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The Journal of Immunology, 1999, 162: 3802-3813.
Copyright © 1999 by The American Association of Immunologists

TCR Signaling Thresholds Regulating T Cell Development and Activation Are Dependent upon SHP-11

Kenneth G. Johnson, Frances G. LeRoy, Leszek K. Borysiewicz and R. James Matthews2

Department of Medicine, Tenovus Building, University of Wales College of Medicine, Heath Park, Cardiff, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An examination of thymocytes and peripheral T cells from SHP-1-deficient motheaten mice possessing a transgenic MHC class I-restricted TCR has implicated SHP-1 in regulating TCR signaling thresholds at three checkpoints in T cell development and activation. First, in the population of CD4-CD8- double negative thymocytes, SHP-1 appears capable of regulating signals from TCR complexes that control the maturation and proliferation of double negative thymocytes. Second, the loss of SHP-1 increased the number of CD4+CD8+ double positive thymocytes capable of maturing as TCRhigh single positive thymocytes. Third, the loss of SHP-1 altered the basal level of activation of naive lymph node T cells. Accordingly, SHP-1-deficient lymph node T cells bearing the transgenic TCR demonstrated a hyperresponsiveness to stimulation with cognate peptide. However, the loss of SHP-1 did not alter the cytolytic ability of mature effector cytotoxic T lymphocytes. Together these results suggest that SHP-1 contributes to establishing thresholds for TCR signaling in thymocytes and naive peripheral T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interactions between molecules on the surface of developing thymocytes with their local environment are translated into intracellular biochemical signals that result in the eventual maturation of effector T cell responses. Firstly, a complex termed the pre-TCR, composed of a functional TCR ß polypeptide chain in association with a surrogate pre-TCR {alpha}-chain, is required to initiate signals in immature CD4-CD8- double negative (DN)3 thymocytes that prevent additional TCR ß gene rearrangements and result in a large proliferative expansion of the pool of thymocytes that can attempt successful rearrangement of the TCR {alpha} genes (1). Subsequently, signals initiated by TCR complexes composed of functional TCR {alpha} and ß polypeptides directly influence the developmental fates of individual CD4+CD8+ double positive (DP) thymocytes and so shape the final repertoire of TCR specificities and affinities found on mature T cells (2). There appears to be considerable congruity in the nature of the signaling mechanisms used by pre-TCR and TCR complexes as indicated by their functional dependence upon the invariant CD3 and {zeta}-chains for signal transduction (3). Furthermore, genetic and biochemical experimental evidence has implicated members of the Src-like protein tyrosine kinases (PTKs), p56lck and p59fyn, and the ZAP-70 family of intracellular kinases as crucial effectors of signaling from the pre-TCR on DN thymocytes (4, 5, 6, 7, 8) and mature TCR complexes on DP thymocytes (6, 9, 10, 11) and single positive (SP) peripheral T cells (12, 13, 14, 15, 16).

Protein tyrosine phosphorylation is a reversible covalent modification reflecting the counteracting effects of PTKs and protein tyrosine phosphatases (PTPs) (17). While the importance of intracellular PTKs in regulating TCR signaling has been recognized, the roles and identities of the PTPs that must impinge upon the TCR signaling pathway remain to be fully established. CD45, a hemopoietic transmembrane PTP, has been shown to be a positive effector of TCR signaling by its capacity to dephosphorylate an inhibitory phosphotyrosine regulatory site at the carboxyl-termini of p56lck and p59fyn, hence priming these PTKs for full catalytic activation (18). However, little is understood of the tyrosine dephosphorylation events that may determine the threshold and extent of TCR activation. Negative influences on TCR signaling would be predicted to have a substantial bearing upon thymocyte development and the establishment of Ag-primed peripheral T cell responses.

SHP-1 is an intracellular member of the family of PTPs and is expressed predominantly in cells of hemopoietic origin and, significantly, this includes both thymocytes and peripheral T cells (19, 20, 21, 22). By virtue of possessing tandem Src homology-2 (SH-2) domains at its amino-terminus, SHP-1 is an excellent candidate for acting as a negative regulator of the proximal steps in TCR-mediated signaling. A critical component to addressing the potential role of SHP-1 in lymphocyte Ag receptor signaling has been the availability of the spontaneous mutant mouse, motheaten (me/me), that is completely lacking in expression of SHP-1 (23, 24). The phenotype of the motheaten mouse is complex but dominated by the appearance of activated macrophages and granulocytes in tissues such as the skin, lungs, and bone marrow (25). Indeed, the chronically active monocytic lineages appear to be responsible for the premature involution of the thymus (26) and for the decreased levels of peripheral B cells observed in motheaten mice (27).

While SHP-1-deficient lymphocytes appear to have a very limited role in the gross pathology and morbidity of the motheaten mouse (28), this does not exclude a role for SHP-1 in normal lymphocyte function (29). Motheaten mice possess high levels of IgM Igs including many autoantibodies (30, 31, 32), correlating with an enrichment for a population of B1-type B cells (IgMhigh, IgD-, CD5+) believed to be generated following chronic stimulation of the B cell receptor (BCR) (33, 34). An examination of SHP-1-deficient B cells expressing a transgenic BCR in the context of irradiation bone marrow chimeras has demonstrated an intrinsic role for SHP-1 in negatively regulating the signaling thresholds that influence BCR signaling and hence B cell development and activation (35). Consistent with the in vivo observations, SHP-1-deficient B cells demonstrate both enhanced calcium fluxing (35) and proliferative responses (36) following in vitro stimulation. Thymocytes from motheaten mice demonstrate increased proliferation in response to in vitro stimulation with anti-CD3 Abs (37, 38), and this phenotype has been purported to result from increases in the kinase activities of p59fyn and p56lck (38) and/or hyperphosphorylation of the CD3 complex and downstream signaling proteins (37). However, there has been no in vivo evidence to-date to implicate SHP-1 in influencing T cell development.

Transgenic mice expressing clonotypic TCRs have been instrumental in delineating the rigorous selection events that occur during thymocyte differentiation (39). To examine the potential role of SHP-1 in regulating thymocyte development and T cell activation, an MHC class I-restricted transgenic TCR-{alpha}ß (40) derived from a cytotoxic T cell clone, F5 (41), which recognizes a nonamer peptide from the influenza virus nucleoprotein in the context of H-2Db, has been introduced into the motheaten genetic background. To examine the potential role for SHP-1 in T cell lineage formation and activation, we have analyzed thymocytes and lymph node T cells from me/me, me/+ and +/+ mice that are either hemizygous (F5hem) or homozygous (F5hom) for the F5-transgenic TCR. We find that SHP-1 negatively regulates TCR signaling pathways in both immature thymocytes and mature peripheral T cells, and, as such, SHP-1 is implicated in contributing to an activation threshold for the TCR that influences T cell development and peripheral T cell responsiveness.


