Protein Tyrosine Phosphatase That Lack Catalytically Active PTPN3 Normal TCR Signal Transduction in Mice

Normal TCR Signal Transduction in Mice That Lack Catalytically Active PTPN3 Protein Tyrosine Phosphatase

This information is current as Timothy J. Bauler, Elizabeth D. Hughes, Yutaka Arimura, of September 30, 2021. Tomas Mustelin, Thomas L. Saunders and Philip D. King J Immunol 2007; 178:3680-3687; ; doi: 10.4049/jimmunol.178.6.3680 http://www.jimmunol.org/content/178/6/3680 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Normal TCR Signal Transduction in Mice That Lack Catalytically Active PTPN3 Protein Tyrosine Phosphatase1

Timothy J. Bauler,* Elizabeth D. Hughes,† Yutaka Arimura,§ Tomas Mustelin,§ Thomas L. Saunders,†‡ and Philip D. King2*

PTPN3 (PTPH1) is a cytoskeletal protein tyrosine phosphatase that has been implicated as a negative regulator of early TCR signal transduction and T cell activation. To determine whether PTPN3 functions as a physiological negative regulator of TCR signaling in primary T cells, we generated gene-trapped and gene-targeted mouse strains that lack expression of catalytically active PTPN3. PTPN3 phosphatase-negative mice were born in expected Mendelian ratios and exhibited normal growth and development. Furthermore, numbers and ratios of T cells in primary and secondary lymphoid organs were unaffected by the PTPN3 mutations and there were no signs of spontaneous T cell activation in the mutant mice with increasing age. TCR-induced signal transduction, cytokine production, and proliferation was normal in PTPN3 phosphatase-negative mice. This was observed using both quiescent T cells and recently Downloaded from stimulated T cells where expression of PTPN3 is substantially up-regulated. We conclude, therefore, that the phosphatase activity of PTPN3 is dispensable for negative regulation of TCR signal transduction and T cell activation. The Journal of Immunology, 2007, 178: 3680–3687.

cells recognize peptide fragments of foreign Ags together PTK phosphorylate tyrosine residues contained in ITAMs located

