Survival of Naive T Cells for the Β Requirement of Calcineurin A

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Requirement of Calcineurin Aβ for the Survival of Naive T Cells Santhakumar Manicassamy, Sonal Gupta, Zhaofeng Huang, Jeffery D. Molkentin, Weirong Shang and Zuoming Sun This information is current as of September 26, 2021. J Immunol 2008; 180:106-112; ; doi: 10.4049/jimmunol.180.1.106 http://www.jimmunol.org/content/180/1/106 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Requirement of Calcineurin A␤ for the Survival of Naive T Cells1

Santhakumar Manicassamy,† Sonal Gupta,† Zhaofeng Huang,† Jeffery D. Molkentin,‡ Weirong Shang,§ and Zuoming Sun2*†

Calcineurin (Cn) is a Ca2؉/calmodulin-dependent phosphatase that dephosphorylates and activates NFAT, a transcription factor essential for T cell activation. T lymphocytes predominantly express the calcineurin A␤ (CnA␤) isoform, and the deletion of the CnA␤ gene results in defective T cell proliferation and IL-2 production in response to TCR stimulation. In this study, we show that CnA␤ enhances the spontaneous survival of naive T cells by maintaining high levels of Bcl-2, a critical homeostatic survival factor for naive T cells. T cells obtained from CnA␤؊/؊ mice displayed accelerated spontaneous apoptosis. The observed apoptosis .of the CnA␤؊/؊ T cells was prevented by IL-7 and IL-15, two cytokines critical for the homeostatic survival of naive T cells ؉ ؉ ؊ ؊ Furthermore, CD4 or CD8 single positive CnA␤ / thymocytes also underwent accelerated apoptosis. However, no obvious Downloaded from -difference in the apoptosis of CD4؉CD8؉ double positive thymocytes was observed between CnA␤؊/؊and wild-type mice, sug gesting a specific function of CnA␤ in the survival of single positive T cells. Bcl-2 levels were found to be significantly lower in ؊/؊ ␤؊/؊␤ CnA T cells. Transgenic expression of Bcl-xL restored the survival of the CnA T cells. Thus, in addition to its role in mediating TCR signals essential for T cell activation, CnA␤ is also required for the homeostatic survival of naive T cells. The Journal of Immunology, 2008, 180: 106–112. http://www.jimmunol.org/ alcineurin (Cn)3 isaCa2ϩ/calmodulin-dependent phos- cells deficient in CnA␤, but not CnA␣, displayed significant de- phatase consisting of a catalytic and calmodulin-binding fects in T cell activation (4, 5). Thus, CnA␤ mediates TCR signals C subunit A (CnA) and a Ca2ϩ-binding regulatory subunit required for T cell activation. B (CnB) (1). In the resting T cells, enzymatic activity of CnA is In addition to T cell activation, TCR signals are also required for ϩ kept inactive by binding to CnB. Increased intracellular Ca2 con- the homeostatic survival of the naive T cells (6, 7). Adoptive trans- centration following TCR stimulation leads to the activation of fer of naive T cells to recipient mice with mismatched MHC type calmodulin that then binds to CnA, resulting in the activation of or MHC-deficient mice indicated that long-term survival of naive enzymatic activity of CnA. Activated Cn dephosphorylates and T cells requires the interactions of TCR and MHC-peptide com- by guest on September 26, 2021 activates NFAT. Dephosphorylated NFAT translocates to the nu- plexes (8, 9). When the peptides with different affinities for TCR cleus and stimulates its target gene transcription, IL-2, which is were tested in the adoptive transfer experiments, it was found that required for T cell activation. The critical role of calcineurin is also the low affinity peptide that results in weak interactions is required demonstrated by the discovery of the immunosuppressive drugs for naive T cell survival, whereas the high affinity peptide that FK506 and cyclosporin A, which are broadly used to prevent al- results in strong interactions between TCR and MHC complexes is lograft rejection. FK506 and cyclosporin A, when bound to their required for T cell activation (10). These results suggest that trans- respective binding proteins, FKBP12 and cyclophilin A, inhibit Cn duction of different signals through TCR is required to mediate T activity and thus prevent T cell activation (2). Three different iso- cell activation and homeostatic survival. Many signaling mole- forms of CnA transcribed from distinct genes have been identified cules including CnA␤ are known to govern the TCR signaling ␤ (3). T cells predominantly express CnA isoforms. Consistently, T events for T cell activation, but little is known about the signaling events that mediate the survival of naive T cells. Anti-apoptotic Bcl-2 is the first important gene identified to *Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, † regulate programmed cell death (11, 12). During T cell devel- CA 91010; Department of Microbiology and Immunology, Medical School of the ϩ ϩ University of Illinois, Chicago, IL 60612; ‡Division of Molecular Cardiovascular opment, Bcl-2 is predominantly expressed in CD4 or CD8 Biology, Department of Pediatrics, Children’s Hospital Medical Center, Cincinnati, single positive T cells. Bcl-x , another critical anti-apoptotic OH 45229; and §Department of Gynecology and Obstetrics, Emory University School L of Medicine, Atlanta, GA 30308 molecule of the Bcl-2 family, is specifically up-regulated in ϩ ϩ Received for publication July 10, 2007. Accepted for publication October 20, 2007. CD4 CD8 double positive cells (13). Bcl-2 thus primarily The costs of publication of this article were defrayed in part by the payment of page enhances the survival of the single positive cells, and Bcl-xL is charges. This article must therefore be hereby marked advertisement in accordance the survival molecule for double positive cells. The essential with 18 U.S.C. Section 1734 solely to indicate this fact. role of Bcl-xL in the survival of double positive cells was dem- 1 This work was supported by National Institutes of Health R01-AI053147. Ϫ/Ϫ onstrated by the apoptosis of Bcl-xL double positive T cells 2 Address correspondence and reprint requests to Dr. Zuoming Sun, Division of Im- (13, 14). Bcl-2Ϫ/Ϫ mice have reduced peripheral T cells (14), munology, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010. E-mail address: [email protected] suggesting that Bcl-2 plays a role in the homeostatic survival of naive T cells. In this study we show that CD4ϩ or CD8ϩ T cells 3 Abbreviations used in this paper: Cn, calcineurin; AICD, activation-induced cell Ϫ/Ϫ death; CnA, calcineurin subunit A (catalytic and calmodulin binding); CnB, cal- obtained from CnA␤ mice displayed accelerated spontane- ϩ cineurin subunit B (Ca2 binding regulatory); PKC-␪, protein kinase C ␪; Tg, trans- ous apoptosis as well as significantly reduced Bcl-2 levels. genic; WT, wild type. ␤Ϫ/Ϫ Forced expression of Bcl-xL restored survival to CnA T ϩ Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 cells. CnA␤ thus regulates the homeostatic survival of CD4 www.jimmunol.org The Journal of Immunology 107