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

C57BL/6J mice heterozygous at the motheaten locus were obtained from Dr. L. Shultz at The Jackson Laboratory (Bar Harbor, MA) and bred under pathogen-free conditions as a source of me/me mutants or me/+ and +/+ controls. me/+ mice were genotyped by PCR using a slight modification of a protocol described previously (23). A new reverse primer complementary to DNA sequences in intron 3 of the SHP-1 gene was designed. The primers were 5'-TCC CTG GGA GCT TCC TGG CTC-3' and 5'-TAG GCA GCA GGA ACC CTG CAG-3', which amplify a genomic fragment of 738 bp. C57BL/10 mice homozygous for the H-2Db-restricted TCR TCR-{alpha}ß transgene, F5, which recognizes a nonamer peptide, NP-68, from influenza virus A/NT/60/68 nucleoprotein and homozygous for the RAG-1 null mutation (F5homRAG-1-/-) (obtained from Dr. D. Kioussis at National Institute of Medical Research, Mill Hill, London), were mated to C57BL/6J me/+ mice to produce an F1 generation. F1 mice of the genotype, me/+F5hemRAG-1+/- were mated inter alia to generate four experimental backgrounds with which to examine the effect of varying levels of expression of SHP-1. The experimental backgrounds were 1) F5hemRAG-1+, 2) F5homRAG-1+, 3) F5hemRAG-1-/-, and 4) F5homRAG-1-/-. In each experimental background, we examined mice that were me/me, me/+, and +/+. Experimental mice were typed for expression of the F5 TCR-{alpha}ß by fluorescence staining of SP CD8+ thymocytes and lymph node CD8+ T cells with a FITC-conjugated KT11 (anti-Vß11) mAb. The status of the RAG-1 gene was evaluated by the presence or absence of SP CD4+ thymocytes. In initial experiments, the RAG-1 locus was additionally typed by PCR using two independent primer combinations. The RAG-1 gene itself was examined using primers that span the disruption in the coding region of the RAG-1 at amino acid 330. The primers used to generate a 793-bp fragment in the wild-type allele of the RAG-1 gene were 5'-ATC GAC GTG AAG GCA GAT GTT-3' and 5'-CTG CGC CCT TCT CGT CAG TGA-3'. The primers used for identifying the neomycin phosphotransferase gene in disrupted RAG-1 alleles have been described previously (28). All mice used in these studies were aged between 8 and 13 days.

Abs and peptides

For flow cytometric analysis, the following Abs were used in this study: FITC-conjugated KT11 (anti-Vß11; a gift from Dr. D. Kioussis, 42); FITC-conjugated 7D4 (CD25), FITC-conjugated IM-7 (CD44), phycoerythrin (PE)-conjugated 53-6.7 (CD8{alpha}), PE-conjugated 145-2C11 (CD3{epsilon}), PE-conjugated M1/70 (CD11b), PE-conjugated H129.19 (CD4), PE-conjugated PK136 (NK1.1), PE-conjugated RA3-6B2 (CD45R/B220), CyChrome-conjugated RM4-5 (CD4), and biotin-conjugated 7D4 (CD25) (purchased from PharMingen, San Diego, CA); and FITC-conjugated KT15 (CD8{alpha}) and PE-conjugated KT6 (CD4) (purchased from Serotec, Oxford, U.K.). Streptavidin-red 670 conjugate was purchased from Life Technologies (Grand Island, NY). NP-68 and GAG peptides were a gift from Dr. D. Kioussis. For biochemical analyses, 4G10 (anti-phosphotyrosine) was purchased from UBI (Lake Placid, NY). Goat antiserum to CD3{epsilon} was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the mouse mAb to ZAP-70 was obtained from Affiniti (Nottingham, U.K.). 2C-11 (anti-CD3{epsilon}) was a kind gift from Dr. D. Cantrell (Imperial Cancer Research Fund, London, U.K.). Rabbit antiserum to the SH-2 domains of SHP-1 was as previously described (19). Horseradish peroxidase-conjugated secondary Abs were purchased from Bio-Rad (Hercules, CA) (anti-mouse) and Santa Cruz Biotechnology (anti-goat).

Cells and medium

Thymic lobes or mesenteric and inguinal lymph node T cells, aseptically removed from motheaten and control mice, were gently homogenized (Jencons B.24 grade) to single-cell suspensions, passed through a 0.2-mm gauge cell strainer (Greiner, Nurtingen, Germany), counted by trypan blue exclusion, and incubated in RPMI 1640 (Life Sciences, Paisley, U.K.) supplemented with 10% heat-inactivated FCS (Serotec), 20 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 50 mM 2-ME. Feeder cells were prepared by irradiating single-cell suspensions of splenocytes (3000 rad) from C57BL/6J mice. EL-4 and CTLL-2 cells were maintained in exponential growth phase at 37°C in RPMI 1640 plus supplements with the addition of 20 ng/ml rIL-2 (Chiron, Harefield, U.K.) to CTLL-2 cultures. The H-2Db-expressing thymic epithelial cell line, YO1 (43), was generously provided by Dr. D. Kioussis (National Institute of Medical Research, London, U.K.) and was maintained in RPMI 1640 plus supplements in the presence of 100 IU/ml IFN-{gamma} (Genzyme, Boston, MA) at 33°C in 5% CO2.

Flow cytometric analysis

Flow cytometry was performed by staining 1 x 106 thymocytes or 2 x 105 lymph node T cells in 20 µl of PBS containing 1% BSA and 0.02% sodium azide on ice for 30 min. DN thymocytes were analyzed by three-color staining with PE-conjugated CD8{alpha}, CD4, CD3, NK1.1, B220, CD11b, biotin-CD25, and FITC-CD44. The biotin-conjugated CD25 was detected using a streptavidin-red 670 conjugate. DP and SP thymocytes and lymph node T cells were analyzed with CyChrome-conjugated CD4, PE-conjugated CD8{alpha}, and a variety of FITC-conjugated Abs. After three washes with PBS, between 10,000 and 100,000 events were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) and analyzed using CellQuest software (Becton Dickinson).

