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The Journal of Immunology, 2007, 178: 3680-3687.
Copyright © 2007 by The American Association of Immunologists, Inc.

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

Timothy J. Bauler*, Elizabeth D. Hughes{dagger}, Yutaka Arimura§, Tomas Mustelin§, Thomas L. Saunders{dagger},{ddagger} and Philip D. King2,*

* Department of Microbiology and Immunology, {dagger} Transgenic Animal Model Core, and {ddagger} Department of Internal Medicine, Division of Molecular Medicine and Genetics, University of Michigan Medical School, Ann Arbor, MI 48109; and § Infectious and Inflammatory Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells recognize peptide fragments of foreign Ags together with self MHC molecules displayed on the surface of APC (1). This specific recognition is achieved through a clonally distributed, cell surface-expressed TCR. In quiescent T cells, TCR binding to peptide-MHC induces T cell cytokine synthesis, proliferation, and differentiation into effector cells that aid in the elimination of Ag from the host.

Over the past two decades, much has been learned of the intracellular signaling pathways that emanate from the TCR which instruct T cell responses (2, 3, 4, 5). One of the first events after TCR engagement is activation of the Src-family protein tyrosine kinases (PTK),3 LCK and FYN. The precise mechanism by which these PTK become activated is uncertain but is likely to involve locally induced PTK aggregation, PTK-interacting adapter proteins, and the CD45 protein tyrosine phosphatase (PTP) (6, 7, 8, 9). Directly or indirectly, these events lead to phosphorylation of a positive-regulatory tyrosine contained in the kinase domain of the PTK which results in increased kinase activity. Upon activation, Src-family PTK phosphorylate tyrosine residues contained in ITAMs located in the cytoplasmic tails of TCR-signaling chains such as TCR{zeta}. The phosphorylated ITAMs are then recognized by the Src homology-2 domains of the Syk-family PTK, Zap70, which is thus recruited to the TCR-signaling complex and activated, in part, through transphosphorylation by Src-family PTK (10). Activated Zap70 phosphorylates the linker for activation of T cells transmembrane adapter protein which couples the TCR to the activation of transcription factors such as AP1, NF-{kappa}B, and NFAT (3). Together, these transcription factors initiate new programs of gene expression that orchestrate T cell responses (2, 3, 4, 5).

Although much is known of the TCR-induced membrane-proximal signaling events that promote T cell activation, less information is available on the mechanisms by which these events are negatively regulated. Strong candidate negative regulators are PTPs, which have the capacity to impede signaling by dephosphorylating positive-regulatory tyrosine residues in different signaling proteins. Of the ~65 PTPs that are expressed in T cells, several have thus far been implicated as negative regulators of proximal TCR signaling (11, 12, 13). Included among these is Src homology region 2 domain-containing phosphatase 1 (SHP-1) and PEST-domain phosphatase (PEP) which have been shown to dephosphorylate and inactivate LCK (SHP-1 and PEP) and Zap70 (SHP-1). Studies of SHP-1- and PEP-deficient mice support the contention that both PTPs are significant negative regulators of proximal TCR signaling (14, 15, 16).

Another PTP that has been proposed to function as an attenuator of early TCR signaling is PTPN3 (also known as PTPH1 in humans) (17). PTPN3 comprises an NH2-terminal FERM (band 4.1, ezrin, radixin, moesin) domain, a central PDZ (PSD-95, Dlg, ZO-1) domain, and a COOH-terminal PTP domain. Overexpression of PTPN3 in the Jurkat T cell leukemia cell line profoundly inhibits TCR signal transduction leading to activation of the promoter for the T cell growth-promoting cytokine, IL-2 (13, 18). Furthermore, a FERM domain-deleted mutant of PTPN3 is impaired in its ability to inhibit TCR-induced IL-2 promoter activity coincident with an inability of this mutant to localize to the plasma membrane (18). This finding suggests that PTPN3 inhibits TCR signaling in Jurkat by dephosphorylating a plasma membrane-localized substrate. A recent study indicates that one important target of PTPN3 is the TCR{zeta} chain. In this regard, PTPN3 was identified as the only PTP from a large tested panel of rPTPs that is capable of interacting physically with TCR{zeta} and dephosphorylating TCR{zeta} ITAMs in vitro (19). In addition, using an unbiased biochemical fractionation approach, PTPN3 and SHP-1 were identified as essentially the only PTPs expressed in Jurkat that are able to dephosphorylate TCR{zeta} (19). Together with the finding that PTPN3 dephosphorylates TCR{zeta} when both are transfected into COS-7 cells, these studies have contributed to the notion that PTPN3 acts a negative regulator of TCR signaling by dephosphorylating the TCR{zeta} chain.

