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Activation and FunctionMetabolic Programming for PropermTORC1-Coupled Immune Signals and Ancestral T Cells in Fish Require

Jialong YangXiumei Wei, Kete Ai, Huiying Li, Yu Zhang, Kang Li and

http://www.jimmunol.org/content/203/5/1172doi: 10.4049/jimmunol.1900008July 2019;

2019; 203:1172-1188; Prepublished online 26J Immunol 

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8.DCSupplementalhttp://www.jimmunol.org/content/suppl/2019/07/26/jimmunol.190000

Referenceshttp://www.jimmunol.org/content/203/5/1172.full#ref-list-1

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The Journal of Immunology

Ancestral T Cells in Fish Require mTORC1-Coupled ImmuneSignals and Metabolic Programming for Proper Activationand Function

Xiumei Wei,*,1 Kete Ai,*,1 Huiying Li,* Yu Zhang,* Kang Li,* and Jialong Yang*,†

T cells suddenly appeared in jawed fish ∼450 million years ago. Biological studies of fish T cells may provide helpful evidence to

understand evolution of adaptive immune systems. To this end, using a Nile tilapia (Oreochromis niloticus) model, we revealed the

regulatory mechanism of adaptive immunity mediated by ancestral T cells in jawed fish. Nile tilapia T cells as well as a tightly

regulated mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway participate in the cellular adaptive

immune response during Streptococcus agalactiae infection. Blockade of mTORC1 signaling by rapamycin impairs T cell acti-

vation and Ag-induced proliferation in this early vertebrate. More critically, we show that signals from mTORC1 are indispens-

able for primordial effector T cells to eliminate infection by promoting the expression of proinflammatory cytokines,

cytotoxic-related molecules, and proapoptotic genes. Mechanistically, teleost mTORC1 directs effector T cell function by coor-

dinating multiple metabolic programs, including glycolysis, glutaminolysis, and lipogenesis through activating key transcription

factors c-Myc, HIF-1a, and sterol regulatory element–binding proteins, and thus links immune signals to metabolic reprogram-

ming in jawed fish. To our knowledge, these results represent the first description of the regulatory mechanism for T cell–mediated

adaptive immunity in a fish species. From an evolutionary viewpoint, our study suggests that primordial T cells are armed with

sophisticated regulatory strategies like those in modern T cells prior to the divergence of bony fish from the tetrapod lineage.

Therefore, our findings fill in an important gap regarding evolution of the adaptive immune system. The Journal of Immunology,

2019, 203: 1172–1188.

Tcells are principal components of the adaptive immunesystem for vertebrates (1, 2). Despite remarkable progressthrough studies on the T cell–mediated response in

mammals, our knowledge regarding T cell evolution remainslimited. As fish comprise the first evolutionary group with an in-tegral adaptive immune system, they are considered ideal modelsfor determining fundamental immunological events, such as theorigin of T/B lymphocytes as well as the evolution of regulatorymechanisms for adaptive immunity (3–6). Recent advances in avariety of teleost species have revealed that their ancestral adap-tive immune system is armed with the same dedicated array of fun-damental weapons as that of tetrapod, including T/B lymphocytes

with somatically rearranged receptors (TCRs/BCRs), CD4/MHCclass II and CD8/MHC class I molecules, and reminiscent IgsIgM/D/T (7–9). As with modern CD4+ Th cells, CD4+ leukocytesof many bony fish, such as zebrafish and rainbow trout, exhibitAg-induced Th1, Th2, and Th17 cytokine responses (5, 10, 11).These cytokine-producing CD4+ leukocytes are therefore identi-fied as potential equivalents of Th cells in tetrapod. Additionally,primitive CD8+ leukocytes from fugu, rainbow trout, and ginbunacrucian carp undergo prompt expansion in response to pathogeninfection or phytohemagglutinin (PHA) stimulation, express highlevels of IFN-g, perforin A, granzyme B, and granulysin, arecytotoxic to microbes or pathogen-infected cells, and provide ef-ficient protection against secondary infection (12–15). Thus, theT cell–mediated response appears to be a universal feature ofadaptive immunity in all jawed vertebrates. Nonetheless, theunderlying mechanisms responsible for the functions exerted byprimordial T cells are completely unknown and remain to beelucidated.The exquisite activation signaling of modern T cells has been

well illustrated in mammals. Following TCR engagement, fun-damental events, including LCK activation, CD3 ITAMs, ZAP70phosphorylation, and LAT signalosome recruitment/formationoccur. Activated PLCg1, a component of the LAT signalosome,hydrolyzes PIP2 into two important second messengers: IP3 andDAG. IP3 leads to an increase of cytosolic free Ca2+ and trig-gers CaM-NFAT signaling, whereas DAG allosterically activatesRasGRP1 and PKCu, followed by initiation of the Ras-Erk1/2-AP1and CARMA1/Bcl10/MALT-IKK–NF-kB pathways, respectively(16, 17). Collectively, downstream signals from IP3 and DAGlead to the mobilization of multiple transcription factors thatare essential for T cell development, activation, survival, prolif-eration, and differentiation (16, 17). In addition, the mammalian/mechanistic target of rapamycin (mTOR) pathway is also indispensable

*State Key Laboratory of Estuarine and Coastal Research, School of Life Sciences,East China Normal University, Shanghai 200241, China; and †Laboratory for MarineBiology and Biotechnology, Qingdao National Laboratory for Marine Science andTechnology, Qingdao 266071, China

1X.W. and K.A. contributed equally to this work.

ORCID: 0000-0002-3112-5012 (J.Y.).

Received for publication January 3, 2019. Accepted for publication June 7, 2019.

This research was supported by the National Natural Science Foundation of China(Grant 31872591), the Shanghai Pujiang Program (Grant 18PJ1402700), and theFundamental Research Funds for the Central Universities to J.Y.

Address correspondence and reprint requests to Prof. Jialong Yang, East China NormalUniversity, No. 500, Dongchuan Road, Minhang District, Shanghai 200241, China.E-mail address: jlyang@bio.ecnu.edu.cn

The online version of this article contains supplemental material.

Abbreviations used in this article: FSC, forward scatter; mTOR, mammalian/mechanistic target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTORcomplex 2; 2-NBDG, 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-2-deoxyglucose;NCBI, National Center for Biotechnology Information; PFK, phosphofructokinase;PHA, phytohemagglutinin; PKM, pyruvate kinase; SREBP, sterol regulatory element–binding protein.

Copyright� 2019 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900008

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for diverse physiological processes in T cells (18). mTOR sig-naling consists of two distinct complexes: mTOR complex 1(mTORC1) and mTOR complex 2 (mTORC2). These complexesshare the same kinase catalytic subunit mTOR, an evolutionarilyconserved serine/threonine kinase (Akt) but are distinguished bythe signature components Raptor and Rictor, respectively. Fur-thermore, mTORC1 is sensitive to acute rapamycin treatment,whereas mTORC2 is resistant to rapamycin except under per-sistent treatment (18). mTORC1 phosphorylates multiple sub-strates, including the following: S6K1 and 4EBP1 to promoteribosome biogenesis, nucleic acid synthesis, and cap-dependenttranslation; HIF-1a, c-Myc, and sterol regulatory element–bindingprotein (SREBP) 1 to regulate cell metabolism; and ULK1 tosuppress autophagy (19, 20). In T cells, the upstream PI3K/AKT,PKCu-CARMA1, RasGRP1/Ras/Erk1/2 axis is critical for TCR-induced mTORC1 activation (21, 22). In turn, mTORC1 and itstight regulation are required for proper T cell activation (18), ef-fector CD8+ T cell expansion and memory CD8+ T cell formation(23, 24), CD4+ Th cell differentiation (25–27), induced regulatoryT cell function (28), and invariant NKT maturation (29, 30). Al-though mTOR pathway is evolutionarily conserved, the regula-tory mechanism underlying in adaptive immunity has mainlybeen described with mammalian models, and it remains to beestablished whether and how mTOR signaling modulates theadaptive immune response in early nontetrapod vertebrates.Accordingly, regulatory mechanism of the teleost T cell response,

as well as the involvement of mTOR pathway in the adaptive im-munity of early vertebrates, are still enigmas that need to be clearlydefined. To this end, a teleost model, the Nile tilapia (O. niloticus),was used to investigate how mTORC1 signaling modulates theadaptive immune response mediated by primordial T cells inearly nontetrapod. We found an intact and evolutionarily conservedmTORC1 pathway in Nile tilapia that appears to participate in theadaptive immune response. Inhibition of mTORC1 by rapamycinundisputedly impaired multiple immunological processes in primordialT cells, including activation, Ag-induced expansion, and infectionclearance. Moreover, mTORC1 ensured the proper function of teleosteffector T cells via regulating metabolic reprogramming. To ourknowledge, this study represents the first description of the regulatorymechanism of mTOR in the nonmammalian adaptive immune systemand reveals that the mTOR-modulated T cell response is an ancestralstrategy that already existed at the early stage of T cell evolutionpreceding the emergence of tetrapod. Our study fills an important gapin evolutionary knowledge regarding both mTOR pathway and T celllineage and thus is of fundamental importance for understanding theevolution of T cell–mediated immunity.

