And Thymocyte Development Function Role in Coordinating Mitochondrial

Gene Targeting RhoA Reveals Its Essential Role in Coordinating Mitochondrial Function and Thymocyte Development

This information is current as Shuangmin Zhang, Diamantis G. Konstantinidis, Jun-Qi of September 27, 2021. Yang, Benjamin Mizukawa, Khalid Kalim, Richard A. Lang, Theodosia A. Kalfa, Yi Zheng and Fukun Guo J Immunol published online 14 November 2014 http://www.jimmunol.org/content/early/2014/11/14/jimmun

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 14, 2014, doi:10.4049/jimmunol.1400839 The Journal of Immunology

Gene Targeting RhoA Reveals Its Essential Role in Coordinating Mitochondrial Function and Thymocyte Development

Shuangmin Zhang,* Diamantis G. Konstantinidis,* Jun-Qi Yang,* Benjamin Mizukawa,* Khalid Kalim,* Richard A. Lang,† Theodosia A. Kalfa,* Yi Zheng,* and Fukun Guo*

Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival. Nonetheless, the physiological function of RhoA in thymocyte development is not clear. By characterizing a conditional gene targeting mouse model bearing T cell deletion of RhoA, we show that RhoA critically regulates thymocyte de- Downloaded from velopment by coordinating multiple developmental events. RhoA gene disruption caused a strong developmental block at the pre- TCR checkpoint and during positive selection. Ablation of RhoA led to reduced DNA synthesis in CD42CD82, CD4+CD82, and CD42CD8+ thymocytes but not in CD4+CD8+ thymocytes. Instead, RhoA-deficient CD4+CD8+ thymocytes showed an impaired mitosis. Furthermore, we found that abrogation of RhoA led to an increased apoptosis in all thymocyte subpopulations. Impor- tantly, we show that the increased apoptosis was resulted from reduced pre-TCR expression and increased production of reactive oxygen species (ROS), which may be because of an enhanced mitochondrial function, as manifested by increased oxidative http://www.jimmunol.org/ phosphorylation, glycolysis, mitochondrial membrane potential, and mitochondrial biogenesis in RhoA-deficient thymocytes. Restoration of pre-TCR expression or treatment of RhoA-deficient mice with a ROS scavenger N-acetylcysteine partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate, to our knowledge, for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis. The Journal of Immunology, 2014, 193: 000–000.

cell development in the thymus proceeds through a series mocytes. The first process is called repertoire selection including of differentiation stages (1–4). The most immature positive and negative selection. During repertoire selection, thy- by guest on September 27, 2021 T populations in the thymus comprise CD42CD82 double- mocytes that express TCRab of low affinity to self-peptide/MHC negative (DN) thymocytes. DN thymocytes can be subdivided complexes are instructed to undergo positive selection and mat- into four developmental stages: CD44+CD252 (DN1), CD44+ uration to SP cells, whereas thymocytes that express TCRab of high CD25+ (DN2), CD442CD25+ (DN3), and CD442CD252 affinity to self-peptide/MHC complexes and thus are potentially au- (DN4). DN thymocyte development is signified by TCRb gene toreactive are eliminated by negative selection. The second process is rearrangements and b-selection. TCRb gene rearrangements are termed lineage commitment, during which thymocytes that recognize known as V(D)J recombination, which begin at DN2 stage, con- self-peptide/MHC class I become CD8+ SP cells and thymocytes that tinue and complete at DN3 stage. b-Selection occurs at DN3 stage recognize self-peptide/MHC class II become CD4+ SP cells. and is a process in which thymocytes successfully expressing pre- RhoA is an intracellular (Ic) signal transducer of the Rho family TCR complex consisting of rearranged TCRb and pre-Ta are small GTPases that cycles between an inactive GDP-bound form rescued from cell death and allowed to proliferate and differentiate and an active GTP-bound form under tight regulation (5, 6). Mostly to DN4 and subsequent CD4+CD8+ double-positive (DP) cells. At by overexpression of dominant active or negative mutants, RhoA the DP stage, thymocytes undergo two concurrent developmental has been shown to modulate actin cytoskeleton organization, cell processes to mature to CD4+ or CD8+ single-positive (SP) thy- adhesion, migration, proliferation, and survival (7–11). In T cells, overexpression of the dominant mutants suggest that RhoA plays *Division of Experimental Hematology and Cancer Biology, Children’s Hospital a role in T cell polarization, thymocyte adhesion, and thymic egress Research Foundation, Cincinnati, OH 45229; and †Division of Pediatric Ophthalmol- (12–17). Furthermore, inactivation of RhoA by C3 transferase in ogy, Children’s Hospital Research Foundation, Cincinnati, OH 45229 transgenic mice caused thymocyte developmental blocks (18, 19). Received for publication April 1, 2014. Accepted for publication October 10, 2014. However, these approaches are hampered by the nonspecific nature This work was supported by National Institutes of Health Grant GM 108661 (to F.G.). of the dominant RhoA mutants or C3 transferase because they may Address correspondence and reprint requests to Dr. Fukun Guo, Division of Exper- affect other Rho GTPases (20–24). Indeed, distinct cell functions imental Hematology and Cancer Biology, Children’s Hospital Research Foundation, of RhoA have been observed in studies of RhoA knockout mouse 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: [email protected] models. For example, contrary to the conventional view that RhoA Abbreviations used in this article: Cox, cyclooxygenase; DN, double negative; DP, double positive; ECAR, extracellular acidification rate; ETP, early T cell precursor; is essential for actin cytoskeleton rearrangement and cell adhesion, Ic, intracellular; NAC, N-acetylcysteine; OCR, oxygen consumption rate; OXPHOS, RhoA-deficient primary mouse embryonic fibroblasts display nor- oxidative phosphorylation; ROS, reactive oxygen species; SP, single positive; Tg, mal actin stress fiber and focal adhesion complex formation (25). It transgenic; WT, wild-type. is therefore highly desirable to use a gene targeting strategy to as- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 sess the physiological functions of RhoA in T cells.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400839 2 RhoA IN THYMOCYTE DEVELOPMENT

