Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis This information is current as of September 25, 2021. Daniel Radtke, Sonja M. Lacher, Nadine Szumilas, Lena Sandrock, Jochen Ackermann, Lars Nitschke and Elisabeth Zinser J Immunol published online 26 February 2016 http://www.jimmunol.org/content/early/2016/02/25/jimmun Downloaded from ol.1501764

Supplementary http://www.jimmunol.org/content/suppl/2016/02/25/jimmunol.150176 http://www.jimmunol.org/ Material 4.DCSupplemental

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 26, 2016, doi:10.4049/jimmunol.1501764 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

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 Downloaded from 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 http://www.jimmunol.org/ 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: 000–000.

he small ubiquitously expressed adaptor protein, growth and B cells (2–4). The analysis of Grb2fl/fl Lckcretg T cell–specific factor receptor–bound protein 2 (Grb2), integrates signals Grb2-deficient mice revealed that Grb2 is important for positive and from the outside of cells to inner signaling pathways via negative selection in the thymus, and it enhances tyrosine phos-

T by guest on September 25, 2021 its central SH2 and the two flanking SH3 domains (1, 2). Grb2 was phorylation downstream of the TCR, including phosphorylation of first described to be a positive regulator of the Ras signaling LAT, PLCg1, and CD3z chain. As a consequence of impaired se- pathway downstream of growth factor receptors (1). Surprisingly, lection processes, Grb2fl/fl Lckcretg mice show reduced T cell Grb2 seems to be dispensable for Ras signaling in CD4+CD8+ numbers in the periphery (2). It was reported recently that Grb2 double-positive (DP) thymocytes (2). However, Grb2 was also recruits THEMIS and SHP1 via its SH3 domains to phosphorylated showntoregulateCa2+ signaling and PI3K/Akt signaling LAT after TCR stimulation (5, 6). Thereby, it directs them to the in lymphocytes via incompletely understood mechanisms (3). immunological synapse where, in turn, the complex with THEMIS Grb2 also plays an important role in lymphocyte development in T 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 *Division of Genetics, Department of Biology, University of Erlangen, 91058 Erlan- thymocyte development (5). However, this model is not mutually gen, Germany; and †Department of Immune Modulation, University Hospital Erlan- exclusive because a complex consisting of two Grb2 molecules and gen, 91052 Erlangen, Germany one Sos1 molecule (2:1 complex) is able to cluster LAT proteins, 1 L.N. and E.Z. contributed equally to this work. which are phosphorylated after TCR engagement. Furthermore, ORCIDs: 0000-0002-5145-1157 (S.M.L.); 0000-0002-4369-0668 (E.Z.). LAT clustering could also be promoted by 2:1 Grb2–Cbl or 2:1 Received for publication August 4, 2015. Accepted for publication January 25, 2016. Grb2–Gab1 complexes. After phosphorylated LAT is clustered, it is This work was supported by the Deutsche Forschungsgemeinschaft (Ni 549/8-1 and able to activate downstream signaling pathways and, thereby, con- SFB643 Project B7 to L.N. and SFB643 Project B9 to E.Z.). tributes to T cell activation. In this model, Grb2 binds to phos- Address correspondence and reprint requests to Prof. Dr. Lars Nitschke or Dr. Elisabeth phorylated tyrosine residues of LAT via its SH2 domain, whereas its Zinser, Division of Genetics, Department of Biology, University of Erlangen, Staudt- strasse 5, 91058 Erlangen, Germany (L.N.) or Department of Immune Modulation at the SH3 domains bind to SOS1, Cbl, or Gab1. Because LAT, SOS1, Department of Dermatology, Universita¨tsklinikum Erlangen, Hartmannstasse 14, 91052 Cbl, and Gab1 have more than one Grb2 binding site, a large Erlangen, Germany (E.Z.). E-mail addresses: [email protected] (L.N.) or elisabeth. multiprotein cluster can be formed (8–10). [email protected] (E.Z.) Despite its described function in T cell development, little is The online version of this article contains supplemental material. known about the role of Grb2 in peripheral naive and effector Abbreviations used in this article: DC, dendritic cell; DN, double negative; DP, + double positive; EAE, experimental autoimmune encephalomyelitis; fwd, forward; T cells. When naive CD4 T cells encounter their Ag in the context Grb2, growth factor receptor–bound protein 2; HSA, heat-stable Ag; MOG, myelin of an MHC class II molecule presented on APCs, such as dendritic oligodendrocyte glycoprotein; rev, reverse; SP, single positive; TCM, central memory cells (DCs), they start to proliferate and differentiate into Th cells. T cell; T , effector memory T cell; Treg, regulatory T cell. EM These cells are important mediators of adaptive immunity. There Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 are four widely accepted Th cell populations: Th1 cells mediate

