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The Journal of Immunology, 2006, 177: 5990-5996.
Copyright © 2006 by The American Association of Immunologists, Inc.

Conditional Deletion of Shp2 Tyrosine Phosphatase in Thymocytes Suppresses Both Pre-TCR and TCR Signals1

Thanh V. Nguyen, Yuehai Ke, Eric E. Zhang and Gen-Sheng Feng2

Programs in Signal Transduction and Stem Cells and Regeneration, Burnham Institute for Medical Research, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well known that T cell differentiation and maturation in the thymus is tightly controlled at multiple checkpoints. However, the molecular mechanism for the control of this developmental program is not fully understood. A number of protein tyrosine kinases, such as Zap-70, Lck, and Fyn, have been shown to promote signals required for thymocyte development, whereas a tyrosine phosphatase Src homology domain-containing tyrosine phosphatase (Shp)1 has a negative effect in pre-TCR and TCR signaling. We show in this study that Shp2, a close relative of Shp1, plays a positive role in T cell development and functions. Lck-Cre-mediated deletion of Shp2 in the thymus resulted in a significant block in thymocyte differentiation/proliferation instructed by the pre-TCR at the beta selection step, and reduced expansion of CD4+ T cells. Furthermore, mature Shp2–/– T cells showed decreased TCR signaling in vitro. Mechanistically, Shp2 acts to promote TCR signaling through the ERK pathway, with impaired activation of ERK kinase observed in Shp2–/– T cells. Thus, our results provide physiological evidence that Shp2 is a common signal transducer for pre-TCR and TCR in promoting T cell maturation and proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tcell development in the thymus proceeds in multiple and successive maturation steps associated with differential gene expression and gene rearrangement, which are orchestrated by signals provided by pre-TCR and then TCR as well as cytokine receptors (1, 2). Immature T cells are double-negative (DN)3 for CD4 and CD8 expression, and the DN population is further divided into four distinct developmental stages based on CD44 and CD25 expression (DN1, CD25–CD44+; DN2, CD25+CD44+; DN3, CD25+CD44–; and DN4, CD25–CD44–). TCR gene rearrangement is coincident with the onset of CD25 expression (DN2) and completed at the DN3 stage. The successfully rearranged beta-chain forms a heterodimer with the pre-T{alpha}-chain and display on the cell surface as the pre-TCR. Signaling through pre-TCR at the DN3 stage (called "beta selection") facilitates cell progression to the DN4 stage. The pre-TCR signaling also provides cues for proliferation, survival, allelic exclusion at the beta locus, induction of TCR{alpha} rearrangement, and subsequent development to the CD4+CD8+ double-positive (DP) stage (1, 2). Mediated by TCR signaling, the DP cell population undergoes vigorous negative and positive selection and down-regulates the expression of CD4 or CD8 to become single-positive (SP), MHC-self-restricted, mature T lymphocytes, which then leave the thymus and occupy peripheral lymphoid tissues, where they function in host immunity.

Signaling from the pre-TCR and TCR represents two successive and important waves during T cell development, which are highly coordinated through mechanisms that are not fully understood. Analyses of gene knockout (KO) and knockin mice have greatly advanced our understanding of signaling molecules that participate in the thymocyte maturation process (3, 4). For example, deletion of Lck severely suppresses thymocyte development at the pre-TCR stage, and Lck is also required for efficient TCR signaling in mature T lymphocytes (5). Thymocytes in Zap-70-deficient mice develop normally to the DP stage, with a defect in selection of SP lineages (6), but the Zap-70/Syk double KO mice exhibit a complete arrest of thymocyte development at the DN stage (7). The transmembrane tyrosine phosphatase CD45 is required for progression from DN to DP stage and also for subsequent maturation from DP to SP T cells (8, 9). The Ras-ERK pathway was demonstrated to function downstream of pre-TCR, regulating beta selection, and was also shown to act downstream of TCR, participating in negative/positive selection and CD4/CD8 lineage differentiation (10, 11, 12). Elucidating the common and distinct signaling components for pre-TCR and TCR will be crucial for advancing T cell biology.

