Mice homozygous for the viable motheaten mutation (me^v) were obtained by mating C57BL/6J me^v/+ breeding pairs derived from breeding stock maintained at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital (Toronto, ON, Canada). The study was approved by the local Ethics Committee of Mount Sinai Hospital. Mice carrying an H-Y-specific TCR transgene, which recognizes the H-Y male-specific antigen presented on H-2D^b (24), were crossed with me^v/+ hetero zygotes/+ heterozygotes and the H-Y TCR/me^v/+ progeny selected and backcrossed with me^v/+ mice to obtain H-Y TCR/me^v homozygotes. For the derivation of CD5 transgenic mice, a huCD2-CD5 transgene was derived as previously detailed by substituting the murine CD5 coding sequence for the TCRζ cDNA sequence in the construct ζ-CT108 (25). Founder lines were identified by Southern blotting, screened for expression of CD5 by Northern blotting and flow cytometry analysis and the mice then backcrossed to C57BL/6J through six generations. The mice were then mated with H-Y TCR transgenic mice to generate H-Y TCR/CD5 transgenics. To derive H-Y TCR/CD5/me^v mice, the H-Y TCR/CD5 transgenics were mated to me^v/+ mice and the F1 H-Y TCR/CD5 transgenic viable motheaten heterozygote progeny then backcrossed with me^v/+ mice. The mice were typed for expression of the H-Y TCR and CD5 transgenes using PCR amplification with the primer pairs: 5′-CAGACCCTCCT TGATCCTGGCCCTCCAGT-3′ (forward) and 5′-CAGTCC GTGGACCAGCCTGATGCTCATGT-3′ (reverse); 5′-GGA GCACATCAGAAGGGCTGGCTT-3′ (forward) and 5′-CGG AGATCCTTGGGCAGAAGACCTG-3′ (reverse), respectively. The PCR amplification cycle (denaturation for 15 sec at 94°C, annealing for 20 sec at 64°C and elongation for 30 sec at 72°C) was repeated 35 times. H-Y TCR and CD5 transgene expression was also confirmed by surface staining of peripheral blood lymphocytes (26). The mice were studied at the ages of 2-3 weeks.

Antibodies used for these studies included FITC-conjugated anti-CD8 (cat. no. 553031; 1:1,000) and anti-CD5 (cat. no. 553020; 1:1,000) antibodies, PE-conjugated anti-CD4 antibody (cat. no. 557307; 1:1,000), and biotin-conjugated monoclonal rat anti-mouse CD5 (clone 53–7.3; cat. no. 553018; 1:1,000), anti-TCR (αβ), and anti-CD4 antibodies all obtained from Pharmingen (La Jolla, CA, USA). Purified monoclonal rat ant-mouse CD5 (clone 53–7.3) was generously provided by Dr L.A. Herzenberg (Stanford University, Stanford, CA, USA) or purchased from Pharmingen. Rat anti-mouse IgG, goat ant-rat IgG and streptavidin and avidin were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Anti-phosphotyrosine monoclonal antibody 4G10, protein A and sheep anti-mouse antibody conjugated to horseradish peroxidase were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). Rabbit polyclonal anti-SHP-1 antibody recognizing the tandem SH2 domains of SHP-1 was generated in the laboratory as previously described (27). Polyclonal anti-CD5 (R5) rabbit serum to the highly conserved peptide sequence, TASHVDNEYSQPPR, in the CD5 cytoplasmic domain was generously provided by Drs Greg Appleyard and Bruce Wilkie (Department of Pathobiology, University of Guelph, Guelph, ON, Canada). Chemicals used for immunoblotting/immunoprecipitation were purchased from Sigma Chemical Corp. (St. Louis, MA, USA).

