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The Journal of Immunology, 1999, 162: 6359-6367.
Copyright © 1999 by The American Association of Immunologists

The Src-Homology Domain 2-Bearing Protein Tyrosine Phosphatase-1 Inhibits Antigen Receptor-Induced Apoptosis of Activated Peripheral T Cells1

Jinyi Zhang*, Ally-Khan Somani*, Stephen Watt{dagger}, Gordon B. Mills{dagger} and Katherine A. Siminovitch2,*

* Departments of Immunology, Medicine, and Molecular and Medical Genetics, University of Toronto, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; and {dagger} Department of Medicine, M.D. Anderson Cancer Center, University of Texas, Houston, TX


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Restimulation of Ag receptors on peripheral T lymphocytes induces tyrosine phosphorylation-based signaling cascades that evoke Fas ligand expression and induction of Fas-mediated programmed cell death. In view of the role for the Src homology domain 2-bearing protein tyrosine phosphatase-1 (SHP-1) in modulating TCR signaling, we investigated the influence of SHP-1 on TCR-mediated apoptosis by assaying the sensitivity of peripheral T cells from SHP-1-deficient viable motheaten (mev) mice to cell death following TCR restimulation. The results of these studies revealed mev peripheral T cells to be markedly more sensitive than wild-type cells to induction of cell death following TCR stimulation. By contrast, PMA/ionophore and anti-Fas Ab-induced apoptotic responses were no different in mev compared with wild-type activated cells. Enhanced apoptosis of TCR-restimulated mev lymphocytes was associated with marked increases in Fas ligand expression as compared with wild-type cells, but was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Thus, the increased sensitivity of mev T cells to apoptosis following TCR restimulation appears to reflect a TCR-driven phenomenon mediated through up-regulation of Fas-Fas ligand interaction and induction of the Fas signaling cascade. These findings, together with the hyperproliferative responses of mev peripheral T cells to initial TCR stimulation, indicate that SHP-1 modulation of TCR signaling translates to the inhibition of both T cell proliferation and activation and, as such, is likely to play a pivotal role in regulating the expansion of Ag-stimulated T cells during an immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Maintenance of immune cellular homeostasis requires fine-tuned regulation of lymphocyte responses to Ag receptor stimulation. In T lymphocytes, such control is realized, at least in part, by the capacity of the TCR and various downstream signaling effectors to translate TCR-ligand engagement not only to T cell proliferation, but also, depending on the developmental and costimulatory contexts, to programmed cell death via apoptosis (1, 2, 3, 4, 5, 6). TCR-evoked apoptosis provides, for example, a fundamental strategy for targeted elimination of autoreactive T cells in the thymus and potentially autoreactive peripheral T cells that have escaped intrathymic censorship processes (7, 8, 9). The pivotal role for TCR-mediated apoptosis in negative selection has been well appreciated for many years and is recognized as an integral mechanism for shaping the T cell repertoire and sustaining tolerance (8, 9, 10, 11). More recently, however, data revealing the induction of apoptosis in mature T cells restimulated through their Ag receptors have identified TCR-induced T cell death as an important process for terminating and controlling the expansion of activated T cell populations following antigenic stimulation (12, 13, 14, 15). T cell death in this latter context is referred to as activation-induced cell death (AICD)3 and has been shown to depend upon up-regulated Fas ligand (FasL)-Fas receptor and, at the later stages of cell activation, TNF-TNF receptor interactions and the consequent induction of the Fas-associated death domain-like IL-1-converting enzyme (FLICE)/caspase proteolytic cascade (16, 17, 18, 19, 20).

