Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy This information is current as of September 25, 2021. Neil B. Lineberry, Leon L. Su, Jack T. Lin, Greg P. Coffey, Christine M. Seroogy and C. Garrison Fathman J Immunol 2008; 181:1622-1626; ; doi: 10.4049/jimmunol.181.3.1622 http://www.jimmunol.org/content/181/3/1622 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge

Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy1 Neil B. Lineberry,* Leon L. Su,* Jack T. Lin,* Greg P. Coffey,† Christine M. Seroogy,2* and C. Garrison Fathman3*

Activation of naive T lymphocytes is regulated through as an initial signal while CD28 interactions with the inducible a series of discrete checkpoints that maintain unrespon- costimulatory molecules CD80 and CD86 provide a second siveness to self. During this multistep process, costimu- signal. However, engagement of TCR alone in the absence of

latory interactions act as inducible signals that allow costimulation can initiate T cell anergy, a state of induced un- Downloaded from APCs to selectively mobilize T cells against foreign Ags. responsiveness to proliferation and IL-2 production (1). Be- In this study, we provide evidence that the anergy-asso- cause T cells with reactivity to self can escape clonal deletion ciated E3 ubiquitin ligase GRAIL ( related to an- (2), anergy could serve as a component of peripheral tolerance ergy in lymphocytes) regulates expression of the co- that inhibits self-reactive T cells from initiating autoimmunity stimulatory molecule CD40L on CD4 T cells. Using its due to the absence of costimulatory signaling. luminal protease-associated domain, GRAIL binds to Whereas early molecular research into clonal T cell anergy http://www.jimmunol.org/ the luminal/extracellular portion of CD40L and facili- suggested defects in signaling and transcription factors, tates transfer of ubiquitin molecules from the intracel- the role of stability controlled by the ubiquitin-protea- lular GRAIL RING (really interesting new gene) finger some pathway in anergy induction and peripheral tolerance has recently been investigated (3, 4). The gene related to anergy in to the small cytosolic portion of CD40L. Down-regula- 4 tion of CD40L occurred following ectopic expression of lymphocytes (GRAIL ; Rnf128) is a type 1 transmembrane E3 ؊ ؊ GRAIL in naive T cells from CD40 / mice, and ex- ubiquitin ligase identified in a screen for transcripts differen- pression of GRAIL in bone marrow chimeric mice was tially up-regulated in T cell clones following anergy induction

by incubation with MHC:peptide alone (5). Naive CD4 T cells by guest on September 25, 2021 associated with diminished lymphoid follicle formation. expressing GRAIL via bone marrow chimera production re- These data provide a model for intrinsic T cell regula- duced their proliferation and IL-2 secretion in response to ac- tion of costimulatory molecules and a molecular frame- tivation signals, suggesting that GRAIL acts as a potent, intrin- work for the initiation of clonal T cell anergy. The sic anergy promoting factor (6). Endogenous GRAIL Journal of Immunology, 2008, 181: 1622–1626. expression was subsequently found in multiple model systems of anergy induction, including anti-CD3 stimulation, ionomy- he immune system uses an efficient dual-tiered system cin treatment, and in vivo adaptive tolerance (5–7). for the discrimination of Ags as nonself. Central toler- Because costimulation abrogates anergy induction and T ance selects developing lymphocytes that bind self- GRAIL is differentially expressed 4–6 h following the initia- MHC but do not react strongly against host-derived Ags, tion of anergy, we hypothesized that an inducible molecule whereas peripheral tolerance encompasses a wide array of pro- downstream of TCR engagement and upstream of CD28 co- cesses in the secondary lymphoid organs that complement cen- stimulatory signaling could serve as a target for GRAIL tral tolerance and help prevent immune response to self. As a regulation. One potential candidate whose degradation could result, full activation of naive T cells requires coordinate signal- block T cell activation is CD40 ligand (CD40L; CD154), a ing with APCs, where binding of TCR and MHC:peptide acts type 2 transmembrane protein of the TNF superfamily (8).