In this study, we have examined the physiological contribution In vitro proliferation assay of RhoA in thymocyte development by characterizing a T cell– DP thymocytes were sorted by flow cytometry, plated on 48-well plates specific RhoA conditional knockout mouse model. We demon- with or without anti-CD3/-CD28 Abs, and cultured for 3 d. Cell growth strate that RhoA is required for thymocyte development by co- rates were assayed by a nonradioactive cell proliferation assay kit ordinating DN thymocyte b-selection, DP thymocyte positive (Promega). selection, SP thymocyte lineage commitment, thymocyte prolif- Immunoblotting eration and survival, and importantly, mitochondrial function. Our data suggest that RhoA couples metabolic homeostasis to thy- Total thymocytes or flow cytometry–sorted DP thymocytes were lysed, and protein content was normalized by Bradford assay. Lysates were separated mocyte development. by 10% SDS-PAGE. The expression or activation (phosphorylation) of RhoA, ZAP70, ERK, and JNK was probed by Western blot using corre- Materials and Methods sponding Abs (Cell Signaling Technology). Mouse gene targeting Transcript expression analysis Conditionally targeted RhoAflox/flox mice were generated as described pre- RNA was isolated from thymocytes using RNeasy Micro Kit (Qiagen) and viously (25). The floxed allele contains loxP sites flanking exon 3 of the flox/flox converted to cDNA using a High Capacity cDNA Reverse Transcription Kit RhoA allele. To delete RhoA in vivo in the T cell lineage, RhoA mice (Applied Biosystems). Real-time PCR was performed with SYBR Green were mated with mice expressing Cre recombinase under the control of dye or TaqMan assay on a 7900HT Real-Time machine (Applied Bio- a CD2 or Lck proximal promoter (The Jackson Laboratory). RhoAflox/flox; 2 2 systems). Data were analyzed using SDS 2.3 software (Applied Biosystems) CD2-Cre mice (hereafter referred to as RhoA / ) were crossed with p14 2/2 and normalized to GAPDH. mice expressing transgenic TCRVa2Vb8 to generate RhoA ;p14TCR 9 The primer sequences were as following: Nrf1, 5 -TATGGCGGAAG- Downloaded from transgenic (Tg) compound mice. Mice used for experiments ranged in age TAATGAAAGACG-39 (forward) and 59-CAACGTAAGCTCTGCCTTG- from 4 to 8 wk. Animals were housed under specific pathogen-free con- TT-39 (reverse); Atp5I, 59-GAGAAGGCACCGTCGATGG-39 (forward) and ditions in the animal facility at Cincinnati Children’s Hospital Research 59-ACACTCTGAATAGCTGTAGGGAT-39 (reverse); Cox5a, GCCGCTG- Foundation in compliance with the Cincinnati Children’s Hospital Medical TCTGTTCCATTC-39 (forward) and 59-GCATCAATGTCTGGCTTGTTG- Center Animal Care and Use Committee protocols. AA-39 (reverse); Ndufa2, 59-TTGCGTGAGATTCGCGTTCA-39 (forward) Flow cytometry analysis by cell surface and Ic staining and 59-ATTCGCGGATCAGAATGG GC-39 (reverse); HK2 F, 59-TGAT- CGCCTGCTTATTCACGG-39 (forward) and 59-AACCGCCTAGAAATC- Single-cell suspensions were prepared from thymus. Cells were incubated TCCAGA-39 (reverse); slc2a, 59-CAGTTCGGCTATAACACTGGTG (forward) http://www.jimmunol.org/ with various combinations of fluorophore-conjugated Abs against the fo- and 59-GCCCCCGACAGAGAAGATG-39 (reverse); PDK1, 59-GGACTTCG- llowing cell surface markers (BD Biosciences) at room temperature for GGTCAGTGAATGC-39 (forward) and 59-TCCTGAGAAGATTGTCGGGGA- 20 min: Thy1.2, CD28, c-Kit, CD4, CD8, CD44, CD25, CD69, TCRb, 39 (reverse); and Pgm1, 59-CAGAACCCTTTAACCTCTGAGTC-39 (forward) IL-7Ra, HSA, L-selectin, and TCR Va2. Immunolabeled cells were ana- and 59-CGAGAAATCCCTGCTCCCATAG-39 (reverse). The TaqMan primer/ lyzed by flow cytometry. probe sets for Notch 3, Hes1, Notch1, PreTa, Deltex1 were Mm00435270_ m1, IcTCRb staining was performed as described previously (26). Briefly, Mm01342805, Mm00435249_m1, Mm00492291_m1, and Mm01281478_m1, cells were incubated with fluorophore-conjugated Abs to cell surface mar- respectively. kers (anti-CD4, anti-CD8, anti-Thy1.2, anti-CD44, anti-CD25, and anti- CD28) as well as with a saturating amount of anti-TCRb to block cell V(D)J recombination surface TCRb. Cells were then fixed at room temperature for 20 min with b by guest on September 27, 2021 1% paraformaldehyde. After washing in PBS, the cells were incubated TCR gene rearrangement was detected as described previously (27). In with 10 mM glycine for 10 min to quench autofluorescence. Cells were brief, genomic DNA was isolated from flow cytometry–sorted DN3 cells b b b b b b b b then permeabilized using 0.5% saponin, 5% FCS, and 10 mM HEPES and amplified by PCR. PCR for D 2-J 2, V 5-J 2, V 8-J 2, V 11 -J 2, a (pH 7.4) at room temperature for 10 min and stained with fluorophore- and eF-1 was performed for 31 cycles with a 1-min annealing time at b b b b conjugated Ab to TCRb at room temperature for 45 min. Ic staining 63˚C. PCR for D 1.1-J 1.7 and D 2.1-J 2.7 was first performed for 20 m of Bcl2 was carried out similarly, except that the Bcl2 Ab was revealed cycles with external primers. A total of 0.5 l from the first amplification with secondary fluorophore-conjugated goat anti-mouse IgG (Jackson was used for a second PCR for an additional 31 cycles with nested ImmunoResearch Laboratories). internal primers. Primer sequences for detecting Db2-Jb2, Vb5-Jb2, Vb8-Jb2, and PCR genotyping to detect RhoA gene deletion Vb11-Jb2 recombination, and for eF-1a were as follows: Db2, 59-G- TAGGCACCTGTGGGGAAGAAACT-39 (forward); Vb5, 59-CCCAGC- Thymocyte subsets were sorted by flow cytometry and genomic DNA from AGATTCTCTCAGTCCAACAG-39 (forward); Vb8, 59-GCATGGGCT- the sorted cells was isolated and analyzed by PCR using primers 59- GAGGCTGATCCATTA-39 (forward); Vb11, 59-TGCTG GTGTCATCC- TCTCTGCACTGAGGGAGTTAGG-39 (forward) and 59-GTACATACA- AAACACCTAG-39 (forward); Jb2, 59-TGAGAGCTGTCTCCTACTA TC- GGGAATGGAAAC AAGG-39 (reverse) to detect floxed allele and primers GATT-39 (reverse); and eF-1a,59-CTGCTGAGATGGGAAA GGGCT-39 59-GCACTGAGGGAGTTAGG-39 (forward) and 59-CTACACTAGCTG- (forward) and 59-TTCAGGATAATCACCTGA GCA-39 (reverse) (28). GGCAC-39 (reverse) to detect knockout allele. For detecting floxed allele, Primer sequences for detecting Db1.1-Jb1.7 and Db2.1-Jb2.7 recombina- PCR was carried out for 33 cycles with a 30-s annealing time at 58˚C. For tion were as following: external primers: Db1.1ext, 59-GAGGAGCAGCT- detecting knockout allele, PCR was carried out for 30 cycles with a 30-s TATCTGG TG-39 (forward); Jb1.7ext, 59-AAGGGA CGACTCTGT- annealing time at 60˚C. PCRs were analyzed by agarose gel electropho- CTTAC-39 (reverse); Db2.1ext, 59-TAGGCAACCTGTGGG GAAGAA resis. The reaction gave rise to a 633-bp fragment for floxed allele and AC-39 (forward); and Jb2.7ext, 59-TGAGAGCTGTCTCC TACTAT C-39 a 667-bp fragment for knockout allele. (reverse); and nested internal primers: Db1.1int, 59-GGTAGACCTATGGG AGGGC-39 (forward); Jb1.7int, 59-ACCATGGTCATCCAACACA G-39 Cell apoptosis analysis (reverse); Db2.1int, 59-GTATCACGATGTA ACATTGTG-39 (forward); Freshly isolated thymocytes were incubated with fluorophore-conjugated and Jb2.7int, 59-GGAAGCGAGAGATGT GAATC-39 (reverse) (29). anti-CD4, anti-CD8, anti-CD44, and anti-CD25 Abs for 20 min. Cells X were washed, incubated with fluorophore-conjugated Annexin V (BD Bio- High-speed cell imaging analysis in flow using Imagestream sciences) for 20 min, and then analyzed by flow cytometry on a FACSCanto The nuclear contents and the distribution of F-actin and b-tubulin within DP system using FACSDiVa software (BD Biosciences). thymocytes were studied by ImagestreamX (Amnis), which combines flow In vivo BrdU incorporation assay cytometry and microscopy (360/numerical aperture 0.9 objective lens) capabilities. Thymocytes were harvested and cultured with anti-CD3/- Mice were injected i.p. with 500 mg BrdU. Twelve hours after injection, CD28 Abs for 3 d. The cells were fixed with 4% formaldehyde. Formal- thymocytes were isolated, immunolabeled with fluorophore-conjugated dehyde was removed by centrifugation, and the cell pellet was cooled on Abs against CD4, CD8, CD44, and CD25, fixed, permeabilized, and then ice for at least 15 min, before permeabilization by consecutive suspensions incubated with fluorophore-conjugated anti-BrdU Ab, according to the in ice-cold 50% acetone, 100% acetone, and again 50% acetone solution. manufacturer’s protocol (BD Biosciences). Immunolabeled cells were then Cells were then incubated with fluorophore-conjugated Abs to CD4 and analyzed by flow cytometry analysis. CD8 and either fluorophore-conjugated anti–b-tubulin Ab (Cell Signaling The Journal of Immunology 3

