CD45 can act as a negative regulator for the transition from early to late CD4+CD8+ thymocytes

Takehito Sato, Satoshi Nunomura, Chiharu Sato, Katsuto Hozumi, Yoshihiro Kumagai1, Takashi Nishimura, Tak W. Mak2, Kenji Kishihara3 and Sonoko Habu

Department of Immunology, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-11, Japan
1 Inheritance and Variation, PRESTO, JRDC, Taya-cho, Sakae-ku, Yokohama, Kanagawa 244, Japan
2 The Amgen Institute and Ontario Cancer Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario M4X 1K9, Canada
3 Department of Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan

Correspondence to: S. Habu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The differentiation process from CD4CD8 double-negative (DN) thymocytes to CD4+CD8+ double-positive (DP) stage is accompanied by vigorous proliferation. The resulting DP cells contain a sizable proportion of large cycling cells, but most DP cells are small resting cells. To explore the molecular mechanisms which regulate cell proliferation of DP thymocytes prior to further development, we used TCR-transgenic (Tg) mice with non-selecting MHC (Tg-Neut), which contain almost exclusively DP thymocytes that are not subject to either positive or negative selection. In Tg-Neut, the thymus contained DP cells of relatively large size, which showed higher extracellular signal-regulated kinase activity and enhanced responsiveness to mitogen compared to small DP cells. This indicates that all the large DP cells in the thymus are not positively selected and that they possess proliferative potential. When Tg-Neut mice were backcrossed with CD45 knockout mice (CD45–/– Tg-Neut), the thymus showed an increase of large DP cells and cycling cells, but a decrease of apoptotic cells. Furthermore, Bcl-2 expression and Jun N-terminal kinase activity, which are associated with resistance to apoptosis, were enhanced. These observations suggest that thymocyte proliferation in the DP stage is suppressed by a CD45-related process with regulation of mitogen-activated protein kinase and Bcl-2 unless DP cells receive TCR-mediated signals.

Keywords: apoptosis, cellular proliferation, thymus


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Orchestrated regulation of cell proliferation and death generally occurs in ontogenical development (1). In the thymus, the developing process from CD4CD8 double-negative (DN) to CD4+CD8+ double-positive (DP) cells, which is induced by pre-TCR-mediated signaling, is accompanied by vigorous proliferation (24). At the resultant DP stage, however, most DP thymocytes cease proliferation and undergo programmed cell death, whereas only a small proportion of DP cells with low-affinity TCR for self-MHC develop into CD4+ or CD8+ single-positive (SP) cells. The latter is a process which is referred to as positive selection and the former is referred as default death (5). It is known that DP cells consist of a sizable proportion of large cycling cells, but most are small and non-dividing cells (6,7). Although most small DP cells have been reported to be dead-end cells (8), other studies showed that small DP cells can mature into SP cells when they are intrathymically transferred (9,10). Ernst et al. demonstrated that cell proliferation is not essential for positive selection in an in vitro re-aggregation culture system (6). Furthermore, Huesmann et al. showed that positively selected cells are not dividing cells, using an in vivo DNA-labeling technique (11). From these studies, it can be surmised that DP cells stop their proliferation to further development or cell death. However, the regulation mechanism for cell proliferation in the DP stage is not well understood.

Current mutant mice showed that several cytoplasmic molecules such as Lck and Ras are located down-stream of pre-TCR-mediated signals for the DN to DP transition, termed the ß-selection process (1215). Furthermore, Crompton et al. reported in an in vitro system using embryonic thymus culture that activation of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) superfamily, is also responsible for transition from DN to DP cells (16). However, it is still unclear whether these molecules are involved in regulating cell proliferation of DP thymocytes as they do in the DN to DP transition.

CD45 is a membrane protein whose cytoplasmic region possesses protein tyrosine phosphatase activity (17). Although CD45 is known to activate Lck by dephosphorylating C-terminal tyrosine (1822), several in vitro studies have shown that Lck activity is greater in CD45-deficient mutant cell lines than in CD45-bearing parent cell lines (19,23,24). The suppressive function of CD45 is also reported for MAPK; purified MAPK can be tyrosine dephosphorylated and inactivated by purified CD45 (25). At the cell level, several studies have shown that MAPK activation via CD2, CD3 or insulin-receptor was suppressed in the presence of CD45 (2628). These reports raise the possibility that CD45 possesses the potential of both negative and positive regulators for signaling, probably depending on cell types or developing stages. Therefore, it is postulated that CD45 could act as a negative modulator for thymocyte development through, for example, regulation of MAPK activities.

In this study, we used TCR-transgenic (Tg) mice without TCR-mediated signals for positive and negative selections in order to determine the molecules regulating cell proliferation of DP thymocytes (29,30). These thymocytes stopped cell proliferation at the DP stage and subsequently died because of the absence of negative and positive selections. Accordingly, these thymocytes are useful for determining the mechanism of regulation of the proliferation at the DP stage. When these mice were backcrossed with CD45 exon 6-deficient mice (31), their thymocytes showed an increase of cycling cells and a decrease of apoptotic cells, with enhanced activity of MAPK, suggesting that CD45 is involved in the cessation of thymocyte proliferation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
I-Ad + ovalbumin (OVA)323–339-specific TCR-Tg mice, OVA23-3, were produced as previously reported (29). These mice were backcrossed with BALB/c and C57BL/6. Their founders were designated Tg-Posi and Tg-Neut respectively. Tg-Posi and Tg-Neut were backcrossed with RAG2-deficient and CD45 exon 6-deficient mice (31).