FIGURE 1. CnA␤Ϫ/Ϫ T cells undergo increased apoptosis. a, T cells purified from WT (CnA␤ϩ) and CnA␤Ϫ/Ϫ (CnA␤Ϫ) mice were left in medium Downloaded from (None) or subjected to stimulation with anti-CD3 (1 ␮g/ml) and CD28 (2 ␮g/ml) Abs or in the presence of IL-2 for 36 h. T cells were then pulsed with p Ͻ 0.001; significantly differs from WT. b, CnA␤ϩ and CnA␤Ϫ CD4ϩ T cells were left in medium or subjected ,ء .3H]thymidine for DNA incorporation] to treatment with IL-2. After 36 h, apoptotic cells were detected by flow cytometric analysis of annexin V staining cells. The percentage of annexin V-positive cells was measured. Effects of stimulation with anti-CD3 (1 ␮g/ml) and anti-CD28 (2 ␮g/ml) Abs on apoptosis of the T cells were also p Ͻ 0.001; significantly differs from WT. c, Similar apoptosis assays ,ء .determined. T cells were stimulated immediately after plating on 96-well plate p Ͻ 0.001; significantly differs from WT. d, WT T cells were left in medium alone or treated with FK506 for ,ء .as described in b using CD8ϩ T cells http://www.jimmunol.org/ 36 h. Apoptotic cells were then detected as described. Data shown are representative of at least three independent experiments. Error bars indicate SD.

