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Susceptibility of naïve and subsets of memory T cells to apoptosis via multiple signaling pathways
Autoimmunity Reviews 6 (2007) 476 – 481 www.elsevier.com/locate/autrev
Susceptibility of naïve and subsets of memory T cells to apoptosis via multiple signaling pathways Sudhir Gupta ⁎, Sastry Gollapudi Division of Basic and Clinical Immunology, Medical Sciences I, C-240, University of California, Irvine, CA 92697, United States Received 5 December 2006; accepted 6 February 2007 Available online 7 March 2007
Abstract Apoptosis is mediated via death receptor, the mitochondrial, and the endoplasmic reticulum pathway. Following activation of naïve T cells with antigens, different subsets of memory T cells are generated. In this review we have discussed relative sensitivity/ resistance of naïve and different subsets of memory T cells to apoptosis via different signaling pathways. Molecular basis for differential sensitivity to apoptosis is discussed. © 2007 Elsevier B.V. All rights reserved. Keywords: TNF-α receptors; Oxidative stress; Endoplasmic reticulum; NF-κB; FLIP; VDAC
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . 2. Death receptor pathway of apoptosis . . . . . . 3. Mitochondrial pathway of apoptosis . . . . . . 4. Endoplasmic reticulum stress-induced apoptosis Acknowledgement . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction There is only one way to conceive but multiple ways to die. Cell death occurs by necrosis, autophagy, and apoptosis. Apoptosis plays an important role in cellular homeostasis and in deletion of self-reactive lymphocytes and removal of effector cells following termination of an immune response. There are three major signaling path⁎ Corresponding author. Tel.: +1 949 824 5818; fax: +1 949 824 4362. E-mail address: [email protected] (S. Gupta). 1568-9972/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2007.02.005
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ways of apoptosis (Fig. 1): the death receptor pathway [1–3], the mitochondrial pathway [3–5], and a recently recognized the endoplasmic reticulum (ER) stress pathway [6,7]. Caspases, which are cysteine proteases, serve as molecular chainsaws to cleave a number of membrane and cytoplasmic substrates, inducing morphological and biochemical features of apoptosis [2]. In all three different apoptosis pathways, common executioner caspases are activated; however, apoptosis via different pathways is associated with activation of distinct initiator caspases [8].
S. Gupta, S. Gollapudi / Autoimmunity Reviews 6 (2007) 476–481
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Fig. 1. Three different pathways of apoptosis. Extrinsic pathway (death receptor pathway) is mediated by interaction between death receptor and death receptor ligand. Mitochondrial pathway is mediated by release of cytochrome c (cyto c) and the endoplasmic reticulum (ER) stress pathway is mediated by calcium release. There is a cross-talk between various pathways of apoptosis.
Following exposure to antigen, naïve T cells (TN) unõdergo clonal expansion followed by a phase of contraction during which antigen-specific T cells undergo apoptosis, and then a small number of antigen-specific T cells are retained as memory T cells. The development of immunological memory provides a long-lasting defense to the host. The memory T cells display differential expression of adhesion molecules (CD62L) and chemokine receptors (CCR-7), which allow them to home into lymph nodes, non-lymphoid tissue and mucosal sites, and to respond to microbes at peripheral tissue sites [9–12]. CCR7+ and CD62high T cells are found in lymph nodes (central memory), whereas CCR7− and CD62Llow (effector memory) are found in extranodal sites such as in the liver and lung. These subpopulations of naïve, central and effector memory T cells are identified by a number of cell surface proteins [12,13]. Recently, we have further characterized these subsets of CD8+ T cells [14]. Naïve CD8+ T cells, in addition to expression of CD45RA and CCR7, also express CD27 and CD28, whereas central memory (TCM) CD8+ T cells retain these cell surface antigens except CD45RA. Effector memory CD8+ T cells are further subdivided into two subsets. One subset of effector memory (TEM) is CCR7−CD45RA−, whereas a second set of effector memory CD8+ T cells reexpress CD45RA (TEMRA). TEMRA CD8+ T cells are CD27− and CD28. Although it is generally considered