Single-cell suspensions of thymocytes (3×10⁷/condition) obtained from wild-type or me^v mice were resuspended in 150 µl of phosphate-buffered saline (PBS) and incubated for 30 min at 4°C in the presence or absence of 2.5 µg biotin-conjugated anti-mouse TCR antibody. Following several washes to remove any unbound antibody, the cells were resuspended in 40 µl PBS and incubated at 37°C for various time points with 50 µg/ml avidin or 25 µg/ml streptavidin. The cells were then pelleted by 30-sec centrifugation and lysed in 400 µl of cold lysis buffer supplemented with protease inhibitors [1% Nonidet P-40, 50 mM HEPES (pH 7.2), 150 mM NaCl, 50 µM NaF, 50 µM 0-phosphate, 50 µM ZnCl₂, 2 mM EDTA, 2 mM Na₃VO₄, 2 mM PMSF, 10 µg/ml leupeptin and 10 µg/ml aprotinin] for 30 min on ice. Nuclei and unlysed cells were removed by centrifugation at 14,000 × g for 10 min at ~4°C and protein concentrations were determined by means of the bicinchoninic acid (BCA) assay (Pierce Biochemicals, Rockford, IL, USA). Equal amounts of lysates (300–500 µg) were incubated for 2 h at 4°C with the appropriate antibody (anti-CD5, or anti-IgG isotype control) and 30 µl of 50% protein G Sepharose beads (Pharmacia, Toronto, Canada) was added and the samples agitated at 4°C for an additional hour. Immunocomplexes were collected by centrifugation and washed five times in 1 ml of cold lysis buffer and then boiled for 5 min in reduced SDS-gel sample buffer. The samples were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). Blots were blocked for ≥1 h in TBS-T containing 3% gelatin or 5% non-fat milk, and incubated for 1 h at room temperature with optimal concentrations of the primary antibody [anti-CD5 (R5), anti-SHP-1 or anti-phosphotyrosine 4G10]. The blots were then incubated with the appropriate secondary antibody conjugated to horseradish peroxidase and subjected to enhanced chemiluminescence (ECL; Amersham Corp., Arlington Heights, IL, USA). Where indicated, the immunoblots were stripped and reprobed with anti-CD5 or anti-SHP-1 antibody.

For analysis of CD5-associated phosphatase activity, anti-CD5 immunoprecipitates were prepared. Briefly, wild-type and me^v-derived thymocytes (1×10⁸), unstimulated or stimulated with biotinylated antibodies to anti-TCR and anti-CD4 at 10 µg/ml plus streptavidin at 25 µg/ml for 5 min at 37°C, were lysed into 400 µl of cold lysis buffer, as described earlier, without sodium orthovanadate. An equal amount of lysates was then subjected to CD5 immunoprecipitation using anti-CD5 (53–7.3) antibody or rat anti-mouse IgG isotype control. Immunoprecipitates were incubated at 37°C for 90 min with 1 mM phosphopeptide RRLIEDAEY-pAARG (Upstate Biotechnology, Lake Placid, NY, USA) in 10 mM Tris-HCl (pH 7.4) phosphatase buffer. Free phosphate detection was carried out as specified by the manufacturer. To standardize for the non-specific phosphatase the activity associated with IgG, relative phosphatase activity was determined by dividing the measured absorbance values with those from unstimulated IgG negative controls.

Thymocytes (5×10⁶ cell/ml) were labeled with Indo-1 (5 µM) and incubated at 37°C in the dark for 30 min. Thymocytes were washed, resuspended in RPMI-1640 containing 2% fetal bovine serum (FBS) and 10 mM HEPES (pH 7.4), and incubated on ice with biotinylated anti-TCR alone (2.0 or 0.3 µg), or in combination with either biotinylated (2.5 µg) or non-biotinylated anti-CD5 (2.5 µg) for 30 min. After washing, the cells were resuspended in RPMI-1640 buffer at a concentration of ×10⁷ cells/ml and stimulated with streptavidin (5 µg/ml). Goat anti-rat antibody (16 µg) was also used to crosslink samples containing non-biotinylated anti-CD5 antibody. The cells were analyzed on a flow cytometer and calcium levels were detected by analysis of the Indo-1 violet-blue fluorescence ratio.

Cells (5×10⁶/sample) were resuspended in 100 µl immunofluorescent staining buffer (PBS containing 1% BSA and 0.05% sodium azide) and incubated with the appropriate fluorochrome-conjugated antibodies (FITC-conjugated anti-CD8 or PE-conjugated anti-CD4) for 30 min at 4°C. For CD5 staining, the cells were incubated with biotinylated anti-CD5 for 30 min at 4°C, washed and then incubated with FITC-conjugated streptavidin. Stained cells were analyzed using a FACScan flow cytometer with CellQuest software (Becton-Dickinson, San Diego, CA, USA).