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501764 2 Grb2 T CELL FUNCTIONS responses against intracellular pathogens, Th2 cells promote re- scatter prior to further gating of fluorescent-labeled populations. The fol- sponses against extracellular parasites, and Th17 cells are asso- lowing Abs were used: anti-CD4–PE GK1.5, anti-Gr-1–FITC RB6-8C5, ciated with autoimmunity and responses against extracellular anti-CD25–FITC 7D4, anti-CD4–PerCP–RM4-5, anti-IL-17–FITC-TC11- 18H10 (all from BD), anti-CD24–FITC heat-stable Ag (HSA) 30-F1, anti- bacteria, as well as fungi. Finally, induced regulatory T cells CD62L–bio MEL-14, anti-CD69–bio H1 cF3, anti-CD8a–FITC 53-6.7, (Tregs) can be induced to inhibit immune responses (11, 12). anti-CD11b–FITC M1/70, anti-TCR-b–bio H57-597, anti-CD44–allo- It is known that the quality and quantity of signaling, especially phycocyanin IM7, anti-CD25–bio PC61.5, anti-IL-17–PE eBio17B7, 2 by the TCR, which is influenced by Grb2 (2), are critical for the anti-IFN-g–allophycocyanin XMG1.2, anti IL-4–allophycocyanin 11B11, anti-Foxp3–allophycocyanin FJK-16s, and streptavidin PerCP-Cy5.5 (all from development and maintenance of peripheral T cell subsets (12– eBioscience). 16). In general, weak signals by the TCR favor Th2 cell responses, whereas strong signals promote Th1 cell responses (14). Th1 cell In vitro T cell differentiation responses are promoted by JNK2 and P38 activation in combi- A 96-well tissue culture round-bottom plate (Falcon or BD) was incubated nation with JNK1 activation that inhibits Th2 responses (17, 18). with 50 ml Tris buffer (pH 9.5) with 1 mg/ml anti-CD3 Ab (145-2C11; JNK and P38 phosphorylation, as well as general tyrosine phos- eBioscience) for 3 h at 37˚C, and each well was washed two times with 13 5 + + phorylation downstream of the TCR, are reduced in Grb2fl/fl PBS with 2% FCS and 2 mM EDTA. A total of 1 3 10 CD4 CD62L Lckcretg mice, suggesting a role for Grb2 in Th cell equilibrium. 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 Th cell effector functions are often studied in animal models for nonessential amino acids, and 10,000 U/ml penicillin/streptomycin, was inflammatory diseases. Experimental autoimmune encephalomy- added. The CD4+CD62L+ T cells were obtained from spleen and lymph elitis (EAE) is an animal model for the early inflammatory phase of node of naive mice using a CD4+CD62L+ T cell Isolation Kit II (Miltenyi human multiple sclerosis, but it is also useful to deepen the un- Biotec), according to the manufacturer’s instructions. Cells were differ- Downloaded from entiated into Th1 cells by adding 1.3 mg/ml anti-CD28 (37.51; eBio- derstanding of Th cell functions in a disease situation in vivo (19, science), 4 ng/ml IL-12, and 50 U/ml IL-2; cells were differentiated into 20). To induce EAE, an immune reaction is initiated against a part Th2 cells by adding 1.3 mg/ml anti-CD28, 5 mg/ml anti–IFN-g (XMG1.2; of the myelin that surrounds nerve cells. This leads to inflam- eBioscience), 50 U/ml IL-2, and 10 ng/ml IL-4; cells were differentiated mation in the CNS. Freund’s adjuvant is used to boost the immune into induced Tregs by adding 1.3 mg/ml anti-CD28, 5 mg/ml anti–IFN-g, response against myelin oligodendrocyte glycoprotein (MOG); by 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, using this immunization protocol, the disease is primarily driven and 2 ng/ml TGF-b (all cytokines were from PeproTech). Cells were in- http://www.jimmunol.org/ by Th1 and Th17 cells (21, 22). cubated for 72 h at 37˚C and 5% CO2 and washed two times with 13 PBS In this study, we investigated the role of Grb2 on Th cell with 2% FCS and 2 mM EDTA. Cells forced to become Th2 cells were function and lineage commitment ex vivo, in vitro, and in the EAE 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 model. We found that Grb2-deficient T cells had an increased steps. After 72 h, or in case of Th2 cells, 120 h of stimulation, cells were potential to differentiate into Th17 cells in vitro and that naive resuspended in 50 ml medium with 1 mg/ml ionomycin and 20 ng/ml PMA, T cell–specific Grb2-deficient mice had a higher proportion of Th1 as well as 1 mg/ml Brefeldin A (BD GolgiPlug), and incubated for 6 h at + + + cells within the CD4 T cell population when analyzed ex vivo. To 37˚C and 5% CO2. Differentiation efficiency of CD4 CD62L T cells analyze the relevance of these findings in vivo, we used the murine toward Th cell populations was analyzed by flow cytometry. by guest on September 25, 2021 EAE model. Despite the shift in Grb2-deficient T cells toward Experimental autoimmune encephalomyelitis 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 Mice were immunized s.c. with 50 mg MOG35–55 peptide (Charite) emulsified in IFA, which was enriched with 10 mg/ml Mycobacterium form of EAE compared with control mice. The cellular mecha- tuberculosis (H37Ra; Difco/BD) to induce EAE. Additionally, 200 ng nism responsible for this impaired disease progression in the EAE pertussis toxin (List/Quadratec) was injected i.p. at days 0 and 2 of EAE. model was examined in this study. 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 experi- Materials and Methods ments, spleen and lymph node cells were harvested from Grb2fl/fl mice Mice on day 19 after immunization. Cells were restimulated with 30 mM MOG peptide for 72 h. After restimulation, CD4+ T cells were purified fl/fl Grb2 mice were described previously (3) and crossed to heterozygous using a CD4+ T Cell Isolation Kit II (Miltenyi Biotec), according to the tg tg fl/f tg Lckcre (23) or CD4cre (24) mice. Grb2 Lckcre mice were on a manufacturer’s instructions. Then 3 3 106 CD4+ Tcells/mousewere fl/fl tg mixed BALB/c/C57BL/6 background, whereas Grb2 CD4cre mice transferred i.v. into Rag12/2 mice, and disease progression was scored. 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, In vitro MOG restimulation whenever possible, littermates were used for all experiments. Animal experiments were approved by a local ethics committee (Regierung Mittel- At day 17 or 30 after MOG injection, splenic cells from mice were har- franken). vested, 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 Flow cytometry (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 Single-cell suspensions were incubated with 10% goat serum in PBS 50 mM 2-ME (all additives were from Sigma-Aldrich) per well in a 96- containing 0.1% (w/v) BSA and 0.05% (w/v) Azid (natriumazid) for 20 min well tissue culture plate for 72 h at 37˚C and 5% CO2. Then 0.1 mCi/well at 4˚C to avoid nonspecific binding. Then cells were incubated with [3H]methyl-thymidine (Hartmann Analytic) was added, and cells were fluorochrome-conjugated Abs (FITC, PE, PeCy5.5, PerCPCy5.5, allo- incubated for an additional 12 h. Subsequently, the plates were harvested phycocyanin) or with biotin-conjugated Abs in PBS containing 0.1% (w/v) with an IH-110 harvester (InnoTech), and filters were counted in a 1450 BSA and 0.05% (w/v) Azid (natriumazid) for 20 min at 4˚C. Cells incu- Microplate Counter (Wallac) to detect incorporated radioactivity as a bated with biotin-conjugated Abs were incubated again with fluorochrome- readout for proliferation. Supernatants from all restimulation cultures were conjugated streptavidin. For intracellular staining, with the exception of harvested to determine the ex vivo cytokine production. Foxp3 staining, a BD Cytofix/Cytoperm Fixation and Permeabilization Solution Kit with BD GolgiPlug (containing Brefeldin A) was used, Cytokine measurements according to the manufacturer’s instructions. For Foxp3 staining, the Anti- Mouse/Rat Foxp3 Staining Set APC (eBioscience) was used, according to To determine cytokine production in supernatants of MOG-restimulated the manufacturer’s instructions. Stained cells were analyzed using BD DCs in T cell cocultures, a commercially cytometric bead array (BD) FACSCalibur and FACSAria II flow cytometers and FlowJo software was used to detect IL-2, IL-17A, IFN-g, IL-6, and TNF-a, according to the (TreeStar). Lymphocytes were gated based on forward scatter and side manufacturer’s instructions. The Journal of Immunology 3