that TEMRA subset is lacking from CD4+ T cells, we have observed a very small subset of TEMRA CD4+ T cells. 2. Death receptor pathway of apoptosis Death receptors belong to a large family of tumor necrosis factor receptor (TNFR), including CD95, TNFR, TRAIL and others [15]. Although we have observed similar differential sensitivity of naïve and memory subsets to CD95-mediated apoptosis (unpublished observations), TNF-α-induced apoptosis has been extensively studied. Therefore, we will review relative sensitivity/resistance of naïve and different memory subsets to TNF-α-induced apoptosis. TNF-α exerts its biological activity by binding to type I and type II receptors (TNFR-I and TNFR-II) and activating several signaling pathways [16]. These receptors share common extracellular domains; however, TNFR-I contains death domain (DD) whereas TNFR-II lacks DD. Although TNFR-II lacks a cytoplasmic DD, several investigators have reported that TNFR-II may induce/potentiate TNF-αinduced apoptosis [17,18]. Both cell survival and cell death signals mediated by TNF-α require distinct sets of adapters and other downstream signaling molecules. Upon ligation with TNF-α, TNFR-I undergoes trimerization of its receptor death domains, which in turn recruits an adapter protein, TNFR-associated death domain
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(TRADD). TRADD then may recruit another adapter molecule, the Fas-associated death domain (FADD). FADD then recruits pro-caspase-8, which becomes active by its dimerization. The remaining downstream signaling steps are similar to those described above for CD95mediated apoptosis. Alternatively, TRADD may recruit distinct sets of adapter proteins, TRAF-2 (TNFR-associated factor-2) and receptor interactive protein (RIP). TRAF-2 and RIP stimulate pathways leading to activation of MAP kinase and NF-κB respectively. MAPK may inhibit [19] or promote apoptosis [20]. NF-κB is a repressor of apoptosis [21,22]. NF-κB exists as either a heterodimer or homodimer of a subfamily of Rel family of proteins. The predominant form of NF-κB is a heterodimer comprising of p50 (NF-κB1) and p65 (RelA). When cells are exposed to TNF-α, IκB is phosphorylated at two specific serine residues, which is a signal for ubiquitination and degradation of IκB by the 26S proteosome. Free NF-κB dimers are released and translocated to the nucleus, where they activate transcription of a number of anti-apoptotic genes including cIAPs (inhibitor of apoptosis proteins), FLIP, TRAF-1, TRAF-2, Bcl-2, and Bcl-xL [23,24]. An inhibition of NF-κB is associated with upregulation of Bax, suggesting that Bax is negatively regulated by NF-κB [25]. The protein kinase complex that phosphorylates IκB in response to TNF-α contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ [21,22]. IKKβ is essential for the activation of IKK by TNF-α. Furthermore, it has been demonstrated that IKKβ is essential for protecting cells from apoptosis, including T cells from TNF-α-induced apoptosis [26,27]. Previously, we have reported TNF-α-induced and CD95-induced apoptosis in CD45RA+ naïve and CD45RA− memory CD4+ and CD8+ T cells [28,29]. However, CD45RA is also present in TEMRA memory T cells and CD45RA− memory cells contain both TCM and TEM. Therefore, we have examined the relative sensitivity of naïve and various memory CD8+ T cell subsets to TNFα-induced apoptosis [30,31]. Our data show that TN and TCM CD8+ T cells were sensitive whereas TEM and TEMRA CD8+ T cells were resistant to TNF-α-induced apoptosis. Apoptosis profile correlated with the profile of activation of caspase-8 and caspase-3. However, no correlation was observed between relative sensitivity of four CD8+ T cell subsets to TNF-α-induced apoptosis and the expression of TNFR-I or TNFR-II, suggesting that downstream signaling molecules may be responsible for differential sensitivity to apoptosis. We have shown that phosphorylation of IKKα/β and IκB and activation of NF-κB following activation with TNF-α, was higher in TEM and TEMRA CD8+ T cells as compared to TN and TCM CD8+ T cells. Since NF-κB
