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Grb2 Is Important for T Cell Development,
Th Cell Differentiation, and Induction of
Experimental Autoimmune
Encephalomyelitis
Daniel Radtke, Sonja M. Lacher, Nadine Szumilas, Lena
Sandrock, Jochen Ackermann, Lars Nitschke and Elisabeth
Zinser
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Copyright © 2016 by The American Association of
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2016; 196:2995-3005; Prepublished online 26
February 2016;
doi: 10.4049/jimmunol.1501764
http://www.jimmunol.org/content/196/7/2995
The Journal of Immunology
Grb2 Is Important for T Cell Development, Th Cell
Differentiation, and Induction of Experimental Autoimmune
Encephalomyelitis
Daniel Radtke,* Sonja M. Lacher,* Nadine Szumilas,* Lena Sandrock,†
Jochen Ackermann,* Lars Nitschke,*,1 and Elisabeth Zinser†,1
T
he small ubiquitously expressed adaptor protein, growth
factor receptor–bound protein 2 (Grb2), integrates signals
from the outside of cells to inner signaling pathways via
its central SH2 and the two flanking SH3 domains (1, 2). Grb2 was
first described to be a positive regulator of the Ras signaling
pathway downstream of growth factor receptors (1). Surprisingly,
Grb2 seems to be dispensable for Ras signaling in CD4+CD8+
double-positive (DP) thymocytes (2). However, Grb2 was also
shown to regulate Ca2 + signaling and PI3K/Akt signaling
in lymphocytes via incompletely understood mechanisms (3).
Grb2 also plays an important role in lymphocyte development in T
*Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlangen, Germany; and †Department of Immune Modulation, University Hospital Erlangen, 91052 Erlangen, Germany
1
L.N. and E.Z. contributed equally to this work.
ORCIDs: 0000-0002-5145-1157 (S.M.L.); 0000-0002-4369-0668 (E.Z.).
Received for publication August 4, 2015. Accepted for publication January 25, 2016.
This work was supported by the Deutsche Forschungsgemeinschaft (Ni 549/8-1 and
SFB643 Project B7 to L.N. and SFB643 Project B9 to E.Z.).
Address correspondence and reprint requests to Prof. Dr. Lars Nitschke or Dr. Elisabeth
Zinser, Division of Genetics, Department of Biology, University of Erlangen, Staudtstrasse 5, 91058 Erlangen, Germany (L.N.) or Department of Immune Modulation at the
Department of Dermatology, Universitätsklinikum Erlangen, Hartmannstasse 14, 91052
Erlangen, Germany (E.Z.). E-mail addresses: [email protected] (L.N.) or elisabeth.
[email protected] (E.Z.)
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; DN, double negative; DP,
double positive; EAE, experimental autoimmune encephalomyelitis; fwd, forward;
Grb2, growth factor receptor–bound protein 2; HSA, heat-stable Ag; MOG, myelin
oligodendrocyte glycoprotein; rev, reverse; SP, single positive; TCM, central memory
T cell; TEM, effector memory T cell; Treg, regulatory T cell.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501764
and B cells (2–4). The analysis of Grb2fl/fl Lckcretg T cell–specific
Grb2-deficient mice revealed that Grb2 is important for positive and
negative selection in the thymus, and it enhances tyrosine phosphorylation downstream of the TCR, including phosphorylation of
LAT, PLCg1, and CD3z chain. As a consequence of impaired selection processes, Grb2fl/fl Lckcretg mice show reduced T cell
numbers in the periphery (2). It was reported recently that Grb2
recruits THEMIS and SHP1 via its SH3 domains to phosphorylated
LAT after TCR stimulation (5, 6). Thereby, it directs them to the
immunological synapse where, in turn, the complex with THEMIS
and SHP1 sets the threshold for selection processes (6, 7). When the
Grb2-dependent recruitment of THEMIS is abrogated, T cell development is impaired; this highlights the importance of Grb2 in
thymocyte development (5). However, this model is not mutually
exclusive because a complex consisting of two Grb2 molecules and
one Sos1 molecule (2:1 complex) is able to cluster LAT proteins,
which are phosphorylated after TCR engagement. Furthermore,
LAT clustering could also be promoted by 2:1 Grb2–Cbl or 2:1
Grb2–Gab1 complexes. After phosphorylated LAT is clustered, it is
able to activate downstream signaling pathways and, thereby, contributes to T cell activation. In this model, Grb2 binds to phosphorylated tyrosine residues of LAT via its SH2 domain, whereas its
SH3 domains bind to SOS1, Cbl, or Gab1. Because LAT, SOS1,
Cbl, and Gab1 have more than one Grb2 binding site, a large
multiprotein cluster can be formed (8–10).
Despite its described function in T cell development, little is
known about the role of Grb2 in peripheral naive and effector
T cells. When naive CD4+ T cells encounter their Ag in the context
of an MHC class II molecule presented on APCs, such as dendritic
cells (DCs), they start to proliferate and differentiate into Th cells.
These cells are important mediators of adaptive immunity. There
are four widely accepted Th cell populations: Th1 cells mediate
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The small adaptor protein growth factor receptor–bound protein 2 (Grb2) modulates and integrates signals from receptors on
cellular surfaces in inner signaling pathways. In murine T cells, Grb2 is crucial for amplification of TCR signaling. T cell–specific
Grb2fl/fl Lckcretg Grb2-deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study,
we found that T cell numbers in Grb2fl/fl CD4cretg mice were normal in the thymus and were only slightly affected in the
periphery. Ex vivo analysis of CD4+ Th cell populations revealed an increased amount of Th1 cells within the CD4+ population
of Grb2fl/fl CD4cretg mice. Additionally, Grb2-deficient T cells showed a greater potential to differentiate into Th17 cells in vitro.
To test whether these changes in Th cell differentiation potential rendered Grb2fl/fl CD4cretg mice more prone to inflammatory
diseases, we used the murine Th1 cell– and Th17 cell–driven model of experimental autoimmune encephalomyelitis (EAE). In
contrast to our expectations, Grb2fl/fl CD4cretg mice developed a milder form of EAE. The impaired EAE disease can be explained
by the reduced proliferation rate of Grb2-deficient CD4+ T cells upon stimulation with IL-2 or upon activation by allogeneic
dendritic cells, because the activation of T cells by dendritic cells and the subsequent T cell proliferation are known to be crucial
factors for the induction of EAE. In summary, Grb2-deficient T cells show defects in T cell development, increased Th1 and Th17
cell differentiation capacities, and impaired proliferation after activation by dendritic cells, which likely reduce the clinical
symptoms of EAE. The Journal of Immunology, 2016, 196: 2995–3005.
2996
Materials and Methods
Mice
Grb2fl/fl mice were described previously (3) and crossed to heterozygous
Lckcretg (23) or CD4cretg (24) mice. Grb2fl/f Lckcretg mice were on a
mixed BALB/c/C57BL/6 background, whereas Grb2fl/fl CD4cretg mice
were backcrossed for 10 generations to the C57BL/6 background. Rag12/2
mice were bred in-house. Age-matched mice on the same background and,
whenever possible, littermates were used for all experiments. Animal experiments were approved by a local ethics committee (Regierung Mittelfranken).