To ascertain whether PTPN3 acts as a physiological negative regulator in T cells, we have generated two different strains of mice that do not express catalytically active PTPN3. T cell development, activation, and TCR signal transduction have each been investigated in these mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of PTPN3-deficient mice

PTPN3 PTP domain-negative (PTPN3 {Delta}PTP) gene-trapped mice were generated from gene-trapped 129P2/Ola Hsd E14Tg2a.4 embryonic stem (ES) cell lines (RRR012, RRR573, and RRS555) purchased from BayGenomics. The position of the gene trap within the Ptpn3 locus was determined by sequencing of PCR products generated from genomic DNA template using Ptpn3 and targeting vector-based primers. In all three ES cell lines, the gene trap is located within exon 27, and for two of the ES cell lines (RRR012 and RRR573) the insertion is in the identical position (further details can be provided upon request). The three new Ptpn3 alleles are referred to as Ptpn3RRR012PdK, Ptpn3RRR573PdK, and Ptpn3RRS555PdK. ES cells were injected into C57BL/6J x (C57BL/6J x DBA/2) blastocysts to generate chimeras which were bred with C57BL/6J mice to achieve germline transmission of the trapped alleles. Experiments were performed with littermate F2 animals generated from the intercross of C57BL/6J x 129P2 Ola Hsd+/Ptpn3 {Delta}PTP F1 mice.

A targeting vector for the generation of Ptpn3 gene-targeted mice was constructed by inserting genomic DNA fragments from a Ptpn3 genomic BAC clone into p-loxP-2FRT-PGKneo. Exon 27 was thus flanked by loxP sites, with the neomycin-resistance (NeoR) selection cassette (flanked by FRT sites) inserted into intron 27 (see Fig. 4, further details available upon request). Linearized vector was electroporated into the Bruce4 ES cell line of C57BL/6J origin (20). Correctly targeted ES cell clones were identified by Southern blotting and injected into C57BL/6J Tyrc-2J/c-2J blastocysts. Resultant chimeras were then bred to C57BL/6J Tyrc-2J/c-2J mice to achieve germline transmission of the targeted Ptpn3tm1PdK allele. Heterozygotes were intercrossed to generate homozygote Ptpn3tm1PdK animals and littermate controls.


Figure 4
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  		FIGURE 4. Normal cytokine synthesis, proliferation, and TCR signal transduction in restimulated PTPN3 {Delta}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 {Delta}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 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{zeta} was detected by Western blotting using a phosphotyrosine Ab. Blots were reprobed with a Zap70 Ab to show equivalent immunoprecipitation of Zap70. E, TCR{zeta} phosphorylation in PTPN3 {Delta}PTP and littermate control wild-type thymocytes was determined as in D.

 
Unless otherwise noted, all mice were 6–8 wk of age at the time of experimentation. All experiments were performed in compliance with University of Michigan guidelines and were approved by the University Committee on the Use and Care of Animals.

Genotyping

Genomic DNA from ES cell clones or tail biopsies was digested overnight with restriction enzymes, run on 0.5% agarose/Tris-acetate-EDTA gels and transferred to positively charged nylon membranes (Ambion) for Southern blotting. Probes were generated by PCR from a Ptpn3 genomic BAC and 32P-labeled by random priming. Following overnight hybridization, membranes were washed under high stringency conditions and exposed to film. In some experiments, the genotype of F1 mice generated from crosses of heterozygote Ptpn3tm1PdK mice was determined by PCR of tail genomic 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, respectively, and generate a 770-bp product from the wild-type allele (a product of greater than 3 kbp might also be generated from the targeted allele under optimal conditions). Primers for the detection of the targeted allele are based within the NeoR gene and generate a product of 330 bp (see Fig. 5).


