Biosci. Biotechnol. Biochem., 74 (9), 1788–1793, 2010

Regulation of Activation and Anergy by the Intensity of the Ca2þ Signal in Cooperation with Other Signals

y Yu ADACHI, Makoto HATTORI, and Tadashi YOSHIDA

Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

Received February 15, 2010; Accepted June 5, 2010; Online Publication, September 7, 2010 [doi:10.1271/bbb.100107]

The results of our previous in vitro study indicated murine models that had been induced with tolerance that the intensity of the Ca2þ signal could determine T in vivo.1–5) It has not been clarified, however, how cell activation and anergy. We show here that the T cell anergy is induced in T cells in vivo. response of mice that had been treated with cyclo- The Ca2þ signal induced by anergic stimulation sporine A during oral tolerance induction was higher (signal 1 alone) has been reported to play a key role in than that of control mice, indicating that the Ca2þ signal inducing T cell anergy. On the other hand, the Ca2þ could also determine T cell activation and tolerization signal is required for anergy and for activation in T in vivo. However, T cell activation was not apparent at cells.8) The treatment of T cells with cyclosporin A, a any concentration of ionomycin, although a low dose of inhibitor, has abolished the induction of T anti-CD3 monoclonal (mAb) induced activa- cell activation as well as anergy,9) indicating that the tion, while a high dose induced anergy in vitro. These Ca2þ signal did not determine the induction of T cell results indicate that the balance between the Ca2þ signal activation and anergy. It has been considered that T cells and other signals which can also be induced by anti-CD3 are activated by the Ca2þ signal in the presence of stimulation, but not the actual intensity of the Ca2þ MAPK signals induced by signal 2, while being signal or the presence of co-stimulation, played an anergized in their absence. However, the mechanism important role in regulating T cell activation and for regulating this activation or anergy by MAPK signals anergy. has not been clarified. Several previous studies have shown that the induction of anergy did not depend on the Key words: anergy; anti-CD3 response to the first stimulation.10,11) The oral admin- (mAb); Ca2þ signal; ionomycin; oral istration of a high dose of an induced vigorous T tolerance cell proliferation and subsequent tolerization.10) A similar phenomenon has also been reported in the study Immunological tolerance is a mechanism that makes about anti-CD3 mAb-induced anergy.11) We therefore the unresponsive to specific have to analyze the response of T cells to restimulation to such as self-antigens. Immunological tolerance is the evaluate whether the first stimulation induced productive essential mechanism for maintaining immune homeo- activation or anergy to the T cells. stasis; therefore, if immunological tolerance breaks We have shown in our previous study the possibility down, autoimmune diseases will develop. Self-antigens that the intensity of the Ca2þ signal could determine T as well as orally administered protein antigens as food cell activation or anergy regardless of the MAPK signals antigens can induce systemic antigen-specific immune in vitro.12) T cell anergy is known as the major tolerance, called oral tolerance. Many studies have been mechanism for oral tolerance, this being the major conducted to better understand the mechanism for oral immunological tolerance that has been observed in tolerance, because such understanding is vital for vivo.2) We therefore examine in this study whether this resolving food and also for preventing auto- regulation is applied for the oral tolerance induced in immune diseases and graft rejection. vivo. We also examine how the Ca2þ signal determines T cell anergy is one of the major mechanisms for the induction of this activation or anergy. Our results immunological tolerance and has been reported to be provide new information about the mechanism for involved in self- and super-antigen tolerance.1–5) T cell anergy induction and contribute to a better understand- anergy, a state in which T cells lose the capacity to ing of the clinical application of oral tolerance for produce IL-2 and to proliferate in response to antigen treating allergy and autoimmune diseases. stimulation, was first reported in an experiment on cloned T cells in vitro called clonal anergy.6) It has been Materials and Methods reported that T cells were anergized by stimulating the Mice. T-cell receptor (TCR; signal 1) in the absence of BALB/c mice (5 weeks old) were purchased from Clea Japan 7) (Tokyo, Japan) and used at 6–20 weeks of age. Ovalbumin (OVA)- costimulation (e.g., CD28; signal 2) in vitro. Many specific TCR-transgenic DO.11.10 mice were obtained from Jackson studies have demonstrated that T cells showing a similar Laboratory (Bar Harbor, ME, USA) and used at 6–20 weeks of age. state to that of clonal anergy could be found in several The T cells of these mice recognize OVA323-339 restricted to I-Ad.