*Division of Immunology and Rheumatology and †Division of Oncology, Department of 3 Address correspondence and reprint requests to Dr. C. Garrison Fathman, Stanford Uni- Medicine, Stanford University School of Medicine, Stanford, CA 94305 versity School of Medicine, Division of Immunology and Rheumatology, Center for Clin- ical Sciences Research Building, Room 2225, Stanford, CA 94305-5166. E-mail address: Received for publication November 27, 2007. Accepted for publication June [email protected] 10, 2008. 4 Abbreviations used in this paper: GRAIL, gene related to anergy in lymphocytes; CD40L, The costs of publication of this article were defrayed in part by the payment of page CD40 ligand; HA, hemagglutinin; PA, protease-associated domain; QPCR, real-time charges. This article must therefore be hereby marked advertisement in accordance quantitative PCR; RING, really interesting new gene. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants CA 65237-17, T32- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 AI07290-21, and U19-AI070352 and the Tom and Susan Ford Stanford Graduate Fel- lowship (to N.L.). 2 Current address: Department of Pediatrics, Division of Immunology and Rheumatology, University of Wisconsin, H4/474 Clinical Sciences Center, 600 Highland Avenue, Mad- ison, WI 53792-4108. www.jimmunol.org The Journal of Immunology 1623

CD40L expression is low and intracellular on naive CD4 T whether ionomycin treatment of recently activated naive T cells cells, but following TCR stimulation cell surface CD40L would correlate GRAIL expression with a deficiency in CD40L. increases rapidly by 6–8 h (9, 10). CD40L trimerization of Purified BALB/c CD4 T cells were stimulated with plate bound CD40 on mature APCs further elevates cell surface CD80 and anti-CD3 and soluble anti-CD28 for 48 h, washed and rested in CD86, which in turn bind CD28 on T cells and provide recip- vitro for 72 h, and then treated with increasing concentrations of rocal bidirectional costimulatory signals for full T cell activation ionomycin overnight. GRAIL expression levels were then assayed (11). Therefore, modulating cell surface CD40L on the T cell by QPCR (Fig. 1A) or Western blotting (Fig. 1B). These data dem- could prevent APC-derived costimulatory signaling and pro- onstrate that endogenous GRAIL expression is induced at the mRNA and protein level by ionomycin treatment and that GRAIL mote anergy induction. Previous evidence lends support to this expression directly correlates with the ionomycin concentration hypothesis, as anti-CD3 anergized T cells display a deficiency in added during anergy induction. In contrast, the addition of PMA CD40L expression, suggesting the presence of an anergy-spe- with ionomycin prevented both anergy induction and up-regulation cific factor that prevents CD40L up-regulation (12). of GRAIL mRNA and protein (Fig. 1, A and B). To examine CD40L regulation in this context, we either incubated ionomycin- Materials and Methods anergized primary CD4 T cells in medium alone or exposed them Reagents and Abs to recall stimulation and then analyzed the cell surface expression Brij96, IPEGAL (Nonidet P-40), mouse anti-FLAG-HRP (clone M2), mouse of CD40L (Fig. 1C). CD40L up-regulation on ionomycin-aner- anti-FLAG-conjugated agarose (M2), and rabbit anti-V5 were from Sigma Al- gized CD4 T cells was markedly reduced following recall activa- drich; anti-hemagglutinin (HA)-HRP (clone F7), anti-HA conjugated agarose tion. In addition, the absence of CD40L on anergized CD4 T cells Downloaded from (clone F7), from Santa Cruz Biotechnology; PE-conjugated anti-mouse CD40L (clone MR1) from BD Pharmingen; allophycocyanin-conjugated anti- without recall stimulation demonstrated that ionomycin treatment CD4 (GK1.5) from eBioscience; and PMA, ionomycin, and N-acetyl-leucinyl- during anergy induction did not up-regulate CD40L itself or pre- leucinyl-norleucinal from Calbiochem. vent its return to baseline (Fig. 1D). Overall, the percentage of Cell culture and transfection CD40L seen on anergized CD4 T cells inversely correlated with GRAIL expression, as increasing amounts of ionomycin led to 293T Phoenix cells were grown in DMEM plus 5% heat-inactivated FBS sup- more GRAIL up-regulation and less CD40L expressed on the cell http://www.jimmunol.org/ plemented with L-glutamine and penicillin/streptomycin. For transfection, TransIT-LT1 transfection reagent was used according to the manufacturer’s surface following recall stimulation (Fig. 1D). Finally, the strength standard protocol (Mirus Bio). Cells were lysed with Brij96 lysis buffer (20 