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Cutting Edge: The Tyrosine Phosphatase SHP-1 Regulates Thymocyte Positive Selection1 David R. Plas,2*† Calvin B. Williams,† Gilbert J. Kersh,† Lynn S. White,*† J. Michael White,† Silke Paust,*† Tatiana Ulyanova,*† Paul M. Allen,† and Matthew L. Thomas3*†
The binding kinetics of the TCR for its interacting ligand and the nature of the resulting signal transduction event determine the fate of a developing thymocyte. The intracellular tyrosine phosphatase SHP-1 is a potential regulator of the TCR signal transduction cascade and may affect thymocyte development. To assess the role of SHP-1 in thymocyte development, we generated T cell-transgenic mice that express a putative dominant negative form of SHP-1, in which a critical cysteine is mutated to serine (SHP-1 C453S). SHP-1 C453S mice that express the 3.L2 TCR transgene are increased in CD4 single positive cells in the thymus and are increased in cells that express the clonotypic TCR. These data suggest that the expression of SHP-1 C453S results in increased positive selection in 3.L2 TCRtransgenic mice and support a role for SHP-1 thymocyte development. The Journal of Immunology, 1999, 162: 5680 – 5684.
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uccessful thymocyte development is dependent on the expression of newly rearranged TCRs, and the subsequent induction of TCR signaling events. The nature of the TCR signal determines the ultimate fate of the thymocyte (1). Strong TCR signals induce thymocyte apoptosis, while very weak signals fail to induce events necessary for thymocyte survival. TCR signals within a defined window promote thymocyte maturation. The TCR signaling threshold is set by a dynamic balance of positive and negative regulatory components, including tyrosine kinases and phosphatases (2, 3). Alterations in either kinase or phosphatase activity are likely to affect positive and negative selection in the
*Howard Hughes Medical Institute, and †Center for Immunology, Department of Pathology, Washington University, St. Louis, MO 63110 Received for publication February 3, 1999. Accepted for publication March 19, 1999. 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 in part by grants from the National Institutes of Health. M.L.T. is an investigator of the Howard Hughes Medical Institute. 2 Current address: Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637 3 Address correspondence and reprint requests to Dr. Matthew L. Thomas, Department of Pathology, Box 8118, Washington University, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address:
[email protected]
Copyright © 1999 by The American Association of Immunologists
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thymus. In support of this concept, gene ablation and transgenic models have demonstrated that the tyrosine kinases p56lck, Csk, and ZAP-70, and the tyrosine phosphatase CD45 are necessary for thymocyte development (4 – 8). The protein tyrosine phosphatase SHP-1 has been shown to regulate TCR signal transduction, and is therefore a candidate for regulating thymocyte development (9 –11). SHP-1 is a cytoplasmic protein tyrosine phosphatase that contains two Src homology 2 (SH2)4 domains at the amino terminus (12). The SH2 domains function to recruit the enzyme to appropriately tyrosine phosphorylated sites and, additionally, to allosterically regulate phosphatase activity (13). SHP-1 is expressed in all hemopoietic cells and is a negative regulator of signal transduction from a number of surface receptors, including cytokine receptors and Ag receptors (14), Motheaten (me) and viable motheaten (mev) mice contain mutations within the SHP-1 gene, hcph, rendering them deficient in SHP-1 enzymatic activity (15, 16). These mice have multiple hemopoietic and immunological disorders and die within 6 to 30 wk of age, principally from a progressive inflammatory disease (17, 18). The thymus in mev mice involutes prematurely, hampering the study of SHP-1 in thymocyte development (16). In addition, SHP-1 deficiency in other cell types affects lymphocyte differentiation and complicates the study of lymphocytes in me or mev mice. For instance, B cell development in mev mice is inhibited due to the dysregulation of other cell types (19). Nonetheless, the alterations in lymphocyte development in me and mev mice suggest that SHP-1 may be important in Ag receptor signaling. SHP-1 has been implicated in the regulation of TCR signaling. Expression of catalytically inactive SHP-1 in Jurkat T cells or T cell hybridomas results in elevated IL-2 promoter activity (9). In addition, thymocytes from newborn mev mice, which express low levels of SHP-1, have elevated levels of Src family kinase activity, and TCR-mediated proliferation is enhanced (10, 11). SHP-1 has been shown to be associated with numerous signal transduction molecules in both B cells and T cells, including ZAP-70, Syk, Vav, and Grb-2 (9, 20, 21). Furthermore, ZAP-70 has been identified as a potential SHP-1 substrate (9). To address whether SHP-1 regulates thymocyte development, we generated a transgenic mouse expressing catalytically inactive