Flow cytometry analysis and cell sorting
Phycoerythrin (PE)–anti-mouse CD4 and streptavidin–Red670 were purchased from Caltag (San Francisco, CA) and Life Technologies (Gaithersburg, MD) respectively. PE–anti-mouse CD25 and biotin–anti-mouse CD69 were purchased from PharMingen (San Diego, CA). Biotin–anti-Ly1(CD5) was purchased from Becton Dickinson (Mountain View, CA). Surface-stained thymocytes were analyzed by a FACScan (Becton Dickinson) or sorted on a FACStar Plus flow cytometer.

Proliferation assay
Thymocytes were cultured in 96-well plate for 48 h in the presence of indicated concentrations of phorbol myristate acetate (PMA) and ionomycin. Either 1x105 or 5x105 cells/well were cultured in round-bottomed or flat-bottomed 96-well plates respectively. Then 1 µCi/well [3H]thymidine was added during the final 18 h and incorporated radioactivities were counted.

Cell cycle analysis
Mice were i.p. injected with the thymidine analogue BrdU. At 2 h after injection, the thymus was removed and fixed with 70% ethanol. Fixed thymocytes were treated with 2 N HCl plus 0.5% Triton X-100 and neutralized with 0.1 M Na2B4O7. After washing with PBS, FITC–anti-BrdU (Becton Dickinson) was added. Following incubation with 5 µg/ml propidium iodine, stained cells were analyzed on a FACScan by the CellFIT program.

In vitro kinase assay
Between 5 and 50x105 cells were lysed with 0.5 ml of lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 1 mM Na3VO4, 0.1 mM EGTA, 1 mM DTT, 0.1 M NaF, 1% Triton X-100, 1 mM PMSF, 20 µg/ml aprotinin and 20 ng/ml leupeptin). After incubation on ice for 30 min, the sample was centrifuged (10,000 r.p.m., 4°C) and the supernatant was collected. The lysate was pre-cleared with Protein G–Sepharose and immunoprecipitated with anit-ERK-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Jun N-terminal kinase (JNK)-1 (Santa Cruz)–Protein G–Sepharose. After washing with lysis buffer 3 times, the immunoprecipitate was rinsed with kinase buffer (50 mM Tris–HCl, pH 7.4, 10 mM MgCl2, 2 mM EGTA and 1 mM DTT). Kinase reaction was performed at room temperature for 10 min (ERK) or at 30°C for 30 min (JNK) in 25 µl of kinase buffer containing 1 pg/ml protein kinase inhibitor (Sigma), 2 µM ATP, 1 µCi [{gamma}-32P]ATP and myelin basic protein (MBP; Sigma) or c-jun-GST (Santa Cruz Biotechnology) as substrate for ERK and JNK respectively. When the sample was small scale (5x105 cells/sample), cold ATP was not added. After 25 µl of sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2 g/ml glycerol) was added and boiled for 5 min, the sample was separated by SDS–PAGE. The dried gel was exposed to Scientific Imaging Film (Kodak, Rochester, NY).

Immunoblotting
Between 2.5 and 20x105 cells were lysed with sample buffer and boiled for 5 min. The lysates was separated by SDS–PAGE and transferred onto Clear-blot Membrane (Atto, Tokyo, Japan). After blocking with BSA, the membrane was incubated with anti-ERK-1, anti-JNK-1, anti-p27kip1 (Santa Cruz) or anti-Bcl-2 (PharMingen, San Diego, CA). Detection was performed using ECL Western blotting reagent (Amersham, Amersham, UK) and Hyperfilm ECL (Amersham).

Detection of apoptotic cells
Formalin-fixed sections of thymuses at day 2 after birth were treated with proteinase K (final 20 µg/ml) at room temperature for 15 min. After washing, tissue sections were incubated with 2% H2O2 diluted in methanol for 5 min to neutralize endogenous peroxidases. After washing with H2O 4 times, sections were incubated with 5 U TdT (Boehringer Mannheim, Mannheim, Germany) and 0.5 nmol biotin–dUTP (Boehringer Mannheim) in 30 µl of TdT buffer (Boehringer Mannheim) at 37°C for 60 min. Sections were incubated with TB buffer (300 mM NaCl/30 mM citrate), 2% BSA in PBS and then with horseradish peroxidase (HRP)–streptavidin. Bound HRP was detected with substrate DBA and sections were lightly counterstained with methylgreen or haematoxylin.

