Naive CD4 T Cell Proliferation Is Controlled by Mammalian
Target of Rapamycin Regulation of GRAIL Expression1
Jack T. Lin,* Neil B. Lineberry,* Michael G. Kattah,* Leon L. Su,* Paul J. Utz,*
C. Garrison Fathman,2* and Linda Wu†
In this study, we demonstrate that the E3 ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) is expressed in quiescent
naive mouse and human CD4 T cells and has a functional role in inhibiting naive T cell proliferation. Following TCR engagement,
CD28 costimulation results in the expression of IL-2 whose signaling through its receptor activates the Akt-mammalian target of
rapamycin (mTOR) pathway. Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL,
Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation. The activation of mTOR
appears to be the critical component of IL-2R signaling regulating GRAIL expression. CTLA4-Ig treatment blocks CD28 co-
stimulation and resultant IL-2 expression, whereas rapamycin and anti-IL-2 treatment block mTOR activation downstream of
IL-2R signaling. Thus, all three of these biotherapeutics inhibit mTOR-dependent translation of mRNA transcripts, resulting in
blockade of Otub1 expression, maintenance of GRAIL, and inhibition of CD4 T cell proliferation. These observations provide a
mechanistic pathway sequentially linking CD28 costimulation, IL-2R signaling, and mTOR activation as important requirements
for naive CD4 T cell proliferation through the regulation of Otub1 and GRAIL expression. Our findings also extend the role of
GRAIL beyond anergy induction and maintenance, suggesting that endogenous GRAIL regulates general cell cycle and prolif-
eration of primary naive CD4 T cells. The Journal of Immunology, 2009, 182: 5919–5928.
bined system involving central (1) and peripheral tolerance (2).
Among several mechanisms to ensure peripheral tolerance is
anergy, a state of unresponsiveness induced in CD4 T cells
upon activation in the absence of costimulatory signals (3, 4). In
addition to naive CD4 TCR binding to antigenic peptide in the
context of MHC, CD28 binding to B7 provided on mature APC
allows IL-2 production, a necessary component of naive CD4 T
cell activation (5). The necessity for naive CD4 T cells to re-
ceive costimulation and signaling through the IL-2R in addition
to TCR ligation serves to create a threshold within the periph-
eral immune system that both ensures the continued survival
and sentry functions of the T cells while also maintaining an
immune environment free from autoimmunity.
Members of the E3 ubiquitin ligase family have been demon-
strated to be important molecular mediators of T cell anergy and
peripheral tolerance. The ubiquitination process requires the E1
enzyme to activate ubiquitin, an E2 enzyme to act as a transferase,
and an E3 ligase to direct substrate specificity for ubiquitination
(6). The E3 ubiquitin ligases Cbl-b, Itch, and gene related to an-
olerance mechanisms play an important role in prevent-
ing unwanted immune responses including autoimmu-
nity. T cells are rendered tolerant to self through a com-
ergy in lymphocyte (GRAIL),3have all been described as playing
a functional role in T cell anergy (7–10). Additionally, Itch has
been shown to prevent autoimmune activation of peripheral T cells
toward a Th2 bias (11), and Cbl-b attenuates T cell hyperrespon-
sive activation absent CD28 costimulation (12, 13).
GRAIL was first detected during the induction of anergy in
CD4 T cell clones (14). These and subsequent experiments,
where GRAIL was ectopically expressed in CD4 T cell clones
(14), or in peripheral T cells following bone marrow reconsti-
tution with transgenic GRAIL-expressing hemopoietic stem
cells (15), demonstrated that GRAIL expression rendered the
CD4 T cells anergic as measured by impaired proliferation and
IL-2 production. Recently, Rho guanine dissociation inhibitor,
involved in actin cytoskeleton rearrangement (16), CD40L, a
receptor that drives B cell class switching and APC activation
(17), and multiple members of the tetraspanin family (18) have
been identified as GRAIL substrates. Otubain-1 (Otub1), a deu-
biquitinating enzyme (19, 20), was initially identified as a bind-
ing partner and subsequently as an epistatic regulator that de-
GRAIL to become degraded in the proteosome (21).
Although a role for GRAIL in regulating CD4 T cell prolifer-
ation has been demonstrated in clones and in transgenic expression
systems, little is known about the expression, regulation, or func-
tion of endogenous GRAIL or Otub1 in naive CD4 T cells. In this
study, we investigated how the expression of GRAIL and Otub1 is
regulated during mouse and human naive CD4 T cell activation.
Our findings demonstrate that Otub1 is expressed and GRAIL is
degraded when naive CD4 T cells are productively activated to
undergo proliferation. The loss of GRAIL is mechanistically
*Stanford University School of Medicine, Department of Medicine, Division of Im-
munology and Rheumatology, Stanford, CA 94305; and†Medarex, Milpitas, CA
Received for publication December 1, 2008. Accepted for publication February
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 grants from the National Institutes of Health Grants
CA065237 (to C.G.F.) and AI07290 (to J.T.L.).
2Address correspondence and reprint requests to Dr. C. Garrison Fathman, Stanford
University School of Medicine, 269 Campus Drive West, CCSR Building Room
2225, Department of Medicine, Division of Immunology and Rheumatology, Stan-
ford, CA 94305. E-mail address: email@example.com
3Abbreviations used in this paper: GRAIL, gene related to anergy in lymphocyte;
mTOR, mammalian target of rapamycin; Otub1, Otubain-1; pOVA, peptide
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
controlled through a pathway involving CD28 costimulation, IL-2
production and IL-2R signaling, and ultimately, mTOR-dependent
translation of select mRNA. Interference of this pathway using
CTLA4-Ig, anti-IL-2, or rapamycin prevents Otub1 protein expres-
sion and maintains GRAIL expression, which inhibits T cell pro-
liferation. These findings implicate Otub1 and GRAIL as impor-
tant components governing T cell unresponsiveness and highlights
them as potential therapeutic targets in regulating immune
Materials and Methods
BALB/c, DO11, NOD, and NOD.B10 female mice were purchased from
The Jackson Laboratory. DO11 CD28?/?female mice were a gift from
Drs. A. Abbas and L. Barron (University of California, San Francisco,
CA). All procedures involving mice were conducted in accordance with
Institutional Animal Care and Use Committee policies as set forth by Stan-
ford University’s Administrative Panel on Laboratory Animal Care, as ac-
credited by the Association for Assessment and Accreditation of Labora-
tory Animal Care International.
