Impaired Ca/calcineurin pathway in in vivo anergized CD4 T cells

Motoko Kimura, Masakatsu Yamashita1, Masato Kubo2, Makio Iwashima4, Chiori Shimizu, Koji Tokoyoda, Joe Chiba3, Masaru Taniguchi, Makoto Katsumata5 and Toshinori Nakayama

Department of Molecular Immunology, Graduate School of Medicine, Chiba University and
1 Department of Developmental Immunology, Chiba University School of Medicine, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan
2 Research Institute for Biological Sciences and
3 Department of Bioengineering, Science University of Tokyo, 2669 Yamazaki, Chiba 279-0022, Japan.
4 Program in Molecular Immunology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2600, USA
5 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6082, USA.

Correspondence to: T. Nakayama


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Clonal anergy is one of the mechanisms that may account for self tolerance induced in T cells in the periphery. In this study we used the well-documented system of in vivo administration of a superantigen, staphylococcal enterotoxin B (SEB), to induce a state of hyporesponsiveness (anergy) in murine peripheral T cells to decipher the intracellular biochemical basis for this process. The TCR-induced Ca response of in vitro activated T cells was found to be impaired with significant defects in the phosphorylation of phospholipase C-{gamma}1. Experiments with calcium ionophore and newly established transgenic mouse lines that express an active form of calcineurin suggested that in vivo SEB-induced anergy is established and/or maintained by a selective impairment in the TCR-induced activation of the Ca/calcineurin pathway.

Keywords: active calcineurin transgenic, anergy, calcineurin, calcium, peripheral tolerance, superantigen


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
One of the mechanisms that may explain the self tolerance induced state in T cells in the periphery is clonal anergy (1,2). TCR stimulation in the absence of co-stimulatory signals induced a state of long-term unresponsiveness in cloned T cells (3,4). Jenkins and Schwartz demonstrated that such an anergic state can be induced in murine T cell clones when they are stimulated by antigen and chemically modified antigen-presenting cells that do not provide appropriate co-stimulatory signals (5). Among various co-stimulatory signals, CD28/CD80 and CD86 are thought to be the most important (68). The anergized T cells were characterized by the impairment in IL-2 production and subsequent proliferation following TCR re-stimulation in the presence of appropriate co-stimulatory signals (3,4,916). Recent work with cloned T cells suggested that the impaired transcription of the IL-2 gene in anergic T cells was due to a failure in TCR-induced activation of p21ras (17) and its downstream mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated protein kinases (ERK) and c-Jun N-terminal kinases (JNK) (18,19). In addition, the involvement of AP1 (14), NF-{kappa}B and NF-AT (20), c-Fos, FosB and JunB (21), and negative regulatory factors (22,23) and Rap1 (24) has been suggested.

The in vivo administration of the superantigen, staphylococcal enterotoxin B (SEB), is well documented to result in a dramatic clonal expansion of SEB-reactive TCR Vß8-bearing T cells and subsequent cell death by apoptosis, and surviving Vß8T cells show a significant decrease in IL-2 production and a reduced capacity for proliferation after TCR stimulation (2530). A similar type of anergy is also induced by endogenous superantigens (31,32). Recently, defects in early tyrosine phosphorylation events (33) and transcription factors (34) in in vivo anergized T cells have been reported; however, the intracellular biochemical basis underlying the in vivo induced anergy is still largely unknown.

Here, we sought evidence for the signaling defect in in vivo SEB-induced anergic T cells using a Vß8-bearing TCR {alpha}ß transgenic mouse system in which most of the peripheral T cells responded to SEB and with the consequence that surviving T cells exist in an anergic state. It was found that the anti-TCR-induced Ca responses were consistently impaired as a result of significant defects in the activation of phospholipase C (PLC)-{gamma}1. The addition of a small amount of calcium ionophore was able to rescue the anergic state and normal proliferative response was detected in transgenic T cells expressing an active form of calcineurin. The results of this study suggest that in vivo SEB-induced anergic state of T cells is established and maintained by a selective impairment in the TCR-induced activation of the Ca/calcineurin pathway.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Animals
Anti-ovalbumin TCR {alpha}ß transgenic (DO10 Tg) mice (35) were kindly provided by Dr Dennis Loh (Nippon Roche Research Center, Kanagawa, Japan). To establish a transgenic mouse with an active form of the calcineurin {alpha} chain (dCaM Tg), a constitutively active form of truncated calcineurin DNA (dCaM, #1–1188) was generated by PCR amplification of wild-type calcineurin cDNA as described (36). A BamHI site was added to the 5' and 3' end. Then, a 1.2 kb dCaM gene was ligated with the lck proximal promoter (37) by using the BamHI site and the resulting 6.5 kb construct was microinjected into fertilized C57BL/6 (B6) eggs. All mice used in this study were maintained under specific pathogen-free conditions.