and CD8ϩ single positive T cells by maintaining high levels of Western blot analysis Bcl-2. The protein levels of Bcl-2 and ␤-actin were analyzed by Western blotting. Enriched T cells were puriﬁed as described above. Enriched T cells (2 ϫ Materials and Methods 106) from WT or CnA␤Ϫ/Ϫ mice were lysed by 1% Nonidet P-40-containing buffer as described previously (16). Anti-Bcl-2 (BD Pharmingen) and

Mice by guest on September 26, 2021 anti-␤-actin Abs were used for Western blot analysis. CnA␤Ϫ/Ϫ mice were originally described by Bueno et al. (4). Mice were kept in the animal facility of the Biological Resource Laboratory at the University of Illinois (Chicago, IL) following the university guide- Tg lines. Bcl-xL mice (where Tg is transgenic) have been previously de- ␤Ϫ/Ϫ Tg scribed (15). CnA and Bcl-xL mice were intercrossed to generate ␤Ϫ/Ϫ Tg CnA /Bcl-xL . Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory. Preparation of lymphocytes Thymus, spleens, or mesenteric lymph nodes were harvested from 8- to ␤Ϫ/Ϫ Tg ␤Ϫ/Ϫ Tg 12-wk-old WT, CnA , Bcl-xL , and CnA /Bcl-xL mice and dis- sociated into single-cell suspensions. RBCs were lysed by adding 5 ml of RBC lysing buffer. CD4ϩ or CD8ϩ T cells were puriﬁed from splenocytes using a CD4ϩ or CD8ϩ isolation kit (Miltenyi Biotec) according to the manufacturer’s protocol. CD4ϩ or CD8ϩ T cells were separated using an autoMACS magnetic cell sorter (Miltenyi Biotec). The purity of the T cells, determined by a ﬂow cytometry, was Ͼ90%. Proliferation assays Enriched T cells (0.3 ϫ 105 cells per well) were cultured in 96-well plates in the absence or presence of 10 ng/ml recombinant mouse IL-2, IL-7, or IL-15 (R&D Systems) and stimulated for 48 h in the presence or absence of anti-CD3 and CD28 Abs. After the 48 h of stimulation the cultures were pulsed with 1 ␮Ci of [3H]thymidine per well for 12 h.

Ϫ/Ϫ Apoptosis assay FIGURE 2. Spontanous apoptosis of the CnA␤ T cells is inhibited by IL-7 and IL-15. a, CnA␤ϩ and CnA␤Ϫ T cells were left in medium or ϩ ϩ Purified splenic CD4 or CD8 T cells were cultured in the absence or treated with IL-7 or IL-15. After 36 h, apoptotic cells were detected by flow presence of 10 ng/ml recombinant mouse IL-2 or IL-7 or IL-15 (R&D cytometric analysis of annexin V staining cells. Effects of stimulation with Systems), and stimulated as described in the proliferation assay for various anti-CD3 (1 ␮g/ml) and CD28 (2 ␮g/ml) Abs on apoptosis of the T cells time points. Cells were washed once with ice-cold annexin V binding p Ͻ 0.005; significantly differs from cytokine ,ء .were also determined buffer (BD Pharmingen) and cell pellets were stained with PE-conjugated ϩ annexin V and 7-aminoactinomycin D 7-AAD (BD Pharmingen) according treatment. b, Similar apoptosis assays as described in a using CD8 T p Ͻ 0.005; significantly differs from cytokine treatment. Data ,ء .to the manufacturer’s protocol. Analyses were performed by a flow cy- cells tometer with CELLQuest software. Viable cells were analyzed by exclud- shown are representative of at least three independent experiments. Error ing dead cells using low forward light scatter. bars, SD. 108 REGULATION OF NAIVE T CELL SURVIVAL BY CALCINEURIN A␤

