Immunology Letters, 26 (1990) 99-109
Elsevier IMLET 01480
Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells Takehito Sato 1, 2, H i d e k a z u T a m a u c h i 3, H i d e o Yagita 4, Yoji A r a t l, Ko O k u m u r a 4 and Sonoko Habu 2 IDepartment of Pharmaceutical Sciences, University of Tokyo, 2Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, 3Department of Microbiology, Kitasato University School of Medicine, Sagamihara, and 4Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
(Received 6 June 1990; accepted 28 June 1990)
1. Summary To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1 ÷ CD3T C R - cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1 ÷ CD3 ÷ cells from normal thymus or spleen did show a response to them. Since Thy-l-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and antiCD3-e (2Cll). Under the presence of PMA or IL-2, accessory cell-depleted splenic T ceils were cultured with G7 or 2Cll. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2Cll. IL-2, however, showed
Key words: Mouse; Thy-1; CD3; Phorbol myristate acetate;
Interleukin-2 Correspondence to: SonokoHabu, Departmentof Immunology, TokaiUniversity,Boseidai, Isehara, KanagawaPrefecture,Japan. Abbreviations: FCS, fetal calf serum; FITC, fluorescein
isothiocyanate; [3H]Tdr, [3H]thymidine;IL-2R, IL-2 receptor; mAb, monoclonal antibody; NWC, nylon wool column; PE, phycoerythrin;PI, phosphatidylinositol;PKC, protein kinase C; PMA, phorbol myristateacetate; TCR, T cell receptor
reciprocal effect on the proliferation of G7 and 2C11treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-e may provide a distinct intracellular pathway for T cell activation. 2. Introduction In the intrathymic differentiation pathway, T cell antigen receptor (TCR) plays an important role in the generation of mature T cells. In the early stage before TCR expression on thymocytes, it is unclear what molecules on the immature thymocytes are involved for their proliferation and differentiation. Ontogenic studies o f murine thymus showed that CD2, Thy-1 and IL-2 receptor (IL-2R) are cell surface molecules on the most immature embryo thymocytes before TCR is expressed [1-3]. Although there is increasing evidence that these molecules may transduce activation signals into mature T fells [4-10], it is unclear whether they are involved in intrathymic differentiation at an early stage. In Thy-l-mediated activation, a requirement for CD3 co-expression has been reported in studies of Thy-1 gene-transfected human lymphomas or murine hybridomas [11, 12] and the presence of the Thy1-specific signal transduction is still controversial. To better understand immature thymocyte differentiation, we examined Thy-l-mediated activation in mature and immature T cells in relation to CD3mediated activation.
0165-2478 / 90 / $ 3.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
99
3. Materials and Methods
4. Results and Discussion
3.1. Mice
To evaluate whether Thy-1 acts as a signal transduction molecule in the immature T cells, the proliferative potential of Thy-1 + cells was determined by [3H]TdR incorporation of these cells in culture with mAb G7 (anti-Thy-1). As source of ThyI+CD3 - cells, thymocytes from BALB/c 15-day embryos and SCID adult mice were used and their proliferative responses were compared with those of normal adult thymocytes. After 3 days of culture with G7 plus PMA, normal adult thymocytes responded with proliferation, but Thy-I+CD3 thymocytes from embryos and SCID did not (Fig. 1). However, the immature thymocytes from embryos and SCID showed high thymidine incorporation by IL-2 (data not shown). The observation implies that the immature Thy-1 +CD3- thymocytes are not activated by a Thy-l-mediated signal but are induced for proliferation by IL-2, through IL-2R which are physiologically expressed on their cell surface. We further examined proliferative response of Thy1 + CD3- spleen cells of athymic nude mice. The nude spleen contains a certain proportion of Thy1 + cells, which are divided into CD3- and CD3 +