4 Abbreviations used in this paper: SH2, Src homology 2; CD4SP, CD4 single positive; me, motheaten; mev, viable me.
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SHP-1 C453S under the control of the CD2 promoter and enhancer. It is likely that SHP-1 C453S acts as a dominant negative protein by forming a stable interaction with SHP-1 SH2 domain docking sites and substrates and, therefore, prevents the recruitment of the endogenous SHP-1 (9, 22, 23). Our studies support a role for SHP-1 in TCR signal transduction and demonstrate the importance in regulating thymocyte development.
Materials and Methods Transgenic mice A cDNA-encoding murine SHP-1 was altered by point mutation to change the essential catalytic cysteine to serine, and an epitope tag derived from c-myc was appended to the carboxyl terminus (SHP-1 C453S) (21). SHP-1 C453S was cloned by blunt-end ligation into the XhoI site into the transgene vector pTEX, which contains the CD2 promoter and enhancer elements (SHP-1 C453S construct) (24). Transgenic mice were generated by standard methods and screened by PCR using the following oligonucleotides: sense 59-TGGTTTCACCGGGACCTC-39; antisense 59-TCTCACT GGTGGGGTCCG-39. All founders were confirmed by Southern blot analysis. Of the eleven founders, two lines were selected for study. Transgenic mice containing the 3.L2 TCR on the B6.AKR background were previously described (25, 26). The B6.AKR mouse, congenic with the C57BL/6 mouse at the H 2 locus, expresses the H 2k allele, which is necessary for 3.L2 positive selection. The 3.L2 transgene and the H 2k allele were determined by PCR using the primers 59-GCAGTCAC CCAAAGCCC-39 and 59-ACCGCCAGCTTTGAGCC-39 for 3.L2, and 59AGTCTTCCCAGCCTTCACACTCAGAGGTAC-39 and 59-CATAGC CCCAAATGTCTGACCTCTGGAGAG-39 for I Eb. The H-2 and 3.L2 genotypes of mice were always confirmed by flow cytometry. The presence of epitope-tagged SHP-1 C453S was confirmed by immunoblot analysis.
Antibodies CD8-FITC, CD4-PE, and I-Ab-FITC were obtained from PharMingen (San Diego, CA). Biotinylated clonotypic Ab specific for the 3.L2 TCR (CAb) was purified from tissue culture supernatant using protein A-Sepharose (Sigma, St. Louis, MO) (25). Streptavidin-Cy5 was obtained from Dako (Carpinteria, CA). For immunoprecipitations, anti-myc ascites was used (9E10, CRL-1729; American Type Culture Collection (ATCC), Manassas, VA). SHP-1 immunoblots were performed using a previously described rabbit antiserum specific for the SHP-1 SH2 domains (9).
Immunoprecipitation and immunoblot analysis Cells at a concentration of 108 cells/ml were lysed using a buffer containing 1% Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 10 mM Tris (pH 8.0), 20 mg/ml aprotinin, 10 mg/ml soybean trypsin inhibitor, and 1 mM PMSF (all from Sigma). Immunoprecipitations were performed by adding 2 ml of anti-myc ascites to lysates, tumbling for 1 h at 4°C, followed by an additional hour of tumbling with protein G-Sepharose (Pharmacia, Uppsala, Sweden). Immunoprecipitates were washed three times in lysis buffer. Immunoblot analysis was performed using anti-SHP-1 serum diluted 1:3000, 10 mM Tris (pH 8.0), 0.1% Tween 20, and 3% BSA. Immunoblots were washed twice in 1% Nonidet P-40, 10 mM Tris (pH 8.0), and 150 mM NaCl followed by one wash in 0.1% Tween 20, 10 mM Tris (pH 8.0), and 150 mM NaCl. Immunoblots were detected with peroxidase-conjugated protein A (Boehringer-Mannheim, Indianapolis, IN) using the ECL kit according to the manufacturer’s instructions (Amersham, Buckinghamshire, England).