Shp2 is a widely expressed cytoplasmic phosphotyrosine phosphatase with two SH2 domains at the N terminus (13, 14). Homozygous deletion of Shp2 in mice leads to embryonic lethality at mid-gestation (15), precluding the means to study Shp2 function in differentiated cell types in adult animals. In previous work, we generated chimeric mice by injecting Shp2-deficient embryonic stem cells into Rag-2–/– blastocysts and failed to detect even Thy-1+ or B220+ precursor lymphocytes of Shp2–/– origin in the chimeras (16). More recently, we demonstrated that normal Shp2 function is critical for the initial step of embryonic stem cell differentiation to mesoderm and to hemangioblasts (17). Thus, the Rag-2 rescue assay did not allow us to evaluate the specific functions of Shp2 during the multiple stages of thymocyte development or in mature lymphocytes. To address the issue, we have generated T cell-specific Shp2 KO mice by crossing conditional Shp2 mutant mice (Shp2flox) with Lck-Cre transgenic mice. Our results indicate that Shp2 participates in signal relay for both pre-TCR and TCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of T cell-specific Shp2 KO mice

Generation of a conditional Shp2 mutant allele (Shp2flox, Shp2F) in mice was published recently (18). Shp2T–/– mice were generated by crossing Shp2flox/flox mice with Lck-Cre transgenic mice (19, 20). For PCR analysis, genomic DNA was extracted from various tissues of animals and used as PCR templates. The following primers were used: 5' primer, 5'CAG TTG CAA CTT TCT TAC CTC; 3' primer, GCA GGA GAC TGC AGC TCA GTG ATG. The PCR conditions were as follows: 94°C, 30 s; 66.6°C, 30 s; 72°C, 1 min 45 s, 35 cycles.

Flow cytometry

Single-cell suspension of thymocytes and splenocytes were prepared from freshly dissected organs following standard procedures, and were stained with appropriate Abs in staining buffer (1% BSA and 0.1% NaN3 in PBS). Cells were washed and analyzed on a FACSCalibur with CellQuest software (BD Biosciences). The following Abs (BD Pharmingen) were used: FITC-, PE-, PerCP-, or biotin-conjugated anti-CD8 (Ly-2); FITC-, PE-, or biotin-conjugated anti-CD4 (L3T4); FITC- or PE-conjugated anti-CD25 (7D4); FITC-, PE-, or biotin-conjugated anti-CD44 (Ly-24); biotin-conjugated anti-mouse lineage panel (including Ly-6G, B220, CD11b, TER119, and CD3{alpha}); FITC- or biotin-conjugated anti-TCRb (H57-597); FITC- or PE-conjugated anti-mouse IgG2a for isotype control Abs (R35.95); anti-mouse CD16/CD32 for blocking Abs; FITC- or PE-conjugated streptavidin; and RED670-conjugated streptavidin (Invitrogen Life Technologies). For intracellular TCRbeta staining, freshly prepared thymocytes were stained with PE-anti-CD25 Ab and a panel of lineage markers, including CD3, CD4, CD8, B220, TER-119, Gr-1, and Mac (all biotinylated and revealed with streptavidin-RED670 from Invitrogen Life Technologies). Cells were washed, fixed in 1% paraformaldehyde, permeabilized in 0.5% saponin, and stained with FITC-TCRbeta Ab (H57-597). TCRbeta-positive cells were examined on CD25+Lin–-gated cells.

Anti-CD3 Ab treatment

Mice at the age of 4–6 wk were injected i.p. with 50 µg of anti-CD3 mAb, clone 145.2C11 (BD Pharmingen) in a total volume of 200 µl in PBS. Four days postinjection, mice were sacrificed, and the thymocytes were isolated for analysis.