y To whom correspondence should be addressed. Tel: +81-42-367-5711; Fax: +81-42-360-8830; E-mail: [email protected] Abbreviations: APC, antigen-presenting cells; CsA, cyclosporin A; mAb, monoclonal antibody; OVA, ovalbumin; TCR, T-cell receptor Regulation of T Cell Activation and Anergy by the Ca2þ Signal 1789 The TCR-transgenic mice were maintained on irradiated food and accordance with the protocol for the BrdU kit for cell proliferation autoclaved distilled water in our animal facilities. All the mice were ELISA (Roche Diagnostics, Basel, Switzerland). The culture super- treated in accordance with the guidelines for the care and use of natant was collected from each well for a assay before the experimental animals of Tokyo University of Agriculture and proliferation assay. The concentration of IFN- in each culture Technology. supernatant was detected by sandwich ELISA. Briefly, Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with the rat Reagents, antigen, and . Mitomycin C, cyclo- anti-mouse IFN- mAb (R4-6A2; BD Pharmingen, San Diego, CA, sporin A (CsA) and ionomycin were purchased from Sigma (St. Louis, USA). After washing and blocking, the culture supernatant and MO, USA) and OVA was obtained from Seikagaku Kogyo (Tokyo, standard were added to the plate. Bound cytokine was detected with the Japan). The anti-CD3 monoclonal antibody (anti-CD3 mAb, 145- biotin-labeled rat anti-mouse IFN- mAb (XMG1.2; BD Pharmingen), 2C11) was purchased from Cedarlane (Hornby, Ontario, Canada), and before incubating with alkaline phosphatase-streptavidin (Zymed, San recombinant mouse interleukin-12 (IL-12) was purchased from Francisco, CA, USA). A p-nitrophenyl-phosphate substrate was added Genzyme Techno (Cambridge, MA, USA). after washing, and the absorbance at 405 nm was determined.