Flow cytometry Single cell suspensions from freshly harvested thymus and spleen were prepared. Red blood cells were lysed, and lymphocytes were washed in PBS supplemented with 0.02% BSA and 0.01% NaN3 (staining buffer). Staining reactions containing 1 3 106 cells and 1 mg of the indicated Ab were incubated for 1 h in ice cold staining buffer. Two-step staining reactions were performed for three color flow cytometry using CD8-FITC, CD4-PE, and cAb-biotin, followed by Avidin-Cy5. All flow cytometry experiments were performed with at least one 3.L2 control mouse. Data were collected for 1 3 105 gated live cells.
Statistical analysis Two-tailed t tests were performed using the statistical analysis package in Microsoft Excel (Redmond, WA).
FIGURE 1. Expression of myc-SHP-1 C453S in 4420 SHP-1 C453S and 4463 SHP-1 C453S mice. Lines 4420 SHP-1 C453S and 4463 SHP-1 C453S were crossed with the 3.L2 TCR-transgenic mouse, and thymocytes and splenocytes were analyzed for the expression of the SHP-1 C453S transgene. Equal numbers of cells were used in anti-c-myc immunoprecipitations, followed by SHP-1 immunoblot analysis. Lanes 1-3, thymocytes. Lanes 4-6, splenocytes.
Results and Discussion To examine whether SHP-1 affects T cell development, a catalytically inactive form of SHP-1 (SHP-1 C453S) was expressed in thymocytes and T cells using the human CD2 promoter and enhancer (24). The CD2 control elements have been shown to mediate tissue-specific expression of reporter constructs in T cells (27). To distinguish the transgene from endogenous SHP-1, the transgene was appended at the carboxyl terminus with an epitopetag derived from c-myc (21). Expression of a dominant negative form of SHP-1 allows the study of SHP-1 function in the T cell lineage in the absence of complicating extrinsic effects that are present in mev mice (19). Of eleven founders, 4420 and 4463 were selected for further analysis based on expression levels of the transgene. Immunoprecipitation and immunoblot analysis of lysates from equal numbers of thymocytes and splenocytes confirms that the SHP-1 C453S protein is expressed in both tissues in lines 4420 and 4463 (Fig. 1). Comparison of the intensity of the bands indicates that there is greater expression of the transgene protein in 4420 than 4463 SHP-1 C453S mice. Comparison of immunoblot analysis indicates a 25 and 10% increase in total SHP-1 expression for the 4420 and 4463 transgenic lines, respectively. As expected, there were no gross changes in thymocyte number or developmental marker expression in either line of SHP-1 C453S mice (data not shown). It is likely that the dominant negative SHP-1 C453S does not completely inhibit SHP-1 function, and thus alterations in signaling will be obscured by changes in the repertoire of TCRs. This has been observed in other studies using a dominant negative approach (28). SHP-1 C453S mice show no evidence of thymic involution, in contrast to mev mice, which exhibit a reduction in thymocyte number and premature thymic involution by the age of 3 wk (18). The difference between the SHP-1 C453S mice and mev mice could be due to insufficient expression of catalytically inactive SHP-1, or alternatively, due to the indirect effects of the multiple hemopoietic abnormalities in mev mice. In support of the latter idea, treatment of mev mice with anti-Mac-1, which inhibits macrophage function, has been shown to rescue T cell development (29). To determine whether the expression of catalytically inactive SHP-1 affects T cell development, we generated mice that express both the clonotypic 3.L2 TCR and the SHP-1 C453S transgenes. The 3.L2 TCR was derived from a Th1 T cell clone that is specific