with self MHC molecules displayed on the surface of in the cytoplasmic tails of TCR-signaling chains such as TCR␨. http://www.jimmunol.org/ T APC (1). This specific recognition is achieved through a The phosphorylated ITAMs are then recognized by the Src homol- clonally distributed, cell surface-expressed TCR. In quiescent T ogy-2 domains of the Syk-family PTK, Zap70, which is thus re- cells, TCR binding to peptide-MHC induces T cell cytokine syn- cruited to the TCR-signaling complex and activated, in part, thesis, proliferation, and differentiation into effector cells that aid through transphosphorylation by Src-family PTK (10). Activated in the elimination of Ag from the host. Zap70 phosphorylates the linker for activation of T cells trans- Over the past two decades, much has been learned of the intra- membrane adapter protein which couples the TCR to the activation cellular signaling pathways that emanate from the TCR which in- of transcription factors such as AP1, NF-␬B, and NFAT (3). To- struct T cell responses (2–5). One of the first events after TCR gether, these transcription factors initiate new programs of gene by guest on September 30, 2021 engagement is activation of the Src-family protein tyrosine kinases expression that orchestrate T cell responses (2–5). 3 (PTK), LCK and FYN. The precise mechanism by which these Although much is known of the TCR-induced membrane-prox- PTK become activated is uncertain but is likely to involve locally imal signaling events that promote T cell activation, less informa- induced PTK aggregation, PTK-interacting adapter proteins, and tion is available on the mechanisms by which these events are the CD45 protein tyrosine phosphatase (PTP) (6–9). Directly or negatively regulated. Strong candidate negative regulators are indirectly, these events lead to phosphorylation of a positive-reg- PTPs, which have the capacity to impede signaling by dephos- ulatory tyrosine contained in the kinase domain of the PTK which phorylating positive-regulatory tyrosine residues in different results in increased kinase activity. Upon activation, Src-family signaling proteins. Of the ϳ65 PTPs that are expressed in T cells, several have thus far been implicated as negative regulators of proximal TCR signaling (11–13). Included among these *Department of Microbiology and Immunology, †Transgenic Animal Model Core, and ‡Department of Internal Medicine, Division of Molecular Medicine and Genetics, is Src homology region 2 domain-containing phosphatase 1 University of Michigan Medical School, Ann Arbor, MI 48109; and §Infectious and (SHP-1) and PEST-domain phosphatase (PEP) which have been Inflammatory Disease Center, Burnham Institute for Medical Research, La Jolla, CA shown to dephosphorylate and inactivate LCK (SHP-1 and PEP) and 92037 Zap70 (SHP-1). Studies of SHP-1- and PEP-deficient mice support Received for publication October 5, 2006. Accepted for publication December 21, 2006. the contention that both PTPs are significant negative regulators of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance proximal TCR signaling (14–16). with 18 U.S.C. Section 1734 solely to indicate this fact. Another PTP that has been proposed to function as an attenuator 1 This work was supported by American Heart Association Grant 0555597Z and by of early TCR signaling is PTPN3 (also known as PTPH1 in hu- National Institutes of Health National Research Service Award 5-T32-GM07544 from mans) (17). PTPN3 comprises an NH -terminal FERM (band 4.1, the National Institute of General Medical Sciences. 2 ezrin, radixin, moesin) domain, a central PDZ (PSD-95, Dlg, 2 Address correspondence and reprint requests to Dr. Philip D. King, Department of Microbiology and Immunology, University of Michigan Medical School, 6606 Med- ZO-1) domain, and a COOH-terminal PTP domain. Overexpres- ical Science II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0620. E-mail sion of PTPN3 in the Jurkat T cell leukemia cell line profoundly address: [email protected] inhibits TCR signal transduction leading to activation of the pro- 3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PTP, protein ty- moter for the T cell growth-promoting cytokine, IL-2 (13, 18). rosine phosphatase; SHP-1, Src homology region 2 domain-containing phosphatase 1; ES, embryonic stem; NeoR, neomycin resistance; LN, lymph node; DN, double-neg- Furthermore, a FERM domain-deleted mutant of PTPN3 is im- ative thymocyte; SEB, staphylococcal enterotoxin B; TACE, TNF-␣-convertase; paired in its ability to inhibit TCR-induced IL-2 promoter activity VCP, Valosin-containing protein; PEP, PEST-domain phosphatase. coincident with an inability of this mutant to localize to the plasma Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 membrane (18). This finding suggests that PTPN3 inhibits TCR www.jimmunol.org The Journal of Immunology 3681 signaling in Jurkat by dephosphorylating a plasma membrane-lo- (CD69), IM7-FITC (CD44), MEL-14-biotin (CD62L), GK1.5-PE and calized substrate. A recent study indicates that one important target H129.19-CyChrome (CD4), 53-6.7-CyChrome (CD8), and PC61-PE of PTPN3 is the TCR␨ chain. In this regard, PTPN3 was identified as (CD25). Cell staining was analyzed by flow cytometry using a FACScan (BD Biosciences). the only PTP from a large tested panel of rPTPs that is capable of interacting physically with TCR␨ and dephosphorylating TCR␨ T cell cytokine production and proliferation ITAMs in vitro (19). In addition, using an unbiased biochemical Splenocytes were stimulated with varying amounts of 145-2C11 (CD3␧; fractionation approach, PTPN3 and SHP-1 were identified as es- eBioscience) and 0.5 ␮g/ml 37.51 (CD28; BD Biosciences) or with varying sentially the only PTPs expressed in Jurkat that are able to de- amounts of staphylococcal enterotoxin B (SEB) in 96-well round-bottom phosphorylate TCR␨ (19). Together with the finding that PTPN3 plates in complete medium (RPMI 1640 supplemented with 10% FCS, 25 nM dephosphorylates TCR␨ when both are transfected into COS-7 2-ME, 50 U/ml penicillin, 50 mg/ml streptomycin, and 2 mM L-glutamine). Concentrations of cytokines in culture supernatant were determined by ELISA cells, these studies have contributed to the notion that PTPN3 acts after 24–48 h of culture. To assess T cell proliferation, splenocyte cultures a negative regulator of TCR signaling by dephosphorylating the were first labeled with 1 ␮M CFSE (Molecular Probes). After 72–96 h, CFSE TCR␨ chain. fluorescence intensity was analyzed by flow cytometry. To ascertain whether PTPN3 acts as a physiological negative In some experiments, recently stimulated T cells were used. For this purpose, splenocytes were first activated with 0.5 ␮g/ml anti-CD3 and regulator in T cells, we have generated two different strains of anti-CD28 in complete medium in 6-well plates. After 48 h, recombinant mice that do not express catalytically active PTPN3. T cell devel- human IL-2 (10 U/ml) was added and cells were cultured for a further 48 h. opment, activation, and TCR signal transduction have each been Flow cytometric analysis showed these cells were Ͼ98% TCR␤ϩ. These investigated in these mice. activated T cells were then restimulated using identical conditions as above for analysis of cytokine production and proliferation. Downloaded from Materials and Methods Western blotting and coimmunoprecipitation Generation of PTPN3-deficient mice Thymocytes, lymph node (LN) T cells or recently stimulated splenic T PTPN3 PTP domain-negative (PTPN3 ⌬PTP) gene-trapped mice were cells were activated by incubation on ice with anti-CD3 and anti-CD28 Abs generated from gene-trapped 129P2/Ola Hsd E14Tg2a.4 embryonic (each 0.2 ␮g/106 cells) followed by cross-linking with anti-Armenian ham- stem (ES) cell lines (RRR012, RRR573, and RRS555) purchased from ster IgG (0.5 ␮g/106 cells; Jackson ImmunoResearch Laboratories) at 37°C for the indicated times. Cells were then lysed in 1% Nonidet P-40, 0.5%