Materials and MethodsEthics statement

All fish care and experimental procedures were performed in accordance withthe Guide for the Care and Use from Laboratory Animals of the Ministry ofScience and Technology of China andwere approved by the East ChinaNormalUniversity Experimental Animal Ethics Committee with an approval numberF20140101. All efforts were made to minimize the suffering of the animals.

Fish maintenance

Nile tilapia larvae (∼3 cm) were obtained from an aquatic farm in Guangzhou,Guangdong Province, China, and maintained in a freshwater system with bio-filters and continuous aeration at 28˚C at the Biological Station of East ChinaNormal University. Fish were fed commercial pellets daily. Only healthy fishwith body lengths of 8–10 cm were used for the study.

Sequence, structure, and phylogenic analysis

The whole genomic sequence of Nile tilapia O. niloticus assembled byanother research group is available on the National Center for Biotechnology

Information (NCBI) GenBank (https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Oreochromis_niloticus/104/), whose annotation is referredto as “NCBI Oreochromis niloticus Annotation Release 104” and with theassembly identification of GCA_001858045.3 (31). cDNA and amino acidsequences of Nile tilapia and other species used in the present study weresearched in NCBI GenBank (https://www.ncbi.nlm.nih.gov) or Ensembl(http://asia.ensembl.org/index.html) and analyzed using the BLASTalgorithm. Protein domains were predicted with the simple modulararchitecture research tool (SMART) version 4.0, and sketch maps ofdomain organization were prepared with DOG version 2.0 software.Homology analysis was conducted using Ident and Sim Analysis pro-vided at http://www.bioinformatics.org/sms/. Multiple sequence align-ment was performed with the ClustalW Multiple Alignment program.Presumed tertiary structures were established using the SWISS-MODELprediction algorithm and displayed by PyMOL version 0.97. A phylo-genetic tree was constructed based on deduced amino acid sequencesusing the neighbor-joining algorithm with MEGA4.1 software. The ac-cession numbers of all selected genes in the present study are listed inSupplemental Table I and Supplemental Table II.

S. agalactiae infection

S. agalactiae was cultured on brain-heart infusion agar. Bacteria in theexponential phase were collected for the CFU assay and resuspended inPBS for infection. Nile tilapia individuals were i.p. injected with the in-dicated concentration of S. agalactiae in 100 ml of PBS. Control fish weremaintained in similar tanks and i.p. injected with the same volume of PBS.Throughout the experiment, fish were kept at 28˚C with aeration and feddaily.

Rapamycin treatment

Rapamycin from Streptomyces hygroscopicus (Sigma) was dissolved inDMSO at 10 mg/ml and diluted with PBS before use. For in vivo inhibi-tion, Nile tilapia was i.p. injected with 75 mg/kg rapamycin for threeconsecutive days before leukocytes were isolated for experiments. Forin vivo inhibition during infection, 2 d after S. agalactiae infection, fishwere i.p. injected with 75 mg/kg rapamycin for three consecutive days andfollowed by one chase every 2 d with the same dose of rapamycin. Thesame volume of PBS was injected as a control. For in vitro inhibition,leukocytes isolated from fish that received 3 d of rapamycin injection werecultured in DMEM containing 10% FBS and 1% penicillin/streptomycin inthe presence of 10 nM rapamycin.

Leukocyte isolation

Leukocytes from the blood and spleen were isolated according to a previousreport, with slight modifications (9). Briefly, blood was collected from Niletilapia through the caudal vessel and immediately diluted with a 2-foldvolume of prechilled anticoagulant (8.17 g/L NaCl, 19.8 g/L glucose,5.46 g/L citric acid, 8.82 g/L sodium citrate, 3.72 g/L EDTA, pH 6.6)before centrifuging and resuspending in DMEM. The spleen was thor-oughly macerated in DMEM, and the cell suspension was passed throughnylon mesh. The blood and splenocyte suspension were then layeredonto a 51/34% discontinuous Percoll (GE Healthcare) density gradientand centrifuged at 500 3 g for 30 min at room temperature with nobreak. Cells accumulated at the interface were collected, washed, pel-leted, and resuspended in DMEM (10% FBS) for further analysis.

In vitro proliferation

For proliferation assays, leukocytes isolated from fish that received 3 d ofrapamycin injection were labeled with 10 mMCFSE (Invitrogen) accordingto the manufacturer’s protocol for 9 min at room temperature and washedtwice with DMEM. The labeled cells were then cultured in DMEM(10% FBS) containing 1 mg/ml PHA in the presence of 0, 10, 100, or1000 nM of rapamycin at 28˚C for 48 or 72 h. CFSE dilution amongviable lymphocytes was analyzed by flow cytometer as a measure of celldivision.

BrdU incorporation

S. agalactiae–infected Nile tilapia individuals were i.p. injected with0.75 mg of BrdU in 200 ml of PBS 1 d before sacrifice, and spleenleukocytes were isolated for assays on days 4, 5, or 6 postinfection. Cellswere fixed with BD Cytofix/Cytoperm Buffer on ice for 30 min andwashed twice with BD Perm/Wash Buffer. Followed by 10-min per-meabilization with BD Cytoperm Plus Buffer on ice and two times’ wash,the cells were subsequently refixed with BD Cytofix/Cytoperm Buffer onice for another 5 min and washed twice again. The cells were then treatedwith 300 mg/ml DNase at 37˚C for 1 h. After that, intracellular staining

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was then performed with an FITC–anti-BrdU Ab (1:100 diluted; BD) inPerm/Wash buffer at room temperature for 20 min before the samples wereanalyzed by flow cytometry.

Death and apoptosis assay

For the cell death assay, isolated leukocytes were stained by the Live/DeadFixable Violet Dead Cell Stain (Invitrogen) in FACS buffer according to themanufacturer’s protocol and were analyzed by flow cytometer. For apo-ptosis assays, freshly isolated spleen leukocytes were stained with AnnexinV (BioLegend) in Annexin V binding buffer (0.01 M HEPES/NaOH, pH7.4, 0.14 M NaCl, 2.5 mM CaCl2) at room temperature for 15 min; 7AAD(Life Technologies) was added to the samples shortly before collection byflow cytometer.

Glucose uptake assay

Spleen leukocytes were plated in 48-well plates at 13 107 cells per well inPBS. The cells were incubated with 100 mM fluorescent 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-2-deoxyglucose (2-NBDG; Life Technolo-gies) at 28˚C for 30 min. The reaction was stopped by removing the culturemedium and washing twice with prechilled PBS. Uptake of 2-NBDG wasanalyzed by flow cytometer after lymphocyte population gating.

Flow cytometer and cell sorting

Isolated leukocytes, CFSE-labeled proliferated cells, BrdU-stained cells,Live/Dead stained cells, and 2-NBDG uptaken cells were resuspended inPBS containing 2% FBS for FACS analysis. For intracellular LCK staining,leukocytes were fixed with BD Cytofix/Cytoperm Buffer and washed withBD Perm/Wash solutions. The fixed cells were then stained with AlexaFluor 647–conjugated–LCK Ab (LCK-01; BioLegend) on ice for 30 min.All the cells were collected using a BD FACSCalibur flow cytometer. Datawere analyzed using FlowJo software. For fresh cell sorting, isolatedleukocytes were collected using a BD FACSAria II flow cytometer. Thesorted cells were centrifuged and resuspended in DMEM (10% FBS) forfuture assays. When sorting LCK+ or LCK2 lymphocytes, freshly isolatedleukocytes were fixed with BD Biosciences Cytofix/Cytoperm in thepresence of RNase Inhibitor (400 U/ml; TaKaRa) and stained with AlexaFluor 647–conjugated–LCK Ab diluted in BD Biosciences Perm/Washsolutions containing 400 U/ml Rnase Inhibitor. The stained cells werethen sorted with a BD FACSAria II flow cytometer.