mM of recall stimulation had no apparent effect on the relative reduc- Tris-HCl (pH 7.5), 150 mM NaCl, 1% Brij96, and protease inhibitors) and tion of cell surface CD40L expression seen (Fig. 1D). These data protein concentration was measured by Bradford assay (Bio-Rad). For ubiquiti- demonstrate a correlation between GRAIL expression and cell sur- nation assays, cells were incubated with N-acetyl-leucinyl-leucinyl-norleucinal face CD40L levels during anergy induction of primary T cells. The (10 ␮g/ml) for 1.5–2 h at 37°C before lysis. After cell lysis in 1% Brij buffer, lysates were denatured with 0.5% SDS at 100°C for 10 min to remove possible biological relevance of these findings was strengthened by dem- CD40L interacting proteins. Lysates were diluted to 0.1% SDS in Brij96 buffer onstrating endogenous GRAIL up-regulation and CD40L down- before immunoprecipitation. regulation in DO11.10 CD4 T cells provided OVA peptide on by guest on September 25, 2021 Ionomycin-induced anergy APCs with costimulatory signals blocked by the addition of solu- ble CTLA4-Ig (data not shown). Purified CD4 T cells from BALB/c mice were stimulated for 48 h with plate bound anti-CD3 (1 ␮g/ml) and soluble anti-CD28 (1 ␮g/ml). Cells were then GRAIL binds and ubiquitinates CD40L in vitro washed and rested in fresh medium for 72 h in vitro. Cells were then incubated with vehicle, ionomycin, or PMA and ionomycin at the indicated concentra- GRAIL is a type 1 transmembrane protein that contains a luminal tions for 18 h and then washed. protease-associated (PA) domain and cytosolic RING (really in- teresting new gene) finger (Fig. 2A). The PA domain currently Real-time quantitative PCR (QPCR) does not possess a described canonical function but is proposed to RNA was harvested and amplified as described previously (6) with actin as a serve as a protein-protein interaction motif (14, 15). The RING Ј normalizing gene. Primers used were as follows: GRAIL forward primer, 5 - finger confers E3 ubiquitin ligase activity by recruiting E2 trans- AGAGAGAGGGGCTTCTGGAG-3Ј; GRAIL reverse primer, 5Ј-CGATGA CCATTGTGACTTGG-3Ј; ␤-actin forward primer, 5Ј-CAGGCATTGCTG ferase loaded with activated ubiquitin molecules for con- ACAGGATGCA-3Ј;and␤-actinreverseprimer,5Ј-GGCCAGGATGGAGCC jugation to substrate proteins (5, 16). CD40L is a type 2 trans- ACCGATC-3Ј. All samples were analyzed in triplicate, and GRAIL mRNA membrane protein that contains a large extracellular domain of the arbitrary units are expressed as the mean of triplicate normalized values against TNF superfamily and a small cytoplasmic tail with unknown func- ␤-actin. tion (Fig. 2A). Although nearly every cellular protein is regulated Bone marrow chimeras and tissue sections by a component of the ubiquitin-proteasome pathway, it would Generation of bone marrow chimeric mice was performed as described (6). appear difficult for cytosolic E3 ligases to simultaneously bind the Spleen and lymph nodes were removed and fixed in buffered formalin followed 22-aa cytosolic tail of CD40L and retain space for conjugation of by paraffin embedding. Tissues sections were stained with H&E. Imaging was the 76-aa ubiquitin molecule. Thus, we hypothesized that the PA done using an inverted Nikon scope equipped with a Spot camera and software. domain of GRAIL might provide an interaction interface for bind- ing CD40L on the luminal/extracellular side of the membrane and Results and Discussion facilitate ubiquitination of the intracellular tail of CD40L by the Up-regulation of endogenous GRAIL correlates with CD40L cytosolic RING domain of GRAIL. down-regulation To first test for an interaction between GRAIL and CD40L, A recently described model system for clonal T cell anergy induc- 293T cells were transfected with N-terminal 3ϫ FLAG-tagged tion used unopposed calcium flux via ionomycin treatment to up- CD40L and various C-terminal V5-tagged GRAIL constructs. Fol- regulate a cohort of E3 ligases in CD4 T cells. These enzymes, lowing immunoprecipitation of CD40L, both wild-type and an en- including Cbl-b, GRAIL, and Itch, were implicated in controlling zymatically inactive RING finger mutant of GRAIL (H2N2) were the expression of proteins involved in T cell activation via the able to coimmunoprecipitate with CD40L (Fig. 2B). However, a ubiquitin-proteasome pathway (13). Because anergized T cell GRAIL mutant lacking the PA domain (⌬PA) repeatedly failed to clones also exhibit reduced CD40L expression (12), we asked coimmunoprecipitate with CD40L, indicating that an intact 1624 CUTTING EDGE: GRAIL UBIQUITINATES CD40L Downloaded from