BayGenomics. The position of the gene trap within the Ptpn3 locus was http://www.jimmunol.org/ determined by sequencing of PCR products generated from genomic DNA n-dodecyl-␤-D-maltoside buffer. For analysis of protein tyrosine phosphor- template using Ptpn3 and targeting vector-based primers. In all three ES ylation, lysates were run on SDS-PAGE gels and transferred to polyvinyli- cell lines, the gene trap is located within exon 27, and for two of the ES cell dene diﬂuoride membranes (PerkinElmer). Membranes were then probed lines (RRR012 and RRR573) the insertion is in the identical position (fur- with an anti-phosphotyrosine Ab (PY99; Santa Cruz Biotechnology) before ther details can be provided upon request). The three new Ptpn3 alleles are stripping and reprobing with an ERK-2 Ab (C-14; Santa Cruz Biotechnol- referred to as Ptpn3RRR012PdK, Ptpn3RRR573PdK, and Ptpn3RRS555PdK.ES ogy) to determine equal loading. To ascertain the extent of TCR␨ phos- cells were injected into C57BL/6J ϫ (C57BL/6J ϫ DBA/2) blastocysts to phorylation, Zap70 was immunoprecipitated from lysates using a Zap70 generate chimeras which were bred with C57BL/6J mice to achieve germ- Ab (99F2; Cell Signaling Technology). Coimmunoprecipitated tyrosine- line transmission of the trapped alleles. Experiments were performed with phosphorylated TCR␨ was then detected by Western blotting using an anti- ϫ littermate F2 animals generated from the intercross of C57BL/6J 129P2 phosphotyrosine Ab as above. ϩ ⌬

/Ptpn3 PTP by guest on September 30, 2021 Ola Hsd F1 mice. In separate experiments, expression of PTPN3 in whole brain lysates A targeting vector for the generation of Ptpn3 gene-targeted mice was and purified unstimulated and recently activated splenic CD4 and CD8 T constructed by inserting genomic DNA fragments from a Ptpn3 genomic cells (purified by negative selection) was determined by Western blotting BAC clone into p-loxP-2FRT-PGKneo. Exon 27 was thus flanked by loxP using a PTPN3 mAb. This Ab was produced by immunization of mice with sites, with the neomycin-resistance (NeoR) selection cassette (flanked by a human PTPN3 peptide (residues 392–503) corresponding to the PDZ FRT sites) inserted into intron 27 (see Fig. 4, further details available upon domain. T cell blots were reprobed with a control HePTP Ab (21). request). Linearized vector was electroporated into the Bruce4 ES cell line of C57BL/6J origin (20). Correctly targeted ES cell clones were identified by Results c-2J/c-2J Southern blotting and injected into C57BL/6J Tyr blastocysts. Resultant Generation of PTPN3 ⌬PTP gene-trapped mice chimeras were then bred to C57BL/6J Tyrc-2J/c-2J mice to achieve germline transmission of the targeted Ptpn3tm1PdK allele. Heterozygotes were inter- Three independent Ptpn3 gene-trapped murine ES cell clones of crossed to generate homozygote Ptpn3tm1PdK animals and littermate controls. 129P2/Ola Hsd strain origin (RRR012, RRR573, and RRS555) Unless otherwise noted, all mice were 6–8 wk of age at the time of were obtained from the BayGenomics gene trap resource. Data experimentation. All experiments were performed in compliance with Uni- Ј versity of Michigan guidelines and were approved by the University Com- from 5 RACE experiments provided by the resource indicated that mittee on the Use and Care of Animals. in Ptpn3 RNA transcripts from these cell lines, Ptpn3 exon 26 is spliced to the splice acceptor upstream of the ␤geo sequence of the Genotyping gene trap. Therefore, because exon 27 of the Ptpn3 gene encodes Genomic DNA from ES cell clones or tail biopsies was digested overnight the nucleophilic cysteine residue of the PTPN3 PTP domain, such with restriction enzymes, run on 0.5% agarose/Tris-acetate-EDTA gels and transcripts would direct the expression of a PTPN3 protein that transferred to positively charged nylon membranes (Ambion) for Southern blotting. Probes were generated by PCR from a Ptpn3 genomic BAC and completely lacks catalytic activity (Fig. 1A). Southern blotting 32P-labeled by random priming. Following overnight hybridization, mem- analysis of genomic DNA prepared from the ES cell clones con- branes were washed under high stringency conditions and exposed to film. firmed that in each case the gene trap was inserted into the Ptpn3 In some experiments, the genotype of F1 mice generated from crosses of locus downstream of exon 26 (Fig. 1, B and C). To determine the tm1PdK heterozygote Ptpn3 mice was determined by PCR of tail genomic precise position of the gene trap, we performed PCRs upon DNA. Forward and reverse primers for the detection of the wild-type Ptpn3 allele were based at the beginning of exon 27 and the end of exon 28, respec- genomic DNA using primers based within the gene trap together tively, and generate a 770-bp product from the wild-type allele (a product of with primers based within the Ptpn3 genomic locus. Sequencing of greater than 3 kbp might also be generated from the targeted allele under the PCR products revealed that for each cell line, the gene trap was optimal conditions). Primers for the detection of the targeted allele are based inserted within exon 27. Furthermore, for two of the cell lines within the NeoR gene and generate a product of 330 bp (see Fig. 5). (RRR012 and RRR573), the gene trap was inserted in the same Flow cytometry position. The location of the gene trap within exon 27 (rather than Splenocytes and thymocytes were blocked with murine IgG (Sigma-Aldrich) intron 26) eliminates the possibility that any catalytically active and then stained with the following conjugated mAbs (BD Biosciences): H57- PTPN3 protein could be produced in these cell lines (or animals 597-CyChrome (TCR␤ chain), RA3-6B2-PE (CD45R/B220), H1.2F3-FITC derived from them) as a result of RNA splicing. 3682 PTPN3 AND TCR SIGNAL TRANSDUCTION