BALB/c normal and athymic nude mice were obtained from Japan CLEA (Tokyo, Japan). They were maintained under specific pathogen-free conditions in our own colony. SCID mice were kindly provided by M. Bosma, Institute for Cancer Research, Philadelphia. We maintained the progeny under specific pathogen-free conditions in the Central Institute for Experimental Animals, Kawasaki, Japan. 3.2. Antibodies Biotin or fluorescein isothiocyanate (FITC)labeled anti-Thy-l.2 (30-H12) and PE-labeled streptavidin were purchased from Becton Dickinson (Mountain View, CA). Anti-CD3-E (145-2Cll) were kindly donated by J. Bluestone [13] and were labeled with FITC in our own laboratory. Anti-Thy-1 (G7) were generously provided by E. Shevach [6, 7]. 3.3. Cell preparation and proliferation assay Spleen cells were applied to a nylon wool column (NWC) and passed fractions were collected by washing with RPMI-1640 medium containing 10°70 fetal calf serum (FCS). In some experiments, these cells were stained with labeled antibodies and sorted out by FACStar (Becton Dickinson). These spleen cells or thymocytes (2 x 104 - 2 x 105 cells/well, as indicated) were cultured in RPMI-1640 containing 10070 FCS, 2 mM glutamine, 1 mM sodium pyruvate, 5 × l0 -5 M 2-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin for 72-120 h in 96well round-bottomed plates in 5070 CO2 at 37°C. Various reagents, such as anti-Thy-1 (G7) ascites, crude purified anti-CD3-e (2Cll), rIL-2 (Takeda Chemical Industries, Osaka, Japan) and PMA (Sigma, St. Louis, MO) at the concentrations indicated were added to the culture. To measure induced proliferation, a standard [3H]thymidine ([3H]TdR) incorporation assay was used. 1 #Ci of [3H]TdR was added to each well, and 18 h after cells were harvested on day 3 or 5, and processed for liquid scintillation counting.
100
a H-TdR incorporation 10 3
10 4
10 5 cpm.
Emb, None PMA G7 + P M A
~-
SClD None PMA G7 + P M A
Normal None PMA G7 + P M A
Fig. 1. Thy-l-mediated proliferative responses of SCID and fetal thymocytes. Embryonic thymocytes prepared from BALB/c at gestation day 15, thymocytes of CB-17 SCID and o f their littermates were cultured (2× 105 cells/well) with x 100 ascites of G7 and P M A (5 ng/ml) for 3 days. Incorporated radioactivity was measured after 18 h pulse with [3H]TdR (l #Ci/well). Data present mean cpm in triplicate and are expressed on a logarithmic scale.
3 H - T d R incorporation 10 3
10 4
10 5
h
cpm.
I
Nude CD3 + None PMA G7 + PMA
I
Nude CD3None PMA G7 + PMA
Normal
I
CO3 +
None PMA G7 + PMA
I
Fig. 2. Thy-l-mediated proliferative response of nude and normal Thy-l-positive (Thy-1 +) splenocytes. Spleen cells of 8-weekold BALB/c nude and normal female mice were prepared and passed through a nylon wool column (NWC). After staining with FITC-anti-CD3-e (2Cll), biotin-anti Thy-l.2 and PEstreptavidin, they were isolated into Thy-I +CD3 + and Thy1 + C D 3 - cells by FACStar. These cells (2x104 cells/well) were incubated with × 500 dilution o f G7 ascites and 5 ng/ml of P M A for 5 days. Incorporated radioactivity was measured as in Fig. 1.
The two populations of nude splenic cells stained with two labeled mAbs, anti-Thy-1 (30-H12, this mAb was not mitogenic) and anti-CD3-e (2Cll), and were separated by FACStar. These separated cells were cultured with G7 plus PMA and were compared for their [3H]TdR incorporation with those of Thy1+ CD3 ÷ normal splenic cells. As shown in Fig. 2, Thy-1+ CD3- cells in nude mice did not respond to G7 plus PMA, whereas the nude mouse Thy-1 +CD3 + cells showed proliferation to G7 plus PMA, although the response was less than that of normal mice. The non-responsiveness of the Thy-1 ÷ CD3- cells to G7 plus PMA may be due to their immaturity or a developmental defect. Alternatively, as proposed in the CD2 activation pathway, in which some component of CD3-TCR complex is required as a transducer molecule for the mitogenesis of T cells [14-16], CD3 co-expression may be required for Thy-l-mediated activation [6, 7]; antibodies binding to Thy-1 antigen may induce some perturbation of the CD3 complex which may be located in the vicinity of Thy-l. If this is the case, the CD3 + cells of the nude and normal mice cultured with PMA would show [3H]TdR incorporation as much as that of G7 plus PMA-cultured CD3 + cells, because the separated CD3 ÷ cells may
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Fig. 3. G7 and 2Cll-mediated proliferative responses of Thy-l-positive splenocytes. Spleen cells were prepared from 8-week-old BALB/c female mice and passed through an NWC. After staining with FITC-anti Thy-l.2 (30-H12; this antibody was not mitogenic in our system), they were isolated into Thy-1 + cells by FACStar. The sorted Thy-1 + cells (2× 104 cells/well) were incubated with varying concentrations of G7 (left) or 2Cll (right) in combination with 5 ng/ml PMA ( o ), 20 ng/ml rIL-2 ( • ), 50 ng/ml rII-2 ( a ) or none ( • ). Incorporated radioactivity was measured at day 5 after 18 h pulse with [3H]thymidine (1/~Ci/well). Data represent mean cpm in triplicate.