Transmission electron microscopy assay

Sorted cells were fixed overnight in 2.5% glutaraldehyde, washed with0.1 M sodium cacodylate, and postfixed with 1% osmium tetroxide for 1 h.After washing with sodium cacodylate and dehydrating in graded acetone,the cells were embedded in graded Epon 812 and kept at 60˚C for 2 d.Sections of 50–70-nm thickness were prepared and stained with 2% uranylacetate and lead citrate before observation with a transmission electronmicroscope (HT7700; Hitachi).

Leukocyte stimulation

For biochemistry analyses, we rested spleen leukocytes in Dulbecco’s PBSwith Ca2+ and Mg2+ for 30 min and then stimulated them with PMA(50 ng/ml) plus ionomycin (500 ng/ml) for different times. Immediatelyafter stimulation, the cells were lysed in lysis buffer (1% NP-40, 150 mMNaCl, 50 mM Tris, pH 7.4) with freshly added protease and phosphorylaseinhibitors. The cell lysates were subjected to immunoblotting analysis.Meanwhile, stimulated cells were harvested for real-time RT-PCR andimmunofluorescence studies. For T cell activation and proliferation assays,spleen leukocytes were cultured in DMEM (10% FBS) and stimulated with1 mg/ml PHA for different times. The stimulated cells were harvested forthe indicated assay.

Western blot analysis

Freshly isolated leukocytes, in vitro–stimulated leukocytes, and tissues cutinto small pieces were lysed in lysis buffer (1% NP-40, 150 mM NaCl,50 mM Tris, pH 7.4) with freshly added protease and phosphorylase in-hibitors. After centrifugation at 10,000 3 g for 10 min, the supernatantswere subjected to SDS-PAGE followed by Western blot analysis usingindicated primary Abs. The Abs for mTOR (2983), Raptor (2280), GbL(3274), S6 (2217), 4EBP1 (9644), phospho-mTOR S2448 (5536),phospho-S6K1 Thr421/Ser424 (9204), phospho-S6 Ser240/244 (5364),phospho-4EBP1 Thr37/46 (2855), Hexokinase 2 (HK2) (2867),c-Myc (13987), HIF-1a (14179), and b-actin (4970) were purchased fromCell Signaling Technology, and Ab for SREBP1 (Sc-365513) was fromSanta Cruz Biotechnology. The binding of primary Ab was detected by

goat anti-rabbit or mouse IgG H&L Alexa Fluor 790 or 680 (Abcam) andobserved by Odyssey CLx Image Studio. The b-actin was selected as in-ternal control. The sizes of targeting proteins were listed in SupplementalTable I.

Immunofluorescence studies

Freshly harvested tissues were embedded in OCT Compound (Leica) andfrozen immediately at 220˚C for 1 d before 7-mm frozen sections wereprepared. The sections were fixed in prechilled acetone for 15 min. Afterblocking with PBS containing 3% BSA for 1 h at room temperature,samples were stained using a primary rabbit anti-mTOR Ab (Cell Sig-naling Technology), followed by a secondary Alexa Fluor 594–conjugatedgoat anti-rabbit IgG (Abcam). The samples were mounted with mountingsolution containing DAPI, and images were acquired using a ZeissApoTome Microscope and analyzed using Photoshop CS4 software. Thespleen leukocytes stimulated with PMA plus ionomycin were collectedonto slides using Cytospin 4 before being fixed and blocked as above. Thecells were stained using a primary rabbit anti–phospho-mTOR S2448, rabbitanti–phospho-S6 Ser240/244, or rabbit anti–phospho-4EBP1 Thr37/46 plus amouse anti–b-actin Ab (Cell Signaling Technology). After staining withsecondary Alexa Fluor 488–conjugated goat anti-rabbit IgG (Abcam) plusAlexa Fluor 594–conjugated goat anti-mouse IgG (Abcam), the samples weremounted, imaged, and analyzed as above.

Histological studies

Livers harvested from control or S. agalactiae–infected fish on day8 postinfection were fixed in 10% formalin solution for 1 d, which wasthen changed to 70% ethanol. The fixed tissues were dewatered in gradedethanol series, cleaned in xylene, and then embedded in paraffin. Sectionsof 7 mm were prepared and stained with H&E according to standardprotocols for histological observation. The stain sections were observedunder Olympus BX53 microscope using the AxioVision software.

Real-time RT-PCR

Total RNAwas extracted from freshly isolated or in vitro–stimulated spleenleukocytes with TRIzol reagent (Invitrogen). After treating total RNAwithDNase I (Promega), first-strand cDNAwas synthesized using total RNA asthe template, oligonucleotide-adaptor primer (59-GGCCACGCGTCGAC-TAGTACT17-39), and M-MLV reverse transcriptase (Promega). The real-time RT-PCR was performed with a Mastercycler ep realplex (Eppendorf)using SYBR Green Mix (Thermo Fisher Scientific). b-Actin was selectedas internal control to normalize the template for corresponding samples, andthe expression level of target genes was analyzed by the 22OOcycle threshold

method (27). The gene-specific primers are listed in Supplemental Table II.

Examination of enzyme activity

Liver harvested from Nile tilapia with or without rapamycin treatment onday 5–7 after S. agalactiae infection was smashed in provided extractionbuffer using a grinding miller at 4˚C. After being centrifuged at 8000 rpm,4˚C for 10 min, the supernatant was used for examination of proteinconcentration and enzyme activities. Activities of phosphofructokinase(PFK), pyruvate kinase (PKM), glutaminase, and alanine aminotransferasewere measured with commercial assay kits (Nanjing Jiancheng Bioengi-neering Institute, China) according to the instructions of the manufacturer.The enzyme activity was calculated as per milligram of tissue protein.

Statistical analysis

Data are presented as the mean 6 SEM, and statistical significance wasdetermined by a two-tailed Student t test. The p values are defined asfollows: *p , 0.05, **p , 0.01, ***p , 0.001.

ResultsT lymphocytes play a pivotal role in the adaptive immunity ofNile tilapia

There are no reports to date about Nile tilapia lymphocytes andtheir immune function. As an initial step to characterize these cells,we isolated leukocytes from peripheral blood and the lymphoidorgan spleen. Two obvious cell layers were obtained after densitygradient centrifugation (Fig. 1A); the top layer contained mostlycell debris, and the bottom layer was composed mainly of intactcells. Based on flow cytometry forward scatter (FSC) and sidescatter analyses, three visible populations (R1–3) from PBLs and

1174 FISH NEED mTORC1 FOR PROPER T CELL ACTIVATION AND FUNCTION

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one dominant population (R1) from spleen leukocytes wereidentified, respectively (Fig. 1A). Giemsa staining showed thatcells in population R1 had a high nucleus/cytoplasm ratio, whichis consistent with typical lymphocyte morphology (Fig. 1B).The same was true when these cells were examined by trans-mission electron microscopy (Fig. 1C). These morphologicalfeatures, as well as a previous report of zebrafish leukocytes (6),strongly suggest the cells in population R1 to be lymphocytes.Additionally, R2 and R3 from PBLs were suspected to bemonocyte/macrophage and precursor cell populations, respec-tively (Fig. 1B, 1C).To investigate the potential participation of Nile tilapia lymphocytes

in antibacterial immunity, we infected fish with the pathogenS. agalactiae (32); 7 3107 CFU in a volume of 100 ml wasemployed because this dose resulted in gradual and moderatemortality (∼30%) within 10 d (Fig. 1D). Eight days postinfection,the frequency of lymphocytes among PBLs increased significantlycompared with the control (Fig. 1E, 1F). Although spleenlymphocyte frequencies were comparable between infected andcontrol animals (Fig. 1E, 1F), the absolute number of lymphocytesexpanded substantially in response to infection (Fig. 1G). Coin-cidentally, bacterial infection caused severe inflammatory infil-tration in the liver (Fig. 1H). Thus, teleost lymphocytes underwentrobust expansion upon pathogen infection and might play a pivotalrole in adaptive immunity.To further determine the involvement of T cell lineage during