Although AICD represents an important modality for limiting the expansion of activated mature T cells, the molecular events whereby TCR restimulation evokes death rather than proliferation-inducing signaling cascades are not well understood. However, recent data concerning the biochemistry of AICD in T cells suggested that at least some of the membrane proximal events coupling activated TCRs to cell death are similar to those linking these receptors to proliferation (21, 22). Thus, for example, induction of the Lck, Fyn, ITK/EMT, and ZAP70 protein tyrosine kinases (PTKs) and consequent increases in protein tyrosine phosphorylation appear to be required for expression of either proliferative or apoptotic responses following TCR stimulation (23, 24, 25, 26, 27, 28, 29). This pivotal role for phosphotyrosine-based signaling events in induction of T cell apoptosis raises the possibility that protein tyrosine phosphatases (PTPs) also play a role in this phenomenon and possibly in modulating TCR signal relay toward an apoptotic rather than proliferative response. One PTP of particular interest in this regard is SHP-1, a cytosolic Src homology domain-2 containing protein, which has been shown to exert a predominantly negative effect on the signaling events linking TCR engagement to proliferation (30, 31, 32, 33, 34). This inhibitory influence of SHP-1 on TCR-induced proliferation is evidenced by the hyperproliferative responses of thymocytes and T cells from SHP-1-deficient motheaten (me) and viable motheaten (mev) mice to TCR stimulation (31, 32) and is realized, at least in part, by SHP-1-mediated dephosphorylation of the Lck and ZAP70 PTKs, as well as TCR subunits and other cytosolic signaling effectors (31, 32, 33, 35). Although the precise mechanisms whereby SHP-1 negatively regulates TCR-driven T cell proliferative responses require further investigation, the available data indicate a pivotal role for SHP-1 in modulating TCR-signaling and, as such, imply the involvement of SHP-1 in the signaling cascades that link activated TCRs to other biological outcomes, such as cell death. This latter possibility is consistent with previous data, showing SHP-1-deficient self-reactive B cells to be unusually susceptible to clonal deletion triggered by binding a low valency form of autoantigen (36). Thus, in contrast to the CD45 receptor PTP, which has been shown to promote Ag receptor-triggered apoptosis (37, 38), SHP-1 may serve to negatively modulate this process, most probably by raising the threshold required for TCR signal relay following ligand binding. To investigate this possibility, we have assessed peripheral T cells from SHP-1-deficient mev mice with respect to their propensity to apoptotic cell death following TCR restimulation. The results of these studies confirmed that proliferative responses and induction of mitogen-activated protein kinase (MAPK) activity were increased in mev T cells after initial TCR stimulation. Restimulation of these cells in vitro by anti-CD3 Ab TCR cross-linking revealed the induction of apoptotic cell death to also be dramatically enhanced in the mev compared with wild-type cells. By contrast, the mev and wild-type cells responded equivalently to treatment with PMA/calcium ionophore or anti-Fas Ab. Enhanced apoptosis of the TCR-restimulated mev cells was associated with markedly increased expression of FasL, as compared with wild-type cells, and was almost abrogated in both mev and wild-type cells by Fas-Fc treatment. Together, these data confirm the association of SHP-1 deficiency with enhanced TCR signaling and indicate that SHP-1 inhibits the biochemical events coupling TCR stimulation to apoptotic cell death during the activation of mature T cells, an effect that is achieved in part by modulation of FasL expression and induction of the Fas death signaling cascade.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice for these studies were obtained by mating C57BL/6J mev/+ and +/+ breeding pairs from stock maintained at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital (Toronto, Ontario, Canada). All mice used in the study were 3–5 wk old. The mev mutation was detected as previously described (39) by PCR amplifying a 69-bp fragment encompassing the site of the T->A transversion in the mev SHP-1 gene using the primer pair 5'-CGTGTCATCGTCATGACT-3' and 5'-AGGAAGTTGGGGCTTTGCCGT-3'. Following RsaI digestion, the amplified products were resolved by electrophoresis through 6% agarose gels and the wild-type and mev alleles determined by visualization of either a 69-bp RsaI (mev) or 48- and 21-bp RsaI (wild-type) fragments.

Reagents

Abs used for these studies included FITC-conjugated anti-Thy 1.2, FITC-conjugated anti-CD8, PE-conjugated anti-CD4, biotin-conjugated anti-TCR{alpha}ß and anti-Fas (Jo6) Abs purchased from PharMingen (San Diego, CA); monoclonal hamster anti-TCR (ß-chain) produced by the H57-597 hybridoma (provided by P. Marrack, Department of Medicine, National Jewish Center, Denver, CO); monoclonal hamster anti-mouse CD3{epsilon} produced by the 145-2C11 hybridoma (provided by R. Miller, Department of Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario, Canada) and purified from hybridoma supernatant by protein G chromatography; rabbit polyclonal anti-Erk 2 (MAPK), anti-murine ß-actin, and the 4G10 monoclonal anti-phosphotyrosine Abs purchased from Upstate Biotechnology (Lake Placid, NY); rabbit anti-hamster and anti-mouse IgG purchased from Jackson ImmunoResearch (West Grove, PA); and rabbit anti-FasL Ab (N-20) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fas-Fc fusion protein (40) was kindly provided by Dr. Shyr-Te Ju (Department of Medicine, Boston University School of Medicine, Boston, MA). PMA, A23187, and other chemicals used for immunoblotting/immunoprecipitation were purchased from Sigma (St. Louis, MO).

Proliferation assay

Single cell suspensions prepared from mev and wild-type congenic wild-type axillary and inguinal lymph nodes were subjected to erythrocyte lysis in ammonium chloride. The membrane Ig-positive cells and macrophages were then depleted by repetitive panning on rabbit anti-mouse IgG-coated tissue culture plates. T cells were further purified by negative affinity selection using a mouse T cell enrichment column (R&D Systems, Minneapolis, MN), which generated purified T cell populations >90% positive for Thy 1.2 expression as assessed by fluorescence cytometry using a FACScan analyzer (Becton Dickinson, Mountain View, CA). The purified T cells were then cultured in 96-well flat-bottom microtiter plates (5 x 104 cells/well) for 72 h in culture medium alone (RPMI 1640 containing 10% heat-inactivated FCS, 50 µM 2-ME, and penicillin/streptomycin) or supplemented with various amounts of anti-CD3 Ab and secondary rabbit anti-hamster IgG (4 µg/ml) to cross-link the primary Ab. Cultured cells were pulsed with [3H]thymidine (1 µCi/well, Dupont/NEN, Boston, MA) 10 h before terminating incubation, and incorporated radioactivity was measured using an automated ß scintillation counter.

