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).
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