Figure 5
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  		FIGURE 5. Generation of Ptpn3tm1PdK mice. A, Shown is part of the wild-type genomic Ptpn3 locus with targeting vector and expected organization of the targeted allele. Positions of HindIII (H) restriction sites and probes used in Southern blotting are indicated. B, Southern blots of HindIII-digested genomic DNA from different ES cell clones probed with 5' and 3' probes. Positions of bands from wild-type and targeted alleles are shown. C, Tail DNA from progeny of F1 mice generated from one of the ES lines (4E4) was used as a template in PCRs with primers that detect wild-type or targeted alleles (positions indicated by arrows in A). Mouse genotypes are indicated at top. D, Western blot of whole brain lysates from homozygote Ptpn3tm1PdK mice and littermate controls using a PTPN3 mAb. Note the absence of wild-type PTPN3 at 110 kDa in the homozygote Ptpn3tm1PdK mice.

 
Flow cytometry

Splenocytes and thymocytes were blocked with murine IgG (Sigma-Aldrich) and then stained with the following conjugated mAbs (BD Biosciences): H57-597-CyChrome (TCRbeta chain), RA3-6B2-PE (CD45R/B220), H1.2F3-FITC (CD69), IM7-FITC (CD44), MEL-14-biotin (CD62L), GK1.5-PE and H129.19-CyChrome (CD4), 53-6.7-CyChrome (CD8), and PC61-PE (CD25). Cell staining was analyzed by flow cytometry using a FACScan (BD Biosciences).

T cell cytokine production and proliferation

Splenocytes were stimulated with varying amounts of 145-2C11 (CD3{epsilon}; eBioscience) and 0.5 µg/ml 37.51 (CD28; BD Biosciences) or with varying amounts of staphylococcal enterotoxin B (SEB) in 96-well round-bottom plates in complete medium (RPMI 1640 supplemented with 10% FCS, 25 nM 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 after 24–48 h of culture. To assess T cell proliferation, splenocyte cultures were first labeled with 1 µM CFSE (Molecular Probes). After 72–96 h, CFSE fluorescence intensity was analyzed by flow cytometry.

In some experiments, recently stimulated T cells were used. For this purpose, splenocytes were first activated with 0.5 µg/ml anti-CD3 and anti-CD28 in complete medium in 6-well plates. After 48 h, recombinant human IL-2 (10 U/ml) was added and cells were cultured for a further 48 h. Flow cytometric analysis showed these cells were >98% TCRbeta+. These activated T cells were then restimulated using identical conditions as above for analysis of cytokine production and proliferation.

Western blotting and coimmunoprecipitation

Thymocytes, lymph node (LN) T cells or recently stimulated splenic T cells were activated by incubation on ice with anti-CD3 and anti-CD28 Abs (each 0.2 µg/106 cells) followed by cross-linking with anti-Armenian hamster IgG (0.5 µg/106 cells; Jackson ImmunoResearch Laboratories) at 37°C for the indicated times. Cells were then lysed in 1% Nonidet P-40, 0.5% n-dodecyl-beta-D-maltoside buffer. For analysis of protein tyrosine phosphorylation, lysates were run on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (PerkinElmer). Membranes were then probed with an anti-phosphotyrosine Ab (PY99; Santa Cruz Biotechnology) before stripping and reprobing with an ERK-2 Ab (C-14; Santa Cruz Biotechnology) to determine equal loading. To ascertain the extent of TCR{zeta} phosphorylation, Zap70 was immunoprecipitated from lysates using a Zap70 Ab (99F2; Cell Signaling Technology). Coimmunoprecipitated tyrosine-phosphorylated TCR{zeta} was then detected by Western blotting using an anti-phosphotyrosine Ab as above.