FIGURE 2. GRAIL binds and ubiquitinates CD40L. A, Schematic display of the domains and orientation of GRAIL and CD40L. B, 293T cells were transfected with 3ϫ FLAG-tagged human CD40L (hCD40L) and the indi- cated V5-tagged GRAIL constructs. Eluted proteins from anti-FLAG conju- gated agarose were separated by SDS-PAGE and blotted with the indicated Abs. http://www.jimmunol.org/ IB, Immunoblot; IP, immunoprecipitation. C, 293T cells were transfected with 3ϫ FLAG-tagged hCD40L, V5-tagged GRAIL constructs, and HA-tagged ubiquitin. Eluted proteins from anti-FLAG-conjugated agarose were separated by SDS-PAGE and blotted with the indicated Abs. D, 293T cells were trans- fected with 0.2 ␮gof3ϫ FLAG-tagged hCD40L and V5-tagged GRAIL at 0.4, 0.8, 1.2, and 1.6 ␮g (empty V5 vector was added for a total of 2 ␮g of total V5 plasmid for each transfection). Cell lysates were separated by SDS-PAGE and blotted with the indicated Abs. by guest on September 25, 2021

quitinate the related TNF superfamily member OX40L (OX40 li- FIGURE 1. Ionomycin-up-regulated GRAIL expression is associated with gand) in cellular ubiquitination assays, suggesting that the CD40L reduced CD40L expression. A and B, MACS-purified CD4 T cells from ubiquitination activity by GRAIL is specific (data not shown). ␮ ␮ BALB/c mice were anergized with a low (0.1 M), medium (0.5 M), or high Next, we asked whether the consequence of GRAIL-mediated ␮ (1.5 M) ionomycin dose with or without PMA (200 ng/ml) overnight. Cells ubiquitination would lead to a lower steady state level of CD40L. were then harvested for QPCR (A) or Western blot analysis (B) of GRAIL ex- Titrating the amount of GRAIL transfected into a fixed amount of pression. N.S., nonspecific band used as a loading control; IB, immunoblot; transfected CD40L caused a reduction in the amount of CD40L Norm., normalized. C, MACS-purified BALB/c CD4 T cells were anergized with 0 ␮M(left) or 1.5 ␮M(right) ionomycin overnight. Live cells were then present in whole cell lysates (Fig. 2D). Thus, the predominant washed, stimulated, and stained for cell surface CD40L. D, Histograms repre- consequence of GRAIL expression is the degradation of CD40L at senting the percentage of cell surface CD40L expressed on gated CD4 T cells the steady state level without any posttranslational modification with varying conditions of ionomycin anergy treatment and recall stimulation. required for substrate binding. Altogether, these biochemical data Black histogram represents CD4 T cells treated with 0 ␮M ionomycin, dark demonstrate that GRAIL uses a unique system for the capture and gray histogram denotes CD4 T cells treated with 0.5 ␮M ionomycin, and light ubiquitination of substrates in which both functional elements are ␮ gray histogram represents CD4 T cells treated with 1.5 M ionomycin. separated across a lipid bilayer. This model can be extrapolated to suggest how transmembrane substrates with small cytosolic do- mains that are not sufficiently large to support both E3 ligase bind- GRAIL N-terminal PA domain was required for CD40L binding ing and ubiquitin conjugation can be regulated at the protein level. (Fig. 2B). Because the H2N2 mutant is more stable due to a marked reduction in autoubiquitination activity, the higher degree GRAIL down-regulates endogenous CD40L in T cells of association seen between CD40L and H2N2 results from the We next wanted to confirm that GRAIL could regulate CD40L larger protein pool of H2N2 compared with GRAIL (Fig. 2B). To expression in primary CD4 T cells. To avoid the degradation of assess whether binding of the PA and TNF superfamily domains GRAIL seen following T cell stimulation without ionomycin an- could then provide a favorable orientation for ubiquitination of the ergy treatment (Fig. 1, A and B), which is required for CD40L small cytosolic tail of CD40L by the intracellular GRAIL RING up-regulation, we used the observation that naive CD4 T cells from finger, we performed cellular ubiquitination assays by expressing CD40Ϫ/Ϫ mice express CD40L at levels well above those seen in epitope-tagged GRAIL, CD40L, and ubiquitin in 293T cells. A naive CD4 T cells from wild-type mice (17). T cells from characteristic high m.w. ubiquitin ladder was seen on CD40L only CD40Ϫ/Ϫ mice provide a full compartment of CD40L expression in the presence of wild-type GRAIL and not with the PA or RING both intracellularly and at the cell surface as measured in fixed and finger domain mutants (Fig. 2C). In addition, GRAIL did not ubi- live cells, respectively (Fig. 3A). Any decrease in CD40L levels in The Journal of Immunology 1625