Histology Statistical analysis Brains and spinal cords of mice were harvested at day 17 of EAE, fixed in Results are expressed as mean and SD, unless stated otherwise in the figure liquid nitrogen, and stored at 280˚C. Organs were embedded in Tissue- legends. A two-tailed Student t test (for two groups) and two-way ANOVA Tek (Sakura), and 5-mm sections were made using a cryotome (Kryocut with the Bonferroni posttest (for multiple groups) were used to determine CM 2000; Leica). Immunohistological staining was performed using an statistical significance, unless stated otherwise in the figure legends. A immunoperoxidase detection system in a humid incubation chamber. nonparametric Mann–Whitney U test was used to analyze clinical EAE Acetone-fixed sections were incubated in PBS, and endogenous per- scoring. Differences with p values , 0.05 were considered statistically oxidase activity was blocked by incubating sections in 3% H2O2. significant. Statistic analyses were performed with Excel or Prism 4.03 or Sections were incubated with a primary anti-CD45 Ab (clone 30G12; 5.0 (GraphPad, La Jolla, CA). 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 Results AEC Chromogen Substrate (Vector Laboratories). Sections were Mild reduction in peripheral T cells in Grb2fl/fl CD4cretg stained with hematoxylin (Sigma-Aldrich), mounted in Aquatex T cell–specific Grb2-deficient mice (Merck), covered with a coverslip, and examined by light microscopy (Leica). To analyze the role of Grb2 at different stages of T cell development, we compared Grb2fl/fl Lckcretg mice that delete Grb2 at RNA isolation and quantitative real-time PCR analysis the transition from the double-negative (DN)2 stage to the DN3 Splenic CD4+ T cells from Grb2fl/fl or Grb2fl/fl CD4cretg mice were iso- stage in early T cell development (23, 26) with Grb2fl/fl CD4cretg + lated using the CD4 T Cell Isolation Kit (Miltenyi Biotec), according to mice that delete Grb2 at the CD4+CD8+ DP stage (24). Successful the manufacturer’s instructions. Cells were stimulated with 1 mg/ml deletion of Grb2 in T cells was confirmed by Western blot Downloaded from ionomycin and 20 ng/ml PMA, as well as 1 mg/ml Brefeldin A (BD (Supplemental Fig. 1A–D). In Grb2fl/fl Lckcretg mice, the cell GolgiPlug), and incubated for 6 h at 37˚C and 5% CO2. Thereafter, RNA was isolated using RNeasy Plus Mini Kit (QIAGEN) reagent, according numbers in the DN1 and DN2 stages were normal. In contrast, the to the manufacturer’s instructions. RNA from spinal cord tissue was re- number of cells in the DN3 stage was increased by 29%, and the 2 moved and stored in RNAlater at 80˚C for analysis. RNA was isolated number of cells in the DN4 stage was decreased by 28%, which using RNeasy Plus Mini Kit reagent, according to the manufacturer’s instructions. cDNA was synthesized with a reverse transcription kit might point to a minor block in T cell development (Supplemental

(Fermentas). For amplification, a SYBR Green mix for qPCR was used Fig. 1E). As also reported by another group (2), the number of http://www.jimmunol.org/ (Bio-Rad). Gene expression was analyzed with a CFX Real-time detec- CD4+CD8+ double positive (DP) cells in the thymus was slightly tion system (Bio-Rad). The relative gene expression levels were calcu- reduced in Grb2fl/fl Lckcretg mice, but the difference in cell lated with the comparative threshold cycle method, and data are 2ΔΔCT numbers was not significant (Supplemental Fig. 2A). Thymic expressed as 2 . Expression was normalized to HPRT. The following + + primer sequences were used: Hprt forward (fwd): 59-GTT GGA TAC AGG CD4 and CD8 single-positive (SP) cells were drastically re- fl/fl tg CCA GAC TTT GTT G-39;reverse(rev):59-GAT TCA ACT TGC GCT CAT duced by 66% in Grb2 Lckcre mice (Supplemental Fig. 2A). CTT AGG C-39; Il17a fwd: 59-TCC AGA AGG CCC TCA GAC TA-39;rev: This is due to defects in positive and negative selection (2). In 59-AGC ATC TTC TCG ACC CTG AA-39; Ifng fwd: 59-GCT TTG CAG CTC accordance with these results, the reduction in T cells in the TTC CTC AT-39,rev:59-GTC ACC ATC CTT TTG CCA GT-39; Tbet fwd: 59- fl/fl tg high/ high 9 9 thymus of Grb2 Lckcre mice is seen in TCR HSA cells, AGC AAG GAC GGC GAA TGT T-3 ;rev:5-GGG TGG ACA TAT AAG high/ low CGG TTC-39; Foxp3 fwd: 59-CCC AGG AAA GAC AGC AAC CTT-39;rev: which are reduced by 32%, and in more mature TCR HSA by guest on September 25, 2021 59-CCT TGC CTT TCT CAT CCA GGA-39; GATA3 fwd: 59-GTC ATC CCT cells, which are reduced by 49% (Supplemental Fig. 2B). GAG CCA CAT CT-39;rev:59-TAG AAG GGG TCG GAG GAA CT- 39; However, no alteration in DN cell numbers or CD4+CD8+ DP or Rorgt fwd: 59-GTG TGC TGT CCT GGG CTA CC-39;rev:59-AGC CTT GCA SP cell numbers was detected in the thymus of Grb2fl/fl CD4cretg CCC CTC ACA G-39;andIl4 fwd: 59-GGT CTC AAC CCC CAG CTA GT-39; rev: 59-GCC GAT GAT CTC TCT CAA GTG AT-39. mice (Fig. 1A, Supplemental Fig. 1E). Only the proportion of TCRhigh/HSAlow cells within the thymocyte population was re- Allogenic T cell proliferation duced (by 49%) (Fig. 1B). This indicates that selection processes fl/fl tg fl/fl BM-derived DCs were generated from Grb2fl/fl, Grb2fl/flCD4cretg,or in Grb2 CD4cre mice were much less affected than in Grb2 tg + + BALB/c mice, as described elsewhere (25). At day 9, bone marrow–DC Lckcre mice. Peripheral CD4 and CD8 SP cells were drasti- cultures were matured with TNF-a or LPS overnight or were left unsti- cally reduced in Grb2fl/fl Lckcretg mice: by 75–77% in the spleen 6 + fl/fl mulated. A total of 3 3 10 allogeneic CD4 T cells, purified from Grb2 and by 94–96% in lymph nodes, in accordance with existing data or Grb2fl/fl CD4cretg mice using a CD4+ T Cell Isolation Kit II (Miltenyi (Supplemental Fig. 2C, 2D) (2). In contrast, peripheral CD4+ and Biotec), were incubated with the indicated numbers of differentially + fl/fl tg stimulated DCs in a 96-well flat-bottom plate (Falcon) for 72 h at 37˚C and CD8 SP cell numbers in Grb2 CD4cre mice were reduced fl/fl 5% CO2. Additionally, the indicated numbers of DCs from Grb2 and only slightly: by 32 or 39%, respectively, in the spleen and by 36 fl/fl tg 6 Grb2 CD4cre mice were incubated with 3 3 10 allogeneic total and 39%, respectively, in the lymph nodes (Fig. 1C, 1D). spleen cells from BALB/C mice in a 96-well flat-bottom plate (Falcon) for 3 72 h at 37˚C and 5% CO2. Then, 1 mCi/well [ H]methyl-thymidine Increased number of effector T cells within peripheral CD4+ (Amersham Biosciences) was added, and cells were incubated for an additional 16 h. The plates were harvested with an IH-110 harvester (Inno- T cells Tech), and filters were counted in a 1450 Microplate Counter (Wallac) to + The number of effector memory T cells (TEMs) within the CD4 detect incorporated radioactivity as a readout for proliferation. T cell population of the spleen was increased by 45% in Grb2fl/fl tg fl/fl tg Stimulation of CD4+CD62L+ T cells and measurement of Lckcre and Grb2 CD4cre mice, whereas naive cells were fl/fl tg tg proliferation reduced by 52% in Grb2 Lckcre mice and by 37% in CD4cre mice (Fig. 1E, Supplemental Fig. 2E). The same was true for the + + CD4 CD62L T cells were obtained from spleen and lymph node of naive CD4+ T cells of the lymph node in Grb2fl/fl Lckcretg mice: T mice using a CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec), EMs according to the manufacturer’s instructions. A total of 3 3 106 cells was were increased by 49%, and naive T cells were reduced by 33.3% seeded and stimulated with 5 mg/ml anti-CD3 (eBioscience), 1 ml/200 ml (Supplemental Fig. 2F), whereas there were no significant changes anti-CD3/anti-CD28 Dynabeads (Dynabeads Mouse T-Activator; Life in these T cell populations in the lymph nodes of Grb2fl/fl 3 Technologies), or 50 U/ml IL-2 for 72 h. Then 1 mCi/well [ H]methyl- CD4cretg mice (Fig. 1F). The preferred differentiation to effector thymidine (Amersham Biosciences) was added, and cells were incubated T cells in T cell–specific Grb2-deficient mice is accompanied by a for an additional 16 h. The plates were harvested with an IH-110 harvester + (InnoTech), and filters were counted in a 1450 Microplate Counter (Wal- higher percentage of CD69-expressing CD4 T cells in the spleen lac) to detect incorporated radioactivity as a readout for proliferation. but not in the lymph node (data not shown). Similarly to CD4+ 4 Grb2 T CELL FUNCTIONS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 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.