In separate experiments, expression of PTPN3 in whole brain lysates and purified unstimulated and recently activated splenic CD4 and CD8 T cells (purified by negative selection) was determined by Western blotting using a PTPN3 mAb. This Ab was produced by immunization of mice with a human PTPN3 peptide (residues 392–503) corresponding to the PDZ domain. T cell blots were reprobed with a control HePTP Ab (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of PTPN3 {Delta}PTP gene-trapped mice

Three independent Ptpn3 gene-trapped murine ES cell clones of 129P2/Ola Hsd strain origin (RRR012, RRR573, and RRS555) were obtained from the BayGenomics gene trap resource. Data from 5' RACE experiments provided by the resource indicated that in Ptpn3 RNA transcripts from these cell lines, Ptpn3 exon 26 is spliced to the splice acceptor upstream of the betageo sequence of the gene trap. Therefore, because exon 27 of the Ptpn3 gene encodes the nucleophilic cysteine residue of the PTPN3 PTP domain, such transcripts would direct the expression of a PTPN3 protein that completely lacks catalytic activity (Fig. 1A). Southern blotting analysis of genomic DNA prepared from the ES cell clones confirmed that in each case the gene trap was inserted into the Ptpn3 locus downstream of exon 26 (Fig. 1, B and C). To determine the precise position of the gene trap, we performed PCRs upon genomic DNA using primers based within the gene trap together with primers based within the Ptpn3 genomic locus. Sequencing of the PCR products revealed that for each cell line, the gene trap was inserted within exon 27. Furthermore, for two of the cell lines (RRR012 and RRR573), the gene trap was inserted in the same position. The location of the gene trap within exon 27 (rather than intron 26) eliminates the possibility that any catalytically active PTPN3 protein could be produced in these cell lines (or animals derived from them) as a result of RNA splicing.


Figure 1
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  		FIGURE 1. Generation of gene-trapped PTPN3 {Delta}PTP mice. A, Domain organization of PTPN3 and exon/intron organization of the Ptpn3 locus corresponding to the PTP domain. Exon 27 encodes the nucleophilic cysteine residue of the PTP active site. B, Representation of wild-type and gene-trapped Ptpn3 alleles showing NcoI (N) restriction sites and probe used in Southern blotting. Gene traps are located within exon 27 of the Ptpn3 gene (see Results for details). SA, splice acceptor; betageo, sequence encoding a beta-galactosidase-neomycin phosphotransferase fusion; pUC, cloning vector component of gene trap. C, Southern blot of NcoI-digested genomic DNA from two independent Ptpn3 gene-trapped ES cell clones (left) and tail DNA from progeny of F1 mice generated from one of the ES cell clones (RRR012) (right). Positions of bands from wild-type (WT) and gene-trapped alleles are indicated. D, Western blot of whole brain lysates from homozygote PTPN3 {Delta}PTP mice and littermate controls using a PTPN3 mAb. Note the absence of a 110-kDa PTPN3 Ab-reactive band in homozygotes which represents catalytically active PTPN3.

 
Ptpn3 gene-trapped ES cell lines were used to produce chimeric mice which were then bred with C57BL/6J mice to generate (C57BL/6 x 129P2/Ola Hsd)F1 mice that carried the gene-trapped allele (Ptpn3RRR012PdK, Ptpn3RRR573PdK, or Ptpn3RRS555PdK). These F1 mice were then intercrossed to generate homozygote PTPN3 {Delta}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 {Delta}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 {Delta}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 {Delta}PTP homozygotes. Theoretically, a high molecular protein of ~250 kDa that contains the PTPN3 FERM and PDZ fused to betageo 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 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 contained the same insertion, further analyses were conducted using Ptpn3RRR012PdK or Ptpn3RRS555PdK homozygote mice only.