A computer-assisted image analysis system with the Interactive Build Analysis System (Carl Zeiss, Zena, Germany) was used to evaluate apoptotic cells. The input image resolution was 512x512 pixels, 8 bits, 256 gray scales. The stained nuclei and DAB reaction products were detected using interference bandpass filter IF 436 and 600 (Olympus, Tokyo, Japan) respectively. The binary images of hematoxylin and DAB were overlaid and segmented images were counted.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
DP large cells in TCR-Tg mice with non-selecting MHC showed increased MAPK activity and enhanced responsiveness to mitogen
The thymus of TCR-Tg mice, OVA23-3 (29,30), with selecting MHC (Tg-Posi) contains large numbers of CD4 SP cells (~20 %, data not shown) and DP cells. On the other hand, the thymus of TCR-Tg mice with non-selecting MHC (Tg-Neut) does not contain SP cells. DP cells of both TCR-Tg mice include a significant number of cells of relatively large cell size. Large DP cells in Tg-Neut did not show characteristics of positive selection, such as high expression of CD69 and CD5 (3234) (Fig. 1Go). Such large DP cells with low CD69 and CD5 were also found in Tg-Neut mice which were backcrossed with RAG-2-deficient mice (35) (data not shown), indicating that these DP cells are not positively selected via endogenous TCR. These results coincide with the previous report by Swat et al. that large DP cells exist in the absence of positive selection using another TCR-Tg mouse system (36). Thus, it is indicated that large DP cells exist even if they are not positively selected.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 1. CD69 and CD5 expression on large and small DP cells. Thymocytes of TCR-Tg mice, OVA23-3 (29, 30), with selecting MHC (I-Ad, Tg-Posi) and non-selecting MHC (I-Ab, Tg-Neut) were stained with FITC–anti-CD8, PE–anti-CD4, biotin–anti-CD69 or biotin–anti-CD5 and Red670–streptavidin. Stained cells were analyzed by a FACScan. Expressions of CD69 (left) and CD5 (right) on gated large DP and small DP cells are shown as solid lines. Dotted lines are non-stained controls.

 
Since DN cells in the thymus develop into DP cells with marked proliferation, a substantial proportion of the DP cells may still be cycling or possess proliferative potential. When the large and small DP cells of Tg-Neut were cultured with PMA and ionomycin, large DP cells showed enhanced aggregation and greater proliferation (Fig. 2Go). We also examined the activity of ERK, a member of MAPK, by an in vitro kinase assay after the lysates of both large and small DP cells were immunoprecipitated with anti-ERK-1 (cross-reactive to ERK-2). In this assay, MBP was utilized as a substrate. As shown in Fig. 3Go, ERK activity was greater in large DP cells than in small DP cells. Furthermore, large DP cells contained large numbers of cycling cells (data not shown). These results suggest that large DP cells still possess proliferating potentials and are presumably recently derived from DN cells. At the same time, the present data are consistent with the reports that ERK is associated with cell proliferation (37,38).



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 2. Large DP cells of TCR-Tg mice with non-selecting MHC (Tg-Neut) showed enhanced aggregation and proliferation responding to PMA plus ionomycin. (a) Thymocytes of Tg-Neut were stained with FITC–anti-CD8 and PE–anti-CD4. Small and large DP cells were fractionated by a FACStar. (b) Phase contrast micrographs of culture after 1 day with PMA (0.2 ng/ml) and ionomycin (1.2 µg/ml). (c) Small and large DP cells were cultured with 0.2 ng/ml PMA and 1.2 µg/ml ionomycin for 48 h in round-bottomed 96-well plates (1x105 cells/well). [3H]Thymidine incorporations (± SD) during the final 18 h are indicated

 


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. ERK activity and p27kip1 expression was greater in large DP cells than in small DP cells. (a) The lysates of 5x105 sorted small DP and large DP cells were immunoprecipitated with anti-ERK-1 and in vitro kinase assay was performed using MBP as a substrate. (b) The lysates of 2.5x105 cells of small DP and large DP cells were separated by SDS–PAGE and transferred to a membrane. The membrane was incubated with anti-p27kip1 and detection was done by ECL. Relative band intensities in arbitrary densiometric units are listed under each lane. Three independent experiments showed similar results. Typical data are presented (a and b).

 
We also tested the expression level of cyclin-dependent kinase inhibitor, p27kip1. Because p27kip1-deficient mice were reported to show thymic hyperplasia, p27kip1 is considered to play a role in the regulation of thymocyte proliferation (3941). Unexpectedly, the expression of p27kip1 was higher in large DP cells than in small cells (Fig. 3Go), suggesting that small DP cells may rest at G0/G1 phase independently of p27kip1.

CD45-deficient Tg-Neut thymus contains increased large DP cells
In our previous study using Tg-Neut mice, we demonstrated that TCR expression is high at the early DP stage even though the cells are not positively selected and the expression is gradually down-regulated thereafter (30). We also reported that the down-regulation of TCR is inhibited in CD45-deficient mice. Then, we examined the role of CD45 in thymocyte proliferation in the absence of TCR-mediated signals. In Tg-Neut whose thymocytes are not positively selected by appropriate MHC, the thymus is composed of mostly DP cells but not SP cells in both CD45-bearing (CD45+/+ Tg-Neut) and CD45-deficient (CD45–/– Tg-Neut) lines. Interestingly, an increased number of DP cells of large size was found in CD45–/– Tg-Neut thymocytes (Fig. 4aGo). In addition, enhancement of proliferation and aggregation was observed in CD45–/– Tg-Neut when they were cultured with PMA plus ionomycin (Fig. 4b and cGo). At the same time, ERK activity was greater in CD45–/– Tg-Neut than in CD45+/+ Tg-Neut, although the amounts of ERK-1 and -2 protein were not different between the two types of mice (Fig. 4dGo).