Ptpn3 gene-trapped ES cell lines were used to produce chimeric mice which were then bred with C57BL/6J mice to generate ϫ (C57BL/6 129P2/Ola Hsd)F1 mice that carried the gene-trapped allele (Ptpn3RRR012PdK, Ptpn3RRR573PdK,orPtpn3RRS555PdK).

These F1 mice were then intercrossed to generate homozygote PTPN3 ⌬PTP F2 mice plus heterozygote and wild-type littermate controls (Fig. 1C). Mice of the three different genotypes were born in ratios that are in accordance with Mendelian inheritance and for PTPN3 ⌬PTP heterozygotes and homozygotes no obvious effects of the mutation upon growth or development were observed. To conﬁrm loss of expression of full-length catalytically active PTPN3 in PTPN3 ⌬PTP homozygotes, we analyzed brain lysates by Western blotting using a PTPN3 mAb directed toward the PDZ domain (Fig. 1D). As shown, the wild-type 110-kDa PTPN3 band could not be detected in PTPN3 ⌬PTP homozygotes. Theoreti- cally, a high molecular protein of ϳ250 kDa that contains the PTPN3 FERM and PDZ fused to ␤geo should be expressed in this mouse, at least in ES cells. Such a product would be generated as

a result of splicing of exon 26 to the gene trap. However, in adult Downloaded from tissues, this product must either not be expressed or be expressed at low levels because we have thus far been unable to detect it in Western blots. Because ES cell lines RRR012 and RRR573 con- FIGURE 1. Generation of gene-trapped PTPN3 ⌬PTP mice. A, Domain tained the same insertion, further analyses were conducted using RRR012PdK RRS555PdK organization of PTPN3 and exon/intron organization of the Ptpn3 locus Ptpn3 or Ptpn3 homozygote mice only. corresponding to the PTP domain. Exon 27 encodes the nucleophilic cys- http://www.jimmunol.org/ teine residue of the PTP active site. B, Representation of wild-type and T cell development and homeostasis in PTPN3 ⌬PTP mice gene-trapped Ptpn3 alleles showing NcoI (N) restriction sites and probe used in Southern blotting. Gene traps are located within exon 27 of the To determine whether the PTPN3 ⌬PTP mutation affected T cell Ptpn3 gene (see Results for details). SA, splice acceptor; ␤geo, sequence development or peripheral T cell homeostasis, numbers and ratios encoding a ␤-galactosidase-neomycin phosphotransferase fusion; pUC, of T cell subsets in thymus and secondary lymphoid organs of 6- cloning vector component of gene trap. C, Southern blot of NcoI-digested to 8-wk-old mice were examined. In the thymus, numbers and Ptpn3 Ϫ Ϫ ϩ ϩ genomic DNA from two independent gene-trapped ES cell clones ratios of CD4 CD8 double-negative (DN), CD4 CD8 double- (left) and tail DNA from progeny of F mice generated from one of the ES 1 positive, and CD4ϩCD8Ϫ or CD4ϪCD8ϩ single-positive thymo- cell clones (RRR012) (right). Positions of bands from wild-type (WT) and ⌬ by guest on September 30, 2021 gene-trapped alleles are indicated. D, Western blot of whole brain lysates cytes were similar between homozygote PTPN3 PTP and wild- from homozygote PTPN3 ⌬PTP mice and littermate controls using a type mice (Fig. 2 and data not shown). Furthermore, levels of PTPN3 mAb. Note the absence of a 110-kDa PTPN3 Ab-reactive band in expression of CD5 upon double-positive thymocytes were the homozygotes which represents catalytically active PTPN3. same between the two groups of mice which is consistent with the