Technology) or phalloidin (Invitrogen). The nuclear stain Draq5 or DAPI As a result, RhoA deletion by CD2-Cre led to an accumulation (Cell Signaling Technology) was added at a concentration of 40 mM, and X of early T cell precursors (ETPs) among DN1 cells, whereas the samples were processed by Imagestream . Approximately 10,000 Lck-Cre–mediated RhoA deletion had no effect on this early events/sample were collected and analyzed with the associated Image Data Exploration and Analysis software (Amnis). thymocyte subset (Fig. 1G). Collectively, these data suggest that RhoA is required for T cell development in the thymus and RhoA Metabolic assays has a T cell intrinsic role in T cell development. 6 For measurement of oxygen consumption rate (OCR), 10 freshly isolated b thymocytes were resuspended in XF assay medium (pH 7.4) containing Defective -selection in the absence of RhoA 2 mM GlutaMax, 1 mM sodium pyruvate and supplemented with 25 mM RhoA deficiency resulted in a reduced cellularity of all DN subsets glucose, plated onto Seahorse Bioscience XF24 cell culture plates using and an increased percentage of DN3 cells at the expense of DN4 Cell Tak (BD Bioscience), and incubated without CO2 at 37˚C. Respiration was measured using the Seahorse XF24 Analyzer (Seahorse Biosciences) and DP cells, suggesting that the developmental transition from 2 2 under basal condition and in the presence of the mitochondrial inhibitor DN3 to DN4 and then DP cells was blocked in RhoA / mice. oligomycin (0.6 mM) and mitochondrial uncoupler FCCP (1 mM). For b 6 DN3 transition to DN4 and DP stage requires successful TCR measurement of extracellular acidification rate (ECAR), 10 freshly iso- gene rearrangements, namely V(D)J recombination, and b-selec- lated thymocytes were resuspended in assay medium (pH 7.4) (Sigma- b b b b Aldrich) containing 2 mM L-glutamin and 2.5 mM glucose, plated onto tion (1–4). We found that V 11-J 2 and V 5-J 2 recombination XF24 cell culture plates, and incubated as for OCR. Extracellular acidi- was moderately impaired upon RhoA deletion, whereas Vb8-Jb2, fication was measured using the Seahorse XF24 Analyzer under basal Db2-Jb2, Db2.1-Jb2.7, and Db1.1-Jb1.7 rearrangements were condition and in the presence of the glycolysis substrate glucose (10 mM), completely intact (Fig. 2A). These results suggest that RhoA mitochondrial inhibitor oligomycin (0.6 mM), and glycolysis inhibitor 2 deficiency does not cause a gross defect in TCRb gene rearrang- Downloaded from deoxy-D-glucose (100 mM) (30, 31). 2/2 For measurements of reactive oxygen species (ROS), mitochondrion ements. In contrast, RhoA mice showed fewer DN3 cells, par- numbers and mitochondrial membrane potential, thymocytes stained for CD4 ticularly DN3b cells, that express Ic and surface TCRb (Fig. 2B, 2C). and CD8 were incubated with 5 mM DCFDA, 100 nM Mitotracker Green, A substantial reduction in the frequency of IcTCRb+ DN4 cells and 50 nM DilC-5 (Invitrogen), respectively, according to the manufacturer’s and DN4 cells expressing surface TCRb was also detected in protocols. The cells were then analyzed by flow cytometry (31). RhoA2/2 mice (Fig. 2B, 2C). Concomitantly, the percentage of For mitochondrial DNA assay, thymocytes were lyzed and homogenized 2 and mitochondrial DNA was then purified with a Mitochondrial DNA IcTCRb DN4 cells was increased in the absence of RhoA http://www.jimmunol.org/ isolation Kit (Biovision), according to the manufacturer’s manual. The (Fig. 2B). However, the total numbers of both IcTCRb+ and purified mitochondrial DNA was quantified by quantitative real-time PCR IcTCRb2 DN4 cells were decreased in RhoA2/2 mice (Fig. 2B). with SYBR Green dye. Mitochondrial DNA content was represented by mitochondrial cyclooxygenase (Cox)2 normalized to nuclear intron of Furthermore, RhoA deficiency caused a reduced expression of b-globin. The primer sequences were as follows: Cox2, 59-GCCGAC- pre-Ta (Fig. 2D). Taken together, these data suggest that RhoA TAAATCAAGCAACA-39 (forward) and 59-CAATGGGCATAAAGCTA- regulates b-selection by governing expression and/or transport of TGG-39 (reverse); and b-globin, 59-GAAGCGATTCTAGGGAGCAG-39 TCRb and pre-Ta but not TCRb gene rearrangements. (forward) and 59-GGAGCAGC GATTCTGAGTAGA-39 (reverse) (32). For ATP assay, thymocytes were lyzed with a cytosol extraction buffer sup- Impaired proliferation and survival in DN cells in the absence plied in the Mitochondrial DNA isolation Kit and homogenized, and then,

