Live Free or Die

[Cell Cycle 5:11, 1171-1174, 1 June 2006]; ©2006 Landes Bioscience

Live Free or Die Perspective

An Immature T cell Decision Encoded in Distinct Bcl-2 Sensitive and Insensitive Ca2+ Signals ABSTRACT

*Correspondence to: Clark W. Distelhorst; Case Western Reserve University; WRB 3133; 10900 Euclid Avenue; Cleveland, Ohio 44106 USA; Tel.: 216.368.4546; Fax: 216.368.8919; Email: [email protected]

Bcl-2, calcium, inositol 1,4,5-trisphosphate receptor, signaling, thymocyte, cell death, apoptosis, T cell receptor, T cell, lymphocyte

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T cell receptor endoplasmic reticulum inositol 1,4,5 trisphosphate

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For over a decade we have advanced the idea that one function of Bcl-2 is to regulate intracellular Ca2+ homeostasis and Ca2+ signaling in immature T cells. This concept gained momentum with our discovery that Bcl-2 interacts with the inositol 1,4,5-trisphosphate (IP3) receptor and regulates its opening, thereby inhibiting T cell receptor (TCR) activation-induced Ca2+ elevation in immature T cells.1 Recently, we found that TCR activation generates distinct patterns of Ca2+ signaling according to the strength of TCR activation and that Bcl-2 selectively inhibits pro-apoptotic Ca2+ elevation induced by strong TCR activation without interfering with pro-survival Ca2+ oscillations induced by weak TCR activation.2 These findings provide novel insight into the mechanism of Bcl-2 action and suggest a mechanism by which Bcl-2 may regulate the processes of positive and negative selection during T cell development in the thymus. The developing T cell passes through a series of successive maturational stages within the thymus gland.3 Each stage is defined by a specific pattern of surface marker expression (Fig. 1). Thymic precursor cells arriving from the bone marrow or fetal liver do not express either the TCR or the CD4 and CD8 antigens (i.e., double negative thymocytes). In the thymic cortex these cells undergo the first maturational step by expressing the TCR and both CD4 and CD8 antigens (i.e., double positive thymocytes). At this stage of development thymocytes undergo positive or negative selection, determining whether they will be free to live and serve the immune system or die by apoptosis. The selection process is stringent, as only a few cells are chosen to live.4,5 The positively selected cells mature to the single positive stage (CD4+CD8- or CD4-CD8+) in the thymic medulla and migrate to the peripheral lymphoid organs where they mount immune responses to foreign antigens. Bcl-2 family members play important roles in thymocyte development.6,7 The antiapoptotic protein Bcl-2 is expressed in thymic precursor cells, downregulated at the double positive stage of thymocyte development and then upregulated with maturation to the single positive stage8-10 (Fig. 1). Bcl-2 downregulation during the double positive stage of T cell development provides a finite window of opportunity for negative selection.11 Accordingly, enforced expression of Bcl-2 in transgenic mice reduces the efficiency of negative selection, causing excessive accumulation of thymocytes.12-14 The major determinant of whether a developing T cell undergoes positive or negative selection during the double positive stage of development is the strength of TCR activation.4,5 Low avidity engagement of the TCR induces positive selection, with subsequent maturation to the single positive (CD4+CD8- or CD4-CD8+) mature T cell stage. Alternatively, high avidity engagement triggers negative selection by apoptosis. How the strength of TCR activation determines cell fate is unknown. But the following evidence suggests that the strength of TCR activation and hence life and death decisions are encoded by distinct Ca2+ signals. The Ca2+ ion is a second messenger responsible for regulating a diverse array of physiological processes.15 Ca2+ signals are generated by elevations in cytoplasmic Ca2+ that in turn activate downstream effectors including Ca2+-sensitive phosphatases, kinases, and

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2778

TCR ER IP3

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Original manuscript submitted: 03/08/06 Manuscript accepted: 04/06/06

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Departments of Medicine and Pharmacology; Comprehensive Cancer Center; Case Western Reserve University and University Hospitals of Cleveland; Cleveland, Ohio USA

In the developing thymus, strong T cell receptor (TCR) activation by self-antigens induces negative selection and weak TCR activation induces positive selection. Both processes are mediated by Ca2+ signals, raising the question of how a single second messenger like Ca2+ can mediate such diverse cell fates. Recent findings indicate that graded TCR activation signals are encoded in distinct patterns of Ca2+ elevation. The anti-apoptotic protein Bcl-2 discriminates between these Ca 2+ signaling patterns, selectively inhibiting pro-apoptotic Ca2+ signals induced by strong TCR activation without suppressing pro-survival Ca2+ signals induced by weak TCR activation.