Preparation of the T-cell growth factor. Erythrocyte-depleted Statistical analysis. Significant differences in the experimental data spleen cells of the BALB/c mice were cultured at 1:5 106 cells/ml were evaluated by the Tukey test or Dunnett test, and are considered to in RPMI-1640 (Nissui Pharmaceutical, Tokyo, Japan) supplemented be significant at p < 0:05. with 5% heat-inactivated fetal calf serum (JRH Bioscience, Lenexa, KS, USA), penicillin, streptomycin, L-glutamine and 2-mercaptoetha- Results nol. Concanavalin A (type IV, Sigma) was added to the culture at 2 mg/ml, and the culture supernatant was collected after 24 h. Methyl The highest production of IFN- from the Th1 cells -D-mannopyranoside (Sigma) was added to the culture supernatant at 20 mg/ml to inactivate the concanavalin A. The culture supernatant was observed after the third stimulation was stirred for 15 min and then centrifuged. The resulting supernatant It has been reported that naı¨ve T cells were resistant to was collected and sterilized by filtration for use as the T-cell growth anergy induction13,14) and that Th1 cells were more factor (TCGF). sensitive to anergic stimulation than Th2 cells.15,16) We therefore used in this study T cells from DO.11.10 TCR- Induction of oral tolerance and CsA treatment. BALB/c mice were transgenic mice after inducing differentiation in Th1 fed with 100 mg of OVA in saline by means of a feeding needle. Those cells by stimulating them with OVA and APC in the mice had been i.p. treated with various concentrations of CsA (0, 20, or 200 mg) dissolved in 100 ml of dimethylsulfoxide (DMSO) for 5 min presence of IL-12. We had to distinguish three different before being rendered tolerant. This set of operations was performed 8 states of the T cells, unstimulated, activated and times every 2 d. anergized, by using the Th1 cells, because the aim of this study was to investigate the difference in the signals Immunization. The mice were immunized s.c. with 100 mg of OVA inducing anergy and activation. We first determined the in complete Freund’s adjuvant (CFA) 7 d after the last feeding of the most appropriate conditions for obtaining Th1 cells in antigen. which these three different states could be clearly distinguished. Th1 induction. Splenocytes isolated from the DO.11.10 mice were IFN- production from the T cells was increased by 6 cultured in 24-well plates at 5 10 cells/well in RPMI-1640 antigen stimulation in the presence of IL-12 (Fig. 1). In containing 10% fetal calf serum in the presence of OVA (100 mg/ml) and IL-12 (210 pg/ml) to induce the T helper type 1 (Th1) particular, IFN- production of Th1 cells that had been cells. Seven days after stimulation, the T cells was restimulated by stimulated three times was at the highest level, and the OVA (100 mg/ml), the antigen-presenting cells (APC, 3:3 106/well), difference between the cells that had been stimulated the T-cell growth factor (10% of the medium) and IL-12 (105 pg/ml). twice and three times was also greatest. On the other Mitomycin C-treated splenocytes of the BALB/c mice were used for hand, IFN- production from the T cells after the fourth APC. Seven days after this restimulation, the living cells were purified stimulation was lower than that from the T cells after the with lympholyte-M (Cedarlane) and used as Th1 cells for anergy induction. third stimulation. We therefore decided to perform anergic stimulation as the third stimulation in this study. Anergy induction. The wells of a 24-well plate were coated with IFN- production of the cells would be lower than that 300 ml of anti-CD3 mAb diluted with phosphate-buffered saline (0, of the T cells that had been stimulated twice when the 0.0001, 0.001, 0.01, 0.1, 1, 5, or 10 mg/ml) at 4 C for 24 h and then stimulation was really anergic, while IFN- production washed with phosphate-buffered saline. The Th1 cells were pre- would be higher if the stimulation was agonistic. We incubated with or without the indicated concentration of CsA (0, 0.05, would also be able to distinguish whether or not 0.1, or 0.5 mg/ml) for 30 min for the experiment shown in Fig. 2A. The stimulation had been effective, because IFN- produc- Th1 cells were anergized by being incubated in the anti-CD3 mAb- tion would be at a similar level to that with T cells that coated wells or treated with various concentrations of ionomycin (0, 0.01, 0.1, 0.5, 1, or 5 mM) in the uncoated wells for 24 h. After these had been stimulated twice if stimulation had not been treatments, the Th1 cells were washed and transferred to the uncoated effective. wells of a 24-well plate and rested for 3–4 d. The intensity of the Ca2þ signal regulated the Restimulation. Seven days after the immunization, lymph node cells induction of anergy and activation in vitro and in vivo isolated from the immunized mice were cultured in a 96-well plate at We have shown in our previous report that both 4 105 cells/well in the presence of OVA (100 mg/ml) for the 5 activation and anergy were induced in T cells by treating experiments shown in Fig. 2B. The anergized Th1 cells (1 10 with the anti-CD3 mAb and CsA depending on the cells/well) were also restimulated by OVA (100 mg/ml, or 1 mg/ml) and APC (4 105 cells/well) in a 96-well plate for the experiments concentration of CsA, indicating that the intensity of the 2þ shown in Figs. 1, 2A, 3 and 4. Ca signal could determine T cell activation and anergy.12) We first reconfirmed this result for T cell Proliferation assay and cytokine detection. Three days after proliferation. The T cell proliferation of Th1 cells that stimulation, the proliferation of the Th1 cells was measured in had been stimulated with the anti-CD3 mAb without a 1790 Y. ADACHI et al. A

B

Fig. 1. IFN- Production by T Cells That Had Been Stimulated Several Times with the Antigen. Naive T cells of DO.11.10 were stimulated with OVA (100 mg/ml) in the presence of IL-12 for the indicated number of times every seven days. After the second stimulation, the cells were cultured in the presence of APC. Seven days after the last stimulation, the established Th1 cells were restimulated with OVA (100 mg/ml) and APC. Three days after this restimulation, IFN- production by those T cells was measured by ELISA. This result is representative of two independent experiments. The graph shows the mean þ SD of triplicate data. Significance by the Tukey test is indicated as ‘‘a and b’’ (p < 0:05).