of RhoA by guest on September 27, 2021 ATP was determined by an ATP Determination Kit (Molecular Probes), according to the manufacturer’s protocol. Successfully rearranged TCRb forms a complex with pre-Ta and Statistical analysis CD3 into pre-TCR on the cell surface of DN3 cells. These DN3 cells are rescued from cell death and committed to proliferate 6 Data are expressed as the mean SD. Data were analyzed by a Student’s and differentiate to DN4 and DP cells (33). Measurement of cell unpaired t test with a two-tailed distribution. Significance was accepted at p , 0.05. proliferation by BrdU incorporation demonstrated that in wild- type (WT) mice, there was a significant increase of cell division in DN4 cells compared with DN3 cells, reflecting successful Results b-selection. However, such an increased proliferation from DN3 Defective thymocyte development in the absence of RhoA to DN4 was abolished in the absence of RhoA (Fig. 3A). More- To delete the RhoA gene in T cell lineage, RhoAflox/flox mice were over, RhoA deficiency diminished the proliferation rate of DN4 cross-bred with CD2-Cre transgenic mice. The RhoAflox/flox;CD2- cells without affecting that of DN3 cells (Fig. 3A). Nonetheless, 2 2 Cre (RhoA / ) mice had a drastic reduction in total thymocyte Annexin V staining revealed that both DN3 and DN4 cells from cellularity (Fig. 1A). The proportion of thymocyte subsets were the mutant were more susceptible to apoptosis compared with WT altered with increased frequency of DN2 and DN3 but decreased counterparts (Fig. 3B). These data provide further evidence that frequency of DN4 and DP thymocytes (Fig. 1B, 1C). The numbers RhoA is required for b-selection. of all thymocyte subsets including DN1–DN4, DP, CD4+ SP, and Cell survival at DN thymocytes is regulated by IL-7R and Notch CD8+ SP thymocytes were markedly decreased in mutant mice signaling (26). Unexpectedly, prosurvival Notch1 and its down- (Fig. 1B, 1C). Analysis of deletion of the RhoA gene and RhoA stream targets Notch3, Hes1, and Deltex1 were elevated in RhoA- protein found that residual thymocytes in mutant mice were par- deficient DN cells (Fig. 3C), possibly reflecting a compensatory tially depleted of RhoA, suggesting that there is strong selection effect of increased cell apoptosis, whereas the expression levels of against the loss of RhoA (Fig. 1D). Because CD2-Cre may also IL-7R and its signaling transducer Bcl2 appeared normal (Fig. 3D). mediate gene deletion in B cell lineage, to achieve T cell–specific We then examined whether the increased apoptosis in RhoA2/2 deletion, we crossed RhoAflox/flox mice with Lck-Cre transgenic DN cells was caused by defective pre-TCR expression. To this mice. We found that RhoA deletion by Lck-Cre also caused end, we restored pre-TCR expression in RhoA2/2 DN cells by a decrease in thymocyte numbers (Fig. 1E). However, the decrease crossing RhoA2/2 mice with p14TCR transgenic mice (Fig. 3E). appears to be less profound compared with that by CD2-Cre– We found that the restoration of pre-TCR expression in resultant mediated RhoA deletion. This might be explained by relatively RhoA2/2;p14TCR Tg mice partially rescued DN cell survival and late deletion of RhoA by Lck-Cre. Indeed, CD2-Cre is known to distribution (Fig. 3F, 3G). Taken together, these data suggest that delete genes earlier than Lck-Cre (26), and CD2-Cre was able to RhoA regulates DN cell survival during b-selection independent of delete RhoA in DN1 cells but Lck-Cre failed to do so (Fig. 1F). IL-7R and Notch signaling but dependent on pre-TCR expression. 4 RhoA IN THYMOCYTE DEVELOPMENT Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. RhoA deficiency causes thymocyte hypocellularity. (A) The numbers of total thymocytes from RhoAflox/flox (WT) and RhoAflox/flox;CD2-Cre (RhoA2/2) mice. (B) Left panel, Representative flow cytometry plots of thymocyte subsets. The percentage of cells in each corresponding quadrant is indicated. Right panel, quantification of the numbers of thymocyte subsets. DN, CD42CD82 DN, DP, CD4+CD8+ DP. (C) Left panel, Representative flow cytometry plots of thymocyte subsets gated from DN cells. The percentage of cells in each corresponding quadrant is indicated. Right panel, Quantification of the numbers of thymocyte subsets within DN cells. DN1, CD44+CD252; DN2, CD44+CD25+; DN3, CD442CD25+; and DN4, CD442CD252.(D) Upper panel, RhoA protein expression. Thymocytes from WT and RhoA2/2 mice were detected for RhoA expression by Western blot. Lower panel, Efficiency of RhoA gene deletion. DNA from flow cytometry–sorted cells was used as PCR template to determine the floxed and knockout (KO) alleles of RhoA. Data are representative of three mice per genotype. (E) Quantification of the numbers of thymocyte subsets in RhoAflox/flox;Lck-Cre2 and RhoAflox/flox;LCK-Cre+ mice. (F) Comparison of RhoA gene deletion in DN1 cells from RhoAflox/flox;LCK-Cre+ mice with that from RhoA2/2 mice. DNA from flow cytometry–sorted DN1 cells was used as PCR template to determine the floxed and KO alleles of RhoA. (G) Comparison of the frequency of ETPs in RhoAflox/flox;LCK-Cre+ mice with that in RhoA2/2 mice. ETPs were identified by flow cytometry as CD42CD82Thy1.2+CD44+c-Kit+CD252 cells. n = 5 mice/genotype for (A)–(C)and(E). For (G), n =4WTand7RhoA2/2 mice; and n = 9 mice for both RhoAflox/flox;LCK-Cre2 and RhoAflox/flox;LCK-Cre+. Error bars represent SD. *p , 0.05, **p , 0.01.