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FIGURE 2. Expression of SHP-1 C453S leads to an increase in 3.L2high cells in the thymus. A, The expression levels of CD4, CD8, and the 3.L2 TCR in thymocytes from 3.L2, 3.L2 3 4420 SHP-1 C453S, and 3.L2 3 4463 SHP-1 C453S mice were analyzed by three color flow cytometry. The data for the 3.L2 and 3.L2 3 4420 SHP-1 C453S mice were acquired in parallel staining reactions and are representative of the average results for CD4SP 3.L2high cells of each genotype. The data for the 3.L2 3 4463 SHP-1 C453S mouse were acquired in a separate experiment. Upper panel, The expression of CD4 and CD8. The box indicates the gate for CD4SP cells. Lower panel, The expression of the 3.L2 TCR in CD4SP cells. The 3.L2 TCR is detected using the clonotypic Ab CAb. B, The results of the analysis described in A are plotted for all mice that have been examined. The means of each group of mice are indicated in the chart. The number of mice in each group and the probability of a statistical difference between the groups are displayed at the bottom of the charts. Left, The percentage of total thymocytes that are CD4SP. Right, The percentage of CD4SP cells that are 3.L2high.
for the 64 –72 peptide from hemoglobin bd, presented by I-Ek (30). The 3.L2 TCR-transgenic mouse is well suited for this study since positive selection of the 3.L2 TCR is sensitive to ligand density (25). Reduction in the number of I-Ek molecules in the thymus reduces 3.L2 positive selection, and the addition of positively se-
lecting altered peptide ligands enhances positive selection (25) (C.B.W. and P.M.A., unpublished data). Thus, the 3.L2 TCRtransgenic mouse provides a model system for thymocyte development wherein positive selection can be both inhibited and enhanced in vivo.
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FIGURE 3. Increased CD4SP3.L2high cells in the spleen. A, Expression of the 3.L2 TCR in CD4SP splenocytes was determined by three color flow cytometry, gating on the CD4SP population. The expression level of the 3.L2 TCR is shown for CD4SP splenocytes. The data are representative of the average results for CD4SP3.L2high cells in mice of each genotype. The data for the 3.L2 control and the 3.L2 3 4420 SHP-1 C453S mice were acquired in parallel, and the data for the 3.L2 3 4463 SHP-1 C453S mouse were acquired in a separate experiment. Note that the overall height of the peak in the 3.L2 3 4463 SHP-1 C453S mouse is lower than in the 3.L2 3 4420 SHP-1 C453S mouse due to the lower number of CD4SP cells in the spleen of that animal. B, The percentage of CD4SP splenocytes that are 3.L2high is plotted for all mice that have been examined. The means for each group of mice are indicated in the chart. The number of mice in each group and the probability of a statistical difference between the groups are displayed at the bottom of the chart.
