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DevelopmentZAP70 Activation during Thymic Nck Recruitment to the TCR Required for

Wolfgang W. Schamel and Balbino AlarcónOrfao,Prouza, Miloslav Suchànek, Manuel Fuentes, Alberto

Aldo Borroto, Irene Arellano, Elaine P. Dopfer, Marek

http://www.jimmunol.org/content/190/3/1103doi: 10.4049/jimmunol.1202055December 2012;

2013; 190:1103-1112; Prepublished online 24J Immunol 

MaterialSupplementary

5.DC1http://www.jimmunol.org/content/suppl/2013/01/02/jimmunol.120205

Referenceshttp://www.jimmunol.org/content/190/3/1103.full#ref-list-1

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The Journal of Immunology

Nck Recruitment to the TCR Required for ZAP70 Activationduring Thymic Development

Aldo Borroto,* Irene Arellano,* Elaine P. Dopfer,† Marek Prouza,‡ Miloslav Suchanek,‡

Manuel Fuentes,x Alberto Orfao,x Wolfgang W. Schamel,†,{ and Balbino Alarcon*

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3« after TCR

engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck

binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4+

CD8+ thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative

mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at

each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of

endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired

tyrosine phosphorylation of CD3z, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our

results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required

for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic

differences in both knockin mice. The Journal of Immunology, 2013, 190: 1103–1112.

In ab T cells, the TCR is composed of the sequence-variableTCRa- and TCRb-chains, which are responsible for ligandrecognition, a peptide derived from the Ag associated to

molecules of the MHC, and the CD3 subunits responsible forsignal transduction (CD3g, CD3d, CD3ε, and CD3z [also namedas CD247]) (1). The TCR has to be able to interpret small dif-ferences in the chemical composition of the peptide Ag bound toMHC as quantitatively and qualitatively different signaling out-comes, although the mechanism underlying this process remainspoorly understood. The most prevalent, simplistic model proposestwo types of cytoplasmic tyrosine kinases as the sole directeffectors of the TCR: Lck and ZAP70 (2). However, direct re-cruitment of other proteins to the CD3 subunits of the TCR hasalso been described (2–5), suggesting that the diversity of sig-

naling outcomes emanating from the TCR may be modulated bythe composition of the “TCR signalosome.” Thus, these mecha-nisms may involve the recruitment and activation of differentcytoplasmic and membrane effectors to the TCR.Nck is a SH2/SH3 adaptor protein that plays a universal role

in coordinating the signaling networks critical for organizing theactin cytoskeleton, cell movement, or axon guidance, connectingtransmembrane receptors to multiple intracellular signaling path-ways (6, 7). Typically, Nck is recruited via its SH2 domain tophosphotyrosine residues in the tail of transmembrane receptorsand then serves as an anchoring site for cytoplasmic effectors viaits three SH3 domains. Nck effectors include proteins that havea pivotal role in the nucleation and polymerization of the actincytoskeleton such as the SCAR/WAVE proteins and the serine/threonine kinase Pak1. In T cells, Nck is not recruited via itsSH2 domain to a membrane receptor but to the cytosolic scaf-folding protein SLP76. Instead, Nck is directly recruited to aproline-rich sequence (PRS) in the cytoplasmic tail of CD3ε uponTCR triggering via its N-terminal SH3 (SH3.1) domain (6–8).Bone marrow reconstitution with CD3ε PRS mutants in CD3ε-

deficient mice, Nck overexpression in primary T cells, Nckknockout mice, and PRS knockin mice have all been used to studythe role of PRS and Nck in T cell development (9–15). Someexperiments have suggested that the PRS is important for thymicmaturation, but not for mature T cell activation in vitro (10), al-though experiments with bone marrow chimeras indicate that thePRS is important to activate mature T cells by weak but not bystrong agonists in vitro (14). Furthermore, Nck recruitment to thePRS seems to regulate TCR levels at the plasma membrane inpreselection double-positive (DP) CD4+CD8+ thymocytes but notat later stages, promoting the degradation of the TCR (10, 16).To evaluate the functional relevance of the Nck-CD3ε interac-

tion, while trying to solve the conflicting data regarding the role ofPRS in thymic maturation, we have generated a novel mouse linewith a conservative germline mutation in the PRS of CD3ε(knockin mouse line [KI]-PRS). We found that Nck recruitment tothe TCR is required at every step during T cell maturation at

*Centro de Biologıa Molecular Severo Ochoa, Consejo Superior de InvestigacionesCientıficas, Universidad Autonoma de Madrid, Cantoblanco, Madrid 28049, Spain;†Centre for Biological Signalling Studies, Faculty of Biology, University of Freiburg,Max Planck Institute for Immunobiology and Epigenetics, 79108 Freiburg, Germany;‡Exbio Praha, a.s., 252 42 Vestec, Czech Republic; xCentro de Investigacion delCancer, Consejo Superior de Investigaciones Cientıficas-University of Salamanca,37007 Salamanca, Spain; and {Centre of Chronic Immunodeficiency, UniversityClinics Freiburg, 79106 Freiburg, Germany

Received for publication July 25, 2012. Accepted for publication November 26,2012.