T cell development and homeostasis in PTPN3 {Delta}PTP mice

To determine whether the PTPN3 {Delta}PTP mutation affected T cell development or peripheral T cell homeostasis, numbers and ratios of T cell subsets in thymus and secondary lymphoid organs of 6- to 8-wk-old mice were examined. In the thymus, numbers and ratios of CD4–CD8– double-negative (DN), CD4+CD8+ double-positive, and CD4+CD8– or CD4–CD8+ single-positive thymocytes were similar between homozygote PTPN3 {Delta}PTP and wild-type mice (Fig. 2 and data not shown). Furthermore, levels of expression of CD5 upon double-positive thymocytes were the same between the two groups of mice which is consistent with the view that there is no hyperactivation of this subset and no increase in the efficiency of positive selection. Among DN thymocytes, ratios of CD44+CD25– (DN1), CD44+CD25+ (DN2), CD44lowCD25+ (DN3), and CD44–CD25– (DN4) thymocytes were the same between the two groups of mice (Fig. 2). In spleen and LN, the number of T cells, their ratio to non-T cells and ratio of CD4+ to CD8+ T cells was unaffected in the homozygote PTPN3 {Delta}PTP mice (Fig. 2 and data not shown). Also, there was no evidence of previous or ongoing spontaneous activation of peripheral T cells in homozygote PTPN3 {Delta}PTP mice as judged by expression of the CD44, CD62L, and CD69 markers (Fig. 2). Apart from T cells, numbers and ratios of other lymphoid populations including B cells, NK cells, macrophages, dendritic cells and granulocytes were all found to be normal in homozygote PTPN3 {Delta}PTP mice (Fig. 2 and data not shown).


Figure 2
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  		FIGURE 2. Normal T cell development in PTPN3 {Delta}PTP mice. Depicted are flow cytometry plots of thymocytes and splenocytes from homozygote PTPN3 {Delta}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.

 
T cell cytokine production and proliferation in PTPN3 {Delta}PTP mice

We examined the ability of T cells from PTPN3 {Delta}PTP mice to synthesize cytokines and proliferate in response to TCR stimulation. Should PTPN3 normally function as a negative regulator of TCR signaling, then it might be expected that PTPN3 {Delta}PTP T cells would synthesize increased quantities of cytokines and proliferate to a greater extent than wild-type T cells. To examine this, splenocytes from homozygous PTPN3 {Delta}PTP mice and wild-type controls were stimulated with a CD3 Ab (directed to the TCR complex) plus an Ab against the CD28 T cell costimulatory receptor in vitro. Alternatively, splenocytes were stimulated with the superantigen, SEB. Concentrations of the cytokines IL-2, IFN-{gamma}, and IL-4 in culture supernatants were then determined after 1–2 days of culture. To measure T cell proliferation, similar cultures were initiated only cells were labeled with CFSE beforehand. Dilution of CFSE fluorescence after 3–4 days of culture was then assessed and taken as an indication of the extent of proliferation. As shown, T cells from homozygous PTPN3 {Delta}PTP mice synthesized similar quantities of IL-2, IFN-{gamma}, and IL-4 as T cells from wild-type littermate mice when stimulated with CD3/CD28 Abs (Fig. 3A). Similarly, PTPN3 {Delta}PTP T cells synthesized comparable amounts of IL-2 (Fig. 3A) and IFN-{gamma} and IL-4 (data not shown) in response to SEB. In addition, the ability of T cells to proliferate to CD3/CD28 Ab or SEB in these in vitro cultures was comparable between the two groups of mice (Fig. 3B).


Figure 3
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  		FIGURE 3. Normal T cell function and TCR signal transduction in PTPN3 {Delta}PTP mice. A, Whole splenocytes were stimulated with the indicated concentrations of CD3 Ab and 0.5 µg/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-{gamma} and IL-4 (at 48 h) were determined by ELISA. Shown are means ± 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 µg/ml) and CD28 (0.5 µg/ml) Abs or with SEB (10 µg/ml). CFSE fluorescence intensity 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 an ERK2 Ab to verify equal loading.