antibacterial immune response, mRNA expression of the TCR andcoreceptor was examined by real-time RT-PCR. Expression ofTCRb, CD8a, CD4-1, and CD4-2 was strikingly induced inspleen lymphocytes at 5 d after S. agalactiae infection (Fig. 2A).Moreover, when applied with T cell–specific inhibitor cyclo-sporine A during S. agalactiae infection, lymphocyte expansionwas effectively suppressed at 7 d postinfection (Fig. 2B). Mean-while, an LCK Ab was used to identify T cells from Nile tilapialeukocyte. On gated lymphocytes population, ∼50 and 70%of lymphocytes were LCK positive in spleen and peripheralblood, respectively (Fig. 2C). Nile tilapia LCK+ but not LCK2

lymphocytes expressed high transcription levels of T cell markerCD3ε and TCRb, whereas B cell–specific marker CD20 wasparticularly expressed in LCK2 but not LCK+ lymphocytes(Fig. 2D), suggesting LCK Ab could be used to identifyT lymphocytes from Nile tilapia. Seven days after S. agalactiaeinfection, frequency of LCK+ lymphocytes was obviously increasedin PBL (Fig. 2E, 2F). Although LCK+ lymphocyte frequencies werecomparable in S. agalactiae infection and control spleen (Fig. 2E,2F), expression of LCK was strikingly enhanced postinfection(Fig. 2G). During the infection, no obvious difference of death ratecould be found between control and infected lymphocytes(Fig. 2H, 2J) or T cells (Fig. 2I, 2J). Altogether, these resultssuggest that ancestral T cells in teleost participate in and play acertain role in adaptive immunity.

An intact and evolutionarily conserved mTORC1 pathway ispresent in Nile tilapia

As mTORC1 signaling plays a central role in mammalian adaptiveimmunity, we sought to determine whether this pathway is also in-volved in the teleost adaptive immune response. Deep exploration ofthe Nile tilapia genome identified all components of the mTORC1pathway, including complex components mTOR, Raptor, GbL,DEPTOR, and PRAS40 (Supplemental Fig. 1A), upstream elementsPI3K, AKT, PDK1, Rheb, PTEN, TSC1, and TSC2 (SupplementalFig. 1B), and downstream elements S6K1 and 4E-BP1 (SupplementalFig. 1C). Discovery of the entire components indicates a conservedmTORC1 signaling in Nile tilapia.

As a central component of mTORC1, Nile tilapia mTOR shareshigh similarity in domain organization with homologs from mouse,oyster, and even Penicillium (Fig. 3A). The N-terminal aminoacids of these mTOR contain a group of tandem HEAT repeats(∼20) (Fig. 3A), which are thought to be essential for mTOR-Raptor interaction (33). The HEAT repeats of all these mTORare followed by FAT and FRB domains; and a catalytic kinasedomain (PI3Kc) and a FATC domain are downstream at theC terminus of the protein (Fig. 3A). In addition to mTOR, othercomponents of the mTORC1 complex, including Raptor,DERTOR, GbL, and PRAS40, also exhibit similar domain orga-nization with that in mammals (Supplemental Fig. 1D). The aminoacid sequences encoded FRB domain of mTOR, which serves as adocking site for rapamycin-FKBP12 complex formation, displayhigh conservation among vertebrate species (Fig. 3B), indicatingthe potential affinity of Nile tilapia mTOR to rapamycin (33).Furthermore, the primary structure of catalytic kinase domainPI3Kc has been well conserved during evolution, particularly withregard to four crucial phosphorylation sites: S2448, which reg-ulates mTOR-intrinsic activity; S2481, which is an autophos-phorylation site; and T2446 and S2478, the functions of whichare not entirely clear (Fig. 3C). Thus, the domain organizationand amino acids of Nile tilapia mTOR are highly conservedthroughout vertebrates.To collect more functional clues, the tertiary structure of Nile

tilapia mTORC1 complex components was predicted based oncorresponding crystal models in mouse. mTOR, as well as Raptorand GbL, share high similarity of tertiary structure with theirmammalian homologs (Fig. 3D, Supplemental Fig. 1E). Moreover,phylogenetic trees revealed that Nile tilapia mTOR and othermTORC1 components cluster with the same molecules from otherteleost (Fig. 3E, Supplemental Fig. 2A–D), indicating that mTORC1components are highly conserved among plants, fungi, invertebrates,and vertebrates.Altogether, our data suggest that Nile tilapia encodes an intact

and evolutionarily conserved mTORC1 pathway.

mTORC1 participates in the adaptive immune response ofNile tilapia

Because Nile tilapia has potential mTORC1 signaling, we sought toknow in which tissue/organ this signaling exists. An immuno-fluorescence assay revealed mTOR to be expressed in almost alltissues detected, including spleen, liver, trunk kidney, gill, intestine,skin, and muscle (Fig. 4A). However, an exception was the headkidney (Fig. 4A), and this result was further confirmed by Westernblotting (Fig. 4B). Thus, mTORC1 is a ubiquitous pathway thatexists in almost all tissues/organs of Nile tilapia.To determine whether mTORC1 signaling participates in

Nile tilapia adaptive immunity, we infected the animals withS. agalactiae. During primary response (5–8 d postinfection),mRNA level of mTOR in spleen lymphocytes was significantlyelevated compared with control (Fig. 4C). The same was truewhen fish were infected with the Gram-negative bacteriumAeromonas hydrophila (Supplemental Fig. 2E). Concordantly,both the protein and phosphorylation levels of mTOR (S2448)in spleen lymphocytes were increased at 5 d postinfection (Fig.4F, 4G). Furthermore, the transcriptional levels of complex com-ponent Raptor, the upstream molecule AKT, and the downstreammolecule S6K1 were all increased by S. agalactiae infection on day5 or 8 (Fig. 4D, 4E). Although the protein levels of S6 and 4EBP1downstream of mTORC1 were comparable between infected andcontrol animals (Fig. 4F), S6 and 4EBP1 phosphorylationin lymphocytes of infected animals were enhanced on day 8 or 5postinfection compared with controls (Fig. 4G). Together, these

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results indicate that mTORC1 signaling participates in the cell-mediated adaptive immune response of Nile tilapia.

Nile tilapia mTORC1 regulates T lymphocyte activation

Next, we sought to figure out how mTORC1 signaling regulatesadaptive immunity of Nile tilapia. We used the agonist PMA and

ionomycin to activate lymphocytes in vitro. Compared with

unstimulated controls, mRNA levels of mTOR and S6K1 were

downregulated, whereas AKT, PDK1, and TSC2 levels were up-

regulated at 2 h after stimulation (Fig. 5A, Supplemental Fig. 2F).

At protein level, mTOR decreased upon lymphocyte activation,but the other two components, GbL and Raptor, remained at alevel similar to the resting conditions (Fig. 5B). These irregularfluctuations in mTORC1 components indicate that this signalingdoes not regulate lymphocyte activation at either mRNA or proteinlevel.Nonetheless, subsequent analysis revealed the phosphorylation

level of mTOR at S2448 and AKT at T308 upstream of mTORC1,and S6K1, S6, and 4EBP1 downstream of mTORC1 were allenhanced upon lymphocyte stimulation (Fig. 5C). Concordantly,

FIGURE 1. Participation of Nile tilapia lymphocytes in adaptive immunity. (A) Isolation of spleen leukocytes or PBLs using Percoll density gradient

centrifugation (left panel); representative dot plots showing cellular populations of leukocytes based on FSC/SSC flow cytometry (right panel). (B) Mi-

croscopy of the morphology of sorted cells in the R1–R3 population by Giemsa staining. (C) TEM assay of sorted cells in R1–R3 populations. (D) Fish were

i.p. injected with 100 ml of the indicated concentration of S. agalactiae. A Kaplan–Meyer survival plot showing the survival percentage of animals

postinfection, n = 20. (E–H) Fish were i.p. injected with 100 ml of 7 3 107 CFU S. agalactiae or PBS, and tissues were harvested on day 8 postinfection for

assay. (E) Representative dot plots of spleen leukocytes and PBLs. (F and G) Percentage (F) and absolute number (G) of lymphocytes within spleen

leukocytes and/or PBLs (n = 5–8). (H) Increased inflammatory infiltration in the bacterial-infected liver by H&E staining. The data shown are representative

of two independent experiments, except for (D), which is representative of at least four independent experiments. **p , 0.01, determined by a two-tailed

Student t-test. SSC, side scatter.