View larger version (72K):
[in this window]
[in a new window]
 
Fig. 4. CD45–/– Tg-Neut thymus contained larger number of large DP cells. (a) Forward scatter (FSC) of DP cells of CD45+/+ and CD45–/–Tg-Neut mice. Percentages of relatively larger cells are indicated. (b) [3H]Thymidine incorporation (± SD) of thymocytes cultured for 48 h in flat-bottomed 96-well plates (5x105 cells/well) with PMA (0.2 ng/ml) and ionomycin (0 to 1.2 mg/ml). (c) Phase contrast micrographs of culture after 1 day with PMA (0.2 ng/ml) and ionomycin (1.2 mg/ml). (d) Anti-ERK-1 immunoprecipitable kinase activity analysis. The lysates of 5x106 cells of CD45+/+ Tg-Neut (left) and CD45–/– Tg-Neut (right) were immunoprecipitated with anti-ERK-1 and MBP kinase assay was performed. Relative band intensities in arbitrary densitometric units are listed under each lane (upper). The expression levels of ERK (p44, ERK-1; p42, ERK-2) in both thymocytes are shown (lower). Three independent experiments showed similar results. Typical data are presented.

 
These features observed in CD45–/– Tg-Neut thymocytes are similar to those of large DP cells in CD45+/+ Tg-Neut (Figs 2 and 3GoGo). Because there is no ligand for TCR in either of the Tg-Neut thymuses, these changes are considered to result from CD45 deficiency independently of TCR-mediated signals. This is the first evidence to show the suppressive effect of CD45 on cell proliferation of non-selected DP cells in the thymus, although CD45 has been reported to be important in both positive and negative selections (18,31,42). CD45 seems to be involved in the negative regulation of DP cell proliferation, as is discussed later.

Cycling cells increase in CD45–/– Tg-Neut in vivo
To ascertain whether large DP cells with proliferative potential substantially increase in the thymus of CD45–/– Tg-Neut, we injected the thymidine analogue BrdU i.p. into CD45–/– and CD45+/+ Tg-Neut mice. Two hours after the injection, the thymocytes were fixed and stained with FITC–anti-BrdU and propidium iodine for analysis by flow cytometry. The number of thymocytes in the S phase of the cell cycle was 2-fold greater in CD45–/– Tg-Neut mice than in CD45+/+ Tg-Neut (Fig. 5Go). A concomitant decrease of the cell number in G0/G1 phase was observed in the CD45–/– Tg-Neut thymus. These findings indicate that CD45 deficiency results in augmentation of thymocyte proliferation in vivo, which is consistent with the increased number of large DP cells in CD45–/– Tg-Neut.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5. Cell cycle analysis of thymocytes of CD45+/+ Tg-Neut (left) and CD45–/– Tg-Neut (right) mice. Two hours after i.p. injection of BrdU, thymocytes were fixed with cold ethanol. The fixed thymocytes were stained with FITC–anti-BrdU and propidium iodine, and analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was calculated on the gated appropriate population. Three mice of each group were examined and the average ± SD is indicated.

 
Apoptotic cells decrease in CD45–/– Tg Neut thymus
In addition to the increase of cycling cells, the decrease of small DP cells by apoptosis may also cause the high frequency of large DP cells in CD45–/– Tg-Neut. Then, we examined apoptotic cells in the fresh thymus of CD45–/– and CD45+/+ Tg-Neut by the TUNEL staining method. In the thymus sections, the frequency of TUNEL-positive cells in total cells was lower in CD45–/– Tg-Neut than in CD45+/+ Tg-Neut (Fig. 6Go). Moreover, both the frequency of DP cells and the total cell number were larger in CD45–/– Tg-Neut than in CD45+/+ Tg-Neut (data not shown). The correlation of the decrease of apoptotic cells with the increase of cycling cells is consistent with the previous report showing that apoptotic cells were more common with small DP cells than with large DP cells when they were cultured in vitro (6).



View larger version (89K):
[in this window]
[in a new window]
 
Fig. 6. Cells with DNA fragmentation detected by TUNEL method. CD45+/+ Tg-Neut (a) and CD45–/– Tg-Neut (b) thymus at 2 days after birth. (c) TUNEL-stained cells and non-stained cells were calculated using a computer-assisted image analysis system as described in Methods. Four independent thymic lobes were examined.

 
Nishina et al. reported that the number of DP thymocytes is decreased in mutant mice lacking the JNK kinase, Sek-1, and that Sek-1-deficient DP cells are more susceptible to Fas- or CD3-mediated apoptosis (43). This raises the possibility that JNK plays a role in the survival of DP thymocytes. Thus, we examined the JNK activity in thymocytes of CD45–/– Tg-Neut. As shown in Fig. 7Go(a), the JNK activity was higher in CD45–/– Tg-Neut than that in CD45+/+ Tg-Neut thymocytes. Moreover, the expression of the anti-apoptotic gene, bcl-2, was higher in DP cells of CD45–/– Tg-Neut than of CD45+/+ Tg-Neut (Fig. 7bGo). These findings are consistent with the decrease of apoptosis in CD45–/– Tg-Neut.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7. (a) Anti-JNK-1 immunoprecipitable kinase activity analysis. The lysates of 5x106 cells of CD45+/+ Tg-Neut (left) and CD45–/– Tg-Neut (right) were immunoprecipitated with anti-JNK-1 and in vitro kinase assay was performed using c-jun-GST as a substrate. (b and c) Expression of Bcl-2 and JNK in DP thymocytes of CD45+/+ Tg-Neut (left) and CD45–/– Tg-Neut (right). Cells (2x106) were lysed, separated by SDS–PAGE and blotted. Three independent experiments showed similar results. Typical data are presented (a–c).