Cell proliferation and apoptosis assay

T cells were purified and enriched to >90% from the spleen using the Pan T Cell Isolation Kit (Miltenyi Biotec). Purified T cells (5 x 104) were added in triplicate to 96-well plates precoated with anti-CD3 alone or together with anti-CD28 Abs (BD Pharmingen). The plate was incubated for 48 h, and 100 µl of the supernatant was used for IL-2 assays before pulsing with 1 µCi of [3H]thymidine overnight and the level of radioactivity incorporation determined. For annexin V staining, freshly isolated thymocytes were incubated with anti-CD3 mAb (5 µg/ml final concentration), or dexamethasone at 1 µM final concentration, or medium alone, overnight, and cells were recovered for apoptotic analysis by flow cytometry using an annexin staining kit (BD Biosciences), following the manufacturer’s instruction.

Molecular analyses

Thymic genomic DNA (20 µg) was digested with XbaI and subjected to Southern blot analysis using a 32P-labeled specific probe. For immunoblotting, thymic protein extracts were probed with specific Abs, and signals were revealed by a secondary HRP-conjugated anti-IgG Ab. The procedure for detection of intracellular phosphorylated ERK (pERK) by FACS was followed essentially as described previously (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Efficiency and specificity of Shp2 deletion

In previous work, we created a conditional Shp2 mutant allele (Shp2flox or Shp2F) in mice, by introducing two loxP sites into introns flanking exon 4 of the Shp2 gene (18). To achieve conditional ablation of Shp2 in the T cell lineage, we crossed Shp2F/F mice with Lck-Cre transgenic mice (19, 20), generating Shp2F/F:Lck-Cre/+ (Shp2T–/–) mice. Southern blot analysis of genomic DNA showed efficient excision of the loxP-flanked DNA sequence in the thymus, giving rise to an expected Shp2 KO band of 0.9 kb (Fig. 1A). Consistent with the literature (19, 20), variations were observed for the efficiency of Cre-mediated DNA recombination in the Southern blot analysis. DNA excision at the Shp2 locus was also confirmed by PCR detection of the floxed and deleted alleles (Fig. 1B). Importantly, the PCR analysis also showed that the Cre-mediated DNA recombination was specific to the thymus and spleen but did not occur in nonlymphoid tissue/organs, such as lung, kidney, liver, and heart. Furthermore, PCR analysis of sorted splenic T and B cells indicated efficient deletion of the Shp2flox allele in the peripheral T cell population but not in B cells (Fig. 1B). Selective deletion of Shp2 in the thymus was verified at the protein level by immunoblotting analysis of various tissue lysates using a specific anti-Shp2 Ab (Fig. 1C).


Figure 1
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  		FIGURE 1. Defective T cell development in Shp2T–/– mice. A, Southern blot analysis on thymic genomic DNA following XbaI digestion, with the WT, Shp2floxed (Flox), and Shp2 deletion (KO) alleles indicated. B, PCR analysis on genomic DNA from various organs of a Shp2T–/– mouse, as well as sorted T or B cells, to detect the Shp2KO and Shp2flox alleles. M denotes 1-Kb Plus DNA Ladder (Invitrogen Life Technologies). C, Protein extracts derived from the same mouse in B were immunoblotted with a Shp2-specific Ab using antitubulin as loading control. D, Total thymocyte numbers from Lck-Cre:Shp2F/F (KO) and Shp2F/F littermate controls (WT) were determined from five independent liters (**, p < 0.01). E, CD4 and CD8 expression profiles. The percentage of cells in each quadrant is shown, and the data are representatives of >10 independent experiments.

 
Deletion of Shp2 reduces thymic cellularity and partially blocks DN thymocyte development

We first compared cell numbers in the thymus between Shp2T–/– and littermate controls. Mice were sacrificed at 6–8 wk of age, and total numbers of thymocytes were determined. On average, the Shp2T–/– mice exhibited a ~50% reduction in total thymocytes as compared with control littermates (Fig. 1D; n = 10). When the percentages of DN, DP, and SP thymocyte subpopulations were compared, there was a 3-fold increase in DN cells in Shp2T–/– mice, accompanied by reduction in the percentage of DP cells (Fig. 1E). Comparison of the absolute cell number for each subpopulation revealed that the reduction in total number of thymocytes in Shp2T–/– mice was primarily due to decreases in DP and SP cells, with a modest increase of DN thymocytes (Table I). Furthermore, the reduction of SP thymocytes was more pronounced in the CD4+ than in CD8+ subpopulation (Table I).