FIGURE 2. Normal T cell development in PTPN3 ⌬PTP mice. De- picted are ﬂow cytometry plots of thymocytes and splenocytes from homozygote PTPN3 ⌬PTP mice and littermate wild-type controls showing expression of the indicated markers on the indicated live cell populations. Percentages of cells that fall within the designated regions of dot plots and percentages of cells that are positive for marker expression in histograms are shown. In CD44/CD62L plots, the boxed population represents CD44highCD62Llow recently activated memory cells. Data are representative of ﬁve repeat experiments. The Journal of Immunology 3683

FIGURE 3. Normal T cell function and TCR signal transduction in PTPN3 ⌬PTP mice. A, Whole splenocytes were stimulated with the indicated concentrations of CD3 Ab and 0.5 ␮g/ml of a CD28 Ab or with the indicated concentrations of SEB. Culture supernatant concentrations of IL-2 (at 24 and 48 h for CD3/CD28 and SEB stimulation, respectively) and IFN-␥ and IL-4 (at 48 h) were determined by ELISA. Shown are means Ϯ 1 SD of triplicate determinations. Similar results were obtained in ﬁve repeat experiments. B, Whole splenocyte cultures were labeled with CFSE and stimulated with CD3 (1 ␮g/ml) and CD28 (0.5 ␮g/ml) ␮

Abs or with SEB (10 g/ml). CFSE ﬂuorescence inten- Downloaded from sity was measured after 72 h (CD3/CD28) or 96 h (SEB) by ﬂow cytometry. Shown are representative experiments of three repeats. C, LN T cells were stimulated with CD3 and CD28 Abs for the indicated times in minutes. Protein tyrosine phosphorylation was then assessed by Western blotting of whole cell lysates using

an anti-phosphotyrosine Ab. Blots were reprobed with http://www.jimmunol.org/ an ERK2 Ab to verify equal loading. by guest on September 30, 2021

view that there is no hyperactivation of this subset and no increase ated only cells were labeled with CFSE beforehand. Dilution of in the efficiency of positive selection. Among DN thymocytes, ratios CFSE fluorescence after 3–4 days of culture was then assessed and of CD44ϩCD25Ϫ (DN1), CD44ϩCD25ϩ (DN2), CD44lowCD25ϩ taken as an indication of the extent of proliferation. As shown, T (DN3), and CD44ϪCD25Ϫ (DN4) thymocytes were the same be- cells from homozygous PTPN3 ⌬PTP mice synthesized similar tween the two groups of mice (Fig. 2). In spleen and LN, the number quantities of IL-2, IFN-␥, and IL-4 as T cells from wild-type lit- of T cells, their ratio to non-T cells and ratio of CD4ϩ to CD8ϩ T cells termate mice when stimulated with CD3/CD28 Abs (Fig. 3A). was unaffected in the homozygote PTPN3 ⌬PTP mice (Fig. 2 and data Similarly, PTPN3 ⌬PTP T cells synthesized comparable amounts not shown). Also, there was no evidence of previous or ongoing spon- of IL-2 (Fig. 3A) and IFN-␥ and IL-4 (data not shown) in response taneous activation of peripheral T cells in homozygote PTPN3 ⌬PTP to SEB. In addition, the ability of T cells to proliferate to CD3/ mice as judged by expression of the CD44, CD62L, and CD69 mark- CD28 Ab or SEB in these in vitro cultures was comparable be- ers (Fig. 2). Apart from T cells, numbers and ratios of other lymphoid tween the two groups of mice (Fig. 3B). populations including B cells, NK cells, macrophages, dendritic cells and granulocytes were all found to be normal in homozygote PTPN3 T cell cytokine production and proliferation of restimulated ⌬ ⌬PTP mice (Fig. 2 and data not shown). PTPN3 PTP T cells Resting peripheral T cells express PTPN3 at only low levels (13, ⌬ T cell cytokine production and proliferation in PTPN3 PTP mice 18). However, we observed that PTPN3 was expressed at substan- We examined the ability of T cells from PTPN3 ⌬PTP mice to tially higher levels in wild-type T cells after 2–3 days stimulation synthesize cytokines and proliferate in response to TCR stimula- with CD3/CD28 Abs and that these elevated levels of expression tion. Should PTPN3 normally function as a negative regulator of were maintained following several days of culture in IL-2 (Fig. TCR signaling, then it might be expected that PTPN3 ⌬PTP T cells 4A). Therefore, we considered the possibility that PTPN3 may func- would synthesize increased quantities of cytokines and proliferate tion as a more significant negative regulator in recently stimulated T to a greater extent than wild-type T cells. To examine this, spleno- cells. To examine this, CD3/CD28-activated splenic T cells were cytes from homozygous PTPN3 ⌬PTP mice and wild-type controls grown in IL-2 for 2 days and then restimulated with CD3 and CD28 were stimulated with a CD3 Ab (directed to the TCR complex) Abs. Cytokine secretion and proliferation was then measured as be- plus an Ab against the CD28 T cell costimulatory receptor in vitro. fore. However, despite the elevated expression levels of PTPN3, re- Alternatively, splenocytes were stimulated with the superantigen, cently stimulated T cells from PTPN3 ⌬PTP mice did not synthesize SEB. Concentrations of the cytokines IL-2, IFN-␥, and IL-4 in any greater amounts of IL-2, IFN-␥, or IL-4 or proliferate more in culture supernatants were then determined after 1–2 days of cul- response to restimulation with CD3/CD28 Abs compared with re- ture. To measure T cell proliferation, similar cultures were initi- cently stimulated T cells from wild-type mice (Fig. 4, B and C). 3684 PTPN3 AND TCR SIGNAL TRANSDUCTION Downloaded from