T cells, within the CD8+ T cell population of Grb2fl/fl Lckcretg T cell population. Furthermore, they showed a small increase in mice and Grb2fl/fl CD4cretg mice we found a reduction in naive Th17 cells (not significant), and the relative number of Th1 cells T cells by 40 and 30%, respectively, and a relative increase in was increased by 55% (Fig. 2A). This indicates that Grb2 defi- + fl/fl TEMs by 111 and 180%, respectively. The percentage of CD62L ciency influences the number of Th1 cells in naive Grb2 + + tg CD44 central memory T cells (TCMs) to CD8 T cells was in- CD4cre mice. To confirm these results, we performed real-time creased by 84% in Grb2fl/fl Lckcretg mice and by 55% in Grb2fl/fl PCR analysis for key genes in Th cell development. Real-time CD4cretg mice (Fig. 1G, Supplemental Fig. 2G). These results PCR analysis of RNA from PMA/ionomycin-stimulated CD4+ indicate that Grb2-deficient T cells have a greater potential to cells from naive Grb2fl/fl CD4cretg mice revealed a higher RNA differentiate into effector T cells. To further analyze the role of level for Th1 cell lineage–driving IFN-g, whereas the RNA level Grb2 in effector T cells, we used Grb2fl/fl CD4cretg mice because for the Th1 cell key transcription factor Tbet was not altered they show a largely normal development and, compared with compared with Grb2fl/fl mice (Fig. 3). We also observed higher Grb2fl/fl Lckcretg mice, mild reductions in total T cell numbers. RNA levels for Th17 cell–specific Rorgt and IL-17A, as well as RNA coding for Treg-specific Foxp3 transcripts, whereas RNA Higher Th1 cell numbers and increased Th17 cell coding for Th2 cell–specific Gata3 and IL-4 was not altered differentiation (Fig. 3). These real-time PCR results confirmed the intracellular To analyze whether the increased number of effector T cells within protein stainings to some extent. The differences between intra- the CD4+ T cell population has an influence on the composition of cellular Ab staining and real-time PCR might be explained by Th cell subsets, splenic CD4+ T cells from Grb2fl/fl CD4cretg mice possible posttranscriptional regulation, different experimental set- were harvested, stimulated with PMA and ionomycin, and incu- ups, or the analysis of RNA from total cell populations as opposed bated with Brefeldin A (BD GolgiPlug) to trap cytokines in the to single-cell analysis by FACS. In summary, the real-time PCR endoplasmic reticulum. The cells were stained for Th1, Th2, or analysis of PMA/ionomycin-stimulated CD4+ T cells of Grb2fl/fl Th17 cell–specific or Treg-specific cell markers. Grb2fl/fl CD4cretg CD4cretg mice showed a shift toward increased IFN-g–producing, mice had normal numbers of Tregs and Th2 cells within the CD4+ IL-17A–producing, and Foxp3+ cells. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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 intra- and 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)andGrb2fl/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.

To analyze the differentiation potential of Grb2-deficient CD4+ which were used in an immature state or were matured with LPS T cells, isolated CD62L+CD4+ T cells, a population that consists or TNF, was clearly reduced compared with controls (Fig. 4A). In primarily of naive T cells, were differentiated in vitro. The cells accordance, IL-2 levels were reduced in supernatants of CD4+ were differentiated toward distinct Th cell populations by adding T cells derived from Grb2-deficient mice (Fig. 4B). In contrast, polarizing cytokines (as described in Materials and Methods). The there was a tendency toward higher IFN-g levels, and IL-17A potential of Grb2-deficient CD62L+CD4+ T cells to differentiate levels were increased in the cell culture supernatants of T cells into Th1 or Th2 cells or Tregs was not altered. In contrast, the derived from Grb2fl/fl CD4cretg mice (Fig. 4C). This indicates that potential of Grb2-deficient CD62L+CD4+ T cells to differentiate either activation by DCs or proliferation after activation is im- into proinflammatory Th17 cells was increased by 41% (Fig. 2B). paired in Grb2-deficient T cells, but the differentiation to effector Taken together, we observed higher numbers of Th1 cells and T cells is not negatively affected, as shown by the in vitro ex- increased numbers of Th17 cells in naive Grb2fl/fl CD4cretg mice periments (Figs. 2, 3). and in vitro CD62L+CD4+ T cells possessed an increased potential To further analyze the observed activation/proliferation defect to differentiate into Th17 cells. in Grb2-deficient CD62L+CD4+ T cells using more confined stimuli, these cells were stimulated in vitro with IL-2, anti-CD3, Reduced potential of Grb2-deficient T cells to proliferate or anti-CD3/anti-CD28. CD4+ Grb2-deficient T cells showed a following activation by DCs significantly reduced proliferation capacity after IL-2 and anti- In addition to changes in Th cell differentiation, we analyzed CD3 stimulation but not after CD3/CD28 costimulation (Fig. 4D). whether Grb2 deficiency also affects the activation potential and This suggests a defective IL-2/TCR signaling in Grb2-deficient proliferation of CD4+ T cells. An MLR experiment was performed T cells. to analyze whether Grb2-deficient CD4+ T cells can proliferate To prove that the alterations seen in Grb2fl/fl CD4cretg mice are efficiently after activation by DCs. The proliferation capacity T cell dependent, we analyzed the potential of DCs, generated of Grb2-deficient CD4+ T cells incubated with allogeneic DCs, from bone marrow of Grb2fl/fl CD4cretg mice, to activate CD4+ 6 Grb2 T CELL FUNCTIONS Downloaded from http://www.jimmunol.org/