Flow cytometry
Single-cell suspensions were incubated with 10% goat serum in PBS
containing 0.1% (w/v) BSA and 0.05% (w/v) Azid (natriumazid) for 20 min
at 4˚C to avoid nonspecific binding. Then cells were incubated with
fluorochrome-conjugated Abs (FITC, PE, PeCy5.5, PerCPCy5.5, allophycocyanin) or with biotin-conjugated Abs in PBS containing 0.1% (w/v)
BSA and 0.05% (w/v) Azid (natriumazid) for 20 min at 4˚C. Cells incubated with biotin-conjugated Abs were incubated again with fluorochromeconjugated streptavidin. For intracellular staining, with the exception of
Foxp3 staining, a BD Cytofix/Cytoperm Fixation and Permeabilization
Solution Kit with BD GolgiPlug (containing Brefeldin A) was used,
according to the manufacturer’s instructions. For Foxp3 staining, the AntiMouse/Rat Foxp3 Staining Set APC (eBioscience) was used, according to
the manufacturer’s instructions. Stained cells were analyzed using BD
FACSCalibur and FACSAria II flow cytometers and FlowJo software
(TreeStar). Lymphocytes were gated based on forward scatter and side
scatter prior to further gating of fluorescent-labeled populations. The following Abs were used: anti-CD4–PE GK1.5, anti-Gr-1–FITC RB6-8C5,
anti-CD25–FITC 7D4, anti-CD4–PerCP–RM4-5, anti-IL-17–FITC-TC1118H10 (all from BD), anti-CD24–FITC heat-stable Ag (HSA) 30-F1, antiCD62L–bio MEL-14, anti-CD69–bio H1 cF3, anti-CD8a–FITC 53-6.7,
anti-CD11b–FITC M1/70, anti-TCR-b–bio H57-597, anti-CD44–allophycocyanin IM7, anti-CD25–bio PC61.5, anti-IL-17–PE eBio17B7,
anti-IFN-g–allophycocyanin XMG1.2, anti2IL-4–allophycocyanin 11B11,
anti-Foxp3–allophycocyanin FJK-16s, and streptavidin PerCP-Cy5.5 (all from
eBioscience).
In vitro T cell differentiation
A 96-well tissue culture round-bottom plate (Falcon or BD) was incubated
with 50 ml Tris buffer (pH 9.5) with 1 mg/ml anti-CD3 Ab (145-2C11;
eBioscience) for 3 h at 37˚C, and each well was washed two times with 13
PBS with 2% FCS and 2 mM EDTA. A total of 1 3 105 CD4+CD62L+
T cells/well, in 100 ml RPMI 1640 (Life Technologies) with 10% PAN
FCS, 1.2 mM L-glutamine, 1 mM sodium pyruvate, 50 mM 2-ME, 0.1 mM
nonessential amino acids, and 10,000 U/ml penicillin/streptomycin, was
added. The CD4+CD62L+ T cells were obtained from spleen and lymph
node of naive mice using a CD4+CD62L+ T cell Isolation Kit II (Miltenyi
Biotec), according to the manufacturer’s instructions. Cells were differentiated into Th1 cells by adding 1.3 mg/ml anti-CD28 (37.51; eBioscience), 4 ng/ml IL-12, and 50 U/ml IL-2; cells were differentiated into
Th2 cells by adding 1.3 mg/ml anti-CD28, 5 mg/ml anti–IFN-g (XMG1.2;
eBioscience), 50 U/ml IL-2, and 10 ng/ml IL-4; cells were differentiated
into induced Tregs by adding 1.3 mg/ml anti-CD28, 5 mg/ml anti–IFN-g,
50 U/ml IL-2, and 3 ng/ml TGF-b; and cells were differentiated into Th17
cells by adding 1.3 mg/ml anti-CD28, 5 mg/ml anti–IFN-g, 30 ng/ml IL-6,
and 2 ng/ml TGF-b (all cytokines were from PeproTech). Cells were incubated for 72 h at 37˚C and 5% CO2 and washed two times with 13 PBS
with 2% FCS and 2 mM EDTA. Cells forced to become Th2 cells were
transferred to noncoated plates and incubated in 50 ml medium plus 50 U
IL-2/well for another 48 h at 37˚C and 5% CO2, followed by two washing
steps. After 72 h, or in case of Th2 cells, 120 h of stimulation, cells were
resuspended in 50 ml medium with 1 mg/ml ionomycin and 20 ng/ml PMA,
as well as 1 mg/ml Brefeldin A (BD GolgiPlug), and incubated for 6 h at
37˚C and 5% CO2. Differentiation efficiency of CD4+CD62L+ T cells
toward Th cell populations was analyzed by flow cytometry.
Experimental autoimmune encephalomyelitis
Mice were immunized s.c. with 50 mg MOG35–55 peptide (Charite)
emulsified in IFA, which was enriched with 10 mg/ml Mycobacterium
tuberculosis (H37Ra; Difco/BD) to induce EAE. Additionally, 200 ng
pertussis toxin (List/Quadratec) was injected i.p. at days 0 and 2 of EAE.
Scoring of EAE symptoms was performed as follows: 0, no disease; 1,
tail weakness; 2, paraparesis; 3, paraplegia; 4, paraplegia with forelimb
weakness; and 5, moribund or dead animals. For transfer-EAE experiments, spleen and lymph node cells were harvested from Grb2fl/fl mice
on day 19 after immunization. Cells were restimulated with 30 mM
MOG peptide for 72 h. After restimulation, CD4+ T cells were purified
using a CD4+ T Cell Isolation Kit II (Miltenyi Biotec), according to the
manufacturer’s instructions. Then 3 3 106 CD4+ T cells/mouse were
transferred i.v. into Rag12/2 mice, and disease progression was scored.
In vitro MOG restimulation
At day 17 or 30 after MOG injection, splenic cells from mice were harvested, and 4 3 105 total spleen cells or purified CD4+ splenic cells were
incubated with different concentrations of MOG peptide or 50 U/ml IL-2
(Proleukin) in 200 ml HL-1 (Lonza) serum-free medium supplemented
with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and
50 mM 2-ME (all additives were from Sigma-Aldrich) per well in a 96well tissue culture plate for 72 h at 37˚C and 5% CO2. Then 0.1 mCi/well
[3H]methyl-thymidine (Hartmann Analytic) was added, and cells were
incubated for an additional 12 h. Subsequently, the plates were harvested
with an IH-110 harvester (InnoTech), and filters were counted in a 1450
Microplate Counter (Wallac) to detect incorporated radioactivity as a
readout for proliferation. Supernatants from all restimulation cultures were
harvested to determine the ex vivo cytokine production.
Cytokine measurements
To determine cytokine production in supernatants of MOG-restimulated
DCs in T cell cocultures, a commercially cytometric bead array (BD)
was used to detect IL-2, IL-17A, IFN-g, IL-6, and TNF-a, according to the
manufacturer’s instructions.
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responses against intracellular pathogens, Th2 cells promote responses against extracellular parasites, and Th17 cells are associated with autoimmunity and responses against extracellular
bacteria, as well as fungi. Finally, induced regulatory T cells
(Tregs) can be induced to inhibit immune responses (11, 12).