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Michael C. Davis Clark W. Distelhorst*

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This work was supported by NIH grant RO1 CA085804 (C.W.D.) and T32 HL07147 (M.C.D.). The authors are extraordinarily grateful to Martin Bootman, Llewelyn Roderick, Michael Berridge and George Dubyak for teaching us about calcium signaling

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Regulation of TCR-Mediated Ca2+ Signals by Bcl-2

proteases. In T cells, the signaling cascade initiated by antigen binding to the TCR activates phospholipase C-γ, generating IP3 which induces Ca2+ release from the endoplasmic reticulum.16,17,18 The resulting elevation of cytoplasmic Ca2+ is shaped by complex processes involving not only release of Ca2+ from the ER, but also extracellular Ca2+ uptake through channels in the plasma membrane and uptake of Ca2+ by mitochondria. The result is a remarkable variety of cytoplasmic Ca2+ response patterns, including transient elevation of Ca2+, repetitive Ca2+ spikes (oscillations) or sustained Ca2+ elevation.19 Different patterns of Ca2+ elevation enable this single second messenger to encode a wide range of cellular processes. Large transient Ca2+ elevations preferentially activate NFκB and JNK, whereas NFAT is preferentially activated by sustained Ca2+ oscillations. In lymphocytes information encoded by the frequency, amplitude and shape of Ca2+ oscillations differentially regulates transcription factors.20,21 The concept that Ca2+ encodes cell fate Figure 1. Stages of thymocyte development. The developing thymocyte undergoes a series of decisions in the thymus is consistent with a previconsecutive maturational stages. Downregulation of Bcl-2 expression during the double positive ously reported correlation between the extent of (CD4+CD8+) stage provides a window of opportunity for negative selection to occur in response Ca2+ elevation following TCR activation and cell to strong TCR activation. This window is closed by Bcl-2 upregulation when double positive thymocytes receive positive selection signals in response to weak TCR activation. death. Among thymocytes expressing a transgenic TCR of defined specificity, a large number had elevated intracellular Ca2+ concentrations in vivo, but only when activation2 (Fig. 2). Thus, Bcl-2 selectively inhibits pro-apoptotic residing in a negatively selecting thymus in which their self-ligand Ca2+ signals without interfering with pro-survival Ca2+ signals. was expressed.22 TCR activation by a high concentration of Downregulation of Bcl-2 expression during the double positive stage anti-CD3 antibody induced cytoplasmic Ca2+ elevation, triggering of T cell development may permit Ca2+ signals induced by strong apoptosis of thymocytes in vitro, whereas lower concentrations of TCR activation to induce negative selection, whereas upregulation anti-CD3 antibody induced lesser cytoplasmic Ca2+ elevations that of Bcl-2 during positive selection closes the window of opportunity did not induce cell death.23 The same held true when the TCR was for negative selection. This action of Bcl-2 in regulating TCR-mediactivated by antigenic peptides under conditions that mimic the ated Ca2+ signals appears to supplement rather than supplant the physiologic thymic environment.24 Thus, negatively selecting established view that Bcl-2 regulates apoptosis by inhibiting antigenic peptides induced a strong Ca2+ flux in immature thymo- mitochondrial pore formation by the Bcl-2 family members Bax and cytes, whereas positively selecting peptides induced a smaller Ca2+ Bak.6 This conclusion is consistent with evidence that transgenic flux. Ca2+ signals can trigger apoptosis by activating a wide range of Bcl-2 inhibits negative selection by a mechanism independent of its cell death pathways.25 Thymocyte negative selection is mediated by ability to antagonize Bax.28,29 According to earlier work in our laboratory, the mechanism of Ca2+-dependent upregulation of the pro-apoptotic Bcl-2 family Bcl-2’s inhibitory effect on TCR-mediated Ca2+ elevation involves a member Bim.26 Recent findings in our laboratory suggest that positive versus neg- physical interaction of Bcl-2 with IP3 receptors that inhibits ative selection decisions in the thymus may be encoded in distinctly IP3-mediated channel opening.1 Interest in the interaction of Bcl-2 different Ca2+ signals governed by the strength of TCR activation.2 with IP3 receptors has escalated rapidly, with other groups subseThese studies employed digital imaging to continuously monitor quently confirming the interaction and investigating its function.30,31 temporal changes in Ca2+ at the single cell level in the immature But how this interaction regulates channel opening is presently double positive WEHI7.2 T cell line. Consistent with earlier findings unknown. Our preliminary unpublished findings suggest that Bcl-2 by Donnadieu et al27 in the Jurkat T cell line, we found that strong may decrease IP3 receptor phosphorylation. This concept is consistent TCR activation by a high concentration of anti-CD3 antibody with evidence from others that Bcl-2 binds the protein phosphatase induced a large transient elevation of Ca2+, whereas weak TCR calcineurin32 and increases the association of calcineurin with IP3 activation by a low concentration of anti-CD3 antibody induced receptors.33,34,35 An intriguing question is how Bcl-2 selectively inhibits Ca2+ sustained Ca2+ oscillations (Fig. 2). The transient Ca2+ elevation 2+ signals induced by strong TCR activation without inhibiting Ca2+ induced by strong TCR activation triggered apoptosis, whereas Ca signals induced by weak TCR activation. We found that knocking oscillations induced by weak TCR activation favored cell survival. Reinforcing the concept that cell fate decisions in the developing down IP3 receptor expression levels by siRNA inhibited the Ca2+ thymus may be encoded by distinct Ca2+ signals, we also discovered elevation induced by strong TCR activation but did not inhibit Ca2+ that the anti-apoptotic protein Bcl-2 selectively inhibits the high oscillations induced by weak TCR activation.2 One possible intertransient elevation of Ca2+ induced by strong TCR activation but pretation of this finding is that only a small contingent of IP3 receptors does not interfere with Ca2+ oscillations induced by weak TCR are needed for initiation of Ca2+ oscillations and hence neither Bcl-2 1172