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. by guest on September 25, 2021 control T cells. This is necessary because CD4 is present on a EAE induction were analyzed with regard to their proinflammatory subset of DCs (27) and, therefore, CD4cre potentially deletes cytokine level. Splenic cells derived from Grb2fl/fl CD4cretg mice Grb2 in this subset. However, the DCs generated from bone produced significantly less disease-promoting IL-17A, IFN-g,IL-2, marrow of Grb2fl/fl CD4cretg mice showed a normal potential to and IL-6, and there was a trend toward reduced TNF-a expression activate T cells (Fig. 4E, Supplemental Fig. 4A, 4B), and Grb2fl/fl levels (Fig. 5D). CD4cretg mice show similar DC numbers as control mice in var- InadditiontotheCD4+ cell compartment, we analyzed CD8+ ious organs (Supplemental Fig. 4C). This suggests that alterations T cell populations, because CD8+ cells can contribute to EAE. in T cell activation and proliferation in Grb2fl/fl CD4cretg mice are However, we did not observe any alterations in the percentage of + likely caused by intrinsic T cell defects. naive cells, TCMs,orTEMs within the CD8 T cell compartment in the spleen at day 30 post-EAE induction (Supplemental Fig. T cell–specific Grb2-deficient mice develop a mild form of EAE 3B). In accordance with the situation in naive CD4+ Grb2fl/fl fl/fl Based on the increased number of Th1 cells in naive Grb2 CD4cretg mice (Fig. 4D), splenic cells derived from Grb2fl/fl tg + + CD4cre mice and the higher differentiation rate of CD62L CD4 CD4cretg mice at day 30 post-EAE induction, which were T cells into proinflammatory Th17 cells, we hypothesized that stimulated with IL-2, showed a reduced proliferation rate (Fig. fl/fl tg Grb2 CD4cre mice are prone to more severe inflammatory 5F). In conclusion, these data obtained from in vivo and ex vivo reactions. To analyze the relevance of this assumption in vivo, we experiments indicate an important role for Grb2 in murine used the inflammation-based EAE model. However, contrary to T cells. our expectations, Grb2fl/fl CD4cretg mice developed a less severe EAE than control mice (Fig. 5A). This was most likely not due to Reduction of proinflammatory milieu in T cell–specific the reduced T cell numbers present in 7–8-wk-old naive Grb2fl/fl Grb2-deficient mice CD4cretg mice, because splenic T cell numbers were similar at Next, Grb2fl/fl CD4cretg mice and control mice were analyzed at day 30 post-EAE induction (Fig. 5B). This is comparable to the day 17 post-EAE induction, at the peak of disease. In line with situation in 15-wk-old naive Grb2fl/fl CD4cretg mice, which show the mild clinical symptoms of Grb2fl/fl CD4cretg mice, they normalized T cell numbers in the spleen (Supplemental Fig. 3A). showed less CD45+ infiltrates in the brain and spinal cord In accordance with the milder course of disease in Grb2fl/fl compared with control mice (Fig. 6A). Notably, also at day 17 CD4cretg mice, splenic cells that were restimulated with MOG post-EAE induction, T cell numbers in the spleen of Grb2fl/fl peptide showed a reduced Ag-specific proliferation capacity (Fig. CD4cretg mice were comparable to the numbers observed in 5C). Diseased Grb2fl/fl CD4cretg mice were also examined with control mice (Fig. 6B). Grb2fl/fl CD4cretg mice also had a re- regard to their cytokine levels. Thus, supernatants of MOG- duced proportion of Th1 cells within spinal cord cells, whereas restimulated splenic cells that were harvested at day 30 post- Th17 cells were not altered (Fig. 6C). In the spinal cord, IL-17A The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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. and IFN-g RNA levels were clearly reduced (Fig. 6D). Inter- in Grb2fl/fl CD4cretg mice, equal numbers of CD4+ T cells from estingly, we found a higher proportion of inhibitory Tregs within Grb2fl/fl CD4cretg or control mice were harvested at the peak of MOG peptide–restimulated T cells of splenic Grb2fl/fl CD4cretg the disease and transferred into Rag12/2 mice lacking B and mice, which were harvested at day 17 post-EAE induction T cells. Rag12/2 mice that received Grb2-deficient CD4+ T cells (Fig. 6E). A similar trend was observed for T cells from the developed significantly less severe transfer EAE at the early stage lymph nodes that were treated in the same way (Fig. 6F). In of disease compared with Rag12/2 mice that received CD4+ addition to CD4+ cells, we analyzed CD8+ T cell populations, control cells. However, at later time points, there was no differ- because CD8+ T cells can contribute to EAE symptoms. We ence between the two groups (Fig. 7A). Also, the survival rate was found that naive T cells were reduced by 41%, whereas TEMs not significantly altered between the groups (Fig. 7B). Thus, once were increased by 171%, within CD8+ cells of the spleen at day EAE symptoms and, thus, the autoimmune reactive T cells are 17 post-EAE induction (Supplemental Fig. 3C). established in donor mice, transferred CD4+ T cells derived from To carefully investigate whether the reduced CD4+ T cell Grb2fl/fl CD4cretg mice can induce comparable EAE-associated numbers are responsible for the milder EAE symptoms observed paralyses as T cells derived from control mice. 8 Grb2 T CELL FUNCTIONS Downloaded from http://www.jimmunol.org/

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 fl/fl fl/fl tg

induce EAE in Grb2 (n = 9) and Grb2 CD4cre mice (n = 11) at day 0. Clinical scoring was performed on the indicated days, as described in Materials by guest on September 25, 2021 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)andGrb2fl/fl CD4cretg (n = 7). Data in (C)–(E)aremean6 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,andE), two-way ANOVA (C).

