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Single-cell observation of calcium signals in T cells and antigen-presenting cells during antigen presentation
Immunology Letters, 46 (1995) 75-79 0165-2478/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved
IMLET 2361
Single-cell observation of calcium signals in T cells and antigen-presenting cells during antigen presentation Chikako
Torigoe
a9* , Takushi
Tadakuma
b and Mamoru
Nakanishi
a
aFaculty of Pharmaceutical Sciences, Nagoya City Unioersi&, Tanabe-don’, Mizuho-ku, Nagoya 467, Japan; b Department of Microbiology, School of Medicine, Keio UrGersi&, Shinjuku-ku, Tokyo 160, Japan
(Received 22 November 1994; revised 18 January 1995; accepted 31 January 1995) Key words: Antigen presentation; Calcium signal; T cell-APC interaction; MHC class II molecule
1. Summary Intracellular calcium ion mobilization in T-cell hybridomas and antigen-presenting cells (APC) during the interaction was observed using confocal fluorescence microscopy. No calcium signal was detected in nonactivated T-cell hybridomas by antigen presentation. However, in activated T-cell hybridomas, intracellular calcium ion concentration rapidly increased by antigen presentation and thereafter apoptosis was induced. On the contrary, during the interaction with T-cell hybridomas, calcium signal was induced in APCs irrespective of the activation of T-cell hybridomas. Chemical modification of APCs with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, which is known to induce T-cell unresponsiveness during antigen presentation, inhibited cap formation of surface MHC class II molecules and suppressed calcium signals during the interaction with T-cell hybridomas.
2. Introduction Antigen presentation is one of the most important processes in immunological events. Direct observation of the initial signal transduction in T cells and APC (antigen-presenting cells) during the interaction has, however, not been conducted because the methods for direct investigation of cell-cell interactions have been quite limited [l]. Antigen presentation causes activation-driven cell death in T-cell hybridomas [2], which is a model of clonal deletion in T-cell differentiation, and antigen Abbreviations: AF’C, antigen-presenting cell; ECDI, 1-ethyl-3-(3-di-
methylaminopropyl) carbodiimide; mAb, monoclonal antibody. * Corresponding author: Chikako Torigoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan. Tel.: (81) 52-8363413; Fax: (81) 52-8349309. SSDI 0165-2478(95)00023-2
presentation with chemically cross-linked APC induces T-cell unresponsiveness 13-51, which is a model of clonal anergy. Calcium ions are thought to be critically responsible for the induction of activation-driven cell death [6], presumably including the activation of some endonucleases [7]. However, calcium signals in T cells during antigen presentation have not been clarified in the context of activation-driven cell death. Cross-linking of MHC antigens on the surface of APC with antibodies causes a rise in intracellular free calcium ion concentration [8,9]. However, calcium signals in APC during the interaction with T cells and antigens has not been reported. We, therefore, studied intracellular calcium signals both in T-cell hybridomas 2-45-12, which specifically recognize azobenzene arsonate+-tyrosine, and APC during the interaction, using confocal fluorescence microscopy. We also clarified the difference in calcium signals between intact and chemically cross-linked APC and assessed the mobility of MHC class II molecules on the cell surface.
3. Materials and Methods
3.1. Reagents ECDI (Sigma) is a condensing reagent of amino and carboxyl groups. Fluo-3-AM was obtained from Dojindo (Kumamoto, Japan). 3.2. Cells Azobenzenearsonate-L-tyrosine-specific T-cell hybridoma was established by fusion of azobenzenearsonate-L-tyrosine-specific T-cell line Al.8 cells and AKR thymoma BW5147 cells [lo]. Cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY) with 75
10% fetal calf serum (FCS; Bocknek, Canada), 5 X 10e5 M 2-ME and antibiotics. BlO.BR/sgsn mice were obtained from Nihon SLC (Shizuoka, Japan) and were used at the age of 6-8 weeks for preparation of splenocytes as APC. Preparation of ECDI-treated splenocytes is described in [3]. 3.3. Antibodies hybridomas [ 111 were Anti-CD3 mAb 1452Cll kindly provided by Dr. Y. Asano. FITC-labeled goat IgG against mouse IgG was obtained from MBL (Nagoya, Japan). FITC-labeled Fab fragments of sheep IgG against mouse IgG were obtained from Biological Specialty (USA). Goat IgG against hamster IgG was obtained from Organon Teknica (Durham, NC). Activation of T cells with cross-linked mAb has been previously described [12].
laser; fluorescence emission was observed above 515 nm. Temperature of the observation chamber was kept at 37°C.