Defective positive selection and lineage commitment in the had fewer TCRloCD692 thymocytes (preselection DP thymocytes), absence of RhoA likely because of defective b-selection. However, the proportion of int + RhoA deficiency led to reduced cellularity of the CD4+ SP and TCR CD69 cells (mainly DP thymocytes at the initial stage of CD8+ SP thymocytes. The decreased numbers of SP thymocytes positive selection) was increased in the absence of RhoA, whereas hi + hi 2 may not only result from defective b-selection but also from im- TCR CD69 (immature SP thymocytes) and TCR CD69 cells 2/2 paired positive selection and/or lineage commitment. To examine (mature SP thymocytes) were lower in RhoA mice. These data positive selection in RhoA2/2 mice, we analyzed DP, CD4+ SP, and suggest that RhoA-deficient thymocytes were blocked at the initial CD8+ SP thymocytes for the surface expression of CD69, upregu- stage of positive selection. The detrimental effect of RhoA defi- 2/2 lation of which is a critical marker of successful positive selection ciency on positive selection was reaffirmed in RhoA ;p14TCR (34). We found that RhoA2/2 mice had more CD69hiCD4+CD8+ Tg mice. p14TCR mice express TCRVa2Vb8 that specifically but less CD69hiCD4+ and CD69hiCD8+ thymocytes than that of the recognizes MHC class I molecule–restricted lymphocytic chorio- WT control mice (Fig. 4A). To determine at which specific point meningitis virus epitope gp33-41 and thus are dominated with mono- DP thymocyte development was blocked in RhoA2/2 mice, we clonal CD8+ T cells (35). Because the majority of DP thymocytes analyzed the expression of TCRb together with CD69 in total recognize self-ligands enabling positive selection, p14TCR mice are 2 2 thymocytes (1, 34) by FACS. As shown in Fig. 4B, RhoA / mice biased toward positive selection. RhoA deficiency in p14TCR mice The Journal of Immunology 5

FIGURE 2. RhoA deficiency causes a defect in b-selection. (A) Genomic DNA PCR analysis of V(D)Jb recombination in flow cytometry–sorted 50,000 DN3 cells. The application of eF-1a was used as input control. Data are representative of three mice per genotype. (B) Flow cytometry analysis of IcTCRb protein expression. Left panel, Representative flow cytometry plots of IcTCRb+ and IcTCRb2 cells gated from DN3a (CD42CD82Thy1.2+CD442CD25+ CD282), DN3b (CD42CD82Thy1.2+CD442 CD25+CD28+), or DN4 (CD42 CD82 Thy1.2+CD442CD252CD28+) cells. The percentage of IcTCRb+ and IcTCRb2 cells is indicated above bracketed lines. Right Downloaded from panel, Mean percentage and numbers of IcTCRb+ and IcTCRb2 cells. (C) Flow cytometry analysis of cell surface protein expression of TCRb. TCRb+ cells were gated from DN3 (CD42CD82Thy1.2+ CD442CD25+)orDN4(CD42CD82Thy1.2+ CD442CD252) cells. Mean percentage of http://www.jimmunol.org/ DN3 and DN4 cells expressing TCRb is shown. (D) mRNA expression of pre-Ta in DN3 (CD42CD8-Thy1.2+CD442CD25+) cells. The data are presented as fold expression relative to one WT mouse. n =4WT and 7–8 RhoA2/2 mice for (B)–(D). Error bars represent SD. *p , 0.05, **p , 0.01. by guest on September 27, 2021

