CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-γ (PLC-γ) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-γ, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8 cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.

CD5, previously called Lyt-1, is a transmembrane protein that is expressed on almost all mature T cells as well as on CD4+CD8+ (double-positive (DP)3) and CD4+CD8/CD4CD8+ (single-positive (SP)) thymocytes (1, 2). Studies on T cell activation have demonstrated that CD5 is not a mere pan-T cell marker, but functions as a costimulatory molecule (3, 4, 5). The activation of resting mature T cells requires two independent signals (6, 7, 8, 9). The first signal stems from the TCR, which is stimulated with Ag peptides plus MHC molecules of APC. The second signal, also called a costimulatory signal, is provided by other receptor-ligand interactions between T cells and APC. Intensive research has characterized the T cell molecule CD28 as the principal receptor that generates costimulatory signals in mature T cells for their activation (7, 8, 9). Multiple molecules on the T cell (3, 4, 5, 10, 11, 12) have also been shown to costimulate resting T cells; in the presence of suboptimal doses of anti-CD3, each of the mAbs against these molecules costimulated resting T cells as potently as anti-CD28 mAb (12, 13). CD5 was included in the group of these costimulatory molecules other than CD28.

The function of CD5 in developing thymocytes was investigated using CD5-deficient (CD5−/−) mice (14). The results showed that the absence of CD5 rendered thymocytes hyper-responsive to TCR stimulation in vitro, as observed by enhanced Ca2+ influx, and that by acting as a negative regulator of TCR-mediated signal transduction, CD5 influences thymocyte development in terms of thymic selection (14). These observations appear to be discordant with the results obtained for the function of CD5 on mature T cells (3, 13). Recent studies on TCR-mediated signal transduction have shown that TCR stimulation generates two branches of signals (15, 16, 17, 18) following the tyrosine phosphorylation of an adaptor protein, LAT (linker for activation of T cells) (19, 20). Ca2+ influx following PLC-γ activation is on one branch, and the Ras pathway leading to ERK activation is on the other branch (15, 16, 17, 18). The studies using CD5−/− mice revealed the effect of CD5 absence on the first branch, i.e., Ca2+ influx. However, it remains unclear whether CD5 influences the second branch of TCR-mediated signal transduction in the thymus.

The present study showed that coligation of CD3 and CD5 in thymocytes resulted in the reduction of Ca2+ influx compared with ligation of CD3 alone, which is consistent with the observation made in CD5−/− thymocytes (14). However, the same treatment induced an enhancement of ERK activation. Consistent with the recent observation (21) that the ERK pathway favors the differentiation of CD4+CD8+ thymocytes into CD4+CD8 cells, coligation of CD3 and CD5 led to enhanced differentiation into the CD4 lineage. The results further showed the mechanism by which ERK is required for this differentiation; ERK activated by CD3/CD5 coligation functions to support the survival of differentiating cells by enhancing Bcl-2 induction rather than to promote the differentiation process. These results indicate that CD5 on developing thymocytes exerts opposite effects on two branches of TCR-mediated signal transduction: reduction of Ca2+ influx in the first branch and enhancement of the ERK pathway in the second. Thus, the results could provide important implications for the TCR-mediated events of thymocyte development involving thymic selection.

BALB/c mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan) and used at 5–8 wk of age.

Anti-CD3 (145-2C11) (22) and anti-CD5 (53-7–313) (1) mAbs were purified from culture supernatants of hybridoma cells. Anti-LAT and anti-PLC-γ Abs were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-Grb2 Ab was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pan-ERK MAP kinase (MAPK) and anti-phospho-ERK Abs were obtained from Transduction Laboratories (Lexington, KY) and New England Biolabs (Beverly, MA), respectively. Biotinylated anti-CD28, biotinylated anti-CD2, and anti-Bcl-2 mAbs were purchased from PharMingen (San Diego, CA). Anti-CD3 and anti-CD5 mAbs were biotinylated in our laboratory. Anti-phosphotyrosine mAb (4G10) was obtained from Upstate Biotechnology. A MEK inhibitor, PD98059, and a p38 MAPK inhibitor, SB203580, were purchased from New England Biolabs and Calbiochem (La Jolla, CA), respectively.

CD4+CD8+ (DP) thymocytes were purified by two cycles of panning on dishes coated with anti-CD8 mAb (83-12-5, anti-Lyt 2.2 mAb) as previously described (23). In some experiments a purified DP population was also prepared by positive selection. Thymocytes were stained with anti-CD8 (2.43) mAb followed by labeling with superparamagnetic microbeads conjugated to mouse anti-rat Ig (Miltenyi Biotec, Sunnyvale, CA). Labeled cells were separated from unlabeled cells by magnetic cell sorting using the MiniMACS (Miltenyi Biotec) according to the technology described previously in detail (24). The magnetic cells were retained in a MiniMACS column inserted into the MiniMACS magnet. Labeled cells were eluted after the column was removed from the magnet. Lymph node cells were depleted of B cells and Ia+ APC by immunomagnetic negative selection to obtain mature T cell populations as previously described (12, 13). The purity of the resulting populations was checked by flow cytometry with anti-CD4/CD8 for DP populations or with anti-CD3 for mature T cell populations. The contaminations of CD4-SP cells were ∼5 and 0.6% in the panning method and positive selection, respectively. Mature T cells prepared from lymph node cells were >98% CD3 positive.