 
When compared with Tg-Neut, JNK activity was much higher in thymocytes of TCR-Tg mice with selecting MHC (Tg-Posi) where all DP cells are considered to have escaped from apoptosis and to develop into SP cells (Sato, unpublished data). Taken together, it is possible that JNK as well as Bcl-2 are down-regulated at the DP stage to make cells prone to die, presumably by CD45-mediated mechanisms, which may be cancelled when DP cells are positively selected via TCR-mediated signals.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We demonstrated that DP thymocytes of relatively large size are present in TCR-Tg mice with non-selecting MHC (Tg-Neut), and that these DP cells do not express cell surface molecules characteristic for positively selected cells such as CD69 and CD5 (3234) (Fig. 1Go). However, they showed the increased activity of MAPK, ERK-1 and -2, compared with small DP cells (Fig. 3Go). This indicated that all the large DP cells in the thymus are not positively selected cells and that such large DP cells without TCR-mediated signals possess potential for proliferation because MAPK was reported to be associated with cell proliferation (37,38). In fact, they proliferated in response to PMA plus ionomycin (Fig. 2Go).

The expression level of ERK in large DP cells was higher than in small cells (data not shown), but the kinase activity per ERK protein could not be estimated clearly from the intensity of blotting bands of the activity and amount of ERK. It cannot be excluded that the increased ERK activity in large DP cells may reflect higher expression of ERK, but it is absolutely clear that in CD45-deficient Tg-Neut thymocytes, the kinase activity of ERK is increased but its expression is not changed (Fig. 4Go). Furthermore, CD45–/– Tg-Neut thymocytes showed enhanced JNK activity and Bcl-2 expression (Fig. 8Go), both of which are thought to be involved in resistance to apoptosis (4345).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 8. CD45 promotes transition from early to late DP cells by negatively regulating the proliferation and presumably promoting apoptosis. This transition is accompanied by down-regulation of the activities of MAPK (ERK and JNK) and Bcl-2 expression. These are prevented by TCR-mediated signals.

 
It is well known that CD45 plays an important role in TCR-mediated signals (4648). Moreover, CD45-deficient mice of both exon 6 and exon 9 knockout lines showed almost complete blockade of positive selection from DP to SP stage, whereas transition from DN to DP stage is slightly inhibited (data not shown and 31,42). However, there is no report of CD45 function in DP thymocytes without TCR-mediated signals as seen in our CD45–/– Tg-Neut mice.

CD45 is also reported to activate Lck and Fyn by dephosphorylation of their C-terminal tyrosine (1822). However, several reports have shown the reverse function of CD45; Lck activity is increased in CD45-deficient mutant cell lines compared to that in CD45-bearing parent cell lines (19,23,24). D'Oro et al. found that CD45 can dephosphorylate Tyr394 of Lck, in addition to C-terminal Tyr505, and that the absence of CD45 drives the hyperphosphorylation of Tyr394 and a resultant increase of the kinase activity of Lck though Tyr505 of Lck was not dephosphorylated (49). Similarly, CD45 can act as a negative regulator for some other kinases (25,50). It is reported that ERK can be tyrosine dephosphorylated and inactivated directly by purified CD45 in vitro (25). Furthermore, several studies have shown that ERK activation via CD2, CD3 or insulin-receptor was suppressed in the presence of CD45 (2628). Cross-linking of CD3 on thymocytes of RAG-deficient mice induced transient up-regulation of ERK activity (16), which was more increased in CD45–/– RAG-deficient mice (T. Sato, unpublished data). Taken together, it is suggested that CD45 may negatively regulate phosphorylation and activation of ERK in DP thymocytes without TCR signals. Similarly, JNK, which is also a member of the MAPK superfamily, could be inactivated by CD45. It is reported that positively selected DP cells increase their TCR expression (51,52). However, all of the TCRhi DP cells were not always post-selected cells. We and Swat et al. found that DP cells with TCRhi exist in TCR-Tg mice with non-selecting MHC, in which DP thymocytes are not positively selected (30,36). We have also reported that TCR expression on DP cells in Tg-Neut at gestation days 16 to 17, when DP cells begin to appear, is at the same high level as that of positively selected DP cells, but that the expression decreases during the further course of gestation (30). This observation suggests that `early DP cells', which recently transited from the DN stage, can express TCR at a higher level. Moreover, the decrease of TCR expression did not occur in the absence of CD45 (30), indicating that there exist some CD45-mediated mechanisms which down-regulate the TCR expression when DP cells do not receive TCR-mediated positive signals. This finding seems to be consistent with the previous reports that Lck regulates TCR surface expression and lysosomal degradation (53,54), because CD45 can positively regulate the kinase activity of Lck.

We found that large DP cells in TCR-Tg mice with non-selecting MHC contained many high TCR-expressing cells, whereas small DP cells did not contain any at all (data not shown). Considering the present findings together with the previous ones of TCR expression, it is strongly suggested that large DP thymocytes without TCR-mediated signals belong to DP cells at the early stage and that in the absence of TCR-mediated signals CD45 is involved in the transition from early to late DP cells by negatively regulating the proliferation and presumably by promoting apoptosis (Fig. 6Go), as well as in TCR down-regulation. This hypothesis is depicted in Fig. 8Go. Early DP cells have greater activity of both ERK and JNK, and express Bcl-2 at a higher level. Then, they are down-regulated and early DP cells become small DP cells at the late stage. CD45 may mediate this transition. When DP cells are positively selected by TCR-mediated signals, JNK activity and Bcl-2 expression are again up-regulated. Subsequently, selected cells acquire a long lifespan, whereas non-selected cells die.