This work was supported by Comision Interministerial de Ciencia y Tecnologia(Grant SAF2010-14912), Redes Tematicas de Investigacion Cooperativa Sanitaria(Grant RD06/0020/1002), “Fundacion Cientıfica” de la “Asociacion Espanola Contrael Cancer,” the European Union (Grant FP7/2007-2013 “SYBILLA”), DeutscheForschungsgemeinschaft (Grant SFB620), Bundesministerium fur Bildung, Wissen-schaft, Forschung und Technologie (Grant 01 EO 0803), and Fundacion RamonAreces (to the Centro de Biologıa Molecular Severo Ochoa).

Address correspondence and reprint requests to Prof. Balbino Alarcon, UniversidadAutonoma de Madrid, Madrid 28049, Spain. E-mail address: balarcon@cbm.uam.es

The online version of this article contains supplemental material.

Abbreviations used in this article: DN, double-negative; DP, double-positive; KI,knockin mouse line; PRS, proline-rich sequence; SP, single-positive; WT, wild type.

This article is distributed under The American Association of Immunologists, Inc.,Reuse Terms and Conditions for Author Choice articles.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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which the pre-TCR or TCR signaling is required, and that it isnecessary for full CD3z phosphorylation and ZAP70 recruitmentto the TCR and activation.

Materials and MethodsGeneration of KI-PRS mice

Knockin mice bearing the PxxP to AxxA double mutation in the PRS ofCD3ε were generated by Genoway. The BAL2-HR targeting vector wasgenerated that contained a neo cassette flanked by flippase recombinationtarget sequences inserted between exons 4 and 5 and two C to G mutationsin exon 5. The mutations were as follows: CCA (CCA . GCA)CCTGTT(CCC . GCC)AAC. The construct was electroporated into C57BL/6 EScells that were selected in G418. Primary screening for 39 homologousrecombination was carried out by PCR, and homologous recombinationwas verified in 59 Southern blots followed by 39 Southern blots. Twelveindependent embryonic stem (ES) clones of 547 were positive for ho-mologous recombination and were injected into blastocysts of C57BL/6mice. Twenty chimeric male mice derived from 8 of the ES clones werecrossed with C57BL/6 Flp deleter females, and 14 mice with germlinetransmission were tested by PCR for excision of the floxed region, whichwas confirmed in Southern blots. A total of four mice were identified asheterozygous for the knockin allele, of which one male was chosen to crosswith C57BL/6 females to generate the KI-PRS colony at the “Centro deBiologıa Molecular Severo Ochoa.”

Cells and transgenic mice

African green monkey COS7 cells were grown in DMEM plus 10% FBS.Thymocytes were maintained in RPMI 10% FBS supplemented with 20mM2-ME and 10 mM sodium pyruvate. KI-PRS mice were crossed with OT-ITCR transgenic mice (OVAp specific, H-2Kb restricted) (17) for the ANDTCR (MCC specific, I-Ek restricted) (18), and for the HY TCR (HY Agspecific, H-2Db restricted) (19). The resulting heterozygous mice werecrossed again to generate TCR transgenic wild type (WT) and knockinhomozygous mice. All experiments involved the use of littermates ho-mozygous for the WT or the knockin alleles. All mice were maintainedunder specific pathogen-free conditions at the animal facility of the“Centro de Biologıa Molecular Severo Ochoa” in accordance with currentnational and European guidelines. All animal procedures were approvedby the ethical committee of the “Centro de Biologıa Molecular SeveroOchoa.”

Flow cytometry

Cells were preincubated with the anti-CD16/32–specific mAb 2.4G2 inPBS, 1% BSA, 0.02% sodium azide before labeling with saturatingamounts of the indicated fluorochrome-labeled or biotinylated mAbs and,where applicable, fluorochrome-labeled streptavidin (reagents purchasedfrom BD Pharmingen, eBioscience, Immunotools, Santa Cruz, and Mil-tenyi). Labeled cells were analyzed on a FACSCalibur or FACSCanto IIflow cytometer (Becton-Dickinson), and the data were analyzed withFlowJo software (Tree Star).

Thymocyte proliferation

A total of 1.5 3 105 thymocytes of each genotype were stimulated atdifferent times with 3 3 105 irradiated spleen cells from CD3ε─/─ mice, asAPCs, preloaded with OVAp (SIINFEKL), with Q4R7 (SIIQFERL), withQ4H7 (SIIQFEHL), or with G4 (SIIGFEKL). Forty-eight hours later, a 12-h pulse with 1 mCi/well [3H]thymidine (Perkin Elmer) was given and ra-dioactivity collected in a glass fiber filter (Perkin-Elmer), and counted ina 1450 microbeta Wallac Trilux liquid scintillation counter.