Activation and Fas-induced apoptosis

Purified lymph node-derived T cells plated at a density of 1 x 106 cells/ml in culture medium were cultured with Con A (2 µg/ml) for 72 h, then washed three times in culture medium alone and subsequently cultured for 48 h in the presence of IL-2 (50 IU/ml) (Sigma). Viable cells recovered by Lymphocyte-M (Cedarlane, Hornsby, Ontario, Canada) and shown by FACScan analysis to be >97% positive for Thy 1.2 and TCR{alpha}ß expression, were recultured at a density of 1 x 106/ml in 24-well plates under the following culture conditions: in the presence of either IL-2 (50 IU/ml) alone or IL-2 with anti-Fas Ab (0.1, 1.0, or 10 µg/ml); in the presence of PMA (2 ng/ml) plus calcium ionophore A23187 (1 µg/ml); in anti-CD3 Ab-coated (1, 10, 100, or 1000 ng/ml) plates; and in anti-CD3 Ab-coated (1 ng/ml) plates in the presence or absence of Fas-Fc (1 µg/ml or 5 µg/ml). (IL-2 was added to all cultures to prevent cytokine deprivation-induced death). Cultures were harvested at 8 h (or 12 or 24 h in the case of anti-Fas Ab-treated cells), washed, and resuspended in 0.5 ml PBS containing 1% BSA, 0.01% sodium azide, and 2 µg/ml propidium iodide (PI; Sigma). Cells were incubated in the dark for 30 min at room temperature and then analyzed for DNA content using the Becton Dickinson FACScan and CELLQUEST software.

Fluorescence staining for cell surface markers

Lymph node-derived cells (1–2 x 105/sample) were suspended in 100 µl ice-cold PBS/1% BSA/0.05% sodium azide and incubated for 30 min at 4°C with FITC or PE-conjugated anti-Thy 1.2, TCR{alpha}ß, CD4, or CD8 Abs. For analysis of Fas receptor expression, purified lymph node-derived T cells were stimulated with Con A and IL-2, as described above, and 2 x 105 cells were then resuspended in incubated 100 µl ice-cold PBS/BSA buffer and incubated for 30 min at 4°C with FITC-conjugated anti-Fas Ab. Following staining, cells were washed three times in PBS/BSA and analyzed on a FACScan flow cytometer.

Flow cytometric analysis of intracellular FasL expression

For analysis of FasL expression, cells were fixed and permeabilized using Fix and Permeabilizing Reagent (Caltag Laboratories, Burlingame, CA) according to manufacturer’s instructions. Briefly, cells were washed with PBS, fixed with 4% paraformaldehyde for 5 min, washed with PBS, and incubated with 0.1% saponin-1% FCS/PBS buffer for 15 min. The cells were then stained with rabbit polyclonal anti-FasL Ab or normal rabbit serum and PE-conjugated goat-anti-rabbit IgG. Following staining, cells were washed three times in PBS/BSA and analyzed on a FACScan flow cytometer.

RT-PCR

Wild-type or mev-purified T cells were prestimulated with Con A/IL-2, then incubated for 8 h in culture medium supplemented with 0, 0.1, or 1.0 µg/ml anti-CD3 Ab, and total RNA was then extracted, using Trizol (Life Technologies/BRL, Rockville, MD), from 5 x 106 cells. Single strand cDNA was prepared from 1 µg of each RNA using 50 pmol oligo(dT)18 and 200 U murine leukemia virus reverse transcriptase (Life Technologies/BRL). Amplification of each cDNA was performed using 2 µl cDNA, 2.5 U Taq polymerase (Pharmacia, Baie d’Urfé, Quebec, Canada), 1 µg [32P]dATP (Dupont/NEN), and the following primer pairs: For FasL, 5'-CAGCTCTTCCACCTGCAGAAGG-3' and 5'-AGATTCCTCAAAATTGATCAGAGAGAG-3' and 5'-ATGAGGTAGTCTGTCAGGT-3'. Samples were amplified using a Perkin-Elmer/Cetus (Norwalk, CT) thermal cycler and the following PCR conditions: 2 min at 94°C followed by 25–30 cycles of 94°C for 1 min, 60°C for 1.5 min, and 72°C for 1 min. Amplification products were resolved by electrophoresis over a 4% nondenaturing polyacrylamide gel followed by autoradiography and densitometry (Molecular Dynamics, Sunnyvale, CA).

Immunoblotting analysis

For analysis of protein tyrosine phosphorylation, purified lymph node-derived T cells (2 x 106) were plated at a density of 1 x 106 cells/ml in culture medium and cultured as described above for 72 h in the presence of Con A (2 µg ml) and IL-2 (50 IU/ml). Cells recovered by lymphocyte-M were then plated at a density of 1 x 106/ml and cultured for 24 h in the presence of IL-2 and anti-Fas Ab (10 µg/ml). Cells were then resuspended in 400 µl cold lysis buffer (1% Nonidet P-40, 50 mM HEPES (pH 7.23), 150 mM NaCl2, 50 mM NaF, 50 mM phosphate, 50 mM ZnCl2, 2 mM EDTA, 2 mM sodium orthovandate, and 2 mM PMSF) and the nuclei and unlysed cells then removed by centrifugation at 4°C for 10 min at 14,000 x g. Protein concentrations were then determined by the bicinochoninic acid technique (Pierce, Rockford, IL) and the lysate proteins then resuspended in SDS buffer, boiled for 5 min, electrophoresed through 12% SDS-polyacrylamide, transferred to nitrocellulose (Schleicher & Schuller, Keene, NH), and incubated at 4°C for at least 1 h in TBST solution (150 mM NaC1, 10 mM Tris-HCl (pH 7.4), 0.05% Tween 20) plus 3% gelatin. Filters were then incubated for 2 h at room temperature with anti-phosphotyrosine Ab in TBS followed by goat anti-mouse antiserum labeled with peroxidase (Amersham, Arlington Heights, IL) and HRP conjugate (Bio-Rad Labs, Hercules, CA). Filters were then stripped as per Amersham’s recommended protocol and reprobed with anti-ß-actin Ab.