101
be stimulated by 2Cll for pre-staining. However, as shown in Fig. 2, co-culture o f CD3-stained cells with P M A did not show proliferation without the presence o f G7, retaining the possibility that an intracellular pathway mediated by Thy-1 and CD3 is distinguished. In order to decide the issue, we c o m p a r e d the proliferative response o f mature T ceils cultured with dose-varied G7 and 2 C l l (Fig. 3). As mature T cells, Thy-1 + spleen cells were separated by FACStar from N W C - p a s s e d spleen ceils stained with anti-Thy-1 (30-H12), but not with 2Cll. In the absence o f accessory cells, neither G7 alone nor 2Cll alone induced a notable increase in [3H]TdR incorporation in a 5day culture o f Thy-1 + splenic cells. The addition o f P M A to G7 or IL-2 to 2 C l l in the culture was able to induce a marked proliferative response, however, the reciprocal c o m b i n a t i o n o f G7 plus IL-2 or 2Cll plus P M A failed to show m u c h increase in [3H]TdR incorporation. Similar results were obtained in both C D 4 + and CD8 + spleen cells, a l t h o u g h C D 4 + cells were activated more t h a n CD8 + cells (data not shown). These results showing the distinct requirement o f IL-2 for the culture with 2 C l l or G7 indicate that the CD3-mediated activation causes IL-2 responsiveness, but that Thy-l-mediated activation does not. It has been reported that anti-CD3 a n t i b o d y induced phosphatidylinositol (PI) turnover and subsequent C-kinase (PKC) activation [17]. There is also evidence to suggest that IL-2 responsiveness may be induced in association with PKC activation via PI breakdown [18]. On the other hand, the present observation showing P M A requirement for T cell activation by G7 implies that Thy-l-mediated triggering does not induce PKC activation in T cells. Taken together, it is suggested that the signals triggered via CD3 and Thy-1 may induce distinct intracellular events. In fact, by use o f the cell monitoring system, in which Ca 2+ mobilization can be observed at single-cell level, the intracellular Ca 2+ concentration o f spleen cells did not change when 2Cll was added, but it did increase when G7 was added (unpublished data). These data do not seem to be inconsistent with the assumption that Thy-l-mediated signals are transduced t h r o u g h the CD3 complex. According to Sussm a n et al. [19], 2 C l l is capable o f P I catalyzation, and IL-2 p r o d u c t i o n is induced in a m u t a n t clone 102
lacking CD3-~" but G7 is not. In addition, recent reports showed that CD3-~'is able to transduce CD2mediated signal on C D 3 - c - cells [20]. Since the m o n o c l o n a l a n t i b o d y 2 C l l is known to react to the c subunit o f the CD3 but not to other subunits [13], it may be possible that G7 binding to Thy-1 induces the perturbation o f CD3, which causes signal transduction via the ~-subunit, not the e subunit, and the signal via c m a y induce PKC activation and IL-2 responsiveness, whereas the signal via ~"may induce Ca 2+ mobilization. The majority o f S C I D t h y m o cytes express neither CD3-TCR nor CD2 [21] but they showed expression o f ~'-chain messenger R N A (manuscript in preparation). Thus, these C D 2 S C I D thymocytes may provide a g o o d model to study a distinct Thy-l-mediated activation pathway.
Acknowledgements We t h a n k Mieko Takei, N a o m i Tsukahara and N o b u m a s a Kobayashi for the FACStar operation, and C h i h a r u Takei, Hiroshi I w a m o t o and Masato Fujii for their technical assistance. We are also grateful to Dr. Takashi Nishimura for helpful discussion. This work was supported by a Grant-in-Aid from the Ministry o f Education and Science, Japan.