It is believed that the lifespan of DP thymocytes is 3–4 days (11,55,56). However, it is not known what molecules set the clock. It is attractive to postulate that CD45 can transduce signals to repress thymocyte proliferation or to induce apoptosis in non-selected DP cells. This hypothesis may be consistent with a recent report that CD45 ligation can induce apoptosis of DP cells (57). On the other hand, Novak et al. proposed the possibility that the shorter isoform, CD45RO, can diffuse more freely in the plane of cell membrane and can interact with substrates more readily than the longer isoform (CD45RABC) does (58). It is reported that the switch from the shorter to longer isoform occurs when DP cells are positively selected (59). Therefore, it can be speculated that the shorter isoforms on non-selected DP cells transduce signals to effectively suppress the cell cycle or to induce apoptosis and that the switch to longer forms prevents this effect when they are positively selected.

On the other hand, Chen et al. reported that the inducibility of the DNA binding activity of AP-1 (38), which is one of the targets of ERK and JNK pathways, is extinguished at most cortical thymocytes, whereas it is found at earlier and full mature stages (60,61). It is believed that the alteration of AP-1 inducibility is associated with mitogen responsiveness of thymocytes. We found that CD45–/– Tg-Neut thymocytes showed enhanced cell division in response to PMA plus ionomycin. It will be of interest to examine whether the DNA binding activity of these transcription factors can be induced at a greater level in CD45–/– Tg-Neut than CD45+/+ thymus.


    Acknowledgments
 
We thank Drs E. V. Rothenberg and M. Raff for helpful discussions. We are grateful to Mr H. Hasegawa for TUNEL staining, to Dr J. Itoh for analyzing the TUNEL-stained sections, and to Mr Okada and Ms Hori for operating the FACStar. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, Japan and CREST.


    Abbreviations
 
DNdouble negative
DPdouble positive
ERKextracellular signal regulated kinase
HRPhorseradish peroxidase
JNKJun N-terminal kinase
MAPKmitogen-activated protein kinase
MBPmyelin basic protein
OVAovalbumin
PEphycoerythrin
PMAphorbol myristate acetate
SPsingle positive
Tgtransgenic