CsA treatment was significantly lower than that of the control (Fig. 2A), indicating that anergy had been induced in those cells. T cell proliferation of Th1 cells Fig. 2. Induction of T Cell Activation and Anergy by the CsA that had been stimulated with the anti-CD3 mAb in the Treatment. presence of 0.05 mg/ml of CsA seemed to be higher than A, Established Th1 cells were stimulated with the anti-CD3 mAb that of cells without the CsA treatment, suggesting that (5 mg/ml) in the presence of various concentrations of CsA (0, 0.05, 0.1, and 0.5 mg/ml) for 24 h. Those Th1 cells were restimulated with anergy induction had been blocked by the CsA treat- OVA (1 mg/ml) and APC after three days of resting. Three days ment. On the other hand, T cell proliferation of Th1 cells after this restimulation, the proliferation of those Th1 cells was that had been stimulated with 0.1 mg/ml of CsA was measured by BrdU ELISA. B, BALB/c mice (n ¼ 5{9) were fed significantly higher than the control, indicating that T with 100 mg of OVA after being treated with various concentrations cell activation rather than anergy had been induced in of CsA (0, 20, and 200 mg). After being fed eight times, lymph node cells were obtained from axillary and inguinal lymph nodes of the these cells. In addition, T cell proliferation of the Th1 mice and cultured with OVA (100 mg/ml). The amount of IFN- cells stimulated with 0.5 mg/ml of CsA was at the same produced in the supernatant was measured by ELISA after three level as the control. These results corresponded well days of culture. These results are representative of three independent experiments. The graph shows the mean þ SD of triplicate data. with the previous results. T cell anergy has been reported as the major Significance by the Dunnett test is indicated as ‘‘ ’’ ( p < 0:05;as 17) compared with untreated Th1 cells), and by the Tukey test is mechanism for oral tolerance. We therefore inhibited indicated as ‘‘a, b, and c’’ (p < 0:05; a comparison among the CsA- the Ca2þ signal during the induction of oral tolerance treated groups). with various concentrations of CsA to examine whether this regulation by the intensity of the Ca2þ signal could tended to be higher than that by the mice without the be applied to induce oral tolerance. The T cell response CsA treatment, suggesting that the Ca2þ signal was of mice rendered tolerant to antigen stimulation was required for the induction of oral tolerance. On the other analyzed, and the tolerance induction was evaluated by hand, IFN- production by lymph node cells obtained comparing with that of the control mice. As shown in from the mice that had been treated with 200 mg of CsA Fig. 2B, IFN- production by lymph node cells obtained was higher than that by the control mice, indicating that from the mice that had been rendered orally tolerant T cell activation rather than tolerance had been induced without the CsA treatment was significantly lower than in these mice. Similar results were obtained from several that by the control mice, indicating that oral tolerance independent experiments. These results strongly support had been induced in these mice. IFN- production by the in vitro observations, showing that the Ca2þ signal lymph node cells obtained from the mice that had been could determine T cell activation and anergy. treated with 20 mg of CsA during tolerance induction Regulation of T Cell Activation and Anergy by the Ca2þ Signal 1791