Discussion CD4cretg mice exhibited no alterations in thymic T cell numbers + + Analysis of Grb2 deficiency in T cell development revealed that based on CD4 and CD8 expression. This is unexpected, because deletion of Grb2 between the DN2 and DN3 stages by Cre under the Grb2fl/fl Lckcretg mice show a drastically reduced number of control of the proximal Lck promoter (23, 26) leads to a slightly CD4+ and CD8+ SP thymocytes due to a crucial role of Grb2 in higher number of DN3 cells and a slightly reduced number of positive and negative selection (2). This was analyzed using DN4 cells, potentially related to defects in pre-TCR or growth TCR-transgenic mouse models and the administration of the factor receptor signaling. In fact, LAT oligomerization and SOS- staphylococcal enterotoxin B superantigen (2). The situation in mediated Ras activation, which are Grb2 dependent (8, 10, 28), Grb2fl/fl CD4cretg mice might reflect an incomplete deletion of were described to be important for b-selection at the DN3 stage. Grb2 in the DP stage, as indicated by the Western blot, or a long This was shown in SOS12/2 mice that were reconstituted with an half-life of the Grb2 protein that is sufficient to sustain a rather SOS mutant, which are not capable of activating Ras, or an SOS1- normal selection. An alternative explanation is that the early dele- SH2 recombinant protein that is unable to cluster phosphorylated tion in Grb2fl/fl Lckcretg mice has an influence on selection LAT. Both mice show a partial block in DN T cell development processes. Mechanistically, the role of Grb2 in negative T cell se- (29), as do Grb2fl/fl Lckcretg mice. This indirectly suggests that lection might be explained by its involvement in Ras activation, 2 2 Ras-activation defects, defects in LAT oligomerization, or a because SOS1 / mice reconstituted with an SOS mutant, which combination could account for the partial block in DN cell stages are not capable of activating Ras, exhibit an impaired negative observed in Grb2fl/fl Lckcretg mice. This partial block may con- selection (29), and SOS recruitment to Ras is Grb2 dependent (28). tribute to the reduced CD4+CD8+ DP T cell numbers (∼30% re- This suggests that Grb2-dependent recruitment of SOS to the duction) in Grb2fl/fl Lckcretg mice (2), because Grb2fl/fl CD4cretg plasma membrane is important for negative selection. However, the mice that delete Grb2 at the CD4+CD8+ DP stage (24) show role of SOS in Ras activation in T cells is controversial (30–32). normal CD4+CD8+ DP T cell numbers. Furthermore, Grb2fl/fl Alternatively, but not mutually exclusively, the role of Grb2 in T cell The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

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 fl/fl fl/fl tg effector populations. (A) At day 17 of EAE, sections of the brain (upper panels) and spinal cord (lower panels) from Grb2 and Grb2 CD4cre mice by guest on September 25, 2021 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. selection might be explained by its ability to recruit a THEMIS- is mutated, positive selection is drastically impaired (5, 7, 33, 34). Grb2-SHP1/2 complex to LAT in the immunological synapse, where The importance of the C-terminal SH3 domain of Grb2 that recruits THEMIS and SHP1/2 set the threshold for positive selection (6, 7). THEMIS (6) is also highlighted by studies in which mutated forms In the absence of THEMIS or if the THEMIS binding site for Grb2 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. 10 Grb2 T CELL FUNCTIONS

Lckcretg mice via retroviral transduction. In the chimeric mice that the generation of EAE-promoting T cells, rather than their activity, were subsequently generated with the transduced cells, SH2 and the is impaired in Grb2fl/fl CD4cretg mice. Reduced T cell numbers or C-terminal SH3 domains of Grb2 were sufficient to restore T cell Grb2-deficient CD8+ T cells may also contribute to the milder development, whereas the N-terminal SH3 domain of Grb2 is dis- EAE observed in Grb2fl/fl CD4cretg mice, but CD8+ T cells are pensable for T cell development (2). However, additional effects of described to be more important in the late phases of EAE rather Grb2 may also influence selection processes, because signaling in than in its induction phase (39). Therefore, it is more likely that Grb2fl/fl Lckcretg mice downstream of the TCR is drastically im- CD4+ cells are the main drivers of the EAE phenotype observed in paired (2). Grb2fl/fl CD4cretg mice. Mechanistically, it was demonstrated that Although the T cell numbers based on CD4 and CD8 expression Grb2 is necessary for the induction of IL-2 secretion via CD28 are normal in Grb2fl/fl CD4cretg mice, they show reduced numbers (40), and it is also necessary to induce a mitogenic response via of TCRhigh/HSAlow cells among the thymocytes. This means that a IL-2R (31, 41). This might explain why proliferation of Grb2- higher proportion of thymocytes in Grb2fl/fl CD4cretg mice are deficient CD4+ T cells is diminished after stimulation with IL-2. TCRhigh/HSAhigh. HSAhigh T cells normally do not reflect func- Additionally, the reduced potential of Grb2-deficient peripheral tional competent mature T cells (35, 36). These cells are more T cells to be activated by DCs in the MLR is likely related to the prone to tolerance induction and apoptosis and are associated with diminished signaling capacity via the TCR in Grb2-deficient negative selection (37, 38), indicating a maturation defect from T cells (2). This, in turn, could also explain the diminished EAE CD4+CD8+ DP T cells to the functional competent SP T cells that symptoms noted in Grb2fl/fl CD4cretg mice compared with con- normally downregulate HSA in Grb2fl/fl CD4cretg mice (38). The trols. In addition to the discussed mouse data, it was reported for reduction in T cells in the thymus of Grb2fl/fl Lckcretg mice is human T cells, in which Grb2 was knocked down via microRNA, Downloaded from reflected by strongly reduced T cell numbers in the periphery, that proximal TCR signaling is enhanced but calcium and MAPK suggesting that the impaired T cell numbers are related to the signaling, as well as IL-2 and IFN-g expression, are reduced as a defects in T cell development. However, homeostasis of T cells result of defects in LAT microcluster assembly after TCR stimu- has not been analyzed. The peripheral T cell numbers in Grb2fl/fl lation (10). Additionally, these data from human cells highlight CD4cretg mice are reduced by ∼30–40% in 7–8-wk-old mice. In the importance of Grb2 in signaling downstream of the TCR. The fl/fl tg fl/fl tg 15-wk-old Grb2 CD4cre mice, cell numbers were normalized signaling defects in Grb2 CD4cre mice seem to be especially http://www.jimmunol.org/ and are comparable to control mice. This suggests a delayed es- important during activation of naive T cells via their TCRs; tablishment of the peripheral T cell pool in Grb2fl/fl CD4cretg mice however, once T cells are activated, the effector functions of due to a reduced generation of T cells in the thymus but, subse- Grb2-deficient T cells seem to be normal in the passive EAE quently, a slow filling of the T cell pool in the periphery upon model. The greater number of Tregs in Grb2fl/fl CD4cretg mice ageing. during EAE could be related to a diminished IL-6 signaling, Grb2fl/fl Lckcretg mice and Grb2fl/fl CD4cretg mice showed a because IL-6–coding mRNA in the spinal cord and IL-6 pro- higher proportion of T effector cells within the CD4+ T cell duction of splenic cells at day 17 post-EAE induction are re- population in the periphery and a reduction in naive cells. Pe- duced and could promote naive T cells to become Tregs rather ripheral T cells were analyzed further only in Grb2fl/fl CD4cretg than Th17 cells. IL-6 is involved in driving naive T cells to by guest on September 25, 2021 mice, because T cells in these mice show a rather normal devel- become Th17 cells rather than Tregs (42). Taken together, Grb2 opmental process compared with T cells in Grb2fl/fl Lckcretg mice. in peripheral T cells is crucial to induce autoimmune responses In ex vivo analyses, splenic CD4+ T cells from Grb2fl/fl CD4cretg in the EAE model, and the importance of Grb2 is especially mice showed a higher proportion of Th1 cells. Furthermore, Grb2- evident during disease induction. deficient CD62L+CD4+ T cells, which represent mostly naive As a ubiquitously expressed adapter protein, Grb2 has various T cells, had a greater potential to differentiate into Th17 cells functions at different stages of T cell life. It probably has many in vitro. In addition to naive T cells, the CD62L+CD4+ population redundant functions in T cells and also competes with its relatives contains TCMs that could account for or contribute to the increase for binding sites as a result of its close homology with other in Th17 cells observed in the context of in vitro–differentiation proteins of the Grb2 family of adapter proteins (43, 44). Therefore, experiments. Moreover, with regard to the lymphopenic situation the effects of Grb2 deletion in T cells may be milder than expected represented by reduced T cell numbers, it cannot be excluded that for such an important signaling protein. Nevertheless, we showed the changes in T effector subsets in naive Grb2fl/fl CD4cretg mice in this study that Grb2 promotes T cell development, modulates are caused by homeostatic-proliferation mechanisms instead of Th cell differentiation, and, by controlling the induction of T cell cell-intrinsic effects due to Grb2 deficiency. Nevertheless, we still effector responses, influences the severity of EAE. observed changes in the expression of key regulators of T effector subsets in 15-wk-old Grb2fl/fl CD4cretg mice that show normal Acknowledgments T cell numbers. However, the changes in effector T cells did not We thank Prof. David Vo¨hringer for discussions and Anne Urbat for tech- fl/fl tg render the Grb2 CD4cre mice more susceptible in the nical help. inflammation-based EAE model that is primarily driven by Th1 and Th17 cells (21, 22). In contrast, Grb2fl/fl CD4cretg mice de- Disclosures veloped less severe EAE-associated symptoms than controls and The authors have no financial conflicts of interest. 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, References Grb2-deficient T cells show a reduced in vitro proliferation po- 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 tential in response to IL-2. SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras + Interestingly, CD4 Grb2-deficient effector T cells, which were signaling. Cell 70: 431–442. isolated from MOG-immunized EAE mice and transferred into 2. Jang, I. K., J. Zhang, Y. J. Chiang, H. K. Kole, D. G. Cronshaw, Y. Zou, and 2/2 H. Gu. 2010. Grb2 functions at the top of the T-cell antigen receptor-induced Rag1 mice, have nearly the same potential to induce a passive tyrosine kinase cascade to control thymic selection. Proc. Natl. Acad. Sci. USA EAE as do cells derived from control animals. This indicates that 107: 10620–10625. The Journal of Immunology 11