4. Results 4.1. Increase of intracellular calcium ion concentration in response to antigen presentation Typical patterns of the change of intracellular calcium ion concentration in T-cell hybridomas in response to stimulations are shown in Fig. 1. In nonactivated T-cell hybridomas, no change was observed in response to antigen and APC (Fig. la). However, a rapid increase in the calcium ion concentration was observed by cross-linking of TCR/CD3 complex with anti-CD3 mAb (Fig. lb). On the contrary, after activa-
3.4. Antigen presentation T-cell hybridomas (6 X 104) were cultured with 6 X lo6 erythrocyte-depleted splenocytes which were treated with mitomycin C (Sigma) (40 pg/ml, 30 min) in Corning 24-well plates. Azobenzenearsonate-L-tyrosine was added to 0.1 mM. After 2 days of antigen presentation, almost all APC treated with mitomycin C were dead and could be easily separated from surviving T-cell hybridomas using the discontinuous density gradient of Percoll. This T-cell hybridoma could also be discriminated from APC by its diameter (N 3 times larger than that of APC). In the case of hapten molecules, pre-loading of APC with antigen is not necessary and re-stimulation was conducted as follows: (a) a mixture of antigen and APC was added and (b) antigen and APC were added separately (the order and time lag of addition were varied). No difference was observed by changing the manner of stimulation. Stimulation of APC with antigen and T-cell hybridomas was conducted in the same manner.
W
(a)
3-
I.
0
loo
200
loo
200
300
Time (see)
3.5. Measurement of intracellular calcium ion and imaging of cell-surface proteins Confocal fluorescence microscopic images of T-cell hybridomas and of APC were taken under a confocal fluorescence microscope system (MRC-600, Bio-Radl with an inverted epifluorescence microscope (TMDEFQ, Nikon) [13]. The pinhole aperture in the detection path of the confocal microscope was 3 mm and we used glycerine-immersed 1.4 NA (numerical aperture) 60 X Nikon Planapo and 0.85 NA 40 X Nikon Fluor objectives. Fluo-3-loaded T-cell hybridomas or FITC-labeled antibodies were excited at 488 nm using an argon ion 76
Fig. 1. Typical time courses of intracellular calcium ion concentration in individual T-cell hybridomas after stimulation. Each stimulation was given at the time indicated by an arrow. a: non-activated T-cell hybridoma stimulated with antigen and APC; non-activated T-cell hybridomas did not respond. b: non-activated T-cell hybridoma stimulated with anti-CD3 antibodies. About 60% of the ceils responded. Here, T-cell hybridomas were pre-incubated with anti-CD3 mAb and, after washing out excess mAb, goat IgG against hamster IgG was added at the indicated time. c-E activated T-cell hybridoma, which was pre-incubated for 2 days with antigen and APC, re-stimulated with antigen and APC. Time lag and pattern of calcium ion mobilization were different in each cell and typical examples are shown. About two-thirds of responding cells showed a large and transient increase in calcium signal as observed in (d) and (f). One-third showed a small and continuous increase as observed in (c) and (e).
by antigen presentation for 3 days (data not shown). We confirmed that [Ca2+li increase by antigen presentation was also observed in T-cell hybridomas activated by immobilized anti-CD3 antibodies before apoptosis was induced. The important difference between activation by antigen presentation and by immobilized anti-CD3 antibodies was the time course. In the case of immobilized anti-CD3 antibodies, it takes about 1 day to induce Ca2+ uptake by antigen presentation and apoptosis was observed the next day. We suspect that such a time lag might result from the quantitative difference in stimulations.
lb)
(a) _
4.2. Increase of intracellular calcium ion concentration in APC
L.
0
I.
loo
I
.I.