Immunoblot analysis of T cell activation

A total of 3 3 107 thymocytes of each genotype were activated at differenttimes with T2Kb APCs preloaded with OVAp peptide (SIINFEKL). Afterdifferent incubation times, the cells were lysed in 1 ml Brij96 lysis buffercontaining protease and phosphatase inhibitors (0.3% Brij96, 140 mMNaCl, 20 mM Tris-HCl [pH 7.8], 10 mM iodoacetamide, 1 mM PMSF, 1mg/ml leupeptin, 1 mg/ml aprotinin, 1 mM sodium orthovanadate, and 20mM sodium fluoride). Immunoprecipitation was performed with anti-CD3z serum 448 (20) or anti-CD3 mAb (145-2C11) and protein ASepharose beads. SDS-PAGE and immunoblotting was performedaccording to standard protocols, and the membranes were probed with theanti-CD3z serum or different anti–phospho-specific Abs, and were visu-alized by ECL. Quantification was performed on ECL autoradiographyfilms using ImageJ software.

Pull-down assay

The construct pGEX-4T1-GST-Nckbtrn [GST-Nck2(DSH2)] was made byPCR of human Nckb. The pGEX-4T1 derivative GST-SH3.1a was kindlyprovided by Dr. R. Geha (Children’s Hospital, Harvard Medical School,Boston, MA). Pull-down assays were performed as described previously(21).

Generation of anti-Nck Abs

BALB/c mice were biweekly immunized s.c. with 30 mg and intra-splenically with 10 mg GST fused to the second SH3 domain (SH3.2) ofhuman Nck1. The first s.c. immunization was performed with CFA, and itwas followed by two injections in IFA. Intrasplenic immunization wasdone in IFA. Ten days after the second and third immunization, the animalswere tail-bled, and the immune response to Ag was measured by ELISA.The mouse selected for generation of mAbs was boosted i.v. with 5 mg Agin saline. Four days later, the spleen was harvested and used for cell fusionwith myeloma cells. A total of 350 3 106 spleen cells were fused to 60 3106 Sp2/0 myeloma cells using polyethyleneglycol 1500 (STEMCELLTechnologies) according to the manufacturer’s recommendations. Thefused cells were initially seeded in tissue culture plates containing semi-solid ClonaCell-HY Selection and Cloning Medium D (STEMCELLTechnologies). About 700 hybridoma clones were picked from semisolidmedium after 14 d of growth. Primary screening to test positive clones forthe production of anti-Nck Abs was performed using an ELISA assay,selecting those reactive with GST-SH3.2 but not with GST. All animalexperiments were performed according to Czech Central Commission forAnimal Welfare guidelines.

Statistical analysis

Quantitative data are shown as the mean6 SD. A nonparametric two-tailedStudent t test was used to assess the confidence intervals.

Abs and other materials

Abs and other materials are described in Table I.

ResultsNck is inducibly recruited to the TCR in DP thymocytes

The cytoplasmic tail of CD3ε contains multiple confirmed andpotential sequences for protein interactions (Fig. 1A). The PRS ofCD3ε casts an elongated footprint on the SH3.1 domain of Nck,interacting with three shallow hydrophobic pockets (22–24). Thecanonical PxxP sequence for PRS-SH3 domain interaction isfollowed by 2 aa at the position +3 that establish interactions withthe third hydrophobic pocket of the SH3.1 domain (SupplementalFig. 1). Thus, the interaction sequence in the PRS of CD3ε isactually defined by the motif PxxPxxDY. To minimize possibleinterference with other proteins and to fully prevent the interactionwith Nck, we generated a knockin mouse bearing a germlinesubstitution of the two central prolines in the PxxPxxDY motifwith alanine (Fig. 1A). This mutation is more conservative thanothers previously used to study the PRS (Fig. 1A).Initially, we verified that no interaction occurred between the

double mutant and the SH3.1 domain in a pull-down assay per-formed in transfected COS cells (Fig. 1B). This analysis was re-peated with thymocyte lysates from knockin (KI-PRS) mice,revealing that TCR does not bind to the SH3.1, irrespective ofanti-CD3 stimulation (Fig. 1C). Conversely, stimulation of WTthymocytes with anti-CD3 clearly provoked TCR binding toSH3.1, in contrast with previous results (10), suggesting that TCRengagement was necessary for the CD3ε PRS to bind SH3.1,probably by inducing the TCR to adopt an active conformation(8). Indeed, the TCR adopted the active conformation in WTthymocytes upon triggering as it did in mature T cells (Fig. 1C,spleen). Because total thymocytes were used in the pull-downassay, the induction of TCR binding to SH3.1 (Fig. 1C) mayoriginate through single-positive (SP) and not through DP thy-mocytes. To determine whether the CD3ε PRS of preselection DP