    Notes
 
Transmitting editor: T. Watanabe

Received 18 July 1998, accepted 1 October 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Raff, M. C. 1996. Size control: the regulation of cell numbers in animal development. Cell 86:173.[ISI][Medline]
  2. Levelt, C. N., Mombaerts, P., Iglesias, A., Tonegawa, S. and Eichmann, K. 1993. Restoration of early thymocyte differentiation in T-cell receptor ß-chain-deficient mutant mice by transmembrane signaling through CD3{varepsilon}. Proc. Natl Acad. Sci. USA 90:11401.[Abstract]
  3. Shinkai, Y. and Alt, F. W. 1994. CD3{varepsilon}-mediated signals rescue the development of CD4+CD8+ thymocytes in RAG-2–/– mice in the absence of TCR ß chain expression. Int. Immunol. 6:995.[Abstract]
  4. Hoffman, E. S., Passoni, L., Crompton, T., Leu, T. M. J., Schatz, D. G., Koff, A., Owen, M. J. and Hayday, A. C. 1996. Productive T-cell receptor ß-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 10:948.[Abstract]
  5. von Boehmer, H. 1994. Positive selection of lymphocytes (review). Cell 76:219.[ISI][Medline]
  6. Ernst, B., Surh, C. D. and Sprent, J. 1995. Thymic selection and cell division. J. Exp. Med. 182:961.[Abstract]
  7. Akashi, K. and Weissman, I. L. 1996. The c-kit+ maturation pathway in mouse thymic T cell development: lineages and selection. Immunity 5:147.[ISI][Medline]
  8. Guidos, C. J., Weissman, I. L. and Adkins, B. 1989. Intrathymic maturation of murine T lymphocytes from CD8+ precursors. Proc. Natl Acad. Sci. USA 86:7542.[Abstract]
  9. Lundberg, K. and Shortman, K. 1994. Small cortical thymocytes are subject to positive selection. J. Exp. Med. 179:1475.[Abstract]
  10. Swat, W., von Boehmer, H. and Kisielow, P. 1994. Small CD4+8+TCRlow thymocytes contain precursors of mature T cells. Eur. J. Immunol. 24:1010.[ISI][Medline]
  11. Huesmann, M., Scott, B., Kisielow, P. and von Boehmer, H. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell 66:533.[ISI][Medline]
  12. Molina, T. J., Kishihara, K., Siderovski, D. P., van Ewijk, W., Narendran, A., Timms, E., Wakeham, A., Paige, C. J., Hartmann, K.-U., Veillette, A., Davidson, D. and Mak, T. W. 1992. Profound block in thymocyte development in mice lacking p56lck. Nature 357:161.[ISI][Medline]
  13. Swat, W., Shinkai, Y., Cheng, H.-L., Davidson, L. and Alt, F. A. 1996. Activated ras signals differentiation and expression of CD4+8+ thymocytes. Proc. Natl Acad. Sci. USA 93:4683.[Abstract/Free Full Text]
  14. van Oers, N. S. C., Lowin-Kropf, B., Finlay, D., Connolly, K. and Weiss, A. 1996. {alpha}ß T cell development is abolished in mice lacking both lck and fyn protein tyrosine kinases. Immunity 5:429.[ISI][Medline]
  15. Mombaerts, P., Anderson, S. J., Perlmutter, R. M., Mak, T. W. and Tonegawa, S. 1994. An activated lck transgene promotes thymocyte development in RAG-1 mutant mice. Immunity 1:261.[ISI][Medline]
  16. Crompton, T., Gilmour, K. C. and Owen, M. J. 1996. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell 86:243.[ISI][Medline]
  17. Trowbridge, I. S. and Thomas, M. L. 1994. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu. Rev. Immunol. 12:85.[ISI][Medline]
  18. Mustelin, T., Coggeshall, K. M. and Altman, A. 1989. Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. Proc. Natl Acad. Sci. USA 86:6302.[Abstract]
  19. Ostergaard, H. L., Shackelford, D. A., Hurley, T. R., Johnson, P., Hyman, R., Sefton, B. M. and Trowbridge, I. S. 1989. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc. Natl Acad. Sci. USA 86:8959.[Abstract]
  20. McFarland, E. D. C., Hurley, T. R., Pingel, J. T., Sefton, B. M., Shaw, A. and Thomas, M. L. 1993. Correlation between Src family member regulation by the protein-tyrosine-phosphatase CD45 and transmembrane signaling through the T-cell receptor. Proc. Natl Acad. Sci. USA 90:1402.[Abstract]
  21. Hurley, T. R., Hyman, R. and Sefton, B. M. 1993. Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases. Mol. Cell. Biol. 13:1651.[Abstract]
  22. Sie, M., Bolen, J. B. and Weiss, A. 1993. CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck. EMBO J. 12:315.[Abstract]
  23. Deans, J. P., Kanner, S. B., Torres, R. M. and Ledbetter, J. A. 1992. Interaction of CD4:lck with the T cell receptor/CD3 complex induces early signaling events in the absence of CD45 tyrosine phosphatase. Eur. J. Immunol. 22:661.[ISI][Medline]
  24. Burns, C. M., Sakaguchi, K., Appella, E. and Ashwell, J. D. 1994. CD45 regulation of tyrosine phosphorylation and enzyme activity of src family kinase. J. Biol. Chem. 269:13594.[Abstract/Free Full Text]
  25. Anderson, N. G., Maller, J. L., Tonks, N. K. and Sturgill, T. W. 1990. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651.[ISI][Medline]
  26. Nel, A. E., Ledbetter, J. A., Williams, K., Ho, P., Akerley, B., Franklin, K. and Katz, R. 1991. Activation of MAP-2 kinase activity by the CD2 receptor in Jurkat T cells can be reversed by CD45 phosphatase. Immunology 73:129.[ISI][Medline]
  27. Pollack, S., Ledbetter, J. A., Katz, R., Williams, K., Akerley, B., Franklin, K., Schieven, G. and Nel, A. E. 1991. Evidence for involvement of glycoprotein-CD45 phosphatase in reversing glycoprotein-CD3-induced microtubule-associated protein-2 kinase activity in Jurkat T-cells. Biochem. J. 276:481.[ISI][Medline]
  28. Kulas, D. T., Freund, G. G. and Mooney, R. A. 1996. The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling. J. Biol. Chem. 271:755.[Abstract/Free Full Text]
  29. Sato, T., Sasahara, T., Nakamura, Y., Osaki, T., Hasegawa, T., Tadakuma, T., Arata, Y., Kumagai, Y., Katsuki, M. and Habu, S. 1994. Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice. Eur. J. Immunol. 24:1512.[ISI][Medline]
  30. Sato, T., Hozumi, K., Kishihara, K., Kametani, Y., Sato, C., Kumagai, Y., Mak, T. W. and Habu, S. 1996. Evidence for down-regulation of highly expressed T cell receptor by CD4 and CD45 on non-selected CD4+CD8+ thymocytes. Int. Immunol. 8:1529.[Abstract]
  31. Kishihara, K., Penninger, J., Wallace, V. A., Kündig, T. M., Kawai, K., Wakeham, A., Timms, E., Pfeffer, K., Ohashi, P. S., Thomas, M. L., Furlonger, C., Paige, C. J. and Mak, T. W. 1993. Normal B lymphocyte development but impaired T cell maturation in CD45-exon6 protein tyrosine phosphatase-deficient mice. Cell 74:143.[ISI][Medline]