I
I..
loo 200 Time (80~)
200
Fig. 2. Typical time courses of intracellular calcium ion concentration in individual APC during the course of interaction with antigen and T-cell hybridomas. Stimulation was given at the time indicated by an arrow. a: ECDI-treated spienocyte stimulated with antigen and T-cell hybridomas; ECDI-treated splenocytes did usually not respond. b-f: untreated splenocyte stimulated with antigen and T-cell hybridomas; about 20% of untreated splenocytes responded. Each cell responded differently. About two-thirds of the responding cells showed a transient increase like (b), (c) and (e). One-third showed a small and continuous increase like (d) or (f).
tion of T-cell hybridomas by antigen presentation for 2 days, re-stimulation with antigen and APC caused an increase in calcium ion concentration in activated T-cell hybridomas (Fig. lc-f). T-cell hybridomas were not activated with APC without antigen nor antigen without APC (we confirmed that IL-2 production was not observed) and calcium signal was not induced in such T-cell hybridomas during antigen presentation. We observed several hundred cells for each stimulation and confirmed that responding cells to anti-CD3 antibody stimulation were about 60% of both non-activated and activated T-cell hybridomas. Responding cells to antigen presentation were about 20% of activated T-cell hybridomas, whereas less than 1% of non-activated hybridomas responded to antigen presentation. Time lag and the pattern of calcium ion mobilization were slightly different in every cell both in the case of antibody and antigen stimulations. In some responding cells, a small and continuous increase in calcium ion concentration was observed. This T-cell hybridoma is known to die during culture with a high density of immobilized antiCD3 antibodies [ll]. It was found that this cell also dies
Fig. 2 shows typical patterns of the change of intracellular calcium ion concentration in APC during interaction with antigen and non-activated T-cell hybridomas. A rapid increase was observed in untreated APC (Fig. 2b-f), wh ereas change of calcium ion concentration was mostly suppressed in ECDI-treated APC (Fig. 2a). The response rate of ECDI-treated APC was about 2-3%, including marginal response which may possibly result from incomplete ECDI fixation. It should be noted that during the interaction between APC and non-activated T-cell hybtidomas, intracellular calcium ion concentration increased only in APC and not in T-cell hybridomas. We examined several hundred cells and responding cells were about 20% of untreated APC. ECDI-treated APC did not induce a calcium signal in T-cell hybridomas, and increase of cell density was observed instead of long-term unresponsiveness in T-cell hybridomas (data not shown). 4.3. Mobility of MHC class II molecules We then investigated the behavior of surface MHC class II molecules to clarify the origin of the difference between ECDI-treated and untreated APC in calcium ion mobilization during antigen presentation. Fig. 3 shows the distribution of surface MHC class II molecules on APC. Without cross-linking, MHC class II molecules were uniformly distributed on the cell surface both in untreated and ECDI-treated APC as shown in Fig. 3 (Ia and Ib). ECDI-treated APC expressed relatively normal levels of Ia molecules on the surface, as detected by FACS analysis (data not shown). However, by extensive cross-linking with second antibodies, MHC class II molecules were fully cross-linked to form caps in untreated splenocytes (Fig. 3, IIa). On the contrary, on ECDI-treated splenocytes MHC class II molecules could not form caps and still remained uni77
I( a >
Fig. 3. MHC class II molecules on the cell surface were stained and observed by a confocal fluorescence microscope. I: anti-I-Ak mAb + FITC-labeled Fab fragments of sheep IgG against mouse IgG. II: anti-I-A’ mAb + FITC-labeled whole molecules of goat IgG against mouse IgG. a: untreated splenocytes; b: ECDI-treated splenocytes.
formly distributed on the cell surface (Fig. 3, IIb). Thus, it is indicated that the mobility of MHC class II molecules is suppressed by ECDI treatment.
5. Discussion During the interaction between T-cell hybridomas and APC, a calcium signal was observed only in APC and not in T-cell hybridomas. Pre-activation was neces78
sary for the induction of calcium signals by antigen presentation in T-cell hybridomas. The minimum size of receptor aggregation for the induction of an intracellular signal is different depending on cells. For example, receptor dimerization is enough for induction in some cells and in other cells more than trimerization is necessary [14]. We suspect that such a difference may result in the difference of calcium signal transduction between non-activated T-cell hybridomas and APC. Some new proteins, such as B7/BBl, are known to be
expressed on the surface of APC when activated by antigen and T cells [15]. Some of such proteins may possibly be essential for signal transduction to T-cell hybridomas 2-45-12. Cell adhesion molecules such as CD2 are up-regulated and other new membrane proteins are also expressed on the surface of activated T cells [16-181. Such proteins may possibly participate in calcium ion mobilization in T-cell hybridomas during the interaction with antigen and APC. Cross-linking of receptors by antibodies is thought to mimic antigen stimulation. However, we showed that antigen presentation and cross-linking of TCR-CD3 complex by antibodies are quantitatively quite different in intracellular calcium ion mobilization. That is presumably because affinity, steric hindrance, mobility and thereby efficiency of cross-linking of receptors differ from each other. By ECDI-treated APC, antigen presentation induced different signals in T-cell hybridomas. Although the conformation and number of MHC class II molecules were not changed by ECDI treatment as shown by mAb binding, flexibility and mobility of MHC class II molecules on the cell membrane may be markedly changed and these factors are critically important for their aggregation. It is known that T cells stimulated by ECDI-treated APC fail to induce IL-2, but IL-3, IFN-y and IL-2 receptors are partially induced and T-cell receptor j3 mRNA is fully induced [4]. In order to stimulate T cells, cross-linking of TCR, i.e., cross-linking of MHC class II molecules on APC, is necessary. Insufficient cross-linking of MHC class II molecules, and accordingly imperfect cross-linking of TCR-CD3 complex during T cell-APC interactions induced different intracellular signals from those induced by normal antigen presentation.