Isolation and stimulation of mouse CD4 T cells
Spleen and lymph nodes were harvested from naive mice and homogenized
through a strainer. RBC were lysed from the suspension using red blood
cell lysing buffer (Sigma-Aldrich). Lymphocytes were isolated by density
centrifugation using Lympholyte-M (Cedarlane Laboratories). CD4?T
cells were sorted via negative selection using an AutoMACS sorter (Mil-
tenyi Biotec). BALB/c CD4?T cells (5 ? 103) were stimulated in 96-well
U-bottom plates with equal numbers of polystyrene latex beads (Interfacial
Dynamics) coated with 1.0 ?g/ml anti-CD3 (145-2C11; eBioscience) and
0.5 ?g/ml anti-CD28 (37.51; eBioscience). For DO11 T cells, 5 ? 103
DO11 CD4?T cells were stimulated in 96-well U-bottom plates with 104
APC and 50 ng/ml peptide OVA323–339(pOVA). Rapamycin (Sigma-
Aldrich) was used at a concentration of 100 nM. CTLA4-Ig (Abatacept;
Bristol-Myers Squibb) was used at a concentration of 10 ?g/ml. Anti-IL-2
Ab (JES6-1A12; eBioscience) was used at a concentration of 10 ?g/ml.
Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with
10% heat-inactivated FCS (Mediatech), 100 nM sodium pyruvate (Invitro-
gen), 2 mM L-glutamine (Invitrogen), 100 nM nonessential amino acids
(Invitrogen), 100 U/ml penicillin/streptomycin (Invitrogen), and 5 nM
Isolation and stimulation of human naive CD4?CD45RA?
Human peripheral blood mononucleated cells from buffy coats of different
donors were obtained from the Stanford Blood Center under Stanford Uni-
versity Institutional Review Board approval. Buffy coats were separated
into leukocytes using Ficoll-Paque Plus (GE Health Sciences). T cells were
prepared using a RosetteSep human CD4?T cell enrichment (Stem Cell
Technologies) followed by a Naive CD4?T cell isolation kit along using
LS MACS columns (Miltenyi Biotec). Negatively selected CD4?CD45RA?
CD45RO?CD25?T cells were isolated at 95–99% purity as confirmed by
flow cytometry using anti-CD4-FITC (OKT4; eBioscience) and anti-
CD45RA-PE (HI100; eBioscience) Ab. CD4?CD45RA?T cells (5 ? 103)
were stimulated in 96-well U-bottom plates with equal numbers of Dyna-
beads CD3/28 T Cell Expander (Invitrogen) or plate-bound anti-CD3 at
1.0 ?g/ml with mitomycin C (Sigma-Aldrich) inactivated APC (anti-CD3/
APC). Rapamycin (Sigma-Aldrich) was used at a concentration of 100 nM,
and CTLA4-Ig (Abatacept, Bristol-Myers Squibb) was used at a concen-
tration of 10 ?g/ml. Agonist anti-CD28 Ab was used at 1.0 ?g/ml
(CD28.2; eBioscience). Anti-IL-2 Ab (5334; eBioscience) was used at a
concentration of 10 ?g/ml. Recombinant human IL-2 (PeproTech) was
used at a concentration of 10 ng/ml. Cells were cultured in X-Vivo 15
medium (Lonza) supplemented with 10% heat-inactivated FCS (Media-
tech), 2 mM L-glutamine (Invitrogen), 100 U/ml penicillin/streptomycin
(Invitrogen), and 5 nM 2-ME (Sigma-Aldrich).
Proliferation and cell division assays
Cells cultured in 96-well U-bottom plate wells were pulsed with 1 ?Ci of
methyl-[3H]thymidine (Amersham Biosciences) for 6 h during the last 72 h
of stimulation and harvested onto filters (Wallac). Filters were wetted with
Betaplate scintillation fluid (PerkinElmer) and counts per minute read on a
1205 Betaplate liquid scintillation counter (Wallac). For CFSE experi-
ments, cells were labeled with 1 ?M CFDA-SE (Sigma-Aldrich) in serum-
free RPMI 1640 medium for 10 min and washed twice before culturing.
CFSE-labeled cells were assayed after 72 h of culture.
Whole-cell lysates were made using lysis buffer consisting of 0.5% Non-
idet P-40, 100 mM sodium chloride, 0.5 mM EDTA, 20 mM Tris (pH
7.6–8.0), with protease inhibitor mixture (Pierce) and phosphatase inhib-
itor mixture (Pierce). Protein samples were loaded on 4–15% Tris-HCl gels
(Bio-Rad) and separated by SDS-PAGE. Protein was transferred from gel
to Immobolin-P polyvinylidene difluoride membrane (Millipore) using
Trans-Blot SD semidry transfer apparatus (Bio-Rad) following the manu-
facturer’s instructions. StartingBlock (Tris-buffered saline with 0.05%
Tween 20) (Pierce) was used to block membranes and was also used during
primary and secondary Ab staining. Secondary Abs were all HRP conju-
gated (Zymed Laboratories). ECL Plus Western blotting reagents (GE
Healthcare) were used for chemiluminescent detection of protein. Chemi-
luminescene signal was exposed onto Amersham Hyperfilm ECL (GE
Healthcare). Membranes were stripped using Restore Western blot strip-
ping buffer (Pierce). Densitometry was performed using ImageJ software
(National Institutes of Health). Primary Abs used were anti-phospho-4E-
BP1 (Thr37/46) (236B4; Cell Signaling Technology), anti-4E-BP1 (53H11;
Cell Signaling Technology), anti-?-actin (ab8226; Abcam), anti-phospho-
Akt (Ser473) (44-623G; Invitrogen), anti-Akt (9272; Cell Signaling Tech-
nology), anti-cyclin D3 (1/cyclin D3; BD Biosciences), anti-GAPDH
(ab9485; Abcam), anti-GRAIL (affinity-purified rabbit polyclonal) or
anti-GRAIL (H11-744; BD Biosciences), anti-Kip1/p27 (57; BD Bio-
sciences), anti-Otub1 (mouse monoclonal, a gift from Berlex Bio-
sciences), anti-phospho-S6K1 (Thr421/Ser424) (9204; Cell Signaling
Technology), anti-S6K1 (9202; Cell Signaling Technology), anti-
phospho-STAT5 (Tyr694/699) (8-5-2; Upstate Biotechnology), and anti-
STAT5 (9363; Cell Signaling Technology).