It is known that the quality and quantity of signaling, especially
by the TCR, which is influenced by Grb2 (2), are critical for the
development and maintenance of peripheral T cell subsets (12–
16). In general, weak signals by the TCR favor Th2 cell responses,
whereas strong signals promote Th1 cell responses (14). Th1 cell
responses are promoted by JNK2 and P38 activation in combination with JNK1 activation that inhibits Th2 responses (17, 18).
JNK and P38 phosphorylation, as well as general tyrosine phosphorylation downstream of the TCR, are reduced in Grb2fl/fl
Lckcretg mice, suggesting a role for Grb2 in Th cell equilibrium.
Th cell effector functions are often studied in animal models for
inflammatory diseases. Experimental autoimmune encephalomyelitis (EAE) is an animal model for the early inflammatory phase of
human multiple sclerosis, but it is also useful to deepen the understanding of Th cell functions in a disease situation in vivo (19,
20). To induce EAE, an immune reaction is initiated against a part
of the myelin that surrounds nerve cells. This leads to inflammation in the CNS. Freund’s adjuvant is used to boost the immune
response against myelin oligodendrocyte glycoprotein (MOG); by
using this immunization protocol, the disease is primarily driven
by Th1 and Th17 cells (21, 22).
In this study, we investigated the role of Grb2 on Th cell
function and lineage commitment ex vivo, in vitro, and in the EAE
model. We found that Grb2-deficient T cells had an increased
potential to differentiate into Th17 cells in vitro and that naive
T cell–specific Grb2-deficient mice had a higher proportion of Th1
cells within the CD4+ T cell population when analyzed ex vivo. To
analyze the relevance of these findings in vivo, we used the murine
EAE model. Despite the shift in Grb2-deficient T cells toward
higher numbers of Th17 cells in vitro and to Th1 cells in an ex
vivo analysis, mice with Grb2-deficient T cells developed a milder
form of EAE compared with control mice. The cellular mechanism responsible for this impaired disease progression in the EAE
model was examined in this study.
Grb2 T CELL FUNCTIONS
The Journal of Immunology
2997
Histology
Statistical analysis
Brains and spinal cords of mice were harvested at day 17 of EAE, fixed in
liquid nitrogen, and stored at 280˚C. Organs were embedded in TissueTek (Sakura), and 5-mm sections were made using a cryotome (Kryocut
CM 2000; Leica). Immunohistological staining was performed using an
immunoperoxidase detection system in a humid incubation chamber.
Acetone-fixed sections were incubated in PBS, and endogenous peroxidase activity was blocked by incubating sections in 3% H2O2.
Sections were incubated with a primary anti-CD45 Ab (clone 30G12;
provided by L. Sorokin, Lund University, Lund, Sweden). The primary
Ab was detected by a streptavidin HRP–coupled goat ant-rat IgG Ab
(Biocare Medical). To visualize Ags, sections were incubated with
AEC Chromogen Substrate (Vector Laboratories). Sections were
stained with hematoxylin (Sigma-Aldrich), mounted in Aquatex
(Merck), covered with a coverslip, and examined by light microscopy
(Leica).
Results are expressed as mean and SD, unless stated otherwise in the figure
legends. A two-tailed Student t test (for two groups) and two-way ANOVA
with the Bonferroni posttest (for multiple groups) were used to determine
statistical significance, unless stated otherwise in the figure legends. A
nonparametric Mann–Whitney U test was used to analyze clinical EAE
scoring. Differences with p values , 0.05 were considered statistically
significant. Statistic analyses were performed with Excel or Prism 4.03 or
5.0 (GraphPad, La Jolla, CA).
RNA isolation and quantitative real-time PCR analysis
Allogenic T cell proliferation
BM-derived DCs were generated from Grb2fl/fl, Grb2fl/flCD4cretg, or
BALB/c mice, as described elsewhere (25). At day 9, bone marrow–DC
cultures were matured with TNF-a or LPS overnight or were left unstimulated. A total of 3 3 106 allogeneic CD4+ T cells, purified from Grb2fl/fl
or Grb2fl/fl CD4cretg mice using a CD4+ T Cell Isolation Kit II (Miltenyi
Biotec), were incubated with the indicated numbers of differentially
stimulated DCs in a 96-well flat-bottom plate (Falcon) for 72 h at 37˚C and
5% CO2. Additionally, the indicated numbers of DCs from Grb2fl/fl and
Grb2fl/fl CD4cretg mice were incubated with 3 3 106 allogeneic total
spleen cells from BALB/C mice in a 96-well flat-bottom plate (Falcon) for
72 h at 37˚C and 5% CO2. Then, 1 mCi/well [3H]methyl-thymidine
(Amersham Biosciences) was added, and cells were incubated for an additional 16 h. The plates were harvested with an IH-110 harvester (InnoTech), and filters were counted in a 1450 Microplate Counter (Wallac) to
detect incorporated radioactivity as a readout for proliferation.
Stimulation of CD4+CD62L+ T cells and measurement of
proliferation
CD4+CD62L+ T cells were obtained from spleen and lymph node of naive
mice using a CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec),
according to the manufacturer’s instructions. A total of 3 3 106 cells was
seeded and stimulated with 5 mg/ml anti-CD3 (eBioscience), 1 ml/200 ml
anti-CD3/anti-CD28 Dynabeads (Dynabeads Mouse T-Activator; Life
Technologies), or 50 U/ml IL-2 for 72 h. Then 1 mCi/well [3H]methylthymidine (Amersham Biosciences) was added, and cells were incubated
for an additional 16 h. The plates were harvested with an IH-110 harvester
(InnoTech), and filters were counted in a 1450 Microplate Counter (Wallac) to detect incorporated radioactivity as a readout for proliferation.
Mild reduction in peripheral T cells in Grb2fl/fl CD4cretg
T cell–specific Grb2-deficient mice
To analyze the role of Grb2 at different stages of T cell development, we compared Grb2fl/fl Lckcretg mice that delete Grb2 at
the transition from the double-negative (DN)2 stage to the DN3
stage in early T cell development (23, 26) with Grb2fl/fl CD4cretg
mice that delete Grb2 at the CD4+CD8+ DP stage (24). Successful
deletion of Grb2 in T cells was confirmed by Western blot
(Supplemental Fig. 1A–D). In Grb2fl/fl Lckcretg mice, the cell
numbers in the DN1 and DN2 stages were normal. In contrast, the
number of cells in the DN3 stage was increased by 29%, and the
number of cells in the DN4 stage was decreased by 28%, which
might point to a minor block in T cell development (Supplemental
Fig. 1E). As also reported by another group (2), the number of
CD4+CD8+ double positive (DP) cells in the thymus was slightly
reduced in Grb2fl/fl Lckcretg mice, but the difference in cell
numbers was not significant (Supplemental Fig. 2A). Thymic
CD4+ and CD8+ single-positive (SP) cells were drastically reduced by 66% in Grb2fl/fl Lckcretg mice (Supplemental Fig. 2A).
This is due to defects in positive and negative selection (2). In
accordance with these results, the reduction in T cells in the
thymus of Grb2fl/fl Lckcretg mice is seen in TCRhigh/HSAhigh cells,
which are reduced by 32%, and in more mature TCRhigh/HSAlow
cells, which are reduced by 49% (Supplemental Fig. 2B).