Acknowledgements This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan.
References [II Nagao,
Y., Yamada, H. and Nakanishi, M. (1992) Immunol. Lett. 34, 151. 121Iwata, M., Hanaoka, S. and Sato, K. (1991) Eur. .I. Immunol. 21, 643. [31 Jenkins, M.K. and Schwartz, R.H. (1987) J. Exp. Med. 165,302. [41 Jenkins, M.K., Pardoll, D.M., Mizuguchi, J., Chused, T.M. and Schwartz, R.H. (1987) Proc. Nat]. Acad. Sci. USA 84, 5409. El Jenkins, M.K., Ashwell, J.D. and Schwartz, R.H. (1988) J. Immunol. 140, 3324. [61McConkey, D.J., HartzelI, P., Amador-Perez, J.F., Orrenius, S. and Jondal, M. (1989) J. Immunol. 143, 1801. 171 Smith, C.A., Williams, G.T., Kingston, R., Jenkinson, E.J. and Owen, J.T. (1989) Nature 337, 181. C., Hivroz. C., Ju, L.-Y. and ml Mooney, N.A., Grillot-Courvalin, Charron, D. (1990) J. Immunol. 145, 2070. 191 Andre, P., Cambier, J.C., Wade, T.K., Raetz, T. and Wade, W.F. (1994) J. Exp. Med. 179, 763. [lOI Saito, S., Tadakuma, T., Inoko, H. and Saito, K. (1988) Mol. Immunol. 25, 569. 1111 Odaka, C., Kizaki, H. and Tadakuma. T. (1990) J. Immunol. 144, 2096. 1121 Leo, O., Foo, M., Sachs, D.H., Samelson, L.E. and Bluestone, J.A. (1987) Proc. Natl. Acad. Sci. USA 84, 1374. 1131 Furuno, T., Hamano, T. and Nakanishi, M. (1993) Biophys. J. 64, 665. [14] Metzger, H. (1992) J. Immunol. 149, 1477. [15] Schwartz, R.H. (1992) Cell 71, 1065. [16] Geppert, T.D. and Lipsky, P.E. (1991) J. Immunol. 146, 3298. [17] Alzona, M., Jack, H.-M., Fisher, R.I. and Ellis, T.M. (1994) J. Immunol. 153, 2861. [18] Izon, D.J., Jones, L.A., Eynon, E.E. and Kruisbeek, A.M. (1994) J. Immunol. 153. 2939.
79
IMLET 2361
Single-cell observation of calcium signals in T cells and antigen-presenting cells during antigen presentation Chikako
Torigoe
a9* , Takushi
Tadakuma
b and Mamoru
Nakanishi
a
aFaculty of Pharmaceutical Sciences, Nagoya City Unioersi&, Tanabe-don’, Mizuho-ku, Nagoya 467, Japan; b Department of Microbiology, School of Medicine, Keio UrGersi&, Shinjuku-ku, Tokyo 160, Japan
(Received 22 November 1994; revised 18 January 1995; accepted 31 January 1995) Key words: Antigen presentation; Calcium signal; T cell-APC interaction; MHC class II molecule
1. Summary Intracellular calcium ion mobilization in T-cell hybridomas and antigen-presenting cells (APC) during the interaction was observed using confocal fluorescence microscopy. No calcium signal was detected in nonactivated T-cell hybridomas by antigen presentation. However, in activated T-cell hybridomas, intracellular calcium ion concentration rapidly increased by antigen presentation and thereafter apoptosis was induced. On the contrary, during the interaction with T-cell hybridomas, calcium signal was induced in APCs irrespective of the activation of T-cell hybridomas. Chemical modification of APCs with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide, which is known to induce T-cell unresponsiveness during antigen presentation, inhibited cap formation of surface MHC class II molecules and suppressed calcium signals during the interaction with T-cell hybridomas.