Samples were stained and washed in PBS with 0.5% BSA and 0.02%
sodium azide. Anti-CD25-PE (PC61; BD Biosciences) staining was used at
(1/100) dilution on ice, in the dark, for 15 min. Samples were acquired
using an LSR flow cytometer (BD Biosciences).
Supernatant was collected 24 h after stimulation. Anti-IL-2 capture Ab
(JES6-1A12; BD Biosciences) and biotinylated detection Ab (JES6-5H4;
BD Biosciences) were used according to the manufacturer’s instructions.
Detection using ExtrAvidin peroxidase conjugate (Sigma-Aldrich) and
3,3?,5,5?-tetramethylbenzidine liquid substrate system (Sigma-Aldrich)
were used according to the manufacturer’s instructions.
Microarray data of NOD vs NOD.B10 pancreatic lymph node mRNA ex-
pression (22) is publicly available at Gene Expression Omnibus (http://
www.ncbi.nim.nih.gov/geo), accession number GSE15150, and was an-
alyzed using Matrix2png software (23).
Real-time quantitative PCR
RNA was collected from samples using RNeasy kit (Qiagen). RNA was
reverse transcribed into cDNA using Omniscript RT kit (Qiagen), with
DNase set (Qiagen). Real-time quantitative PCR was conducted using Bril-
liant qPCR SYBR Green Mastermix (Stratagene) according to the
manufacturer’s instructions, and cDNA samples were run on an Mx4000
thermocycler (Stratagene). Primers used for mouse GRAIL: (F) 5?-GCGC
AGTCAGCAAATGAA-3?, (R) 5?-TGTCAACATGGGGAACAACA-3?;
mouse IL-2: (F) 5?-CCTGAGCAGGATGGAGAATTACA-3?, (R) 5?-TC
CAGAACATGCCGCAGAG-3?; mouse Otub1: (F) 5?-CGACTCCGAA
and mouse ?-actin: (F) 5?-CAGGCATTGCTGACAGGATGCA-3?, (R)
Retroviral transduction was performed as described previously (24). Mu-
rine GRAIL (Rnf128) cDNA was cloned into the MSCV-IRES-GFP vector,
denoted as MSCV-GRAIL-IRES-GFP (GRAIL-expressing). MSCV-
GRAIL-IRES-GFP and MSCV-IRES-GFP (vector control) retroviral vec-
tors were used to generate retrovirus for CD4 T cell transduction experi-
ments. The MSCV-IRES-GFP retroviral vector was a gift from Drs. K.
Murphy and T. Murphy (Washington University, St. Louis, MO).
5920mTOR REGULATION OF GRAIL EXPRESSION CONTROLS PROLIFERATION
GRAIL is expressed in naive CD4 T cells and down-regulated
To ask when and where GRAIL is initially expressed, we exam-
ined T cells from the thymus of BALB/c mice. We found that
GRAIL was expressed abundantly in Qa-2?late-stage, and less so
in Qa-2?early-stage, single-positive CD4 T cells (Fig. 1A) but not
in earlier-stage thymocytes (data not shown). Qa-2 is a nonpoly-
morphic MHC class I Ag that is expressed on the cell surface of all
peripheral CD4 T cells and on the subset of mature single-positive
CD4 T cells in the thymus primed for exit to the periphery (25).
GRAIL protein is also present in peripheral naive mouse CD4 T
cells isolated ex vivo but is lost within 18 h and absent for up to
48 h following anti-CD3/anti-CD28 (anti-CD3/28) activation of
these cells (Fig. 1B). These data show that late-stage, single-pos-
itive CD4 thymocytes and peripheral, naive CD4 T cells express
GRAIL and that GRAIL expression is lost upon activation.
Down-regulation of GRAIL following stimulation of CD4 T cells
is required for optimal proliferation
To demonstrate that the loss of GRAIL expression in naive CD4 T
cells has a functional consequence, we used retroviral transduction
of naive mouse CD4 T cells with GRAIL-expressing or control
vector, both expressing GFP as a reporter, and sorted GFP? cells
for analysis (supplemental Fig. 1).4Twenty-four hours following
activation, when endogenous GRAIL is absent, immunoblots of
cell lysates verified ectopic GRAIL expression in the CD4 T cells
transduced with the GRAIL-expressing vector when compared
with vector control transduced cells (Fig. 2A). As a consequence of
maintaining transgenic GRAIL expression during anti-CD3/28 ac-
tivation, proliferation of CD4 T cells transduced to express GRAIL
was markedly inhibited compared with vector control-trans-
duced CD4 T cells (Fig. 2B). Thus, GRAIL expressed in pe-
ripheral naive CD4 T cells maintains unresponsiveness, and its
down-regulation is functionally required during T cell activa-
tion to allow proliferation.
CD28 costimulation is required for GRAIL down-regulation,
IL-2 production, and CD4 T cell proliferation
Successful activation of naive CD4 T cells requires both produc-
tive TCR/CD3 engagement and CD28 costimulation (26, 27). We
confirmed this using naive CD4 T cells from DO11 CD28?/?or
DO11 CD28?/?transgenic BALB/c mice. As expected, in re-
sponse to Ag-pulsed APC, proliferation of DO11 CD28?/?CD4 T
cells was diminished when compared with that of DO11 CD28?/?
CD4 T cells (Fig. 3A). In the absence of CD28 costimulation, IL-2
production was diminished (Fig. 3B), and this diminished produc-
tion of IL-2 resulted in decreased IL-2R signaling as demonstrated
by reduced STAT5 phosphorylation (Fig. 3C). When GRAIL was
4The online version of this article contains supplemental material.
?-actin immunoblots of Qa-2?or Qa-2?CD4 single-positive thymocytes
from naive BALB/c mice. B, GRAIL and GAPDH immunoblots of CD4 T
cells from naive BALB/c mice at indicated hours of bead stimulation (anti-
CD3/28). GRAIL-transfected Jurkat cells were used as a positive control
(?). Data are representative of more than three experiments with similar
GRAIL expression in naive CD4 T cells. A, GRAIL and
lation diminishes proliferation. A, GRAIL and ?-actin immunoblots of
CD4 T cells from naive BALB/c mice following retroviral transduction in
vitro with GRAIL-GFP or control (vector)-GFP. Protein lysates were made
from sorted GFP?cells from both transduced populations after 24 h of
stimulation (anti-CD3/28). B, Proliferation assay of bead stimulated (anti-
CD3/28) CD4 T cells retrovirally transduced as in A and sorted for GFP?
cells, without (?) or with (?) stimulation (anti-CD3/28). Error bars indi-
cate SD of triplicates. Measured as counts per minute. Data are represen-
tative of two experiments with similar results.