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FIGURE 2. T lymphocytes of Nile tilapia paly a certain role in the adaptive immunity. (A) Relative mRNA levels of the indicated molecules in spleen

lymphocytes with or without S. agalactiae infection on day 5 postinfection by real-time RT-PCR. n = 4. (B) Nile tilapia individuals infected with

S. agalactiae were i.p. injected with cyclosporine A every day; animals were sacrificed on day 7 for assay. Representative dot-plots of spleen leukocytes

and PBLs. (C) Representative dot-plots showing LCK+ cells on gated lymphocytes population from spleen and peripheral blood leukocytes. (D) mRNA

level of indicated molecules in sorted Nile tilapia LCK2 or LCK+ lymphocytes. (E–J) Nile tilapia was infected with (Figure legend continues)

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elevated phosphorylation of mTOR at S2448, S6, and 4EBP1 wasobvious at 15 min after lymphocytes were stimulated by PMA andionomycin by confocal microscopy (Fig. 5D–F). To confirm therole that mTORC1 plays in T lymphocyte activation, we treatedanimals with rapamycin before PMA and ionomycin stimulation.Inhibition of mTORC1 severely impaired phosphorylation of S6and 4EBP1 (Fig. 5G). Meanwhile, blockade of mTORC1 crippledthe upregulation of IFN-g upon PHA-induced T cell activation(Fig. 5H), suggesting that mTORC1 signaling is indispensablefor T lymphocyte activation in Nile tilapia. Together, these re-sults suggest that Nile tilapia mTORC1 signaling regulatesT lymphocyte activation at the phosphorylation level but notmRNA or protein level.

Nile tilapia mTORC1 promotes T lymphocyte proliferation

Ag-induced clonal expansion is a hallmark of adaptive immunity.Once activated by Ag, T lymphocytes undergo prompt proliferationand differentiate into distinct subsets according to the milieu. Torule out a possible function of mTORC1 signaling in lymphocyteexpansion, we treated fish with rapamycin during S. agalactiaeinfection. In contrast to the robust expansion of lymphocytes de-tected in the group without rapamycin treatment, blockade ofmTORC1 signaling impaired lymphocyte expansion, as revealedby decreased frequencies of lymphocytes among PBLs and spleenleukocytes and the reduced absolute number of lymphocytes in thespleen on day 8 postinfection (Fig. 6A–C). T cells, especiallyCD8+ T cells, are critical for controlling and eradicating patho-gens. A dramatic decrease of TCRb, CD8a, and CD4-2 mRNAin lymphocytes that lack of mTORC1 activity was observed onday 5 after S. agalactiae infection (Fig. 6D, 6E). FACS analysisusing LCK Ab also revealed a defect of LCK+ lymphocytes ex-pansion in PBL that lack of mTORC1 activity (Fig. 6F, 6G). Al-though frequencies of LCK+ lymphocytes were comparable inspleen that were with and without mTORC1 activity (Fig. 6F, 6G),LCK express level was obviously decreased when mTORC1 wasinhibited by rapamycin (Fig. 6H). These results suggested a de-fective expansion of effector T cell, especially the CD8+ T cells, inthe absence of mTORC1.To further confirm the regulatory role of mTORC1 in teleost

lymphocyte proliferation, we injected BrdU into S. agalactiae–infected fish with or without rapamycin inhibition 1 d before sacrifice.Four days postinfection, BrdU incorporation into lymphocytesin mTORC1-inhibited fish was notably lower than that in infectedcontrol animals (Supplemental Figs. 2g, 7b). The same was truewhen we examined BrdU+ lymphocytes on day 5 postinfection(Supplemental Figs. 2h, 7b). Remarkably, the frequency of BrdU+

lymphocytes increased on day 6 postinfection in infected controlanimals, whereas very limited expansion of BrdU+ lymphocytes wasobserved when mTORC1 signaling was blocked by rapamycin(Fig. 7A, 7B).To investigate the potential role of mTORC1 in the proliferation

of T cell lineages, CFSE-labeled lymphocytes were stimulated withthe T cell–specific mitogen PHA in the presence or absence ofrapamycin. Nile tilapia T lymphocytes showed vigorous prolifer-ation following 48 h of PHA stimulation; in contrast, cells thatlacked mTORC1 activity displayed an obvious defect in prolifera-tion after stimulation (Fig. 7C). To confirm this result, a rapamycin

dose-dependent inhibitory assay was performed during CFSE di-lution assay. After 3 d of PHA stimulation, most live lymphocytesunderwent robust proliferation; more unproliferated lymphocyteswere identified in the rapamycin-treated group, and the frequencyof unproliferated lymphocytes increased with an increase of rapa-mycin dose (Fig. 7D). Therefore, mTORC1 is crucial for prolifer-ation of the teleost T lymphocyte lineage.In addition, the reduced number of lymphocytes in rapamycin-

treated animals was not caused by enhanced apoptosis, becausethe frequencies of Annexin V+ and 7AAD+ lymphocytes did notincrease compared with uninhibited control (Fig. 7E). Collectively,these results suggest that Nile tilapia mTORC1 promotes pathogen-induced expansion of T lymphocytes.

mTORC1 signaling is indispensable for teleost effector T cellsto eliminate infection

Once activated, naive CD8+ T cells massively expand and dif-ferentiate into cytotoxic, inflammatory cytokine-secreting effec-tor T cells (34). In our study, blockade of mTORC1 by rapamycinimpaired lymphocyte expansion (Fig. 6A–C), and lack of mTORC1signaling decreased inflammatory infiltration in the liver afterS. agalactiae infection (Fig. 8A), rendering the animals morevulnerable to pathogen (Fig. 8B). The high mortality rate of thefish lacking mTORC1 was due to their failure to control theinfection, as reflected by high bacterial burdens in the livercompared with controls (Fig. 8C). These observations demonstratethat mTORC1 signaling is associated with lymphocyte-mediatedinfection elimination.Further investigation revealed that compared with controls,

mRNA levels of proinflammatory cytokine IFN-g, cytotoxic-related gene perforin A and granzyme B, and proapoptoticgene FasL were significantly downregulated in rapamycin-treated lymphocytes on day 8 after S. agalactiae infection (Fig. 8D).Interestingly, transcript levels of another two proinflammatory cy-tokines, TNF-a and LT-a, were comparable between control andmTORC1 inhibition groups (Fig. 8E). Moreover, the mRNA ex-pression levels of CD44 and T-bet, which is an important cellsurface marker and a transcription factor for effector CD8+ T cells,respectively, were coincidentally decreased compared with controls(Fig. 8F), suggesting the dysregulation of effector T cells in theabsence of mTORC1 signaling. Together, these results indicate thatmTORC1 is required for effector T cell–mediated elimination ofinfection in Nile tilapia.

Nile tilapia mTORC1 regulates effector T cell function viametabolic reprogramming

Cell metabolism is appreciated as a key regulator of T cellfunction. In a mouse model, dynamic metabolic switching fromthe TCA cycle and fatty acid b-oxidation to glycolysis, lipogen-esis, and glutamine oxidation was accompanied by activation ofnaive T cells into effector T cells (35–37). To investigate meta-bolic reprogramming during T cell activation in Nile tilapia, westimulated lymphocytes with PHA in vitro. Upon T cell activation,expressions of glucose transporter 1 (Glut1), rate-limiting en-zymes PFK and PKM, which collectively facilitated the glycoly-sis process, were dramatically upregulated at the mRNA level(Fig. 9A), suggesting a possible elevated glycolysis in activated

S. agalactiae, and fish were sacrificed on day 7 for assay. (E) Representative dot-plots shown frequencies of LCK+ lymphocytes in PBL or spleen. (F) Bar

figures shown percentage of LCK+ lymphocytes in PBL or spleen (n = 6). (G) Overlaid histogram shown LCK expression in control and S. agalactiae–

infected spleen lymphocytes. (H and I) Representative dot-plots showing death rate of gated lymphocytes (H) or LCK+ lymphocytes (I) in control and

S. agalactiae–infected animals. (J) Death rate of indicated cells in PBL or spleen of control and S. agalactiae–infected animals (n = 5). The data shown

are representative of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, determined by a two-tailed Student t test.