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  		Table I. Cell numbers of total and different subpopulations of thymocytes

 
Deletion of Shp2 significantly attenuated the DN3 to DN4 transition process, with an accumulation of DN3 thymocytes in Shp2T–/– mice compared with controls (KO, 60% vs wild type (WT), 43%) with a corresponding decrease in DN4 cells (KO, 29% vs WT, 44%) (Fig. 2A). Despite a 50% reduction in total thymocytes, Shp2T–/– mice had a 4-fold increase in the absolute number of DN3 cells, as compared with controls (Fig. 2B). Because pre-TCR signals also lead to proliferative expansion/progression of DN thymocytes to DP cells, we evaluated the proliferative capacity of the DN cell population in Shp2T–/– mice, by comparing the DNA contents of DN3 and DN4 stage thymocytes (CD4–CD8–Lin–CD44–). As shown in Fig. 2C, there was a lower percentage of cells in the proliferative S and G2 phases of cell cycle in Shp2T–/– mice compared with cells from littermate controls, with an average of 2.23 ± 0.3-fold decrease (p < 0.05; n = 3). Because altered apoptosis could also contribute to a change in thymocyte number, we assessed cell apoptosis rates using annexin V staining. Total thymocytes freshly obtained from animals cultured with anti-CD3, dexamethasone, or medium were analyzed. We did not observe a difference in the fraction of annexin V-positive cells between Shp2T–/– and littermate controls (Fig. 2D). Together, these results suggest that the decrease of Shp2-deficient DP thymocyte number was likely due to impaired proliferation/progression from the DN to DP stage rather than increased cell apoptosis.


Figure 2
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  		FIGURE 2. Attenuated pre-TCR signaling in Shp2T–/– mice. A, Surface expression levels of CD44 and CD25. The percentage of cells in each quadrant is shown. B, Comparison of the absolute number of total DN, DN1, DN2, DN3, and DN4 cells (n = 10). C, Total thymocytes were stained with FITC-CD44 mAb and a panel of lineage markers including CD3, CD4, CD8, B200, TER-119, Gr-1, and Mac-1, fixed and stained with propidium iodine. The DNA content of FITC– (CD44–Lin–) thymocytes was analyzed by flow cytometry. Shp2T–/– mice had an average of 2.23 ± 0.3-fold decrease of the S+G2/M phase cells (p = 0.027; n = 3). D, Cell apoptosis was determined by annexin V staining after treatment of thymocytes with medium, dexamethasone, or anti-CD3. E, Intracellular staining for TCRbeta expression in CD25+ DN thymocytes (DN2 and DN3 stages). F, Developmental arrest of DN thymocytes in Shp2T–/– animals was not overcome by anti-CD3 treatment in vivo.

 
To determine the cellular mechanism for the block in transition of DN3 to DN4 stage, we determined the level of intracellular beta-chain, because a failure to properly rearrange beta gene could block this critical step in the differentiation process. We observed that the percentage of cells with intracellular beta-chain staining at the DN2 and DN3 stages (CD4–CD8–Lin–CD25+) was indistinguishable between Shp2T–/– and control mice (Fig. 2E). This result suggests that the observed defect in DN3 to DN4 transition and also in DP cell expansion lies downstream of pre-TCR. To address this issue, we performed forced thymocyte maturation assay in vivo with anti-CD3 Ab treatment. It has been shown that injection of anti-CD3 Ab accelerates thymocyte maturation from DN to DP stages by stimulating the signals downstream of pre-TCR, and expression of a dominant-negative Shc mutant in early thymocytes suppressed pre-TCR signals (22). Mice were injected with 50 µg of anti-CD3 mAb i.p., and thymocytes were harvested for analysis 4 days later. Although control mice showed accelerated progression from DN3 with few cells remained in that stage, the effect of anti-CD3 treatment was not observed in Shp2T–/– mice, with a relatively high number of DN3 cells detected (Fig. 2F). Thus, Shp2 is required for the relay of signals emanating from the pre-TCR.