suppresses apoptosis by inducing anti-apoptotic genes, we examined the expression of anti-apoptotic proteins by Western blotting. We observed relatively increased expression of Bcl-2, Bcl-XL and FLIP, and decreased expression of pro-apoptotic Bax (which is negatively regulated by NF-κB) in TEM and TEMRA CD8+ T cells, as compared to TN and TCM CD8+ T cells. No difference in MAPK activation was observed between various subsets. In summary, anti-apoptotic molecules are upregulated, and pro-apoptotic molecules are downregulated in TEM and TEMRA CD8+ T cells that are relatively resistant to apoptosis. 3. Mitochondrial pathway of apoptosis Several stimuli, including chemotherapeutic agents, UV radiation, oxidative stress and others, appear to mediate apoptosis via the mitochondrial pathway. Mitochondrial pathway of apoptosis and structure of mitochondrial have been reviewed elsewhere [3–5]. Mitochondria contain two well-defined compartments: the matrix, surrounded by the inner membrane (IM), and the intermembrane space, which is surrounded by the outer membrane (OM). The IM contains various molecules, including ATP synthase, electron transport chain, and adenine nucleotide translocator. Under physiological conditions these molecules allow the respiratory chain to create an electrochemical gradient (membrane potential). The OM contains a voltage-dependent anion channel (VDAC). Bcl-2 is located on the IM and appears to play an important role in the maintenance of mitochondrial membrane potential (ΔΨm). The intermembrane space contains holocytochrome c, certain pro-caspases, adenylate kinase 2, Endo G, Daiblo/Smac, and apoptosis-inducing factor. The permeabilization of the OM, therefore, results in the release of these molecules into the cytoplasm. IM permeabilization leads to changes in ΔΨm. Once released from the mitochondria, cytochrome c binds to an adapter molecule Apaf-1 (Apoptotic protease-activating factor) in the presence of ATP/dATP, and recruits pro-caspase 9 to form apoptosome. Pro-caspase-9 is dimerized and activated without undergoing cleavage, and active caspases-9 activate executioner caspases to orchestrate apoptosis. The mitochondrial membrane permeabilization, and the release of cytochrome c, are controlled by members of the Bcl-2 family. We have recently examined the relative sensitivity of TN, TCM, TEM, and TEMRA CD4+ and CD8+ T cells to H2O2-induced apoptosis (manuscript in preparation). We observed that both TN and TCM CD4+ and CD8+ T cells were sensitive to H2O2-induced apoptosis, which was associated with an activation of caspase-9 and caspase-3, release of cytochrome c, and reduction in intracellular glutathione (GSH) levels. Furthermore, TCM CD4+ and
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CD8+ T cells showed greater sensitivity to H2O2-induced apoptosis than TN CD4+ and CD8+ T cells. In contrast, TEM and TEMRA CD4+ and CD8+ T cells were resistant to H2O2-induced apoptosis and to caspase activation. In vitro addition of GSH inhibited H2O2-induced apoptosis in both TN and TCM CD4+ and CD8+ T cells. These data strongly suggest that, similar to death receptor signaling, TN and TCM are sensitive to death signaling via mitochondrial pathway, whereas TEM and TEMRA are relatively resistant to apoptosis. Furthermore, we have investigated various molecular mechanisms of differential sensitivity of naïve and memory CD4+ and CD8+ T cells to oxidative stress. Bcl-XL expression and VDAC expression were overexpressed in TEM and TEMRA CD4+ and CD8+ T cells, whereas Bax overexpression was observed in TN and TCM CD4+ and CD8+ T cells. 4. Endoplasmic reticulum stress-induced apoptosis Ca++ storage and signaling, as well as folding, modification, and sorting of newly synthesized proteins, are among the main functions of the ER [32]. Disturbance of any of these functions can lead to ER stress, which in turn may induce apoptosis [6,7]. Both overload and depletion of the ER Ca++ pool can result in changes in protein folding and in ER stress. Prolonged ER stress stimulates the activation of pro-caspase-12 [33]. Pro-caspase 12 is localized in the ER membrane and is activated and