FIGURE 4. Normal cytokine synthesis, proliferation, and TCR signal transduction in restimulated PTPN3 ⌬PTP T cells. A, Puriﬁed CD4 and CD8 splenic T cells from C57BL/6 wild-type mice were stimulated with CD3/CD28 Abs (1°) for 72 h and then cultured in IL-2 for a further 48 h. T cells were then restimulated with CD3/CD28 Abs (2°). Aliquots were taken from CD3/CD28-stimulated cultures at the indicated time points (h) and lysed. Expression of PTPN3 was then determined by Western blotting. Blots were stripped and reprobed with a HePTP Ab to show equal loading. B and C, CD3/CD28- induced cytokine synthesis and proliferation of recently activated T cells from homozygote PTPN3 ⌬PTP mice and littermate wild-type controls (stimulated with CD3/CD28 Abs for 48 h and IL-2 propagated for a further 48 h) was determined as in Fig. 3. Shown are results of representative experiments from http://www.jimmunol.org/ ﬁve repeats. D, Recently activated T cells were stimulated with Abs to CD3 and CD28 for the indicated times (min) and lysed. Zap70 was then immunoprecipitated from lysates and coimmunoprecipitated tyrosine-phosphorylated TCR␨ was detected by Western blotting using a phosphotyrosine Ab. Blots were reprobed with a Zap70 Ab to show equivalent immunoprecipitation of Zap70. E, TCR␨ phosphorylation in PTPN3 ⌬PTP and littermate control wild-type thymocytes was determined as in D.

TCR signaling in PTPN3 ⌬PTP T cells PTPN3 has been proposed to function at an early step in the TCR signal transduction cascade acting to dephosphorylate the TCR␨ by guest on September 30, 2021 chain (19). As such, it would be predicted that in T cells from PTPN3 ⌬PTP mice a number of signaling proteins should become hyperphosphorylated on tyrosine residues in response to CD3/ CD28 stimulation. We examined this by Western blotting using a protein phosphotyrosine-specific Ab. In T cells that had not been recently stimulated, no increased protein tyrosine phosphorylation was observed (Fig. 3C). Likewise, no increased tyrosine phosphorylation was observed when the same experiments were performed using recently stimulated T cells (data not shown). Using recently stimulated T cells, we examined TCR␨ chain tyrosine phosphorylation directly. For this purpose, we immunoprecipitated Zap70 from T cell lysates and then detected any coimmunoprecipitated tyrosine-phosphorylated TCR␨ by Western blotting using a phosphotyrosine Ab as previously described (22). Results from these experiments showed that TCR␨ is tyrosine phosphorylated and as- sociates with Zap70 to a similar degree in homozygote PTPN3 ⌬PTP and wild-type T cells (Fig. 4D). Similar results were also obtained with thymocytes (Fig. 4E). FIGURE 5. Generation of Ptpn3tm1PdK mice. A, Shown is part of the Generation of PTPN3 gene-targeted mice wild-type genomic Ptpn3 locus with targeting vector and expected orga- To guard against the potential of early embryonic lethality of ho- nization of the targeted allele. Positions of HindIII (H) restriction sites and mozygote PTPN3 ⌬PTP mice, we also embarked upon the pro- probes used in Southern blotting are indicated. B, Southern blots of duction of conditional PTPN3 ⌬PTP mutant mice. To this end, we HindIII-digested genomic DNA from different ES cell clones probed with Ј Ј used the technique of homologous recombination to generate 5 and 3 probes. Positions of bands from wild-type and targeted alleles are shown. C, Tail DNA from progeny of F mice generated from one of the Ptpn3 gene-targeted clones in the Bruce4 ES cell line of C57BL/6J 1 ES lines (4E4) was used as a template in PCRs with primers that detect origin (Fig. 5A). Several correctly targeted clones were produced wild-type or targeted alleles (positions indicated by arrows in A). Mouse in which exon 27 of the Ptpn3 gene was flanked by loxP sequences genotypes are indicated at top. D, Western blot of whole brain lysates from R and a Neo cassette flanked by FRT sequences remained within homozygote Ptpn3tm1PdK mice and littermate controls using a PTPN3 intron 27 (Fig. 5, A and B). Chimeric mice were produced from mAb. Note the absence of wild-type PTPN3 at 110 kDa in the homozygote two such targeted clones (4D4 and 4E4) and were bred with Ptpn3tm1PdK mice. The Journal of Immunology 3685