References [1] Yagita, H., Asakawa, J., Tansyo, S., Nakamura, T., Habu, S. and Okumura, K. (1989) Eur. J. Immunol. 19, 2211. [2] Habu, S., Okumura, K., Diamantstein, T. and Shevach, E. (1985) Eur. J. Immunol. 15, 456. [3] Takacs, L., Osawa, H. and Diamantstein, T. (1984) Eur. J. Immunol. 14, 1152. [4] Meuer, S. C., Hussey, R. E., Fabbi, M., Fox, D., Acuto, O., Fitzgerald, K. A., Hodgdon, J. C., Protentis, J. P., Schlossman, S. E and Reinherz, E. L. (1984) Cell 36, 897. [5] Leonard, W. J., Depper, J. M., Uchiyama, Y., Smith, K., Waldmann, T. A. and Greene, W. C. (1982)Nature 300, 267. [6] Gunter, K. C., Malek, T. R. and Shevach, E. M. (1984) J. Exp. Med. 159, 716. [7] Kroczek, R. A., Gunter, K. C., Seligmann, B. and Shevach, E. M. (1986) J. Immunol. 136, 4379. [8] MacDonald, H. R., Bron, C., Rousseaux, M., Horvath, C. and Cerottini, J. C. (1985) Eur. J. Immunol. 15, 495. [9] Pont, S., Regnier-Vigouroux, A., Naquet, R, Blanc, D., Pierres, A., Marchetto, S. and Pierres, M. (1985) Eur. J. lmmunol. 15, 1222. [10] Kroczek, R.A., Gunter, K.C., Germain, R.N. and Shevach, E. M. (1986) Nature 322, 181. [11] Gunter, K. C., Germain, R. N., Kroczek, R. A., Saito, T., Yokoyama, W. M., Chan, C., Weiss, A. and Shevach, E. M.
(1987) Nature 326, 505. [12] Sussman, J. J., Saito, T., Shevach, E. M., Germain, R. N. and Ashwell, J. D. (1988) J. Immunol. 140, 2520. [13] Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. and Bluestone, J. A. (1987) Proc. Natl. Acad. Sci. USA 84, 1374. [14] Breitmeyer, J. B., Daley, J. E, Levine, H. B. and Schlossman, S. E (1987) J. Immunol. 139, 2899. [15] Alcover, A., Chang, H. C., Sayre, E H., Hussey, R. E. and Reinherz, E. L. (1988) Eur. J. Immunol. 18, 363. [16] Alcover, A., Alberini, C., Acuto, O., Clayton, L. K., Transy, C., Spagnoli, G. C., Moingeon, P., Lopez, E and Reinherz,
E. L. (1988) EMBO J. 7, 1973. [17] Imboden, J. B. and Stobo, J. D. (1985) J. Exp. Med. 161,446. [18] Kakiuchi, T., Mizuguchi, J. and Nariuchi, H. (1988) J. Immunol. 141, 3278. [19] Sussman, J. J., Bonifacino, J. S., Lippincott-Schwartz, J., Weissman, A. M., Saito, T., Klausner, R. D. and Ashwell, J. D. (1988) Cell 52, 85. [20] Anderson, P., Caligiuri, M., Ritz, J. and Schlossman, S. E (1989) Nature 341, 159. [21] Habu, S., Norihisa, Y., Sato, T., Yagita, H. and Okumura, K. (1989) Curr. Topics Microbiol. Immunol. 152, 27.