 
T cell cytokine production and proliferation of restimulated PTPN3 {Delta}PTP T cells

Resting peripheral T cells express PTPN3 at only low levels (13, 18). However, we observed that PTPN3 was expressed at substantially higher levels in wild-type T cells after 2–3 days stimulation with CD3/CD28 Abs and that these elevated levels of expression were maintained following several days of culture in IL-2 (Fig. 4A). Therefore, we considered the possibility that PTPN3 may function as a more significant negative regulator in recently stimulated T cells. To examine this, CD3/CD28-activated splenic T cells were grown in IL-2 for 2 days and then restimulated with CD3 and CD28 Abs. Cytokine secretion and proliferation was then measured as before. However, despite the elevated expression levels of PTPN3, recently stimulated T cells from PTPN3 {Delta}PTP mice did not synthesize any greater amounts of IL-2, IFN-{gamma}, or IL-4 or proliferate more in response to restimulation with CD3/CD28 Abs compared with recently stimulated T cells from wild-type mice (Fig. 4, B and C).

TCR signaling in PTPN3 {Delta}PTP T cells

PTPN3 has been proposed to function at an early step in the TCR signal transduction cascade acting to dephosphorylate the TCR{zeta} chain (19). As such, it would be predicted that in T cells from PTPN3 {Delta}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{zeta} chain tyrosine phosphorylation directly. For this purpose, we immunoprecipitated Zap70 from T cell lysates and then detected any coimmunoprecipitated tyrosine-phosphorylated TCR{zeta} by Western blotting using a phosphotyrosine Ab as previously described (22). Results from these experiments showed that TCR{zeta} is tyrosine phosphorylated and associates with Zap70 to a similar degree in homozygote PTPN3 {Delta}PTP and wild-type T cells (Fig. 4D). Similar results were also obtained with thymocytes (Fig. 4E).

Generation of PTPN3 gene-targeted mice

To guard against the potential of early embryonic lethality of homozygote PTPN3 {Delta}PTP mice, we also embarked upon the production of conditional PTPN3 {Delta}PTP mutant mice. To this end, we used the technique of homologous recombination to generate Ptpn3 gene-targeted clones in the Bruce4 ES cell line of C57BL/6J origin (Fig. 5A). Several correctly targeted clones were produced in which exon 27 of the Ptpn3 gene was flanked by loxP sequences and a NeoR cassette flanked by FRT sequences remained within intron 27 (Fig. 5, A and B). Chimeric mice were produced from two such targeted clones (4D4 and 4E4) and were bred with C57BL/6J mice to generate heterozygote Ptpn3tm1PdK mice. The NeoR cassette of the targeted allele contains a strong cryptic splice acceptor sequence. Therefore, if the NeoR cassette is allowed to remain within a targeted intron then this commonly results in a null allele as upstream exons are spliced to the NeoR cassette in RNA transcripts (23). For the Ptpn3tm1PdK targeted allele, if exon 27 or any upstream exon were to splice to the NeoR cassette, then transcripts would direct the expression of a catalytically inactive PTPN3 protein because an in-frame stop codon would be encountered within the NeoR cassette which would prevent the translation of the majority of the PTP domain. Based on these considerations, therefore, heterozygote Ptpn3tm1PdK mice were not immediately crossed with Flp recombinase-transgenic mice (to delete the NeoR cassette) but instead were intercrossed to generate homozygous Ptpn3tm1PdK mice and littermate heterozygote Ptpn3tm1PdK and wild-type controls (Fig. 5C). This approach would allow a ready means to examine the influence of loss of catalytically active PTPN3 on a pure-bred C57BL/6J genetic background.

Homozygote Ptpn3tm1PdK, heterozygote Ptpn3tm1PdK, and wild-type mice were born in expected Mendelian ratios. Furthermore, as with gene-trapped PTPN3 mice, there were no obvious defects in growth or development of homozygote or heterozygote Ptpn3tm1PdK mice. To confirm loss of normal PTPN3 protein expression in homozygous Ptpn3tm1PdK mice, brain lysates were analyzed by Western blotting using a PTPN3 mAb (Fig. 5D). As shown, full-length catalytically active PTPN3 protein at 110 kDa could not be detected in the Ptpn3tm1PdK homozygotes. Furthermore, no additional reactive bands at lower molecular masses could be detected in heterozygote or homozygote Ptpn3tm1PdK mice compared with wild-type mice even upon long exposure. Because the PTPN3 Ab used reacts with the PDZ domain of the phosphatase, this indicates that if any PTPN3 proteins are produced in these mice then they either lack the PDZ domain as well as the PTP domain or, if they contain the PDZ domain, are expressed at very low undetectable levels (note that the expected molecular mass of a FERM plus PDZ domain-containing protein is at least 50 kDa).