Thymocytes or mature T cells were washed and resuspended at a concentration of 1 × 107/ml in RPMI 1640 medium containing 5 μg/ml biotinylated anti-CD3 mAb and 5 μg/ml biotinylated mAb against various T cell molecules, and cells were incubated for 30 min at 4°C. The cells were washed with RPMI 1640 and incubated with 20 μg/ml streptavidin for 5–15 min at 37°C. After stimulation, cells were pelleted and lysed with RIPA buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1% Nonidet P-40, 1% deoxycholate, 1% SDS, and 1 mM Na3VO4). After a 30-min incubation at 4°C, the insoluble material was removed by centrifugation at 15,000 rpm. Postnuclear supernatants were eluted by SDS sample buffer.

Thymocytes or mature T cells were lysed with lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1% Nonidet P-40, 1% N-octyl-β-d-glucoside, and 1 mM Na3VO4). After a 30-min incubation at 4°C, the lysates were immunoprecipitated by adding 10 μg/ml of the indicated mAbs plus either protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) or protein G-Sepharose beads (Pharmacia Biotech). After a 2-h incubation at 4°C under constant agitation, the beads were washed four times in lysis buffer containing only 0.5% N-octyl-β-d-glucoside.

Proteins were separated by SDS-PAGE in reducing conditions and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) in 25 mM Tris, 192 mM glycine, and 20% methanol, and the membrane was blocked overnight in Tris-buffered saline with 0.05% Tween 20 containing 0.2% OVA. Specific molecules and phosphorylated proteins were probed by the corresponding Abs or anti-phosphotyrosine mAb (4G10), respectively, followed by HRP-conjugated protein A (Amersham, Aylesbury, U.K.) or HRP-conjugated sheep anti-mouse IgG (Amersham). The proteins were revealed by enhanced chemiluminescence (Amersham).

Thymocytes or mature (lymph node) T cells were suspended at 1 × 107/ml in 2% FCS/PBS containing 3 μM fura-2/AM (DOJINDO, Kumamoto, Japan) and incubated at 37°C for 30 min. Fura-2-loaded cells were pelleted and washed twice, then resuspended at 5 × 106/ml in PBS containing 0.5 mM CaCl2. The calcium response was initiated by biotinylated anti-CD3 mAb (0.25 or 2.5 μg/ml) and biotinylated mAb (5 μg/ml) against various T cell molecules plus streptavidin (20 μg/ml). Cells were analyzed for free calcium ion by measurement of fura-2 fluorescence emission on a fluorescence photometer (F-3000; Hitachi, Tokyo, Japan).

Thymocytes (1 × 106/well) were cultured in 24-well culture plates in 1 ml of RPMI 1640 medium supplemented with 10% FCS and 2-ME and stimulated with biotinylated anti-CD3 (2.5 μg/ml) alone or together with biotinylated anti-CD5 mAb (2.5 μg/ml) followed by cross-linking with streptavidin. Cells were harvested after 12–48 h.

The surface expression of CD8, CD4, or CD69 molecules was analyzed by direct staining with PE-labeled anti-CD8, allophycocyanin-labeled anti-CD4, and FITC-labeled anti-CD69 mAbs (PharMingen). In some experiments cells were stained with annexin V (PharMingen). The stained cells were analyzed by FACSCalibur (Becton Dickinson, Mountain View, CA). Among harvested cells (∼10,000 cells/group), viable cells were gated using forward and side scatter. The gated cells were analyzed for the expression of CD4, CD8, or CD69.

Pronase treatment was performed as previously described (25). Briefly, thymocytes were resuspended at a concentration of 5 × 106/ml in PBS containing 100 μg/ml Pronase (Calbiochem-Novabiochem, San Diego, CA) for 15 min at 37°C and washed three times with complete culture medium.

We examined the effect of CD5 engagement in thymocytes on the activation of various molecules involved in TCR-mediated signal transduction. Unfractionated thymocytes from BALB/c mice were stimulated with biotinylated anti-CD3 (5.0 μg/ml) plus biotinylated anti-CD5 (5.0 μg/ml) followed by cross-linking with streptavidin for 5 min at 37°C. Cells were lysed, and the lysate was subjected to SDS-PAGE and analyzed by anti-phosphotyrosine immunoblotting (Fig. 1). The results show that coligation of CD3 and CD5 induces enhanced levels of tyrosine phosphorylation in several molecules, including the ∼36-kDa protein, compared with those observed by stimulation with anti-CD3 alone (Fig. 1).

FIGURE 1.

Effect of CD5 engagement on TCR-triggered tyrosine phosphorylation of signaling molecules in thymocytes. Unfractionated BALB/c thymocytes were stimulated with biotinylated anti-CD3 and/or biotinylated anti-CD5 mAb (5.0 μg/ml each) followed by cross-linking with 20 μg/ml streptavidin for 5 min at 37°C. The lysates from these stimulated cells were resolved on SDS-PAGE and immunoblotted with anti-phosphotyrosine mAb.

FIGURE 1.

Effect of CD5 engagement on TCR-triggered tyrosine phosphorylation of signaling molecules in thymocytes. Unfractionated BALB/c thymocytes were stimulated with biotinylated anti-CD3 and/or biotinylated anti-CD5 mAb (5.0 μg/ml each) followed by cross-linking with 20 μg/ml streptavidin for 5 min at 37°C. The lysates from these stimulated cells were resolved on SDS-PAGE and immunoblotted with anti-phosphotyrosine mAb.