3. Ackermann, J. A., D. Radtke, A. Maurberger, T. H. Winkler, and L. Nitschke. 25. Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Ro¨ssner, F. Koch, N. Romani, and 2011. Grb2 regulates B-cell maturation, B-cell memory responses and inhibits G. Schuler. 1999. An advanced culture method for generating large quantities of B-cell Ca2+ signalling. EMBO J. 30: 1621–1633. highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223: 4. Gong, Q., A. M. Cheng, A. M. Akk, J. Alberola-Ila, G. Gong, T. Pawson, and 77–92. A. C. Chan. 2001. Disruption of T cell signaling networks and development by 26. Shimizu, C., H. Kawamoto, M. Yamashita, M. Kimura, E. Kondou, Y. Kaneko, Grb2 haploid insufficiency. Nat. Immunol. 2: 29–36. S. Okada, T. Tokuhisa, M. Yokoyama, M. Taniguchi, et al. 2001. Progression of 5. Paster, W., C. Brockmeyer, G. Fu, P. C. Simister, B. de Wet, A. Martinez-Rianõ, T cell lineage restriction in the earliest subpopulation of murine adult thymus J. A. Hoerter, S. M. Feller, C. Wulfing,€ N. R. J. Gascoigne, and O. Acuto. 2013. visualized by the expression of lck proximal promoter activity. Int. Immunol. 13: GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte 105–117. development. J. Immunol. 190: 3749–3756. 27. Vremec, D., J. Pooley, H. Hochrein, L. Wu, and K. Shortman. 2000. CD4 and 6. Paster, W., A. M. Bruger, K. Katsch, C. Gre´goire, R. Roncagalli, G. Fu, N. R. J. Gascoigne, CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J. K. Nika, A. Cohnen, S. M. Feller, et al. 2015. A THEMIS:SHP1 complex promotes T-cell Immunol. 164: 2978–2986. survival. EMBO J. 34: 393–409. 28. Buday, L., and J. Downward. 2008. Many faces of Ras activation. Biochim. 7. Fu, G., J. Casas, S. Rigaud, V. Rybakin, F. Lambolez, J. Brzostek, J. A. Hoerter, Biophys. Acta 1786: 178–187. W. Paster, O. Acuto, H. Cheroutre, et al. 2013. Themis sets the signal threshold 29. Kortum, R. L., L. Balagopalan, C. P. Alexander, J. Garcia, J. M. Pinski, for positive and negative selection in T-cell development. Nature 504: 441–445. R. K. Merrill, P. H. Nguyen, W. Li, I. Agarwal, I. O. Akpan, et al. 2013. The 8. Houtman, J. C., H. Yamaguchi, M. Barda-Saad, A. Braiman, B. Bowden, ability of Sos1 to oligomerize the adaptor protein LAT is separable from its E. Appella, P. Schuck, and L. E. Samelson. 2006. Oligomerization of signaling guanine nucleotide exchange activity in vivo. Sci. Signal. 6: ra99. complexes by the multipoint binding of GRB2 to both LAT and SOS1. Nat. 30. Dower, N. A., S. L. Stang, D. A. Bottorff, J. O. Ebinu, P. Dickie, Struct. Mol. Biol. 13: 798–805. H. L. Ostergaard, and J. C. Stone. 2000. RasGRP is essential for mouse thy- 9. McDonald, C. B., J. El Hokayem, N. Zafar, J. E. Balke, V. Bhat, D. C. Mikles, mocyte differentiation and TCR signaling. Nat. Immunol. 1: 317–321. B. J. Deegan, K. L. Seldeen, and A. Farooq. 2013. Allostery mediates ligand 31. Warnecke, N., M. Poltorak, B. S. Kowtharapu, B. Arndt, J. C. Stone, binding to Grb2 adaptor in a mutually exclusive manner. J. Mol. Recognit. 26: B. Schraven, and L. Simeoni. 2012. TCR-mediated Erk activation does not de- 92–103. pend on Sos and Grb2 in peripheral human T cells. EMBO Rep. 13: 386–391. 10. Bilal, M. Y., and J. C. Houtman. 2015. GRB2 Nucleates T Cell Receptor- 32. Poltorak, M., I. Meinert, J. C. Stone, B. Schraven, and L. Simeoni. 2014. Sos1 Mediated LAT Clusters That Control PLC-g1 Activation and Cytokine Pro- regulates sustained TCR-mediated Erk activation. Eur. J. Immunol. 44: 1535– Downloaded from duction. Front. Immunol. 6: 141. 1540. 11. Kanno, Y., G. Vahedi, K. Hirahara, K. Singleton, and J. J. O’Shea. 2012. 33. Patrick, M. S., H. Oda, K. Hayakawa, Y. Sato, K. Eshima, T. Kirikae, S. Iemura, Transcriptional and epigenetic control of T helper cell specification: molecular M. Shirai, T. Abe, T. Natsume, et al. 2009. Gasp, a Grb2-associating protein, is mechanisms underlying commitment and plasticity. Annu. Rev. Immunol. 30: critical for positive selection of thymocytes. Proc. Natl. Acad. Sci. USA 106: 707–731. 16345–16350. 12. Zhou, L., M. M. Chong, and D. R. Littman. 2009. Plasticity of CD4+ T cell 34. Johnson, A. L., L. Aravind, N. Shulzhenko, A. Morgun, S.-Y. Choi, lineage differentiation. Immunity 30: 646–655. T. L. Crockford, T. Lambe, H. Domaschenz, E. M. Kucharska, L. Zheng, et al.