However, no alteration in DN cell numbers or CD4+CD8+ DP or
SP cell numbers was detected in the thymus of Grb2fl/fl CD4cretg
mice (Fig. 1A, Supplemental Fig. 1E). Only the proportion of
TCRhigh/HSAlow cells within the thymocyte population was reduced (by 49%) (Fig. 1B). This indicates that selection processes
in Grb2fl/fl CD4cretg mice were much less affected than in Grb2fl/fl
Lckcretg mice. Peripheral CD4+ and CD8+ SP cells were drastically reduced in Grb2fl/fl Lckcretg mice: by 75–77% in the spleen
and by 94–96% in lymph nodes, in accordance with existing data
(Supplemental Fig. 2C, 2D) (2). In contrast, peripheral CD4+ and
CD8+ SP cell numbers in Grb2fl/fl CD4cretg mice were reduced
only slightly: by 32 or 39%, respectively, in the spleen and by 36
and 39%, respectively, in the lymph nodes (Fig. 1C, 1D).
Increased number of effector T cells within peripheral CD4+
T cells
The number of effector memory T cells (TEMs) within the CD4+
T cell population of the spleen was increased by 45% in Grb2fl/fl
Lckcretg and Grb2fl/fl CD4cretg mice, whereas naive cells were
reduced by 52% in Grb2fl/fl Lckcretg mice and by 37% in CD4cretg
mice (Fig. 1E, Supplemental Fig. 2E). The same was true for the
CD4+ T cells of the lymph node in Grb2fl/fl Lckcretg mice: TEMs
were increased by 49%, and naive T cells were reduced by 33.3%
(Supplemental Fig. 2F), whereas there were no significant changes
in these T cell populations in the lymph nodes of Grb2fl/fl
CD4cretg mice (Fig. 1F). The preferred differentiation to effector
T cells in T cell–specific Grb2-deficient mice is accompanied by a
higher percentage of CD69-expressing CD4+ T cells in the spleen
but not in the lymph node (data not shown). Similarly to CD4+
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Splenic CD4+ T cells from Grb2fl/fl or Grb2fl/fl CD4cretg mice were isolated using the CD4+ T Cell Isolation Kit (Miltenyi Biotec), according to
the manufacturer’s instructions. Cells were stimulated with 1 mg/ml
ionomycin and 20 ng/ml PMA, as well as 1 mg/ml Brefeldin A (BD
GolgiPlug), and incubated for 6 h at 37˚C and 5% CO2. Thereafter, RNA
was isolated using RNeasy Plus Mini Kit (QIAGEN) reagent, according
to the manufacturer’s instructions. RNA from spinal cord tissue was removed and stored in RNAlater at 280˚C for analysis. RNA was isolated
using RNeasy Plus Mini Kit reagent, according to the manufacturer’s
instructions. cDNA was synthesized with a reverse transcription kit
(Fermentas). For amplification, a SYBR Green mix for qPCR was used
(Bio-Rad). Gene expression was analyzed with a CFX Real-time detection system (Bio-Rad). The relative gene expression levels were calculated with the comparative threshold cycle method, and data are
expressed as 22ΔΔCT. Expression was normalized to HPRT. The following
primer sequences were used: Hprt forward (fwd): 59-GTT GGA TAC AGG
CCA GAC TTT GTT G-39; reverse (rev): 59-GAT TCA ACT TGC GCT CAT
CTT AGG C-39; Il17a fwd: 59-TCC AGA AGG CCC TCA GAC TA-39; rev:
59-AGC ATC TTC TCG ACC CTG AA-39; Ifng fwd: 59-GCT TTG CAG CTC
TTC CTC AT-39, rev: 59-GTC ACC ATC CTT TTG CCA GT-39; Tbet fwd: 59AGC AAG GAC GGC GAA TGT T-39; rev: 59-GGG TGG ACA TAT AAG
CGG TTC-39; Foxp3 fwd: 59-CCC AGG AAA GAC AGC AAC CTT-39; rev:
59-CCT TGC CTT TCT CAT CCA GGA-39; GATA3 fwd: 59-GTC ATC CCT
GAG CCA CAT CT-39; rev: 59-TAG AAG GGG TCG GAG GAA CT- 39;
Rorgt fwd: 59-GTG TGC TGT CCT GGG CTA CC-39; rev: 59-AGC CTT GCA
CCC CTC ACA G-39; and Il4 fwd: 59-GGT CTC AAC CCC CAG CTA GT-39;
rev: 59-GCC GAT GAT CTC TCT CAA GTG AT-39.
Results
2998
Grb2 T CELL FUNCTIONS
T cells, within the CD8+ T cell population of Grb2fl/fl Lckcretg
mice and Grb2fl/fl CD4cretg mice we found a reduction in naive
T cells by 40 and 30%, respectively, and a relative increase in
TEMs by 111 and 180%, respectively. The percentage of CD62L+
CD44+ central memory T cells (TCMs) to CD8+ T cells was increased by 84% in Grb2fl/fl Lckcretg mice and by 55% in Grb2fl/fl
CD4cretg mice (Fig. 1G, Supplemental Fig. 2G). These results
indicate that Grb2-deficient T cells have a greater potential to
differentiate into effector T cells. To further analyze the role of
Grb2 in effector T cells, we used Grb2fl/fl CD4cretg mice because
they show a largely normal development and, compared with
Grb2fl/fl Lckcretg mice, mild reductions in total T cell numbers.
Higher Th1 cell numbers and increased Th17 cell
differentiation
To analyze whether the increased number of effector T cells within
the CD4+ T cell population has an influence on the composition of
Th cell subsets, splenic CD4+ T cells from Grb2fl/fl CD4cretg mice
were harvested, stimulated with PMA and ionomycin, and incubated with Brefeldin A (BD GolgiPlug) to trap cytokines in the
endoplasmic reticulum. The cells were stained for Th1, Th2, or
Th17 cell–specific or Treg-specific cell markers. Grb2fl/fl CD4cretg
mice had normal numbers of Tregs and Th2 cells within the CD4+
T cell population. Furthermore, they showed a small increase in
Th17 cells (not significant), and the relative number of Th1 cells
was increased by 55% (Fig. 2A). This indicates that Grb2 deficiency influences the number of Th1 cells in naive Grb2fl/fl
CD4cretg mice. To confirm these results, we performed real-time
PCR analysis for key genes in Th cell development. Real-time
PCR analysis of RNA from PMA/ionomycin-stimulated CD4+
cells from naive Grb2fl/fl CD4cretg mice revealed a higher RNA
level for Th1 cell lineage–driving IFN-g, whereas the RNA level
for the Th1 cell key transcription factor Tbet was not altered
compared with Grb2fl/fl mice (Fig. 3). We also observed higher
RNA levels for Th17 cell–specific Rorgt and IL-17A, as well as
RNA coding for Treg-specific Foxp3 transcripts, whereas RNA
coding for Th2 cell–specific Gata3 and IL-4 was not altered
(Fig. 3). These real-time PCR results confirmed the intracellular
protein stainings to some extent. The differences between intracellular Ab staining and real-time PCR might be explained by
possible posttranscriptional regulation, different experimental setups, or the analysis of RNA from total cell populations as opposed
to single-cell analysis by FACS. In summary, the real-time PCR
analysis of PMA/ionomycin-stimulated CD4+ T cells of Grb2fl/fl
CD4cretg mice showed a shift toward increased IFN-g–producing,
IL-17A–producing, and Foxp3+ cells.