T cell development and function in PTPN3 gene-targeted mice

Analysis of T cell development and function was performed using Ptpn3tm1PdK mice and control mice generated from one of the targeted ES cell lines (4E4). Numbers and ratios of T cell subsets to one another and to other leukocytes were determined in thymus and peripheral lymphoid organs of 6- to 8-wk-old mice as before. In thymus, no differences in the number or ratio of DN (DN 1–4), double-positive and single-positive thymocytes were noted between wild-type, heterozygote, and homozygote Ptpn3tm1PdK mice (Fig. 6A and data not shown). Similarly, the number and ratio of TCR+, CD4+, and CD8+ T cells in spleen and LN was comparable between the three groups of mice (Fig. 6A and data not shown). All other examined leukocyte populations were also present in lymphoid organs in expected numbers and ratios (data not shown).


Figure 6
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  		FIGURE 6. Normal T cell development, function, and TCR signal transduction in Ptpn3tm1PdK mice. A, Percentages of the indicated thymocyte (left) and splenocyte populations (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 representative 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.

 
We next examined CD3/CD28-induced T cell cytokine synthesis in Ptpn3tm1PdK mice (Fig. 6B). As with homozygote Ptpn3 gene-trapped mice, T cells from homozygote Ptpn3tm1PdK mice synthesized similar quantities of IL-2, IFN-{gamma}, and IL-4 in response to CD3/CD28 as wild-type littermates. Furthermore, when recently 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 {Delta}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, 25, 26, 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 {Delta}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, numbers and ratios of T cell subsets were normal and there was no 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 serological evidence of autoimmunity (data not shown).


Figure 7
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  		FIGURE 7. Phenotypic analysis of splenocytes from aged gene-trapped PTPN3 {Delta}PTP mice. A, Percentage representation of the indicated splenocyte populations from aged mice (10.5 to 12 mo) was determined by flow cytometry. Data are represented as means plus 1 SD from analyses of six mice of each genotype. B and C, Expression of CD44/CD62L and CD69 upon CD4+ or TCRbeta+ splenic T cells from aged mice was determined by flow cytometry. Shown are representative histograms from four repeat experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
PTPN3 has been suggested to function as an important negative regulator of TCR signal transduction acting to dephosphorylate the TCR{zeta} chain (13, 18, 19). However, this conclusion has been based upon studies which have examined the effect of overexpression of 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 regulator of TCR signaling in primary T cells. For this purpose, we produced two independent strains of PTPN3 mutant mice. One strain carries a gene-trapped Ptpn3 allele and other contains a gene-targeted Ptpn3 allele. Catalytically active PTPN3 is not expressed in either strain of mice. Both strains of mice were viable and fertile and exhibited normal growth and development showing that catalytically active PTPN3 is dispensable in these regards. More pertinent to the current study, T cell development, T cell activation in the periphery, and TCR signal transduction were unaffected in both strains. Thus, catalytically active PTPN3 does not appear to have an essential function as a negative regulator of TCR signal transduction in primary T cells.

One potential explanation for the lack of a requirement for catalytically active PTPN3 in negative regulation of TCR signaling is functional redundancy with a closely related PTP known as PTPMEG1 (PTPN4) (30). Like PTPN3, PTPMEG1 comprises of a FERM domain, a PDZ domain and a PTP domain and shows 50% overall sequence identity with PTPN3. Moreover, PTPMEG1 is well expressed in T cells and similar to PTPN3 is able to inhibit TCR-induced IL-2 promoter activation when overexpressed in Jurkat (13). Therefore, in T cells, as well as in other cell types, loss of catalytically active PTPN3 may be compensated for by PTPMEG1. To address this issue of redundancy, it will be necessary to generate PTPMEG1-deficient mice and PTPN3/PTPMEG1-double-deficient mice. Of course, as an alternative explanation for the lack of an apparent influence of the loss of catalytically active PTPN3, we cannot exclude the possibility that PTPN3 performs some scaffolding function in T cells independent of the catalytic domain. However, because PTP domain-negative PTPN3 variants are at best expressed at very low levels in mutant mice, this possibility seems unlikely.