  32. Swat, W., Dessing, M., von Bohemer, H. and Kisielow, P. 1993. CD69 expression during selection and maturation of CD4+8+ thymocytes. Eur. J. Immunol. 23:739.[ISI][Medline]
  33. Dutz, J. P., Ong, C. J., Marth, J. and Teh, H.-A. 1995. Distinct differentiative stages of CD4+CD8+ thymocyte development defined by the lack of coreceptor binding in positive selection. J. Immunol. 154:2588.[Abstract/Free Full Text]
  34. Yamashita, I., Nagata, T., Tada, T. and Nakayama, T. 1993. CD69 cell surface expression identifies developing thymocytes which audition for T cell antigen-mediated positive selection. Int. Immunol. 5:1139.[Abstract]
  35. Shinkai, Y., Rathbun, G., Lam, K., Oltz, E. M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M. and Alt, F. A. 1992. Rag-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68:855.[ISI][Medline]
  36. Swat, W., Dessing, M., Baron, A., Kisielow, P. and von Boehmer, H. 1992. Phenotypic changes accompanying positive selection of CD4+CD8+ thymocytes. Eur. J. Immunol. 22:2367.[ISI][Medline]
  37. Marshall, C. J. 1995. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179.[ISI][Medline]
  38. Su, B. and Karin, M. 1996. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8:402.[ISI][Medline]
  39. Nakayama, K., Ishida, N., Shirane, M., Inomata, A., Inoue, T., Shishido, N., Horii, I., Loh, D. Y. and Nakayama, K.-I. 1996. Mice lacking p27Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85:707.[ISI][Medline]
  40. Kiyokawa, H., Kineman, R. D., Manova-Todorova, K. O., Soares, V. C., Hoffman, E. S., Ono, M., Khanam, D., Hayday, A. C., Frohman, L. A. and Koff, A. 1996. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell 85:721.[ISI][Medline]
  41. Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K., Tsai, L.-H., Broudy, V., Perlmutter, R. M., Kaushansky, K. and Roberts, J. M. 1996. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell 85:733.[ISI][Medline]
  42. Byth, K. F., Conroy, L. A., Howlett, S., Smith, A. J. H., May, J., Alexander, D. R. and Holmes, N. 1996. CD45-null transgenic mice reveal a positive regulatory role for CD45 in early thymocyte development, in the selection of CD4+CD8+ thymocytes, and in B cell maturation. J. Exp. Med. 183:1707.[Abstract]
  43. Nishina, H., Fischer, K. D., Radvanyi, L., Shahinian, A., Hakem, R., Rubie, E. A., Bernstein, A., Mak, T. W., Woodgett, J. R. and Penninger, J. M. 1997. Stress-signalling kinase Sek1 protects thymocytes from apoptosis mediated by CD95 and CD3. Nature 385:350.[ISI][Medline]
  44. Cory, S. 1995. Regulation of lymphocyte survival by the bcl-2 gene family. Annu. Rev. Immunol. 13:513.[ISI][Medline]
  45. Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, O. and Korsmeyer, S. J. 1991. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell, 67:879.[ISI][Medline]
  46. Pingel, J. T. and Thomas, M. L. 1989. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell 58:1055.[ISI][Medline]
  47. Weaver, C. T., Pingel, J. T., Nelson, J. O. and Thomas, M. L. 1991. CD8+ T-cell clones deficient in the expression of the CD45 protein tyrosine phosphatase have impaired responses to T-cell receptor stimuli. Mol. Cell. Biol. 11:4415.[ISI][Medline]
  48. Desai, D. M., Sap, J., Silvennoinen, O., Schlessinger, J. and Weiss, A. 1994. The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation. EMBO J. 13:4002.[Abstract]
  49. D'Oro, U., Sakaguchi, K., Appella, E. and Ashwell, J. 1996. Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity. Mol. Cell. Biol. 16:4996.[Abstract]
  50. Katagiri, T., Ogimoto, M., Hasegawa, K., Mizuno, K. and Yakura, H. 1995. Selective regulation of Lyn tyrosine kinase by CD45 in immature B cells. J. Biol. Chem. 270:27987.[Abstract/Free Full Text]
  51. Ohashi, P. S., Pircher, H., Bürki, K., Zinkernagel, R. M. and Hengartner, H. 1990. Distinct sequence of negative or positive selection implied by thymocyte T-cell receptor densities. Nature 346:861.[ISI][Medline]
  52. Ghendler, Y., Hussey, R. E., Witte, T., Mizoguchi, E., Clayton, L. K., Bhan, A. K., Koyasu, S., Chang, H.-C. and Reinherz, E. L. 1997. Double-positive T cell receptorhigh thymocytes are resistant to peptide/major histocompatibility complex ligand-induced negative selection. Eur. J. Immunol. 27:2279.[ISI][Medline]
  53. Ericsson, P. O. and Teh, H. S. 1995. The protein tyrosine kinase p56lck regulates TCR expression and T cell selection. Int. Immunol. 7:617.[Abstract]
  54. D'Oro, U., Vacchio, M. S., Weissman, A. M. and Ashwell, J. D. 1997. Activation of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lysosomal degradation. Immunity 7:619.[ISI][Medline]
  55. Penit, C. 1990. Positive selection is an early event in thymocyte differentiation: high TCR expression by cycling immature thymocytes precedes final maturation by several days. Int. Immunol. 2:629.[ISI][Medline]
  56. Lucas, B., Vasseur, F. and Penit, C. 1993. Normal sequence of phenotypic transitions in one cohort of 5-bromo-2'-deoxyuridine-pulse-labeled thymocytes. J. Immunol. 151:4574.[Abstract/Free Full Text]
  57. Lesage, S., Steff, A.-M., Philippoussis, F., Pagé, M., Trop, S., Mateo, V. and Hugo, P. 1997. CD4+CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway. J. Immunol. 159:4762.[Abstract]
  58. Novak, T. J., Farber, D., Leitenberg, D., Hong, S., Johnson, P. and Bottomly, K. 1994. Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition. Immunity 1:109.[ISI][Medline]
  59. Wallace, V. A., Fung-Leung, W., Timms, E., Gray, D., Kishihara, K., Loh, D. Y., Penninger, J. and Mak, T. W. 1992. CD45RA and CD45RBhigh expression induced by thymic selection events. J. Exp. Med. 176:1657.[Abstract]
  60. Chen, D. and Rothenberg, E. V. 1993. Molecular basis for developmental changes in interleukin-2 gene inducibility. Mol. Cell. Biol. 13:228.[Abstract]
  61. Rothenberg, E. V. and Ward, S. B. 1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc. Natl Acad. Sci. USA 93:9358.[Abstract/Free Full Text]