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Elsevier IMLET 01480
Dissociation of signal transduction via Thy-1 and CD3 antigens in murine T cells Takehito Sato 1, 2, H i d e k a z u T a m a u c h i 3, H i d e o Yagita 4, Yoji A r a t l, Ko O k u m u r a 4 and Sonoko Habu 2 IDepartment of Pharmaceutical Sciences, University of Tokyo, 2Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, 3Department of Microbiology, Kitasato University School of Medicine, Sagamihara, and 4Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan
(Received 6 June 1990; accepted 28 June 1990)
1. Summary To understand the proliferation/differentiation of immature thymocytes which have not express T cell antigen receptor (TCR), we studied whether Thy-1 has signal-transducing capacity. Thy-1 ÷ CD3T C R - cells including thymocytes from BALB/c embryos and SCID mice and nude mouse splenic cells did not show proliferative responses in the culture with anti-Thy-1 (G7) plus phorbol myristate acetate (PMA), whereas Thy-1 ÷ CD3 ÷ cells from normal thymus or spleen did show a response to them. Since Thy-l-mediated activation is suggested to require co-expression of the CD3-TCR complex, we compared the T cell proliferative response in mature T cells stimulated with anti-Thy-1 (G7) and antiCD3-e (2Cll). Under the presence of PMA or IL-2, accessory cell-depleted splenic T ceils were cultured with G7 or 2Cll. PMA augmented the proliferative response of splenic T cells cultured with G7 much more than that with 2Cll. IL-2, however, showed
Key words: Mouse; Thy-1; CD3; Phorbol myristate acetate;
Interleukin-2 Correspondence to: SonokoHabu, Departmentof Immunology, TokaiUniversity,Boseidai, Isehara, KanagawaPrefecture,Japan. Abbreviations: FCS, fetal calf serum; FITC, fluorescein
isothiocyanate; [3H]Tdr, [3H]thymidine;IL-2R, IL-2 receptor; mAb, monoclonal antibody; NWC, nylon wool column; PE, phycoerythrin;PI, phosphatidylinositol;PKC, protein kinase C; PMA, phorbol myristateacetate; TCR, T cell receptor
reciprocal effect on the proliferation of G7 and 2C11treated splenic T cells. These data suggest that signals triggered via Thy-1 and CD3-e may provide a distinct intracellular pathway for T cell activation. 2. Introduction In the intrathymic differentiation pathway, T cell antigen receptor (TCR) plays an important role in the generation of mature T cells. In the early stage before TCR expression on thymocytes, it is unclear what molecules on the immature thymocytes are involved for their proliferation and differentiation. Ontogenic studies o f murine thymus showed that CD2, Thy-1 and IL-2 receptor (IL-2R) are cell surface molecules on the most immature embryo thymocytes before TCR is expressed [1-3]. Although there is increasing evidence that these molecules may transduce activation signals into mature T fells [4-10], it is unclear whether they are involved in intrathymic differentiation at an early stage. In Thy-l-mediated activation, a requirement for CD3 co-expression has been reported in studies of Thy-1 gene-transfected human lymphomas or murine hybridomas [11, 12] and the presence of the Thy1-specific signal transduction is still controversial. To better understand immature thymocyte differentiation, we examined Thy-l-mediated activation in mature and immature T cells in relation to CD3mediated activation.
0165-2478 / 90 / $ 3.50 © 1990 ElsevierSciencePublishers B.V.(BiomedicalDivision)
99
3. Materials and Methods
4. Results and Discussion
3.1. Mice
To evaluate whether Thy-1 acts as a signal transduction molecule in the immature T cells, the proliferative potential of Thy-1 + cells was determined by [3H]TdR incorporation of these cells in culture with mAb G7 (anti-Thy-1). As source of ThyI+CD3 - cells, thymocytes from BALB/c 15-day embryos and SCID adult mice were used and their proliferative responses were compared with those of normal adult thymocytes. After 3 days of culture with G7 plus PMA, normal adult thymocytes responded with proliferation, but Thy-I+CD3 thymocytes from embryos and SCID did not (Fig. 1). However, the immature thymocytes from embryos and SCID showed high thymidine incorporation by IL-2 (data not shown). The observation implies that the immature Thy-1 +CD3- thymocytes are not activated by a Thy-l-mediated signal but are induced for proliferation by IL-2, through IL-2R which are physiologically expressed on their cell surface. We further examined proliferative response of Thy1 + CD3- spleen cells of athymic nude mice. The nude spleen contains a certain proportion of Thy1 + cells, which are divided into CD3- and CD3 +