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LAT plays an exclusive role in generating two branches of signals following TCR stimulation (15, 16, 17, 18). To determine whether the most strikingly activated protein in Fig. 1 (∼36-kDa protein) represents LAT, the lysates from thymocytes stimulated with anti-CD3 and/or anti-CD5 were immunoprecipitated with anti-LAT Ab. Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine mAb. As shown in Fig. 2 A, tyrosine phosphorylation of LAT was strikingly enhanced by coligation of CD3 and CD5 compared with ligation of CD3 alone. The same blot was stripped of detecting Ab and reprobed with anti-LAT to confirm equal loading of LAT protein on each lane.

FIGURE 2.

Enhanced LAT phosphorylation and association of PLC-γ/Grb2 with LAT following coligation of CD3 and CD5. The lysates from anti-CD3- and/or anti-CD5-stimulated thymocytes were immunoprecipitated with anti-LAT Ab. Immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb and then stripped and reblotted with anti-LAT Ab (A). A portion of the same immunoprecipitates as those used in A was immunoblotted with anti-PLC-γ or anti-Grb2 Ab (B).

FIGURE 2.

Enhanced LAT phosphorylation and association of PLC-γ/Grb2 with LAT following coligation of CD3 and CD5. The lysates from anti-CD3- and/or anti-CD5-stimulated thymocytes were immunoprecipitated with anti-LAT Ab. Immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb and then stripped and reblotted with anti-LAT Ab (A). A portion of the same immunoprecipitates as those used in A was immunoblotted with anti-PLC-γ or anti-Grb2 Ab (B).

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Recent studies have shown that after TCR stimulation, phosphorylated LAT associates with various molecules, including PLC-γ and Grb2 (19, 26, 27). The association of these molecules with LAT was compared between stimulation with anti-CD3 and anti-CD3/anti-CD5. Portions of the same anti-LAT immunoprecipitates as those used in Fig. 2,A were analyzed by immunoblotting with anti-PLC-γ and anti-Grb2 Ab. Fig. 2 B shows enhanced association of LAT with PLC-γ and Grb2 following coligation of CD3 and CD5. These results indicate that CD5 costimulation of thymocytes results in the up-regulation of LAT phosphorylation and the enhanced recruitment of PLC-γ and Grb2 that are required for downstream TCR signaling events.

Although the kinase(s) responsible for phosphorylation of PLC-γ is unknown, the recruitment of PLC-γ to LAT appears to be required for PLC-γ phosphorylation (17). We examined the phosphorylation levels of PLC-γ in thymocytes following stimulation with anti-CD3 alone or anti-CD3 plus anti-CD5. As shown in Fig. 3,A, coligation of CD3 and CD5 in thymocytes induced higher levels of PLC-γ phosphorylation than CD3 ligation alone. Fig. 3 B shows that coligation of CD3 and CD5 induces enhanced PLC-γ phosphorylation in thymocytes and mature T cells with similar time courses.

FIGURE 3.

Enhanced PLC-γ phosphorylation in thymocytes and mature T cells following coligation of CD3 and CD5. A, The lysates from anti-CD3- and/or anti-CD5-stimulated thymocytes were immunoprecipitated with anti-PLC-γ Ab. Immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb and then reprobed with anti-PLC-γ Ab. B, Thymocytes and mature T cells were stimulated for various times with anti-CD3 alone or with anti-CD5.

FIGURE 3.

Enhanced PLC-γ phosphorylation in thymocytes and mature T cells following coligation of CD3 and CD5. A, The lysates from anti-CD3- and/or anti-CD5-stimulated thymocytes were immunoprecipitated with anti-PLC-γ Ab. Immunoprecipitates were immunoblotted with anti-phosphotyrosine mAb and then reprobed with anti-PLC-γ Ab. B, Thymocytes and mature T cells were stimulated for various times with anti-CD3 alone or with anti-CD5.

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We next examined whether increased phosphorylation of PLC-γ leads to the elevation of Ca2+ influx in mature T cells as well as thymocytes. Our previous results showed that costimulation of resting mature T cells with anti-CD5 plus suboptimal doses of anti-CD3 results in a striking enhancement of T cell activation compared with that induced with anti-CD3 alone (13). Consistent with this, coligation of mature T cells with anti-CD5 and a suboptimal dose (0.25 μg/ml) of anti-CD3 induced high levels of Ca2+ mobilization under conditions in which stimulation with anti-CD3 (Fig. 4) or anti-CD5 (data not shown) mAb alone mobilized Ca2+ to a lesser or marginal extent, respectively. A higher dose (2.5 μg/ml) of anti-CD3 alone induced potent Ca2+ influx without requiring costimulation. While thymocytes stimulated with anti-CD3 at a lower (0.25 μg/ml) dose failed to display Ca2+ responses (data not shown), stimulation with a higher (2.5 μg/ml) dose of anti-CD3 induced an appreciable level of Ca2+ influx. In contrast to the effect observed in mature T cells, CD5 costimulation in thymocytes apparently reduced Ca2+ influx (Fig. 4). Thus, while CD5 costimulation up-regulates the phosphorylation of LAT and PLC-γ, a branch of TCR-mediated signaling as observed by Ca2+ influx is unexpectedly down-regulated.