Anergy induction
DO10 Tg mice were injected i.p. with 50 µg of SEB. Sixteen days later, mice were sacrificed and the responsiveness of the splenic CD4 T cells was individually examined before analysis.

Cell purification and cell culture
The purification of CD4 T cells from spleen cells was performed using an anti-CD8 mAb and subsequent panning with plastic dishes coated with goat anti-mouse Ig as described (38). The purity of cell preparations was checked by flow cytometry analysis. The contamination of CD8 T cells was <5%. More than 95% of CD4 T cells express Vß8 transgenic TCR in DO10 Tg mice with SEB treatment. Numbers of CD4+ T cells were appropriately adjusted before the cells were added to stimulation cultures.

Proliferation assay
Purified splenic CD4 T cells (2x105) were stimulated in 200 µl cultures for 40 h with immobilized anti-TCR Vß8 mAb (F23.1; 100 µg/ml), or 10 or 30 µg/ml of SEB with irradiated BALB/c spleen cells (3000 rad, 5x105). Where indicated, recombinant IL-2 (100 U/ml) or ionomycin (3 or 10 nM) was added at the beginning of the stimulation culture. [3H]Thymidine (0.5 µCi/well) was added to the stimulation culture for the last 16 h and the incorporated radioactivity measured by using a ß-plate (38).

ELISA for measuring IL-2 production
Purified splenic CD4 T cells (2x105) were stimulated in 200 µl cultures with immobilized anti-TCR Vß8 mAb (F23.1; 100 µg/ml) and culture supernatants were collected 24 h later. The same numbers of CD4+ cells from PBS- or SEB-treated DO10 Tg mice were used in the stimulation culture. IL-2 production was measured by ELISA as described (38).

Measurement of intracellular free calcium ion concentration ([Ca2+]i)
Purified splenic T cells were loaded with Indo-1 (Indo-1 AM; Molecular Probes, Eugene, OR) in the presence of F127 (39). After washing, the cells were incubated with anti-CD8– FITC (53.6-72–FITC), anti-CD4–phycoerythrin (RM4-5–phycoerythrin; PharMingen, San Diego, CA) and anti-TCRß–biotin or anti-TCRß–biotin plus anti-CD4–biotin (PL-172–biotin) at 4°C. The binding of RL-172 to CD4 molecules was not blocked by another anti-CD4 mAb (RM4-5) used (data not shown). The stained cells were washed and subjected to Ca analysis on FACS Vantage (Becton Dickinson, Mountain View, CA). TCR was cross-linked with avidin. The [Ca2+]i was monitored for 512 s and results were analyzed MULTITIME software (Phoenix Flow Systems, San Diego, CA).

Measurement of IP3 production
IP3 release was analyzed by a method previously described (40). In brief, purified splenic CD4 T cells were treated with anti-Vß8 mAb at 4°C, washed and then cross-linked with anti-hamster IgG (100 µg/ml) at 37°C for 1 min. IP3 levels were assessed using an IP3 radioreceptor assay kit (Amersham, Little Chalfont, UK) following the manufacturer's protocol. The lowest detection level was 0.19 pmol/0.1 ml in this assay system.

Luciferase assay
Luciferase assay with NF-AT-Luc construct was performed as described (41). In brief, transfected T cells were lysed in a lysis buffer (25 mM Tris–phosphate, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol and 1% Triton X-100). The extracts were mixed with luciferase substrate solution (Promega, Madison, WI) and the emitted light was measured with a luminometer.

