J. Immunol. 170, 4127–4133 (2003). drugs on many individual synapses could be determined. The quantitative 26. Kaji, T., Hachimura, S., Ise, W. & Kaminogawa, S. Proteome analysis reveals cas- data reflect the percentage of intact LFA-1–ICAM-1 rings after carrier or drug pase activation in hyporesponsive CD4 T induced in vivo by the oral treatment on 103 contact areas. In separate experiments, the effects of U73343 administration of antigen. J. Biol. Chem. 278, 27836–27843 (2003). 27. Rizo, J. & Sudhof, T.C. C2-domains, structure and function of a universal Ca2+- and U73122 were stable for up to 1 h and U73122-dependent destruction of binding domain. J. Biol. Chem. 273, 15879–15882 (1998). the LFA-1 adhesion ring was not dependent on prior treatment with U73343. 28. Rotin, D., Staub, O. & Haguenauer-Tsapis, R. Ubiquitination and endocytosis of We found these effects in three independent experiments with U73122 con- plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein lig- centrations from 0.1 to 1 µM (data not shown). ases. J. Membr. Biol. 176, 1–17 (2000). 29. Plant, P.J. et al. Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb. J. Cell. Biol. 149, 1473–1484 (2000). Note: Supplementary information is available on the Nature Immunology website. 30. Seto, E.S., Bellen, H.J. & Lloyd, T.E. When cell biology meets development: endo- cytic regulation of signaling pathways. Genes Dev. 16, 1314–1336 (2002). ACKNOWLEDGMENTS 31. Katzmann, D.J., Babst, M. & Emr, S.D. Ubiquitin-dependent sorting into the multi- We thank members of the Rao and Dustin laboratories for discussions, and T. Starr vesicular body pathway requires the function of a conserved endosomal protein sort- for the preparation of ICAM-1 and I-Ek for planar bilayer experiments. We also ing complex, ESCRT-I. Cell 106, 145–155 (2001).

264 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY ARTICLES

32. Rao, N., Dodge, I. & Band, H. The Cbl family of ubiquitin ligases: critical negative 44. Reedquist, K.A. et al. The small GTPase, Rap1, mediates CD31-induced integrin regulators of tyrosine kinase signaling in the immune system. J. Leukoc. Biol. 71, adhesion. J. Cell. Biol. 148, 1151–1158 (2000). 753–763 (2002). 45. Sebzda, E., Bracke, M., Tugal, T., Hogg, N. & Cantrell, D.A. Rap1A positively regu- 33. Dustin, M.L., Bromley, S.K., Kan, Z., Peterson, D.A. & Unanue, E.R. Antigen recep- lates T cells via integrin activation rather than inhibiting lymphocyte signaling. Nat. tor engagement delivers a stop signal to migrating T lymphocytes. Proc. Natl. Acad. Immunol. 3, 251–258 (2002). Sci. USA 94, 3909–3913 (1997). 46. Katagiri, K. et al. Rap1 is a potent activation signal for leukocyte function-associ- 34. Negulescu, P.A., Krasieva, T.B., Khan, A., Kerschbaum, H.H. & Cahalan, M.D. Polarity ated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase. of T cell shape, motility, and sensitivity to antigen. Immunity 4, 421–430 (1996). Mol. Cell. Biol. 20, 1956–1969 (2000). 35. Dustin, M.L. & Springer, T.A. T-cell receptor cross-linking transiently stimulates 47. Katagiri, K., Hattori, M., Minato, N. & Kinashi, T. Rap1 functions as a key regulator adhesiveness through LFA-1. Nature 341, 619–624 (1989). of T-cell and antigen-presenting cell interactions and modulates T-cell responses. 36. Sun, Z. et al. PKC-θ is required for TCR-induced NF-κB activation in mature but not Mol. Cell. Biol. 22, 1001–1015 (2002). immature T lymphocytes. Nature 404, 402–407 (2000). 48. Suga, K. et al. CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via 37. Yokoi, N. et al. Cblb is a major susceptibility gene for rat type 1 diabetes mellitus. activation of Rap1. FEBS Lett. 489, 249–253 (2001). Nat. Genet. 31, 391–394 (2002). 49. Ishida, D. et al. Antigen-driven T cell anergy and defective memory T cell response 38. Courbard, J.R. et al. Interaction between two ubiquitin-protein isopeptide ligases of via deregulated Rap1 activation in SPA-1-deficient mice. Proc. Natl. Acad. Sci. USA different classes, CBLC and AIP4/ITCH. J. Biol. Chem. 277, 45267–45275 (2002). 100, 10919–10924 (2003). 39. Kowanetz, K. et al. Identification of a novel proline-arginine motif involved in 50. Fang, D. et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in CIN85-dependent clustering of Cbl and downregulation of EGF receptors. J. Biol. TH2 differentiation. Nat. Immunol. 3, 281–287 (2002). Chem. 278, 39735–39746 (2003). 51. Gajewski, T.F., Qian, D., Fields, P. & Fitch, F.W. Anergic T-lymphocyte clones have 40. Lechner, O. et al. Fingerprints of anergic T cells. Curr. Biol. 11, 587–595 (2001). altered inositol phosphate, calcium, and tyrosine kinase signaling pathways. Proc. 41. Korthauer, U. et al. Anergic T lymphocytes selectively express an integrin regulatory Natl. Acad. Sci. USA 91, 38–42 (1994). protein of the cytohesin family. J. Immunol. 164, 308–318 (2000). 52. Khoshnan, A., Bae, D., Tindell, C.A. & Nel, A.E. The physical association of protein α β κ 42. Kolanus, W. et al. L 2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a kinase C theta with a lipid raft-associated inhibitor of B factor kinase (IKK) com- cytoplasmic regulatory molecule. Cell 86, 233–242 (1996). plex plays a role in the activation of the NF-κB cascade by TCR and CD28. 43. Boussiotis, V.A., Freeman, G.J., Berezovskaya, A., Barber, D.L. & Nadler, L.M. J. Immunol. 165, 6933–6940 (2000). Maintenance of human T cell anergy: blocking of IL-2 gene transcription by acti- 53. Grynkiewicz, G., Poenie, M. & Tsien, R.Y. A new generation of Ca2+ indicators with vated Rap1. Science 278, 124–128 (1997). greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450 (1985). http://www.nature.com/natureimmunology © 2004 Nature Publishing Group

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 265