13. Walsh, K. P., and K. H. Mills. 2013. Dendritic cells and other innate determi- 2009. Themis is a member of a new metazoan gene family and is required for the http://www.jimmunol.org/ nants of T helper cell polarisation. Trends Immunol. 34: 521–530. completion of thymocyte positive selection. Nat. Immunol. 10: 831–839. 14. Yamane, H., and W. E. Paul. 2013. Early signaling events that underlie fate 35. Ramsdell, F., M. Jenkins, Q. Dinh, and B. J. Fowlkes. 1991. The majority of decisions of naive CD4(+) T cells toward distinct T-helper cell subsets. Immunol. CD4+82 thymocytes are functionally immature. J. Immunol. 147: 1779–1785. Rev. 252: 12–23. 36. Nikolic´-Zugic´, J., and M. J. Bevan. 1990. Functional and phenotypic delineation 15. Nakayamada, S., H. Takahashi, Y. Kanno, and J. J. O’Shea. 2012. Helper T cell of two subsets of CD4 single positive cells in the thymus. Int. Immunol. 2: 135– diversity and plasticity. Curr. Opin. Immunol. 24: 297–302. 141. 16. O’Shea, J. J., and W. E. Paul. 2010. Mechanisms underlying lineage commitment 37. Kishimoto, H., and J. Sprent. 1997. Negative selection in the thymus includes and plasticity of helper CD4+ T cells. Science 327: 1098–1102. semimature T cells. J. Exp. Med. 185: 263–271. 17. Rinco´n, M., R. A. Flavell, and R. A. Davis. 2000. The JNK and P38 MAP kinase 38. Hough, M. R., F. Takei, R. K. Humphries, and R. Kay. 1994. Defective devel- signaling pathways in T cell-mediated immune responses. Free Radic. Biol. opment of thymocytes overexpressing the costimulatory molecule, heat-stable Med. 28: 1328–1337. antigen. J. Exp. Med. 179: 177–184.

18. Rinco´n, M., D. Conze, L. Weiss, N. L. Diehl, K. A. Fortner, D. Yang, 39. Koh, D. R., W. P. Fung-Leung, A. Ho, D. Gray, H. Acha-Orbea, and T. W. Mak. by guest on September 25, 2021 R. A. Flavell, H. Enslen, A. Whitmarsh, and R. J. Davis. 2000. Conference 1992. Less mortality but more relapses in experimental allergic encephalomy- highlight: do T cells care about the mitogen-activated protein kinase signalling elitis in CD8-/- mice. Science 256: 1210–1213. pathways? Immunol. Cell Biol. 78: 166–175. 40. Watanabe, R., Y. Harada, K. Takeda, J. Takahashi, K. Ohnuki, S. Ogawa, 19. Stromnes, I. M., and J. M. Goverman. 2006. Active induction of experimental D. Ohgai, N. Kaibara, O. Koiwai, K. Tanabe, et al. 2006. Grb2 and Gads exhibit allergic encephalomyelitis. Nat. Protoc. 1: 1810–1819. different interactions with CD28 and play distinct roles in CD28-mediated co- 20. Rangachari, M., and V. K. Kuchroo. 2013. Using EAE to better understand stimulation. J. Immunol. 177: 1085–1091. principles of immune function and autoimmune pathology. J. Autoimmun. 45: 41. Evans, G. A., M. A. Goldsmith, J. A. Johnston, W. Xu, S. R. Weiler, R. Erwin, 31–39. O. M. Howard, R. T. Abraham, J. J. O’Shea, W. C. Greene, et al. 1995. Analysis 21. Tigno-Aranjuez, J. T., R. Jaini, V. K. Tuohy, P. V. Lehmann, and M. Tary- of interleukin-2-dependent signal transduction through the Shc/Grb2 adapter Lehmann. 2009. Encephalitogenicity of complete Freund’s adjuvant relative to pathway. Interleukin-2-dependent mitogenesis does not require Shc phosphory- CpG is linked to induction of Th17 cells. J. Immunol. 183: 5654–5661. lation or receptor association. J. Biol. Chem. 270: 28858–28863. 22. Batoulis, H., K. Addicks, and S. Kuerten. 2010. Emerging concepts in autoim- 42. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 Cells. mune encephalomyelitis beyond the CD4/T(H)1 paradigm. Ann. Anat. 192: 179– Annu. Rev. Immunol. 27: 485–517. 193. 43. Feng, G. S., Y. B. Ouyang, D. P. Hu, Z. Q. Shi, R. Gentz, and J. Ni. 1996. Grap is 23. Orban, P. C., D. Chui, and J. D. Marth. 1992. Tissue- and site-speciﬁc DNA a novel SH3-SH2-SH3 adaptor protein that couples tyrosine kinases to the Ras recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89: 6861–6865. pathway. J. Biol. Chem. 271: 12129–12132. 24. Ye, S. K., Y. Agata, H. C. Lee, H. Kurooka, T. Kitamura, A. Shimizu, T. Honjo, 44. Liu, S. K., and C. J. McGlade. 1998. Gads is a novel SH2 and SH3 domain- and K. Ikuta. 2001. The IL-7 receptor controls the accessibility of the containing adaptor protein that binds to tyrosine-phosphorylated Shc. Oncogene TCRgamma locus by Stat5 and histone acetylation. Immunity 15: 813–823. 17: 3073–3082.