Immunoprecipitation and immunoblotting
Purified splenic CD4 T cells were treated with anti-TCRß mAb (H57-597) or anti-TCRß mAb (H57-597) plus anti-CD4 mAb (GK1.5) and cross-linked with anti-hamster IgG (anti-HIg; 100 µg/ml) at 37°C. After washing, the stimulated cells were solubilized as described (42). Anti-PLC-{gamma}1 antibody (Upstate Biotechnology, Lake Placid, NY; cat. no. 06-152) was added to the lysates and allowed to incubate at 4°C for 1 h. Subsequently, 50 µl of Protein G–Sepharose was added and samples were tumbling for another 1 h at 4°C. The immunoprecipitates were applied to SDS–PAGE under reducing conditions and then subjected to electrotransfer to PVDF membranes. The membranes were incubated with anti-phosphotyrosine mAb, 4G.10 (Upstate Biotechnology), or anti-PLC-{gamma}1 antibody (Upstate Biotechnology; cat. no. 06-159), washed and then incubated with horseradish peroxidase-conjugated Protein A (Cappel, Durham, NC). The band intensities were measured by a densitometer and an arbitrary densitometric unit was assigned to each band.

MAPK assay
Purified splenic CD4 T cells were treated with anti-TCRß mAb (H57-597) and cross-linked with anti-hamster IgG (100 µg/ml) at 37°C for 2 or 5 min. The cells were solubilized and cell lysates were applied to a 10% SDS–PAGE. Proteins were transferred to a PVDF membrane and phosphorylation of MAPK (ERK1) was visualized by using phospho-MAPK detection kit (NEB, Boston, MA; cat. no. 9100).


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Anergy induction in Vß8-bearing TCR{alpha}ß transgenic T cells in DO10 Tg mice
In this paper, we investigated the molecular mechanisms underlying a superantigen, SEB-induced T cell anergy using a Vß8-bearing TCR{alpha}ß transgenic (DO10 Tg) mouse model system (35) in which the majority (~90%) of peripheral T cells express SEB-reactive TCR Vß8. In DO10 Tg mice, a similar mode of clonal anergy to that observed in normal BALB/c mice (2529) was induced as evidenced by the following observations: (i) the number of CD4 T cells in the spleen increased in the first 3 days and then it decreased during next a few days (data not shown), (ii) the absolute number of splenic CD4 T cells was decreased significantly (~50–60% level) (Fig. 1AGo), (iii) neither TCR ß nor CD4 expression on the CD4 T cells was down-modulated (Fig. 1AGo), (iv) the surviving CD4 T cells showed decreased proliferative responses upon anti-Vß8 stimulation (Fig. 1BGo) or SEB stimulation (Fig. 1CGo), (v) anti-Vß8-mAb-induced IL-2 production was impaired (Fig. 1BGo), and (vi) the hyporesponsiveness was rescued by the addition of recombinant IL-2 to proliferation cultures (Fig. 1DGo). In addition, the majority of CD4 T cells (>85%) expressed CD69 and CD25 activation markers 12 h after SEB treatment (Fig. 1EGo), suggesting that most of the CD4 T cells were activated as a consequence of the SEB treatment. Thus, the observed anergic state is a consequence of T cell response to SEB, rather than a simple survival of a poorly responding T cell subpopulation. Collectively, these results demonstrate the appropriateness of the DO10 Tg mouse model for the proposed investigation.



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Fig. 1. Anergy induction in Vß8-bearing TCR{alpha}ß transgenic (DO10 Tg) mice. DO10 Tg mice, 6–8 weeks old, were injected i.p. with 50 µg of SEB. (A) Sixteen days later, splenocytes were stained with anti-CD4 and anti-CD8 mAb or anti-CD4 and anti-Vß8 mAb. Yields of cells are shown as boxed numbers. Percentages of cells present in each area are also shown. Purified splenic CD4 T cells from PBS-treated (open bar) or SEB-treated (hatched bar) mice were stimulated with immobilized anti-Vß8 antibody (B and D) or SEB (10 and 30 µg/ml) and antigen-presenting cells (APC) (C). The amounts of [3H]thymidine uptake and IL-2 production are shown. Where indicated, recombinant mouse IL-2 (100 U/ml) was added. (E) Twelve hours after PBS (solid line) or SEB treatment (dashed line), DO10 Tg spleen cells were stained with anti-CD4 and anti-CD69 mAb or anti-CD4 and anti-CD25 mAb. Representative profiles of CD69 and CD25 expression on the electronically gated CD4 T cells are shown with background stainings (shaded area).