resulted in a marked decrease in CD8+ SP thymocytes and lower CD4+ SP and CD8+ SP, but not in DP, thymocytes. Because RhoA expression of Va2 TCR on CD8+ SP thymocytes (Fig. 4C). Taken has been shown to play an important role in the M phase (mitosis) together, these results indicate that RhoA is required for thymo- of the cell cycle (25, 37, 38), we examined whether mitosis was cyte positive selection. defective in RhoA-deficient DP thymocytes. Analysis of nuclear After the initiation of positive selection, DP thymocytes contents by using Imagestreamx imaging flow cytometer (Amnis) downregulate CD4 and CD8, passing through a CD4+CD8int found that RhoA deficiency resulted in a significant elevation of transitional stage before committing to mature CD4+ SP and multinucleated cells in DP thymocytes when the cells were cultured CD8+ SP thymocytes (1). We found that RhoA2/2 mice had with anti-CD3/-CD28 Abs (Fig. 5C–E). Both F-actin and b-tubulin fewer CD4+CD8int cells (Fig. 4D). However, the frequency of assemblies, two critical structural components of mitosis (39, 40), mature (HSA2CD62Lhi)CD4+ SP and CD8+ SP thymocytes were abolished in RhoA-deficient multinucleated cells (Fig. 5D, was not altered by RhoA deficiency (Fig. 4E), suggesting that 5E). RhoA2/2 DP thymocytes bearing a single nucleus also showed RhoA deficiency causes defects in the early, but not the late, an impaired b-tubulin staining, compared with WT counterparts stage of lineage commitment. Thus, RhoA is not only required for (Fig. 5F). These data suggest that RhoA is critical for regulating thymocyte-positive selection but also for lineage commitment. mitosis of DP thymocytes through controlling the actin and tubulin cytoskeleton. Consistent with defective mitosis, RhoA-deficient DP Impaired survival and proliferation in DP and CD4+ and thymocytes proliferated slower than WT control cells in response to CD8+ SP thymocytes in the absence of RhoA TCR cross-linking with anti-CD3/-CD28 Abs (Fig. 5G). Because Positive selection is accompanied by cell survival and proliferation survival and proliferation activities depend on TCR signaling in (36). It is thus logical to postulate that impaired survival and pro- developing DP thymocytes, we analyzed Ic signaling events in + + liferation could contribute to the decreased DP, CD4 SP, and CD8 sorted DP thymocytes from RhoA2/2 and WT mice. As shown 2/2 SP thymocytes in RhoA mice. Indeed, Annexin V staining in Fig. 5H, the phosphorylation of ZAP70, ERK, and JNK was + revealed that there was an increased apoptosis in DP, CD4 SP impaired in the absence of RhoA, suggesting that RhoA is essential + and CD8 SP mutant thymocytes (Fig. 5A). Interestingly, although for TCR signaling. RhoA2/2 CD4+ SP and CD8+ SP thymocytes had less BrdU incorporation, DP thymocytes from RhoA2/2 mice had comparable Altered mitochondrial function in RhoA-deficient thymocytes BrdU incorporation to that of WT counterparts (Fig. 5B), suggest- Cell survival and proliferation are regulated by mitochondrial ing that the S phase (DNA synthesis) of the cell cycle is impaired in energy metabolism (41, 42). In thymocytes, it has been indicated 6 RhoA IN THYMOCYTE DEVELOPMENT

FIGURE 3. RhoA deﬁciency causes an impaired proliferation and/or survival in DN3 and DN4 thymocytes that can be rescued by restoration of pre-TCR expression. (A) Flow cytometry analysis of BrdU incorporation. BrdU+ cells were gated from DN3 or DN4 cells. Mean percentage of BrdU+ cells is shown. (B) Flow cytometry analysis of apoptosis. Annexin V+ cells were gated from DN3 or DN4 cells. Mean frequency of Annexin V+ cells is shown. (C) mRNA expression of Notch signaling molecules in DN4 cells. The data are presented as fold expression relative to one WT mouse. (D) Flow cytometry analysis of protein expression a of cell surface IL-7R and Ic Bcl-2 in DN3, Downloaded from IcTCRb+ DN4, and IcTCRb2 DN4 cells. Mean ﬂuorescence intensity (MFI) is shown. (E) Flow cytometry analysis of IcTCRb protein expression in DN3 and DN4 cells from WT;p14TCR Tg and RhoA2/2;p14TCR Tg mice. Mean percentage of IcTCRb+ cells is shown. (F) Flow cytometry analysis of apoptosis in DN3 or DN4 http://www.jimmunol.org/ cells from the non-Tg and Tg mice is indicated. Mean frequency of Annexin V+ cells is shown. (G) Flow cytometry analysis of the frequency of DN3 and DN4 cells in the non-Tg and Tg mice is indicated. n = 3–6 mice/genotype. The results from a representative experiment of two (A, C, and D) or three (B) independent experiments are shown. Error bars represent SD. **p , 0.01. by guest on September 27, 2021

that Notch promotes DN cell survival by regulating glucose stantiate this hypothesis, we first measured OCR, an indicator of metabolism (33). Because RhoA deficiency caused defects in oxidative phosphorylation (OXPHOS), and ECAR, an indicator thymocyte survival and proliferation, we hypothesized that de- of aerobic glycolysis, by Seahorse XF Cell Mito Stress Test and pletion of RhoA may affect mitochondrial function. To sub- XF Glycolysis Test, respectively. To our surprise, RhoA-deficient

FIGURE 4. RhoA deﬁciency causes an impaired positive selection. (A) Flow cytometry analysis of CD69+ cells gated from DP, CD4+ SP, or CD8+ SP thymocytes. Mean frequency of CD69+ cells is shown. (B) Flow cytometry analysis of CD69 and TCRb expression in total thymocytes. (C) Flow cytometry analysis of the frequency of CD8+ SP and TCRVahiCD8+ SP thymocytes in WT;p14TCR Tg and RhoA2/2;p14TCR Tg mice. (D) Flow cytometry analysis of CD4+CD8int cells gated from TCRhi CD69+ population. Mean frequency of CD4+CD8int cells is shown. (E) Flow cytometry analysis of HSA2CD62Lhi cells gated from CD4+ SP or CD8+ SP thymocytes. Mean frequency of HSA2CD62Lhi cells is shown. n = 3 mice/genotype. The results from a representative experiment of two independent experiments are shown. Error bars represent SD. *p , 0.05, **p , 0.01. The Journal of Immunology 7