Cell sorting

For proliferation assays, CD8+ lymph node T cells were immunomagnetically purified from single-cell suspensions of freshly isolated lymph nodes with anti-CD8{alpha}-conjugated microbeads and "MS+" immunomagnetic cell sorting columns (Miltenyi Biotec, Sunnyvale, CA) according to manufacturer’s instructions. SP CD8+ thymocytes were purified on a Becton Dickinson FACS440 following thymocyte staining with CD8{alpha}-FITC and CD4-PE in the absence of sodium azide. For biochemical analyses, DP thymocytes from F5homRAG-1-/- animals were immunomagnetically purified with anti-CD4-conjugated microbeads and "LS+" immunomagnetic cell sorting columns (Miltenyi Biotec) according to manufacturer’s instructions.

Proliferation, IL-2 release, and cell-surface marker expression assays

Purified lymph node or thymic CD8+ T cells were incubated in triplicate on 96-well flat-bottom plates at 5 x 103 cells/well containing 2 x 105 irradiated feeder cells in the presence of cognate (NP-68) or control (GAG) peptide that binds to H-2Kb but is not recognized by the F5 TCR (43). After 20 h, [3H]thymidine (Amersham International, Aylesbury, U.K.) was added at 1 µCi/well, and the cultures were incubated for an additional 8 h before harvesting and proliferation assessed by measuring the incorporated radioactivity. For IL-2 measurement, purified CD8+ lymph node T cells (10 x 103) were similarly cultured, and supernatants were harvested after 28 h. Extensively washed CTLL-2 cells (5,000) were added to diluted supernatants (1 in 2) and incubated for 48 h with [3H]thymidine (1 µCi/well) present for the last 24 h of culture before harvesting, and the incorporated radioactivity was assessed. For the induction of activation marker expression, lymph node cells (0.5 x 106/ml) or thymocytes (1 x 106/ml) were incubated for 24 h with irradiated splenocyte feeders (2 x 106/ml) that had been previously pulsed for 1 h at room temperature with 2 µM NP-68. The expression of CD25, CD44, and CD5 upon SP CD8+ T cells was examined by flow cytometric analysis.

CTL Assay

CTL assays were conducted as previously described (44) with slight modifications. Lymph node T cells (0.5 x 106/ml) were primed for 4 days with irradiated feeder cells (3 x 106/ml) and NP-68 peptide (1 x 10-8 M) in the presence of IL-2 (10ng/ml) and were then washed and added to a round-bottom 96-well plate. EL-4 (H-2b) targets were pulsed with 5 x 10-5M NP-68 (cognate) or GAG (control) peptide for 1 h at 37°C before 51Cr labeling (Amersham International), added to effectors at 1000 cells/well, incubated at 37°C for 4 h, and 51Cr release in supernatants was determined. Specific percent lysis = [(cpmsamples - cpmmedium)/(cpmmaximum - cpmmedium)] x 100.

Analysis of phosphotyrosine-containing proteins

The thymic epithelial cell line, YO1, was used to stimulate thymocytes expressing the F5 TCR as previously described (45). Briefly, purified DP thymocytes from F5homRAG-1-/- mice were centrifuged onto YO1 cells that had been previously pulsed with either 10 µM agonist (NP-68) or control (GAG) peptide for 2 h at 37°C. After 5 min at 37°C, thymocytes were removed and resuspended in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 25 mM NaF, 1 mM sodium orthovanadate, 0.5% Nonidet P-40) containing protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µM EDTA, 1 mM PMSF). Immunoprecipitations were performed for 1 h at 4°C, captured with protein-A Sepharose for a further hour at 4°C, and washed extensively in Nonidet P-40 lysis buffer. Immunoprecipitations and cell lysates were resolved by 12% SDS-PAGE, Western blotted onto polyvinylidene diflouride membranes (Millipore, Bedford, MA), and probed for phosphotyrosine-containing proteins with an anti-phosphotyrosine Ab (4G10). Blots were stripped for 30 min at 55°C (100 mM 2-ME, 2% SDS, 62.5 mM Tris-HCl, pH 6.8) before reprobing. Proteins were detected with horseradish peroxidase-conjugated secondary Abs and enhanced chemiluminescence (Amersham International).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased number of mature CD25- CD44- DN thymocytes in motheaten mice possessing the MHC class I-restricted F5 TCR

To examine thymocyte differentiation and T cell activation with respect to the level of expression of SHP-1, a number of experimental mice were generated (for details see Materials and Methods). Mice either hemizygous or homozygous for the F5 {alpha}ß-transgenic TCR were generated in RAG-1-deficient or -sufficient genetic backgrounds. These were 1) F5hemRAG-1+, 2) F5homRAG-1+, 3) F5hemRAG-1-/-, and 4) F5homRAG-1-/-, in which thymocyte subpopulations and lymph node T cells from mice that were either me/me, me/+, or +/+ were examined. The F5 TCR uses the V{alpha}4 and Vß11 gene segments of the TCR {alpha}- and ß-chain genes, respectively (46). An increased expression of Vß11 was detected on thymocyte subpopulations in the F5hom vs F5hem experimental backgrounds, although for each thymocyte subpopulation (including DN, DP, and SP) equivalent levels of expression of Vß11 were detected in me/me, me/+, and +/+ mice (data not shown). The total number of thymocytes in me/me mice expressing the F5 TCR ranged from 44–77% that of age-matched controls (Tables I and II), and we attributed this premature involution of the thymus to secondary macrophage-mediated effects as has been previously reported for nontransgenic motheaten mice (26).

Developing thymocytes are subjected to two major developmental check-points. The first check-point is mediated by the pre-TCR complex composed of the pre-TCR {alpha}-chain associated with a functional TCR ß-chain on a population of DN thymocytes that are further distinguished as being CD25+ CD44- (47). Pre-TCR signaling on this population induces their proliferation and further enables their differentiation to a more mature DN population that are characterized as being CD25-CD44- (47). In nontransgenic me/me and control (me/+ and +/+) mice, the percentages of CD25-CD44- and CD25+CD44- DN thymocytes were approximately equal (Fig. 1GoA). However, analysis of DN thymocytes from F5 TCR-transgenic mice revealed significant differences in the relative proportions of the immature CD25+CD44- and more mature CD25-CD44- populations when comparing me/me and control mice (me/+ and +/+). First, the expression of the F5 TCR-{alpha}ß in DN thymocytes from +/+ mice led to a skewing toward the more mature CD25-CD44- population such that the level of expression of Vß11 correlated with the extent of the skewing (as illustrated in Fig. 1GoA for the F5hemRAG-1+ and F5homRAG-1+ experimental backgrounds). Second, the skewing toward the more mature CD25-CD44- population was much more pronounced in me/me vs +/+ mice in all four F5 TCR-transgenic experimental backgrounds (Fig. 1GoA and Table IGo), although no significant differences in the proportions of DN thymocytes were detected between me/+ and +/+ mice (data not shown). While the total numbers of DN thymocytes were increased in control (+/+ and me/+) mice in the F5homRAG-1+ vs F5hemRAG-1+ experimental backgrounds, this effect was much greater for me/me mice (Table IGo). A similar effect was also noted for DN thymocytes from me/me mice in the F5homRAG-1-/- vs F5hemRAG-1-/- experimental backgrounds (Table IGo). Importantly, the absolute numbers of CD25-CD44- DN thymocytes were significantly increased in me/me vs control mice (me/+ and +/+) in the F5homRAG-1+ (p < 0.01) and F5homRAG-1-/- (p < 0.05) experimental backgrounds (Table IGo and Fig. 1GoB). In F5 TCR-transgenic as compared with nontransgenic mice, we presume that signaling through TCR complexes on DN thymocytes is enhanced, leading to an increased proliferation of the CD25-CD44- population and/or an accelerated maturation of the CD25+CD44- population of DN thymocytes or a combination of both phenomena. In the context of this enhanced signaling, a role for SHP-1 in regulating TCR complexes on DN thymocytes is revealed.