CnA␤Ϫ/Ϫ mice (Fig. 1b). CD4ϩ T cells were left in medium alone or treated with IL-2 for 36 h. Apoptotic cells were then detected with a ﬂow cytometer. Approximately 30% of the WT CD4ϩ T cells were found dead after 36 h in medium. Compared with the WT T cells, CnA␤Ϫ/Ϫ T cells displayed greatly increased apoptosis as indicated by the fact that ϳ60% of the CnA␤Ϫ/Ϫ T cell underwent apoptosis. IL-2 treatment could not prevent accelerated spontaneous apoptosis of the CnA␤Ϫ/Ϫ FIGURE 3. CD4ϩ and CD8ϩ single positive, but not CD4ϩCD8ϩ dou- cells. These results suggest that CnA␤ is a critical signaling ble positive, thymocytes from CnA␤Ϫ/Ϫ mice undergo accelerated apopto- molecule required for maintaining the spontaneous survival of ϩ ϩ ϩ ϩ sis. Spontaneous apoptosis of the CD4 , CD8 , and CD4 CD8 was the naive CD4ϩ T cells. TCRs deliver signals required for stim- Ϫ/Ϫ analyzed as described using thymocytes obtained from WT and CnA␤ ulating proliferation and enhancing survival (25, 26). Such sur- mice. Data shown are representative of at least three independent vival signals ensure the completion of the T cell activation pro- experiments. cess essential for differentiating naive T cells to effectors that mediate actual immune responses (27). We therefore deter- Luciferase assays mined whether stimulation of TCR can prevent the apoptosis of the CnA␤Ϫ/Ϫ T cells (Fig. 1b). Compared with the unstimulated A 2.6-kb Bcl-2 promoter element was cloned upstream of a luciferase gene ϩ 7 CD4 T cells (30% apoptotic cells), CD3/CD28 stimulation did (pGL2-Basic vector) (17). Jurkat or Jurkat-Tag cells (10 /ml) were trans- ϩ enhance the survival of the WT CD4 T cells (20% apoptotic Downloaded from fected by electroporation with 5 ␮g of the Bcl-2 luciferase reporter plasmid ␤Ϫ/Ϫ together with 15 ␮g of the constitutively active or the inhibitory calcineurin cells). However, the survival of the CnA T cells was not B subunit expression plasmid. Identical amounts of the corresponding pa- signiﬁcantly changed by CD3/CD28 stimulation even in the rental vectors were used as control. For normalization, 100 ng of the Re- presence of IL-2, indicating that CnA␤ mediates the TCR sig- nilla luciferase reporter vector pTK-Renilla-LUC was used. After 36 h, nals required for enhancing T cell survival. Similar results were cells were lysed and assayed for dual luciferase activity (Promega). obtained from analyzing the apoptosis of CD8ϩ T cells (Fig.

Statistical analysis 1c). Altogether, CnA␤ mediates the critical survival signals for http://www.jimmunol.org/ CD4ϩ and CD8ϩ T cells. We performed Student’s t test for statistical analysis. The preceding experiments used defective CnA␤Ϫ/Ϫ T cells; Results we next determined the effects of blocking the Cn pathway on CnA␤Ϫ/Ϫ T cells undergo accelerated spontaneous apoptosis the survival of the WT T cells. FK506, a specific inhibitor for Cn, was used to treat the purified CD4ϩ and CD8ϩ T cells (Fig. Previous studies including ours have shown that protein kinase ϩ 1d). Indeed, inhibition of Cn by FK506 greatly increased spon- ␪ ␪ 2 ϩ ϩ C (PKC- ) regulates the Ca /NFAT pathway (18–20) as taneous apoptosis both in CD4 and in CD8 T cells from well as T cell survival (16, 21, 22); we thus intended to deter- ϳ30% to 60%, confirming the critical role of Cn in the survival