 
Impaired Ca responses and normal activation of ERK/MAPK pathway upon TCR stimulation in the SEB-induced anergic T cells
We began the analysis with the measurement of the Ca response after TCR cross-linking using flow cytometry. Splenic T cells from SEB-treated DO10 Tg mice were first loaded with Indo-1, and then stained with anti-CD4 mAb, anti-CD8 mAb and anti-TCR Vß8–biotin. Vß8 TCR was cross-linked with avidin. As shown in Fig. 2Go(A), the anti-TCR Vß8 mAb-induced rapid elevation in [Ca2+]i of CD4 T cells was significantly reduced in the SEB-induced anergic T cells. The decrease was more dramatic at the lower dose of avidin as demonstrated by either mean Indo-1 ratio or percent responding cells. A total of four independent experiments with seven individual SEB-treated mice was performed with similar results (data not shown). Consistent with this result, the impaired proliferation of the SEB-induced anergic T cells was completely restored by the addition of low doses of Ca ionophore (Fig. 2BGo), indicating that the increased [Ca2+]i was sufficient for rescue of the SEB-induced anergic state of the T cells. Thus, the critical signaling defect in the SEB-induced anergic T cells appeared to be the impairment in Ca response after TCR stimulation.




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Fig. 2. Impaired anti-TCR-induced Ca responses in the SEB-induced anergic T cells of DO10 Tg mice. (A) Purified splenic T cells from PBS- (solid line) or SEB- (dotted line) treated DO10 Tg mice were first loaded with Indo-1, and then stained with anti-CD4–phycoerythrin, anti-CD8–FITC and anti-TCRß–biotin. The cells were stimulated with avidin (arrows) at 37°C. Mean Indo-1 ratio and percent responding cells of electronically gated CD4 T cells are shown. (B) Purified splenic CD4 T cells from PBS- (open bar) or SEB- (hatched bar) treated mice were stimulated with immobilized anti-Vß8 mAb in the presence of low doses of ionomycin (3 and 10 nM). (C) Phosphorylation status and protein levels of PLC-{gamma}1 in purified splenic CD4 T cells were assessed after anti-TCR cross-linking. (D) IP3 production induced by TCR cross-linking was assessed in purified splenic CD4 T cells from PBS- (open bar) or SEB- (hatched bar) treated mice. ND, not detectable. (E) Anti-TCR-induced activation of ERK/MAPK pathway was assessed by immunoblotting with anti-phospho-ERK antibody. Representative results are shown with arbitrary densitometric units.

 
Next, the activation of PLC-{gamma}1, an upstream signaling component of anti-TCR-induced Ca elevation (4345), was assessed by measuring the phosphorylation status of PLC-{gamma}1. As shown in Fig. 2Go(C), the increase in PLC-{gamma}1 phosphorylation after TCR cross-linking was significantly impaired in the anergic T cells. The amount of PLC-{gamma}1 protein was not significantly decreased (Fig. 2CGo, lower panel). Thus, the impaired Ca response of the anergic T cells appears to be accompanied by some defect in PLC-{gamma}1 activation. The levels of IP3 release from SEB-treated CD4 T cells after cross-linking of TCR with anti-TCR Vß8 mAb and anti-hamster Ig were also measured (Fig. 2DGo). A significant increase in IP3 levels was observed in CD4 T cells of the PBS-treated group, while the level of IP3 in CD4 T cells of the SEB-treated group remained at the background level.

There have been reports of studies with cloned T cells that suggested that the anergic state is due to a failure of TCR-induced activation of p21ras (17) and MAPK pathways (18,19). Therefore, we investigated the TCR-induced activation of the ERK/MAPK pathway in the SEB-induced anergic T cells. The phosphorylation status of MAPK (ERK1) in in vivo SEB-induced anergic T cells after TCR cross-linking was measured and no significant decrease in phosphorylation was detected (Fig. 2EGo). Five independent experiments with anti-TCR mAb titration were performed and similar results were obtained in all (data not shown). These results would indicate that the intracellular biochemical basis of the in vivo SEB-induced anergy is different from that of anergic T cell clones in other mouse systems.