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FIGURE 1. Reduced number of T cells in Grb2fl/fl CD4cretg mice. Thymic T cell maturation was analyzed by staining for CD3, CD4, and CD8 (A) and
HSA (CD24) and TCRb (B). Peripheral T cell subsets were analyzed based on CD4 and CD8 expression for spleen (C) and lymph node (D). CD4+
peripheral T cells (E and F) or CD8+ peripheral T cells (G) were further analyzed for naive T cells, TCM, and TEM subsets of the spleen (E and G) or the
lymph node (F). In (A)–(C), (E), and (G), Grb2fl/fl CD4crewt (n = 13) and Grb2fl/fl CD4cretg (n = 8). In (D), Grb2fl/fl CD4crewt (n = 8) and Grb2fl/fl CD4cretg
(n = 6). In (F), Grb2fl/fl CD4crewt (n = 10) and Grb2fl/fl CD4cretg (n = 5). Mice were 7–8 wk of age. Pooled data from three independent experiments are
shown. *p # 0.05, ***p # 0.001, Student t test.
The Journal of Immunology
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To analyze the differentiation potential of Grb2-deficient CD4+
T cells, isolated CD62L+CD4+ T cells, a population that consists
primarily of naive T cells, were differentiated in vitro. The cells
were differentiated toward distinct Th cell populations by adding
polarizing cytokines (as described in Materials and Methods). The
potential of Grb2-deficient CD62L+CD4+ T cells to differentiate
into Th1 or Th2 cells or Tregs was not altered. In contrast, the
potential of Grb2-deficient CD62L+CD4+ T cells to differentiate
into proinflammatory Th17 cells was increased by 41% (Fig. 2B).
Taken together, we observed higher numbers of Th1 cells and
increased numbers of Th17 cells in naive Grb2fl/fl CD4cretg mice
and in vitro CD62L+CD4+ T cells possessed an increased potential
to differentiate into Th17 cells.
Reduced potential of Grb2-deficient T cells to proliferate
following activation by DCs
In addition to changes in Th cell differentiation, we analyzed
whether Grb2 deficiency also affects the activation potential and
proliferation of CD4+ T cells. An MLR experiment was performed
to analyze whether Grb2-deficient CD4+ T cells can proliferate
efficiently after activation by DCs. The proliferation capacity
of Grb2-deficient CD4+ T cells incubated with allogeneic DCs,
which were used in an immature state or were matured with LPS
or TNF, was clearly reduced compared with controls (Fig. 4A). In
accordance, IL-2 levels were reduced in supernatants of CD4+
T cells derived from Grb2-deficient mice (Fig. 4B). In contrast,
there was a tendency toward higher IFN-g levels, and IL-17A
levels were increased in the cell culture supernatants of T cells
derived from Grb2fl/fl CD4cretg mice (Fig. 4C). This indicates that
either activation by DCs or proliferation after activation is impaired in Grb2-deficient T cells, but the differentiation to effector
T cells is not negatively affected, as shown by the in vitro experiments (Figs. 2, 3).
To further analyze the observed activation/proliferation defect
in Grb2-deficient CD62L+CD4+ T cells using more confined
stimuli, these cells were stimulated in vitro with IL-2, anti-CD3,
or anti-CD3/anti-CD28. CD4+ Grb2-deficient T cells showed a
significantly reduced proliferation capacity after IL-2 and antiCD3 stimulation but not after CD3/CD28 costimulation (Fig. 4D).
This suggests a defective IL-2/TCR signaling in Grb2-deficient
T cells.
To prove that the alterations seen in Grb2fl/fl CD4cretg mice are
T cell dependent, we analyzed the potential of DCs, generated
from bone marrow of Grb2fl/fl CD4cretg mice, to activate CD4+
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FIGURE 2. Increased Th1 CD4+ T cell population and enhanced Th17 differentiation of Grb2fl/fl CD4cretg mice. (A) Splenic cells of Grb2fl/fl CD4cretg
mice and controls were incubated for 4–6 h with 1 mg/ml ionomycin, 20 ng/ml PMA, and 1 mg/ml Brefeldin A (BD GolgiPlug). Cells were stained intraand extracellularly to determine the percentage of Th1, Th2, and Th17 cells and Tregs among the CD4+ cells. (B) Splenic cells of Grb2fl/fl CD4cretg mice
and controls were enriched for CD62L+CD4+ cells, which primarily contain naive T cells, by MACS. Cells were differentiated for 3 d or, in case of Th2 cell
differentiation, for 5 d, toward Th1, Th2, or Th17 cells or Tregs. The differentiation potential was analyzed by flow cytometry. In (A) Grb2fl/fl CD4crewt (n = 10)
and Grb2fl/fl CD4cretg (n = 5). In (B), Grb2fl/fl CD4crewt (n = 5) and Grb2fl/fl CD4cretg (n = 6). Pooled data (mean 6 SD) from two (A) and four (B) independent
experiments are shown. *p # 0.05, **p # 0.01, Student t test.
3000
Grb2 T CELL FUNCTIONS
control T cells. This is necessary because CD4 is present on a
subset of DCs (27) and, therefore, CD4cre potentially deletes
Grb2 in this subset. However, the DCs generated from bone
marrow of Grb2fl/fl CD4cretg mice showed a normal potential to
activate T cells (Fig. 4E, Supplemental Fig. 4A, 4B), and Grb2fl/fl
CD4cretg mice show similar DC numbers as control mice in various organs (Supplemental Fig. 4C). This suggests that alterations
in T cell activation and proliferation in Grb2fl/fl CD4cretg mice are
likely caused by intrinsic T cell defects.
T cell–specific Grb2-deficient mice develop a mild form of EAE
Based on the increased number of Th1 cells in naive Grb2fl/fl
CD4cretg mice and the higher differentiation rate of CD62L+CD4+
T cells into proinflammatory Th17 cells, we hypothesized that
Grb2fl/fl CD4cretg mice are prone to more severe inflammatory
reactions. To analyze the relevance of this assumption in vivo, we
used the inflammation-based EAE model. However, contrary to
our expectations, Grb2fl/fl CD4cretg mice developed a less severe
EAE than control mice (Fig. 5A). This was most likely not due to
the reduced T cell numbers present in 7–8-wk-old naive Grb2fl/fl
CD4cretg mice, because splenic T cell numbers were similar at
day 30 post-EAE induction (Fig. 5B). This is comparable to the
situation in 15-wk-old naive Grb2fl/fl CD4cretg mice, which show
normalized T cell numbers in the spleen (Supplemental Fig. 3A).
In accordance with the milder course of disease in Grb2fl/fl
CD4cretg mice, splenic cells that were restimulated with MOG
peptide showed a reduced Ag-specific proliferation capacity (Fig.
