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
a series of discrete checkpoints that maintain unrespon-
siveness to self. During this multistep process, costimu-
latory interactions act as inducible signals that allow
APCs to selectively mobilize T cells against foreign Ags.
In this study, we provide evidence that the anergy-asso-
ciated E3 ubiquitin ligase GRAIL (gene related to an-
ergy in lymphocytes) regulates expression of the co-
stimulatory molecule CD40L on CD4 T cells. Using its
luminal protease-associated domain, GRAIL binds to
the luminal/extracellular portion of CD40L and facili-
tates transfer of ubiquitin molecules from the intracel-
lular GRAIL RING (really interesting new gene) finger
to the small cytosolic portion of CD40L. Down-regula-
tion of CD40L occurred following ectopic expression of
GRAIL in naive T cells from CD40?/?mice, and ex-
pression of GRAIL in bone marrow chimeric mice was
These data provide a model for intrinsic T cell regula-
tion of costimulatory molecules and a molecular frame-
work for the initiation of clonal T cell anergy. The
Journal of Immunology, 2008, 181: 1622–1626.
MHC but do not react strongly against host-derived Ags,
whereas peripheral tolerance encompasses a wide array of pro-
cesses in the secondary lymphoid organs that complement cen-
tral tolerance and help prevent immune response to self. As a
result, full activation of naive T cells requires coordinate signal-
ing with APCs, where binding of TCR and MHC:peptide acts
he immune system uses an efficient dual-tiered system
for the discrimination of Ags as nonself. Central toler-
ance selects developing lymphocytes that bind self-
as an initial signal while CD28 interactions with the inducible
costimulatory molecules CD80 and CD86 provide a second
signal. However, engagement of TCR alone in the absence of
costimulation can initiate T cell anergy, a state of induced un-
responsiveness to proliferation and IL-2 production (1). Be-
cause T cells with reactivity to self can escape clonal deletion
(2), anergy could serve as a component of peripheral tolerance
that inhibits self-reactive T cells from initiating autoimmunity
due to the absence of costimulatory signaling.
Whereas early molecular research into clonal T cell anergy
the role of protein stability controlled by the ubiquitin-protea-
some pathway in anergy induction and peripheral tolerance has
recently been investigated (3, 4). The gene related to anergy in
lymphocytes (GRAIL4; Rnf128) is a type 1 transmembrane E3
ubiquitin ligase identified in a screen for transcripts differen-
tially up-regulated in T cell clones following anergy induction
expressing GRAIL via bone marrow chimera production re-
duced their proliferation and IL-2 secretion in response to ac-
tivation signals, suggesting that GRAIL acts as a potent, intrin-
sic anergy promoting factor (6). Endogenous GRAIL
expression was subsequently found in multiple model systems
of anergy induction, including anti-CD3 stimulation, ionomy-
cin treatment, and in vivo adaptive tolerance (5–7).
Because costimulation abrogates anergy induction and
GRAIL is differentially expressed 4–6 h following the initia-
tion of anergy, we hypothesized that an inducible molecule
downstream of TCR engagement and upstream of CD28 co-
stimulatory signaling could serve as a target for GRAIL
regulation. One potential candidate whose degradation could
block T cell activation is CD40 ligand (CD40L; CD154), a
type 2 transmembrane protein of the TNF superfamily (8).
*Division of Immunology and Rheumatology and†Division of Oncology, Department of
Medicine, Stanford University School of Medicine, Stanford, CA 94305
Received for publication November 27, 2007. Accepted for publication June
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants CA 65237-17, T32-
AI07290-21, and U19-AI070352 and the Tom and Susan Ford Stanford Graduate Fel-
lowship (to N.L.).
University of Wisconsin, H4/474 Clinical Sciences Center, 600 Highland Avenue, Mad-
ison, WI 53792-4108.