Sustained GRAIL expression following CD4 T cell stimu-
5921 The Journal of Immunology
examined following activation of naive DO11 CD28?/?or DO11
CD28?/?CD4 T cells, GRAIL expression was markedly dimin-
ished in the activated DO11 CD28?/?cells, while, in comparison,
GRAIL expression was maintained in CD4 T cells from the DO11
CD28?/?mice (Fig. 3D). This follows other reports implicating
CD28 costimulation during T cell activation as a necessary com-
ponent in triggering the loss of inhibitory E3 ubiquitin ligases (12,
13), in this case leading to GRAIL degradation.
IL-2R signaling down-regulates GRAIL, allowing CD4 T cell
An important function of TCR/CD3 engagement is up-regula-
tion of the IL-2R?-chain (CD25) to form the high-affinity het-
erotrimeric IL-2R (28). CD28 costimulation triggers CD4 T cell
production of the growth-promoting cytokine IL-2 (29, 30).
Thus, following full activation of CD4 T cells, in an autocrine
or paracrine fashion, IL-2 engages the high-affinity IL-2R and
uses STAT5 and Akt signaling to drive CD4 T cell proliferation
and differentiation (31–33). As the absence of CD28 costimu-
lation led to diminished IL-2 production and IL-2R signaling,
we investigated the role of IL-2 in modulating GRAIL expres-
sion. Blocking IL-2R engagement during mouse naive CD4 T
cell activation, using a neutralizing anti-IL-2 Ab, inhibited CD4
T cell proliferation (Fig. 4A) and blocked phosphorylation of
STAT5 and Akt (Fig. 4B). As Akt phosphorylation was defi-
cient in the absence of IL-2R signaling, we also observed di-
minished mTOR activity assessed by decreased phosphoryla-
tion of S6K1 and 4E-BP1 (Fig. 4C). This suggested as one
possibility that inhibition of IL-2R signaling might result in
maintenance of GRAIL expression that would inhibit CD4 T
cell proliferation. In support of this possibility, GRAIL expres-
sion was maintained following naive CD4 T cell activation in
the presence of anti-IL-2 (Fig. 4D). These data suggest that
during naive CD4 T cell activation, IL-2 production and IL-2R
engagement are necessary for GRAIL degradation.
mTOR inhibition prevents Otub1 protein expression and
maintains GRAIL, resulting in diminished cell proliferation
mTOR is a signal transduction kinase whose phosphorylation and
subsequent kinase activity promote both overall protein translation
and augment specific protein translation of a subset of mRNA (34).
mTOR phosphorylation depends on the input of growth factor sig-
nals received by the cell, and mTOR kinase activity can be mon-
itored by phosphorylation of S6K1 and 4E-BP1 (35). When T cells
are productively stimulated, mTOR is activated through phosphor-
ylation via a pathway involving phosphorylated Akt (36). Acti-
vated mTOR phosphorylates its targets S6K1 and 4E-BP1 (35).
mTOR activated through phosphorylation is involved in T cell
activation (37–40) and trafficking (41). Because CD28 costimula-
tion drives production of IL-2, and IL-2R engagement and signal-
ing are both important growth signals for CD4 T cells and activate
Akt and mTOR, we directly assessed the involvement of mTOR by
using the small molecule mTOR inhibitor, rapamycin. As ex-
pected, treatment with rapamycin during activation of mouse naive
tion. A, Proliferation assay of APC and peptide OVA (pOVA) stimulated
DO11 CD4 T cells isolated from naive DO11 CD28?/?(WT DO11) or
DO11 CD28?/?(CD28- DO11) mice. Error bars indicate SD of triplicates.
Measured as counts per minute. Data are representative of three experi-
ments with similar results. B, IL-2 ELISA of supernatants collected from
DO11 CD4 T cells as in A after 24 h of stimulation (APC/pOVA), mea-
sured as ng/ml (n.d., not detected). Error bars indicate SD of triplicates. C,
Phospho-STAT5 (Tyr694/699) and total STAT5 immunoblots of re-sorted
DO11 CD4 T cells as in A after 48 h of stimulation (APC/pOVA). Data are
representative of two experiments with similar results. D, GRAIL and
?-actin immunoblots of re-sorted DO11 CD4 T cells as in A after 48 h of
stimulation (APC/pOVA). Numbers below blots indicate relative densi-
tometry levels for GRAIL. Data are representative of two experiments with
CD28 costimulation is necessary for GRAIL down-regula-
assay of CD4 T cells isolated from naive BALB/c mice with (?) or without
(?) bead stimulation (anti-CD3/28), including anti-IL-2 Ab (?IL-2) con-
dition. Error bars indicate SD of triplicates. Measured as counts per minute.
B, Phospho-STAT5 (Tyr694/699), total STAT5, phospho-Akt (Ser473), and
total Akt immunoblots of CD4 T cells as in A after 48 h of bead stimula-
tion. Data are representative of three experiments with similar results. C,
Phospho-S6K1 (Thr421/Ser424), total S6K1, phospho-4E-BP1 (Thr37/46),
and total 4E-BP1 immunoblots of CD4 T cells as in A after 48 h of bead
stimulation. On total 4E-BP1 immunoblot, top arrow indicates hyperphos-
phorylated form and bottom arrow indicates hypophosphorylated form.
Data are representative of three experiments with similar results. D,
GRAIL and ?-actin immunoblots of CD4 T cells as in A, ex vivo (0) or
after 48 h of bead stimulation. Numbers below blots indicate relative den-
sitometry levels for GRAIL. Data are representative of three experiments
with similar results.