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  		FIGURE 1. Increased proliferation and/or accelerated maturation of DN thymocytes in motheaten mice expressing the F5 TCR-{alpha}ß. Thymocytes from nontransgenic and F5 TCR-{alpha}ß mice aged 8–13 days were stained with PE-conjugated anti-CD3{epsilon}, anti-CD4, anti-CD8{alpha}, anti-CD11b, anti-NK1.1, and anti-B220 to define a DN population of immature thymocytes. Cells were simultaneously stained with Ab conjugates of CD44-FITC and CD25-biotin followed by streptavidin-red 670 and analyzed by three-color flow cytometry using electronic gates set around PE-negative DN thymocytes. A, Representative profiles of CD44/CD25 staining in the nontransgenic, F5hemRAG-1+ and F5homRAG-1+ experimental backgrounds are shown. The percentage of live cells in each quadrant is listed. Quadrants were set such that in +/+ nontransgenic mice approximately equivalent percentages of CD25+CD44- and CD25-CD44- thymocytes were detected. B, Bar graphs show mean numbers of CD25+CD44- and CD25-CD44- DN thymocytes from nine control (me/+ and +/+) and eight me/me mice in the F5homRAG-1+ experimental background and three control (me/+ and +/+) and four me/me mice in the F5homRAG-1-/- experimental background. Error bars represent SDs. me/me DN thymocytes showed a significant increase in the number of more mature CD25-CD44- fraction.

 

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  		Table I. Total numbers of DN (CD4-CD8-) thymic subpopulations in class I-restricted F5 TCR-transgenic mice1

 
Increased number of SP CD8+ thymocytes in SHP-1-deficient mice possessing the MHC class I-restricted F5 TCR

The second major check-point in thymocyte development is at the level of the TCR-{alpha}ß-expressing DP thymocyte. Due to the macrophage-mediated loss of thymocytes (26), we restricted our analysis wherever possible to me/me mice in which the total number of thymocytes was at least 60% that of controls. Furthermore, as there was an indication of an intermediate phenotype in thymocytes from me/+ mice (data not shown), we restricted our analysis of SP thymocytes to a comparison of me/me and +/+ mice. In each of the four experimental backgrounds, the percentage of SP CD8+ thymocytes was significantly increased in me/me vs +/+ mice, although in both the F5hemRAG-1+ and F5homRAG-1+ backgrounds the percentage of SP CD4+ thymocytes was not similarly increased (Fig. 2Go, A and B). Despite the decreases in mean total numbers of thymocytes for me/me mice in each of the four experimental backgrounds, the absolute numbers of SP CD8+ TCRhigh thymocytes in me/me vs control (+/+) mice were significantly increased (p < 0.01) in three of the experimental backgrounds (F5hemRAG-1+, F5homRAG-1+, and F5homRAG-1-/-) (Table IIGo). No significant difference in the number of SP CD8+ thymocytes was observed in F5hemRAG-1-/- mice and this presumably reflected the more severe thymic involution of the me/me mice examined in this background (Table IIGo). A previous report has indicated that very few SP CD8+ thymocytes from wild-type mice expressing the F5 TCR-{alpha}ß are proliferating in vivo (48). Likewise, cell cycle analysis of SP CD8+ thymocytes in me/me and +/+ mice in each of the four experimental backgrounds revealed a comparable and very limited number of cells in G2/S (data not shown). Furthermore, no differences in the number of apoptotic SP CD8+ thymocytes were detected in me/me vs control (+/+) mice (data not shown). Levels of CD44 and CD25 were equivalently low on me/me and +/+ SP CD8+ thymocytes (data not shown), indicating that mature peripheral T cells had not re-entered the thymi of me/me mice. Finally, it appears there were no defects in the ability of me/me SP thymocytes to migrate to the periphery as increased numbers of SP CD8+ T cells were detected in the lymph nodes of me/me vs +/+ mice (as described below, Fig. 3Go). Thus, the increased numbers of SP CD8+ thymocytes in me/me mice most likely represent an increase in positive selection.



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  		FIGURE 2. Increased positive selection of SP CD8 thymocytes in me/me mice possessing F5 TCR. Thymocytes from F5 TCR-expressing animals aged 8–11 days were stained with anti-CD8{alpha}-PE, anti-CD4-CyChrome, and anti-Vß11-FITC conjugates and analyzed by three-color flow cytometry. A, Bar graphs show mean percentages of SP CD4+ and CD8+ thymocytes from me/me and +/+ mice in each of the four experimental backgrounds. Error bars represent SDs. B, Representative profiles of CD4/CD8 staining from mice in the F5hemRAG-1+ experimental background are shown. Numbers represent the percentage of live cells in the indicated regions.

 

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  		Table II. Total numbers of CD4+ and CD8+ SP and DP thymic subpopulations in class I-restricted F5 TCR-transgenic mice1

 


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  		FIGURE 3. Skewing of the ratio of CD8:CD4 T cells in lymph nodes of F5 TCR me/me mice. Lymph node cells from me/me and +/+ mice in the F5hemRAG-1+ and F5homRAG-1+ backgrounds were stained with anti-CD8{alpha}-PE and anti-CD4-CyChrome conjugates and analyzed by two-color flow cytometry. Bar graphs show the ratio of individual percentages of CD8+ and CD4+ T lymphocytes in lymph nodes. Data shown is drawn from pools of at least six individual mice of each genotype. Error bars represent SDs.