5C). Diseased Grb2fl/fl CD4cretg mice were also examined with
regard to their cytokine levels. Thus, supernatants of MOGrestimulated splenic cells that were harvested at day 30 post-
EAE induction were analyzed with regard to their proinflammatory
cytokine level. Splenic cells derived from Grb2fl/fl CD4cretg mice
produced significantly less disease-promoting IL-17A, IFN-g, IL-2,
and IL-6, and there was a trend toward reduced TNF-a expression
levels (Fig. 5D).
In addition to the CD4+ cell compartment, we analyzed CD8+
T cell populations, because CD8+ cells can contribute to EAE.
However, we did not observe any alterations in the percentage of
naive cells, TCMs, or TEMs within the CD8+ T cell compartment
in the spleen at day 30 post-EAE induction (Supplemental Fig.
3B). In accordance with the situation in naive CD4+ Grb2fl/fl
CD4cretg mice (Fig. 4D), splenic cells derived from Grb2fl/fl
CD4cretg mice at day 30 post-EAE induction, which were
stimulated with IL-2, showed a reduced proliferation rate (Fig.
5F). In conclusion, these data obtained from in vivo and ex vivo
experiments indicate an important role for Grb2 in murine
T cells.
Reduction of proinflammatory milieu in T cell–specific
Grb2-deficient mice
Next, Grb2fl/fl CD4cretg mice and control mice were analyzed at
day 17 post-EAE induction, at the peak of disease. In line with
the mild clinical symptoms of Grb2fl/fl CD4cretg mice, they
showed less CD45+ infiltrates in the brain and spinal cord
compared with control mice (Fig. 6A). Notably, also at day 17
post-EAE induction, T cell numbers in the spleen of Grb2fl/fl
CD4cretg mice were comparable to the numbers observed in
control mice (Fig. 6B). Grb2fl/fl CD4cretg mice also had a reduced proportion of Th1 cells within spinal cord cells, whereas
Th17 cells were not altered (Fig. 6C). In the spinal cord, IL-17A
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FIGURE 3. Grb2fl/fl CD4cretg mice show higher RNA levels of IFN-g, Rorgt, Il-17A, and Foxp3. Splenic cells of 15-wk-old Grb2fl/fl CD4cretg mice and
controls were incubated for 4–6 h with 1 mg/ml ionomycin, 20 ng/ml PMA, and 1 mg/ml Brefeldin A (BD GolgiPlug). CD4+ cells were enriched by MACS,
followed by RNA purification and a reverse-transcription step. Quantitative real-time PCR was performed for Th cell lineage–specific transcription factors
Tbet (Th1), Rorgt (Th17), Gata3 (Th2), and Foxp3 (Treg) (A), as well as for RNA of typical cytokines for different Th cell lineages: IFN-g (Th1), IL-17
(Th17), and IL-4 (Th2) (B). Expression was normalized to Hprt and calculated as 22ΔΔCT. For Tbet, Rorgt, Gata3, Foxp3, and IFN-g: Grb2fl/fl CD4crewt (n =
5) and Grb2fl/fl CD4cretg (n = 5). For IL-17A and IL-4: Grb2fl/fl CD4crewt (n = 4) and Grb2fl/fl CD4cretg (n = 4). Pooled data (mean 6 SD) from two
independent experiments are shown. *p # 0.05, **p # 0.01, Mann–Whitney U test.
The Journal of Immunology
3001
and IFN-g RNA levels were clearly reduced (Fig. 6D). Interestingly, we found a higher proportion of inhibitory Tregs within
MOG peptide–restimulated T cells of splenic Grb2fl/fl CD4cretg
mice, which were harvested at day 17 post-EAE induction
(Fig. 6E). A similar trend was observed for T cells from the
lymph nodes that were treated in the same way (Fig. 6F). In
addition to CD4+ cells, we analyzed CD8+ T cell populations,
because CD8+ T cells can contribute to EAE symptoms. We
found that naive T cells were reduced by 41%, whereas TEMs
were increased by 171%, within CD8+ cells of the spleen at day
17 post-EAE induction (Supplemental Fig. 3C).
To carefully investigate whether the reduced CD4+ T cell
numbers are responsible for the milder EAE symptoms observed
in Grb2fl/fl CD4cretg mice, equal numbers of CD4+ T cells from
Grb2fl/fl CD4cretg or control mice were harvested at the peak of
the disease and transferred into Rag12/2 mice lacking B and
T cells. Rag12/2 mice that received Grb2-deficient CD4+ T cells
developed significantly less severe transfer EAE at the early stage
of disease compared with Rag12/2 mice that received CD4+
control cells. However, at later time points, there was no difference between the two groups (Fig. 7A). Also, the survival rate was
not significantly altered between the groups (Fig. 7B). Thus, once
EAE symptoms and, thus, the autoimmune reactive T cells are
established in donor mice, transferred CD4+ T cells derived from
Grb2fl/fl CD4cretg mice can induce comparable EAE-associated
paralyses as T cells derived from control mice.
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FIGURE 4. Reduced capacity of Grb2-deficient T cells to be activated by DCs. (A) A total of 3 3 105 CD4+ T cells from Grb2fl/fl and Grb2fl/fl CD4cretg
mice were incubated for 3 d with different numbers of matured DCs generated from BALB/c progenitors in a MLR. DCs were left unstimulated or were
matured with 100 ng/ml LPS or with 500 U/ml TNF-a before cocultivation. Proliferation of allogeneic T cells was analyzed by [3H]methyl-thymidine
uptake of proliferating cells. (B) Secreted IL-2 concentrations were analyzed in the supernatants derived from the different cell cocultures. (C) Secreted
IFN-g and IL-17 levels were analyzed in the supernatants of LPS-stimulated DC–T cell cocultures. (D) CD62L+CD4+ T cells (3 3 105) were stimulated for
3 d with 50 U/ml IL-2, 1 mg/ml anti-D3, or 2 ml/200 ml anti-CD3/anti-CD28 Dynabeads. Proliferation of cells was analyzed by [3H]methyl-thymidine
uptake of proliferating cells. (E) Splenic cells (3 3 105) from BALB/c mice were incubated for 3 d with different numbers of matured DCs generated from
Grb2fl/fl and Grb2fl/fl CD4cretg progenitors in a MLR. DCs were left unstimulated or were stimulated with LPS or with TNF-a. Proliferation of cells was
analyzed by [3H]methyl-thymidine uptake of proliferating cells. In (A)–(C) and (E), Grb2fl/fl (n = 3) and Grb2fl/fl CD4cretg (n = 3); pooled data from three
independent experiments are shown. In (D), Grb2fl/fl (n = 4) and Grb2fl/fl CD4cretg (n = 4); the experiment was performed two times, and data represent one
experiment, which is also representative of the other. Data are mean 6 SEM. *p # 0.05, **p # 0.01, Student t test.
3002
Grb2 T CELL FUNCTIONS
Discussion
Analysis of Grb2 deficiency in T cell development revealed that
deletion of Grb2 between the DN2 and DN3 stages by Cre under the
control of the proximal Lck promoter (23, 26) leads to a slightly
higher number of DN3 cells and a slightly reduced number of
DN4 cells, potentially related to defects in pre-TCR or growth
factor receptor signaling. In fact, LAT oligomerization and SOSmediated Ras activation, which are Grb2 dependent (8, 10, 28),
were described to be important for b-selection at the DN3 stage.