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thymocytes was induced or constitutively exposed, we depletedthe TCRhigh thymocytes, thereby obtaining a 94% pure pop-ulation of preselection DP thymocytes with low levels of TCR(Fig. 1D). Binding of the TCR to SH3.1 in the preselection DPpopulation above background levels was induced upon TCRengagement (Fig. 1D), in agreement with a recent report (25).The pull-down assay determines whether the TCR can bind theSH3.1 domain but not whether Nck is indeed recruited to themutant TCR. To clarify this issue, we used immunoblottingwith a new anti-Nck mAb (Supplemental Fig. 2) after immu-noprecipitation with anti-CD3 from cell lysates of TCR-triggeredT cells from nontransgenic and OT-I TCR transgenic mice.Endogenous Nck was recruited to the TCR after stimulation withanti-CD3 of WT but not homozygous KI-PRS thymocytes (Fig.1E). Similar outcomes were observed in OT-I transgenic mice:induction of endogenous Nck binding was detected in WT thy-mocytes within 30 s after stimulation with OVAp (Table I) Ag-loaded Ag-presenting T-2Kb cells, but not in KI-PRS thymocytes(Fig. 1F). These results demonstrate that Nck is induciblyrecruited to the TCR during thymic differentiation in a PRS-dependent manner.There were no apparent differences between KI-PRS and WT

mice in terms of the distribution of the double-negative (DN), DP,and SP stages of thymocyte differentiation (Fig. 2A). However, wedetected increased TCR expression in DP thymocytes, but not inmature SP thymocytes in KI-PRS versus WT mice (Fig. 2A). The

increase in TCR expression in DP thymocytes is consistent withprevious observations in knockin mice bearing the 8-aa replace-ment in the PRS (10), where the PRS of CD3ε was thought to playan important role in the degradation of the TCR in DP thymocytes,perhaps by promoting the recruitment of the SLAP adaptor proteinto the TCR. Indeed, defective ubiquitylation of the CD3z chainhas been described (16) in those knockin mice (10), further sug-gesting that the PRS, and perhaps Nck recruitment, favors thepolyubiquitylation and degradation of the TCR in DP thymocytes.Therefore, we analyzed how the TCR in thymocytes of our KI-PRS mice became ubiquitylated and degraded in comparison withWT thymocytes. After immunoprecipitation with anti-CD3z andimmunoblotting with anti-ubiquitin, we detected two major bands,one corresponding to a CD3z homodimer in which each subunit ismodified with one ubiquitin molecule and a larger one corre-sponding to bis-ubiquitylated CD3z (Fig. 2B). Stimulation of WTthymocytes with anti-CD3 resulted in increased ubiquitylation,starting 1 h after stimulation, and an increased rate of CD3zdegradation, which was almost complete 4 h after stimulation.The basal degradation rate of CD3z in WT thymocytes was verylow and surprisingly, mutation of the PRS augmented rather thanreduced ubiquitylation and degradation of CD3z. Indeed, ubiq-uitylation in basal conditions was higher in KI-PRS versus WTthymocytes, whereas stimulation with anti-CD3 resulted in aninitial burst of mono- and bis-ubiquitylation followed by a loss ofthe ubiquitylation signal, probably caused by accelerated CD3z

Table I. Abs and other materials

Clone or Code Description Use Origin

AbAnti-CD3z 448 Rabbit polyclonal WB/IF San Jose et al., 1998 (20)Anti-mouse CD3 145-2C11 Hamster mAb Stimulation/IP J. Bluestone, UCSFH57-FITC, biotinylated H57-597 Hamster mAb FC Immunotools BioscienceCD4-PE-PerCP-biotinylated RM4-5 Rat mAb FC BD PharmingenCD8a-FITC, -PE, -PerCP,

-biotinylated53-6.7 Rat mAb FC BD Pharmingen

CD5-biotinylated 53-7.3 Rat mAb FC eBioscienceCD24-FITC 01574-D Rat mAb FC BD PharmingenCD44-FITC KM81 Rat mAb IF Immunotools BioscienceCD25-allophycocyanin PC61.5 Rat mAb IF eBioscienceVb3 TCR-PE–biotinylated KJ25 Mouse mAb FC BD PharmingenT3.70 (TCR)-biotinylated T3.70 Mouse mAb FC eBioscienceVa2 TCR-PE B20.1 Rat mAb FC BD PharmingenCD69-FITC–biotinylated H1.2F3 Hamster mAb FC BD PharmingenPhospho-Zap70 (Tyr319) 65E4 Rabbit mAb WB/IF Cell SignalingPhospho-Zap70 (Tyr292) Ab12868 Rabbit mAb WB/IF AbCamPhospho-CD3z (Tyr Y1) EM-26 Mouse mAb WB Exbio4G10 4G10 Mouse mAb WB MilliporeUbiquitin sc8017 Mouse mAb WB Santa Cruz BiotechnologyZAP70 (total) 2705 Rabbit mAb WB Cell SignalingAlexa 488 A-21206 Donkey anti-rabbit IgGs IF InvitrogenAlexa 488 A-21202 Donkey anti-mouse IgGs IF/FC InvitrogenAlexa 555 A-31572 Donkey anti-rabbit IgGs IF InvitrogenAlexa 555 A-31570 Donkey anti-mouse IgGs IF InvitrogenAlexa 555 S-32355 Streptavidin IF InvitrogenAlexa 488 S-11223 Streptavidin IF InvitrogenAlexa 594 A-21207 Donkey anti-rabbit IgGs IF InvitrogenAlexa 594 A-21203 Donkey anti-mouse IgGs IF InvitrogenAlexa 647 A-31573 Donkey anti-rabbit IgGs IF InvitrogenAnti-Flag M2 Mouse mAb WB Sigma