Establishment of transgenic mice with an active form of the calcineurin {alpha} chain (dCaM Tg)
In order to assess the role of calcineurin activation in the SEB-induced anergic state, we generated transgenic mouse lines with an active form of the calcineurin {alpha} chain (dCaM) using a well-known T cell-specific transgenic system with the proximal promoter of lck (37) (Fig. 3AGo). A T cell line 68-41 transfected with the dCaM construct showed a certain level of promoter activity of NF-AT in the absence of anti-TCR stimulation as well as a significant increase after stimulation (Fig. 3BGo). A similar pattern of increased promoter activity was detected upon stimulation with phorbol myristate acetate (PMA) and ionomycin (data not shown). Two dCaM Tg lines with five copies of transgenic integration in B6 and dCaMxD10 double-Tg mice showed no significant differences in phenotypic features of thymocytes and splenocytes as compared to normal littermates (Fig. 3CGo), suggesting that T cell differentiation in the thymus was not perturbed. Since the activation of calcineurin is thought to be required for positive selection in the thymus (46,47), this result was somewhat unexpected. This could perhaps be explained the modest increase in calcineurin activity in the dCaM transgenic thymocytes. In fact, the expression levels of dCaM Tg mRNA and protein in thymocytes were ~5% compared to those of endogenous calcineurin (data not shown). The TCR-induced phosphatase activity of calcineurin in dCaM Tg thymocytes is 1.5 times greater than that of wild-type normal thymocytes (data not shown). To address the effect of the active form of calcineurin in thymic selection, more comprehensive analyses are required.



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Fig. 3. Establishment of transgenic mice with an active form of calcineurin {alpha} chain (dCaM Tg). (A) A schematic representation of the dCaM Tg construct. (B) Anti-TCR-induced activation of NF-AT promoter in a T cell line transfected with dCaM. Mean luciferase light units of cell lysates from a T cell line 68-41 transfected with dCaM after anti-TCR stimulation are shown with SD. (C) CD4/CD8 staining profiles of thymocytes and splenocytes from dCaM Tg mice with B6 background and dCaMxDO10 double-Tg mice with (B6xBALB/c)F1 background. Percentages of cells present in each area are shown. (D) Proliferative responses of purified dCaM Tg CD4 T cells were assessed upon stimulation with a small amount of PMA (3 nM) with or without a low dose (30 nM) of ionomycin.

 
Similarly, the CD4+ splenic T cells express small but significant levels of dCaM Tg mRNA (~3% compared to endogenous calcineurin mRNA). Equivalent levels of proliferative response of the dCaM Tg CD4 T cells as compared to those of littermates were induced with immobilized anti-TCR mAb (100 µg/ml, data not shown). To detect functional changes of the dCaM Tg, proliferative responses were measured following stimulation with a small amount of PMA (3 nM) with or without a low dose of ionomycin. Significantly higher response was induced by stimulation with PMA alone and a similar increased response was observed in the presence of a low dose of ionomycin (Fig. 3DGo). These results suggest that calcineurin is activated more easily in dCaM Tg CD4 T cells.

Impaired proliferation of the SEB-induced anergic T cells was rescued by the presence of an active form of calcineurin (dCaM)
Consequently, this dCaM Tg line was mated with DO10 Tg mice and the double Tg mice were used to ascertain whether the presence of an active form of calcineurin would rescue the SEB-induced anergy in these mice. After SEB treatment, the behavior of dCaM Tg Vß8+ T cells (initial expansion and following decrease in number) was similar to that of DO10 Tg or non-transgenic BALB/c mice (data not shown). This suggests that dCaM Tg Vß8 T cells responded to SEB in a similar fashion. As shown in Fig. 4Go(A), comparable impairment in the TCR-induced Ca response of surviving DO10xdCaM double-Tg T cells is seen. The result suggests that a defect leading to an impaired Ca response characteristic of SEB-induced anergic T cells was induced even in the presence of the dCaM Tg. These normal responses to SEB seem to be due to the fact that the level of increased phosphatase activity is moderate. Interestingly, however, the surviving CD4 T cells in the double-transgenic mice showed equivalent levels of proliferation upon anti-TCR Vß8 stimulation in vitro (Fig. 4BGo). These results suggest that the presence of the dCaM Tg can influence the anergic state of CD4 T cells.



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Fig. 4. Impaired proliferation of the SEB-induced anergic T cells was rescued by the presence of an active form of calcineurin (dCaM). (A) Anti-TCR-induced Ca responses of surviving splenic CD4 T cells from PBS- (solid line) or SEB- (dotted line) treated DO10xdCaM double-Tg mice were assessed. The cells were stimulated with avidin (arrows) at 37°C. Representative results of two independent sets of SEB-treated animals are shown. (B) Purified splenic T cells from PBS- (open) or SEB- (hatched) treated DO10 Tg+, dCaM Tg littermate (LM) and DO10xdCaM double-Tg mice (dCaM) were stimulated with immobilized anti-Vß8 antibody, and [3H]thymidine uptake was measured. Three independent experiments with seven SEB-treated dCaM Tg mice were done and similar results were obtained.