Immunoprecipitation and assay of MAPK activity

For analysis of MAPK activity, 1 x 107 mev or wild-type lymph node T cells were resuspended in 300 µl PBS incubated for 30 min at 4°C in the presence or absence of 5 µg biotin-conjugated anti-mouse TCR Ab. Following several washes to remove unbound Ab, the cells were resuspended in 40 µl PBS and incubated at 37°C for 1, 5, or 10 min with 10 µg/ml strepavidin. Cells were then pelleted by 30 s of centrifugation and lysed by 20 min of resuspension in 400 µl cold Nonidet P-40 lysis buffer. Following 10 min centrifugation at 4°C at 14,000 x g, lysate protein concentrations were evaluated by a bicinochoninic acid assay and lysates containing 50 mg protein then precleared by incubation with protein A-Sepharose (Pharmacia) for 1 h at 4°C. Lysate proteins were then incubated with anti-Erk2 Ab or rabbit IgG for 1 h at 4°C followed by 1 h of incubation with 25 µl packed protein A-Sepharose beads pretreated with 1 mg/ml BSA. The immune complexes were collected by centrifugation, washed sequentially in Nonidet P-40 lysis buffer and MAPK buffer (5 mM HEPES (pH 7.4), 10 mM MgCl2, 100 mM Na3VO4), and then resuspended in 50 µl reaction buffer (30 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 2 mM MnCl2 containing 5 µg myelin basic protein (MBP; Upstate Biotechnology), 1 mM cold ATP and 10 uCi [{gamma}-32P]ATP (Dupont/NEN). After 15 min of incubation at 30°C, reactions were terminated by addition of 12 µl 5x SDS-PAGE loading buffer and the samples then boiled, electrophoresed through 12% polyacrylamide gels, and transferred to nitrocellulose. The phosphorylated MBP bands were visualized by autoradiography and levels of Erk2 expression determined by anti-Erk2 immunoblotting using an enhanced chemiluminescence system (Amersham). Incorporation of 32P by the MBP substrate was also quantified by densitometric analysis (Molecular Dynamics PhosphorImager).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SHP-1-deficient peripheral T cells show enhancement in TCR-triggered proliferative responses and MAPK activation compared with wild-type cells

Previously, we (31) and others (32) have shown that thymocytes from me and mev mice, which essentially lack SHP-1 activity (41), are hyperresponsive to mitogenic stimulation through the TCR. This observation appears to reflect SHP-1 effects on both the TCR and downstream signaling effectors that link TCR engagement to Ras activation and induction of the MAPK cascade (31, 32, 33). To assess whether SHP-1 affects TCR signaling in mature peripheral T cells in a manner analogous to thymocytes, T cells purified from lymph nodes of mev and wild-type mice were evaluated with respect to induction of proliferative responses and activation of the Erk2 MAPK following anti-CD3 Ab stimulation. The results of this analysis revealed the mev T cell proliferative response to TCR stimulation to be markedly enhanced compared with that detected in wild-type T cells (Fig. 1GoA). TCR stimulation also evoked levels of Erk2 activation that were much higher in mev than wild-type cells, at least in the period immediately following TCR ligation (Fig. 1Go, B and C). These differences could not be ascribed to variability in the T cell subpopulations assayed as the T cell populations purified from lymph nodes of the mev and wild-type mice were not different with respect to proportions of CD4 and CD8 single positive cells (data not shown). These results are therefore consistent with an inhibitory role for SHP-1 in relation to the coupling of TCR stimulation to mature, peripheral T cell, as well as thymocyte, proliferation.



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  		FIGURE 1. TCR signaling is increased in SHP-1-deficient T cells. A, T lymphocytes purified from lymph nodes of C57BL/6 mev/mev (Mev) and wild-type (+/+) mice were incubated for 72 h at 2.5 x 105 cells/ml in culture medium in the presence of 0, 0.1, or 1.0 µg/ml anti-CD3 Ab and 4.0 µg/ml rabbit anti-hamster IgG and proliferation evaluated after a 10-h pulse with [3H]thymidine. Data are expressed as cpm x 10-4 and represent means (±SEM) of triplicate cultures and three independent experiments. B, Cell lysates were prepared from purified lymph node T cells (1 x 107) treated with 5 µg biotinylated anti-TCR Ab and incubated for the indicated times with 5 µg strepavidin. Lysate proteins were then immunoprecipitated with anti-Erk2 Ab, and the ability of the immune complexes to phosphorylate MBP then evaluated using SDS-PAGE and autoradiography (upper panel). Levels of Erk2 immunoprecipitated from respective cell lysates were evaluated by anti-Erk2 immunoblotting equivalent aliquots of each anti-Erk2 immunoprecipitate (lower panel). C, Histogram representation of MAPK activity in viable motheaten vs wild-type T cells at varying times after TCR ligation. The intensities of the bands shown in B were quantified by densitometric analysis and the results then expressed as intensity of phosphorylated MBP bands relative to bands representing immunoprecipitated Erk-2.