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T cells of Nile tilapia. Meanwhile, rapid upregulation of tran-scription factor SREBP1 and 2 (Fig. 9B) as well as sodium-coupled neutral amino acid transporter 2 (SNAT2), neutralamino acid transporter B(0) (ASCT2), and glutamate dehydroge-nase 1 (GDH) (Fig. 9C) suggested the strikingly enhanced lipo-genesis and glutamine oxidation programs accompanied Nile

tilapia T cell activation. Thus, these observations indicate multipledynamic metabolic reprogramming upon T cell activation in a fishspecies.Recent data in mammals have highlighted key roles of mTOR

pathway in intimately linking T cell function and metabolism(35, 36). Thus, we further investigated the potential regulatory

FIGURE 3. Evolutionary conservation of the Nile tilapia mTOR molecule. (A) Comparison of the domain organization of mTOR proteins from indicated

species. (B and C) Multisequence alignment analysis of the FRB domain (B) and catalytic kinase domain (C) from Nile tilapia mTOR with homologs in

other animals. Amino acid residues with 100% identity are in pink, and similar amino acids are in yellow. The four conserved phosphorylation sites are

boxed. (D) Prediction of the tertiary structure of mTOR from Nile tilapia and mouse by SWISS-MODEL. (E) Phylogenetic trees constructed with the amino

acid sequences of mTOR from the indicated species. The tree was constructed using the neighbor-joining (NJ) algorithm with the Mega 4.1 program based

on multiple sequence alignment by ClustalW. Bootstrap values of 1000 replicates (%) are indicated for the branches. The accession numbers of selected

sequences are listed in Supplemental Table I.

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roles of mTORC1 in these metabolic programs preferentiallyexhibited by teleost effector T cells. After S. agalactiae infection,a dramatic increase in cell size indicated the strong anabolicin activated lymphocytes (Fig. 9D). Additionally, blockade ofmTORC1 signaling during infection strikingly dampened the ro-bust aerobic oxidation occurring in lymphocytes, as revealed bythe failure in cell size increase and glucose uptake upon activation(Fig. 9D, 9E). Furthermore, rapamycin treatment reduced themRNA levels of glucose transporter Glut1, rate-limiting enzymesHK2, and PFK (Fig. 9F). More importantly, the enzyme activity ofPFK was also impaired when mTORC1 was inhibited by rapa-mycin (Fig. 9G). These observations suggest an important rolefor mTORC1 signaling in promoting the glycolysis program of

effector lymphocytes in teleost fish. However, both mRNA ex-pression and enzyme activity of another rate-limiting enzymePKM was not affected by the lack of mTORC1 activity (Fig. 9F,9G). In contrast, reduced expression of the lipid synthesis regu-lator SREBP1 in mTORC1 lacked effector lymphocytes (Fig. 9H),suggesting the potential contribution of mTORC1 to the lipo-genesis program followed by lymphocyte activation. Additionally,inhibition of mTORC1 also reduced glutamine oxidation, withconcordantly decreased mRNA expression of glutamine oxida-tion genes, including SNAT2, ASCT2, glutaminase liver isoform(GLS2), and GDH (Fig. 9I). Meanwhile, loss of mTORC1 activityimpaired the enzyme activity of alanine aminotransferase, whichtransforms glutamate into a-ketoglutarate (a-KG) but not the

FIGURE 4. mTORC1 signaling is involved in the adaptive immune response of Nile tilapia. (A) mTOR distribution revealed by immunofluorescence

microcopy. Cryosections of the indicated tissues were stained with a primary rabbit anti-mTOR Ab followed by a secondary Alexa Fluor 594-labeled goat

anti-rabbit Ab, scale bar, 20 mm. (B) mTOR expression in the indicated tissues was determined by Western blotting using a primary rabbit anti-mTOR Ab.

(C–G) Fish were i.p. injected with S. agalactiae or PBS, and spleen lymphocytes were harvested on the indicated days postinfection. (C–E) Relative mRNA

levels of mTOR (C), Raptor (D), and AKT and S6K1 (E) in lymphocytes with or without infection was examined by real-time RT-PCR (n = 4–5). (F and G)

Immunoblotting analysis was examined using the indicated Abs in lymphocytes with or without infection. The data shown are representative of at least two

independent experiments. *p , 0.05, **p , 0.01, determined by two-tailed Student t test.

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activity of glutaminase, which transforms glutamine into glu-tamate (Fig. 9J). Collectively, mTORC1 signaling coordinates

multiple metabolic reprogramming events during ancestral T cell

activation.

mTORC1 coordinates metabolic programs through keytranscription factors

Metabolic reprogramming during mammalian T cell activation isaccompanied by upregulation of several key transcription factors.

In a mouse model, the oncogene c-Myc is responsible for initiating

glycolysis and glutamine oxidation (38), HIF-1a is indispensablefor glycolysis (39, 40), and LIPIN 1–regulated SREBP1/2 arecrucial for lipogenesis (41). Remarkably, we found that expressionof these transcription factors was also elevated following Niletilapia T cell activation (Figs. 9B, 10A), suggesting their potentialinvolvement in corresponding metabolic programs in ancestralT cells. Emerging evidences from a mouse model has highlightedthe cross-talk of mTORC1 signaling with these transcriptionfactors during T cell metabolic reprogramming (35–37). Inhibitionof mTORC1 activity by rapamycin decreased levels of c-Myc

FIGURE 5. mTORC1 regulates T lymphocyte activation in Nile tilapia. (A–F) Nile tilapia lymphocytes were stimulated with PMA and ionomycin

in vitro. (a) Relative mRNA levels of the indicated molecules in lymphocytes with or without stimulation were examined by real-time RT-PCR (n = 4).

(B and C) Immunoblotting analysis showing protein (B) or phosphorylation levels of the indicated mTORC1 components in lymphocytes with or without

stimulation. (D–F) Immunofluorescence analysis showing phosphorylation of mTOR at S2448 (D), S6 (E), and 4EBP1 (F) with or without stimulation.

(G and H) Nile tilapia individuals were treated with rapamycin for three consecutive days before lymphocytes were stimulated with PMA and ionomycin for

examination of S6 and 4EBP1 phosphorylation (G) or stimulated with PHA for real-time RT-PCR assay (H). The data shown in (A) and (D)–(H) are

representative of two independent experiments; the data in (B) and (C) are representative of more than four independent experiments. *p , 0.05, **p , 0.01,

determined by a two-tailed Student t test.

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protein but not mRNA (Fig. 10A, 10C), indicating that mTORC1enhances expression of c-Myc by promoting its translation ratherthan its transcription. Concordantly, both mRNA and protein ex-pression of HIF-1a were reduced in activated lymphocytes lackingmTORC1 (Fig. 10A, 10C). Thus, mTORC1 signaling promotesglycolysis and glutamine oxidation in teleost effector T cells bycontrolling c-Myc and HIF-1a. In contrast, the fact that mTORC1signaling blockade by rapamycin impaired transcription factorSREBP1 (Figs. 9G, 10C) but elevated its inhibitory molecule

LIPIN 1 (Fig. 10B) suggests that teleost mTORC1 also modulateslipogenesis in activated lymphocytes through LIPIN 1–regulatedSREBP. Thus, metabolic reprogramming following Nile tilapia T cellactivation is highly dependent on the induction of mTORC1-driven transcription factors.Altogether, in a teleost model, once native T cells encounter

presented Ag, the mTORC1 signaling, which is represented byS6K1/S6 and 4E-BP1 phosphorylation, is activated. Then mTORC1coordinates glycolysis, lipogenesis, and glutaminolysis metabolic

FIGURE 6. mTORC1 signaling is indispensable for Ag-induced expansion of Nile tilapia lymphocyte. Nile tilapia individuals infected with S. agalactiae

were i.p. injected with rapamycin on days 2, 3, 4, and 6, and animals were sacrificed on indicated days for the assay. (A–C) Representative dot-plots

(A), percentage (B), and absolute number (C) of lymphocytes within spleen leukocytes and/or PBLs on day 8 postinfection (n = 5–7). (D and E) Real-time

RT-PCR (D) or semi–RT-PCR (e) revealed the mRNA levels of the indicated genes in lymphocytes with or without rapamycin treatment on day 5 after

S. agalactiae infection (n = 5–6). (F–H) Fish were sacrificed on day 7 postinfection. (F) Representative density plots show frequencies of LCK+ lym-

phocytes in PBL or spleen. (G) Bar figures show percentage of LCK+ lymphocytes in PBL or spleen (n = 6). (H) Overlaid histograms show LCK expression

in spleen lymphocytes with or without rapamycin treatment. The data shown are representative of two independent experiments. *p , 0.05, **p , 0.01,

***p , 0.001, determined by a two-tailed Student t test.

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programs via controlling c-myc, HIF-1a, and SREBP transcriptionfactors to ensure the proper activation and subsequent expansion ofT cells. Furthermore, mTORC1 activity is also crucial for effectorT cells to exert cytotoxic function during infection elimination(Fig. 11).