ERK activation is attenuated in Shp2–/– thymocytes

To understand the molecular basis for the observed thymocyte developmental defects, we first assessed the ERK signaling pathway. Total thymocytes freshly obtained from animals were stimulated with anti-CD3, PMA, or anti-CD3/PMA in vitro, and protein extracts were immunoblotted with anti-pERK or anti-ERK Abs (Fig. 3A). Although a significant induction of pERK signal was observed at 2, 5, and 10 min in control cell lysates, pERK levels were dramatically decreased at these time points in preparations from a Shp2T–/– mouse, in response to anti-CD3 or anti-CD3/PMA treatment but not PMA alone. However, there was no significant difference in p-p38 levels between control and mutant. Consistently, flow cytometric analysis also detected lower pERK levels in stimulated DN thymocytes of mutant origin than that found in controls following anti-CD3/PMA stimulation for 5 and 10 min (Fig. 3B). Thus, Shp2 has a positive role in mediating TCR-triggered ERK activation in thymocytes, consistent with an observation made by many groups who found that Shp2 acts to promote signaling through the ERK pathway in a variety of cell types (13, 14).


Figure 3
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  		FIGURE 3. Shp2-deficient thymocytes exhibit reduced ERK phosphorylation. A, Thymocytes were stimulated with the anti-CD3, anti-CD3+ PMA, or PMA for the indicated time periods, and cell lysates were prepared and analyzed by immunoblotting with Abs to pERK, ERK, phospho-p38 (pp38) and p38. B, pERK levels in sorted DN thymocytes were analyzed by flow cytometry following treatment of anti-CD3+ PMA for 5 or 10 min. Each experiment in Fig. 3 was performed two to three times, and representative results were shown.

 
Ablation of Shp2 reduces splenic cellularity and suppresses T lymphocyte proliferation

Because Shp2 was also efficiently deleted in peripheral T cells (Fig. 1B), we examined the impact of Shp2 ablation on mature T cell function. A significant decrease of cell numbers in the spleens of Shp2T–/– mice was observed as compared with littermate controls (Fig. 4A). On average, the total number of splenic cells in mutant mice was ~57% of the control (KO, 27 x 106 vs WT, 47 x 106; n = 10). The Shp2 deletion had a more profound effect on the CD4+ than the CD8+ cell subpopulations (Fig. 4B), consistent with the analysis on thymocytes (Table I). When the splenocytes were analyzed for CD19 and TCR markers, there was also a decrease in TCR-positive cells, although no change was observed for the expression level of CD19, the B cell marker (Fig. 4C).


Figure 4
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  		FIGURE 4. Shp2T–/– mice exhibit reduced splenic cellularity. A, Total splenocytes after RBC lysis were averaged at 27 x 106 (KO) vs 47 x 106 (WT) (n = 10; *, p < 0.05). B, Total splenocytes were gated for live and CD3+ cells and analyzed for CD4 and CD8 markers. Numbers in quadrants are percentages for each population. C, Total splenocytes were gated for live cells and analyzed for CD19 and TCR markers. Numbers in quadrants are percentages for each population.

 
The reduced splenic T cell number suggests a positive role for Shp2 in T lymphocyte proliferation and/or development, and thus we investigated whether deletion of Shp2 affected T cell responses to various mitogenic stimuli in vitro. T cells were purified from the spleens and stimulated with anti-CD3 with or without costimulation of anti-CD28 Abs or PMA. The proliferative response of T cells from the mutant mice was consistently 2- to 3-fold lower than that of littermate controls, as measured by thymidine incorporation assay (Fig. 5A). However, we did not observe a difference in cell death between the two genotypes of splenic T cells (data not shown), consistent with the similar apoptosis rates seen in thymocytes (Fig. 2C). Therefore, the reduced proliferative response seen in mutant cells is likely due to defects in cell proliferation rather than increased cell apoptosis. Consistent with this theory, we detected significantly lower amounts of secreted IL-2 from Shp2 mutant cells than in controls, following stimulation of CD3, CD3+CD28, or CD3+PMA (Fig. 5B). Addition of IL-2 into the culture medium did not rescue the defective response to anti-CD3 stimulation (Fig. 5A). We also examined CD25 and CD69 profiles in separated CD4 and CD8 cell subpopulations. Following anti-CD3 stimulation, expression of the activation marker CD25 was decreased in both CD4 and CD8 cells isolated from the KO mice, compared with the WT control (Fig. 5C). However, decrease of the CD69 expression was detected in CD4 cells but not in CD8 cells. Thus, Shp2 is a positive regulator of TCR signaling in mature T lymphocytes and may have distinct functions in CD4 and CD8 cells.