FIGURE 5. RhoA deficiency causes an impaired survival and/or proliferation in DP and CD4+ SP and CD8+ SP thymocytes. (A) Flow cytometry analysis of apoptosis. Annexin V+ cells were gated from DP, CD4+ SP, or CD8+ SP cells. Mean frequency of Annexin V+ cells is shown. (B) Flow cytometry analysis of BrdU incorporation. BrdU+ cells were gated from DP, CD4+ SP, or CD8+ SP cells. Mean percentage of BrdU+ cells is shown. (C) Imagestream analysis of nuclear contents in DP thymocytes cultured with anti-CD3/- CD28 Abs. Mean frequency of multinucleated cells is shown. (D and E) Imagestream analysis of F-actin (D) and b-tubulin (E) distribution in DP Downloaded from thymocytes cultured with anti-CD3/- CD28 Abs. Images (original magnifi- cation 360) shown in each panel are from samples pooled from at least three mice of same genotypes. (F) Quantifi- cation of mean fluorescence of F-actin http://www.jimmunol.org/ and b-tubulin staining in DP thymocytes bearing single nucleus. (G) Pro- liferation rate of DP thymocytes cultured with anti-CD3/-CD28 Abs. Fold growth relative to WT cells cultured without anti-CD3/-CD28 is shown. (H) Western blot analysis of TCR signaling events in DP thymocytes. The results are from samples pooled from at least three mice of same genotypes and by guest on September 27, 2021 represent two independent experiments. For (A) and (B), n = 3 mice/genotype and the results are from a representative experiment of two (A) or three (B) independent experiments. For (C), n =4 WTand6RhoA2/2 mice. For (G), n =3 mice/genotype. Error bars represent SD. *p , 0.05, **p , 0.01.

thymocytes showed an increase in both basal and maximal electron ATPproductioninRhoA2/2 thymocytes may be due to the transport chain accelerator FCCP-induced respiration (Fig. 6A), increased ROS. Indeed, flow cytometry analysis of ROS levels which coincided with an upregulation of a number of mitochondrial found that consistent with the increased OXPHOS, ROS were genes involved in OXPHOS, including Nrf1, Atp5I, Ndufa2, and significantly elevated in all RhoA2/2 thymocyte subsets (Fig. 7A). Cox5a (Fig. 6B) (43). Consistent with the increased OXPHOS, ROS can be deleterious to cells (42, 44). To test whether the RhoA2/2 thymocytes exhibited higher ECAR/glycolysis and in- increased ROS are responsible for the defective thymocyte creased expression of Hk2, Slc2a, Pdk1, and Pgm1, all of which development in RhoA2/2 mice, we treated the mice with anti- have been shown to be important for glycolysis (Fig. 6C, 6D) (43). oxidant N-acetylcysteine (NAC) to scavenge ROS. NAC re- We subsequently measured mitochondrion mass and mitochondrial pressed ROS production in RhoA2/2 thymocyte subsets to levels membrane potential by flow cytometry with Mito Tracker Green comparable to that in NAC-treated WT thymocytes (Fig. 7B). and DILC-5, respectively. RhoA deficiency led to an increase in mito- The increase in cell apoptosis in RhoA2/2 thymocytes was par- chondrion contents and mitochondrial membrane potential in all tially reversed by NAC treatment (Fig. 7C). NAC also partially thymocyte subsets (DN, DP, CD4+ SP, and CD8+ SP) (Fig. 6E, 6F). reversed the increase in the frequency of DN thymocytes and Furthermore, mitochondrial DNA copy numbers were found the decrease in the frequency of DP thymocytes in RhoA2/2 increased by .200-fold in the absence of RhoA (Fig. 6G). However, mice (Fig. 7D). In addition, the hypocellularity in total thy- ATP production in RhoA-deficient thymocytes was reduced (Fig. 6H). mocytes and thymocyte subpopulations in RhoA2/2 mice Because mitochondrial oxidation may generate ROS to sup- was partially rescued by NAC treatment (Fig. 7E). Taken to- press ATP production (44, 45), we reasoned that the decline in gether, these data suggest that RhoA represses mitochondrial 8 RhoA IN THYMOCYTE DEVELOPMENT

FIGURE 6. RhoA deficiency causes an enhanced mitochondrial function in thymocytes. (A) OCR in total thymocytes under basal condition and in response to oligomycin and FCCP. (B) mRNA expression of genes involved in oxidative phosphorylation in total thymocytes. The data are presented as fold expression relative to one WT sample. (C) ECAR in total thymocytes under basal condition and in response to glucose, oligomycin, and 2 deoxy-D- glucose (2-DG). (D) mRNA expression of genes involved in glycolysis in total thymocytes. The data are presented as fold expression relative to one WT sample. (E) Quantification of mitochondrion numbers/mass by flow Downloaded from cytometry analysis of Mitotracker Green staining in total thymocytes and thymocyte subsets. Mean fluorescence intensity (MFI) is shown. (F) Mitochondrial membrane potential assayed by flow cytometry of DilC-5 staining in total thymocytes and thymocyte subsets. MFI G is shown. ( ) Mitochondrial DNA contents in http://www.jimmunol.org/ total thymocytes. Mitochondrial DNA was represented by mitochondrial Cox2 normalized to nuclear b-globin. The data are presented as mitochondrial DNA relative to one WT mouse. (H) ATP levels in total thymocytes. In (A)–(D), the results are averaged from three samples per genotype with each sample pooled from at least three mice of same genotypes. In (E)–(H), n =5 mice/genotype. Error bars represent SD. *p , 0.05, **p , 0.01. by guest on September 27, 2021

metabolism and ROS production, contributing to its regulation of optosis, the increased apoptosis in RhoA2/2 DN4 cells likely thymocyte development. reflects the increased frequency of IcTCRb2 DN4 cells in the mutant mice. Discussion Survival and proliferation of thymocytes at the pre-TCR In this study, we report that RhoA is essential for thymocyte de- checkpoint are not only regulated by pre-TCR but also by IL-7R velopment, with RhoA deletion leading to multiple defects during and Notch (26, 46). The expressions of IL-7R and its signaling thymopoiesis. RhoA regulates a complex mechanism governing transducer Bcl2 remained unchanged in RhoA-deficient DN cells, b-selection, positive selection, cell survival and proliferation, and whereas Notch1 and a few Notch1 targets were upregulated in the pre-TCR and TCR signaling. Importantly, RhoA integrates energy absence of RhoA. The intact IL-7R signaling may reflect an metabolism in this process. overlapping role of RhoA with the closely related RhoB and/or During thymocyte development, the pre-TCR checkpoint is RhoC, whereas the increased Notch signaling may be a compen- crucial for DN cell transition to DP cells (26). DN cells that satory effect of the increased apoptosis. However, we cannot ex- successfully express pre-TCR are prevented from programmed clude the possibility that the elevated Notch signaling is intrinsic cell death and are committed to proliferate and differentiate to to RhoA deletion (i.e., RhoA directly negatively regulates Notch DP cells (26, 33). We show that RhoA2/2 DN3 cells express signaling). Indeed, Rac1/Rac2 and Cdc42 of the Rho GTPase reduced Ic and cell surface TCRb. Consistent with this, we family have been shown to inhibit Notch signaling during mouse found fewer IcTCRb+ DN4 cells in RhoA2/2 mice. RhoA2/2 T cell development and Drosophila wing growth/development, DN cells also had less pre-Ta expression. These data suggest respectively (26). that RhoA is crucial for pre-TCR expression. By expressing It is interesting that RhoA regulates proliferation of DP and transgenic TCR to restore pre-TCR expression in RhoA2/2 DN SP cells through different mechanisms. RhoA deficiency resulted cells, we demonstrate that RhoA-regulated pre-TCR expression in a reduced BrdU incorporation in CD4+ SP and CD8+ SP contributes to DN cell survival. Furthermore, because DN4 thymocytes. In contrast, the levels of BrdU incorporation in cells that do not express TCRb are usually eliminated by ap- RhoA2/2 DP thymocytes were indistinguishable to those in WT The Journal of Immunology 9