␪ by guest on September 26, 2021 mine whether Cn plays a role in PKC- -regulated T cell sur- of naive T cells. vival. Mice deficient in catalytic subunit A␤ (CnA␤Ϫ/Ϫ) were used in this study because, similar to PKC-␪, CnA␤ also me- ␤Ϫ/Ϫ diates TCR signals required for T cell activation (4). We first IL-7 and IL-15 prevent the apoptosis of the CnA T cells examined T cell proliferation in response to TCR stimulation IL-7 and IL-15 have been shown to be critical for the homeostatic using a [3H]thymidine incorporation assay (Fig. 1a). Consistent survival of the naive T cells (28, 29). We therefore determined with previous results (4), CnA␤Ϫ/Ϫ T cells displayed significant whether these two cytokines can prevent spontaneous apoptosis of defects in CD3/CD28 stimulation-induced proliferation, and the CnA␤Ϫ/Ϫ CD4ϩ T cells (Fig. 2a). Consistent with their roles such defects could not be prevented by exogenous IL-2. This in the homeostatic survival of T cells, IL-7 and IL-15 enhanced result confirmed a critical role of CnA␤ in T cell activation. To the survival of WT T cells. IL-7 treatment inhibited apoptosis of determine whether cell death contributed to the significantly the CnA␤Ϫ/Ϫ CD4ϩ T cells from ϳ60 to ϳ40%. IL-15 treat- reduced [3H]thymidine incorporation by CnA␤Ϫ/Ϫ T cells, we ment further inhibited apoptosis of the CnA␤Ϫ/Ϫ CD4ϩ T cells performed a apoptosis assay using annexin V and 7-aminoacti- to ϳ30%, which is equivalent to that observed in WT CD4ϩ T nomycin D as described previously (16, 23, 24). We first ana- cells. These results suggest that IL-7 and IL-15 can compensate lyzed apoptosis of the CD4ϩ T cells obtained from WT and for CnA␤-mediated survival. Furthermore, because IL-7 and

FIGURE 4. CnA␤Ϫ/Ϫ T cells had signiﬁcantly reduced Bcl-2 levels. a, Bcl-2 levels were determined by Western blot analysis of WT and CnA␤Ϫ/Ϫ T cells. Ac- tin serves as a control for equal loading. Data shown are representative of at least three independent experiments. b, Cn regulates Bcl-2 reporter activity. A luciferase reporter under the control of a Bcl-2 promoter was transfected into Jurkat cells alone (reporter) or together with an expression plasmid encoding the inhibitory B subunit (CnB) or constitutively active Cn. Bcl-2 luciferase reporter activity is indicated as fold induction relative to the activity obtained from unstimulated cells in the control group. Data shown are representative of at least three independent experiments. The Journal of Immunology 109