FIGURE 4.

Coligation of CD3 and CD5 results in the inhibition of Ca2+ influx in thymocytes, but not in lymph node T cells. Intracellular free calcium levels in fura-2-loaded cells were monitored by a spectrophotometer after stimulation with 2.5 (upper panels) or 0.25 (lower panels) μg/ml biotinylated anti-CD3 and/or with 5 μg/ml biotinylated anti-CD5 followed by streptavidin (20 μg/ml). The first and second arrows in each panel indicate the time points for the addition of biotinylated mAbs and streptavidin, respectively.

FIGURE 4.

Coligation of CD3 and CD5 results in the inhibition of Ca2+ influx in thymocytes, but not in lymph node T cells. Intracellular free calcium levels in fura-2-loaded cells were monitored by a spectrophotometer after stimulation with 2.5 (upper panels) or 0.25 (lower panels) μg/ml biotinylated anti-CD3 and/or with 5 μg/ml biotinylated anti-CD5 followed by streptavidin (20 μg/ml). The first and second arrows in each panel indicate the time points for the addition of biotinylated mAbs and streptavidin, respectively.

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We examined whether enhanced activation of LAT by CD3-CD5 coligation leads to the up-regulation of the ERK pathway. As shown in Fig. 5 A, coligation of CD3 and CD5 in thymocytes resulted in much higher levels of ERK activation than CD3 ligation alone, as detected by immunoblotting of thymocyte lysates with anti-phospho-ERK MAPK Ab. Thus, CD5 costimulation in thymocytes down-regulates one branch of TCR signaling (Ca2+ influx), but at the same time induces the up-regulation of another branch of TCR signaling (ERK pathway).

FIGURE 5.

Enhanced activation of ERK following coligation of CD3 and CD5. Thymocytes were stimulated with anti-CD3 and/or anti-CD5 for 5 min (A) or with anti-CD3 with the indicated mAbs for 5–15 min (B). The lysates from these stimulated cells were immunoblotted with anti-phospho ERK Ab and then reprobed with anti-ERK Ab.

FIGURE 5.

Enhanced activation of ERK following coligation of CD3 and CD5. Thymocytes were stimulated with anti-CD3 and/or anti-CD5 for 5 min (A) or with anti-CD3 with the indicated mAbs for 5–15 min (B). The lysates from these stimulated cells were immunoblotted with anti-phospho ERK Ab and then reprobed with anti-ERK Ab.

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We compared the effects of coligation of CD5 and other costimulatory molecules on two branches of TCR signaling (Ca2+ influx and ERK activation). Two costimulatory molecules were chosen: one is CD28, a principal costimulatory molecule, and the other is CD2, a representative among non-CD28 costimulatory molecules. The results in Fig. 5 B show that costimulation of all three molecules (CD5, CD2, and CD28) resulted in enhanced ERK activation at an early time point (5 min after costimulation). However, it should be noted that the ERK activation induced by CD5 costimulation was observed for as long as 15 min with a gradual decline, whereas that induced by CD2 or CD28 costimulation declined rapidly.

Fig. 6 shows differential effects of CD5 vs. CD2/CD28 costimulation on Ca2+ influx. CD5 costimulation again reduced Ca2+ influx. In contrast, such a reduction was not induced by CD2 or CD28 coligation. Particularly CD2 coengagement led to the peak of Ca2+ influx at an earlier time point than that in anti-CD3 ligation alone. Thus, the results indicated that CD5 costimulation induces a unique pattern of TCR signaling in thymocytes.

FIGURE 6.

Differential effects of CD5 vs. CD2 and CD28 costimulation on Ca2+ influx in thymocytes. Thymocytes were stimulated with biotinylated anti-CD3 alone (2.5 μg/ml) or together with biotinylated anti-CD5, anti-CD2, or anti-CD28 mAb (5 μg/ml) followed by streptavidin.

FIGURE 6.

Differential effects of CD5 vs. CD2 and CD28 costimulation on Ca2+ influx in thymocytes. Thymocytes were stimulated with biotinylated anti-CD3 alone (2.5 μg/ml) or together with biotinylated anti-CD5, anti-CD2, or anti-CD28 mAb (5 μg/ml) followed by streptavidin.