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:
CD40 ligand; HA, hemagglutinin; PA, protease-associated domain; QPCR, real-time
quantitative PCR; RING, really interesting new gene.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
CD40L expression is low and intracellular on naive CD4 T
cells, but following TCR stimulation cell surface CD40L
increases rapidly by 6–8 h (9, 10). CD40L trimerization of
CD40 on mature APCs further elevates cell surface CD80 and
CD86, which in turn bind CD28 on T cells and provide recip-
(11). Therefore, modulating cell surface CD40L on the T cell
could prevent APC-derived costimulatory signaling and pro-
mote anergy induction. Previous evidence lends support to this
CD40L expression, suggesting the presence of an anergy-spe-
cific factor that prevents CD40L up-regulation (12).
Materials and Methods
Reagents and Abs
Brij96, IPEGAL (Nonidet P-40), mouse anti-FLAG-HRP (clone M2), mouse
anti-FLAG-conjugated agarose (M2), and rabbit anti-V5 were from Sigma Al-
drich; anti-hemagglutinin (HA)-HRP (clone F7), anti-HA conjugated agarose
(clone F7), from Santa Cruz Biotechnology; PE-conjugated anti-mouse
leucinyl-norleucinal from Calbiochem.
Cell culture and transfection
293T Phoenix cells were grown in DMEM plus 5% heat-inactivated FBS sup-
plemented with L-glutamine and penicillin/streptomycin. For transfection,
TransIT-LT1 transfection reagent was used according to the manufacturer’s
Tris-HCl (pH 7.5), 150 mM NaCl, 1% Brij96, and protease inhibitors) and
nation assays, cells were incubated with N-acetyl-leucinyl-leucinyl-norleucinal
(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
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
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-
tions for 18 h and then washed.
Real-time quantitative PCR (QPCR)
RNA was harvested and amplified as described previously (6) with actin as a
normalizing gene. Primers used were as follows: GRAIL forward primer, 5?-
AGAGAGAGGGGCTTCTGGAG-3?; GRAIL reverse primer, 5?-CGATGA
CCATTGTGACTTGG-3?; ?-actin forward primer, 5?-CAGGCATTGCTG
ACCGATC-3?. All samples were analyzed in triplicate, and GRAIL mRNA
arbitrary units are expressed as the mean of triplicate normalized values against
Bone marrow chimeras and tissue sections
Generation of bone marrow chimeric mice was performed as described (6).
by paraffin embedding. Tissues sections were stained with H&E. Imaging was
Results and Discussion
Up-regulation of endogenous GRAIL correlates with CD40L
A recently described model system for clonal T cell anergy induc-
tion used unopposed calcium flux via ionomycin treatment to up-
regulate a cohort of E3 ligases in CD4 T cells. These enzymes,
including Cbl-b, GRAIL, and Itch, were implicated in controlling
the expression of proteins involved in T cell activation via the
ubiquitin-proteasome pathway (13). Because anergized T cell
clones also exhibit reduced CD40L expression (12), we asked
whether ionomycin treatment of recently activated naive T cells
would correlate GRAIL expression with a deficiency in CD40L.
Purified BALB/c CD4 T cells were stimulated with plate bound
anti-CD3 and soluble anti-CD28 for 48 h, washed and rested in
vitro for 72 h, and then treated with increasing concentrations of
ionomycin overnight. GRAIL expression levels were then assayed
by QPCR (Fig. 1A) or Western blotting (Fig. 1B). These data dem-
onstrate that endogenous GRAIL expression is induced at the
mRNA and protein level by ionomycin treatment and that GRAIL
expression directly correlates with the ionomycin concentration
added during anergy induction. In contrast, the addition of PMA
with ionomycin prevented both anergy induction and up-regulation
of GRAIL mRNA and protein (Fig. 1, A and B). To examine
CD40L regulation in this context, we either incubated ionomycin-
anergized primary CD4 T cells in medium alone or exposed them
to recall stimulation and then analyzed the cell surface expression
of CD40L (Fig. 1C). CD40L up-regulation on ionomycin-aner-
gized CD4 T cells was markedly reduced following recall activa-
tion. In addition, the absence of CD40L on anergized CD4 T cells
without recall stimulation demonstrated that ionomycin treatment
during anergy induction did not up-regulate CD40L itself or pre-
vent its return to baseline (Fig. 1D). Overall, the percentage of
CD40L seen on anergized CD4 T cells inversely correlated with
GRAIL expression, as increasing amounts of ionomycin led to
more GRAIL up-regulation and less CD40L expressed on the cell
surface following recall stimulation (Fig. 1D). Finally, the strength
of recall stimulation had no apparent effect on the relative reduc-
tion of cell surface CD40L expression seen (Fig. 1D). These data
demonstrate a correlation between GRAIL expression and cell sur-
face CD40L levels during anergy induction of primary T cells. The
biological relevance of these findings was strengthened by dem-
onstrating endogenous GRAIL up-regulation and CD40L down-
regulation in DO11.10 CD4 T cells provided OVA peptide on
APCs with costimulatory signals blocked by the addition of solu-
ble CTLA4-Ig (data not shown).