A 100 B 100 80 80 60 60

40 40 Count Count 20 20

0 0 100 101 102 103 104 100 101 102 103 104 CD40L CD40L FIGURE 3. GRAIL expression down-regulates endogenous CD40L expres- sion. A, Freshly MACS-purified CD4 T cells from C57BL/6 and CD40Ϫ/Ϫ mice were stained for CD40L. Gray histogram represents unstimulated wild- type CD4 T cells, thick line denotes live CD40Ϫ/Ϫ CD4 T cells, and dotted line represents intracellular staining of CD40Ϫ/Ϫ CD4 T cells. B, MACS purified CD4 T cells from CD40Ϫ/Ϫ mice were stimulated overnight in vitro and then infected with expressing the indicated GRAIL construct. After 36 h, cells were stained for total CD40L and gated GFPϩ cells are shown. Thick line represents vector-transduced CD4 T cells, gray line denotes GRAIL transduced FIGURE 4. CD4 T cells, and dotted line represents ⌬PA-transduced CD4 T cells. GRAIL expression in naive CD4 T cells results in diminished

follicle formation in vivo. DO11.10 bone marrow chimeric mice were gener- Downloaded from ated as described (6). Mice were sacrificed 28 days after injection of transduced CD40Ϫ/Ϫ CD4 T cells transduced to express GRAIL should be hematopoietic cells and lymphoid tissue was processed for histological analysis. indicative of GRAIL-mediated ubiquitination and degradation of A, GFP vector control. B, Wild-type GRAIL. C, Dominant negative H2N2 GRAIL. D, Otubain-1 ARF-1 (epistatic protein stabilizer of GRAIL). Original endogenous CD40L. Following transduction of vector alone, wild- ϫ ଁ ⌬ magnification was 10; denotes follicular zone. One representative mouse is type GRAIL, or PA constructs, each containing internal ribo- shown from two to three mice per group from three independent experiments.