Flow cytometry analysis of CD4 and CD8 expression in 3.L2 and 3.L2,SHP-1 C453S thymocytes revealed an increase in the percentage of CD4 single positive (CD4SP) thymocytes in both SHP-1 C453S transgenic lines (Fig. 2A, upper panel). This represents an increase in the number of CD4SP cells in the thymus, since there were no differences in thymocyte numbers between the different transgenic mouse strains (3.L2, 4.93 3 107 6 2.24 3 107 (n 5 12); 3.L2, SHP-1 C453S (4420), 4.04 3 107 6 2.43 3 107 (n 5 12); 3.L2, SHP-1 C453S (4463), 4.53 3 107 6 3.38 3 107 (n 5 6)). The increased numbers of CD4SP cells can be explained as an increase in the positive selection of the 3.L2 TCR, since there is a greater percentage of 3.L21 T cells within the CD4SP population (Fig. 2A, lower panel). The differences in CD4SP cells and the CD4SP3.L2high cells were reproducible and statistically significant, as determined by Student’s t test (Fig. 2B). There is no significant difference in the number of CD4 CD8 double negative thymocytes nor in the number of 3.L2 TCR-expressing double negative thymocytes between the different genotypes. Since the 3.L2 control mice and the 3.L2,SHP-1 C453S mice express similar levels of I-Ek, the increase in CD4SP3.L2high thymocytes is likely to reflect a change in the TCR signaling pathway. A possible explanation for this finding is that the expression of
SHP-1 C453S has rendered the T cells more sensitive to antigenic stimulation, allowing an increase in positive selection of the 3.L2 TCR in the absence of an increase in ligand density. This hypothesis is supported by our previous studies, in which expression of SHP-1 C453S in the 3.L2 hybridoma resulted in increased IL-2 production in response to stimulation with antigenic peptide (9). Furthermore, stimulation of 3.L2,SHP-1 C453S thymocytes by CD3 cross-linking results in a 12% increase in the maximum rise in intracellular calcium when compared with 3.L2 thymocytes (3.L2, 264 nM calcium; 3.L2, SHP-1 C453S, 295 nM calcium). However, other developmental markers, such as CD2, CD5, CD24, and CD69 are unchanged between the two transgenic lines, indicating no gross alterations in development that may have occurred if the SHP-1 C453 transgene was affecting other signaling pathways. Taken together, the most likely explanation for the increase in CD4SP3.L2high cells in 3.L2, SHP-1 C453S mice is that TCR signal transduction has been altered, leading to more efficient positive selection in response to TCR stimulation. The changes in thymocyte development in 3.L2, SHP-1 C453S mice are also reflected in the periphery. Both lines of mice revealed an increase in the number of CD4SP3.L2high cells in the spleen (Fig. 3A). This difference is reproducible and statistically
5684 significant, as assessed by Student’s t test (Fig. 3B). There was not a statistically significant difference in the percentage of total splenocytes that are CD4SP, perhaps indicating a steady state regulation of the total number of CD4SP cells in the spleen. There was no significant difference in 3.L2 positive selection between the two SHP-1 C453S transgenic lines, even though the expression level of SHP-1 C453S is different between the lines. It may be that both mice express sufficient levels of SHP-1 C453S to enhance positive selection. This concept is supported by studies of 3.L2 positive selection in the context of positive selecting altered peptide ligands, where the levels of CD4SP3.L2high cells are similarly increased (C.B.W. and P.M.A., unpublished data). Thus, the level of positive selection in both lines of 3.L2, SHP-1 C453S mice is equivalent to the highest level of positive selection that has been observed. The analysis of TCR-transgenic mice expressing catalytically inactive SHP-1 confirms the proposed role of SHP-1 in T cell development. While it is possible that the effects observed are due to SHP-1 regulating other signaling pathways, a more likely explanation is that the effects are due to SHP-1 regulating TCR signal transduction during thymocyte positive selection. We favor this interpretation for the following reasons: 1) we have previously demonstrated that SHP-1 C453S regulates ZAP-70 in response to engagement of the 3.L2 TCR (9); and 2) the effects of the SHP-1 C453S transgene were not obvious in non-TCR-transgenic mice, possibly because the threshold for positive selection had changed without changing the number of positively selected thymocytes. It is possible that the repertoire of TCR usage may be altered, but the overall percentage of CD4SP and CD8SP T cells is unaffected. This is reminiscent of the phenotype of transgenic mice expressing dominant negative Ras, in which the effects of dominant negative Ras were best observed in the context of the H-Y TCR (28). If the change in positive selection as observed in this study were due to changes in cytokine signaling rather than TCR signaling, an effect should be observed regardless of the TCR being expressed. We generated SHP-1 C453S mice to study SHP-1 function in T cell development in the absence of activated macrophages that are present in SHP-1-deficient me and mev mice (19, 29). Our studies in SHP-1 C453S mice support the proposed role of SHP-1 as a regulatory enzyme in the TCR signal transduction pathway. The studies demonstrate that SHP-1 regulation is important during thymocyte positive selection.
Acknowledgments We thank Julie Blasioli for critical review of the manuscript. We thank Mike Owen for the use of the pTEX vector.
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