2. Introduction Antigen presentation is one of the most important processes in immunological events. Direct observation of the initial signal transduction in T cells and APC (antigen-presenting cells) during the interaction has, however, not been conducted because the methods for direct investigation of cell-cell interactions have been quite limited [l]. Antigen presentation causes activation-driven cell death in T-cell hybridomas [2], which is a model of clonal deletion in T-cell differentiation, and antigen Abbreviations: AF’C, antigen-presenting cell; ECDI, 1-ethyl-3-(3-di-
methylaminopropyl) carbodiimide; mAb, monoclonal antibody. * Corresponding author: Chikako Torigoe, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan. Tel.: (81) 52-8363413; Fax: (81) 52-8349309. SSDI 0165-2478(95)00023-2
presentation with chemically cross-linked APC induces T-cell unresponsiveness 13-51, which is a model of clonal anergy. Calcium ions are thought to be critically responsible for the induction of activation-driven cell death [6], presumably including the activation of some endonucleases [7]. However, calcium signals in T cells during antigen presentation have not been clarified in the context of activation-driven cell death. Cross-linking of MHC antigens on the surface of APC with antibodies causes a rise in intracellular free calcium ion concentration [8,9]. However, calcium signals in APC during the interaction with T cells and antigens has not been reported. We, therefore, studied intracellular calcium signals both in T-cell hybridomas 2-45-12, which specifically recognize azobenzene arsonate+-tyrosine, and APC during the interaction, using confocal fluorescence microscopy. We also clarified the difference in calcium signals between intact and chemically cross-linked APC and assessed the mobility of MHC class II molecules on the cell surface.
3. Materials and Methods
3.1. Reagents ECDI (Sigma) is a condensing reagent of amino and carboxyl groups. Fluo-3-AM was obtained from Dojindo (Kumamoto, Japan). 3.2. Cells Azobenzenearsonate-L-tyrosine-specific T-cell hybridoma was established by fusion of azobenzenearsonate-L-tyrosine-specific T-cell line Al.8 cells and AKR thymoma BW5147 cells [lo]. Cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY) with 75
10% fetal calf serum (FCS; Bocknek, Canada), 5 X 10e5 M 2-ME and antibiotics. BlO.BR/sgsn mice were obtained from Nihon SLC (Shizuoka, Japan) and were used at the age of 6-8 weeks for preparation of splenocytes as APC. Preparation of ECDI-treated splenocytes is described in [3]. 3.3. Antibodies hybridomas [ 111 were Anti-CD3 mAb 1452Cll kindly provided by Dr. Y. Asano. FITC-labeled goat IgG against mouse IgG was obtained from MBL (Nagoya, Japan). FITC-labeled Fab fragments of sheep IgG against mouse IgG were obtained from Biological Specialty (USA). Goat IgG against hamster IgG was obtained from Organon Teknica (Durham, NC). Activation of T cells with cross-linked mAb has been previously described [12].
laser; fluorescence emission was observed above 515 nm. Temperature of the observation chamber was kept at 37°C.
4. Results 4.1. Increase of intracellular calcium ion concentration in response to antigen presentation Typical patterns of the change of intracellular calcium ion concentration in T-cell hybridomas in response to stimulations are shown in Fig. 1. In nonactivated T-cell hybridomas, no change was observed in response to antigen and APC (Fig. la). However, a rapid increase in the calcium ion concentration was observed by cross-linking of TCR/CD3 complex with anti-CD3 mAb (Fig. lb). On the contrary, after activa-
3.4. Antigen presentation T-cell hybridomas (6 X 104) were cultured with 6 X lo6 erythrocyte-depleted splenocytes which were treated with mitomycin C (Sigma) (40 pg/ml, 30 min) in Corning 24-well plates. Azobenzenearsonate-L-tyrosine was added to 0.1 mM. After 2 days of antigen presentation, almost all APC treated with mitomycin C were dead and could be easily separated from surviving T-cell hybridomas using the discontinuous density gradient of Percoll. This T-cell hybridoma could also be discriminated from APC by its diameter (N 3 times larger than that of APC). In the case of hapten molecules, pre-loading of APC with antigen is not necessary and re-stimulation was conducted as follows: (a) a mixture of antigen and APC was added and (b) antigen and APC were added separately (the order and time lag of addition were varied). No difference was observed by changing the manner of stimulation. Stimulation of APC with antigen and T-cell hybridomas was conducted in the same manner.