DiscussionT cells have been discovered for half a century, and cumulativeevidences regarding these cells in phylogenetically divergentspecies have vastly contributed to our understanding of theirsubsets, functions, and evolutionary origins. Fish species are thefirst evolutionary group to produce T cells, and recent studies onT cells from teleost models have illustrated several fundamentalimmunological events. For example, characterization of cell typesbearing CD4-1 and CD4-2 in rainbow trout have shed light on theevolutionary origins and primordial roles of CD4+ lymphocytesand CD4+ monocytes/macrophages (5). Although the potentphagocytic and Ag-presenting abilities of gd T cells from zebra-fish have also provided valuable insight into the evolutionaryhistory of T cell subsets (6). Although adaptive immunity featuresof T cells, such as Th1/2/17 cytokine responses and cytotoxicability (5, 7, 11, 13–15), have been revealed in teleost species,

their modulation is still largely unknown. Our studies illustrate theregulatory mechanism of T cell–driven response in a fish speciesand provide valuable evidence for solving the enigma regardingevolution of T cell immunity.mAbs are considered indispensable tools in the study of cellular

immunology. mAbs against T cell coreceptors such as CD4-1,CD4-2, and CD8a have been developed for a few species of tel-eost fish (5, 15, 42); however, mAbs of CD3ε for total T cells orTCRb for ab T cells are not yet available. Although mammalianZap-70 Ab has been used to identify T cells in fixed cells andtissues of several fish species (7, 43, 44), it could not cross withNile tilapia lymphocytes (data not shown). However, we foundanother mammalian Ab (human LCK mAb) could specificallyidentify Nile tilapia T cell in the present study. In view of thissituation, we used the LCK mAb to identified fish total T cells;meanwhile we used T cell–specific inhibitor cyclosporin A, mi-togen PHA, lymphocytes signaling agonist PMA, and ionomycinto exclude interference from other cell lineages. Although furtherstudy needs to be performed in the future, our results regardingT cell function and biological processes have provided valuableevidence to understand regulatory mechanisms of T cell immunityin a fish species.

FIGURE 7. Nile tilapia lymphocyte requires mTORC1 signaling for proper proliferation. (A and B). Nile tilapia individuals infected with S. agalactiae

were i.p. injected or not with rapamycin on days 2, 3, and 4; the animals were i.p. injected with BrdU 1 d before sacrifice. BrdU incorporation in

lymphocytes was examined by flow cytometry 24 h later. (A) Representative contour plots showing BrdU staining of gated lymphocytes. (B) Percentages of

BrdU+ lymphocytes on the indicated days postinfection; each symbol represents one animal. The whole experiments in (A) and (B) are repeated twice

independently. (C and D) CFSE-labeled Nile tilapia lymphocytes were stimulated with PHA for 48 or 72 h with or without rapamycin. (C) Overlaid

histograms show CFSE intensity of gated lymphocytes at 48 h after stimulation. (D) Overlaid histograms show CFSE intensity of gated live lymphocytes at

72 h after stimulation with the indicated concentration of rapamycin. (E) Representative contour plot of 7AAD and Annexin V staining of gated

lymphocytes on day 8 after S. agalactiae infection. The data shown are representative of two independent experiments.

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The activation of mammalian mTOR signaling has been widelyappreciated; however, it remains unclear whether the mTORpathway is regulated in the same way in early vertebrates. Ourstudy identified an intact mTORC1 pathway in Nile tilapia,whereby all the mTORC1 complex components identified inmouse, including the scaffolding protein Raptor, constitutivecomponent GbL, and potent inhibitor DEPROE and PRAS40 arepresent in this teleost. However, precise interaction among thesecomponents requires further elucidation. Importantly, mTORC1itself as well as elements of the PI3K-AKT-TSC1/2-RheB axisupstream of mTORC1 and two best-characterized downstreamtargets, S6K1 and 4E-BP1, are conserved in teleost. Both patho-gen infection and in vitro TCR signaling activated teleostmTORC1 pathway via phosphorylation of mTOR at Ser2448 andare followed by downstream S6K1/S6 and 4EBP1 phosphoryla-tion. The high conservation of FRB domain, which is crucialfor rapamycin binding (33), suggests that this macrolide canbe used as a potent inhibitor of mTORC1 signaling in Nile tilapia.As expected, acute treatment with rapamycin severely damp-ened mTORC1 activation, as revealed by reduced S6 and 4EBP1phosphorylation. Thus, the existence of all mTORC1 pathway

components and their phosphorylation collectively suggests anmTORC1 activation strategy similar to that of mammals existingin teleost. To our knowledge, this study is the first to demonstratethe existence of mTORC1-S6K1-S6 and mTORC1-4EBP1-eIF4Eregulatory strategy in a fish species. Our results thus indicate thatnot only the mTORC1 pathway but also its regulation is highlyconserved among jawed vertebrates. Teleost mTOR signalingappears to exist broadly in both lymphoid and nonlymphoid tis-sues; however, with exception in head kidney. Because headkidney of teleost is an important organ for immature lymphocytesdevelopment (45), lack of mTOR signaling might be associatedwith lymphocytes development. However, future work is war-ranted to illustrate the potential mechanism for this interestingexpression pattern.mTOR pathway is extensively involved in T cell biology, with

activation by both TCR and costimulatory signals. Downstream ofTCR signaling, IP3 and DAG generated by PLCg1 initiate Ca2+-CaM-NFAT, RasGRP1-Ras-Erk1/2-AP1, and PKCu-IKK–NF-kBpathway and thus collectively contribute to the proper activationand function of T cells (2, 16, 17). Synchronously, downstreamof DAG, signals from both the RasGRP1-Ras-Erk1/2-AP1 and

FIGURE 8. Critical role of mTORC1 for tilapia effector cells to eliminate infection. (A–C) Nile tilapia individuals infected with S. agalactiae were

i.p. injected or not with rapamycin on day 2, 3, 4, and 6. (A) H&E staining showing inflammatory infiltration in the liver on day 8 postinfection.

(B). Kaplan–Meyer survival plot showing the survival percentage of Nile tilapia with or without rapamycin treatment postinfection, n = 20. The data shown

are representative of at least four independent experiments. (C) S. agalactiae titers in the liver on day 6 postinfection, n = 5. (D–F) Relative mRNA levels of

the indicated molecules in lymphocytes with or without rapamycin treatment on day 8 after S. agalactiae infection (n = 4). The data shown in (A) and

(C)–(F) are representative of two independent experiments. p , 0.05, **p , 0.01, determined by a two-tailed Student t test.

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PKCu-IKK–NF-kB axis may in turn stimulate TCR-induced ac-tivation of mTORC1 (19). In addition, TCR-proximal events drivePI3K-PDK1-AKT signaling to induce TSC2 degradation andRheB activation, which are tightly associated with subsequentmTORC1 activation (19). As stimulation of the DAG analoguePMA together with ionomycin lead to thorough TCR activation ina short time, these regents have been extensively used in studies ofTCR signaling and T cell function. Because long-time PMA andionomycin stimulation will cause the cell death, we examined theprotein and phosphorylation level of mTORC1 signaling at 5, 15,

or 45 min after stimulation and detected transcription level ofmTORC1 signaling at 2 h poststimulation for its relative slowerresponse than phosphorylation. In addition, T cell–specific mito-gen PHA was also used in the present study to induce T cell ac-tivation and proliferation. Because relative mild effect of PHAcompares with PMA and ionomycin, we are allowed to examinethe mRNA expression or cell proliferation at 12 h, 24 h, or even3 d poststimulation. Our results reveal that upon PMA-inducedTCR activation, mTORC1 signaling is robustly activated inearly vertebrate teleost. Furthermore, inhibition of mTORC1 by

FIGURE 9. Teleost mTORC1 regulates effector T cell function through metabolic reprogramming. (A–C) Nile tilapia lymphocytes were stimulated with

PHA for 24 h in vitro. The relative mRNA levels of the indicated molecules in lymphocytes with or without stimulation were examined by real-time

RT-PCR, n = 4–6. (D and E) Nile tilapia individuals infected with S. agalactiae were i.p. injected with rapamycin on days 2, 3, 4, and 6, and animals were

sacrificed on day 7 for the assay. (D) Flow cytometry analysis of lymphocyte size by quantification of the FSC mean fluorescence intensity (MFI), n = 5.