 
Skewing in the ratios of mature SP CD4+ and CD8+ T cells in the lymph nodes of SHP-1-deficient mice possessing the MHC class I-restricted F5 TCR

In the F5hemRAG-1+ and F5homRAG-1+ experimental backgrounds, the ratio of SP CD8+ and CD4+ mature T cells in the lymph nodes of me/me and control mice (+/+) is reversed compared with nontransgenic mice such that there are proportionately more SP CD8+ T cells (40). However, in each of the F5hemRAG-1+ and F5homRAG-1+ backgrounds the skewing toward SP CD8+ T cells was significantly more pronounced (p < 0.01) in me/me vs +/+ mice (Fig. 3Go). Cell cycle analysis of SP CD8+ lymph node T cells in each of the four experimental backgrounds revealed no significant differences in the percentage of T cells at each stage of the cell cycle (including the apoptotic subG0 population) when comparing me/me and control (+/+) mice (data not shown). Therefore, the increased ratio of SP CD8+ to CD4+ T cells in lymph nodes from me/me mice in the F5hemRAG-1+ and F5homRAG-1+ backgrounds would be consistent with an enhanced positive selection of SHP-1-deficient SP CD8+ thymocytes.

Increased in vivo activation of resting lymph node T cells from motheaten mice possessing the MHC class I-restricted F5 TCR

The levels of expression of TCR as represented by Vß11 staining were equivalent between me/me and control (me/+ and +/+) lymph node T cells within each of the four transgenic F5 experimental backgrounds (Table IIIGo and Fig. 4Go for the F5hemRAG-1-/- background). The stimulation of the TCR-{alpha}ß on mature T cells leads to numerous downstream phenotypic changes, including the induced cell-surface expression of the receptors CD44 and CD25 (49). An increased (p < 0.01) basal level of expression of CD44 was detected on resting transgenic F5 TCR CD8+ lymph node T cells from me/me vs control mice (me/+ and +/+) in all four experimental backgrounds, while basal levels of expression of CD25 were elevated (p < 0.01) on resting transgenic F5 TCR CD8+ lymph node T cells from me/me vs control mice (me/+ and +/+) in both F5hem backgrounds (Table IIIGo and Fig. 4Go). In addition, in each of the four transgenic F5 TCR experimental backgrounds the mean size of the me/me vs control lymph node T cells was also increased (data not shown). No alterations in the expression of any activation markers were detected in me/+ compared with +/+ lymph node T cells (data not shown). Increased levels of expression of CD5 were also detected on SP CD8+ lymph node T cells from me/me vs control mice (me/+ and +/+) in all experimental backgrounds (Table IIIGo and Fig. 4Go for the F5hemRAG-1-/- background). In contrast, equivalent levels of expression of CD25 and CD5 were detected on SP CD8+ lymph node T cells from nontransgenic me/me and control (me/+) mice (Fig. 4Go). We infer from these results that the loss of SHP-1 in resting lymph node T cells bearing the F5 TCR leads to an increased basal level of activation that must be mediated through the transgenic TCR.


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  		Table III. Expression of cell-surface markers on peripheral CD8+ T cells from class I-restricted F5 TCR-transgenic mice1

 


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  		FIGURE 4. Resting peripheral T lymphocytes from F5 TCR me/me mice show increased expression of activation markers. Lymph node cells from me/me or control mice (me/+ or +/+) aged 8–13 days in either nontransgenic or F5 TCR transgenic backgrounds were stained with anti-CD8{alpha}-PE and one of three FITC-conjugates to CD25, CD44, and CD5, and green channel fluorescence was assessed on electronically gated populations of CD8+ cells. Representative histograms depicting activation marker expression from me/me vs me/+ mice in the F5hemRAG-1-/- or nontransgenic backgrounds are shown (see Table IIIGo for statistical analysis). The differences in the basal expression levels of activation markers in T cells from F5 TCR me/me vs control (me/+ and +/+) mice as estimated by Student’s ttest were significant. No significant differences in the expression of activation markers were detected between lymph node T cells from F5 TCR me/+ and +/+ mice. The marker region, M1, on the histograms show the populations of T cells analyzed to calculate the mean fluorescence intensities indicated in Table IIIGo.

 
Hyperproliferation but equivalent cytolytic activity of motheaten lymph node CD8+ T cells in response to stimulation with cognate peptide

A previous report has shown that unseparated lymph node T cells from nontransgenic viable motheaten mice, primed in vivo with OVA, hyperproliferated in comparison to controls when restimulated in vitro (37). We were interested in evaluating whether F5 TCR-{alpha}ß T cells from me/me mice also demonstrated an increased sensitivity to cognate Ag. Purified SP CD8+ lymph node T cells from me/me F5 TCR-{alpha}ß-transgenic mice were stimulated in vitro with APCs and a titration of cognate peptide (NP-68). Lymph node T cells from me/me mice proliferated more strongly to all concentrations of the cognate peptide but had no detectable proliferation toward a control H-2Db binding peptide (GAG) that is not recognized by the F5 TCR-{alpha}ß (Fig. 5GoA). The increased proliferative responsiveness of SP CD8+ lymph node T cells from me/me mice was shown to correlate with an increased secretion of IL-2 (Fig. 5GoB). Likewise, an increased number of CD8+ lymph node T cells from me/me mice were shown to up-regulate the activation markers, CD25 (70% and 41% for me/me and control, respectively), CD44 (72% and 49% for me/me and control, respectively) and CD5 (73% and 44% for me/me and control, respectively) following stimulation in vitro with cognate peptide (Fig. 5GoC). In addition, purified SP CD8+ thymocytes from me/me F5 TCR-{alpha}ß-transgenic mice also demonstrated a hyperproliferative response to cognate Ag (Fig. 5GoD). However, it was interesting to note that SP CD8+ thymocytes from me/me mice expressing the F5 TCR-{alpha}ß did not demonstrate elevated levels of activation marker expression both before and following stimulation in vitro with cognate peptide (data not shown). The increased sensitivity of me/me T cells to stimulation with cognate peptide with regards to IL-2 secretion and proliferation provides further direct evidence for a lowered threshold of responsiveness through the TCR in SHP-1-deficient T cells. Therefore, we were interested in detecting the presence and level of NP-68-specific cytolytic activity of mature CTLs generated from the lymph nodes of me/me mice. As shown in Fig. 5GoE, lymph node T cells from me/me mice were indeed capable of lysing target cells in an Ag-specific fashion following 4 days priming with cognate peptide in vitro, although the cytolytic activity of the mature effector CD8+ T cells generated from me/me and +/+ control mice was demonstrated to be equivalent.