W
(a)
3-
I.
0
loo
200
loo
200
300
Time (see)
3.5. Measurement of intracellular calcium ion and imaging of cell-surface proteins Confocal fluorescence microscopic images of T-cell hybridomas and of APC were taken under a confocal fluorescence microscope system (MRC-600, Bio-Radl with an inverted epifluorescence microscope (TMDEFQ, Nikon) [13]. The pinhole aperture in the detection path of the confocal microscope was 3 mm and we used glycerine-immersed 1.4 NA (numerical aperture) 60 X Nikon Planapo and 0.85 NA 40 X Nikon Fluor objectives. Fluo-3-loaded T-cell hybridomas or FITC-labeled antibodies were excited at 488 nm using an argon ion 76
Fig. 1. Typical time courses of intracellular calcium ion concentration in individual T-cell hybridomas after stimulation. Each stimulation was given at the time indicated by an arrow. a: non-activated T-cell hybridoma stimulated with antigen and APC; non-activated T-cell hybridomas did not respond. b: non-activated T-cell hybridoma stimulated with anti-CD3 antibodies. About 60% of the ceils responded. Here, T-cell hybridomas were pre-incubated with anti-CD3 mAb and, after washing out excess mAb, goat IgG against hamster IgG was added at the indicated time. c-E activated T-cell hybridoma, which was pre-incubated for 2 days with antigen and APC, re-stimulated with antigen and APC. Time lag and pattern of calcium ion mobilization were different in each cell and typical examples are shown. About two-thirds of responding cells showed a large and transient increase in calcium signal as observed in (d) and (f). One-third showed a small and continuous increase as observed in (c) and (e).
by antigen presentation for 3 days (data not shown). We confirmed that [Ca2+li increase by antigen presentation was also observed in T-cell hybridomas activated by immobilized anti-CD3 antibodies before apoptosis was induced. The important difference between activation by antigen presentation and by immobilized anti-CD3 antibodies was the time course. In the case of immobilized anti-CD3 antibodies, it takes about 1 day to induce Ca2+ uptake by antigen presentation and apoptosis was observed the next day. We suspect that such a time lag might result from the quantitative difference in stimulations.
lb)
(a) _
4.2. Increase of intracellular calcium ion concentration in APC
L.
0
I.
loo
I
.I.
I
I..
loo 200 Time (80~)
200
Fig. 2. Typical time courses of intracellular calcium ion concentration in individual APC during the course of interaction with antigen and T-cell hybridomas. Stimulation was given at the time indicated by an arrow. a: ECDI-treated spienocyte stimulated with antigen and T-cell hybridomas; ECDI-treated splenocytes did usually not respond. b-f: untreated splenocyte stimulated with antigen and T-cell hybridomas; about 20% of untreated splenocytes responded. Each cell responded differently. About two-thirds of the responding cells showed a transient increase like (b), (c) and (e). One-third showed a small and continuous increase like (d) or (f).