GRAIL binds and ubiquitinates CD40L in vitro
GRAIL is a type 1 transmembrane protein that contains a luminal
protease-associated (PA) domain and cytosolic RING (really in-
teresting new gene) finger (Fig. 2A). The PA domain currently
does not possess a described canonical function but is proposed to
serve as a protein-protein interaction motif (14, 15). The RING
finger confers E3 ubiquitin ligase activity by recruiting E2 trans-
ferase enzymes loaded with activated ubiquitin molecules for con-
jugation to substrate proteins (5, 16). CD40L is a type 2 trans-
membrane protein that contains a large extracellular domain of the
TNF superfamily and a small cytoplasmic tail with unknown func-
tion (Fig. 2A). Although nearly every cellular protein is regulated
by a component of the ubiquitin-proteasome pathway, it would
appear difficult for cytosolic E3 ligases to simultaneously bind the
22-aa cytosolic tail of CD40L and retain space for conjugation of
the 76-aa ubiquitin molecule. Thus, we hypothesized that the PA
domain of GRAIL might provide an interaction interface for bind-
ing CD40L on the luminal/extracellular side of the membrane and
facilitate ubiquitination of the intracellular tail of CD40L by the
cytosolic RING domain of GRAIL.
To first test for an interaction between GRAIL and CD40L,
293T cells were transfected with N-terminal 3? FLAG-tagged
CD40L and various C-terminal V5-tagged GRAIL constructs. Fol-
lowing immunoprecipitation of CD40L, both wild-type and an en-
zymatically inactive RING finger mutant of GRAIL (H2N2) were
able to coimmunoprecipitate with CD40L (Fig. 2B). However, a
GRAIL mutant lacking the PA domain (?PA) repeatedly failed to
coimmunoprecipitate with CD40L, indicating that an intact
1623The Journal of Immunology
GRAIL N-terminal PA domain was required for CD40L binding
(Fig. 2B). Because the H2N2 mutant is more stable due to a
marked reduction in autoubiquitination activity, the higher degree
of association seen between CD40L and H2N2 results from the
larger protein pool of H2N2 compared with GRAIL (Fig. 2B). To
assess whether binding of the PA and TNF superfamily domains
could then provide a favorable orientation for ubiquitination of the
small cytosolic tail of CD40L by the intracellular GRAIL RING
finger, we performed cellular ubiquitination assays by expressing
epitope-tagged GRAIL, CD40L, and ubiquitin in 293T cells. A
characteristic high m.w. ubiquitin ladder was seen on CD40L only
in the presence of wild-type GRAIL and not with the PA or RING
finger domain mutants (Fig. 2C). In addition, GRAIL did not ubi-
quitinate the related TNF superfamily member OX40L (OX40 li-
gand) in cellular ubiquitination assays, suggesting that the CD40L
ubiquitination activity by GRAIL is specific (data not shown).
Next, we asked whether the consequence of GRAIL-mediated
ubiquitination would lead to a lower steady state level of CD40L.
Titrating the amount of GRAIL transfected into a fixed amount of
transfected CD40L caused a reduction in the amount of CD40L
present in whole cell lysates (Fig. 2D). Thus, the predominant
consequence of GRAIL expression is the degradation of CD40L at
the steady state level without any posttranslational modification
required for substrate binding. Altogether, these biochemical data
demonstrate that GRAIL uses a unique system for the capture and
ubiquitination of substrates in which both functional elements are
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-
ing and ubiquitin conjugation can be regulated at the protein level.