BALB/c normal and athymic nude mice were obtained from Japan CLEA (Tokyo, Japan). They were maintained under specific pathogen-free conditions in our own colony. SCID mice were kindly provided by M. Bosma, Institute for Cancer Research, Philadelphia. We maintained the progeny under specific pathogen-free conditions in the Central Institute for Experimental Animals, Kawasaki, Japan. 3.2. Antibodies Biotin or fluorescein isothiocyanate (FITC)labeled anti-Thy-l.2 (30-H12) and PE-labeled streptavidin were purchased from Becton Dickinson (Mountain View, CA). Anti-CD3-E (145-2Cll) were kindly donated by J. Bluestone [13] and were labeled with FITC in our own laboratory. Anti-Thy-1 (G7) were generously provided by E. Shevach [6, 7]. 3.3. Cell preparation and proliferation assay Spleen cells were applied to a nylon wool column (NWC) and passed fractions were collected by washing with RPMI-1640 medium containing 10°70 fetal calf serum (FCS). In some experiments, these cells were stained with labeled antibodies and sorted out by FACStar (Becton Dickinson). These spleen cells or thymocytes (2 x 104 - 2 x 105 cells/well, as indicated) were cultured in RPMI-1640 containing 10070 FCS, 2 mM glutamine, 1 mM sodium pyruvate, 5 × l0 -5 M 2-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin for 72-120 h in 96well round-bottomed plates in 5070 CO2 at 37°C. Various reagents, such as anti-Thy-1 (G7) ascites, crude purified anti-CD3-e (2Cll), rIL-2 (Takeda Chemical Industries, Osaka, Japan) and PMA (Sigma, St. Louis, MO) at the concentrations indicated were added to the culture. To measure induced proliferation, a standard [3H]thymidine ([3H]TdR) incorporation assay was used. 1 #Ci of [3H]TdR was added to each well, and 18 h after cells were harvested on day 3 or 5, and processed for liquid scintillation counting.
100
a H-TdR incorporation 10 3
10 4
10 5 cpm.
Emb, None PMA G7 + P M A
~-
SClD None PMA G7 + P M A
Normal None PMA G7 + P M A
Fig. 1. Thy-l-mediated proliferative responses of SCID and fetal thymocytes. Embryonic thymocytes prepared from BALB/c at gestation day 15, thymocytes of CB-17 SCID and o f their littermates were cultured (2× 105 cells/well) with x 100 ascites of G7 and P M A (5 ng/ml) for 3 days. Incorporated radioactivity was measured after 18 h pulse with [3H]TdR (l #Ci/well). Data present mean cpm in triplicate and are expressed on a logarithmic scale.
3 H - T d R incorporation 10 3
10 4
10 5
h
cpm.
I
Nude CD3 + None PMA G7 + PMA
I
Nude CD3None PMA G7 + PMA
Normal
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Fig. 2. Thy-l-mediated proliferative response of nude and normal Thy-l-positive (Thy-1 +) splenocytes. Spleen cells of 8-weekold BALB/c nude and normal female mice were prepared and passed through a nylon wool column (NWC). After staining with FITC-anti-CD3-e (2Cll), biotin-anti Thy-l.2 and PEstreptavidin, they were isolated into Thy-I +CD3 + and Thy1 + C D 3 - cells by FACStar. These cells (2x104 cells/well) were incubated with × 500 dilution o f G7 ascites and 5 ng/ml of P M A for 5 days. Incorporated radioactivity was measured as in Fig. 1.
The two populations of nude splenic cells stained with two labeled mAbs, anti-Thy-1 (30-H12, this mAb was not mitogenic) and anti-CD3-e (2Cll), and were separated by FACStar. These separated cells were cultured with G7 plus PMA and were compared for their [3H]TdR incorporation with those of Thy1+ CD3 ÷ normal splenic cells. As shown in Fig. 2, Thy-1+ CD3- cells in nude mice did not respond to G7 plus PMA, whereas the nude mouse Thy-1 +CD3 + cells showed proliferation to G7 plus PMA, although the response was less than that of normal mice. The non-responsiveness of the Thy-1 ÷ CD3- cells to G7 plus PMA may be due to their immaturity or a developmental defect. Alternatively, as proposed in the CD2 activation pathway, in which some component of CD3-TCR complex is required as a transducer molecule for the mitogenesis of T cells [14-16], CD3 co-expression may be required for Thy-l-mediated activation [6, 7]; antibodies binding to Thy-1 antigen may induce some perturbation of the CD3 complex which may be located in the vicinity of Thy-l. If this is the case, the CD3 + cells of the nude and normal mice cultured with PMA would show [3H]TdR incorporation as much as that of G7 plus PMA-cultured CD3 + cells, because the separated CD3 ÷ cells may
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Fig. 3. G7 and 2Cll-mediated proliferative responses of Thy-l-positive splenocytes. Spleen cells were prepared from 8-week-old BALB/c female mice and passed through an NWC. After staining with FITC-anti Thy-l.2 (30-H12; this antibody was not mitogenic in our system), they were isolated into Thy-1 + cells by FACStar. The sorted Thy-1 + cells (2× 104 cells/well) were incubated with varying concentrations of G7 (left) or 2Cll (right) in combination with 5 ng/ml PMA ( o ), 20 ng/ml rIL-2 ( • ), 50 ng/ml rII-2 ( a ) or none ( • ). Incorporated radioactivity was measured at day 5 after 18 h pulse with [3H]thymidine (1/~Ci/well). Data represent mean cpm in triplicate.
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be stimulated by 2Cll for pre-staining. However, as shown in Fig. 2, co-culture o f CD3-stained cells with P M A did not show proliferation without the presence o f G7, retaining the possibility that an intracellular pathway mediated by Thy-1 and CD3 is distinguished. In order to decide the issue, we c o m p a r e d the proliferative response o f mature T ceils cultured with dose-varied G7 and 2 C l l (Fig. 3). As mature T cells, Thy-1 + spleen cells were separated by FACStar from N W C - p a s s e d spleen ceils stained with anti-Thy-1 (30-H12), but not with 2Cll. In the absence o f accessory cells, neither G7 alone nor 2Cll alone induced a notable increase in [3H]TdR incorporation in a 5day culture o f Thy-1 + splenic cells. The addition o f P M A to G7 or IL-2 to 2 C l l in the culture was able to induce a marked proliferative response, however, the reciprocal c o m b i n a t i o n o f G7 plus IL-2 or 2Cll plus P M A failed to show m u c h increase in [3H]TdR incorporation. Similar results were obtained in both C D 4 + and CD8 + spleen cells, a l t h o u g h C D 4 + cells were activated more t h a n CD8 + cells (data not shown). These results showing the distinct requirement o f IL-2 for the culture with 2 C l l or G7 indicate that the CD3-mediated activation causes IL-2 responsiveness, but that Thy-l-mediated activation does not. It has been reported that anti-CD3 a n t i b o d y induced phosphatidylinositol (PI) turnover and subsequent C-kinase (PKC) activation [17]. There is also evidence to suggest that IL-2 responsiveness may be induced in association with PKC activation via PI breakdown [18]. On the other hand, the present observation showing P M A requirement for T cell activation by G7 implies that Thy-l-mediated triggering does not induce PKC activation in T cells. Taken together, it is suggested that the signals triggered via CD3 and Thy-1 may induce distinct intracellular events. In fact, by use o f the cell monitoring system, in which Ca 2+ mobilization can be observed at single-cell level, the intracellular Ca 2+ concentration o f spleen cells did not change when 2Cll was added, but it did increase when G7 was added (unpublished data). These data do not seem to be inconsistent with the assumption that Thy-l-mediated signals are transduced t h r o u g h the CD3 complex. According to Sussm a n et al. [19], 2 C l l is capable o f P I catalyzation, and IL-2 p r o d u c t i o n is induced in a m u t a n t clone 102
lacking CD3-~" but G7 is not. In addition, recent reports showed that CD3-~'is able to transduce CD2mediated signal on C D 3 - c - cells [20]. Since the m o n o c l o n a l a n t i b o d y 2 C l l is known to react to the c subunit o f the CD3 but not to other subunits [13], it may be possible that G7 binding to Thy-1 induces the perturbation o f CD3, which causes signal transduction via the ~-subunit, not the e subunit, and the signal via c m a y induce PKC activation and IL-2 responsiveness, whereas the signal via ~"may induce Ca 2+ mobilization. The majority o f S C I D t h y m o cytes express neither CD3-TCR nor CD2 [21] but they showed expression o f ~'-chain messenger R N A (manuscript in preparation). Thus, these C D 2 S C I D thymocytes may provide a g o o d model to study a distinct Thy-l-mediated activation pathway.
Acknowledgements We t h a n k Mieko Takei, N a o m i Tsukahara and N o b u m a s a Kobayashi for the FACStar operation, and C h i h a r u Takei, Hiroshi I w a m o t o and Masato Fujii for their technical assistance. We are also grateful to Dr. Takashi Nishimura for helpful discussion. This work was supported by a Grant-in-Aid from the Ministry o f Education and Science, Japan.
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