We previously demonstrated the ability of SHP-1 to associate with CD5 in activated mouse thymocytes, association that was somewhat enhanced following CD5 tyrosine phosphorylation, suggesting that the interaction may be SH2 domain is mediated (28). Since then there have been several studies supporting this contention. Bikah et al have reported observing CD5 association with SHP-1 in resting but not activated T cells and in BKS-2 B lymphoma cells (29). Sen et al have also reported that phosphorylated CD5 is associated with SHP-1 in B-1 cells (30). Recently, Perez-Villar et al have also shown a constitutive association of CD5 with SHP-1 in Jurka T cells and PHA-expanded T lymphoblasts, which increased following TCR stimulation (21). In addition, mapped Tyr-378 in the ITIM-like sequence of CD5 was found to be essential for SHP-1 binding to CD5 in Jurkat T cells (21). However, the association of SHP-1 with CD5 as well as the SH2 mechanism for mediating SHP-1 interaction has been previously addressed (16,31,32). We therefore examined the association profile of SHP-1 and CD5 in resting and TCR-stimulated wild-type thymocytes. Immunoprecipitates of CD5 when immunoblotted with SHP-1 revealed a strong association with SHP-1 following TCR activation (Fig. 1). The association of SHP-1 with CD5 occured rapidly following this stimulation and directly correlated to the level of CD5 phosphorylation (interaction decreased as the level of CD5 was reduced). Thus, association of SHP-1 with CD5 in thymocytes was not constitutive but rather dependent on TCR activation and the level of CD5 tyrosine phosphorylation.

Given that the profile of SHP-1 binding to CD5 was found to be dependent on the level of CD5 phosphorylation (Fig. 1), together with recent data demonstrating a correlation between the phosphatase activity of SHP-1 and the status of CD5 phosphorylation (involving SHP-1 as the phosphatase responsible for CD5 dephosphorylation) (22), we addressed whether CD5 is a substrate for SHP-1 activity. Previously it was shown that many molecules representing direct or indirect potential substrates for SHP-1 activity, were either constitutively hyper-phosphorylated and/or exhibited enhanced and prolonged activation-induced tyrosine phosphorylation profiles in SHP-1 deficient compared to wild-type cells (28). Of note, the tyrosine phosphorylation status of CD5 in the resting and TCR-stimulated thymocytes from normal compared to SHP-1 deficient (me^v) mice was unchanged (Fig. 2). The lack of constitutive and hyper-phosphorylated CD5 in the context of SHP-1-deficiency, together with a normal phosphorylation profile following TCR stimulation, strongly argues against a role for SHP-1 in the dephosphorylation of CD5.

CD5 expression is closely regulated throughout T-cell development. CD5 is expressed at low levels on immature CD4⁻CD8⁻ (DN) thymocytes and becomes increasingly expressed on CD4⁺CD8⁺ (DP) and single-positive (SP) CD4⁺ or CD8⁺ thymocytes (3), with the mature peripheral T cells expressing high levels (33). Recently, Azzam et al showed that CD5 expression was regulated by the strength and avidity of TCR signals (3). The lack of any observable differences in the thymic CD5 tyrosine phosphorylation profiles in me^v mice led us to examine thymic CD5 surface expression levels. In contrast to the findings in peripheral T cells isolated from SHP-1-deficient mice, which express elevated basal levels of CD5 (34), we detected equivalent CD5 surface levels in me^v compared to wild-type thymocytes (Fig. 3). Therefore, the lack of SHP-1 did not affect levels of CD5 surface expression during early T-cell development.

Previous studies in the human T-cell Jurkat lymphoma cell line demonstrated a moderate tyrosine phosphatase activity associated with CD5 immunoprecipitates (21). This CD5-associated PTP activity was shown to substantially increase following TCR-stimulation (21). Perez-Villar et al elucidated the molecular basis for this CD5-associated phosphatase activity and attributed this function to the interaction of CD5 with SHP-1 but not SHP-2 (21). Supporting this view are recent data by Sen et al, examining CD5-associated PTP activity in wild-type B-1 cells (30). Sen et al also found CD5 to be associated with PTP activity, which could be completely eliminated by the prior immunodepletion of SHP-1 but not SHP-2, supporting the hypothesis that the phosphatase activity associated with CD5 was derived mainly from SHP-1 (30). Given that CD5 is not a direct substrate for SHP-1 action, the functional association of SHP-1 with CD5 supports a model whereby the negative regulatory function of CD5 is mediated by the recruitment of SHP-1 into the antigen-receptor complex (21,28–30). By contrast, Gary-Gouy et al suggest that the effect of CD5, at least, on BCR signaling was independent of SHP-1. In that study, the authors did not show any physical interaction with SHP-1, SHP-2 or SHIP concluding that other inhibitory phosphatases may exist that carry out the negative function of CD5 (31). Given those findings, we examined and compared CD5-associated PTP activity from wild-type and SHP-1-deficient (me^v) thymocytes. The results showed that SHP-1-deficient thymocytes also retained wild-type compared to CD5-associated PTP activity (Fig. 4). This PTP activity (as in the case of wild-type) increased following TCR stimulation. Therefore, our findings suggested that there is a redundancy with respect to SHP-1 function, or more likely, that there are other phosphatases besides SHP-1 that are responsible for mediating the negative modulatory effects of CD5 in thymocytes.