Apart from early TCR signal transduction, PTPN3 has also been implicated as a regulator of other cellular signaling processes. Notably, PTPN3 has been described to interact physically with the cytoplasmic domain of TNF-{alpha}-convertase (TACE) (31). TACE is a metalloprotease disintegrin involved in ectodomain shedding of several proteins including not only TNF-{alpha} but also TGF-{alpha}, L-selectin, TNFRs I and II and IL-1RII (32, 33). In cotransfection experiments performed in COS-7 cells, PTPN3 was shown to inhibit cell surface expression of TACE in a manner that was dependent upon PTPN3 catalytic activity. As a consequence, PTPN3 inhibited phorbol ester-induced secretion of TNF-{alpha} when coexpressed with TACE in COS-7 (31). The inference is that PTPN3 functions as a physiologic negative regulator of TACE and ectodomain shedding of the aforementioned proteins. However, we have not observed any increased or accelerated loss of L-selectin from the surface of T cells from PTPN3 PTP domain-negative mice when stimulated with CD3/CD28 Abs in vitro (data not shown). This finding argues against a role for PTPN3 in regulating TACE expression levels in primary cells.

Another function that has been ascribed to PTPN3 is dephosphorylation of the ubiquitously expressed molecular chaperone, Valosin-containing protein (VCP). VCP was originally identified as a ligand of the PTPN3 PTP domain by a substrate-trapping technique (34). Later, PTPN3-mediated dephosphorylation of VCP was shown to promote the formation of transitional endoplasmic reticulum formation in vitro (35). However, while we cannot exclude a role for PTPN3 in transitional endoplasmic reticulum formation per se, in T cells at least we have not observed any altered tyrosine phosphorylation of VCP either before or after activation with CD3 and CD28 Abs (data not shown). This demonstrates that catalytically active PTPN3 is not an essential regulator of VCP tyrosine phosphorylation in this cell type.

Finally, in a recent study, PTPN3 was identified as 1 of 6 PTPs of all 87 that are encoded in the human genome to be frequently mutated in colorectal cancer cell lines (36). Interestingly, of the other 5 PTPs, 1, PTPL1/FAP-1 (PTPN13), also contains FERM and PDZ domains and another, PTP36/PEZ (PTPN14), contains a FERM domain. Thus, of the only 5 PTPs that contain FERM domains, 3 are found to be mutated in colorectal cancer cell lines. This finding implies that PTPN3 (as well as the other 2 FERM domain-containing PTPs) may function as important tumor suppressors in colorectal cancer. Thus far, we have not observed spontaneous intestinal tumors in PTPN3 PTP domain-negative mice up to 1 year of age. However, it will be important to determine whether loss of catalytically active PTPN3 accelerates or enhances tumor formation in established murine colorectal cancer models such as the APCMin/+ mouse. Such studies are ongoing in the laboratory.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by American Heart Association Grant 0555597Z and by National Institutes of Health National Research Service Award 5-T32-GM07544 from the National Institute of General Medical Sciences. Back

2 Address correspondence and reprint requests to Dr. Philip D. King, Department of Microbiology and Immunology, University of Michigan Medical School, 6606 Medical Science II, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0620. E-mail address: kingp{at}umich.edu Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SHP-1, Src homology region 2 domain-containing phosphatase 1; ES, embryonic stem; NeoR, neomycin resistance; LN, lymph node; DN, double-negative thymocyte; SEB, staphylococcal enterotoxin B; TACE, TNF-{alpha}-convertase; VCP, Valosin-containing protein; PEP, PEST-domain phosphatase. Back

Received for publication October 5, 2006. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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