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  		FIGURE 5. Peripheral CD8+ T cells from me/me mice expressing the F5 TCR show enhanced in vitro proliferation and IL-2 production but equivalent cytolytic activity following stimulation with cognate Ag. A, Sorted CD8+ lymph nodes were incubated with a titration of NP-68 or a control Db binding peptide, GAG, in the presence of irradiated splenocytes for 28 h, and proliferation was assessed by [3H]thymidine incorporation. Data is representative of three independent experiments. B, Sorted CD8+ lymph node T cells were incubated with irradiated splenocytes and either NP-68 or GAG peptide for 28 h, and IL-2 production was assessed as a function of CTLL-2 proliferation. Data is representative of three independent experiments. C, Lymph node T cells were activated for 24 h with irradiated splenocytes previously pulsed with NP-68, and cell-surface marker expression on anti-CD8{alpha}-PE-positive cells was assessed with Ab-FITC conjugates to CD25, CD44, and CD5. Cells in the region, M2, are defined as CD25high, CD44high, or CD5high. Data is representative of two motheaten and four littermate controls. D, Sorted SP CD8+ thymocytes were incubated with NP-68 in the presence of irradiated splenocytes for 28 h, and proliferation was assessed by [3H]thymidine incorporation. E, Lymph node T cells were primed for 96 h with NP-68 (10-8 M) and rIL-2 (10 ng/ml), and the CTL activity to EL-4 cells presenting either NP-68 or GAG peptide was assessed. Data is representative of six independent experiments. All data shown is for the F5hemRAG+ background. Error bars represent SDs.

 
Equivalent tyrosine phosphorylation of the CD3 complex in motheaten and control thymocytes expressing the F5 TCR

As an approach to determining the molecular basis for the lowered TCR signaling threshold of motheaten T cells expressing the F5 TCR, we examined the phosphotyrosine content of proteins present in total cell lysates isolated from purified motheaten and control DP thymocytes that had been stimulated in vitro with the cognate peptide (Fig. 6GoA). Such an analysis revealed equivalent patterns of phosphotyrosine-containing proteins in motheaten and littermate control thymocytes, indicating that SHP-1 must dephosphorylate a very limited subset of the phosphotyrosine-containing proteins involved in regulating TCR signaling. To examine more directly the tyrosine phosphorylation state of the TCR signaling complex, we also performed anti-CD3{epsilon} immunoprecipitations from motheaten and control DP thymocytes following activation with the cognate peptide (Fig. 6GoB). It was apparent from this analysis that the basal tyrosine phosphorylation of the {zeta}-chain was equivalent in motheaten and control thymocytes. Likewise, stimulation with the cognate peptide resulted in an equivalent induction of tyrosine phosphorylation on the {zeta}-chain and a very similar recruitment of the intracellular PTK, ZAP-70, to the CD3 complex. In addition, the phosphotyrosine content of the ZAP-70 associated with the CD3 complex was equivalent when comparing motheaten and littermate control thymocytes following stimulation (Fig. 6GoB).



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  		FIGURE 6. Equivalent tyrosine phosphorylation of the CD3 complex in motheaten and control thymocytes expressing the F5 TCR. Purified DP thymocytes from F5homRAG-1-/- mice were stimulated for 5 min with YO1 cells preloaded with 10 µM of either control GAG (-) or cognate NP-68 (+) peptide, and thymocytes were lysed in 0.5% Nonidet P-40 lysis buffer as described in Materials and Methods. Total cell lysates of 1 x 106 thymocytes (A) or CD3{epsilon} immunoprecipitations from 1 x 107 thymocytes (B) were analyzed by anti-phosphotyrosine immunoblotting. Blots were reprobed for ZAP-70 or CD3{epsilon} as indicated. The migration of molecular mass markers is indicated on the left. Data is representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This genetic study has implicated the PTP, SHP-1, as a negative regulator of signals generated by TCR complexes on developing thymocytes and naive peripheral T cells.

SHP-1 regulates TCR signaling complexes in DN thymocytes

The intracellular PTKs, p56lck, p59fyn, ZAP-70, and Syk (4, 5, 6, 7, 8), and the receptor PTP, CD45 (50), have all previously been shown to be positive mediators of signaling from the pre-TCR complex, thereby confirming the importance of tyrosine phosphorylation to pre-TCR signaling and emphasizing the similarities in the functional operation of the pre-TCR and mature TCR signaling complexes. However, until now there has been no indication that signaling from the pre-TCR may be subject to any negative regulation (51). Loss of SHP-1 resulted in an increase in the ratio of the mature CD25-CD44- to immature CD25+CD44- populations of DN thymocytes in F5 TCR-transgenic mice. This may reflect an accelerated maturation toward the more mature CD25-CD44- population of DN thymocytes and/or an increased proliferative expansion of the CD25-CD44- DN thymocytes. As this phenotype was more pronounced in me/me mice in the F5hom (in which transgenic ß-chain expression is increased) vs F5hem experimental backgrounds, it is consistent with SHP-1 regulating signals from TCR complexes on DN thymocytes. In DN thymocytes from nontransgenic mice, the pre-TCR complex is composed of the surrogate pre-TCR {alpha}-chain found in association with a functional TCR ß-chain. However, in this study we have examined mice expressing an {alpha}ß-transgenic TCR and hence we cannot distinguish between the possibility that SHP-1 is regulating signals from a TCR complex composed of the transgenic ß-chain associated with either the endogenous pre-TCR {alpha}-chain or the transgenic TCR {alpha}-chain. Interestingly, it has been reported in another class I-restricted TCR-{alpha}ß system that expression of the transgenic {alpha}-chain was found on only a fraction of the DN thymocytes expressing the transgenic ß-chain (52). However, an appropriate mAb is not available to evaluate the expression on DN thymocytes of the V{alpha}4 chain used by the F5 TCR. Nevertheless, it would appear that if SHP-1 is regulating signals from an TCR-{alpha}ß complex (instead of/in addition to pre-TCR complexes) on DN thymocytes in F5 {alpha}ß-transgenic mice, then the TCR-{alpha}ß complex is at least mimicking signaling that would normally be associated with the pre-TCR complex. The important implication from this study is that SHP-1 must be expressed in DN thymocytes and must be capable of dephosphorylating one or more components of the transgenic TCR signaling complex or downstream signaling molecules in DN thymocytes to effectively increase the threshold for signaling.