Other materialsOVA peptide SIINFEKL OT-I stimulation CBMSO peptide synthesis facilityQ4R7 SIIQFERL OT-I stimulation CBMSO peptide synthesis facilityQ4H7 SIIQFEHL OT-I stimulation CBMSO peptide synthesis facilityG4 SIIGFEKL OT-I stimulation CBMSO peptide synthesis facilityMethyl-[3H]thymidine NET027Z005MC T cell proliferation Perkin Elmer

BD, Becton Dickinson; CBMSO, Centro de Biologıa Molecular Severo Ochoa; FC, flow cytometry; IF, immunofluorescence; IP, immunoprecipitation; UCSF, University ofCalifornia, San Francisco; WB, Western blot.

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FIGURE 1. Mutation of the two central prolines in CD3ε abolishes Nck recruitment to the TCR. (A) Cartoon of the cytoplasmic tail of CD3ε illustratingdifferent sequence motifs. A cartoon indicating the mutations in the PRS previously described (in boldface) is shown (right panel). (B) Pull-down (pd) of

lysates of COS cells transfected with either Flag-tagged WTor CD3ε double proline mutant (fεwt or fεmut) with GST-SH3.1 analyzed in immunoblots (IB)

probed with anti-Flag. Relative densitometric values of three samples run in parallel are shown (right panel). Data are representative of two experiments.

(C) Thymocytes and splenocytes isolated from either WT or KI-PRS mice were left unstimulated or stimulated with anti-CD3 for 5 min before lysis and

GST-SH3.1 pd. Relative densitometric values of three samples run in parallel are shown (right panels). Data are representative of three experiments. (D)

Preselection DP thymocytes from WT mice were enriched by panning on anti-TCRb–coated plates at 0˚C. The double-color plot shows that 94% of the

nonattached thymocytes were DP for CD4 and CD8, whereas the single-color histogram demonstrates that all TCRbright and TCRint were removed. The

purified preselection DP WT thymocytes were stimulated with anti-CD3 for the times indicated and lysed. Subsequently, a GST-SH3.1 pd and anti-CD3z IB

were performed. Relative densitometric values of three samples run in parallel are shown (right panel). Data are representative of two experiments. (E)

Nontransgenic WTand KI-PRS total thymocytes were stimulated with anti-CD3, and the lysates precipitated with anti-CD3 (Ip) were analyzed in IB probed

with anti-Nck (top). Anti-CD3z served as a loading control. Likewise, total cell lysates were immunoblotted with anti-Nck to reveal equal levels of ex-

pression. Relative densitometric values of three samples run in parallel are shown (right panel). (F) A similar experiment was carried out with thymocytes

from OT-I TCR transgenic mice that were stimulated with T2-Kb APCs loaded with OVAp for the times indicated. Relative densitometric values of three

samples run in parallel are shown (right panel). Data are representative of three experiments. BRS, basic amino acid–rich sequence; EC, extracellular

domain; ER-RS, endoplasmic reticulum retention sequence; IC, intracellular domain; PTB, phosphotyrosine-binding motif; PxxP, consensus SH3 domain-

binding motif; TM, transmembrane domain.

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degradation (Fig. 2B). Therefore, the increased TCR expressiondetected in DP thymocytes of our KI-PRS mice could not beexplained by decreased rates of TCR ubiquitylation and degra-dation. These observations raised the question why DP thymo-cytes exhibit higher levels of TCR expression in our KI-PRS miceif the PRS mutation and blockage of Nck recruitment acceleratesTCR degradation.

Blocking Nck recruitment to the TCR impairs DP thymocytedifferentiation at each of the TCR-dependent selection stages

The OT-I TCR transgenic mice provided a clue to resolve the abovequestion, as anti-CD4 and anti-CD8 staining revealed an abnormaldistribution of thymic subpopulations in KI-PRS mice. Preselec-tion DP thymocytes with high levels of both CD4 and CD8 werestrongly diminished, with thymocytes accumulating at interme-diate stages of differentiation from DP to CD8SP (i.e., CD4+

CD8low and CD4lowCD8low; Fig. 3A) (26). This result indicatedthat the PRS mutation provoked a partial arrest beyond the positiveselection checkpoint at the DP stage. The DP thymocyte pop-ulation can be subdivided in three different stages (DP1-DP3),classified according to the expression of the TCR and CD5 (27).Accordingly, OT-I DP thymocytes from KI-PRS mice accumulateat the DP3 stage, visualized both in percentages and in the absolutenumber of thymocytes (Fig. 3B). Therefore, the accumulation ofthymocytes at the DP3 stage and not a reduction in TCR degra-dation apparently accounts for the potential increase in TCR ex-pression in DP thymocytes induced by the PRS mutation.