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cleaved by m-calpain during ER stress, or in response to mobilization of intracellular Ca++ stores. Activated caspase-12 then activates caspase-3. Several ER member proteins interact with Bcl-2 family members and influence apoptosis [34,35]. ER stress can be induced by changes in Ca++ contents within the ER [6]. Although a role of intracellular Ca++ in apoptosis is well known, a role of ER stress has not been investigated. Recently, we have used a number of approaches to study the effect of ER stress on apoptosis of various subsets of CD8+ T cells (unpublished data). Thapsigargin, which increases intracellular calcium, is an ER stressor. We examined the effect of thapsigargin on TN, TCM, TEM, and TEMRA CD8+ T cell apoptosis. Thapsigargin, in a concentration-dependent manner, induced apoptosis in all four subsets of CD8+ T cells; however, TN and TCM showed significantly greater sensitivity to thapsigargin-induced apoptosis, as compared to TEM and TEMRA; albeit TEMRA was almost completely resistant to thapsigargin-induced apoptosis. Furthermore, we observed that thapsigargin enhanced TNF-α-induced apoptosis in TN and TCM to a greater extent than in TEM and none in TEMRA. This correlated with an increase in intracellular Ca+, which was higher in TN and TCM, as compared to TEM and TEMRA CD8+ T cells (unpublished observations). It is unclear why TN and TCM T cells are sensitive to apoptosis, whereas TEM and TEMRA T cells are resistant to
Fig. 2. Phenotypes and changes in apoptotic pathways in naïve (TN), central memory (TCM), and two types of effector memory (TEM and TEMRA) T cells/VDAC=voltage-dependent anion channel.
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apoptosis. Furthermore, it is not known whether TEM and TEMRA T cells can be rendered sensitive to apoptosis. The relative sensitivity/resistance to apoptosis may be due to differential replicative capacity of these subsets; TN and TCM T cells display high replicative properties, whereas TEM and TEMRA show features of replicative senescence, i.e. short telomere length and poor replicative capacity [36–38]. Therefore, to maintain cellular homeostasis TN and TCM T cells must undergo apoptosis at a much greater rate than poorly replicating TEM and TEMRA T cells. In summary, TN and TCM are sensitive, whereas TEM and TEMRA T cell subsets are relatively resistant to apoptosis by signals provided by multiple distinct pathways of apoptosis. This would suggest that the balance between anti-apoptotic and pro-apoptotic molecules, including Bcl-2 family proteins, FLIP, and IAP may be critical in conferring sensitivity, or resistance, to apoptosis in naïve and different memory subsets of T cells. Data are summarized in Fig. 2. Acknowledgement The work cited was in part supported by grant AG18313. Take-home messages • Naïve and central memory subsets of CD4+ and CD8+ T cells are sensitive to apoptosis by signals provided by death receptor, the mitochondrial, and the endoplasmic reticulum pathways. Sensitivity of naïve and central memory cells to apoptosis via TNF receptor (TNFR) appears to be due to decreased signaling downstream of TNFR, including decreased phosphorylation of IKKα/β and IκB, which results in decreased activation of NF-κB (a survival signal). • Naïve and central memory CD4+ and CD8+ T cells are also sensitive to H2O2-induced apoptosis (the mitochondrial pathway) and the calcium stress (the endoplasmic reticulum pathway)-induced apoptosis. • Effector CD4+ and CD8+ T cells are resistant to apoptosis by multiple pathways, which is associated with increased activation of NF-κB and increased expression of anti-apoptotic proteins (Bcl-2/Bcl-xL, FLIP and IAPs). References [1] Krammer PH. CD95's deadly mission in the immune system. Nature 2000;407:789–95. [2] Gupta S. Decision between life and death during TNF-induced signaling. J Clin Immunol 2002;22:270–8.