GRAIL down-regulates endogenous CD40L in T cells
We next wanted to confirm that GRAIL could regulate CD40L
expression in primary CD4 T cells. To avoid the degradation of
GRAIL seen following T cell stimulation without ionomycin an-
ergy treatment (Fig. 1, A and B), which is required for CD40L
up-regulation, we used the observation that naive CD4 T cells from
CD40?/?mice express CD40L at levels well above those seen in
naive CD4 T cells from wild-type mice (17). T cells from
CD40?/?mice provide a full compartment of CD40L expression
both intracellularly and at the cell surface as measured in fixed and
live cells, respectively (Fig. 3A). Any decrease in CD40L levels in
reduced CD40L expression. A and B, MACS-purified CD4 T cells from
BALB/c mice were anergized with a low (0.1 ?M), medium (0.5 ?M), or high
(1.5 ?M) ionomycin dose with or without PMA (200 ng/ml) overnight. Cells
were then harvested for QPCR (A) or Western blot analysis (B) of GRAIL ex-
pression. N.S., nonspecific band used as a loading control; IB, immunoblot;
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
washed, stimulated, and stained for cell surface CD40L. D, Histograms repre-
senting the percentage of cell surface CD40L expressed on gated CD4 T cells
with varying conditions of ionomycin anergy treatment and recall stimulation.
Black histogram represents CD4 T cells treated with 0 ?M ionomycin, dark
gray histogram denotes CD4 T cells treated with 0.5 ?M ionomycin, and light
gray histogram represents CD4 T cells treated with 1.5 ?M ionomycin.
Ionomycin-up-regulated GRAIL expression is associated with
of the domains and orientation of GRAIL and CD40L. B, 293T cells were
transfected with 3? FLAG-tagged human CD40L (hCD40L) and the indi-
cated V5-tagged GRAIL constructs. Eluted proteins from anti-FLAG conju-
3? 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-
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.
GRAIL binds and ubiquitinates CD40L. A, Schematic display
1624CUTTING EDGE: GRAIL UBIQUITINATES CD40L
CD40?/?CD4 T cells transduced to express GRAIL should be
indicative of GRAIL-mediated ubiquitination and degradation of
endogenous CD40L. Following transduction of vector alone, wild-
type GRAIL, or ?PA constructs, each containing internal ribo-
some entry site (IRES)-enhanced GFP as a reporter, total CD40L
on transduced T cells was reduced only in those cells expressing
wild-type GRAIL (Fig. 3B). These data demonstrate that GRAIL
down-regulates expression of the total cellular pool of endogenous
CD40L in CD4 T cells, most likely CD40L molecules trafficking
through the GRAIL-positive endosomal compartments. Targeting
nascent CD40L molecules would be preferred for anergy induction
instead of removing CD40L from the cell surface following CD40
binding and providing activation signals to the APC (18). Even
though GRAIL does not appear to drastically reduce CD40L ex-
pression following retroviral transduction in T cells, the mecha-
nism of action for diminished CD40L expression could be one of
many elements that act in concert to modulate the activation sig-
nals being transmitted between the T cells and APC to create the
GRAIL expression in vivo reduces lymphoid follicle formation
To ask whether GRAIL expression in naive CD4 T cells could
result in a similar phenotype compared with the reduced lymphoid
follicle formation seen in CD40L?/?mice, we generated bone
marrow chimeric mice by infecting DO11.10 bone marrow cells
with retrovirus expressing vector alone, wild-type GRAIL, H2N2,
or a dominant negative mutant of Otubain-1, a negative regulator
of GRAIL expression (19). Following transfer of GFP-sorted bone
marrow into BALB/c mice, the spleen and lymph nodes were re-
moved from reconstituted recipients for analysis. H&E staining
showed defined primary follicles in vector control chimeras (Fig.