 
`Calcium-blocked' anergy
The present study demonstrates that TCR-mediated activation of the Ca/calcineurin pathway is crucial for in vivo SEB-induced T cell anergy. Both the Ca/calcineurin and Ras/MAPK pathways are required for IL-2 transcription and secretion (48,49), and therefore defects in either pathway may result in T cell unresponsiveness. Recent biochemical studies using anergized mouse T cell clones have revealed so called `Ras-blocked' anergy (15), where preferential blockade in the activation of Ras, ERK/MAPK pathway and JNK/MAPK pathway is observed (1719). In contrast, our results strongly suggest the existence of `calcium-blocked' anergy. A significant difference in experimental systems appears to be the source of T cells. Most of the results that support `Ras-blocked' anergy are derived from analyses with cultured T cell clones, such as A.E7 and pGL10 (1719). Although the results from cultured T cells are compelling, it is uncertain whether they are reflective of molecular events occurring in the resident T cells in vivo. Thus, we have used the in vivo system described here in order to clarify molecular mechanisms governing anergic state in vivo. The in vivo SEB-induced anergy is a well-documented anergic system. From this study, `calcium-blocked' anergy has been revealed. More recently, we have been investigating other anergic T cell models, such as anergy induced by orally administered soluble protein antigen ovalbumin. Intriguingly, similar `calcium-blocked' anergy was observed in this ovalbumin system. No impairment in the Ras/MAPK signaling pathway was detected (manuscript in preparation). Similarly, anergic T cells induced in vivo by MHC molecules, Ld in the 2C TCR transgenic system, exhibit a defect in calcium mobilization and no impairment in ERK1/2 activation (50). Thus, the `calcium-blocked' anergy may not to be limited to superantigen-induced anergy, although further investigations are required to determine the general occurrence of the `calcium-blocked' anergy.

We detected defects in the activation of PLC-{gamma}1 (Fig. 2CGo). This is good agreement with the results that suggest that Ca responses were largely dependent on PLC-{gamma}1 activation (4345). We detected a slight but significant defect in the activation of Fyn but not of Lck after TCR cross-linking (data not shown). Although extensive analyses with various different TCR stimuli were performed, no defect in TCR{zeta} phosphorylation was observed (data not shown). The reason for the discrepancy between our results and those of Migita et al. (33), in which TCR {zeta} phosphorylation was impaired, is not clear In our DO10 Tg mouse system, large numbers of relatively pure anergic T cells can be obtained without additional purification steps, that may include the use of anti-Vß8 mAb which has a potential to stimulate T cells, to generate sufficient numbers of cells for further analysis. Large numbers of T cells are activated, and large amounts of various cytokines such as IL-2, IL-4 and tumor necrosis factor-{alpha} are produced from T cells in the DO10 Tg system, and, therefore, a different mode of anergy from so called `superantigen-induced anergy' could be the consequence. This possibility cannot be excluded; however, we feel this is unlikely because the behavior of SEB-reactive Vß8 T cells in the spleen was quite similar to transgenic-negative litermates and also we found that anergic T cells in either non-transgenic BALB/c or DO10 Tg mice show a similar specific defect in IL-2 production that could be rescued by the addition of recombinant IL-2.

In summary, the activation of the Ca/calcineurin pathway is a critical process that if altered may result in the induction of peripheral T cell anergy. In addition, the effects resulting from modest expression of the dCaM transgene in this system raise the interesting prospect that variations in calcineurin expression and activation may affect susceptibility to autoimmune and infectious diseases in man.


    Acknowledgments
 
The authors are grateful to Dr Ralph T. Kubo for helpful comments and constructive criticisms in the preparation of the manuscript. This work was supported by grants from the Ministry of Education, Science and Culture (Japan), by the Ministry of Health and Welfare (Japan), and by the Kanagawa Academy of Science and Technology.


    Abbreviations
 
[Ca2+]i intracellular free calcium ion concentrations
dCaM active form of calcineurin {alpha} chain
DO10 Tg anti-OVA TCR{alpha}ß transgenic
ERK extracellular signal-regulated protein kinases
JNK c-Jun N-terminal kinases
MAPK mitogen-activated protein kinase
PLC phospholipase C
PMA phorbol myristate acetate
SEB staphylococcal enterotoxin B

    Notes
 
Transmitting editor: A. Singer

Received 1 November 1999, accepted 11 February 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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