Figure 5
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  		FIGURE 5. Reduced T cell activation in Shp2T–/– mice. A, Purified splenic T cells stimulated with PBS or plate-bound anti-CD3 (1 mg/ml) with or without plate-bound anti-CD28 (5 mg/ml), IL-2 (20 ng/ml), PMA (5 ng/ml) or PMA alone, and thymidine incorporation were determined. (**, p < 0.01). B, Levels of IL-2 secreted in supernatants were determined after stimulation of purified T cells as in A. All proliferation assays were done in triplicates and shown as averages with SD (**, p < 0.01). C, Splenocytes were stimulated with plate-bound anti-CD3 (1 µg/ml) for 14 h. Cells were harvested and assayed for T cell-activated markers CD25 or CD69 marker on CD4 or CD8-gated cells. Both CD4+ and CD8+ cells isolated from KO mice showed impaired expression of CD25 marker as compared with that derived from the WT animal. However, the decrease of CD69 expression was more profound on CD4+ than CD8+ T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
By creating a new mouse model with selective Shp2 deletion in thymocytes, we have uncovered a requirement for this cytoplasmic tyrosine phosphatase in promoting T cell differentiation and maturation. The gene-targeting technology in mice has revolutionized our understanding of T cell biology in the past decade (4). In particular, this effective experimental approach has allowed determination of critical roles for a number of intracellular signaling molecules in T cell differentiation at distinct steps. On the growing list of molecules are several protein tyrosine kinases, such as Lck/Fyn, Zap-70/Syk as well as adaptor proteins including SLP-76, LAT, Vav, and Shc (3, 4, 22). The positive roles played by Lck/Fyn and Zap-70/Syk in mediating pre-TCR and TCR signaling clearly illustrate the importance of tyrosine phosphorylation as a common molecular mechanism in T cell development and functions.

The above mechanism naturally predicts the involvement of tyrosine phosphatases as negative regulators of signal relay from pre-TCR and TCR complexes. Indeed, Shp1, a tyrosine phosphatase that is predominantly expressed in cells of hemopoietic origin and is structurally similar to Shp2, has been shown to play such a negative role (23, 24, 25, 26). In particular, breeding of the Shp1-deficient motheaten mice with TCR transgenic mice revealed Shp1 functions in setting up the TCR signal thresholds in both DN and DP thymocytes (25, 26, 27). Shp1 deficiency leads to an increase in the ratio of mature DN4 (CD25–CD44–) vs immature DN3 (CD25+CD44–) populations, and also an expansion of the CD4+CD8+ DP thymocyte subpopulation. Using different TCR transgenic mice, researchers also uncovered Shp1 functions in negative and/or positive selection of thymocytes (25, 26, 27). Shp1 deficiency also results in hyperresponsiveness of a transgenic TCR to stimulation with cognate peptide in thymocytes or peripheral T cells (25, 26).

Data obtained in this study support the notion that Shp2 acts to promote T cell maturation/proliferation at both the DN and DP cell stages. The Shp2T–/– mice exhibited a high efficiency and fidelity of Shp2 gene deletion in thymocytes, mediated by the Cre recombinase, consistent with previous reports using this Cre transgenic mouse line (19, 20). The Shp2 deletion significantly suppressed progression of DN3 thymocytes to the DN4 stage, as evidenced by DN3 cell accumulation and decrease of DN4 cell numbers. The reduced DN cell number in S and G2 phases of cell cycle also illustrates a role of Shp2 in amplifying proliferative signals emanating from the pre-TCR. Thus, the Shp2 mutation impairs thymocyte differentiation and proliferation, by affecting the critical beta selection step instructed by the pre-TCR signals, leading to reduction of thymic cellularity observed in Shp2T–/– animals.