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A recent study showed that simultaneous stimulation of TCR and various surface molecules, such as CD2, on DP thymocytes with culture plate-immobilized mAb signals responsive thymocytes to differentiate into CD4+CD8 cells in the absence of thymic stromal cells (28). We examined whether such differentiation is also induced by costimulation with biotinylated anti-CD3 and biotinylated anti-CD5 followed by cross-linking with streptavidin. A DP thymocyte population prepared by panning with anti-CD8 was unstimulated or stimulated with anti-CD3 alone (2.5 μg/ml) or together with anti-CD5 (2.5 μg/ml) for 48 h. As shown in Fig. 7,A, ligation with anti-CD3 alone did not drive DP cells for differentiation. Anti-CD5 treatment also failed to induce a change in the expression of CD4 and CD8 (data not shown). However, coligation of CD3 and CD5 molecules resulted in a striking increase in the number of CD4+CD8 cells and a simultaneous decrease in the proportion of CD4+CD8+ cells. The levels of CD4 expression in the majority of CD4+CD8 cells generated were lower than those observed on mature CD4+CD8 cells. In contrast, the generation of CD4CD8+ cells was only marginally enhanced by coligation of CD3 and CD5. Although similar patterns of differentiation were observed upon coengagement of CD2 or CD28, the effect was the strongest for CD5 coengagement (Fig. 7,B). Among CD4+CD8 cells generated after CD5 coengagement, the major population was CD4dullCD8, but appreciable numbers of CD4highCD8 cells were also observed. Because 5% of CD4highCD8 cells were present in a starting DP population (Fig. 7,A), we prepared a highly purified DP population by positive selection. The contamination of CD4+CD8 cells in this population was ∼1/10th that in the DP population prepared by panning (Fig. 7,C). Only few CD4highCD8 cells were generated from a highly purified DP population, suggesting that the generation of CD4highCD8 cells in Fig. 7, A and B, is due to the expansion of mature SP (CD4highCD8) thymocytes contained in the starting population. Total cells generated from culture of a purified DP population were stained with annexin V. Fig. 7 D shows the annexin V staining of cells set on viable and nonviable (control) gates. Almost all cells on the viable gate were annexin V negative, indicating that they are viable, but not apoptotic, cells.

FIGURE 7.

Coligation of CD3 and CD5 drives DP thymocytes for differentiation to the CD4 lineage. A, A DP-enriched population was obtained by two cycles of panning using anti-CD8 mAb. This population was not stimulated or was stimulated with biotinylated anti-CD3 (2.5 μg/ml) alone or together with biotinylated anti-CD5 (2.5 μg/ml) followed by streptavidin (20 μg/ml) for 48 h. B, Similarly prepared DP-enriched populations were stimulated with biotinylated anti-CD3 (2.5 μg/ml) together with biotinylated anti-CD2, anti-CD28, or anti-CD5 (2.5 μg/ml) followed by streptavidin. C, A DP population was prepared by positive selection as described in Materials and Methods and was stimulated with anti-CD3 plus anti-CD5. Among harvested cells, viable cells were gated using forward and side scatter, and gated cells were analyzed for the expression of CD4 and CD8. D, Cells harvested from culture in the protocol of C, right, were stained with annexin V. Cells on the viable and nonviable gates were analyzed for staining with annexin V.

FIGURE 7.

Coligation of CD3 and CD5 drives DP thymocytes for differentiation to the CD4 lineage. A, A DP-enriched population was obtained by two cycles of panning using anti-CD8 mAb. This population was not stimulated or was stimulated with biotinylated anti-CD3 (2.5 μg/ml) alone or together with biotinylated anti-CD5 (2.5 μg/ml) followed by streptavidin (20 μg/ml) for 48 h. B, Similarly prepared DP-enriched populations were stimulated with biotinylated anti-CD3 (2.5 μg/ml) together with biotinylated anti-CD2, anti-CD28, or anti-CD5 (2.5 μg/ml) followed by streptavidin. C, A DP population was prepared by positive selection as described in Materials and Methods and was stimulated with anti-CD3 plus anti-CD5. Among harvested cells, viable cells were gated using forward and side scatter, and gated cells were analyzed for the expression of CD4 and CD8. D, Cells harvested from culture in the protocol of C, right, were stained with annexin V. Cells on the viable and nonviable gates were analyzed for staining with annexin V.

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Fig. 8 shows the time course of the change in the expression of CD4 and CD8 following coligation of CD3 and CD5. The population that has apparently reduced the levels of CD4 and CD8 expression appeared as early as 12 h after CD3/CD5 coligation. Most cells in this population expressed CD69, a marker of positively selected thymocytes. With the progress of culture, CD8 was almost completely lost. In contrast, the major population did not lose CD4, but remained CD4dull. Cibotti et al. (28) reported that enhanced TCR signals in the absence of lineage-specific signals initially induced transformation of CD4+CD8+ thymocytes into CD4low(dull)CD8 cells and that CD4 was then selectively reexpressed upon release of the cells from surface signals. To determine the capacity of CD4dullCD8 cells in our cultures to newly produce CD4 and CD8 molecules, CD4dullCD8 cells were treated with pronase and then rested without stimulation. The results in Fig. 9 show that pronase treatment removed existing surface CD4/CD8 molecules in both unstimulated and anti-CD3/anti-CD5-stimulated thymocytes. After 14-h culture, pronase-stripped cells in the former culture re-expressed both CD4 and CD8 proteins. In contrast, propase-treated cells in the latter culture reexpressed only CD4 proteins. A small number of CD4+CD8+ cells was regenerated in this culture, but these cells are considered to be derived from CD4+CD8+ cells existing in the population before pronase treatment. These results suggest that CD4dullCD8 cells generated after coligation of CD3 and CD5 precursors of CD4+CD8 cells.

FIGURE 8.

Time course of the generation of CD4dullCD8CD69+ cells following coengagement of CD3 and CD5. DP cells were stimulated with anti-CD3 plus anti-CD5. Cells harvested after various times were stained triply for CD4, CD8, and CD69. The cells were gated by the intensities of CD4 and CD8 expression, and the gated cells were analyzed for the expression of CD69.

FIGURE 8.