some entry site (IRES)-enhanced GFP as a reporter, total CD40L http://www.jimmunol.org/ on transduced T cells was reduced only in those cells expressing wild-type GRAIL (Fig. 3B). These data demonstrate that GRAIL only abrogated the effects seen with wild-type GRAIL, but also down-regulates expression of the total cellular pool of endogenous enlarged the primary follicles above that seen in vector control CD40L in CD4 T cells, most likely CD40L molecules trafficking mice. In addition, stabilization of endogenous GRAIL by a dom- through the GRAIL-positive endosomal compartments. Targeting inant negative Otubain-1 mutant was sufficient to reduce primary nascent CD40L molecules would be preferred for anergy induction follicle formation to that seen in chimeric mice expressing wild- instead of removing CD40L from the cell surface following CD40 type GRAIL. Together, these data also suggest that both ectopic binding and providing activation signals to the APC (18). Even and endogenous GRAIL can reduce primary follicle formation though GRAIL does not appear to drastically reduce CD40L ex- in vivo. by guest on September 25, 2021 pression following retroviral transduction in T cells, the mecha- Previous work suggested that diminished cell surface CD40L nism of action for diminished CD40L expression could be one of occurred following anergy induction, suggesting the possibility many elements that act in concert to modulate the activation sig- that an anergy-specific factor regulates CD40L expression (12). nals being transmitted between the T cells and APC to create the Because CD40L-CD40 interactions supply maturation signals for anergic phenotype. APCs to provide, in turn, costimulatory signals that prevent anergy induction, proteolytic regulation of CD40L during anergy induc- GRAIL expression in vivo reduces lymphoid follicle formation tion provided a rational hypothesis for investigation. Furthermore, To ask whether GRAIL expression in naive CD4 T cells could the small intracellular CD40L region suggested that cytosolic pro- result in a similar phenotype compared with the reduced lymphoid teins would have difficulty mediating this effect, and a transmem- follicle formation seen in CD40LϪ/Ϫ mice, we generated bone brane E3 ubiquitin ligase associated with anergy induction would marrow chimeric mice by infecting DO11.10 bone marrow cells instead be an ideal candidate. In this study, we demonstrate that with retrovirus expressing vector alone, wild-type GRAIL, H2N2, GRAIL expression in CD4 T cells following ionomycin-mediated or a dominant negative mutant of Otubain-1, a negative regulator anergy induction is associated with decreased expression of of GRAIL expression (19). Following transfer of GFP-sorted bone CD40L. Biochemical evidence demonstrates that GRAIL uses a marrow into BALB/c mice, the spleen and lymph nodes were re- novel split substrate capture ubiquitination model for regulation of moved from reconstituted recipients for analysis. H&E staining CD40L and possibly other transmembrane proteins with very showed defined primary follicles in vector control chimeras (Fig. small intracellular regions. Retroviral transduction of GRAIL into 4A), whereas wild-type GRAIL-expressing chimeras exhibited T cells resulted in diminished expression of total CD40L, and a smaller, diffuse primary follicles (Fig. 4B). Chimeric mice express- GRAIL mutant lacking a substrate capture domain blocked this ing the dominant negative H2N2 mutant contained extensive pri- effect. GRAIL expression in vivo in bone marrow chimeras re- mary follicles that were larger than the vector control (Fig. 4C). In sulted in diminished follicle formation. Although GRAIL appears addition, bone marrow chimeric mice expressing a dominant neg- to be selective for CD40L among other TNF superfamily members ative mutant of Otubain 1, which stabilizes endogenous GRAIL assayed, E3 ligases target multiple substrates. Therefore, it is pos- levels, displayed very small and disorganized primary follicles sible that modulation of other GRAIL targets also play a signifi- (Fig. 4D), even smaller than those seen in wild-type GRAIL-ex- cant role in anergy induction in addition to the effects seen with pressing chimeric mice (Fig. 4B). These data demonstrate that ex- CD40L expression. These data suggest a molecular model for CD4 pression of GRAIL as a transgene in vivo results in a phenotype T cell anergy induction in which TCR signaling in the absence of consistent with reduced CD40L expression by peripheral CD4 T costimulation leads to GRAIL expression, reduced CD40L up-reg- cells. Outcompeting endogenous GRAIL with the dominant neg- ulation, and inhibition of the bidirectional costimulatory signaling ative H2N2 mutant lacking an E3 ubiquitin ligase function not cascade required for full CD4 T cell activation. 1626 CUTTING EDGE: GRAIL UBIQUITINATES CD40L

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