As compared with littermate controls, Shp2T–/– mice possessed a reduced number of CD4+ thymocytes, with no dramatic changes in the CD8+ subpopulation. This result suggests a selective role of Shp2 in influencing lineage commitment of DP thymocytes to the CD4+ cell lineage. Several groups have reported that the intensity of the ERK signal is more critical to the development of CD4+ than CD8+ thymocytes (10, 28, 29, 30). Consistently, we detected reduced pERK levels in thymocytes from Shp2T–/– mice as compared with that of controls following in vitro stimulation with anti-CD3, although the kinetics of ERK activation was not altered. Thus, one important biochemical mechanism for Shp2 action in T cells is to control the strength of signals flowing through the ERK pathway.

Although the number of splenic cells is dramatically reduced, deletion of both Shp2flox alleles were detected in mature splenic T lymphocytes of homozygous Shp2T–/– mutant mice. Therefore, despite a suppressive effect of the Shp2 mutation on both thymocyte differentiation and proliferation, T cell maturation was not completely blocked in Shp2T–/– mice. This could be possibly due to a progressive expression of the Cre recombinase in developing thymocytes under control of the lck promoter. Also, because Shp2 acts to enhance signals, such as the ERK pathway, in orchestrating the thymocyte development program, we may not expect to see a complete block of T cell development in Shp2T–/– mice.

Consistent with the reduced splenic cellularity, isolated Shp2–/– T lymphocytes display decreased cell proliferation and IL-2 production in response to TCR activation and other mitogens in vitro. Notably, Shp2–/– thymocytes and mature T cells do not show a survival problem. Thus, even with impaired proliferative capacity, Shp2–/– T cells were not selected against by any residual Shp2F/F T cells. Otherwise, one would see accumulation of mature Shp2F/F T cells in the spleen as compared with the thymus, similar to that seen in mice with Lck-Cre-mediated deletion of SC35, a splicing factor, in thymocytes (20).

In essence, this study provides a fresh view on Shp2 functions in promoting thymocyte differentiation, proliferative expansion, and lineage commitment. Interestingly, deficiency of Shp1 or Shp2 leads to opposite phenotypes at several points, suggesting their opposing activities, direct or indirect, in thymocyte development/function. 1) Loss of Shp1 led to an increased ratio of DN4:DN3 cell populations, whereas a significant block to the DN3-DN4 transition/maturation was observed in Shp2T–/– mice. 2) A selective expansion of the CD4+ cell population was detected in motheaten mice under different transgenic TCR background, whereas deletion of Shp2 had a more profound suppressive effect on development of CD4+ compared with CD8+ cells. 3) The proliferative signals triggered by TCR, such as production of IL-2, were augmented in the absence of Shp1 but attenuated by ablation of Shp2 in T lymphocytes. It will be interesting to determine how and where Shp1 and Shp2 operate in thymocytes at various developmental stages. Notably, there are also examples showing a negative effect of Shp2 in regulating TCR signaling through interaction with CTLA-4 (31, 32), but the mechanism of this interaction needs to be further investigated. Creation of the Shp2T–/– mouse model now offers an invaluable tool to understand how the pre-TCR and TCR signals are modulated and coordinated by Shp2 tyrosine phosphatase.


   Acknowledgments
 
We thank Drs. X. Fu and J. D. Marth at the University of California, San Diego (La Jolla, CA) for the lck-cre mice, and our colleagues for helpful discussion and critical comments on the manuscript.


   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 National Institutes of Health Grant R01 CA78606 (to G.-S.F.). Back

2 Address correspondence and reprint requests to Dr. Gen-Sheng Feng, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: gfeng{at}burnham.org Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; KO, knockout; pERK, phosphorylated ERK; WT, wild type. Back

Received for publication June 28, 2005. Accepted for publication August 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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