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interactions with stromal cells.36 Also, Ca2+ oscillations activate the Ca2+-calmodulin-dependent protein phosphatase calcineurin.5 Calcineurin dephosphorylates and thereby activates NFAT, leading to increased production of the cytokine interleukin-2. Because NFAT is rephosphorylated and thereby deactivated in the nucleus, nuclear retention of NFAT requires sustained activation of calcineurin which is most efficiently achieved by Ca2+ oscillations.17,19,37 In summary, our recent findings indicate that the strength of TCR activation is encoded in distinct Bcl-2-sensitive and insensitive Ca2+ signals that determine whether immature T cells will live or die. Because these findings are based on studies in an immature T cell line with TCR activation triggered by anti-CD3 antibody, an ultimate test of their applicability to cell fate decisions during thymic development must await in vivo studies of antigen-mediated TCR activation. Ca2+ signals in vivo may be more complex than those generated in vitro, as costimulatory signals combine with TCR activation to shape Ca2+ response patterns and determine cell fate.38 The concept that Bcl-2 2+ Figure 2. Cell fate decisions encoded by distinct Bcl-2 sensitive and insensitive Ca signals. functions as a regulator of Ca2+ signals is indeed Strong TCR activation induces a transient, Bcl-2 sensitive Ca2+ elevation that leads to negative novel and it is presently unknown whether Bcl-2 selection by apoptosis, whereas weak TCR activation induces Ca2+ oscillations that are not has a generalized role in regulating Ca2+ signals or associated with apoptosis and not inhibited by Bcl-2. a highly specific role in regulating Ca2+ signals nor siRNA-mediated suppression of IP3 receptor levels are sufficient generated in response to TCR activation. to impede oscillatory Ca2+ responses following weak TCR activation. 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Andjelic S, Jain N, Nikolic-Zugic J. Immature thymocytes become sensitive to calcium-mediated apoptosis with the onset of CD8, CD4, and the T cell receptor expression: the critical determinant of whether or not TCR induces apoptosis A role for bcl-2? J Exp Med 1993; 178:1745-51. 2+ appears to lie in both the duration and amplitude of the Ca elevation. 12. Strasser A, Harris AW, Cory S. bcl-2 transgene inhibits T cell death and perturbs thymic 2+ In contrast to the Ca elevation induced by strong TCR activation, self-censorship. Cell 1991; 67:889-99. the oscillatory Ca2+ elevation induced by weak TCR activation 13. Siegel RM, Katsumata M, Miyashita T, Louie DC, Greene MI, Reed JC. Inhibition of thymocyte apoptosis and negative antigenic selection in bcl-2 transgenic mice. Proc Natl signals cell survival and proliferation. The periodic brief spikes of Acad Sci USA 1992; 89:7003-7. Ca2+ that compose the Ca2+ oscillatory response to weak TCR 14. Strasser A, Harris AW, von Boehmer H, Cory S. Positive and negative selection of T cells 2+ activation are an efficient way for the T cell to sustain Ca signals in T-cell receptor transgenic mice expressing a bcl-2 transgene. Proc Natl Acad Sci USA 1994; 91:1376-80. without activating death pathways. Ca2+ oscillations induced by 15. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. weak TCR activation contribute to positive selection in the thymus Nature Rev Mol Cell Biol 2000; 1:11-21. by at least two mechanisms. For one, Ca2+ oscillations regulate 16. Berridge MJ. Lymphocyte activation in health and disease. Critical Reviews in thymocyte motility during positive selection, thereby modulating Immunology 1997; 17:155-78. www.landesbioscience.com

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