 
TCR-induced apoptosis following restimulation is enhanced in SHP-1-deficient peripheral T cells

In view of the inhibitory influence of SHP-1 on TCR-mediated proliferation of peripheral T cells, the possibility that SHP-1 inhibits the induction of apoptosis following TCR restimulation was investigated. To specifically address SHP-1’s role in AICD of peripheral T cells, T cells purified from mev and wild-type lymph nodes were treated with Con A and IL-2, the activated cells then purified and subjected to TCR/CD3 cross-linking by 8 h of exposure to immobilized anti-CD3 Ab, and cell death then evaluated by flow cytometric analysis of PI-stained cells. As illustrated in Fig. 2Go, the results of this analysis revealed TCR restimulation of both wild-type and mev T cells to be associated with substantive increases in PI staining, a phenomenon well-recognized as being indicative of apoptotic T cell death (19). However, as indicated by this assay, as well as by evaluation of DNA fragmentation (data not shown), induction of apoptotic cell death was clearly much greater in the mev than the wild-type cells. This differential sensitivity of the mev cells to TCR-mediated apoptosis was evident at all concentrations of stimulatory (anti-CD3) Ab studied (Fig. 2GoB), but was particularly apparent at low concentrations of Ab (1 ng/ml), at which apoptotic death was detected only in mev and not in wild-type cells (Fig. 2GoB). As induction of increases in intracellular calcium concentration and protein kinase C (PKC) activation are required for TCR-induced apoptosis of mature T cells (42), the capacity of PKC activation by PMA/ionomycin treatment to induce apoptosis of Con A/IL-2-treated mev and wild-type cells was also examined. As shown in Fig. 2GoC, PMA/ionomycin induced apoptosis of the mev cells to a level comparable to that engendered by anti-CD3 Ab treatment. However, in contrast to restimulation through the TCR, PMA/ionomycin stimulation was associated with an amount of cell death in the mev cells that was comparable to that detected in wild-type cells. Similarly, and as is consistent with previous data pertaining to SHP-1 effects on Ag-receptor-induced proliferation (31, 43), mev and wild-type cells showed no differences in their susceptibility to cell death triggered by anti-Fas Ab treatment or IL-2 withdrawal, maneuvers that evoke apoptosis independently of Ag-receptor activation (Fig. 2GoC). Together, these data indicate that SHP-1 negatively regulates TCR-induced apoptosis of peripheral T cells and imply that this role for SHP-1 reflects its capacity to modulate signal transduction via the TCR.



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  		FIGURE 2. TCR-mediated apoptosis is increased in SHP-1-deficient T cells. Purified lymph node T cells (5 x 106 cells) were activated with 2 µg/ml Con A, cultured for 72 h, and then cultured for an additional 48 h with IL-2 (50 U/ml). The viable cells were isolated and cultured (2 x 106 cells/ml) in the presence of IL-2 for 8 h in 24-well plates coated with varying concentrations of anti-CD3{epsilon} Ab or in noncoated plates in the presence of anti-Fas Ab or PMA/ionophore. Cells were harvested and analyzed for PI staining by flow cytometry. A, Flow cytometric analysis showing PI staining of wild-type (+/+) vs viable motheaten (Mev) cells following incubation with medium alone or 1 µg/ml plate-bound anti-CD3 Ab in the presence of IL-2. Numbers of positively staining, nonviable (D) and nonstaining, viable (L) cells are indicated in the upper and lower quadrants, respectively. The results are representative of four independent experiments. B, Histogram representation of the percentage of wild-type (+/+) and viable motheaten (Mev) PI-stained T cells following 8 h of restimulation with varying amounts of anti-CD3 Ab. C, Histogram representation of the percentage of wild-type (+/+) and viable motheaten (Mev) T cells staining positively for PI following 10 h of restimulation with anti-CD3 Ab (1 µg/ml), anti-Fas Ab (10 µg/ml), or PMA (2 ng/ml) ionomycin (1 µg/ml). Values shown in B and C represent means (±SEM) of triplicate cultures.