Time course of the generation of CD4dullCD8CD69+ cells following coengagement of CD3 and CD5. DP cells were stimulated with anti-CD3 plus anti-CD5. Cells harvested after various times were stained triply for CD4, CD8, and CD69. The cells were gated by the intensities of CD4 and CD8 expression, and the gated cells were analyzed for the expression of CD69.

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FIGURE 9.

Re-expression of CD4, but not of CD8, molecules by CD4dullCD8 cells treated with pronase. DP thymocytes were unstimulated (upper panel) or stimulated with anti-CD3 plus anti-CD5 for 36 h. Cells were treated with pronase as described in Materials and Methods. After washing, the cells were recultured in the absence of stimulating reagents for 14 h.

FIGURE 9.

Re-expression of CD4, but not of CD8, molecules by CD4dullCD8 cells treated with pronase. DP thymocytes were unstimulated (upper panel) or stimulated with anti-CD3 plus anti-CD5 for 36 h. Cells were treated with pronase as described in Materials and Methods. After washing, the cells were recultured in the absence of stimulating reagents for 14 h.

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In fetal thymus organ culture (FTOC), the differentiation of DP cells into CD4+CD8, but not into CD4CD8+, SP cells was shown to depend on the activation of the ERK pathway (21). We examined whether the ERK pathway activated by coligation of CD3 and CD5 is responsible for the differentiation of DP thymocytes to the CD4 lineage. As shown in Fig. 10 (the percentages of CD4dullCD8 (lower right) and percent viable cell recovery in the lower left parentheses), addition of SB203580 (an inhibitor for another type of MAPK, p38) to culture of CD5-costimulated DP cells did not inhibit the generation of CD4dullCD8 cells. In contrast, an ERK MAPK inhibitor, PD98059, influenced the differentiation of DP cells. However, most cells did not necessarily remain CD4+CD8+, but a considerable proportion of cells reduced their levels of CD4 and CD8.

FIGURE 10.

Inhibition of the generation of CD4dullCD8 cells by an ERK inhibitor, but not by a p38 MAPK inhibitor. DP thymocytes were stimulated with anti-CD3 plus anti-CD5 in the presence of 20 μM PD98059 or SB203580 (control) for 48 h. Cells were harvested, and ∼10,000 cells/group were examined. Among these, viable cells were gated using forward and side scatter, and gated cells were analyzed for the expression of CD4 and CD8. The numbers at the lowerright of each figure are the percentages of cells in the indicated region. The percentage of viable cell recovery [(the number of recovered viable cells/input cells) × 100] is also shown in the parentheses at the lower left of each figure.

FIGURE 10.

Inhibition of the generation of CD4dullCD8 cells by an ERK inhibitor, but not by a p38 MAPK inhibitor. DP thymocytes were stimulated with anti-CD3 plus anti-CD5 in the presence of 20 μM PD98059 or SB203580 (control) for 48 h. Cells were harvested, and ∼10,000 cells/group were examined. Among these, viable cells were gated using forward and side scatter, and gated cells were analyzed for the expression of CD4 and CD8. The numbers at the lowerright of each figure are the percentages of cells in the indicated region. The percentage of viable cell recovery [(the number of recovered viable cells/input cells) × 100] is also shown in the parentheses at the lower left of each figure.

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To more carefully examine the effect of PD98059 on the differentiation of DP cells, a time-course analysis of the differentiation in the presence of PD98059 was performed. The results in Fig. 11 show that the reduction of CD4/CD8 expression did not differ greatly between cultures with and without PD98059 at an earlier time point (12 h after culture). More than 24 h of culture in the presence of PD98059 not only failed to generate CD4lowCD8 cells, but also strikingly reduced the recovery of viable cells. PD98059 did not affect the survival of DP cells when they were not stimulated with anti-CD3 and anti-CD5 (Fig. 11, third line). These results suggest that the ERK activated following CD3-CD5 coligation is not necessarily required for the differentiation of DP cells, but, rather, contributes to maintaining thymocytes differentiating into the CD4 lineage.

FIGURE 11.

Time course of inhibition of generation of CD4dullCD8 cells. DP cells were unstimulated or were stimulated with anti-CD3 plus anti-CD5 in the presence or the absence of 20 μM PD98059 for various times. The percentages of CD4dullCD8 (in the indicated region) and the percent cell recovery are shown in A and B, respectively.

FIGURE 11.

Time course of inhibition of generation of CD4dullCD8 cells. DP cells were unstimulated or were stimulated with anti-CD3 plus anti-CD5 in the presence or the absence of 20 μM PD98059 for various times. The percentages of CD4dullCD8 (in the indicated region) and the percent cell recovery are shown in A and B, respectively.

Close modal

Bcl-2 has been shown not only to protect immature thymocytes from multiple death stimuli (29, 30), but also to promote thymic maturation (31). Bcl-2 is highly expressed in CD4CD8 DN cells, but is low in the CD4+CD8+ cells (32). Subsequently, positively selected thymocytes up-regulate Bcl-2 expression (31). We examined the levels of Bcl-2 expression in DP thymocytes stimulated with anti-CD3/CD5 in the presence or the absence of PD98059. As shown in Fig. 12, coligation of CD3 and CD5 in DP thymocytes resulted in Bcl-2 up-regulation, and such an up-regulation was potently prevented in the presence of an ERK inhibitor, PD98059. These results indicate that the ERK pathway activated by CD5 costimulation in DP cells is responsible for the induction of Bcl-2 in DP cells. Taken together, the CD5-mediated activation of ERK is associated with the up-regulation of an important biological effect in DP thymocytes.