IL-2R signaling down-regulates GRAIL. A, Proliferation
5922 mTOR REGULATION OF GRAIL EXPRESSION CONTROLS PROLIFERATION
CD4 T cells resulted in the inhibition of mTOR activity as dem-
onstrated by lack of phosphorylation of S6K1 and 4E-BP1
(Fig. 5A). As inhibition of IL-2R signaling during activation
coincided with diminished mTOR activity and maintenance of
GRAIL expression, we reasoned that rapamycin blockade of
mTOR activity might lead to a decrease in Otub1 expression,
accounting for the continued presence of GRAIL. When assess-
ing Otub1 mRNA levels during mouse naive CD4 T cell acti-
vation in the presence or absence of mTOR inhibition, however,
there was no demonstrable correlation between Otub1 mRNA
levels and GRAIL expression. Activation of the T cells in-
creased the level of Otub1 mRNA with or without the addition
of rapamycin (Fig. 5B, left panel), whereas GRAIL mRNA lev-
els were decreased regardless of the absence or presence of
rapamycin (Fig. 5B, right panel). However, GRAIL protein was
shown to be absent when mTOR was active and present when
mTOR was inactive, and direct inhibition of mTOR activity by
rapamycin inhibited proliferation (Fig. 5C) and cell division
(supplemental Fig. 2).4Expression of cyclin D3, a pro-cell cy-
cle molecule (42), and p27/Kip1, an anti-cell cycle molecule
(43), were diminished and increased, respectively, with rapa-
mycin treatment (Fig. 5D). These discordant results were re-
solved by investigating Otub1 protein expression levels follow-
ing rapamycin treatment. We had previously demonstrated that
human Otub1 protein leads to degradation of human GRAIL
protein (21), and in a similar manner, murine Otub1 protein
leads to murine GRAIL protein degradation (supplemental Fig.
3).4Ex vivo-isolated naive CD4 T cells express no Otub1 pro-
tein, thus allowing GRAIL expression (Fig. 5E, first lane), de-
spite the presence of detectable Otub1 mRNA as mTOR-medi-
ated protein translation is inactive in these cells. Upon
stimulation, mTOR is activated and Otub1 protein is expressed,
leading to the degradation of GRAIL (Fig. 5E, second lane).
When mTOR is inhibited by rapamycin treatment during stim-
ulation, although Otub1 mRNA is up-regulated, Otub1 protein
is not expressed, allowing GRAIL to be maintained (Fig. 5E,
third lane). Although regulation of GRAIL mRNA levels may
be involved in GRAIL protein down-regulation upon stimula-
tion, protein expression is dominantly influenced by regulatory
factors at the protein level. These findings show that mTOR
activation is required for naive CD4 T cell proliferation by per-
mitting Otub1 protein expression and GRAIL degradation. Con-
sistent with the mTOR function of promoting mRNA translation
through activation of its downstream targets S6K1 and 4E-BP1,
these results support our hypothesis that regulation of GRAIL
expression by mTOR is at the Otub1 protein expression level.
immunoblots of CD4 T cells isolated from naive BALB/c mice ex vivo (0), or after 48 h of bead stimulation (anti-CD3/28), including rapamycin (RAPA). On
total 4E-BP1 immunoblot, top arrow indicates hyperphosphorylated form and bottom arrow indicates hypophosphorylated form. Data are representative of more
than three experiments with similar results. B, Otub-1 (left panel) or GRAIL (right panel) expression levels of CD4 T cells as in A, ex vivo (0) or after 24 h bead
stimulation (anti-CD3/28), including RAPA. Otub-1 or GRAIL expression levels were normalized to ?-actin expression levels. Error bars indicate SD of triplicates.
Data are representative of two experiments with similar results. C, Proliferation assay of CD4 T cells as in A, without (?) or with (?) bead stimulation
(anti-CD3/28), including RAPA. Error bars indicate SD of triplicates. Measured as counts per minute. Data are representative of three experiments with similar
results. D, Cyclin D3, Kip1/p27, and ?-actin immunoblots of CD4 T cells as in A after 48 h of bead stimulation (anti-CD3/28), including RAPA. Data are
representative of three experiments with similar results. E, GRAIL, Otub1, and ?-actin immunoblots of CD4 T cells as in A, ex vivo (0), or after 48 h of bead
stimulation (anti-CD3/28), with RAPA. Numbers below blots indicate relative densitometry levels for GRAIL or Otub1. Data are representative of three exper-
iments with similar results.
mTOR inhibition maintains GRAIL expression. A, Phospho-S6K1 (Thr421/Ser424), total S6K1, phospho-4E-BP1 (Thr37/46), and total 4E-BP1
5923 The Journal of Immunology
mTOR is the downstream critical component of IL-2R signaling
regulating Otub1 and GRAIL
Inhibition of mTOR activity is sufficient to block Otub1 protein
expression and maintain GRAIL, resulting in diminished cell pro-
liferation. However, direct inhibition of mTOR activity by rapa-
mycin may have been indirectly due to diminished IL-2 production
and IL-2R signaling. As evidence of this possibility, we observed
decreased IL-2 mRNA levels (Fig. 6A) and IL-2 production (Fig.
6B) by naive CD4 T cells stimulated in the presence of rapamycin.
This diminution of IL-2 may have had a quantitative effect on
IL-2R signaling or may have arisen through a delay in IL-2 pro-
duction during an early critical phase, subsequently affecting
GRAIL expression and proliferation. The activation-induced com-
ponent of the high-affinity IL-2R, CD25 (IL-2R?), was increased
to similar levels when stimulated in the presence or absence of
rapamycin (Fig. 6C). This indiscriminate up-regulation of CD25
enables potentially equivalent IL-2R signaling; however, dimin-
ished IL-2 production in the presence of rapamycin may still have
accounted for the observed differences. We reasoned that addition
of exogenous IL-2 at the start of activation could compensate for
either diminished or delayed IL-2 production in the presence of
rapamycin; however, addition of exogenous IL-2 did not overcome
the rapamycin induced inhibition of cell proliferation (Fig. 6D).
Following exogenous IL-2 addition, rapamycin did not inhibit
phosphorylation of STAT5 or Akt but specifically inhibited mTOR
activity as demonstrated by decreased phosphorylation of S6K1
and 4E-BP1, reduced Otub1 protein, and maintenance of GRAIL
(Fig. 6E). The intact phosphorylation of Akt was seen at both
Ser473and Thr308(data not shown), suggesting the absence of any
secondary effects by rapamycin inhibition of mTOR on the ability
of the T cells to activate Akt. Thus, the critical component of
IL-2R signaling regulating Otub1 and GRAIL, and their subse-
quent effects on proliferation, appears to be mTOR. Inhibition of
mTOR, even in the presence of phosphorylated STAT5 and Akt,
blocked Otub1 protein expression and maintained GRAIL expres-
sion, resulting in the inhibition of cell proliferation.