Biochemical observations thus far in SHP-1-deficient thymocytes suggest that CD5 does not require SHP-1 activity, as there were no differences in the CD5 phosphorylation status, CD5 surface expression or CD5-associated PTP activity. However, these findings still do not exclude a possible requirement of SHP-1 in CD5 signaling and function. One of the earliest biochemical events to occur following TCR stimulation is the enhanced mobilization of calcium (35–38). Studies examining CD5 function have identified an important role for CD5 signaling in the calcium pathway (11,15). Peripheral T-cell co-stimulation through anti-CD5 antibodies has been shown to increase TCR/CD3-induced intracellular Ca²⁺ concentration (39), and this increase is entirely due to an influx of extracellular calcium (40-42). By contrast, CD5 acts as a negative regulator of antigen receptor-mediated calcium mobilization in thymocytes and B-1 cells (15,29) since thymocytes and B-1 cells from CD5-deficient mice exhibit a moderate increase in Ca²⁺ mobilization following antigen receptor activation (15,29). The differences suggest that CD5 possesses a dual function, providing either positive or negative modulatory signals depending on the cell type and maturational stage (43). However, the calcium mobilization profiles associated with CD5 and antigen receptor stimulation depend on the method of antibody crosslinking. In particular, co-crosslinking the antigen-receptor with CD5 compared to a separate crosslinking of the two receptors generates a qualitatively different calcium mobilization profile that can be explained in a manner consistent with a negative regulatory function for CD5 (21,30,31).

Therefore, to examine the functional contribution of SHP-1 in CD5/TCR-mediated Ca²⁺ mobilization, we produced a stimulation protocol based on those studies (Fig. 5). As shown in the top panel of Fig. 5, TCR crosslinking induces a rapid recruitment of CD5 to the TCR/CD3 complex, as has already been demonstrated by co-capping studies (44). Similarly, the coligation of TCR and CD5 resulted in a similar or increased recruitment of CD5 to the TCR complex (Fig. 5, middle panel), as this treatment has been reported to reduce the Ca²⁺ influx when compared to the ligation of TCR/CD3 alone (21). By contrast, when the TCR and CD5 were separately cross linked (Fig. 5, bottom panel) the recruitment of CD5 to the TCR complex was inhibited or significantly decreased (44). In keeping with the negative role of CD5, separate crosslinking of the antigen receptor and CD5 has been demonstrated to increase the Ca²⁺ mobilization and proliferative response in B-1 cells, and this has been explained to occur as a result of CD5 sequestration from the BCR (29,30). We hypothesized that if SHP-1 is required in the CD5-mediated calcium mobilization pathway, then the lack of SHP-1 activity may significantly affect the calcium influx profiles in thymocytes from SHP-1-deficient me^v compared to wild-type mice. As expected, the co-ligation of TCR and CD5 resulted in a slightly decreased or unchanged calcium influx in wild-type thymocytes when compared to the ligation of TCR alone (Figs. 6A and 5). Similarly, the inhibitory effects of CD5 were intact and readily observable in the me^v mice (Fig. 6B). Although, the initial upswing in calcium influx occurred at relatively the same time after the addition of the crosslinking agent (~40 sec), there was a definite decrease in the slope and amount of Ca²⁺ influx in thymocytes from SHP-1-deficient mice following CD5 and TCR coligation versus TCR ligation alone (Fig. 6B), suggesting that the CD5 receptor retains functionality even in the absence of SHP-1 activity. We also examined, whether the calcium mobilization in thymocytes could be enhanced by sequestering or inhibiting the recruitment of CD5 to the antigen receptor complex (Fig. 5), as previously demonstrated by Sen et al in B-1 cells (30). The separate crosslinking of the TCR and CD5 resulted in a moderate increase in calcium mobilization, as witnessed by a definite change in the slope and amount of Ca²⁺ influx, in the wild-type and me^v thymocytes when compared to the ligation of TCR alone (Fig. 6C and D). Our results support a negative regulatory role for CD5 in thymocytes, which is consistent with the observations made in CD5-deficient thymocytes (15), and addresses the involvement of SHP-1 in CD5 signaling.