FIGURE 12.

ERK activation by CD3/CD5 coengagement is responsible for enhanced Bcl-2 induction. DP thymocytes were stimulated with anti-CD3 and/or anti-CD5 in the presence of 20 μM PD98059 for 18 h (A) or for 12 and 24 h (B).

FIGURE 12.

ERK activation by CD3/CD5 coengagement is responsible for enhanced Bcl-2 induction. DP thymocytes were stimulated with anti-CD3 and/or anti-CD5 in the presence of 20 μM PD98059 for 18 h (A) or for 12 and 24 h (B).

Close modal

Interaction of TCR with Ag initiates an intracellular cascade of signaling events that culminates in T cell activation. Both elevation of intracellular Ca2+ and stimulation of the Ras pathway leading to ERK activation are required for the induction of T cell activation (15, 16, 17, 18, 19). This is based on the ability of these two signaling pathways to activate critical transcriptional factors, such as NF-AT and AP-1, required for the expression of IL-2. However, there has long been significant gaps in a basic understanding of many events induced following TCR engagement. In this context, the recently cloned adapter protein LAT proved to be the molecule that has been proposed to bridge the two branches of TCR signaling events mentioned above (19, 20). Thus, LAT was shown to play an essential and exclusive role in TCR-mediated activation of both the Ca2+ response and the Ras/ERK pathway (17).

The present study showed that coengagement of TCR (CD3) and CD5 on DP thymocytes induces a striking enhancement of LAT activation, but results in differential effects on two branches of TCR signaling events: the reduction of Ca2+ influx despite enhanced PLC-γ phosphorylation and the up-regulation of the ERK pathway. Moreover, CD5 costimulation induced the differentiation of CD4+CD8+ thymocytes to the CD4 lineage, resulting in an increase in the generation of CD4dullCD8CD69+ cells. The up-regulation of the ERK pathway by CD5 coengagement was responsible for the induction of a cell survival protein, Bcl-2, and underlie the differentiation of DP thymocytes by supporting the survival of differentiating cells. These results indicate that CD5 does not simply function as a molecule that negatively regulates TCR signaling in developing thymocytes, but enhances a branch of TCR signaling and supports the differentiation of DP thymocytes to the CD4 lineage.

Although CD28 has been recognized as the principal costimulatory receptor for T cell activation (7, 8, 9), a number of other molecules have also been described to possess costimulatory capacity, including CD5 (3, 4, 5), CD2 (10), CD9 (12), CD43 (33), and CD44 (11). Among these, CD5 was shown to exert its distinct costimulatory effects depending on whether responding cells are mature T cells or developing thymocytes. In mature T cells, CD5 costimulation increases Ca2+ influx in T cells triggered with suboptimal doses of anti-CD3 (Fig. 4), resulting in enhanced T cell activation as measured by [3H]TdR uptake (3, 4, 5, 13). In contrast, Ca2+ influx was considerably stronger in thymocytes from CD5−/− mice than in those from wild-type mice (14). Consistent with this, coengagement of normal thymocytes with anti-CD3 and anti-CD5 mAbs reduced Ca2+ influx (Fig. 4). Thus, it is obvious that CD5 costimulation induces opposite effects on TCR-stimulated mature T cells and developing thymocytes. This contrasted with the function of other costimulatory molecules, such as CD2 and CD28 (Fig. 6). Studies are currently being performed in our laboratory to investigate the mechanisms by which CD5 costimulation of thymocytes leads to down-regulation of Ca2+ influx. These include the measurement of inositol trisphosphate levels, as well as the determination of inositol trisphosphate receptor phosphorylation and phosphoinositide 3-kinase activation.

The most important aspect of the present study concerns the effect of CD5 costimulation on another branch of TCR signaling in developing thymocytes. In contrast to negative regulation of Ca2+ responses, CD5 positively costimulated developing thymocytes for ERK activation downstream from the Ras pathway. If the ERK pathway enhanced by CD5 costimulation contributes to positive events such as thymocyte differentiation/activation, CD5 should not necessarily be regarded as a molecule for the negative regulation of TCR signaling in developing thymocytes. In this context, our results showed that enhanced ERK activation in CD3/CD5-costimulated DP cells contributes to promoting the differentiation of DP thymocytes to the CD4 lineage.

A number of studies have shown the requirement for the Ras/ERK pathway in thymocyte development (34, 35, 36, 37). More recently, a study using FTOC demonstrated that ERK is required for differentiation from DP thymocytes to CD4, but not CD8, SP cells (21). Addition of a MEK inhibitor, PD98059, to FTOC resulted in a considerable decrease in the generation of CD4+CD8 cells (21). The inhibition of the differentiation may be explained by the following two possibilities: ERK activation is required for generating a differentiation signal itself or for maintaining cells that have started to differentiate into CD4+ cells.