Human naive CD4 T cells require CD28 co-stimulation and
IL-2R signaling during stimulation to down-regulate GRAIL
GRAIL and Otub1 interactions were originally identified in a yeast
two-hybrid screen using a human genomic library (21). Recent
studies have demonstrated that GRAIL expression is associated
quires mTOR activation to regulate
GRAIL. A, IL-2 expression levels of
CD4 T cells isolated from naive
ex vivo (0), or after 24 h bead stimula-
tion (anti-CD3/28), including Rapamy-
cin (RAPA). IL-2 expression levels
were normalized to ?-actin expression
levels. Error bars indicate SD of tripli-
periments with similar results. B, IL-2
ELISA of supernatants collected from
CD4 T cells as in A, unstimulated (?)
or after 24 h of bead stimulation, in-
cluding RAPA, measured as ng/ml. Er-
ror bars indicate SD of triplicates. C,
CD25 cell surface staining by flow cy-
tometry of CD4 T cells as in A, un-
stimulated (?), or after 24 h of bead
stimulation, including RAPA and IL-2.
Numbers indicate percent CD25 posi-
tive. D, Proliferation assay of CD4 T
cells as in A, without (?) or with (?)
bead stimulation (anti-CD3/28), includ-
ing RAPA. Error bars indicate SD of
triplicates. Measured as counts per
minute. Data are representative of three
experiments with similar results. E,
STAT5, phospho-Akt (Ser473), total
Akt, phospho-S6K1 (Thr421/Ser424), to-
tal S6K1, phospho-4E-BP1 (Thr37/46),
total 4E-BP1, GRAIL, Otub1, and
?-actin immunoblots of CD4 T cells as
in A, after 48 of bead stimulation (anti-
CD3/28), including RAPA and IL-2.
On total 4E-BP1 immunoblot, top ar-
form and bottom arrow indicates hypo-
phosphorylated form. Numbers below
blots indicate relative densitometry lev-
els for GRAIL or Otub1.
5924 mTOR REGULATION OF GRAIL EXPRESSION CONTROLS PROLIFERATION
with anergy and inhibition of proliferation during human CD4 T
cell activation (44). Thus, we asked whether human GRAIL and
Otub1 regulation used the same pathway as that described in
mouse naive CD4 T cells by examining activation of human naive
CD4?CD45RA?T cells (supplemental Fig. 4).4We initially dem-
onstrated that GRAIL was expressed in human naive CD4?
CD45RA?T cells isolated ex vivo and, following stimulation us-
ing plate-bound anti-CD3 and APC (added to supply B7 for CD28
costimulation; supplemental Fig. 5i),4GRAIL expression was lost,
and Otub1 protein expression was observed (Fig. 7A). The prolif-
eration of human naive CD4?CD45RA? T cells, activated in this
manner, was inhibited when CTLA4-Ig was included in the cul-
ture, but proliferation was restored if agonist anti-CD28 Ab was
added to the CTLA4-Ig containing cultures (Fig. 7B and supple-
mental Fig. 5ii).4CTLA4-Ig inhibition of CD28 costimulation re-
sulted in diminished cyclin D3 and increased Kip1/p27, which was
also reversed by the addition of agonist anti-CD28 Ab (Fig. 7C).
When CD28 costimulation was blocked by CTLA4-Ig treatment,
IL-2R signaling was impaired as seen by a decrease in phosphor-
ylation of STAT5 and reversed by the addition of agonist anti-
CD28 Ab (Fig. 7D). Addition of exogenous IL-2 to CTLA4-Ig
treatment reversed the inhibition of proliferation (supplemental
Fig. 6),4suggesting that the lack of CD28 costimulation did not
inhibit IL-2R up-regulation but that IL-2 production was impaired.
The inhibition of CD28 costimulation by CTLA4-Ig treatment
blocked Otub1 expression and sustained GRAIL expression, and
this effect could be reversed by the addition of agonist anti-CD28
Ab (Fig. 7E). These findings in human naive CD4 T cells mirror
our findings in mouse naive CD4 T cells. We conclude that CD28
CD28 costimulation. A, GRAIL, Otub1, and ?-actin immunoblots of hu-
man naive CD4?CD45RA?ex vivo (0), or after 48 h of bead stimulation
(anti-CD3/28). Data are representative of three experiments from different
donors with similar results. B, Proliferation assay of CD4 T cells as in A,
without (?) or with (?) stimulation (anti-CD3/APC), including CTLA4-Ig
and anti-CD28. Error bars indicate SD of triplicates. Measured as counts
per minute. Data are representative of two experiments from different do-
nors with similar results. C, Cyclin D3, Kip1/p27, and ?-actin immuno-
blots of CD4 T cells as in A, after 48 h of stimulation (anti-CD3/APC),
including CTLA4-Ig and anti-CD28 (?28). Data are representative of two
experiments from different donors with similar results. D, Phospho-STAT5
(Tyr694/699) and total STAT5 immunoblots of CD4 T cells as in A, after
48 h of stimulation (anti-CD3/APC), including CTLA4-Ig and anti-CD28
(?28). Data are representative of two experiments from different donors
with similar results. E, GRAIL, Otub1, and ?-actin immunoblots of CD4 T
cells as in A, after 48 h of stimulation (anti-CD3/APC), including
CTLA4-Ig and anti-CD28 (?28). Data are representative of two experi-
ments from different donors with similar results.
Human naive CD4 T cells down-regulate GRAIL through
cells to down-regulate GRAIL. A, Phospho-Akt (Ser473) and total Akt im-
munoblots of human naive CD4?CD45RA?T cells after 48 h of stimu-
lation (anti-CD3/APC), including CTLA4-Ig and anti-CD28 (?28). Data
are representative of two experiments from different donors with similar
results. B, Phospho-S6K1 (Thr421/Ser424), total S6K1, phospho-4E-BP1
(Thr37/46), total 4E-BP1 immunoblots of CD4 T cells as in A, after 48 h of
stimulation (anti-CD3/APC), including CTLA4-Ig and anti-CD28 (?28).
On total 4E-BP1 immunoblot, top arrow indicates hyperphosphorylated
form and bottom arrow indicates hypophosphorylated form. Data are rep-
resentative of two experiments from different donors with similar results.