(E) Glucose uptake, as measured by 2-NBDG labeling in gated spleen lymphocytes. (F, H, and I). Real-time RT-PCR showed the mRNA levels of the

indicated genes in lymphocytes with or without rapamycin treatment on day 7 after S. agalactiae infection, n = 5. (G and J) Activities of indicated enzyme

in liver tissues with or without rapamycin treatment on day 5–7 after S. agalactiae infection, n = 5. The data shown are representative of two independent

experiments. *p , 0.05, **p , 0.01, ***p , 0.001 determined by a two-tailed Student t test.

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rapamycin reduced IFN-g production, suggesting an impairedT cell activation. Although the precise pathway by which TCRaxis controls teleost mTORC1 requires further clarification, ourresults show a solid connection between mTORC1 signaling andteleost T cell activation. Interestingly, early TCR activation inmammals also depends on intact mTORC1 signaling, becauserapamycin treatment impairs phosphorylation of LCK and ZAP-70(46), suggesting a positive feedback between mTORC1 andTCR-proximal signaling. It remains to be determined whetherthis feedback regulation exists in fish species.Naive T cells patrol within peripheral lymphoid organs in a

quiescent state. Upon Ag stimulation through TCRs, these cells areactivated and exit quiescence, leading to clonal expansion andeffector differentiation (20). The strength of mTORC1 signaling iscritical for maintaining quiescence of peripheral T cells. T cellsthat are deficient of TSC1 elevated mTORC1 signaling but losequiescence, indicating by the spontaneous entry into a state ofclonal expansion (47). In contrast, genetic deletion of Raptor inT cells abrogated TCR-induced proliferation both in vivo andin vitro (48). In this study, we show that mTORC1-dependentquiescence exiting and clonal expansion of lymphocytes is alsoa pivotal mechanism for adaptive immunity in early vertebratesbecause proliferation was severely impaired in T lymphocyteslacking mTORC1, as determined by both in vivo BrdU incorpo-ration and in vitro CFSE dilution. Accompanied by Ag-inducedexpansion, mammalian T cells differentiate into effector subsets,and many cascades, including mTOR, are responsible for thisprocess. Our results highlight that during S. agalactiae infection,weakened mTORC1 signaling causes a reduction of Nile tilapiaeffector T cells, especially CD8a-expressing effector cells, whichare necessary for acute infection clearance. The mTORC1-drivenT-bet, a critical transcription factor for CD8+ effector T cells(24), accounts for the decrease of this subset of Niletilapia lymphocytes at the peak of infection. Concordantly, this

reduction of teleost CD8-expressing effector cells was correlatedwith reduced proinflammatory cytokine and cytotoxic genes, im-paired pathogen clearance, and aggravated the infection. Becausemany recent studies have revealed the significant roles of cyto-toxic T cells and INF-g–producing Th1 cells to control andeliminate the infection caused by extracellular Streptococcuspathogens (49, 50), it is not so surprising that S. agalactiae in-fection could trigger CD8+ T cell–like response of Nile tilapia.Although blockade of mTORC1 signaling by rapamycin cannotexclude effects from other cell lineages, our study as a forerunneris of great reference value for studies of the regulatory mechanismof adaptive immunity mediated by ancestral T cells. Gene editingbased on CRISPR/Cas9 is expected to further elucidate themTORC1-mediated regulation of teleost immunity in a T cell–intrinsic manner.Effector T cell differentiation is accompanied by striking met-

abolic reprogramming (39). Resting naive T cells have low met-abolic requirements that depend on lipid oxidation to fuel basalenergy generation. Upon activation, T cells rapidly grow, prolif-erate, and exert effector functions. During this transition, T cellsprovide essential intermediate metabolites by dramatically in-creasing aerobic glycolysis, glutamine metabolism, and lipogen-esis to support the biosynthesis of required proteins, nucleic acids,and lipid membranes (17, 18, 35, 39). In this study, we note thatactivation of primordial T cells was also accompanied by dramaticmetabolic reprogramming, as revealed by increased lymphocytesize, enhanced glucose uptake, and elevated expression of a seriesof genes related to glycolysis, glutamine metabolism, and lipo-genesis. In accordance with recent findings in mouse models(35–37), our results regarding the transcription properties ofmetabolic genes in rapamycin-treated Nile tilapia emphasize theintimate coordination of mTORC1 signaling and T cell meta-bolism in fish species, suggesting that mTORC1-driven metabolicreprogramming is a central regulator in teleost effector T cells. In

FIGURE 10. Teleost mTORC1 regulates metabolic programs through key transcription factors. (A) Nile tilapia lymphocytes were stimulated with PHA

for 24 h in vitro. The relative mRNA levels of the indicated molecules in lymphocytes with or without stimulation were examined by real-time RT-PCR,

n = 4–6. (B) Real-time RT-PCR showed the mRNA levels of the indicated genes in lymphocytes with or without rapamycin treatment on day 7 after

S. agalactiae infection, n = 5. (C) Spleen lymphocytes isolated from rapamycin-treated or control Nile tilapia were stimulated with PHA for 24 h in the

presence or absence of additional rapamycin. Western blotting analysis revealed the level of the indicated molecules in lymphocytes with or without

mTORC1 inhibition. The data shown are representative of two independent experiments. *p , 0.05 determined by a two-tailed Student t test.

FIGURE 11. Teleost T cells require mTORC1-

coupled immune signals and metabolic programming

for proper activation and function. In Nile tilapia, once

native T cells encounter Ag, mTORC1 signaling that is

represented by S6K1/S6 phosphorylation is activated.

Then mTORC1 coordinates glycolysis, lipogenesis, and

glutaminolysis metabolic programs through control-

ling c-myc, HIF-1a, and SREBP transcription factors

to ensure the proper activation and subsequent expansion

of T cell. Meanwhile, mTORC1 signaling is indispens-

able for effector T cells to exert cytotoxic function and

eliminate infection.

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mammals, metabolic reprogramming is preceded by upregulationof several key transcription factors, and one of the earliest elevatedfactors is the oncogene c-Myc, which is responsible for initiatingT cell activation–induced glycolysis and glutaminolysis (38).Acute deletion of c-Myc impairs both glycolysis and gluta-minolysis by reducing expression of rate-limiting enzymes andthe glutamine antiporter (38). In addition, rapamycin treatmentor Raptor deletion impairs c-Myc upregulation, suggesting thatmTORC1 signaling controls expression of this oncogene (38).During the metabolic reprogramming that accompanies T cellactivation, mTORC1 also directs glucose uptake and glycolyticmetabolism in effector T cells partially through HIF-1 expressionand promotes T cell lipogenesis through activation of SREBPs(39–41). Strikingly, these metabolic programs directed by tran-scription factors were also found to be mTORC1-dependent inteleost lymphocytes. To our knowledge, these results represent thefirst description of the regulatory mechanism of T lymphocytemetabolic reprogramming in nonmammalian species. mTORC1drives the metabolic reprogramming that controls T cell activa-tion, and function is thus a sophisticated and evolutionarily con-served strategy throughout jawed vertebrates.In summary, our study has identified an intact and conserved

mTORC1 signaling in an early vertebrate Nile tilapia. Morecritically, we show that this signaling plays pivotal roles in teleostadaptive immunity by regulating T cell activation, clonal expan-sion, and infection clearance, thus providing a regulatory mech-anism for T cell biological processes in a fish species. Moreover,our results with this phylogenetically primitive vertebrate illustratethat mTORC1 directs effector T cell function through several keytranscription factor–controlled metabolic reprogramming and thusconstructs an intimate link between immune signals and metabolicprograms in teleost. Altogether, our study provides importantevidence that preceding the emergence of tetrapod, primordialT cells in jawed fish were armed with sophisticated strategiessimilar to those modern T cells to modulate adaptive immunity.These findings offer critical insight into the evolutionary origins ofT cell–mediated immune responses.

DisclosuresThe authors have no financial conflicts of interest.

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