FIGURE 6. Normal T cell development, function, and TCR signal transduction in Ptpn3tm1PdK mice. A, Percent- ages of the indicated thymocyte (left) and splenocyte pop- Downloaded from ulations (right) in homozygote Ptpn3tm1PdK and littermate wild-type mice were determined by ﬂow cytometry. Data are represented as mean percentage plus 1 SD and are derived from four mice of each genotype. B, Synthesis of cytokines by quiescent (left panels) and recently stimulated (as in Fig. 4) (right panels) splenic T cells was determined as in Figs. 3 and 4. Data are represen- http://www.jimmunol.org/ tative of three repeat experiments. C, Proliferation of quiescent splenic T cells was determined by dilution of CFSE ﬂuorescence as in Fig. 3. The same results were obtained in three repeat experiments. D, The CD3/ CD28-induced protein tyrosine phosphorylation response of quiescent splenic T cells was determined by Western blotting as in Fig. 3. Any small differences in the phosphotyrosine signal are seen to correlate with the amount of protein loaded. by guest on September 30, 2021

C57BL/6J mice to generate heterozygote Ptpn3tm1PdK mice. The cassette) but instead were intercrossed to generate homozygous NeoR cassette of the targeted allele contains a strong cryptic splice Ptpn3tm1PdK mice and littermate heterozygote Ptpn3tm1PdK and acceptor sequence. Therefore, if the NeoR cassette is allowed to wild-type controls (Fig. 5C). This approach would allow a ready remain within a targeted intron then this commonly results in a null means to examine the inﬂuence of loss of catalytically active allele as upstream exons are spliced to the NeoR cassette in RNA PTPN3 on a pure-bred C57BL/6J genetic background. transcripts (23). For the Ptpn3tm1PdK targeted allele, if exon 27 or Homozygote Ptpn3tm1PdK, heterozygote Ptpn3tm1PdK, and wild- any upstream exon were to splice to the NeoR cassette, then tran- type mice were born in expected Mendelian ratios. Furthermore, as scripts would direct the expression of a catalytically inactive with gene-trapped PTPN3 mice, there were no obvious defects PTPN3 protein because an in-frame stop codon would be encoun- in growth or development of homozygote or heterozygote tered within the NeoR cassette which would prevent the translation Ptpn3tm1PdK mice. To conﬁrm loss of normal PTPN3 protein ex- of the majority of the PTP domain. Based on these considerations, pression in homozygous Ptpn3tm1PdK mice, brain lysates were an- therefore, heterozygote Ptpn3tm1PdK mice were not immediately alyzed by Western blotting using a PTPN3 mAb (Fig. 5D). As crossed with Flp recombinase-transgenic mice (to delete the NeoR shown, full-length catalytically active PTPN3 protein at 110 kDa 3686 PTPN3 AND TCR SIGNAL TRANSDUCTION

stimulated T cells from the two groups of mice were examined there was also no apparent difference in the quantity of cytokines that were synthesized. Consistent with these results, T cells from homozygote Ptpn3tm1PdK mice proliferated normally in response to CD3/CD28 and showed a similar CD3/CD28-induced protein tyrosine phosphorylation response as T cells from wild-type mice (Fig. 6, C and D).

T cell numbers and function in aged gene-trapped PTPN3 ⌬PTP mice Mice with defects in T cell negative-regulatory pathways commonly accumulate large numbers of activated T cells in peripheral lymphoid organs with age (24–27). Furthermore, this lymphoid hyperplasia is frequently associated with the development of systemic autoimmune disease (24, 28, 29). Therefore, we examined older gene-trapped PTPN3 ⌬PTP mice (up to 1 year of age) for any evidence of T cell dysregulation or generalized autoimmunity. No such signs were observed. In peripheral lymphoid organs, num-

bers and ratios of T cell subsets were normal and there was no Downloaded from altered expression of CD44, CD62L, or CD69 activation markers (Fig. 7). T cell production of cytokines in response to CD3/CD28 stimulation was also normal (data not shown). Mice remained healthy and there was no morphological, histological, or serolog- ical evidence of autoimmunity (data not shown).