[3] Gupta S. Molecular signaling in death receptor and mitochondrial pathways of apoptosis. Int J Oncol 2003;22:15–20. [4] Green DR, Evan GI. A matter of life and death. Cancer Cell 2002;1:19–30. [5] Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora's box opens. Nat Rev Mol Cell Biol 2001;2:67–71. [6] Orrenius S, Zhivotovsky B, Nicotera N. Regulation of cell death: the calcium–apoptosis link. Nat Rev Mol Cell Biol 2003;4: 552–65. [7] Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol 2001;3:E255–66. [8] Gupta S. Death of lymphocytes: a clue to immune deficiency in human aging. Disc Med 2005;5:298–302. [9] Lefrancois L, Marzo AL. The descent of memory T-cell subsets. Nat Rev Immunol 2006;6:618–23. [10] Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 2004;22:745–63. [11] Kataoka T, Budd RC, Holler N, Thome M, Martinon F, Irmler M, et al. Preferential localization of effector memory cells in nonlymphoid tissue. Science 2001;291:2413–7. [12] Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708–12. [13] Tomiyama H, Matsuda T, Takiguchi M. Differentiation of CD8+ T cells from a memory to memory/effector phenotype. J Immunol 2002;168:5538–50. [14] Gupta S, Bi R, Su K, Yel L, Chiplunkar S, Gollapudi S. Characterization of naïve, memory, and effector CD8+ T cells: effect of age. Exp Gerontol 2004;39:545–50. [15] Locksley RM, Kileen N, Lenardo MJ. The TNF and TNF receptor superfamilies. Cell 2001;104:487–501. [16] Wallach D, Boldin M, Varfolomeev E, Beyaert R, Vandenabeele P, Fiers W. Cell death induction by receptors of the TNF family: towards a molecular understanding. FEBS Lett 1997;410:96–106. [17] Declercz W, Denecker G, Fiers W, Vandenabeele P. Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75. J Immunol 1998;161:390–9. [18] Vandenabeele P, Declercq W, Vanhaesebroeck B, Grooten J, Fiers W. Both TNF receptors are required for TNF-mediated induction of apoptosis in PC60 cells. J Immunol 1995;154:2904–13. [19] Natoli G, Costanzo A, Ianni A, Templeton DJ, Woodgett JR, Balsano C, et al. Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF-2-dependent pathway. Science 1997;275:200–3. [20] Ichijo N, Nishida E, Irie K, Ten Dijke P, Saitoh M, Moriguchi T, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 1997;275:90–4. [21] Karin M, Lin A. NF-κB at the crossroads of life and death. Nat Immunol 2002;3:221–7. [22] Ghosh S, Karin M. Missing pieces in the NF-kB puzzle. Cell 2002;109:S81–96. [23] Pahl HL. Activators and target genes of Rel/NF-kB transcription factors. Oncogene 1999;18:6855–66. [24] Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, et al. Tumor necrosis factor-induces Bcl-2 and Bcl-x expression through NF-κB activation in primary hippocampus neurons. J Biol Chem 1999;274:8531–8. [25] Bentires-Alj M, Dejardin E, Viatour P, Van Lint C, Froesch B, Reed JC, et al. Inhibition of the NF-κB transcription factor
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increases Bax expression in cancer cell lines. Oncogene 2001;20: 2805–13. Li ZW, Chu WM, Hu YL, Delhase M, Deerinck T, Ellisman M, et al. The IKKβ subunit of IκB kinase (IKK) is essential for nuclear factor-κ activation and prevention of apoptosis. J Exp Med 1999;89:1839–45. