The src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) plays an important role in development and in growth factor receptor signaling pathways, yet little is known of its role in the immune system. We generated mice expressing a dominant-negative version of the protein, SHP2(CS), specifically in T cells. In SHP2(CS) mice, T cell development appears normal with regard to both negative and positive selection. However, SHP2(CS) T cells express higher levels of activation markers, and aged mice have elevated serum Abs. This is associated with a marked increase in IL-4, IL-5, and IL-10 secretion by SHP2(CS) T cells in vitro. In addition, primary thymus-dependent B cell responses are deficient in SHP2(CS) mice. We show that whereas TCR-induced linker for activation of T cells phosphorylation is defective, CTLA-4 and programmed death-1 signaling are not affected by SHP2(CS) expression. Our results suggest that a key action of wild-type SHP2 is to suppress differentiation of T cells to the Th2 phenotype.

The cytoplasmic src homology 2 domain-containing tyrosine phosphatase 2 (SHP2)3 consists of a single phosphotyrosine phosphatase (PTPase) domain and tandem SH2 domains. SHP2 is ubiquitously expressed and is, in general, thought to act as a positive regulator of signal transduction (1). Missense mutations in PTPN11, the human gene encoding SHP2, result in Noonan syndrome (2). The mutations are gain-of-function changes resulting in hyperactive SHP2, and contribute to characteristic facial and cardiac abnormalities. Homozygotic deletion of exon 3 of the Shp2 gene in mice results in embryonic lethality as a consequence of multiple developmental defects (3). Studies of chimeric mice, generated by injection of SHP2-deficient embryonic stem cells into RAG-2-deficient blastocysts, have shown that SHP2 is required in the earliest hemopoietic progenitors (4), while analysis of fibroblast cell lines, derived from Shp2 exon 3-deficient embryos, indicates that SHP2 is required for signaling downstream of a number of growth factor receptors (1).

Activation of SHP2 is mediated by binding to tyrosine-phosphorylated binding partners. Structural analysis shows that, in the resting state, the N-terminal SH2 domain of SHP2 blocks the catalytic domain (5). Interaction of the SH2 domains with phosphotyrosine peptides induces a conformational change in SHP2, resulting in stimulation of phosphatase activity (6). SHP2 function in many cell systems requires interaction through its SH2 domains with members of the Grb2-associated binders (GAB) family of docking proteins (7, 8, 9). It is likely that tyrosyl phosphorylation of SHP2 itself contributes to its function in some, but not all, signaling pathways (10).

SHP2 is required for the optimal activation of the Ras/ERK pathway in several different physiological contexts. It is possible that important substrates for SHP2 in fibroblasts include Sprouty (11) and phosphoprotein associated with glycosphingolipid-enriched domains (12). Additional targets for SHP2-mediated dephosphorylation may also include cell surface receptors. For example, it has been reported that SHP2 dephosphorylates a tyrosine residue in the epidermal growth factor receptor (EGFR), preventing recruitment of RasGAP to the EGFR signaling complex (13). Therefore, it is likely that SHP2 acts to relieve inhibition of the Ras pathway and is required for the sustained phase, rather than the initiation, of Ras activation. SHP2 may also regulate PI3K activity. In this regard, it has been reported that SHP2 potentiates PI3K activation downstream of the insulin receptor (14). Conversely, SHP2 has been shown to negatively regulate PI3K in EGFR signaling (15). Together, these data indicate that the precise role of SHP2 in signaling pathways is highly dependent on the cell type and receptor studied.

Although it is clear that SHP2 is important for development and growth factor signaling, little is known of its role in T cells. SHP2 has been implicated as a positive regulator of ERK in IL-2 signaling (16) and downstream of the TCR in Jurkat cells (17), but may also be involved in the negative regulation of T cell activation by inhibitory receptors such as CTLA-4 (18) and programmed death (PD)-1 (19). We therefore sought to investigate the role of SHP2 in primary T cells by developing a transgenic mouse system in which a catalytically inactive version of the PTPase, SHP2(CS), was overexpressed specifically in T cells. SHP2(CS) has been shown to act as a dominant-negative protein for endogenous SHP2 in many systems (14, 15, 20, 21). Furthermore, other groups have found that a floxed allele of Shp2 cannot be effectively deleted by any T cell-directed Cre recombinase transgene, although it is deleted well by other Cre lines (B. Neel, unpublished observations), suggesting that a transgenic approach may represent the best means of studying SHP2 in T cells in vivo. Our data show that SHP2(CS) mice have normal T cell development, yet display increased T cell activation in vivo, as they age. This is associated with an increased propensity of SHP2(CS) T cells to produce IL-4, but not IFN-γ, upon antigenic stimulation in vitro. Surprisingly, and in contrast to these findings, primary T-dependent B cell responses are markedly deficient in vivo in SHP2(CS) mice. We show that phosphorylation of the adaptor linker for activation of T cells (LAT), downstream of TCR stimulation, is defective in SHP2(CS) T cells, whereas CTLA-4 and PD-1 signaling remain unaffected. Our results suggest that a key action of wild-type (WT) SHP2 is in the regulation of Th2-type responses.

Murine SHP2 cDNA, encoding the major transcript of SHP2, was isolated from the pBluescript KS SHP2 vector (a gift from T. Yi, Taussig Cancer Center, Cleveland, OH). A Cys459→Ser mutation was introduced and an N-terminal polyglutamine (EE) tag was added by standard methods. EE SHP2(CS) was cloned into the hCD2 minigene cassette (22) (a gift from D. Kioussis, National Institute of Medical Research, Mill Hill, U.K.). A 13.3-kb fragment containing the promoter, the SHP2 cDNA, and the 3′ LCR was cut from the vector, gel purified, and microinjected into 329 pronuclei (day 2.5 embryos). Embryos were transferred to the ovarian ducts of pseudopregnant recipients. Transgenic mice were backcrossed to the B10.BR and BALB/c backgrounds for at least six generations. DO11.10 (23) and BM3 RAG2−/− (24) TCR transgenic mice were provided by M. Turner (Babraham Institute, Babraham, Cambridge, U.K.). Mice were maintained under specific pathogen-free conditions at The Babraham Institute, and all procedures were conducted under United Kingdom Home Office guidelines. In all experiments, age (±2 wk) and sex-matched WT and SHP2(CS) mice were compared. The responses of female and male mice were comparable in all cases.

Mice were genotyped from tail tissue biopsies. Founder mice were genotyped using a 2.1-kb HindIII fragment of the hCD2 vector as a probe for Southern blotting. Subsequent genotyping was conducted by PCR using primers specific for transgenes: SHP2(CS) forward, 5′-CGAGAGAGCCAGAGCCACCC-3′; SHP2(CS) reverse, 5′-GTAATCAGAAACAGGCTCATTGGG-3′; BM3 TCR forward, 5′-CTTGTTGCCACTGCCCCCAT-3′; BM3 TCR reverse, 5′-CTGTGATGGAGAAGACAACG-3′; DO11.10 TCR forward, 5′-TGGCTCTACAGTGAGTTTGGTGCCA-3′; DO11.10 TCR reverse, 5′-TGCAGCTGGATGGGATGAGCCAAGG-3′.

Cells were cultured in RPMI 1640 (Invitrogen Life Technologies), supplemented with 5 × 10−5 M 2-ME, 5% FCS, 50 mM HEPES, and antibiotics. WT and SHP2(CS) DO11.10 T cells were cultured in the presence of irradiated (10 Gy) BALB/c splenocytes loaded with OVA323–339 peptide (Department of Biochemistry, Southampton University) at a ratio of 1:3 responder:APC. rIL-2 (BD Pharmingen) was used at a concentration of 20 ng/ml. Following 4-day culture, cells were washed and rested for 6 h in serum-free medium. CD25 expression levels were assessed by flow cytometry (see below). A total of 2 × 107 blast cells was stimulated with 20 ng/ml IL-2 for the indicated time intervals, and cells were lysed in distilled water containing 1% Nonidet P-40, 20 mM HEPES, and 10 mM 2-ME. Supernatants were precleared with 10 μl of a 50% protein G-Sepharose beads slurry for 1 h at 4°C, and then incubated with anti-GAB2 (a gift from B. Neel, Harvard Medical School, Boston, MA) + protein G beads overnight at 4°C. Immunoprecipitates were washed in five changes of assay buffer (20 mM HEPES, 10 mM 2-ME), and resuspended in assay buffer. A total of 50 μl of sample was incubated at 37°C for 30 min in the presence of 100 μM phosphopeptide (TSTEPQpYQPGENL; Upstate Biotechnology), and malachite green buffer was added. Absorbance was read at 655 nm, and free phosphate was calculated by comparison with phosphate standards.

Flow cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences). Abs to the following cell surface markers were used (all BD Pharmingen, unless stated otherwise): CD3ε (145-2C11; in-house hybridoma), CD4 (YTA3.1.2; in-house), CD8 (53.6.7), CD25 (PC61), CD44 (IM7), B220 (RA3-6B2), CD69 (H1-2F3), DO11.10 TCR (KJ-126), and IL-4Rα (mILR4-M1). FITC, PE, tricolor, or allophyocyanin conjugates were used. For Vβ TCR analysis, a mouse Vβ TCR Screening Panel was used. At least 10,000 events were captured and analyzed using CelQuest software (BD Biosciences).

Samples of preimmune sera were taken 7 days before the initial immunization. Groups of female mice were immunized i.p. with 100 μg of trinitrophenyl (TNP)-keyhole limpet hemocyanin (KLH) or 100 μg of TNP-AECM-FICOLL (both obtained from Biosearch Technologies) emulsified in alum (Alu-Gel-S; Serva Electrophoresis). Samples of blood from tail veins were taken on days 7 and 11 postimmunization. For secondary responses, mice received a further 100 μg of TNP-KLH/alum on day 21. Tail bleeds were taken on day 28.

For analysis of serum anti-TNP Abs, Nunc Maxisorp plates were coated with 5 μg/ml TNP-OVA (Biosearch Technologies) overnight. Plates were blocked by addition of PBS/1% BSA for 1 h. Serial dilutions of sera were added. To detect specific Ab isotypes, secondary Abs were used (Clonotyping System-HRP; Southern Biotechnology Associates). For total serum Abs, anti-λκ Ig was used as capture Abs, and then the Clonotyping System was used to detect specific isotypes. For detection of IgE, OptEIA Mouse IgE set was used (BD Pharmingen). EC50 values were calculated by nonlinear regression analysis using Prism software (Microsoft).

Cytokine ELISA was performed using the following kits: IL-2, IL-4, IL-5, and IFN-γ (eBioscience); IL-10 (BioSource International). Recombinant murine cytokines were used as controls.

CD4+ T cells were purified from spleens using Spin-Sep murine T cell enrichment kits (StemCell Technologies). Purity of CD4+ cells was >90%, as assessed by FACS analysis. For the experiments documented in Fig. 1 A, T and B cells were sorted using a FACS Aria flow cytometer (BD Biosciences) by positive selection using CD3 and CD19 Abs, respectively. Cells were cultured in the conditions described above. WT or SHP2(CS) B10.BR cells were cultured in 96-well plates coated with CD3ε and CD28 mAbs at the indicated concentrations. For analysis of DO11.10 cells, CD4+ cells were incubated with irradiated splenocytes loaded with OVA peptide, as described above. Proliferation was assessed by pulsing cells with 1 μCi of [3H]thymidine (Amersham Biosciences), and measuring incorporation during the last 16 h of a 60-h incubation. Alternatively, supernatant was taken at the indicated time intervals and used for ELISA. For T cell differentiation studies, cells were incubated with APC + OVA for 96 h, washed thoroughly, rested in fresh medium for 16 h, then restimulated with either fresh APC:peptide or 6 μm latex beads (Sigma-Aldrich) precoated with 10 μg/ml anti-CD3 + 10 μg/ml anti-CTLA-4 (9H10), anti-PD-1 (RMP1-30) (both obtained from eBioscience), or control Ab (anti-KLH; BD Biosciences) at a cell:bead ratio of 1:2. Culture supernatants were collected after a further 24 h. The effects on cytokine secretion of CTLA-4 or PD-1 Abs were calculated as percentage of inhibition relative to control beads (CD3 + control Ab).

FIGURE 1.

Expression and function of SHP2(CS). A, Western blot analysis of SHP2(CS) in T and B cells from WT and transgenic mice (L1, L4, L5). Transgene expression was determined by blotting membranes with EE mAb. Blots were stripped and reprobed with SHP2 (C18) and tubulin Abs. B, Association of SHP2 with GAB2 following IL-2 stimulation of blasts cells, and C, inhibition of IL-2-induced SHP2 activation by SHP2(CS). PTPase activity of GAB2 immunoprecipitates was assessed, as detailed in Materials and Methods. Data represent the average of three measurements of PTPase activity ± SD. D, Inhibition of IL-2-induced ERK, but not STAT5, by SHP2(CS). Values in all panels correspond to relative levels of phosphorylated proteins normalized for loading. All data represent one of two separate experiments.

FIGURE 1.

Expression and function of SHP2(CS). A, Western blot analysis of SHP2(CS) in T and B cells from WT and transgenic mice (L1, L4, L5). Transgene expression was determined by blotting membranes with EE mAb. Blots were stripped and reprobed with SHP2 (C18) and tubulin Abs. B, Association of SHP2 with GAB2 following IL-2 stimulation of blasts cells, and C, inhibition of IL-2-induced SHP2 activation by SHP2(CS). PTPase activity of GAB2 immunoprecipitates was assessed, as detailed in Materials and Methods. Data represent the average of three measurements of PTPase activity ± SD. D, Inhibition of IL-2-induced ERK, but not STAT5, by SHP2(CS). Values in all panels correspond to relative levels of phosphorylated proteins normalized for loading. All data represent one of two separate experiments.

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For analysis of TCR signals, 107 lymph node (LN) cells from WT or SHP2(CS) mice (70% CD3+) were rested in serum-free medium for 3 h, and then stimulated with 10 μg/ml 2C11. For analysis of IL-4R signaling, WT or SHP2(CS) DO11.10 blast cells were rested as above, and then stimulated with 50 ng/ml rIL-4 (BD Pharmingen). Cells were lysed in buffer (1% Nonidet P-40, 60 μM octyl-glucoside, 200 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 20 mM EDTA, 1 mM sodium orthovanadate, and complete protease inhibitors) on ice for 10 min. Lysates were centrifuged, and insoluble material was discarded. Samples were resolved by SDS-PAGE, and proteins were transferred to nitrocellulose membranes. Membranes were blocked in PBS/0.05% Tween 20 (PBS-T) containing 5% BSA for 1 h, and then incubated with primary Abs diluted in PBS-T. The following Abs were used: phosphotyrosine (4G10; in house), SHP2 (C-18), ERK2 (C-14), and STAT6 (M-20) (all obtained from Santa Cruz Biotechnology); LAT and phosphoLAT Tyr191 (both obtained from Upstate Biotechnology); and phosphop44/42 MAPK Thr202/Tyr204, phosphoAkt Ser473, Akt, and phosphoSTAT6 (all obtained from New England Biolabs). Anti-phospho protein kinase D (PKD) Ser196 was a gift from D. Cantrell (University of Dundee, Dundee, U.K.). Anti-mouse and anti-rabbit Igs-HRP were used as secondary Abs (DakoCytomation), and bands were visualized using ECL (Amersham Biosciences). Densitometry of blots or film was conducted using a phosphor imager (Fuji LAS100), followed by analysis with AIDA software. Western blots were stripped at room temperature for 30 min using a low pH stripping buffer (pH 2.3) before reblocking and reprobing, as documented above.

For immunoprecipitation, 2 × 107 cells were stimulated as above, and postnuclear extracts were prepared. Lysates were precleared with 10 μl of a 50% protein G-Sepharose beads slurry for 1 h at 4°C, and then incubated with immunoprecipitating Ab + 10 μl of protein G beads overnight at 4°C. Immunoprecipitates were washed in five changes of lysis buffer. SDS-PAGE and Western blotting were conducted, as described above.

A total of 7 × 106 LN cells from WT or SHP2(CS) B10.BR mice was rested in complete medium at room temperature for 1 h. Cells were loaded with 5 μg/ml cell-permeable Indo-1 (Molecular Probes) for 30 min at 37°C, and then washed in medium. Cells were stained with anti-CD4 FITC and anti-CD8 PE, and then were washed and rested for 30 min at room temperature. A FACSVantage flow cytometer (BD Pharmingen) was used to analyze background calcium levels for 30 s; then cells were stimulated by the addition of 50 μg/ml 2C11. Data were analyzed using FlowJo software, and values for maximal calcium peaks were calculated relative to baseline values (Tree Star).

Two-tailed unpaired Student’s t test was used for statistical analysis. Values of p < 0.05 were considered to be significant.

A vector was constructed incorporating murine SHP2 cDNA encoding full-length SHP2 with a mutation in the catalytic site Cys459 residue (SHP2(CS)) and an N-terminal polyglutamine tag, under the regulation of the human CD2 promoter. Microinjections resulted in the birth of 51 pups: 5 carried the transgene as determined by Southern blot analysis (data not shown). Germline transmission of the transgene was obtained for 3 of 5 transgene-positive mice, and three independent SHP2(CS) mouse lines were established by backcrossing founder mice to the B10.BR strain. The three lines were further backcrossed to the I-Ad-restricted DO11.10 (23) and H-2Kk-restricted BM3 (24) TCR transgenic strains. Expression of endogenous and transgenic SHP2 in peripheral T cells and B cells of B10.BR mice was assessed by Western blotting using polyglutamine mAb (EE) and SHP2 polyclonal Abs. Fig. 1 A shows that SHP2(CS) is detectable in T cell lysates from all three lines of mice (designated L1, L4, and L5), but not in lysates of WT cells. Quantification of band intensities, relative to tubulin reprobes, showed that similar levels of SHP2(CS) were present in L1 and L4 T cells, with L5 expressing ∼50% that of the other lines. Reprobing the membranes for total levels of SHP2 revealed that SHP2(CS) was expressed at ∼7- and 4-fold higher levels than endogenous SHP2 in lines L1 or L4 and L5, respectively. A similar pattern of transgene expression was apparent in thymus lysates (data not shown). SHP2(CS) was also expressed in B cells of L4, but not L1 or L5, mice. For all subsequent experiments, we analyzed L1 or L5 mice in which transgene expression was T cell restricted.

To determine that expression of SHP2(CS) acted to inhibit endogenous SHP2, an assay for GAB2-associated PTPase activity was performed. Following IL-2 stimulation of T cells, GAB2 is phosphorylated and associates with SHP2 (25). Because IL-2R and GAB2 expression are low in naive cells, CD4+ cells from WT or SHP2(CS) DO11.10 mice were cultured for 96 h with peptide-loaded APCs and exogenous IL-2. Cells were rested and restimulated with IL-2, and GAB2 complexes were then immunoprecipitated. GAB2 association with SHP2 and GAB2-associated PTPase activity was enhanced following IL-2 stimulation of WT DO11.10 T cells (Fig. 1, B and C). In contrast, there was a striking increase in SHP2/SHP2(CS) association with GAB2 under both basal and stimulated conditions in SHP2(CS) DO11.10 cells, whereas the increase in PTPase activity was completely abrogated, with a similar level of inhibition apparent in cells from both L1 and L5 transgenic mice (Fig. 1 C). Importantly, IL-2R levels were similar in IL-2 blast T cells from transgenic and WT lines (data not shown). Given that the levels of inhibition of PTPase activity were equivalent in both lines, L1 and L5 mice were used interchangeably in all additional experiments, with comparable results achieved with both.

To confirm that SHP2(CS) expression interferes with downstream IL-2 signaling pathways, Western blots were performed. Activation of STAT5 by IL-2, as assessed by levels of STAT5 Tyr694 phosphorylation relative to loading controls, was comparable between SHP2(CS) and WT cells (Fig. 1,D). By contrast, ERK activation was substantially reduced in SHP2(CS) T cells. Basal levels of ERK phosphorylation were slightly increased in SHP2(CS) blasts, whereas IL-2 induction of ERK activation was inhibited by 30–60% (Fig. 1 D). The level of inhibition of phosphorylation of ERK1 and ERK2 in the presence of SHP2(CS) was equivalent. These data are consistent with previous reports, based on cell lines, implicating SHP2 as a positive regulator of ERK, but not STAT5 activation downstream of the IL-2R (26), and indicate that expression of SHP2(CS) inhibits endogenous SHP2 function in primary T cells.

T cell development in SHP2(CS) mice was assessed. Thymi were taken from 8-wk-old WT or SHP2(CS) B10.BR mice, and cells were stained with CD4 and CD8 mAbs. FACS analysis indicated that normal proportions of double-negative (DN), double-positive, and single-positive (SP) populations were found in SHP2(CS) thymi (Fig. 2,A, upper panel). To determine whether early maturation of thymocytes was affected by SHP2(CS) expression, the DN population was further analyzed. CD4+ and CD8+ cells were excluded from analysis using a FITC gate, and DN1-DN4 populations were assessed by expression of CD44 and CD25. SHP2(CS) thymi were shown to have similar proportions of all DN populations (Fig. 2 A, lower panel). Despite these analyses, a small, but consistent reduction in the cellularity of the SHP2(CS) B10.BR thymi, relative to WT controls, was noted (WT, 1.429 ± 0.41 × 108; SHP2(CS), 1.139 ± 0.249 × 108; n = 16; p = 0.0218).

FIGURE 2.

Thymocyte development in SHP2CS mice. A, FACS profiles of thymocytes from WT and SHP2CS B10.BR mice. Thymocyte populations were stained with Abs to CD4 and CD8, or DN thymocytes (CD4CD8) with Abs to CD44 and CD25. Values correspond to the percentage of cells falling within the corresponding quadrants. B, FACS profiles of WT and SHP2CS DO11.10 thymi. Cells were stained with CD4 and CD8 Abs or with the clonotypic TCR mAb KJ-126. C, FACS profiles for WT and SHP2CS BM3 thymi. Cells were stained as for A and B. A–C, All data shown are representative of at least six separate mice of each genotype. D, Deletion of Vβ5-, 11-, and 12-bearing cells in WT (n = 7) and SHP2CS (n = 11) B10.BR, but not C57BL/6 (n = 5), thymi. Data shown are the average percentage of CD4+ SP thymocytes bearing specific Vβ chains ± SD.

FIGURE 2.

Thymocyte development in SHP2CS mice. A, FACS profiles of thymocytes from WT and SHP2CS B10.BR mice. Thymocyte populations were stained with Abs to CD4 and CD8, or DN thymocytes (CD4CD8) with Abs to CD44 and CD25. Values correspond to the percentage of cells falling within the corresponding quadrants. B, FACS profiles of WT and SHP2CS DO11.10 thymi. Cells were stained with CD4 and CD8 Abs or with the clonotypic TCR mAb KJ-126. C, FACS profiles for WT and SHP2CS BM3 thymi. Cells were stained as for A and B. A–C, All data shown are representative of at least six separate mice of each genotype. D, Deletion of Vβ5-, 11-, and 12-bearing cells in WT (n = 7) and SHP2CS (n = 11) B10.BR, but not C57BL/6 (n = 5), thymi. Data shown are the average percentage of CD4+ SP thymocytes bearing specific Vβ chains ± SD.

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Positive selection was assessed by analyzing SHP2(CS) T cell development in the context of TCR transgenes. Similar proportions of thymic subpopulations were measured in SHP2(CS) DO11.10 RAG2+/+ and SHP2(CS) BM3 RAG2−/− mice relative to DO11.10 RAG2+/+ and BM3 RAG2−/− mice, respectively (Fig. 2, B and C). Transgenic TCR expression in the thymi of DO11.10 and SHP2(CS) DO11.10 mice was assessed and found to be very similar (Fig. 2 B). Furthermore, the cellularity of age-matched DO11.10 and SHP2(CS) DO11.10 thymi (WT, 1.084 ± 0.185 108 (n = 6); SHP2(CS), 1.184 ± 0.275 × 108 (n = 8); p = 0.536) or BM3 and SHP2(CS) BM3 thymi (WT, 1.09 ± 0.7 × 108 (n = 7); SHP2(CS), 0.854 ± 0.604 × 108 (n = 16); p = 0.38) was not significantly different.

Expression of superantigens encoded by mouse mammary tumor viruses leads to I-E-dependent deletion of double-positive thymocytes bearing Vβ5, 11, and 12 in B10.BR mice (27), and can be used as a model for negative selection. CD4+ SP thymocytes from WT B10.BR and SHP2(CS) B10.BR mice were assessed for expression of Vβ5, 11, and 12. Expression of Vβ8 on CD4+ SP cells, and thymi from C57BL/6 mice, which express mouse mammary tumor viruses, but do not delete Vβ5, 11, and 12 due to the absence of I-E molecules, was analyzed as controls. Vβ5-, 11-, and 12-bearing CD4+ SP thymocytes were almost completely absent in WT B10.BR and SHP2(CS) B10.BR thymi (Fig. 2 D). As expected, Vβ5-, 11-, and 12-bearing CD4+ SP thymocytes were readily detectable in C57BL/6 thymi. Similar levels of Vβ8+ CD4+ SP thymocytes were detected in all three mouse strains. Consistent with effective superantigen-mediated deletion, no mature T cells bearing Vβ5, 11, or 12 could be detected in the LN or spleens of WT or SHP2(CS) B10.BR mice (data not shown).

Taken together, the data obtained by analysis of SHP2(CS) B10.BR, SHP2(CS) DO11.10, and SHP2(CS) BM3 mice indicate that the SHP2(CS) transgene has no detectable effect on T cell development in the thymus.

Additional experiments were undertaken to characterize peripheral T cells in SHP2CS mice. Spleen and LN cell numbers were comparable between young adult (6- to 12-wk) WT and SHP2CS B10.BR mice (data not shown). Furthermore, similar proportions of CD3+, CD4+, and CD8+ T cells and B220+ B cells were present in WT and SHP2CS LN and spleens, as assessed by flow cytometry (Fig. 3, A and B, left panels). Similar numbers and proportions of peripheral T cells were also present in TCR transgenic SHP2(CS) mice relative to congenic control mice (data not shown). Analysis of B cell subsets indicated normal numbers and proportions of marginal zone and follicular B cells in the spleen and peritoneal B1 cells in SHP2CS B10.BR mice (data not shown). However, expression of activation markers was enhanced on SHP2CS B10.BR T cells derived from young adult mice. In this regard, both CD4+ and particularly CD8+ T cells in LN from SHP2CS B10.BR mice demonstrated higher levels of expression of CD44 (30 and 100% higher than WT, respectively; Fig. 3,A). Furthermore, a 2-fold higher proportion of SHP2CS B10.BR LN T cells had lost expression of CD62L (Fig. 3,A). Elevated expression of CD44 was also seen on splenic CD8+ T cells from SHP2CS mice (Fig. 3 B). Surface expression of early activation markers such as CD69 was often raised on SHP2CS B10.BR T cells, although this phenotype was less consistent (data not shown).

FIGURE 3.

Increased expression of activation markers on SHP2CS peripheral T cells. FACS profiles of LN (A) or spleen (SPL) (B) cells stained with Abs to CD3, CD4, CD8, CD62L, CD44, or B220. Values represent the percentage of cells falling within the marked regions or quadrants. Data shown are representative of at least 20 mice of each genotype, ranging in age from 6 to 12 wk.

FIGURE 3.

Increased expression of activation markers on SHP2CS peripheral T cells. FACS profiles of LN (A) or spleen (SPL) (B) cells stained with Abs to CD3, CD4, CD8, CD62L, CD44, or B220. Values represent the percentage of cells falling within the marked regions or quadrants. Data shown are representative of at least 20 mice of each genotype, ranging in age from 6 to 12 wk.

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These findings were suggestive of enhanced basal T cell activation, and led us to study the phenotype of aged SHP2(CS) B10.BR mice. Cohorts of SHP2(CS) and WT mice were allowed to age for at least 12 mo. FACS analysis of LN (Fig. 4, A and B) and splenic (data not shown) T cells confirmed that SHP2(CS) T cells expressed 2-fold higher levels of activation markers such as CD44 and CD69. Furthermore, and in contrast to the case in younger mice, the cellularity of mesenteric LN, but not spleens, in SHP2(CS) mice was increased by >3-fold (Fig. 4 C). The increase in cell numbers was attributed to an overall increase in both T and B cells. Thus, both the proportion and absolute numbers of activated phenotype T cells were increased in aged SHP2(CS) B10.BR mice relative to age-matched WT controls. Numbers and cellularity of Peyer’s patches were not affected by SHP2(CS) (data not shown).

FIGURE 4.

Evidence for enhanced immune activation in aged SHP2(CS) mice (age range, 12–18 mo). A, Increased surface expression of activation markers on SHP2(CS) T cells. The data shown represent FACS profiles of gated CD4+, CD8+, or CD3+ T cells counterstained with Abs to CD44, CD69, or CD25, and are representative of at least seven mice of each genotype. Values represent percentages of cells within the marked regions. B, Elevated surface expression of CD44 of SHP2CS T cells, and C, increased cellularity of LN from aged SHP2CS mice. The data shown represent the average values from four female mice of each genotype, analyzed on the same day, ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by Student’s t test. D, Increased levels of serum Abs in aged SHP2CS B10.BR mice (n = 11) relative to WT mice (n = 7). ♦, Represent values for individual mice; —, represent the mean value for each group.

FIGURE 4.

Evidence for enhanced immune activation in aged SHP2(CS) mice (age range, 12–18 mo). A, Increased surface expression of activation markers on SHP2(CS) T cells. The data shown represent FACS profiles of gated CD4+, CD8+, or CD3+ T cells counterstained with Abs to CD44, CD69, or CD25, and are representative of at least seven mice of each genotype. Values represent percentages of cells within the marked regions. B, Elevated surface expression of CD44 of SHP2CS T cells, and C, increased cellularity of LN from aged SHP2CS mice. The data shown represent the average values from four female mice of each genotype, analyzed on the same day, ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 by Student’s t test. D, Increased levels of serum Abs in aged SHP2CS B10.BR mice (n = 11) relative to WT mice (n = 7). ♦, Represent values for individual mice; —, represent the mean value for each group.

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Sera from WT and SHP2CS B10.BR mice were analyzed for levels of Ig. Levels of serum Ig were similar in young WT and SHP2CS B10.BR mice (data not shown). In contrast, sera from aged SHP2(CS) mice (n = 11) contained significantly higher levels of Ig than age-matched controls (n = 7) (Fig. 4 D). This was a result of enhanced levels of IgG1, IgG2a, and, most strikingly, IgE, which was elevated >20-fold. By contrast, serum IgM, IgG2b, and IgG3 were not significantly different between the groups of WT and SHP2(CS) mice. Taken together, the data obtained from analysis of aged mice are consistent with the suggestion that basal T cell activation is enhanced in SHP2(CS) mice.

T cells were purified from the spleens of WT or SHP2(CS) B10.BR mice, and in vitro responses were analyzed. Cells were cultured in the presence of varying doses of plate-bound CD3ε ± CD28 mAbs, and proliferation was assessed. Proliferation of WT and SHP2(CS) T cells was comparable at all doses of CD3ε Ab, both in the presence and absence of anti-CD28 (Fig. 5,A). Furthermore, IL-2 secretion by WT and SHP2(CS) T cells was not significantly different (Fig. 5,B). To assess MHC-peptide-induced T cell responses, CD4+KJ-126+ cells from DO11.10 or SHP2(CS) DO11.10 mice were cultured in the presence of irradiated APCs loaded with OVA peptide. Proliferative responses and IL-2 secretion of DO11.10 and SHP2(CS) DO11.10 cells were found to be similar (Fig. 5, A and B).

FIGURE 5.

Increased secretion of Th2 cytokines by SHP2CS T cells in vitro. Proliferative responses (A) and IL-2 secretion (B) of WT and SHP2CS B10.BR and DO11.10 T cells in response to CD3 ± CD28 mAbs or OVA peptide, respectively. The data shown represent the mean response of three mice of each genotype within one experiment ± SD, and are representative of at least three repeated experiments. C, Secretion of effector cytokines. Data represent the mean of the response of cells from WT (n = 3) or SHP2CS (n = 5) DO11.10 mice ± SD from one of at least four experiments. D, Cytokine secretion profiles following cell culture under Th1- or Th2-polarizing condition. E, Effects of anti-CTLA-4 or anti-PD-1 on IL-4 secretion. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; NS, p > 0.05, by Student’s t test.

FIGURE 5.

Increased secretion of Th2 cytokines by SHP2CS T cells in vitro. Proliferative responses (A) and IL-2 secretion (B) of WT and SHP2CS B10.BR and DO11.10 T cells in response to CD3 ± CD28 mAbs or OVA peptide, respectively. The data shown represent the mean response of three mice of each genotype within one experiment ± SD, and are representative of at least three repeated experiments. C, Secretion of effector cytokines. Data represent the mean of the response of cells from WT (n = 3) or SHP2CS (n = 5) DO11.10 mice ± SD from one of at least four experiments. D, Cytokine secretion profiles following cell culture under Th1- or Th2-polarizing condition. E, Effects of anti-CTLA-4 or anti-PD-1 on IL-4 secretion. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; NS, p > 0.05, by Student’s t test.

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Additional experiments were conducted to analyze Th1/Th2 differentiation of SHP-2(CS) T cells in vitro. CD4+ T cells from WT or SHP2(CS) DO11.10 mice were cultured in the presence of APCs and peptide for 96 h, rested, and then restimulated for 24 h. Cytokine production of differentiated cells was analyzed. Fig. 5 C shows typical results from one such experiment. The production of IL-4 by SHP2(CS) DO11.10 cells was 2-fold higher than WT levels. Similarly, levels of IL-5 and IL-10 were increased by 50–80% in supernatants from SHP2(CS) cultures. Statistical analysis demonstrated that production of IL-4, IL-5, and IL-10 was significantly higher than WT levels. By contrast, there was no significant difference in the levels of IFN-γ secreted by SHP2(CS) cells. Addition of IL-4R-blocking Abs did not affect the overall trend of higher levels of IL-4 in SHP2(CS) T cell supernatants, indicating that this was not a consequence of decreased usage of cytokine, but represented elevated cytokine secretion (data not shown).

Experiments were performed to assess cytokine production under Th1- or Th2-polarizing conditions. WT and SHP2(CS) DO11.10 cells were cultured in the presence of peptide-loaded APCs and either IL-12 (Th1 conditions) or IL-4 (Th2 conditions). When cultured in the presence of IL-12, both WT and SHP2(CS) DO11.10 T cells produced similar, large quantities of IFN-γ, a small amount of IL-10, and undetectable levels of IL-4, upon restimulation with peptide-APC alone (Fig. 5,D). By contrast, under Th2-polarizing conditions, large quantities of IL-4 and IL-10, but no IFN-γ, were produced. Furthermore, the level of IL-4 production by SHP2(CS) cells was ∼70% higher than WT cells, with a similar level of IL-10 production by both groups of mice (Fig. 5 D).

These data were reminiscent of experiments in which CTLA-4−/− mice were reconstituted with either CTLA-4 in which the cytoplasmic domain had been deleted or a mutation in Tyr201 (28). The mutated proteins largely rescue the CTLA-4−/− phenotype, yet with age the mice accumulate activated T cells of a Th2 phenotype. Furthermore, CTLA-4−/− DO11.10 TCR transgenic T cells preferentially develop into Th2 cells when stimulated with peptide Ag in vitro (29). Therefore, we chose to assess the function of CTLA-4 and PD-1 by measuring the inhibition of CD3-induced IL-4 secretion by PD-1 or CTLA-4 mAbs. In these experiments, CTLA-4 and PD-1 mAbs inhibited WT DO11.10 cell IL-4 secretion by ∼50 and 35%, respectively (Fig. 5 E). A proportionally similar level of inhibition of SHP2(CS) DO11.10 T cell IL-4 secretion was also evident.

Overall, these results show that SHP2(CS) CD4+ T cells proliferate normally in response to either CD3 mAb or cognate peptide-APC. However, SHP2(CS) DO11.10 cells produce increased levels of Th2 cytokines, particularly IL-4, under both neutral and Th2-polarizing conditions, yet production of IFN-γ is unaffected. Furthermore, CTLA-4 or PD-1 mAbs are equally effective at inhibiting IL-4 secretion by WT or SHP2(CS) cells.

To assess in vivo responses, groups of six WT or transgenic mice were immunized with the thymus-dependent Ag TNP-KLH in alum. Blood samples were taken from the tail veins of immunized mice on days 7 and 11, and the presence of TNP Abs was assessed. The results showed that TNP Ig levels in WT and SHP2(CS) mice were similar before immunization (data not shown). Surprisingly, analysis of sera taken on day 11 after immunization indicated that total levels of TNP Abs were significantly lower in sera from SHP2(CS) mice relative to WT mice (Fig. 6). Similar levels of anti-TNP IgM were present in sera from SHP2(CS) or control mice, whereas reduced levels of all IgG subclasses were detected in SHP2(CS) sera (Fig. 6 and data not shown). The averages of the total serum Ig, IgG1, and IgG2b anti-TNP responses of SHP2(CS) mice were reduced by 50 ± 19%, 62 ± 13%, and 71 ± 11%, respectively, relative to WT levels.

FIGURE 6.

Decreased primary T-dependent responses in SHP2CS mice. Data represent serum TNP Ab levels from immunized female WT (n = 6) or SHP2CS (n = 6) mice during primary (day 11) or secondary (day 28) responses. ♦, Represent values for individual mice; ▪, represent the mean value for each group. ∗, p < 0.05; ∗∗, p < 0.01; NS, p > 0.05. The data shown represent one of three repeated experiments.

FIGURE 6.

Decreased primary T-dependent responses in SHP2CS mice. Data represent serum TNP Ab levels from immunized female WT (n = 6) or SHP2CS (n = 6) mice during primary (day 11) or secondary (day 28) responses. ♦, Represent values for individual mice; ▪, represent the mean value for each group. ∗, p < 0.05; ∗∗, p < 0.01; NS, p > 0.05. The data shown represent one of three repeated experiments.

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Mice received a second dose of TNP-KLH in alum on day 21 after the initial immunization. Sera were collected after a further 7 days. Interestingly, following this second immunization, the anti-TNP response of SHP2(CS) B10.BR mice was of a similar magnitude to WT mice (Fig. 6). Total levels of anti-TNP Abs and anti-TNP IgM, IgG1, and IgG2b isotypes were not significantly different between the two groups of mice.

To confirm that the reduced primary response of SHP2(CS) mice was a result of defective T cell, rather than B cell, function, groups of mice were immunized with the T-independent type 2 Ag, TNP-Ficoll. Analysis of total serum anti-TNP Ab levels and IgM subclasses from sera taken on day 7 after immunization indicated no significant difference in the response of WT and SHP2(CS) mice (Fig. 7). There was a slight reduction in the production of TNP-specific IgG3 in SHP2(CS) mice, although this isotype was produced at much lower levels than IgM in both WT and transgenic mice. Taken together, these data indicate that, during primary responses, T cell help for T-dependent B cell responses is defective in SHP2(CS) mice. However, this deficiency is overcome by repeated immunization.

FIGURE 7.

Thymus-independent type 2 responses of WT and SHP2(CS) B10.BR mice. Data represent serum anti-TNP Ab levels from immunized female WT (n = 4) or SHP2CS (n = 4) mice during primary responses. ♦, Represent values for individual mice; ▪, represent the mean value for each group. ∗, p < 0.05; NS, p > 0.05.

FIGURE 7.

Thymus-independent type 2 responses of WT and SHP2(CS) B10.BR mice. Data represent serum anti-TNP Ab levels from immunized female WT (n = 4) or SHP2CS (n = 4) mice during primary responses. ♦, Represent values for individual mice; ▪, represent the mean value for each group. ∗, p < 0.05; NS, p > 0.05.

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The effects of transgene expression on TCR signaling were assessed by stimulating WT and SHP2(CS) cells with CD3ε mAb, followed by Western blotting to assess key signaling pathways. Fig. 8,A shows that similar levels of CD3-stimulated tyrosine-phosphorylated proteins were detected in WT and SHP2(CS) cell lysates. However, a consistent reduction in the intensity of the phosphorylation level of a 36- to 38-kDa protein was noted in SHP2(CS) lysates. Reprobing with a LAT Ab showed that the 36- to 38-kDa protein migrates in the same position as LAT. Identification of LAT was confirmed by use of a phospho-specific pTyr191 LAT Ab, which showed that anti-CD3 induced 30–40% lower levels of pY191-LAT in SHP2(CS) T cells relative to WT cells at all time points investigated (Fig. 8 B). Similar results were achieved using a second phospho-specific pTyr226 LAT Ab (data not shown). These data indicate that LAT phosphorylation is defective in SHP2(CS) T cells.

FIGURE 8.

Effects of SHP2(CS) expression on T cell signaling pathways. A, Western blot of phosphotyrosine patterns following CD3 mAb stimulation of WT or SHP2(CS) LN cells. B, Western blots of pY191-LAT, pPKD, pERK1/2, pAkt in WT and SHP-2(CS) cell lysates. C, FACS trace of CD3-induced calcium mobilization in gated CD4+ T cells from WT or SHP2(CS) mice. Data shown represent cells from one of six mice of each genotype. D and E, SHP2 and LAT immunoprecipitates analyzed for associated phosphotyrosyl proteins and SHP2, respectively. F, Western blot analysis of STAT6 phosphorylation following IL-4 stimulation of WT or SHP2(CS) DO11.10 blasts. In all cases, blots represent one of at least three repeated experiments, and values correspond to relative levels of phosphorylated proteins, as determined by densitometric analysis, normalized for loading.

FIGURE 8.

Effects of SHP2(CS) expression on T cell signaling pathways. A, Western blot of phosphotyrosine patterns following CD3 mAb stimulation of WT or SHP2(CS) LN cells. B, Western blots of pY191-LAT, pPKD, pERK1/2, pAkt in WT and SHP-2(CS) cell lysates. C, FACS trace of CD3-induced calcium mobilization in gated CD4+ T cells from WT or SHP2(CS) mice. Data shown represent cells from one of six mice of each genotype. D and E, SHP2 and LAT immunoprecipitates analyzed for associated phosphotyrosyl proteins and SHP2, respectively. F, Western blot analysis of STAT6 phosphorylation following IL-4 stimulation of WT or SHP2(CS) DO11.10 blasts. In all cases, blots represent one of at least three repeated experiments, and values correspond to relative levels of phosphorylated proteins, as determined by densitometric analysis, normalized for loading.

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To determine the effects of SHP2(CS) on downstream signaling pathways, CD3 stimulation of intracellular calcium mobilization was assessed by flow cytometry. Data indicated that the kinetics of calcium flux induced by anti-CD3 were similar in WT or SHP2(CS) cells (Fig. 8,C). However, the magnitude of the SHP2(CS) cells’ response was clearly reduced. Analysis of multiple experiments indicated that the peak calcium response of SHP2(CS) T cells was reduced by 35 ± 9% (n = 6; p < 0.01). By contrast, activation of additional pathways by anti-CD3, including phosphorylation of PKD, ERK, and Akt, appeared normal in SHP2(CS) T cells (Fig. 8 B).

It was possible that SHP2(CS) regulated LAT phosphorylation by a direct interaction. Therefore, experiments were performed to assess SHP2(CS)-binding proteins in primary T cells. Under both basal and stimulated conditions, tyrosine-phosphorylated proteins of ∼70, 130, and 150 kDa were found to coimmunoprecipitate with SHP2 (Fig. 8,D). Reprobes of these blots suggested that the 70-kDa protein was neither SHP2, as SHP2 migrated slightly faster than this pTyr band, nor SHP1 (Fig. 8,D and data not shown). Furthermore, the higher molecular mass bands did not correspond to phospholipase Cγ (data not shown). The identities of these proteins are, as yet, unknown. Importantly, there was no evidence for the presence of LAT in SHP2 immunoprecipitates (data not shown). Furthermore, in reciprocal experiments, SHP2 was not detectable in LAT immunoprecipitates (Fig. 8 E).

Finally, we investigated whether the effects of SHP2(CS) expression on cytokine secretion might be due to a direct effect on IL-4R signaling pathways. Activation of STAT6, by tyrosine phosphorylation, is known to be required for IL-4R signal transduction (30). WT and SHP2(CS) DO11.10 cells were cultured with APCs and peptide under neutral conditions, rested, and stimulated with IL-4, and phosphorylation of STAT6 was assessed by Western blotting. Levels of STAT6 phosphorylation in SHP2(CS) cells were comparable to WT levels at every time point investigated (Fig. 8 F). Likewise, levels of IL-4R expression on the surface of WT and SHP2(CS) cells were equivalent (data not shown). These data suggest that SHP2(CS) expression does not impact upon IL-4R signaling pathways in T cells.

SHP2 is known to play a vital role in mammalian development and signaling pathways. In this study, we have addressed the role of SHP2 in T cells, using two lines of transgenic mice expressing a dominant-negative version of the protein. Characterization of the L1 and L5 lines of mice showed that SHP2(CS) was expressed specifically in T cells and that it functioned to inhibit endogenous SHP2 activity, at least downstream of IL-2R signaling. Nonetheless, we cannot rule out the possibility that some SHP2 activity is retained in additional pathways.

Our studies have shown that maturation of T cells occurs normally in the thymi of SHP2(CS) mice (Fig. 2). Previous studies have shown that SHP2-deficient embryonic stem cells are deficient in their differentiation to hemopoietic progenitor cells (31), resulting in a complete absence of Thy-1+ or B220+ lymphoid progenitors in chimeric mice generated in RAG2−/− blastocyst complementation experiments (4). The CD2 promoter cassette used in this study to drive expression of SHP2(CS) becomes active at a much later stage of T cell development (22); therefore, it can be concluded that SHP2 activity is essential only in the generation of T cell progenitors. In contrast, mice expressing a dominant-negative version of SHP1 display enhanced positive and negative selection of thymocytes (32), indicating clear differences in the roles of these related phosphatases. Furthermore, the difference in the phenotypes of transgenic mice expressing dominant-negative SHP1, as opposed to SHP2, suggests that the SHP2(CS) transgene does not have nonspecific effects on the activity of SHP1.

Increased expression of activation markers such as CD44 represents a striking aspect of the SHP2(CS) T cell phenotype (Figs. 3 and 4). This was not observed in TCR transgenic SHP2(CS) T cells (data not shown), suggesting that SHP2(CS) T cells demonstrate an enhanced sensitivity to environmental or endogenous Ags relative to WT cells, a property lost when their specificity is restricted by expression of a transgenic TCR. Increased expression of activation markers was associated with elevated serum Ig levels in aged mice, particularly IgG1 and IgE, indicative of increased B cell activation. However, histological analysis of organs from aged SHP2(CS) mice appeared normal, and anti-nuclear Abs were not detected in sera from these mice (data not shown). These data suggest that, despite displaying enhanced T and B cell activation, SHP2(CS) B10.BR mice do not develop overt autoimmunity. Because the SHP2(CS) transgene could not be detected by Western blotting of purified B cell lysates from L1 and L5 mice, the most likely explanation for our observations is that increased Th cell activation in SHP2(CS) mice facilitates increased Ig secretion by B cells.

Additional experiments indicated that SHP2(CS) T cells secrete higher levels of Th2 cytokines upon stimulation (Fig. 5). By contrast, the ability of T cells to secrete IFN-γ was unaffected by SHP2(CS) expression. In addition to the cytokine milieu, a variety of factors has been implicated in the induction of Th1 or Th2 responses. These include the strength of TCR signal (33) and, in particular, induction of ERK activation: culture of CD4+ T cells with a high affinity peptide gives rise to a sustained ERK signal and Th1 differentiation, while inhibition of TCR-induced ERK induces IL-4 production (34). However, OVA323–339 represents a high affinity peptide for the DO11.10 TCR, and, in the current work, SHP2(CS) expression did not impact upon TCR-induced ERK activation. Furthermore, SHP2(CS) and WT T cells expressed similar levels of IL-4R, and STAT6 activation by IL-4 was comparable in both cell types, so a direct effect of SHP2(CS) on IL-4 signaling does not appear to be the explanation for the Th2 phenotype. In contrast, it is interesting to note that TCR-induced LAT phosphorylation was reduced in SHP2(CS) T cells (Fig. 8). The 30–40% reduction in LAT phosphorylation was associated with a proportionally similar reduction in the magnitude of peak calcium flux in SHP2(CS) cells (Fig. 8 C). Other pathways assessed did not appear to be affected. Similar findings, albeit more striking, have been reported for transgenic mice in which the phospholipase Cγ1-binding tyrosine residue in LAT (Y132) is mutated. This has been shown to result in the accumulation of Th2 T cells and plasma cells secreting IgG1 and IgE, suggesting that phosphorylation of WT LAT can lead to the suppression of T cell differentiation along the Th2 pathway (35). We have been unable to directly assess the effect of SHP2(CS) expression on phosphorylation of LAT Y132 due to the paucity of Abs. However, our data have shown that the Y191 and Y226 residues are equally affected, suggesting that SHP2 globally regulates LAT phosphorylation rather than specific tyrosine residues. SHP2 has also been shown to interact with LAT indirectly, through a GAB2/Gads/Grb2 complex, in Jurkat T cells (36). We could not detect an interaction between SHP2 and LAT in our primary cells, probably as a result of the low expression levels of GAB2 in naive T cells (24). The precise mechanism by which SHP2(CS) inhibits LAT phosphorylation, and subsequent calcium mobilization, requires further investigation.

SHP2 has been implicated as a mediator of inhibitory coreceptor signaling in T cells (18, 19). Our data suggest that SHP2 is dispensable for the function of both the CTLA-4 and PD-1 inhibitory receptors (Fig. 5 E). The intracellular domain of CTLA-4 also associates with the protein phosphatase 2A serine/threonine phosphatase (37), and CTLA-4 can function in some circumstances in the complete absence of the intracellular domain or when the SHP2-binding tyrosine residues have been mutated (27, 38). CTLA-4 may also regulate T cell function through its effects on Cbl-b expression (39), and both CTLA-4 (40) and PD-1 (19) have been shown to recruit SHP1 in addition to SHP2. These data are consistent with the hypothesis that while SHP2 may contribute to CTLA-4 and PD-1 function, additional mechanisms are available for inhibition mediated by either receptor. However, SHP2 may be involved in inhibitory signaling by receptors such as the B and T lymphocyte attenuator, which is induced on T cells following activation (41). It is possible that dysregulation of B and T lymphocyte attenuator, or of another inhibitory receptor yet to be described, might explain the actions of SHP2(CS) in promoting Th2 cell differentiation.

IL-4 production by Th2 cells is important for B cell isotype switching to IgG1 and IgE (30); therefore, the increase in IL-4 production by SHP2(CS) T cells in vitro is consistent with the observed increase in serum IgG1 and IgE in aged SHP2(CS) mice. Given this increased T cell help to B cells, it might at first appear paradoxical that the primary T-dependent B cell response of SHP2(CS) mice was reduced by 60–70% following the first immunization (Fig. 6). Because SHP2 has been implicated as a positive regulator of cell migration and chemotaxis (42), we considered that the defective immune response might be due to an action of SHP2(CS) on T cell migration. However, CD4+ T cells from SHP2(CS) migrate equally well in response to CCL19 as WT cells (data not shown). Furthermore, SHP2(CS) T cells effectively up-regulate CD40L upon anti-CD3 stimulation in vitro (data not shown), suggesting that the transgene does not perturb this aspect of T cell help to B cells. Instead, the most economical explanation for the defective primary immune response arises from the marked increase in IL-10 secretion that occurs upon SHP2(CS) T cell stimulation (Fig. 5,C). IL-10 is known to have inhibitory effects on dendritic cell function (43), and it is therefore possible that increased IL-10 secretion by SHP2(CS) T cells suppresses the effectiveness of Ag presentation in the primary response. Repeated immunization could then render IL-10 innocuous by lowering the threshold for efficient dendritic cell function, explaining the normal secondary responses observed (Fig. 6).

In summary, our results suggest that WT SHP2 plays an important role in suppressing the differentiation of T cells to a Th2 phenotype, and all of our experimental findings may be interpreted by assuming this single major function. Given the relatively small number of tyrosine phosphatases in the mouse genome, it is surprising that the perturbation of SHP2 in the T-lineage produces such a relatively simple phenotype. Whether the molecular mechanism involves mediation of the signals of an inhibitory receptor and/or regulation of LAT phosphorylation requires further investigation.

We are grateful to Prof. D. Cantrell, Dr. D. Kioussis, Prof. B. Neel, Dr. M. Turner, and Dr. T. Yi for their provision of reagents; to Dr. K. Smith for helpful advice; to G. Morgan for assistance with FACS; and to C. Wilson for animal husbandry.

The authors have no financial conflict of interest.

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 a grant from the Biotechnology and Biological Sciences Research Council. G.H. was a recipient of a European Molecular Biology Organization fellowship.

3

Abbreviations used in this paper: SH2, src homology 2 domain-containing tyrosine phosphatase 2; DN, double negative; EGFR, epidermal growth factor receptor; GAB, Grb2-associated binder; KLH, keyhole limpet hemocyanin; LAT, linker for activation of T cells; LN, lymph node; PD-1, programmed death-1; PKD, protein kinase D; PTPase, phosphotyrosine phosphatase; SP, single positive; TNP, trinitrophenyl; WT, wild type.

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