 
SHP-1 does not alter the signaling capacity of Fas

As Fas/FasL interaction plays a key role in AICD, further studies were undertaken to ascertain whether enhancement of TCR-induced apoptosis in mev T cells reflects a direct effect of SHP-1 deficiency on Fas signaling. To this end, the levels of Fas receptor surface expression were evaluated on mev and wild-type T cells at 72 h following Con A stimulation. As shown in Fig. 3GoA, results of immunofluorescence analysis revealed no difference between these cells in relation to the amount of Fas expression induced at this time point after activation. The sensitivity of mev and wild-type TCR-stimulated cells to Fas-mediated cell death induction was then compared by treating cells for 12 or 24 h after initial stimulation with varying amounts of anti-Fas Ab in the presence of IL-2 (the latter being included so as to circumvent cell death consequent to growth factor deprivation). As illustrated in Fig. 3GoB, the results of this analysis revealed the amount of cell death induced in mev and wild-type cells to be comparable at each differing dose and duration of anti-Fas Ab exposure. Furthermore, although Fas signaling has been reported to be impaired in mev thymocytes (44, 45), our analysis revealed no differences between mev and wild-type thymocytes in terms of sensitivity to Fas-induced apoptosis (data not shown). Similarly, anti-Fas Ab, which has previously been shown to induce protein tyrosine dephosphorylation in peripheral T cells (46), triggered comparable changes in the protein tyrosine phosphorylation profiles of mev and wild-type TCR-stimulated peripheral T cells (Fig. 3GoC). Thus, alteration in the signaling properties of Fas does not appear responsible for the enhancement of TCR-induced apoptosis in SHP-1-deficient mev T cells.



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  		FIGURE 3. Fas-induced apoptosis occurs normally in SHP-1-deficient T cells. A, Purified T cells from wild-type (+/+) and viable motheaten (Mev) mice were Con A/IL-2-stimulated (as described in Fig. 2Go) for 72 h, and the cells then subjected to flow cytometric analysis following staining with FITC-conjugated anti-Fas Ab (clear histogram) or FITC-hamster IgG (shaded histogram). B, Purified T cells from wild-type and viable motheaten mice were Con A/IL-2-stimulated for 72 h, the cells then harvested, washed, and stimulated in the presence of IL-2 with anti-Fas Ab (0.1–10 µg/ml) for 12 or 24 h. Histograms represent the percentage of cells staining positively for PI as evaluated by flow cytometric analysis. C, Cell lysates were prepared from wild-type and viable motheaten T cells stimulated sequentially with Con A/IL-2 and IL-2 plus anti-Fas Ab (10 µg/ml) (as described in B). Lysate proteins where then subjected to SDS-PAGE and the phosphotyrosine-containing proteins then detected by immunoblotting with anti-phosphotyrosine Ab.

 
FasL expression is aberrantly increased in TCR-stimulated SHP-1-deficient T cells

Up-regulation of FasL expression and increased Fas/FasL interactions have been shown to play a pivotal role in translating TCR restimulation to apoptic cell death (15, 16, 17). Thus, while Fas expression and Fas-mediated apoptosis per se are not affected by SHP-1 deficiency, increased sensitivity of mev T cells to TCR-induced apoptosis might reflect enhanced expression of FasL in these cells following TCR engagement. Although up-regulation of TNF-{alpha} expression has also been implicated in induction of AICD (18, 19, 20), TNF-{alpha}/TNFRp75 interactions appear to be most relevant in late stages of TCR-directed apoptosis (19) and are thus less likely to account for the differential amounts of apoptosis in mev compared with wild-type cells observed at 6–10 h after TCR restimulation. Accordingly, the relevance of FasL expression to the increases in TCR-induced apoptosis detected in mev T cells was evaluated by assaying levels of FasL expression in T cell blasts at varying time points following TCR restimulation. To this end, FasL mRNA was RT-PCR amplified from cells treated with varying concentrations of anti-CD3 Ab and the levels of the amplified products then assessed by densitometry and expressed relative to levels of coamplified ß-actin. As illustrated by the representative data shown in Fig. 4Go, A and B, the results of this semiquantitative analysis revealed FasL expression to be at least 2-fold higher in the stimulated mev cells relative to levels detected in wild-type cells. FasL expression in the mev and wild-type TCR-restimulated cells was also evaluated by staining permeabilized cells with an Ab that recognizes an N-terminal epitope of FasL. Again, intracellular levels of FasL expression in T cells restimulated with varying amounts of anti-CD3{epsilon} Ab were consistently about 2-fold greater in the mev than wild-type cells (Fig. 4GoC). To ascertain the extent to which this increase in FasL expression accounts for enhanced sensitivity of SHP-1-deficient cells to AICD, these cells were also evaluated for their response to TCR restimulation conducted in the presence of soluble Fas-Fc fusion protein. This reagent has been previously shown to impede TCR-triggered apoptosis by preventing FasL binding to Fas receptors on the T cell surface (16). As shown in Fig. 5Go, the results of this analysis revealed levels of apoptosis triggered by TCR restimulation to be reduced in both wild-type and mev cells in the context of Fas-Fc treatment. The effects of Fas-Fc on apoptosis induction were dose-dependent and were significantly greater in the mev compared with wild-type cells. Thus, treatment with 5 µg/ml Fas-Fc essentially reduced the amount of mev T cell death to levels only marginally higher than those detected in wild-type cells. Together these findings suggest that increased FasL expression represents a major factor in the heightened susceptibility of mev T cells to TCR-induced apoptosis.