Fig. 3. IFN- Production by Th1 Cells That Had Been Stimulated Fig. 4. IFN- Production by Th1 Cells That Had Been Stimulated with Various Concentrations of Ionomycin. with Various Concentrations of the Anti-CD3 mAb. Established Th1 cells were stimulated with various concentrations Established Th1 cells were stimulated with various concentrations of ionomycin (0, 0.01, 0.1, 0.5, 1, and 5 mM) for 24 h. Those Th1 of the anti-CD3 mAb (0, 0.0001, 0.001, 0.01, 0.1, 1 and 10 mg/ml) cells were re-stimulated with OVA (100 mg/ml) and APC after three for 24 h. Those Th1 cells were restimulated with OVA (100 mg/ml) days of resting. IFN- production by those Th1 cells was measured and APC after three days of resting. IFN- production by those Th1 by ELISA after three days of culture. This result is representative of cells was measured by ELISA after three days of culture. This result two independent experiments. The graph shows the mean þ SD of is representative of two independent experiments. The graph shows triplicate data. Significance by the Dunnett test is indicated as ‘‘’’ the mean þ SD of triplicate date. Significance by the Dunnett test is (p < 0:05; compared with Th1 cells not treated with ionomycin). indicated as ‘‘’’ ( p < 0:05; compared with Th1 cells not treated with the anti-CD3 mAb). Treatment with any concentration of ionomycin could not induce activation of the Th1 cells but induced anergy of T cells in cooperation with other signals. While in a dose-dependent manner ionomycin is a specific activator of the Ca2þ signal, anti- We had shown in our previous study that treating T CD3 mAb stimulation and oral administration of an cells with the anti-CD3 mAb in the presence of CsA antigen introduce many other signals as well as the Ca2þ induced both T cell activation and anergy, depending on signal. The balance between the Ca2þ and some other the dose of CsA.12) The results of this study show that signals might therefore be important to determine the CsA could regulate the induction of oral tolerance and activation and anergy of T cells. We next examined activation, indicating that the intensity of the Ca2þ whether stimulation with various concentrations of the signal could determine T cell activation and anergy anti-CD3 mAb would induce both the activation and in vitro and in vivo. We therefore next examined anergy of Th1 cells. whether stimulation by ionomycin, which induced the As shown in Fig. 4, IFN- production by Th1 cells Ca2þ signal in T cells, could induce activation and that had been treated with 0.1, 1 and 10 mg/ml of the anergy depending on the concentration. anti-CD3 mAb was significantly lower than that of the Th1 cells were stimulated by various doses of control, indicating that anergy had been induced in those ionomycin for 24 h to induce activation or anergy, and cells. IFN- production was gradually increased by IFN- production from the cells after restimulation was decreasing the concentration of the anti-CD3 mAb under analyzed. The results show that IFN- production by the those conditions. Interestingly, the IFN- production by Th1 cells that had been treated with 1 and 5 mM of Th1 cells treated with 0.01 mg/ml of the anti-CD3 mAb ionomycin was significantly lower than that by the was significantly higher than that of the control, control, indicating that anergy had been induced indicating that T cell activation rather than anergy had (Fig. 3). The IFN- production then increased gradually been induced. as the concentration of ionomycin was reduced. How- ever, no activation was apparent at any concentration of Discussion ionomycin, suggesting that the intensity of the Ca2þ signal alone could not determine the induction of anergy The aim of this study was to clarify the mechanism and activation, but that it just determined the intensity of for regulating T cell activation and anergy by the Ca2þ anergy. signal in vitro and in vivo. Our results clearly demon- strate that T cell activation and anergy could be A low dose of the anti-CD3 mAb induced the activation controlled by the intensity of the Ca2þ signal in vitro. of Th1 cells, while a high dose induced anergy We confirmed a similar role of the Ca2þ signal in the The results shown in this report suggest that the Ca2þ induction of T cell tolerance in vivo by using the mice signal might have determined the activation and anergy that had been induced orally tolerant. Furthermore, the 1792 Y. ADACHI et al. results indicate that this regulation was not determined as well as T cell anergy contributed to the establishment by the intensity of the Ca2þ signal alone, but by the of oral tolerance. On the other hand, some reports have balance between the Ca2þ signal and some other signals. strongly indicated that T cell anergy played an important These findings would contribute to a better understand- role in the establishment of oral tolerance.19,20) Ise et al. ing of the mechanism for anergy induction. have shown that the T cell response of mice rendered T cell anergy has been reported as one of the most orally tolerant after removing CD4þCD25þ T cells important mechanisms for several types of immunolog- retained a hyporesponsive state.19) Melamed et al. have ical tolerance.1–5) However, the mechanism for inducing reported that T cells obtained from mice rendered orally anergy has not been elucidated. It was commonly tolerant were hyporesponsive without any suppressive believed in previous studies that anergic stimulation activity.20) Although the involvement of the Ca2þ signal was entirely different from the stimulation inducing T in Treg induction during oral tolerance cannot be cell activation. Those reports have suggested that TCR discounted, our result strongly supports the in vitro stimulation in the absence of co-stimulation induced T observation shown in Fig. 2A, indicating that the cell anergy, while, in the presence of co-stimulation, it intensity of the Ca2þ signal regulated T cell activation induced T