C, Phospho-STAT5 (Tyr694/699), total STAT5, phospho-Akt (Ser473), total
Akt, phospho-S6K1 (Thr421/Ser424), total S6K1, phospho-4E-BP1 (Thr37/46),
and total 4E-BP1 immunoblots of CD4 T cells as in A, after 48 h of bead
stimulation (anti-CD3/28), including rapamycin (RAPA). On total 4E-BP1
immunoblot, top arrow indicates hyperphosphorylated form and bottom
arrow indicates hypophosphorylated form. Data are representative of three
experiments from different donors with similar results. D, Proliferation
assay of CD4 T cells as in A, without (?) or with (?) bead stimulation
(anti-CD3/28), RAPA. Error bars indicate SD of triplicates. Measured as
counts per minute. Data are representative of three experiments from dif-
ferent donors with similar results. E, Cyclin D3, Kip1/p27, and ?-actin
immunoblots of CD4 T cells as in A, after 48 h of stimulation (anti-CD3/
28), including RAPA. Data are representative of three experiments from
different donors with similar results. F, GRAIL, Otu1, and ?-actin immu-
noblots of CD4 T cells as in A, after 48 h of stimulation (anti-CD3/28),
including RAPA. Data are representative of three experiments from dif-
ferent donors with similar results.
Activation of mTOR is required for human naive CD4 T
5925 The Journal of Immunology
costimulation, and resultant IL-2 production and IL-2R signaling,
are important events regulating Otub1 and GRAIL expression and
proliferation in human as well as mouse naive CD4 T cells.
Human naive CD4 T cells require mTOR activation to allow
Otub1 protein expression and GRAIL degradation
Since human naive CD4 T cells require CD28 costimulation and
IL-2R signaling to modulate GRAIL expression, we reasoned that
the mTOR pathway might also control human Otub1 and GRAIL
expression. CD28 costimulation blockade of human naive CD4 T
cell activation resulted in decreased phosphorylation of Akt, an
upstream component within the mTOR activation pathway (Fig.
8A). In examining mTOR activity, phosphorylation of S6K1 and
4E-BP1 were both down-regulated in the presence of CTLA4-Ig
and restored with direct agonist anti-CD28 Ab costimulation (Fig.
8B). Treatment of human naive CD4 T cells with rapamycin did
not affect the phosphorylation of STAT5 or Akt but resulted in the
inhibition of mTOR activity as measured by decreased phosphor-
ylation of S6K1 and 4E-BP1 (Fig. 8C), similar to results seen in
mouse CD4 T cells. Rapamycin treatment inhibited human naive
CD4 T cell proliferation (Fig. 8D) and resulted in decreased Cyclin
D3 and increased Kip1/p27 levels (Fig. 8E). Human naive CD4 T
cells activated in the presence of rapamycin failed to up-regulate
Otub1 protein and maintained GRAIL expression (Fig. 8F). These
results are identical to the effects seen in mouse naive CD4 T cells.
GRAIL is expressed in human and mouse naive CD4 T cells, and
its degradation following TCR/CD3 engagement and costimula-
tion is required for proliferation. These data demonstrate a role for
GRAIL in controlling naive CD4 T cell activation and prolifera-
tion in addition to GRAIL’s role in the induction and maintenance
of anergy. As demonstrated in these studies, not only TCR/CD3
engagement and CD28 costimulation are required for full activa-
tion, IL-2 production and IL-2R signaling are also necessary to
allow proliferation. Phosphorylation of Akt following IL-2R en-
gagement drives mTOR activation leading to Otub1 protein ex-
pression, degradation of GRAIL, and T cell proliferation. These
data demonstrate a pathway of GRAIL regulation that links critical
components of CD4 T cell stimulation to CD4 T cell proliferation.
Interference in this pathway highlights the potential importance of
this pathway in peripheral T cell tolerance and may suggest new
targets for immunotherapeutics (Fig. 9).
Our results link TCR/CD3 engagement and CD28 costimulation
with IL-2 production and IL-2R signaling to activation of mTOR
kinase that is required for activation induced proliferation of hu-
man and mouse naive CD4 T cells. Our studies highlight the im-
portance of IL-2R signaling in sustaining mTOR activation during
naive CD4 T cell activation. However, we also found that at early
times (10 min to 1 h) following activation by CD3 and CD28
signaling, Akt is phosphorylated even in the presence of anti-IL-2
Abs, resulting in mTOR activation independent of IL-2R sig-
naling (our unpublished observations) in agreement with pre-
vious reports (36, 40). This discrepancy is resolved by differ-
entiating the IL-2R signaling requirement at different time
points following naive CD4 T cell activation. At later times
(24–72 h) following activation, IL-2R signaling was required
for sustained mTOR activity as anti-IL-2 Abs blocked phos-
phorylation of Akt and mTOR activation at these later time
points (Fig. 4, B and C), resulting in the sustained presence of
GRAIL (Fig. 4D) and decreased proliferation (Fig. 4A).
Previous studies have implicated S6K1 regulation by mTOR in
CD4 T cell activation (39, 45), identifying a role for this pathway
in directing mTOR-dependent protein translation. In this study, we
cells. CTLA4-Ig, anti-IL-2, and Rapamycin regulation of Otub1 and GRAIL expression controls naive CD4 T cell proliferation. A, Productive activation of naive CD4 T
cells leading to proliferation comes about through TCR engagement and CD28 costimulation, IL-2 production, signaling through the IL-2R leading to phosphorylation of
does not allow IL-2 production, thus prevents Akt phosphorylation, mTOR is inactive, and Otub1 protein is absent, leading to the maintenance of GRAIL, inhibiting
proliferation. C, Anti-IL-2 blocks IL-2R engagement, thus preventing Akt phosphorylation, mTOR is inactive, and Otub1 protein is absent, leading to the maintenance of
GRAIL, inhibiting proliferation. D, Rapamycin blocks the activity of mTOR, prevents protein expression of Otubain-1, leading to the maintenance of GRAIL, inhibiting
The CD28 costimulation, IL-2 signaling, and mTOR pathway regulate Otub1 and GRAIL expression, controlling proliferation in primary naive CD4 T
5926 mTOR REGULATION OF GRAIL EXPRESSION CONTROLS PROLIFERATION
demonstrate that naive CD4 T cells also regulate 4E-BP1 through
the mTOR-dependent pathway via phosphorylation on Thr37/46.