Tg ␤Ϫ/Ϫ FIGURE 5. Bcl-xL restored survival to CnA T cells. a, Spontaneous apoptosis was performed using CD4ϩ T cells obtained from spleens of WT, CnA␤Ϫ/Ϫ, Ͻ ء Tg ␤Ϫ/Ϫ Tg Bcl-xL , and CnA /Bcl-xL mice. , p 0.01; significantly differs from CnA␤Ϫ/Ϫ mice. b, Similar apoptosis assays as described in a using CD8ϩ T cells from p Ͻ 0.01; significantly ,ء .different genotypes of mice differs from CnA␤Ϫ/Ϫ mice. c, CD4ϩ cell number in the ␤Ϫ/Ϫ Tg ␤Ϫ/Ϫ Downloaded from spleens of WT, CnA , Bcl-xL , and CnA /Bcl- Tg ϩ xL mice. d, CD8 cell number in the spleens of WT, ␤Ϫ/Ϫ Tg ␤Ϫ/Ϫ Tg CnA , Bcl-xL , and CnA /Bcl-xL mice. e, Spontaneous apoptosis was performed using CD4ϩ thy- ␤Ϫ/Ϫ Tg mocytes obtained from WT, CnA , Bcl-xL , and Ͻ ء Ϫ/Ϫ Tg␤ CnA /Bcl-xL mice. , p 0.01; significantly dif- ␤Ϫ/Ϫ Tg fers from CnA /Bcl-xL mice. f, Similar apoptosis http://www.jimmunol.org/ assays as described in c using CD8ϩ thymocytes from p Ͻ 0.05; significantly ,ء .different genotypes of mice ␤Ϫ/Ϫ Tg differs from CnA /Bcl-xL mice. g, Total thymocyte ␤Ϫ/Ϫ Tg ␤Ϫ/Ϫ number of WT, CnA , Bcl-xL , and CnA / Ͻ ء Tg Bcl-xL . , p 0.05; significantly differs from CnA␤Ϫ/Ϫ mice. h, CD4ϩCD8ϩ thymocyte number of p Ͻ 0.05; significantly ,ء .different genotypes of mice differs from CnA␤Ϫ/Ϫ mice. i, CD4ϩ thymocyte number -p Ͻ 0.001; signifi ,ء .of different genotypes of mice Ϫ/Ϫ ϩ cantly differs from CnA␤ mice. j, CD8 thymocyte by guest on September 26, 2021 ;p Ͻ 0.001 ,ء .number of different genotypes of mice significantly differs from CnA␤Ϫ/Ϫ mice. Data shown are representative of at least three independent experiments. Error bars, SD.

IL-15 can enhance T cell survival in the absence of CnA␤, IL-7- such as IL-7 and IL-15, is critical for maintaining the survival and IL-15-mediated survival is independent of CnA␤. CD4ϩ T of naive T cells. cells were also stimulated with CD3/CD28 in the presence or CnA␤ is required for the survival of the CD4ϩ or CD8ϩ single absence of IL-7 or IL-15. Consistent with the previous results, ϩ ϩ TCR stimulation could not prevent apoptosis of the CnA␤Ϫ/Ϫ T positive but not the CD4 CD8 double positive thymocytes cells. IL-7 and IL-15 treatment, although inhibit spontaneous The thymus is the location for T cell differentiation and matura- apoptosis, had no significant effects on apoptosis of the tion. Immature CD4ϩCD8ϩ thymocytes are subject to positive and CnA␤Ϫ/Ϫ CD4ϩ T cells induced by TCR stimulation. This re- negative selection in the thymus. Only the cells that meet the se- sult suggests that the spontaneous apoptosis does not likely con- lection criteria are allowed to mature into CD4ϩ or CD8ϩ single tribute to the apoptosis induced by TCR crosslinking. The role positive cells and migrate out of the thymus. We thus also deter- of CnA␤ in the spontaneous survival and the TCR stimulation- mined whether CnA␤ provides survival signals for thymocytes. mediated survival signals are likely two separate events. Similar Spontaneous apoptosis assays were performed using thymocytes results were also