To discriminate the above two possibilities, we took advantage of the thymic stroma-free thymocyte culture system, which was modified from that described by Cibotti et al. (28). They showed that stimulation of DP thymocytes with culture plate-bound anti-CD3 plus various mAbs against T cell surface molecules induces their differentiation into the CD4 lineage of cells (28). Their system consisted of two steps of cultures; in the first culture, DP cells stimulated with a mixture of mAbs were rendered CD4dullCD8. These cells differentiated into CD4+CD8 when they were recultured in the absence of stimulating mAbs. In this study, thymocytes were stimulated with streptavidin/biotin-cross-linked anti-CD3 plus anti-CD5 instead of culture plate-bound mAbs. Our stimulation system induced the differentiation to a similar stage (CD4dullCD8) as that observed in the model reported by Cibotti et al. (28), permitting us to examine the effect of PD98059 on this initial stage of DP cell differentiation. Our results showed that addition of PD98059 markedly decreased the generation of CD4dullCD8CD69+ cells, which is phenomenologically consistent with the observation of Sharp et al. (21).

Our study using a MEK inhibitor provided additional information regarding the mechanism that underlies the inhibition of differentiation. Because the viable cell recovery was not affected throughout culture when DP cells were not stimulated, PD98059 itself was not toxic to DP cells. Therefore, if PD98056 inhibits the differentiation per se, it is assumed that comparable numbers of CD4+CD8+ cells are recovered from unstimulated and stimulated cultures as long as the inhibitor is included. Stimulation with anti-CD3 plus anti-CD5 in the presence of PD98059 gradually decreased the number of CD4+CD8+ cells. The rate of the reduction in this culture was comparable to that observed in the inhibitor-free stimulation culture. Appreciable proportions of cells reducing the intensities of CD4/CD8 appeared after stimulation in the presence of the inhibitor. However, the numbers of differentiating cells gradually decreased, in contrast to the almost constant recovery of these cells in the inhibitor-free stimulation culture. Most of TCR/CD5-triggered cells in the inhibitor-positive culture failed to reach the stage of CD4lowCD8. These observations may favor the possibility that the ERK pathway does not necessarily initiate the differentiation process but, rather, functions to support thymocytes that have started their differentiation to the CD4 lineage.

In the model described by Cibotti et al. (28), coengagement of TCR with various molecules induced several differentiative events during the first signaling culture in which the phenotype of responding cells was altered from CD4+CD8+ to CD4dullCD8. First, TCR signals were previously shown to down-regulate the expression of CD4 and CD8 in mature T cells via transcriptional and post-transcriptional manners (37). Similar mechanisms (termination of coreceptor transcription and destabilization of coreceptor mRNAs) appear to occur in immature thymocytes (28, 38). Cibotti et al. presented an interesting hypothesis regarding the phenotype alteration to CD4dull/+CD8. In their view, TCR signaling not only destabilizes both CD4 and CD8 coreceptor mRNAs, but also terminates CD8 transcription, leaving CD4 transcription intact. These mechanisms generate CD4dullCD8 cells initially, and upon cessation of TCR signaling, CD4 mRNA degradation events cease, resulting in the generation of CD4+CD8 cells. In our model, when CD4dullCD8 cells were stripped of existing surface CD4/CD8 proteins by pronase treatment and recultured in the absence of stimulating reagents, they started to re-express CD4, but not CD8, molecules. These observations are consistent with the above-mentioned hypothesis and support the idea that CD4dullCD8 cells generated after coligation of CD3 and CD5 in our model are also precursors of CD4+CD8 cells.

Second, the initial events induced by costimulation of TCR and other molecules include the expression of various proteins. Among these, induced expression of Bcl-2 protein is particularly important. Most DP thymocytes express very low levels of Bcl-2 protein (32), and induction of Bcl-2 expression strictly correlates with lineage commitment (31, 39). The induction of Bcl-2 protein is important, because Bcl-2 expression protects immature thymocytes from multiple death signals (29, 30) and promotes different steps of thymic maturation (31). Cibbotti et al. found that the first signaling culture of DP thymocytes induces the expression of Bcl-2 protein (28). We have also demonstrated that, like costimulation of various molecules in the Cibotti model, CD5 costimulation leads to the induction of Bcl-2 protein expression, providing differentiating thymocytes with surviving potential. More importantly, activation of the ERK pathway was responsible for Bcl-2 induction. Thus, ERK activation by CD5 costimulation in DP thymocytes does not simply represent an enhancement of a branch of TCR signaling, but has biologically important relevance.

It still remains to be solved how the signal(s) capable of initiating the down-regulation of CD4 and CD8 is generated by TCR/CD5 coengagement. While it is obvious that CD5 costimulation in thymocytes down-regulates Ca2+ influx, it is necessary to investigate its biological significance. Selection of thymocytes expressing distinct transgenic TCRs was shown to be influenced in CD5-deficient mice differently depending on initial differences in TCR ligand affinity (14). This may be due in part to the capacity of CD5 to exert its distinct effects on two branches of TCR signaling in developing thymocytes. Further studies will also be required to investigate the biological mechanism by which CD5 costimulation results in a decrease in Ca2+ influx in DP thymocytes and the mechanism by which such a decreased Ca2+ influx occurs despite increased phosphorylation of PLC-γ.

We thank Dr. Avinash Bhandoola for critical review of this manuscript, and Tomoko Katsuta and Mari Yoneyama for secretarial assistance.

1

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan.

3

Abbreviations used in this paper: DP, double-positive; SP, single-positive; ERK, extracellular signal-regulated kinase; PLC-γ, phospholipase C-γ; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; FTOC, fetal thymus organ culture; LAT, linker for activation of T cells.

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