FIGURE 7. Phenotypic analysis of splenocytes from aged gene-trapped http://www.jimmunol.org/ PTPN3 ⌬PTP mice. A, Percentage representation of the indicated splenocyte Discussion populations from aged mice (10.5 to 12 mo) was determined by flow cytom- PTPN3 has been suggested to function as an important negative etry. Data are represented as means plus 1 SD from analyses of six mice of regulator of TCR signal transduction acting to dephosphorylate the each genotype. B and C, Expression of CD44/CD62L and CD69 upon CD4ϩ TCR␨ chain (13, 18, 19). However, this conclusion has been based or TCR␤ϩ splenic T cells from aged mice was determined by flow cytometry. upon studies which have examined the effect of overexpression of Shown are representative histograms from four repeat experiments. PTPN3 in the Jurkat T leukemia cell line and upon biochemical characterization of PTPN3 activity in vitro. Therefore, we sought to determine whether PTPN3 acts as a physiological negative reg- could not be detected in the Ptpn3tm1PdK homozygotes. Further- ulator of TCR signaling in primary T cells. For this purpose, we by guest on September 30, 2021 more, no additional reactive bands at lower molecular masses produced two independent strains of PTPN3 mutant mice. One could be detected in heterozygote or homozygote Ptpn3tm1PdK strain carries a gene-trapped Ptpn3 allele and other contains a mice compared with wild-type mice even upon long exposure. gene-targeted Ptpn3 allele. Catalytically active PTPN3 is not ex- Because the PTPN3 Ab used reacts with the PDZ domain of the pressed in either strain of mice. Both strains of mice were viable phosphatase, this indicates that if any PTPN3 proteins are pro- and fertile and exhibited normal growth and development showing duced in these mice then they either lack the PDZ domain as well that catalytically active PTPN3 is dispensable in these regards. as the PTP domain or, if they contain the PDZ domain, are ex- More pertinent to the current study, T cell development, T cell pressed at very low undetectable levels (note that the expected activation in the periphery, and TCR signal transduction were un- molecular mass of a FERM plus PDZ domain-containing protein is affected in both strains. Thus, catalytically active PTPN3 does not at least 50 kDa). appear to have an essential function as a negative regulator of TCR signal transduction in primary T cells. T cell development and function in PTPN3 gene-targeted mice One potential explanation for the lack of a requirement for Analysis of T cell development and function was performed using catalytically active PTPN3 in negative regulation of TCR sig- Ptpn3tm1PdK mice and control mice generated from one of the tar- naling is functional redundancy with a closely related PTP geted ES cell lines (4E4). Numbers and ratios of T cell subsets to one known as PTPMEG1 (PTPN4) (30). Like PTPN3, PTPMEG1 another and to other leukocytes were determined in thymus and pe- comprises of a FERM domain, a PDZ domain and a PTP do- ripheral lymphoid organs of 6- to 8-wk-old mice as before. In thymus, main and shows 50% overall sequence identity with PTPN3. no differences in the number or ratio of DN (DN 1–4), double-posi- Moreover, PTPMEG1 is well expressed in T cells and similar to tive and single-positive thymocytes were noted between wild-type, PTPN3 is able to inhibit TCR-induced IL-2 promoter activation heterozygote, and homozygote Ptpn3tm1PdK mice (Fig. 6A and data when overexpressed in Jurkat (13). Therefore, in T cells, as well not shown). Similarly, the number and ratio of TCRϩ, CD4ϩ, and as in other cell types, loss of catalytically active PTPN3 may be CD8ϩ T cells in spleen and LN was comparable between the three compensated for by PTPMEG1. To address this issue of redun- groups of mice (Fig. 6A and data not shown). All other examined dancy, it will be necessary to generate PTPMEG1-deficient mice leukocyte populations were also present in lymphoid organs in ex- and PTPN3/PTPMEG1-double-deficient mice. Of course, as an al- pected numbers and ratios (data not shown). ternative explanation for the lack of an apparent influence of the We next examined CD3/CD28-induced T cell cytokine synthe- loss of catalytically active PTPN3, we cannot exclude the possi- sis in Ptpn3tm1PdK mice (Fig. 6B). As with homozygote Ptpn3 bility that PTPN3 performs some scaffolding function in T cells gene-trapped mice, T cells from homozygote Ptpn3tm1PdK mice independent of the catalytic domain. However, because PTP do- synthesized similar quantities of IL-2, IFN-␥, and IL-4 in response main-negative PTPN3 variants are at best expressed at very low to CD3/CD28 as wild-type littermates. Furthermore, when recently levels in mutant mice, this possibility seems unlikely. The Journal of Immunology 3687

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