A selection in thymocytes from CD5-deficient, α/β-TCR transgenic mice has been shown to be altered in a manner consistent with enhanced TCR signaling (15). As another tool for exploring the functional and physiological role of CD5 in thymocyte development, Azzam et al (3) generated transgenic mice in which there is a T cell-specific, CD2 promoter/enhancer driven CD5 overexpression (CD5OE). CD5 surface expression in thymocytes from CD5OE mice have an ~2.5-fold increase in CD5 expression as compared to wild-type mice (Fig. 7). This increase in CD5 surface expression does not alter the kinetics of TCR-mediated CD5 phosphorylation, except for the observed increase in intensity of CD5 phosphorylation which is explained by the increase in CD5 protein levels (Fig. 7B). This observation suggests that the increased CD5 in the CD5OE mice is fully functional and participates in the CD5 signaling pathway. The CD5OE mice were previously bred onto the H-Y TCR transgenic background and shown to manifest a decrease in the positive and negative selection, thus corroborating the inhibitory role for CD5 in modulating signaling thresholds in T-cell selection (26).

One of the physiologically relevant functions of CD5 is to modulate TCR-mediated signals involved in T-cell development (3). Thus, we examined the ability of CD5 to modulate the selection process in the absence of SHP-1 activity to establish the physiological relevance of SHP-1 in CD5 signaling. In addition to other authors, we previously identified a role for SHP-1 in raising the signaling threshold required for both positive and negative selection (26,34). Since CD5 and SHP-1 downregulate signals involved in T-cell selection, we hypothesized that CD5 overexpression would not be able to downregulate selection in the absence of SHP-1 activity, if SHP-1 is essential for CD5 function.

As shown earlier [Zhang et al (26), unpublished data], the inhibitory effect of CD5 overexpression (CD5OE) on positive selection was readily detected in female thymocytes from H-Y TCR/CD5OE compared to HY-TCR transgenic mice (Fig. 8, upper panel). CD5 overexpression results in an increase in the representation of DP cells as well as a decrease in the number of SP CD8⁺ cells. The overexpression of CD5 in SHP-1-deficient me^v H-Y TCR transgenic mice also increased the size of the DP population and decreased the number of SP CD8⁺ cells compared to me^v H-Y TCR transgenic mice (Fig. 8, lower panel). Therefore, the lack of SHP-1 activity does not impact on the ability of CD5 to increase the threshold for TCR signaling. This finding provides evidence in favor of an SHP-1-independent CD5 receptor capable of downregulating positive selection.

As previously shown (26), the inhibitory effect of CD5 overexpression (CD5OE) on negative selection was also readily detected in the current study, as revealed by the increased representation of DP and SP CD8⁺ cells in the thymuses of H-Y TCR/CD5 compared to H-Y TCR mice (Fig. 9, upper panel). Nevertheless, we previously showed that the introduction of the me^v mutation nullifies the effects of CD5 overexpression, suggesting that SHP-1 deficiency is able to counteract the inhibitory effects of CD5 overexpression on negative selection (26), and raising the possibility that SHP-1 activity is required for CD5 to realize its inhibitory effects on negative selection. Careful re-examination of these data in light of the findings presented above for positive selection, suggests that the lack of any obvious and marked decrease in negative selection conferred by CD5 overexpression is due to the inability of CD5 to compensate for the increased signaling (and negative selection) inherent in SHP-1 deficiency, rather than due to a lack of CD5 function. Supporting this hypothesis is the finding that negative selection in the thymi of me^v H-Y TCR/CD5OE transgenic mice reveal a slight, albeit reproducible increase in the DP and SP CD4^+hi populations when compared with me^v H-Y TCR mice (Fig. 9 lower panel). The rescue of SP CD4⁺ cells observed in the me^v H-Y TCR mice is noteworthy, particularly in light of the study by Page, which found that CD5 was able to block MHC class II-dependent negative selection of CD4⁺ cells rather than CD8⁺ cells. The findings suggest that the effects of CD5 may be less profound on MHC class I-dependent negative selection (45). It is therefore possible that the enhanced TCR signaling imbued by SHP-1 deficiency (26), results in the initial rescue of a small population of CD4⁺ cells (even in the absence of MHC class II antigen presentation) manifesting the appropriate signals necessary for positive selection. Subsequently, a majority of these cells undergo negative selection for the same reasons as outlined above and it is only with CD5 overexpression that this CD4⁺ population is rescued in the me^v H-Y TCR male mice (Fig. 9). One explanation for this result is that when the analysis is restricted to a clonotypic TCR, a partial reduction in signaling ability readily decreases the signals from the positive selection interactions below the required threshold. By contrast, when the negative selection stimulus is strong enough, a partial reduction in signaling ability may not be sufficient to interfere with this process. Thus, CD5 effects on positive and negative T-cell selection are realized independently of SHP-1.