Positive selection is accompanied by downregulation of CD24and upregulation of CD69. Analysis of the DP3 subpopulationrevealed an increase and decrease in the expression of thesemarkers, respectively, in the OT-I KI-PRS versus WT OT-I thy-mocytes (Fig. 3B), indicating defective positive selection in KI-PRS OT-I thymocytes. Defects in positive and negative selectionwere also detected in female and male HY TCR transgenic mice,respectively, on a mutant KI-PRS background (Supplemental Fig.3A, 3B). OT-I and HY are models for CD8 T cell selection.Defects in positive selection were also manifested in the ANDTCR transgenic model for CD4 T cell selection. In this model, theformation of CD4SP thymocytes and the upregulation of TCRtransgene (Vb3) expression were reduced in KI-PRS mice (Sup-plemental Fig. 3C, 3D).Although the PRS mutation reduced the absolute number of total

thymocytes in OT-I TCR transgenic mice (Fig. 3A), we detected nodifferences in the distribution (Fig. 2A) or absolute numbers (Fig.3C) of nontransgenic thymocytes between mutant and WT geno-types. However, a more careful analysis of the DP populationindicated an accumulation of the DP2 subpopulation of thymo-cytes (Fig. 3C). Furthermore, analysis of total thymocytes basedon their expression of CD24 and TCR revealed an accumulation ofthymocytes at a DP stage with partially upregulated TCR levelsand high CD24 expression (population b in Fig. 3D), which cor-responded to the DP2 subpopulation. Thymocytes also accumu-lated at transitional stages in KI-PRS mice, as defined by highlevels of TCR and CD24 expression (population c in Fig. 3D).

FIGURE 2. High TCR expression

in KI-PRS DP thymocytes is not the

result of lower degradation. (A)

Distribution in the four major thy-

mic populations of nontransgenic

WT and KI-PRS mice according to

CD4 and CD8 expression. TCR ex-

pression for WT (shadowed) and KI-

PRS (solid line) mice are shown in

the histograms. Mean fluorescence

intensity (MFI) values for TCRb

expression are indicated in gray

(WT) and black (KI-PRS) type. Data

are representative of six experi-

ments. (B) Total thymocytes were

incubated with or without anti-CD3

for the times indicated, and anti-

CD3z immunoprecipitates were

probed in nonreducing immunoblots

with anti-ubiquitin. Based on their

mobility, the lower band appears to

be the monoubiquitinated and the

upper band the bis-ubiquitinated

CD3z homodimer. The membranes

were reprobed with anti-CD3z (bot-

tom panels), and relative densito-

metric values of three samples run in

parallel are shown (right panels).

Data are representative of three

experiments.

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These results confirm that high TCR expression in the DP pop-ulation of KI-PRS mice occurs because of an arrest of thymicmaturation at an intermediate stage, and not because of decreasedTCR degradation.The accumulation of thymocytes at the DP2 stage in KI-PRS

mice suggests positive selection is impaired. In contrast, the re-duction in DP1 cells compared with WT mice indicates that theinput of DP1 precursors from the DN stages is also impaired in KI-PRS mice. Indeed, the analysis of the DN populations in OT-I TCRtransgenic, as well as nontransgenic mice, of the WT and mutantgenotypes revealed that the PRS mutation caused a partial arrestat the DN3 stage, suggesting that pre-TCR signaling is deficient

(Fig. 4). An accumulation of DN thymocytes at the DN3 stage wasalso observed in mice expressing the AND and HY TCR trans-genes (Supplemental Fig. 3E, 3F). Overall, these results indicatethat the PRS of CD3ε is necessary for full pre-TCR and TCRsignaling at each step during thymic maturation, that is, b-selec-tion, positive and negative selection, and maturation to the CD4and CD8 T cell lineages.

Nck recruitment to the TCR is required for the most immediateTCR signaling events in the thymus

The impairment of thymic differentiation in KI-PRS mice sug-gested that the recruitment of Nck to the PRS is required for TCR

FIGURE 3. Mutation of the PRS arrests thymocyte development at intermediate stages of differentiation. (A) Flow cytometry of OT-I TCR transgenic

WT and KI-PRS thymocytes showing the percentage of DP and postselection transitional (CD4+CD8low and CD4low CD8low) thymocyte populations.

Absolute cell number in each subpopulation counted in three mice per group is shown below. Data are representative of five experiments. (B) CD5 and

transgenic TCR (Va2) analysis of DP subpopulations (top histograms). Lower histograms show the expression of positive selection markers in the total DP

population and in the DP3 subpopulation. Absolute cell number in each DP subpopulation counted in three mice per group is shown to the right. Data are

representative of five experiments. (C) Flow cytometry of nontransgenic WT and KI-PRS thymocytes showing the percentage of DP, transitional (CD4+

CD8low), CD4SP, and CD8SP thymocyte populations. DP thymocytes were analyzed for the expression of CD5 and TCR (CD3) to assess their distribution

in the DP1, DP2, and DP3 subpopulations. Absolute cell number in each thymic subpopulation was counted in four mice per group. Data are representative

of three experiments. (D) Flow cytometry analysis of CD24 and TCR expression in nontransgenic WT and KI-PRS mice allowed the thymocytes to be

divided into four subpopulations (a–d). These subpopulations were reanalyzed according to CD4 and CD8 expression (bottom panels). Absolute cell

number in the a–d subpopulation, counted in four mice per group, is shown (right panel). Data are representative of three experiments.