observed from analyzing the apoptosis of obtained from both WT and CnA␤Ϫ/Ϫ mice (Fig. 3) as described CD8ϩ T cells (Fig. 2b). Thus, CnA␤, together with cytokines previously (23, 24). No obvious differences were detected in the 110 REGULATION OF NAIVE T CELL SURVIVAL BY CALCINEURIN A␤ apoptosis of CD4ϩCD8ϩ thymocytes between WT and CnA␤Ϫ/Ϫ CnA␤Ϫ/Ϫ mice (Fig. 5e), which was also reflected by the reduced T cells. However, increased apoptosis was observed in CD4ϩ CD4ϩCD8ϩ double positive thymocytes in CnA␤Ϫ/Ϫ mice (Fig. (ϳ40%) and CD8ϩ (ϳ65%) single positive T cells deficient in 5f). The most significant reduction was observed in the number of CnA␤ when compared with the corresponding WT CD4ϩ (ϳ20%) CD4ϩ (Fig. 5f) and CD8ϩ (Fig. 5h) single positive cells in and CD8ϩ (ϳ40%) T cells. This result suggests that CnA␤ is CnA␤Ϫ/Ϫ mice. Consistent with the observation in the number of ϩ ϩ Tg required for enhancing the survival of CD4 and CD8 single peripheral T cells, Bcl-xL , although it could slightly increase the positive, but not the CD4ϩCD8ϩ double positive, thymocytes. number of single positive T cells, could not restore the number of ϩ ϩ ␤Ϫ/Ϫ Ϫ/Ϫ CD4 and CD8 thymocytes in CnA mice to the WT levels. CnA␤ T cells express greatly reduced levels of Tg The fact that Bcl-xL restored the survival, but not the number, of anti-apoptotic protein Bcl-2 single positive T cells suggests that CnA␤ regulates additional Bcl-2 is a critical survival molecule for naive T cells, because functions such as the proliferation of the T cells in addition to Bcl-2Ϫ/Ϫ T cells are susceptible to apoptosis and transgenic ex- regulating survival. pression of Bcl-2 protects T cells from apoptosis (30, 31). We thus detected protein levels of Bcl-2 in T cells obtained from spleens Discussion using Western blot analysis (Fig. 4a). Indeed, CnA␤Ϫ/Ϫ T cells Using CnA␤Ϫ/Ϫ mice, we showed that deletion of the CnA␤ gene had greatly reduced levels of Bcl-2 compared with that of the WT leads to an increased apoptosis of naive CD4ϩ and CD8ϩ T cells T cells. CnA␤ is thus required to maintain high levels of Bcl-2 in likely due to the reduced survival molecule Bcl-2. Apoptosis of the naive T cells. CnA␤Ϫ/Ϫ T cells could be prevented by IL-7, IL-15, or the trans- Downloaded from The 2 promoter contains multiple consensus NFAT-binding genic expression of Bcl-xL. The survival of naive T cells depends sites, and the mutation of two critical NFAT-binding sites reduces on the signals generated from the continuous interactions between the Bcl-2 promoter activity in cardiac myocytes (32). We thus TCR and MHC complexes and from cytokines such as IL-7 and decided to determine whether Cn can regulate Bcl-2 promoter ac- IL-15 (33–35). It is likely that CnA␤ mediates the TCR signals tivity in T cells. We obtained a Bcl-2-luciferase reporter containing essential for the homeostatic survival of naive T cells. a 2.6-kb Bcl-2 promoter region described in a previous report (17). After emigrating from the thymus, the naive T cell can survive