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  		FIGURE 4. FasL expression is increased in TCR-activated SHP-1-deficient T cells. Purified lymph node T cells (5 x 106) from wild-type (+/+) and viable motheaten (Mev) mice were stimulated with Con A/IL-2 for 72 h, washed, and then cultured in the presence of IL-2 for 8 h in 24-well plates coated with medium or 0.1 µg/ml, 1.0 µg/ml, or 10 µg/ml anti-CD3 Ab. A, FasL expression was assayed in the TCR-restimulated cells by RT-PCR amplification as described in Materials and Methods. To control for RNA quality/quantity, ß-actin expression was similarly evaluated. B, The intensity of FasL and ß-actin PCR amplification products were evaluated by densitometry and expressed as relative intensities of the FasL to ß-actin species. C, Intracellular expression of FasL was evaluated in fixed and permeabilized in TCR-restimulated mev and wild-type T cells by flow cytometric analysis. Con A/IL-2-treated T cells were washed and restimulated with 0.1, 1.0, or 10 µg/ml anti-CD3{epsilon} Ab (as above) and the cells then fixed and permeabilized and stained with anti-FasL Ab (N-20) and PE-conjugated goat anti-rabbit Ab. The percentage of stained cells is indicated within each histogram.

 


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  		FIGURE 5. TCR-mediated apoptosis of SHP-1-deficient and wild-type cells is inhibited by Fas-Fc fusion protein. Following 72 h of incubation with Con A/IL-2, T cells purified from viable motheaten and wild-type mice were washed and recultured at a density of 2 x 106 cells/ml with 50 U/ml IL-2 or plastic-bound anti-CD3{epsilon} Ab (1 µg/ml) and in the presence of 1 µg/ml Fas-Fc, 5 µg/ml Fas-Fc, or control IgG for 10 h. Cells were then harvested, stained with 2 µg/ml PI, and the percent apoptotic death evaluated by flow cytometric analysis. Values represent the means of (±SEM) of triplicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cumulative data concerning the SHP-1 tyrosine phosphatase functions in lymphocytes have identified a pivotal role for SHP-1 in down-regulation of Ag receptor-driven growth-promoting signaling cascades (30, 47). In thymocytes, SHP-1 inhibitory effects on TCR-elicited proliferation have been linked to inhibited induction of MAPK activation, a finding that appears to reflect SHP-1 modulation of several upstream signaling effectors involved in the activation of Ras or Ras-related proteins (31, 32). Similarly, the data presented here reveal that TCR-induced proliferative responses and MAPK activation are also increased in mev relative to wild-type peripheral T cells, an observation that indicates that the coupling of TCR stimulation to proliferation of peripheral T cells is also inhibited by SHP-1. The current data also reveal mev T cells to manifest heightened sensitivity to TCR-mediated reactivation-induced apoptosis, thus suggesting that, depending on the stimulatory context, SHP-1 negative effects on TCR signaling translate to the inhibition of not only proliferation, but also apoptotic cell death. As no differences were detected between mev and wild-type T cells with respect to the triggering of apoptosis by anti-Fas Ab or other maneuvers that elicit cell death via mechanisms independent of TCR engagement, it appears that the inhibitory influence of SHP-1 on AICD is realized through modulation of TCR-driven signaling events. In view of the capacity of Fas-Fc to essentially abrogate TCR-induced apoptosis of both mev and wild-type cells as well as the relatively enhanced FasL expression detected in mev T cells following TCR engagement, up-regulation of Fas-FasL interaction and by extension the Fas death signaling cascade, appears to play a key role in parlaying SHP-1 inhibitory effects on TCR signaling to suppression of cell death following TCR restimulation.

While the current data provide the first direct evidence for SHP-1 involvement in AICD of T cells, a role for this PTP in modulating Ag receptor-directed apoptosis has been previously suggested by data showing increased susceptibility of SHP-1-deficient immature B cells to clonal deletion/negative selection in the bone marrow (36). Taken together with this latter observation, the current data are consistent with the previously contended role for SHP-1 in increasing Ag receptor signaling thresholds (30, 36, 39) and suggest that this effect of SHP-1 impacts on proliferation, apoptosis, and probably a diversity of other cell behaviors evoked by Ag receptor engagement. The capacity of SHP-1 to modulate B cell receptor (BCR) and TCR thresholds for signaling proliferation has been linked to SHP-1 interactions with the Ag receptors directly (31, 43), with a spectrum of accessory receptor molecules (47, 48, 49), and also with PTKs that act immediately downstream of Ag receptor engagement (32, 33, 35). The extent to which these sets of molecular interactions underlie SHP-1 effects on TCR-driven apoptosis remains to be determined, but based on data implicating two putative SHP-1 substrates, Lck and ZAP70 (32, 33, 35), in the induction of FasL expression and cell death following TCR restimulation (28, 29), it appears likely that at least some of the early molecular interactions whereby SHP-1 influences TCR signaling of apoptosis will be the same as those that couple TCR engagement to proliferation. By contrast, it also seems likely that at least some disparity exists in the proximal as well as downstream signaling events connecting TCR stimulation to proliferation vs apoptosis. This contention is supported by recent data, concerning the interactions of SHP-1 with various receptor comodulators of BCR signaling. Thus, for example, SHP-1 capacity to inhibit BCR-induced B cell activation has been linked to the association of SHP-1 with accessory molecules such as CD22 and the Fc{gamma}RIIB1, interactions mediated by binding of the SHP-1 Src homology 2 domains to phosphotyrosines within immunoreceptor tyrosine inhibitory motifs (ITIMs) (48, 49). However, SHP-1 interactions with another ITIM-containing accessory molecule, CD72, appear to be exclusively associated with the delivery of apoptotic death signals (47). Whether these types of SHP-1-comodulator interactions are also relevant to SHP-1 effects on TCR signaling remains to be determined. However, data revealing that in TCR-stimulated thymocytes SHP-1 interacts with CD5 (31), a putative negative modulator of TCR signaling (50), suggest that in T as well as B cells, the influence of SHP-1 on translation of Ag receptor signaling to variable biological outcomes relates, at least in part, to its differential interactions with TCR accessory molecules. This possibility, however, as well as the amalgam of other molecular interactions that enable SHP-1 to modulate TCR-mediated apoptosis require further investigation.