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signaling. Furthermore, because both positive and negative se-lection are impaired by the PRS mutation (Fig. 3, SupplementalFig. 3), we studied whether the proliferative response of OT-Ithymocytes to a panel of positive- or negative-selecting peptideAgs (28) was affected. The response to the two negatively selectingpeptides (OVAp and Q4R7) was inhibited in cells expressing themutant PRS, whereas the proliferative response to the two positivelyselecting peptides (Q4H7 and G4) was too weak even in WT thy-mocytes to detect significant differences (Supplemental Fig. 4).To evaluate how the PRS mutation affected TCR-proximal

signals that could explain its effects on thymic differentiation,we carried out a number of flow cytometry experiments withZAP70 and phospho-ZAP70–specific Abs. DP thymocyte differ-entiation from the DP1-DP3 stages is accompanied by the up-regulation of ZAP70 (27). Although ZAP70 expression was higherin KI-PRS versus WT DP thymocytes when the total populationwas examined (Fig. 5A), there were no differences in ZAP70levels when the DP1, DP2, and DP3 subpopulations of WT andKI-PRS thymocytes were analyzed, suggesting that the PRS mu-tation affects DP maturation, but not the upregulation of ZAP70.However, a similar analysis using an anti–phospho-Tyr319-ZAP70Ab, which detects active ZAP70, revealed deficient ZAP70 acti-vation at all stages of DP maturation in KI-PRS mice (Fig. 5B).Defective phosphorylation of ZAP70 in Tyr319 was also detectedin all post-DP stages in KI-PRS thymocytes when compared withWT counterparts (Fig. 5B). Similar findings were observed withanother phospho-ZAP70 Ab specific for Tyr292 (Fig. 5C). Hence,Nck binding to CD3ε appears to be required for efficient activationof ZAP70 during thymic differentiation.Because phosphorylation of ZAP70 requires recruitment to the

CD3 phospho-ITAMs (2), we investigated whether deficientphosphorylation of the CD3 ITAMs could underlie the observeddefects in ZAP70 phosphorylation. When total thymocytes fromOT-I transgenic mice were stimulated with T-2Kb APCs loaded

with OVAp, CD3z was rapidly phosphorylated in WT OT-I thy-mocytes, as detected by anti-CD3 immunoprecipitation and im-munoblotting with a pan-phosphotyrosine Ab, peaking 30 s afterstimulation (Fig. 5D, upper row). By contrast, induction of CD3ztyrosine phosphorylation in KI-PRS OT-I transgenic mice wasboth delayed and weaker. When the membranes were reprobedwith a mAb specific for the phosphorylated form of the N-terminaltyrosine of the first ITAM of CD3z (phoshpho-zY1) (29), defec-tive tyrosine phosphorylation of CD3z in thymocytes bearing themutation in the PRS was confirmed (Fig. 5D, second row). Finally,the coimmunoprecipitation of ZAP70 with the TCR was impairedby the PRS mutation (Fig. 5D, third row). Together, these resultsindicate that Nck binding to the PRS sequence of CD3ε is requiredfor full tyrosine phosphorylation of the CD3z ITAMs and forZAP70 recruitment and activation in thymocytes.

DiscussionIn this study, we have generated a genetically modified mouse linebearing alanine replacements for the two central prolines of thecanonical PxxP motif of CD3ε. This mutation impaired T celldevelopment at each individual step at which the pre-TCR or TCRare required: pre-TCR signaling at the DN3-DN4 transition,positive and negative selection at the DP stage, and maturationinto CD4SP and CD8SP thymocytes. Unlike in a previously de-scribed genetically modified mouse line bearing an 8-aa replace-ment in the PRS of CD3ε (10, 16), we did not find that PRSmutation resulted in slower degradation of the TCR. Furthermore,we found that endogenous Nck is not constitutively bound to theTCR, but inducibly recruited to the TCR in WT thymocytes uponTCR triggering. How can we explain the discrepancy in thephenotypes of our KI-PRS with the previous KI? We suggest thatthe discrepancy is explained by the different extent of the PRSmutations used. Although in the previous knockin mutant, 8 aa ofCD3ε containing the PRS were replaced by the sequence con-

DN DN3 DN4

FIGURE 4. Partial arrest of thy-

mocyte differentiation at the pre-

TCR stage in nontransgenic and OT-

I TCR transgenic KI-PRS mice. (A)

Flow cytometry of thymocytes from

nontransgenic WT and KI-PRS mice

showing thymocyte distribution in

the four major DN subpopulations.