SHP-1 regulates TCR signaling complexes in DP thymocytes

DP thymocytes are subjected to rigorous selection whereby thymocytes expressing TCRs with intermediate affinities for self-peptide/MHC complexes are positively selected to ensure their survival and further maturation (53, 54). In contrast, DP thymocytes expressing TCRs with high affinities for self-peptide/MHC complexes are negatively selected by apoptosis (55, 56). Signaling molecules such as CD45, p56lck, p59fyn, Zap-70, Syk, and p95Vav that affect the degree of signal strength or quality of signal originating at the TCR on DP thymocytes have been demonstrated to influence thymocyte selection (6, 9, 10, 11, 50, 57, 58, 59). We conclude from these investigations that SHP-1 also plays a role in regulating signals from the TCR complex on DP thymocytes that direct the developmental decisions for the maturation of SP thymocytes. In three of the four experimental backgrounds, modest but significant (p < 0.01) increases in the absolute numbers of SP CD8+ thymocytes were detected in me/me mice. However, these absolute increases were particularly striking considering that even though a restricted subset of me/me mice were used in these analyses the overall total numbers of thymocytes in these mice were still consistently lower (47–77%) than that of controls. Furthermore, we were obliged to use the me/me mice at day 8–13 even though the percentage of SP CD8+ thymocytes in the thymi of neonatal F5 TCR control (+/+) mice was considerably less than that observed in adult F5 TCR control mice (3% at day 8–13 compared with 15–20% at 6 wk) (40). The loss of DP thymocytes from F5 TCR-transgenic me/me mice is unlikely to have resulted from the partially activated peripheral T cells as similar depletions also occurred in nontransgenic me/me mice in which the resting peripheral T cells did not appear activated. Instead, the large decreases in DP thymocytes from F5 TCR transgenic me/me mice, which greatly exceeded the more modest increases in SP thymocytes, most likely resulted from the adverse effects of the activated macrophage and neutrophil populations (26). As the SP CD8+ thymocytes from me/me mice demonstrated a normal cell cycle profile (data not shown) and as the exit and entry of T cells in the thymus appeared normal, the most straightforward interpretation of the elevated numbers of SP CD8+ thymocytes is increased positive selection. In support of this argument, the absolute numbers of SP CD8+ lymph node T cells were increased in me/me mice in all four F5 TCR experimental backgrounds. However, due to the difficulties of presenting results on absolute numbers of T cells in lymph nodes, we have illustrated these findings as an increased ratio (p < 0.01) of SP CD8+ vs CD4+ lymph node T cells in me/me vs control mice for the F5hemRAG-1+ and F5homRAG-1+ backgrounds. The skewing in the CD8/CD4 ratio in me/me mice is consistent with the prediction that the loss of SHP-1 enhances the transition of DP to SP thymocytes, which then migrate and accumulate in the lymph nodes.

Therefore, it appears that SHP-1 contributes to raising the threshold for signaling through the TCR on DP thymocytes such that the same H-2Db/self-peptide ligands result in stronger TCR signals when presented to me/me vs +/+ DP thymocytes; H-2Db/self-peptide complexes normally incapable of generating a sufficient survival signal through the F5 TCR may now become capable of inducing signals that allow the DP to SP transition. Therefore, the selection of DP thymocytes would be dependent upon not only the availability and quality of self-peptide/MHC complexes together with the affinity and level of cell-surface expression of TCR (TCR avidity) (60, 61, 62, 63) and co-receptors (64, 65) but also the degree of activation of SHP-1.

SHP-1 regulates TCR signaling complexes in resting mature T cells

We also conclude that SHP-1 contributes to a threshold for TCR activation on mature T cells. In the absence of SHP-1, lymph node T cells expressing the F5 TCR-{alpha}ß were enlarged and demonstrated a small but significantly increased basal expression of T cell activation markers despite a lack of exposure to cognate Ag. The basal expression of activation markers was equivalent in nontransgenic me/me and control T cells, suggesting the elevated levels of activation markers in F5 T cells from me/me mice resulted from expression of the transgenic F5 TCR and were a consequence of the intrinsic loss of SHP-1 in the T cells and not due to secondary macrophage-mediated events. The more activated basal phenotype of me/me T cells was particularly striking in the RAG-1-/- experimental backgrounds, in which the interpretation of results is not complicated by the possible association of endogenous TCR {alpha}-chains with the transgenic TCR ß-chain (66). The implications of these observations are twofold. First, there must exist a low-level, spontaneous stimulation of the TCR on resting T cells that presumably involves a minimal cross-reactivity of the F5 TCR-{alpha}ß with cells expressing self-peptides bound to H-2Db molecules, and second, SHP-1 must be constitutively active in resting T cells. This would certainly be consistent with recent results indicating that naive T cells require a minimal survival signal from MHC molecules (67, 68, 69, 70, 71, 72). In this context, the term "resting" must encompass a minimal stimulation of the TCR that is occurring continuously even in the absence of cognate Ag. Until now, the functional consequences of these low-level constitutive signals have not been apparent. However, in the absence of SHP-1 it is clear that these survival signals can be sufficiently strong to cause an up-regulation of cell-surface activation markers. The increased basal expression of activation markers (CD25 and CD44) on lymph node T cells from me/me mice was more pronounced in the F5hem vs F5hom backgrounds and this was particularly apparent for the F5hemRAG-1-/- and F5homRAG-1-/- experimental backgrounds. The reasons for these differences are not clear but may reflect a degree of negative feedback on the expression of activation markers in me/me lymph node T cells expressing the highest levels of F5 TCR.

The elevated basal levels of CD5 in peripheral T cells from me/me mice are also intriguing. CD5 has been demonstrated to play a negative regulatory role on T cells (73), and in p95Vav-deficient mice T cell-surface levels of CD5 are lowered possibly as a compensatory mechanism for compromises in the efficiency of TCR signaling in the absence of p95Vav (59). Therefore, it is intriguing to speculate that the elevated levels of CD5 detected in SHP-1-deficient mature T cells may be performing a related feedback role to compensate for enhanced TCR signaling.

Previous data has demonstrated an increased sensitivity of me/me thymocytes to anti-CD3 (37, 38) but not IL-2 stimulation (38). We also believe the hyperproliferative phenotype of F5 T cells from me/me mice results from enhanced signaling through the TCR and not the IL-2R. First, F5 T cells from me/me vs control mice secreted more IL-2 in response to stimulation with cognate Ag. Second, SP CD8+ thymocytes from F5 me/me mice hyperproliferated in response to Ag stimulation (Fig. 5GoD) even though basal levels of CD25 were equivalent to matched controls (data not shown). Third, Con A T cell blasts generated from F5 me/me and control mice demonstrated an equivalent proliferative response to IL-2 (K.G.J. and R.J.M., unpublished observations).