FIGURE 7. RhoA promotes thymocyte development partially through repressing ROS hyperproduction. (A) RhoA2/2 thymocytes show increased levels of ROS revealed by flow cytometry analysis of DCFDA staining in total thymocytes and thymocytes subsets. Mean fluorescence intensity (MFI) is shown. (B)ROS scavenger NAC treatment reverses the ROS levels in RhoA2/2 thymocytes. WT and RhoA2/2 mice were injected i.p. with 15 mg/g body weight NAC every other day for 14 d. Ten days after last NAC injection, the mice were sacri- ficed, and thymocytes were harvested, stained with CD4, CD8, and DCFDA, and analyzed by flow cytometry. (C) NAC treatment partially rescues cell apoptosis in RhoA2/2 thymocytes. Mice were treated with NAC as described in (B). Apoptosis in thymocyte subsets was assayed by flow cytometry analysis of Annexin V+ cells. (D and E) NAC treatment partially reverses the Downloaded from changes in frequency of DN and DP thymocytes (D) and partially rescues thymocyte hypocellularity (E) in RhoA2/2 mice. Mice were treated with NAC as described in (B). The frequency and numbers of thymocyte subsets were revealed by flow cytometry analysis. In (A), n = 5 mice/genotype. In (B)–(E), n =3mice/ http://www.jimmunol.org/ genotype. The results from a representative experiment of two independent experiments are shown. Error bars represent SD. *p , 0.05, **p , 0.01.

control cells. Nonetheless, RhoA2/2 DP thymocytes contained cellular damage. For example, sustained ROS inhibit peripheral

more multinucleated cells compared with WT counterparts. T cell proliferation and induce apoptosis in thymocytes (50, 51). by guest on September 27, 2021 Therefore, RhoA appears to promote mitosis in DP thymocytes We therefore reasoned that the increased ROS levels in RhoA- but DNA synthesis in CD4+ SP and CD8+ SP thymocytes. The role deficient thymocytes might contribute to the increased apoptosis of RhoA in mitosis of DP thymocytes seems to be broadly con- and dampened thymocyte development. To test this, we treated served because RhoA is also involved in mitosis of a number of RhoA2/2 mice with ROS scavenger NAC and found that re- other cells including keratinocytes, mouse embryonic fibroblasts, moval of excessive ROS by NAC partially restored cell survival and hematopoietic progenitor cells (25, 37, 38). and the frequency and numbers of thymocytes. These results It has been shown that inactivation of RhoA in C3 transferase suggest that RhoA regulates thymocyte survival and develop- transgenic mice blocked thymocyte development (18, 19). However, ment not only through governing pre-TCR expression but also compared with the C3 mice that showed no survival defect in DN4 oxidative stress. Because a low level of ROS can act as signaling cells (19), RhoA2/2 mice exhibited increased apoptosis in this pop- intermediates that in general are beneficial to cells (50, 52), our ulation. Moreover, whereas C3 mice showed normal positive selec- findings indicate, to our knowledge, for the first time that a fine- tion (18), RhoA2/2 mice were impaired at this checkpoint. These tuned ROS production regulated by RhoA through a delicate differences are likely due to the nonspecific nature of C3 transferase, control of metabolic programs is important for thymocyte de- which inactivates RhoA as well as RhoB and RhoC (24). velopment. In sum, our study demonstrates that RhoA coor- Metabolic pathways have been appreciated to be essential for dinates multiple developmental events to regulate thymopoiesis, naive T cell activation and effector T cell differentiation (43, 45, providing a missing link between mitochondrial metabolism and 47, 48). However, it remains poorly understood whether and how thymocyte development. metabolic programs are regulated in thymocyte development. An intriguing finding from our study is that RhoA appears to serve as a coordinator of mitochondrial metabolic programs with thymocyte Disclosures development. RhoA deficiency in thymocytes resulted in an in- The authors have no financial conflicts of interest. creased mitochondrial function. Although it suggests that RhoA represses mitochondrial metabolic programs, we cannot rule out the References 2/2 possibility that the enhanced mitochondrial function in RhoA 1. Wang, D., M. Zheng, L. Lei, J. Ji, Y. Yao, Y. Qiu, L. Ma, J. Lou, C. Ouyang, thymocytes reflects, at least in part, a compensatory effect of the X. Zhang, et al. 2012. Tespa1 is involved in late thymocyte development through increased apoptosis. the regulation of TCR-mediated signaling. Nat. Immunol. 13: 560–568. 2. Lucas, J. A., L. O. Atherly, and L. J. Berg. 2002. The absence of Itk inhibits It is known that mitochondria are a major source of ROS, where positive selection without changing lineage commitment. J. Immunol. 168: they are produced as byproducts of OXPHOS (42, 49). In line 6142–6151. 3. Wakabayashi, Y., H. Watanabe, J. Inoue, N. Takeda, J. Sakata, Y. Mishima, with the increased OXPHOS, ROS production was elevated in J. Hitomi, T. Yamamoto, M. Utsuyama, O. Niwa, et al. 2003. Bcl11b is required RhoA-deficient thymocytes. Excessive ROS can lead to broad for differentiation and survival of ab T lymphocytes. Nat. Immunol. 4: 533–539. 10 RhoA IN THYMOCYTE DEVELOPMENT

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