Thymocytes were gated in the DN

quadrant according to CD4 and CD8

expression, and the selected cells

were reanalyzed for the expression

of CD25 and CD44 markers. The

DN3 and DN4 subpopulations cor-

respond to CD442CD25+ and

CD442CD252 DN thymocytes, re-

spectively. Absolute cell number in

each thymic subpopulation was

counted in four mice per group (right

panel). Data are representative of six

experiments. (B) The analysis de-

scribed in (A) was also performed on

thymocytes from OT-I TCR trans-

genic WT and KI-PRS mice. Abso-

lute cell number in each thymic

subpopulation was counted in three

mice per group (right panel). Data

are representative of six experiments.

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FIGURE 5. PRS mutation impairs early activation events in thymocytes. (A) Intracellular flow cytometry analysis of ZAP70 expression in total DP and DP1-

DP3 subpopulations. Histograms corresponding to WT are shadowed, and those corresponding to KI-PRS are indicated by solid lines. Mean fluorescence intensity

(MFI) values for WTand KI-PRS mice are shown in gray and black type, respectively. Data are representative of three experiments. (B) Intracellular flow cytometry

analysis of phospho-Y319 ZAP70 in total DP, in DP1-DP3 subpopulations, and in transitional (CD4+CD8low and CD4lowCD8low) and mature (CD8SP) thymocytes.

Data are representative of three experiments. (C) Intracellular flow cytometry analysis of phospho-Y292 ZAP70 of thymocyte populations as in (B). Data are

representative of three experiments. (D) Phospho-CD3 was analyzed after stimulation of OT-I thymocytes from WT and KI-PRS mice with T2-Kb APCs loaded

with OVAp, probing anti-CD3 precipitates in immunoblots with anti-phosphotyrosine. The membrane was reprobed with an Ab specific for the phosphorylated

form of the N-terminal tyrosine of the first ITAM of CD3z (EM-26) and with an Ab directed against total ZAP70. Data are representative of three experiments.

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tained in a similar position of the g-chain of the FcεRI (10), ourknockin mice bear a more conservative mutation: the two centralproline residues of the canonical SH3-binding PxxP motif werereplaced by alanine residues. The 8-aa replacement affects thePRS, as well as a potential phospho-tyrosine-binding site thatconforms to the canonical NPxY sequence contained within thePRS. Thus, although our PxxP-to-AxxA mutation exclusivelyabrogates Nck recruitment, the 8-aa replacement might preventthe recruitment of a second protein to the NPxY sequence. Adefective recruitment of the second protein might explain themilder phenotype of the 8-aa replacement mutant. Studies aimedat identifying this second protein are currently under way. Thedifferences between PRS mutations could also explain otherobservations in transgenic and retrogenic mice (9, 13, 14).An important role for Nck during thymic maturation and mature

T cell function has been demonstrated in double-knockout micelacking Nck1 in all tissues, and conditionally lacking Nck2 inT cells only (11, 12). The phenotype of mice lacking Nck inT cells resembles that of our KI-PRS mice, although in someaspects the phenotype of these mice is stronger or milder thanours. This may be explained by the participation of Nck in T cellactivation in both TCR recruitment-dependent and -independentpathways, and by the incomplete elimination of Nck2 through theconditional approach. Therefore, unlike previous articles on Nck-deficient mice (11, 12), this article highlights the importance ofNck in T cell activation by binding to CD3ε.We investigated the effect of inhibiting the Nck-CD3ε interac-

tion on early TCR signaling in KI-PRS TCR transgenic mice.ZAP70 recruitment to the TCR and ZAP70 activation, revealed byits phosphorylation at tyrosine residues 292 and 319, were in-hibited in KI-PRS T cells. This is probably due to an unexpectedeffect of the PRS mutation on CD3z tyrosine phosphorylation. Theanalysis of thymocytes from KI-PRS mice shows impaired CD3ztyrosine phosphorylation, suggesting that Nck binding to CD3ε isrequired for full tyrosine phosphorylation of CD3z. This effect ofNck binding to CD3ε could be explained by Nck mediating therecruitment of priming tyrosine kinase, perhaps Lck, to the TCRor by the stabilization of the active conformation of the TCR afterNck binding to the PRS. Because it has recently been shown thatLck activity remains unaltered after TCR triggering (30), regu-lating the TCR triggering-dependent phosphorylation of the CD3ITAMs may, in fact, be the result of ITAM accessibility rather thanof kinase activity. The conformational change of the TCR may bethe mechanism responsible for placing the cytoplasmic tails of theCD3 subunits in an appropriate position for phosphorylation, andNck binding to the PRS a stabilizing factor. It remains to be de-termined what is the importance of Nck recruitment to the TCRfor mature T cell activation in our KI-PRS mice compared withthe 8-aa replacement mutant.

AcknowledgmentsWe thank Fabien Bertaux and Kader Thiam of Genoway for the generation

of the KI-PRS mice. We also thank Manuel Fresno, Francisco Sanchez-

Madrid, and Mark Sefton for critical reading of the manuscript. We are

indebted to Cristina Prieto, Valentina Blanco, Tania Gomez, and Fernando

Barahona for expert technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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