This was shown in SOS12/2 mice that were reconstituted with an
SOS mutant, which are not capable of activating Ras, or an SOS1SH2 recombinant protein that is unable to cluster phosphorylated
LAT. Both mice show a partial block in DN T cell development
(29), as do Grb2fl/fl Lckcretg mice. This indirectly suggests that
Ras-activation defects, defects in LAT oligomerization, or a
combination could account for the partial block in DN cell stages
observed in Grb2fl/fl Lckcretg mice. This partial block may contribute to the reduced CD4+CD8+ DP T cell numbers (∼30% reduction) in Grb2fl/fl Lckcretg mice (2), because Grb2fl/fl CD4cretg
mice that delete Grb2 at the CD4+CD8+ DP stage (24) show
normal CD4+CD8+ DP T cell numbers. Furthermore, Grb2fl/fl
CD4cretg mice exhibited no alterations in thymic T cell numbers
based on CD4+ and CD8+ expression. This is unexpected, because
Grb2fl/fl Lckcretg mice show a drastically reduced number of
CD4+ and CD8+ SP thymocytes due to a crucial role of Grb2 in
positive and negative selection (2). This was analyzed using
TCR-transgenic mouse models and the administration of the
staphylococcal enterotoxin B superantigen (2). The situation in
Grb2fl/fl CD4cretg mice might reflect an incomplete deletion of
Grb2 in the DP stage, as indicated by the Western blot, or a long
half-life of the Grb2 protein that is sufficient to sustain a rather
normal selection. An alternative explanation is that the early deletion in Grb2fl/fl Lckcretg mice has an influence on selection
processes. Mechanistically, the role of Grb2 in negative T cell selection might be explained by its involvement in Ras activation,
because SOS12/2 mice reconstituted with an SOS mutant, which
are not capable of activating Ras, exhibit an impaired negative
selection (29), and SOS recruitment to Ras is Grb2 dependent (28).
This suggests that Grb2-dependent recruitment of SOS to the
plasma membrane is important for negative selection. However, the
role of SOS in Ras activation in T cells is controversial (30–32).
Alternatively, but not mutually exclusively, the role of Grb2 in T cell
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FIGURE 5. Grb2fl/fl CD4cretg mice develop less severe EAE. (A) MOG peptide emulsified in CFA and enriched with M. tuberculosis was injected s.c. to
induce EAE in Grb2fl/fl (n = 9) and Grb2fl/fl CD4cretg mice (n = 11) at day 0. Clinical scoring was performed on the indicated days, as described in Materials
and Methods. (B) At day 30 of EAE, splenic T cell subsets were analyzed, based on CD4 and CD8 expression, by flow cytometry. (C) Splenic T cells were
restimulated in vitro with MOG peptide at day 30 of EAE, and proliferation was analyzed, after 4 d of restimulation, by [3H]methyl-thymidine uptake of
proliferating cells. (D) Supernatants of MOG-restimulated spleen cells in (C) were analyzed for their protein expression of IL-2, IL-6, IL-17, TNF-a, and
IFN-g in Grb2fl/fl and Grb2fl/fl CD4cretg. (E) Additionally, splenic cells from Grb2fl/fl and Grb2fl/fl CD4cretg mice were restimulated with rIL-2 for 4 d to test
the proliferation capacity at day 30 of EAE induction. Proliferation was measured based on [3H]methyl-thymidine uptake. In (A), (B), and (E), Grb2fl/fl (n = 9)
and Grb2fl/fl CD4cretg (n = 11). In (C) and (D), Grb2fl/fl (n = 6) and Grb2fl/fl CD4cretg (n = 7). Data in (C)–(E) are mean 6 SEM. EAE scoring significances: days
12, 18, 24, 25 (NS); days 20–22 (p # 0.05); days 13–17, 19, 23, 26, 27, 28 (p # 0.01). Pooled data from two independent experiments are shown. *p # 0.05,
**p # 0.01, ***p # 0.001, Mann–Whitney U test (A), Student t test (B, D, and E), two-way ANOVA (C).
The Journal of Immunology
3003
selection might be explained by its ability to recruit a THEMISGrb2-SHP1/2 complex to LAT in the immunological synapse, where
THEMIS and SHP1/2 set the threshold for positive selection (6, 7).
In the absence of THEMIS or if the THEMIS binding site for Grb2
is mutated, positive selection is drastically impaired (5, 7, 33, 34).
The importance of the C-terminal SH3 domain of Grb2 that recruits
THEMIS (6) is also highlighted by studies in which mutated forms
of Grb2 were transduced into bone marrow cells from Grb2fl/fl
FIGURE 7. Grb2-deficient CD4+ T cells exhibit a normal capacity to induce EAE in a transfer EAE model. (A) CD4+ T cells from Grb2fl/fl CD4crewt or
Grb2fl/fl CD4cretg mice were isolated at day 19 of EAE, and a defined number of cells was transferred into Rag12/2 recipient mice. Clinical scoring of EAE.
(B) Survival of mice. Grb2fl/fl (n = 7) and Grb2fl/fl CD4cretg (n = 8). Data are mean 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001, Mann–Whitney U test.
EAE scoring significances: day 4 (NS); days 5, 6, and 7 (p # 0.01); days 8 and 10 (p # 0.001). These experiments were performed at least two times, and
data represent a typical experiment.
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FIGURE 6. Grb2fl/fl CD4cretg–deficient mice show fewer CD45+ cell infiltrates in the brain and the spinal cord and characteristic changes in T cell
effector populations. (A) At day 17 of EAE, sections of the brain (upper panels) and spinal cord (lower panels) from Grb2fl/fl and Grb2fl/fl CD4cretg mice
were stained for CD45+ infiltrates. These experiments were performed at least three times. Data represent a typical experiment. (B) At day 17 of EAE,
splenic T cells were analyzed ex vivo based on CD4 and CD8 expression (left panels) and were quantified (right panel). (C) Spinal cord cells from both
mouse groups were restimulated with MOG peptide for 3 d and analyzed for the expression of CD4 (percentage of positive cells of all cells, left panel),
IFN-g, and IL-17A by flow cytometry. Percentages of IFNg+ and IL-17A+ cells within the CD4+ population (right panel). (D) To detect the amount of RNA
coding for IFN-g or IL-17A in spinal cord, RNA was isolated and transcribed to DNA, and quantitative PCRs were performed for Grb2fl/fl and Grb2fl/fl
CD4cretg mice. Splenic (E) or lymph node (F) cells were restimulated with MOG peptide for 3 d and analyzed for the percentages of CD4+ or CD8+ cells
(left panel), as well as for the percentages of CD4+CD25+Foxp3+ Tregs within the CD4+ population (right panel). In (B)–(F), Grb2fl/fl (n = 6) and Grb2fl/fl
CD4cretg (n = 8). Data in (C–F) are mean 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001, Student t test.