Although a greater number of lymph node T cells from me/me mice demonstrated an up-regulation of cell-surface activation markers following in vitro stimulation, this is likely to be related to the increased basal level of activation marker expression. Conversely, the level of expression of activation markers was equivalent in me/me and control SP CD8+ thymocytes both before and following stimulation. Thus, it appears that the loss of SHP-1 has a differential effect on the up-regulation of activation marker expression vs secretion of IL-2. This is most likely attributed to the less stringent TCR signaling requirements for CD25 cell-surface expression vs IL-2 gene expression (74). It was also intriguing that the cytolytic function of effector F5 CTLs appeared to be independent of SHP-1 (Fig. 5GoE) even though these cells still express SHP-1 (data not shown). The equivalent CTL activities of motheaten and littermate control T cells may also relate to the lower TCR signaling requirements required for CTL killing vs IL-2 secretion (75). Alternatively, SHP-1 may not be constitutively active in primed effector CTLs compared with resting peripheral T cells, thus suggesting that the means by which SHP-1 influences naive T cell activation and CTL killing may be distinct. Nevertheless, as SP CD8+ T cells from me/me mice have an increased proliferative capacity (and by virtue of possessing equivalent lysis activity compared with controls), one can state that SHP-1 activity in CD8+ T cells will be important for the in vivo generation of functional CTLs. Previous reports have implicated a role for SHP-1 in inhibiting target cell lysis by a limited number of human CTLs expressing either the CD94 receptor or killer inhibitory receptors (76, 77). However, these receptors are found expressed mainly on human NK cells and require specific MHC class I engagement for SHP-1 recruitment and activation (78). Likewise, the mouse NK inhibitory receptor, Ly 49, is primarily expressed on NK cells (79) and an extremely limited number of T cells including some CD8+ T cells (80) and requires specific MHC class I binding to trigger inhibitory signals.

In summary, SHP-1 must function to raise the basal threshold for functional signaling through TCR complexes on developing thymocytes and resting naive peripheral T cells. It is also intriguing that lymph node T cells from F5 TCR me/me mice demonstrated an increased magnitude of response in addition to an increased sensitivity to cognate peptide, which may reflect a sustained triggering of the TCR in the absence of SHP-1. We predict SHP-1 is capable of negatively dephosphorylating one or more components of TCR signaling pathways and hence in the absence of SHP-1 this leads to elevated basal levels of tyrosine phosphorylation and hence a lowered threshold for TCR signaling. It has been reported previously that the CD3{epsilon} and {zeta}-chains of the TCR complex, and a Grb2 associated phosphoprotein of 38 kDa (37), demonstrate increased tyrosine phosphorylation in thymocytes from nontransgenic motheaten mice following anti-CD3 stimulation. In addition, it has been suggested that the kinase activities of p56lck and p59fyn are increased in thymocytes from nontransgenic motheaten mice (38). Our own preliminary biochemical analysis of TCR signaling in motheaten F5 thymocytes would indicate that SHP-1 has a limited number of substrates and must be regulating TCR complexes downstream of ZAP-70. Work is currently in progress to identify one or more of the substrates of SHP-1 in the context of TCR signaling.

The phenotype we have described for the me/me T cells expressing the F5-transgenic TCR resembles in several ways the characteristics of mev/mev B cells expressing a transgenic BCR directed against hen egg lysozyme. These mev/mev B cells also appear to have an activated phenotype, yet without having encountered cognate hen egg lysozyme Ag, suggesting that the BCR has been exposed to a chronic low level of stimulation with a cross-reactive ligand and that SHP-1 must also be active in the resting B cell (35).

There is considerable interest in delineating the molecular basis for SHP-1 activation in T cells. The binding of the amino-terminal SH-2 domain of SHP-1 to phosphotyrosine-containing ligands is a stringent requirement for the activation of the PTP domain of SHP-1 (81, 82, 83). In the context of the B cell, SHP-1 has been demonstrated to bind to the B cell-surface receptor CD22 (83, 84, 85) that becomes inducibly tyrosine phosphorylated following triggering of the BCR (86). Furthermore, both CD22 (87, 88) and SHP-1 (36) have been demonstrated to be physically associated with the BCR complex in resting B cells. The binding of SHP-1 to CD22 is mediated via the phosphotyrosine-containing immunoreceptor tyrosine-based inhibitory motifs found in the intracellular domain of CD22 (83). Therefore, it is conceivable that the low basal level of tyrosine phosphorylation that exists on CD22 (85) is responsible for both the activation of SHP-1 and its association with the BCR in resting B cells (33, 34). The involvement of SHP-1 in regulating TCR thresholds suggests that SHP-1 is also physically associated with and activated by an immunoreceptor tyrosine-based inhibitory motif-containing receptor (89) in thymocytes and resting T cells. In addition, it has been reported that following TCR activation SHP-1 can be found associated via its SH-2 domains with a number of phosphotyrosine-containing molecules in activated T cells including CD5 (37), p95Vav (90), and ZAP-70 (91).

The molecular decisions governing thymocyte development and mature T cell activation have been demonstrated to be integrally linked to TCR signaling thresholds and will be extremely important in ensuring a response to an infectious pathogen but not at the cost of autoimmune disease. Indeed, it can be envisaged that a reduction in the threshold for signaling through the TCR may predispose T cells to respond to self-Ags that would normally be ignored. Determining the molecular basis of the pathways that affect TCR signaling thresholds clearly offers the potential of better understanding and identifying new targets for eventual therapeutic intervention in human autoimmune disease.


   Acknowledgments
 
We thank Dr. L. Shultz and Dr. D. Kioussis for the generous provision of motheaten and F5 TCR transgenic mice, respectively. We also thank Dr. D. Kioussis for Ab, peptide reagents, and the Y01 cell line and K. Davies and staff of Biomedical Services, University of Wales College of Medicine for expert animal husbandry. We also thank D. Kioussis, O. Williams, L. Smyth, M. Labéta, S. Man, M. Rowe, S. Tabi, and M. Sims for advice and critical comments on the manuscript.


   Footnotes
 
1 This work was supported by a Research Career Development Fellowship Grant (040127/Z/93/Z/RB/YJ45) to R.J.M. from The Wellcome Trust and a University of Wales Medical Research Scholarship to K.G.J. Back

2 Address correspondence and reprint requests to Dr. R. J. Matthews, Department of Medicine, Tenovus Building, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XX, U.K. E-mail address: Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SH-2, Src homology region-2; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; RAG-1, recombinase activation gene-1; BCR, B cell receptor; PE, phycoerythrin. Back

Received for publication August 17, 1998. Accepted for publication December 30, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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