cell activation.6,7) It is known that TCR and anergy. It has been reported that a low dose of an stimulation could be induced by an anti-CD3 mAb orally administered antigen induced T cell activation treatment as well as antigen stimulation. However, rather than tolerance, while a high dose induced antigen stimulation can stimulate both TCR and co- tolerance without activation.21) The results shown in this stimulatory molecules (e.g., CD28) because antigens are present report, which indicate that a weak Ca2þ signal always presented to T cells by APC expressing ligands was preferential to induce activation, but that a strong of such co-stimulatory molecules,7) although the anti- Ca2þ signal induced anergy, confirm this observation. CD3 mAb can mainly stimulate TCR. This is why the Several reports have demonstrated that the induction of T anti-CD3 antibody is believed to induce T cell anergy. cell anergy and tolerance was abrogated by a treatment Furthermore, the Ca2þ signal induced by TCR stimula- with CsA,12) but there has been no report showing that a tion has been reported to play a key role in both T cell treatment with CsA during the induction of tolerance anergy and activation.8) Conversely, it has been reported induced activation. Our findings provide a new strategy that the Ca2þ signal could not determine the induction of for a better understanding of tolerance induction. anergy and activation, and that some types of signal We have shown in this study that the intensity of the from co-stimulation were involved in this determina- Ca2þ signal could regulate the induction of anergy and tion.6,7) However, how the co-stimulation signal would activation in both in vitro and in vivo systems (Fig. 2). determine the induction of T cell activation and anergy However, there is no report showing that treating T cells remained to be clarified, although it has been suggested with ionomycin, which is a calcium ionophore and is that the signal might strengthen some signals other than known to induce T cell anergy,22) could induce anergy the Ca2þ signal such as MAPK signals.18) The results of and activation depending on the dose. We had con- our previous study surprisingly showed that treating T sequently believed that stimulation with ionomycin cells with the anti-CD3 mAb, which is a well-known alone would induce T cell anergy, but not activation. method for inducing anergy in T cells, could induce We have confirmed in this study that ionomycin did not both T cell anergy and activation, depending on the induce T cell activation at any concentration, and just intensity of the Ca2þ signal. This result suggested a new induced anergy in a dose-dependent manner (Fig. 3). mechanism for determining T cell anergy or activation. This finding indicates that, although some other factors We have already described that our previous study would be needed to induce T cell activation, anergy showed that treating T cells with the anti-CD3 mAb could be induced by the Ca2þ signal alone. Indeed, some in the presence of CsA induced both T cell activation previous studies have shown that treating T cells with and anergy, depending on the dose of CsA.9) We ionomycin alone induced anergy,22,23) while treating first reconfirmed this result in the present study. We with ionomycin and PMA induced activation, supporting evaluated the activation and anergy by IFN- production our observations.8,24) However, our result from the in the previous study, and observed the same phenom- experiments using CsA indicates that the intensity of enon for T cell proliferation in the present work (Fig. 2). the Ca2þ signal could determine T cell activation and However, the level of anergy and activation seemed to anergy. Taken together, all these observations strongly be lower than that evaluated by IFN- production (data suggest that the balance between the Ca2þ signal and not shown). We had already reported that IFN- some other signals, but not the actual intensity of the production was more sensitive for the induction of Ca2þ signal or the presence of co-stimulation, was anergy than proliferation. In particular, the proliferation important to determine the induction of T cell anergy of Th2 cells was hardly reduced by anergy induction.14) and activation. The Th1 cells used in this study might have included Interestingly, we could also show that stimulating T Th2 cells to some degree, and the T cells obtained from cells with the anti-CD3 mAb could induce both T cell mice rendered orally tolerant would contain more Th2. anergy and activation, depending on the dosage (Fig. 4). We therefore decided to evaluate anergy and activation It had been believed that treating T cells with the anti- by IFN- production in the subsequent experiments. CD3 mAb mainly induced the Ca2þ signal and con- We next examined whether this regulation could be sequently anergy. However, stimulating with the anti- applied for oral tolerance. Our results show that a low CD3 mAb actually induced not only the Ca2þ signal, but dose of CsA weakened the induction of oral tolerance, also such others as MAPK signals.25) Both the intensity while a high dose induced T cell activation rather than and balance of these signals would be changed depend- tolerance (Fig. 2B). It is known that the induction of Treg ing on the dose of the anti-CD3 mAb.26) This might be Regulation of T Cell Activation and Anergy by the Ca2þ Signal 1793 one reason why the anti-CD3 mAb could induce both References activation and anergy, but that ionomycin could only induce anergy. 1) Rellahan BL, Jones LA, Kruisbeek AM, Fry AM, and Matis LA, The nuclear factor of activated T-cells (NFAT) is J. Exp. Med., 172, 1091–1100 (1990). 2þ 2) Melamed D and Friedman A, Eur. J. Immunol., 23, 935–942 activated by the Ca /calcineurin signaling path- (1993). 22,24) way. 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This work was supported in part by a grant from Japan Science and Technology Agency.