3004
the generation of EAE-promoting T cells, rather than their activity,
is impaired in Grb2fl/fl CD4cretg mice. Reduced T cell numbers or
Grb2-deficient CD8+ T cells may also contribute to the milder
EAE observed in Grb2fl/fl CD4cretg mice, but CD8+ T cells are
described to be more important in the late phases of EAE rather
than in its induction phase (39). Therefore, it is more likely that
CD4+ cells are the main drivers of the EAE phenotype observed in
Grb2fl/fl CD4cretg mice. Mechanistically, it was demonstrated that
Grb2 is necessary for the induction of IL-2 secretion via CD28
(40), and it is also necessary to induce a mitogenic response via
IL-2R (31, 41). This might explain why proliferation of Grb2deficient CD4+ T cells is diminished after stimulation with IL-2.
Additionally, the reduced potential of Grb2-deficient peripheral
T cells to be activated by DCs in the MLR is likely related to the
diminished signaling capacity via the TCR in Grb2-deficient
T cells (2). This, in turn, could also explain the diminished EAE
symptoms noted in Grb2fl/fl CD4cretg mice compared with controls. In addition to the discussed mouse data, it was reported for
human T cells, in which Grb2 was knocked down via microRNA,
that proximal TCR signaling is enhanced but calcium and MAPK
signaling, as well as IL-2 and IFN-g expression, are reduced as a
result of defects in LAT microcluster assembly after TCR stimulation (10). Additionally, these data from human cells highlight
the importance of Grb2 in signaling downstream of the TCR. The
signaling defects in Grb2fl/fl CD4cretg mice seem to be especially
important during activation of naive T cells via their TCRs;
however, once T cells are activated, the effector functions of
Grb2-deficient T cells seem to be normal in the passive EAE
model. The greater number of Tregs in Grb2fl/fl CD4cretg mice
during EAE could be related to a diminished IL-6 signaling,
because IL-6–coding mRNA in the spinal cord and IL-6 production of splenic cells at day 17 post-EAE induction are reduced and could promote naive T cells to become Tregs rather
than Th17 cells. IL-6 is involved in driving naive T cells to
become Th17 cells rather than Tregs (42). Taken together, Grb2
in peripheral T cells is crucial to induce autoimmune responses
in the EAE model, and the importance of Grb2 is especially
evident during disease induction.
As a ubiquitously expressed adapter protein, Grb2 has various
functions at different stages of T cell life. It probably has many
redundant functions in T cells and also competes with its relatives
for binding sites as a result of its close homology with other
proteins of the Grb2 family of adapter proteins (43, 44). Therefore,
the effects of Grb2 deletion in T cells may be milder than expected
for such an important signaling protein. Nevertheless, we showed
in this study that Grb2 promotes T cell development, modulates
Th cell differentiation, and, by controlling the induction of T cell
effector responses, influences the severity of EAE.
Acknowledgments
We thank Prof. David Vöhringer for discussions and Anne Urbat for technical help.
Disclosures
The authors have no financial conflicts of interest.
References
1. Lowenstein, E. J., R. J. Daly, A. G. Batzer, W. Li, B. Margolis, R. Lammers,
A. Ullrich, E. Y. Skolnik, D. Bar-Sagi, and J. Schlessinger. 1992. The SH2 and
SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras
signaling. Cell 70: 431–442.
2. Jang, I. K., J. Zhang, Y. J. Chiang, H. K. Kole, D. G. Cronshaw, Y. Zou, and
H. Gu. 2010. Grb2 functions at the top of the T-cell antigen receptor-induced
tyrosine kinase cascade to control thymic selection. Proc. Natl. Acad. Sci. USA
107: 10620–10625.
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Lckcretg mice via retroviral transduction. In the chimeric mice that
were subsequently generated with the transduced cells, SH2 and the
C-terminal SH3 domains of Grb2 were sufficient to restore T cell
development, whereas the N-terminal SH3 domain of Grb2 is dispensable for T cell development (2). However, additional effects of
Grb2 may also influence selection processes, because signaling in
Grb2fl/fl Lckcretg mice downstream of the TCR is drastically impaired (2).
Although the T cell numbers based on CD4 and CD8 expression
are normal in Grb2fl/fl CD4cretg mice, they show reduced numbers
of TCRhigh/HSAlow cells among the thymocytes. This means that a
higher proportion of thymocytes in Grb2fl/fl CD4cretg mice are
TCRhigh/HSAhigh. HSAhigh T cells normally do not reflect functional competent mature T cells (35, 36). These cells are more
prone to tolerance induction and apoptosis and are associated with
negative selection (37, 38), indicating a maturation defect from
CD4+CD8+ DP T cells to the functional competent SP T cells that
normally downregulate HSA in Grb2fl/fl CD4cretg mice (38). The
reduction in T cells in the thymus of Grb2fl/fl Lckcretg mice is
reflected by strongly reduced T cell numbers in the periphery,
suggesting that the impaired T cell numbers are related to the
defects in T cell development. However, homeostasis of T cells
has not been analyzed. The peripheral T cell numbers in Grb2fl/fl
CD4cretg mice are reduced by ∼30–40% in 7–8-wk-old mice. In
15-wk-old Grb2fl/fl CD4cretg mice, cell numbers were normalized
and are comparable to control mice. This suggests a delayed establishment of the peripheral T cell pool in Grb2fl/fl CD4cretg mice
due to a reduced generation of T cells in the thymus but, subsequently, a slow filling of the T cell pool in the periphery upon
ageing.
Grb2fl/fl Lckcretg mice and Grb2fl/fl CD4cretg mice showed a
higher proportion of T effector cells within the CD4+ T cell
population in the periphery and a reduction in naive cells. Peripheral T cells were analyzed further only in Grb2fl/fl CD4cretg
mice, because T cells in these mice show a rather normal developmental process compared with T cells in Grb2fl/fl Lckcretg mice.
In ex vivo analyses, splenic CD4+ T cells from Grb2fl/fl CD4cretg
mice showed a higher proportion of Th1 cells. Furthermore, Grb2deficient CD62L+CD4+ T cells, which represent mostly naive
T cells, had a greater potential to differentiate into Th17 cells
in vitro. In addition to naive T cells, the CD62L+CD4+ population
contains TCMs that could account for or contribute to the increase
in Th17 cells observed in the context of in vitro–differentiation
experiments. Moreover, with regard to the lymphopenic situation
represented by reduced T cell numbers, it cannot be excluded that
the changes in T effector subsets in naive Grb2fl/fl CD4cretg mice
are caused by homeostatic-proliferation mechanisms instead of
cell-intrinsic effects due to Grb2 deficiency. Nevertheless, we still
observed changes in the expression of key regulators of T effector
subsets in 15-wk-old Grb2fl/fl CD4cretg mice that show normal
T cell numbers. However, the changes in effector T cells did not
render the Grb2fl/fl CD4cretg mice more susceptible in the
inflammation-based EAE model that is primarily driven by Th1
and Th17 cells (21, 22). In contrast, Grb2fl/fl CD4cretg mice developed less severe EAE-associated symptoms than controls and
had higher numbers of Tregs. This could be due to a diminished
ability of peripheral CD4+ T cells to proliferate after activation by
DCs, as shown in the MLR experiment. In line with these findings,
Grb2-deficient T cells show a reduced in vitro proliferation potential in response to IL-2.
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EAE as do cells derived from control animals. This indicates that
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