The Bcl-2 reporter was transfected into Jurkat cells by electropo- an extended period of time only in the presence of continuous http://www.jimmunol.org/ ration alone or together with an expression plasmid encoding the interaction between TCR and MHC complexes (33–35). By adop- inhibitory B subunit (CnB) or the constitutively active Cn (Active- tive transfer experiments, Takeda et al. (36) have shown that the Cn) (Fig. 4b). CnB inhibited ϳ50% of the Bcl-2 reporter activity. long-term survival of CD4ϩ T cells requires MHC II, because the Constitutively active Cn drastically stimulated Bcl-2 reporter ac- number of CD4 T cells adoptively transferred to MHC II-deﬁcient tivity. This result suggests that Cn can directly stimulate Bcl-2 mice is gradually reduced since the number of CD4 T cells trans- promoter activity. ferred to WT mice. Similarly, survival of the naive CD8ϩ T cells also depends on the presence of MHC I binding to peptide Ags (8). Transgenic expression of Bcl-xL restored the survival of ␣ Ϫ/Ϫ Furthermore, conditional ablation of the TCR -chain gene in the

␤ by guest on September 26, 2021 CnA T cells mature T cell stage leads to a shorter lifespan of naive T cells (37), To determine whether the reduced Bcl-2 observed in CnA␤Ϫ/Ϫ suggesting that TCR-mediated signals are essential for naive T cell mice is responsible for the accelerated apoptosis of the CnA␤Ϫ/Ϫ survival. Therefore, signals resulting from the interaction between ␤Ϫ/Ϫ Tg T cells, CnA mice were crossed with Bcl-xL mice (Bcl- MHC and TCR maintain the survival of naive T cells. In addition Tg xL ). Bcl-xL is another anti-apoptotic member of the Bcl-2 family. to TCR signals, cytokines such as IL-7 and IL-15 are also required Similar to Bcl-2, forced expression of Bcl-xL protects cells from for the survival of naive T cells. Naive T cells survive for only a Tg apoptosis (15). Overexpression of Bcl-xL in Bcl-xL mice is short period of time when adoptively transferred to IL-7-deficient achieved by using a Lck proximal promoter that targets a transgene mice (28, 38). Elevating IL-7 levels either by expressing a trans- specifically to T cell compartments. Apoptosis was compared be- gene or injecting rIL-7 results in a significantly increased T cell tween T cells obtained from the spleens of WT, CnA␤Ϫ/Ϫ, Bcl- population (39, 40). IL-15 seems to have a similar function as IL-7 Tg ␤Ϫ/Ϫ Tg xL , and CnA /Bcl-xL mice (Fig. 5, a and b). Consistent in promoting T cell survival, likely due to the fact that they share with the results observed in Fig. 1, CnA␤Ϫ/Ϫ CD4ϩ T cells un- a common receptor subunit, ␥c (41). Although it is known that derwent increased apoptosis compared with that of the WT CD4ϩ both the TCR and cytokines are required for the survival of naive

T cells. However, forced expression of Bcl-xL restored the survival T cells in vivo, little is known about the molecules mediating the of the CnA␤Ϫ/Ϫ CD4ϩ T cells to levels similar to those observed survival signals. Our results demonstrated that CnA␤ mediates the in the WT CD4ϩ T cells. Similar results were also observed in critical survival signals for naive T cells, because lack of CnA␤ CD8ϩ T cells (Fig. 5b). These results suggest that CnA␤-regulated leads to a signiﬁcantly increased spontaneous apoptosis. CnA␤Ϫ/Ϫ T cell survival is dependent on the Bcl-2 survival factor. We then T cells had increased levels of phosphorylated NFAT, indicating counted the number of CD4ϩ (Fig. 5c) and CD8ϩ (Fig. 5d) T cells the lack of activation of NFAT (4). It is thus likely that CnA␤ in the spleens of different genotypes of mice. The number of both regulates T cell survival via activating NFAT. As both TCR- and CD4ϩ and CD8ϩ T cells in CnA␤Ϫ/Ϫ mice were greatly reduced cytokine-mediated signals are required for the naive T cell sur- Tg ␤ compared with that of the WT mice. Surprisingly, Bcl-xL , which vival, it is not clear whether CnA mediates the survival signals restored survival to CnA␤Ϫ/Ϫ T cells, only slightly increased the for TCR or cytokines or both. Our data indicated that IL-7 or IL-15 number of CD4ϩ and CD8ϩ cells, but far from the levels observed is able to enhance the survival of CnA␤Ϫ/Ϫ mice, suggesting that in the WT mice. IL-7 or IL-15 can deliver survival signals independently of CnA␤. Tg We next examined the effects of Bcl-xL on the survival of It is unlikely that activation-induced cell death (AICD) is respon- CnA␤Ϫ/Ϫ thymocytes (Fig. 5, c and d). Similar to peripheral T sible for the apoptosis of the CnA␤Ϫ/Ϫ T cells, as CnA␤ is required cells, apoptosis of the CnA␤Ϫ/Ϫ CD4ϩ (Fig. 5c) and CD8ϩ (Fig. for T cell activation (4). Consistently, we did not ﬁnd more acti- Tg ␤Ϫ/Ϫ 5d) thymocytes was inhibited by Bcl-xL . We also counted the vated T cells in CnA mice by examining T cell activation number of each subset of thymocytes (Fig. 5, e–h). Compared with markers such as CD25. Furthermore, we have shown that the WT mice, the total thymocyte number was reduced in CnA␤Ϫ/Ϫ T cell apoptosis induced by TCR stimulation cannot be The Journal of Immunology 111

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