Phosphorylation of 4E-BP1 leads to its dissociation from eIF4E,
allowing active eIF4E to bind eIF4G during translation initiation
complex formation (46). A functional consequence attributed to
active eIF4E is preferential translation of specific mRNAs nor-
mally translated into protein at low or absent rates (47, 48). The
phosphorylation of 4E-BP1, and subsequent activation of eIF4E,
may allow protein translation of a subset of mRNAs important for
T cell activation. We propose that Otub1 mRNA is under such
regulation as its protein expression does not appear to be mediated
through changes in mRNA transcript levels yet is sensitive to
mTOR inhibition. The therapeutic effects of rapamycin in the in-
hibition of CD4 T cell activation and proliferation may be due not
only to decreased overall protein translation but also to prevention
of translation of a subset of mRNAs critical for successful
This study is the first demonstration that endogenous GRAIL
protein regulation in primary human and mouse naive CD4 T cells
plays an important role in controlling T cell activation and prolif-
eration. In mice, GRAIL expression can be traced to Qa-2?CD4
single-positive thymocytes poised for export to the periphery; thus,
GRAIL expression may be an important component of peripheral
tolerance in naive CD4 T cells, in addition to its role in CD4 T cell
anergy. Qa-2?CD4 single-positive thymocytes, but not earlier
stage thymocytes, respond to TCR ligation in a manner similar to
peripheral CD4 T cells (49). The observations of GRAIL expres-
sion in Qa-2?CD4 single-positive thymocytes and expression in
peripheral naive CD4 T cells suggest a possible role for GRAIL in
CD4 T cell tolerance to TCR self-peptide/MHC encountered dur-
ing the transition from the thymus to the peripheral environment.
TCR engagement of self selecting-peptide/MHC needs to remain a
nonresponsive event for the naive CD4 T cell, and yet TCR en-
gagement is necessary for maintaining their survival and keeping
them poised for potential activation by non-self (50–55). When
foreign Ag is presented as non-self-peptide in the context of MHC
class II, the increased affinity/avidity of the TCR engagement, as
well as the presence of danger-induced APC costimulatory signals
following B7-CD28 ligation, breaks the quiescent state of the na-
ive CD4 T cell that these data suggest is maintained by GRAIL.
IL-2 signals through the IL-2R on CD4 T cells via mTOR to en-
sure GRAIL degradation to allow proliferation. Thus, maintenance
of GRAIL serves to preserve quiescence of naive CD4 T cells and
its down-regulation is required to allow proliferation.
Anergic CD4 T cells express multiple E3 ubiquitin ligases, sug-
gesting possible unique roles in maintaining cellular nonrespon-
siveness (7, 9). The differential expression of these E3 ubiquitin
ligases in primary CD4 T cells during quiescence and activation
may provide insights into further elucidation of their functions in
periperhal T cell tolerance. We found that while GRAIL was
present in naive quiescent CD4 T cells and down-regulated upon
activation, by contrast, Cbl-b was expressed at low levels in naive
quiescent CD4 T cells and up-regulated upon activation (data not
shown). Another group has recently also reported on the observed
Cbl-b up-regulation upon activation in primary CD4 T cells (56).
Their findings suggest that Cbl-b acts to limit CD4 T cell prolif-
eration following TCR and CD28 activation through Cbl-b ubiq-
uitination and degradation of phospholipase C? (57) and PI3K (58,
59). Cbl-b decrease of PI3K expression diminishes downstream
phosphorylation of ERK and Akt (56). We proposed that GRAIL
and Cbl-b both serve to counteract CD4 T cell activation, however,
at different stages. GRAIL, by maintaining quiescence in the ab-
sence of CD28 costimulation, and Cbl-b, by dampening prolifer-
ation of activated cells. GRAIL and Cbl-b may be mechanistically
linked through Cbl-b down-regulation of Akt phosphorylation. A
decrease in Akt phosphorylation would decrease mTOR activation,
abrogating Otub1 protein expression and thus resulting in the re-
expression of GRAIL and inhibition of cell proliferation. In this
regard, we found that human and mouse naive CD4 T cells acti-
vated and subsequently rested eventually diminished their levels of
phosphorylated Akt, S6K1, and 4E-BP1 (data not shown). The
return of these cells to a nonproliferating quiescent state was cor-
related with the re-expression of GRAIL. Reactivation by TCR/
CD3 and CD28 stimulation again led to Otub1 protein expression
and down-regulation of GRAIL before cell proliferation (data not
shown). An investigation into this proposed interrelationship be-
tween GRAIL and Cbl-b control of CD4 T cell proliferation would
help characterize their overlapping and distinct roles.
NOD mice serve as a murine model of human type 1 diabetes
with increasing incidence of hyperglycemia with age (60). The
disease process is thought to occur initially through autoimmune T
cell activation, possibly in the pancreatic lymph node, followed by
inflammation of the islets of langerhans (insulitis) that, at ?12 wk
of age, leads to islet ?-cell destruction and resultant hyperglycemia
(61). In search of genes differentially expressed during disease
initiation and progression, we examined pancreatic lymph nodes
from NOD and disease-resistant NOD.B10 (H-2b) congenic mice.
We conducted genome-wide analyses of gene expression using
microarrays comparing NOD vs NOD.B10 pancreatic lymph node
RNA (22). At certain ages, including 12 wk, GRAIL expression
was decreased in pancreatic lymph nodes of NOD mice compared
with NOD.B10 mice (supplemental Fig. 7A).4This differential
GRAIL expression was verified by quantitative PCR of pancreatic
lymph node RNA samples from multiple 12-wk-old NOD and
NOD.B10 mice (supplemental Fig. 7B).4Our findings suggest a
potential peripheral tolerance role for GRAIL on naive CD4 T
cells in vivo, which might be lost during NOD disease pathogen-
esis. In a study of primate HIV infection, GRAIL was up-regulated
in anergic CD4 T cells isolated from disease-susceptible SIV-in-
fected rhesus macaques, whereas SIV-resistant sooty mangabey
primates showed no increase in GRAIL (62). A role for GRAIL in
human disease was recently demonstrated in patients successfully
treated for ulcerative colitis: patients in remission expressed higher
levels of GRAIL in CD4 T cells vs patients with ongoing disease
or normal controls (63) These studies and the findings reported
above suggest that regulation of GRAIL and Otub1 may play an
important role in peripheral tolerance.
We are grateful to Cariel Taylor for animal husbandry and laboratory man-
agement. Carol Figueroa provided administrative assistance.
The authors have no financial conflict of interest.
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5928 mTOR REGULATION OF GRAIL EXPRESSION CONTROLS PROLIFERATION