tion of T-cell hybridomas by antigen presentation for 2 days, re-stimulation with antigen and APC caused an increase in calcium ion concentration in activated T-cell hybridomas (Fig. lc-f). T-cell hybridomas were not activated with APC without antigen nor antigen without APC (we confirmed that IL-2 production was not observed) and calcium signal was not induced in such T-cell hybridomas during antigen presentation. We observed several hundred cells for each stimulation and confirmed that responding cells to anti-CD3 antibody stimulation were about 60% of both non-activated and activated T-cell hybridomas. Responding cells to antigen presentation were about 20% of activated T-cell hybridomas, whereas less than 1% of non-activated hybridomas responded to antigen presentation. Time lag and the pattern of calcium ion mobilization were slightly different in every cell both in the case of antibody and antigen stimulations. In some responding cells, a small and continuous increase in calcium ion concentration was observed. This T-cell hybridoma is known to die during culture with a high density of immobilized antiCD3 antibodies [ll]. It was found that this cell also dies
Fig. 2 shows typical patterns of the change of intracellular calcium ion concentration in APC during interaction with antigen and non-activated T-cell hybridomas. A rapid increase was observed in untreated APC (Fig. 2b-f), wh ereas change of calcium ion concentration was mostly suppressed in ECDI-treated APC (Fig. 2a). The response rate of ECDI-treated APC was about 2-3%, including marginal response which may possibly result from incomplete ECDI fixation. It should be noted that during the interaction between APC and non-activated T-cell hybtidomas, intracellular calcium ion concentration increased only in APC and not in T-cell hybridomas. We examined several hundred cells and responding cells were about 20% of untreated APC. ECDI-treated APC did not induce a calcium signal in T-cell hybridomas, and increase of cell density was observed instead of long-term unresponsiveness in T-cell hybridomas (data not shown). 4.3. Mobility of MHC class II molecules We then investigated the behavior of surface MHC class II molecules to clarify the origin of the difference between ECDI-treated and untreated APC in calcium ion mobilization during antigen presentation. Fig. 3 shows the distribution of surface MHC class II molecules on APC. Without cross-linking, MHC class II molecules were uniformly distributed on the cell surface both in untreated and ECDI-treated APC as shown in Fig. 3 (Ia and Ib). ECDI-treated APC expressed relatively normal levels of Ia molecules on the surface, as detected by FACS analysis (data not shown). However, by extensive cross-linking with second antibodies, MHC class II molecules were fully cross-linked to form caps in untreated splenocytes (Fig. 3, IIa). On the contrary, on ECDI-treated splenocytes MHC class II molecules could not form caps and still remained uni77
I( a >
Fig. 3. MHC class II molecules on the cell surface were stained and observed by a confocal fluorescence microscope. I: anti-I-Ak mAb + FITC-labeled Fab fragments of sheep IgG against mouse IgG. II: anti-I-A’ mAb + FITC-labeled whole molecules of goat IgG against mouse IgG. a: untreated splenocytes; b: ECDI-treated splenocytes.
formly distributed on the cell surface (Fig. 3, IIb). Thus, it is indicated that the mobility of MHC class II molecules is suppressed by ECDI treatment.
5. Discussion During the interaction between T-cell hybridomas and APC, a calcium signal was observed only in APC and not in T-cell hybridomas. Pre-activation was neces78
sary for the induction of calcium signals by antigen presentation in T-cell hybridomas. The minimum size of receptor aggregation for the induction of an intracellular signal is different depending on cells. For example, receptor dimerization is enough for induction in some cells and in other cells more than trimerization is necessary [14]. We suspect that such a difference may result in the difference of calcium signal transduction between non-activated T-cell hybridomas and APC. Some new proteins, such as B7/BBl, are known to be
expressed on the surface of APC when activated by antigen and T cells [15]. Some of such proteins may possibly be essential for signal transduction to T-cell hybridomas 2-45-12. Cell adhesion molecules such as CD2 are up-regulated and other new membrane proteins are also expressed on the surface of activated T cells [16-181. Such proteins may possibly participate in calcium ion mobilization in T-cell hybridomas during the interaction with antigen and APC. Cross-linking of receptors by antibodies is thought to mimic antigen stimulation. However, we showed that antigen presentation and cross-linking of TCR-CD3 complex by antibodies are quantitatively quite different in intracellular calcium ion mobilization. That is presumably because affinity, steric hindrance, mobility and thereby efficiency of cross-linking of receptors differ from each other. By ECDI-treated APC, antigen presentation induced different signals in T-cell hybridomas. Although the conformation and number of MHC class II molecules were not changed by ECDI treatment as shown by mAb binding, flexibility and mobility of MHC class II molecules on the cell membrane may be markedly changed and these factors are critically important for their aggregation. It is known that T cells stimulated by ECDI-treated APC fail to induce IL-2, but IL-3, IFN-y and IL-2 receptors are partially induced and T-cell receptor j3 mRNA is fully induced [4]. In order to stimulate T cells, cross-linking of TCR, i.e., cross-linking of MHC class II molecules on APC, is necessary. Insufficient cross-linking of MHC class II molecules, and accordingly imperfect cross-linking of TCR-CD3 complex during T cell-APC interactions induced different intracellular signals from those induced by normal antigen presentation.
Acknowledgements This work was supported by a grant-in-aid from the Ministry of Education, Science and Culture, Japan.
References [II Nagao,
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