The current data revealing increases in FasL expression following TCR-restimulation to be enhanced in the SHP-1-deficient mev T cells and the capacity of Fas-Fc to essentially abrogate induction of apoptosis in both mev and wild-type cells are consistent with previous data indicating a prerequisite role for Fas-FasL interaction in triggering of T cell AICD (15, 16, 17). While FasL expression is known to be negligible in resting T cells (15, 16, 17, 51), at present little is known about the biochemical events connecting TCR stimulation to up-regulated FasL expression. However, induction of FasL expression during TCR activation has been shown to require activity of both ZAP70 and Lck (28, 29, 52). As ZAP70 and Lck have also been shown to represent targets for SHP-1 dephosphorylation and deactivation (32, 33, 35), it appears likely that SHP-1 modulation of these PTK activities represents one of the mechanisms whereby SHP-1 impacts on TCR-induced apoptosis. By contrast, the current data showing Fas expression and function to be normal in mev peripheral T cells suggest that SHP-1 effects on induction of AICD are not realized by modulation of the intrinsic signaling capacity of the Fas receptor or the downstream signaling events evoked by Fas ligation. These findings are in contrast to previous data suggesting that Fas signaling is impaired in motheaten thymocytes and by extension that SHP-1 is required for normal signal relay through this receptor (44, 45). This discrepancy may reflect differences in the experimental systems being used to assay Fas function. In the latter study, for example, IL-2 was not included in the culture medium and, under these conditions, particularly in view of the enhanced responsiveness of motheaten compared with wild-type T cells to TCR stimulation, cell death due to growth factor deprivation notably interfered significantly with the subsequent assay of Fas signaling. This possibility is supported by data from Takayama et al. (53) that indicate that SHP-1 is not required for Fas- or perforin-dependent CTL-induced apoptotic cell death of mev T cell blasts. These observations are consistent with the current data revealing the Fas signaling pathway to be intact in the context of SHP-1 deficiency and with the conclusion that SHP-1 is not required for induction of apoptosis through the Fas receptor.

The demonstration of SHP-1 involvement in modulating TCR-induced apoptosis reveals a potential influence of this PTP on the selection processes shaping the immune repertoire. This contention is supported by many lines of evidence linking the outcome of thymocyte selection to the strength of Ag receptor signaling (54, 55, 56, 57). According to this "signaling threshold" model, lack of TCR/ligand interaction renders thymocytes susceptible to programmed cell death by "default," while low-affinity/avidity interactions of the TCR with MHC/peptide generates an intracellular signal sufficient for survival and the increased intensity of signal evoked by high-affinity/avidity TCR-ligand interactions engenders induction of apoptosis. While recognition of the pivotal role strength of activation signal plays in shaping the outcome of TCR engagement has emerged primarily through studies of thymocyte development and selection, the TCR signaling threshold paradigm is increasingly being invoked to explain peripheral T cell behavior as well. Thus, for example, while appropriate Ag-receptor stimulation results in the clonal expansion and proliferation of mature T cells, high-dose or prolonged Ag stimulation of such cells has been shown to induce their programmed cell death (58). Similarly, the current data revealing the outcome of TCR restimulation to be influenced by SHP-1 effects on TCR signaling support the contention that signaling intensity substantively affects the impact of TCR engagement on mature T cell physiology.

While the biochemical mechanisms whereby such quantitative differences in TCR signaling are translated to variable cell responses remain to be determined, the current data provide further evidence of a crucial role for phosphotyrosine-based signaling events in modulating TCR-induced apoptosis. As AICD provides a potential mechanism for both deleting autoreactive cells and terminating immune responses in the periphery, the demonstrated involvement of SHP-1 in regulating this form of T cell programmed cell death identifies modulation of SHP-1 activity as a potential strategy for inhibiting adverse sequelae of T cell activation as exemplified by autoimmune disease and graft rejection.


   Acknowledgments
 
We thank Drs. Shyr-Je Ju, Phillipa Marrack, and Rick Miller for their generous donation of reagents.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council, National Cancer Institute of Canada (NCIC), Arthritis Society of Canada, and National Institutes of Health. A-K.S. is a recipient of a Steve Fonyo studentship award from the NCIC. K.A.S. is a Research Scientist of the Arthritis Society of Canada. Back

2 Address correspondence and reprint requests to Dr. Katherine Siminovitch, Mount Sinai Hospital, Room 656A, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada. E-mail address: Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; SHP-1, Src homology domain 2-bearing protein tyrosine phosphatase-1; FasL, Fas ligand; MAPK, mitogen-activated protein kinase; mev, viable motheaten; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; MBP, myelin basic protein; BCR, B cell receptor; PI, propidium iodide. Back

Received for publication July 16, 1998. Accepted for publication March 10, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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