4A), whereas wild-type GRAIL-expressing chimeras exhibited
smaller, diffuse primary follicles (Fig. 4B). Chimeric mice express-
ing the dominant negative H2N2 mutant contained extensive pri-
mary follicles that were larger than the vector control (Fig. 4C). In
addition, bone marrow chimeric mice expressing a dominant neg-
ative mutant of Otubain 1, which stabilizes endogenous GRAIL
levels, displayed very small and disorganized primary follicles
(Fig. 4D), even smaller than those seen in wild-type GRAIL-ex-
pressing chimeric mice (Fig. 4B). These data demonstrate that ex-
pression of GRAIL as a transgene in vivo results in a phenotype
consistent with reduced CD40L expression by peripheral CD4 T
cells. Outcompeting endogenous GRAIL with the dominant neg-
ative H2N2 mutant lacking an E3 ubiquitin ligase function not
only abrogated the effects seen with wild-type GRAIL, but also
enlarged the primary follicles above that seen in vector control
mice. In addition, stabilization of endogenous GRAIL by a dom-
inant negative Otubain-1 mutant was sufficient to reduce primary
follicle formation to that seen in chimeric mice expressing wild-
type GRAIL. Together, these data also suggest that both ectopic
and endogenous GRAIL can reduce primary follicle formation
Previous work suggested that diminished cell surface CD40L
occurred following anergy induction, suggesting the possibility
that an anergy-specific factor regulates CD40L expression (12).
Because CD40L-CD40 interactions supply maturation signals for
APCs to provide, in turn, costimulatory signals that prevent anergy
induction, proteolytic regulation of CD40L during anergy induc-
tion provided a rational hypothesis for investigation. Furthermore,
the small intracellular CD40L region suggested that cytosolic pro-
teins would have difficulty mediating this effect, and a transmem-
brane E3 ubiquitin ligase associated with anergy induction would
instead be an ideal candidate. In this study, we demonstrate that
GRAIL expression in CD4 T cells following ionomycin-mediated
anergy induction is associated with decreased expression of
CD40L. Biochemical evidence demonstrates that GRAIL uses a
novel split substrate capture ubiquitination model for regulation of
CD40L and possibly other transmembrane proteins with very
small intracellular regions. Retroviral transduction of GRAIL into
T cells resulted in diminished expression of total CD40L, and a
GRAIL mutant lacking a substrate capture domain blocked this
effect. GRAIL expression in vivo in bone marrow chimeras re-
sulted in diminished follicle formation. Although GRAIL appears
to be selective for CD40L among other TNF superfamily members
assayed, E3 ligases target multiple substrates. Therefore, it is pos-
sible that modulation of other GRAIL targets also play a signifi-
cant role in anergy induction in addition to the effects seen with
CD40L expression. These data suggest a molecular model for CD4
T cell anergy induction in which TCR signaling in the absence of
costimulation leads to GRAIL expression, reduced CD40L up-reg-
ulation, and inhibition of the bidirectional costimulatory signaling
cascade required for full CD4 T cell activation.
sion. A, Freshly MACS-purified CD4 T cells from C57BL/6 and CD40?/?
mice were stained for CD40L. Gray histogram represents unstimulated wild-
represents intracellular staining of CD40?/?CD4 T cells. B, MACS purified
CD4 T cells from CD40?/?mice were stimulated overnight in vitro and then
infected with retrovirus expressing the indicated GRAIL construct. After 36 h,
cells were stained for total CD40L and gated GFP?cells are shown. Thick line
CD4 T cells, and dotted line represents ?PA-transduced CD4 T cells.
follicle formation in vivo. DO11.10 bone marrow chimeric mice were gener-
ated as described (6). Mice were sacrificed 28 days after injection of transduced
hematopoietic cells and lymphoid tissue was processed for histological analysis.
A, GFP vector control. B, Wild-type GRAIL. C, Dominant negative H2N2
GRAIL. D, Otubain-1 ARF-1 (epistatic protein stabilizer of GRAIL). Original
shown from two to three mice per group from three independent experiments.
GRAIL expression in naive CD4 T cells results in diminished
1625The Journal of Immunology
We are indebted to Cariel Taylor for excellent technical assistance. We grate-
fully acknowledge Shoshana Levy for the hCD40L cDNA and Ron Kopito for
HA-tagged ubiquitin. Special thanks for Robyn Rajkovich for administrative
The authors have no financial conflict of interest.
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1626 CUTTING EDGE: GRAIL UBIQUITINATES CD40L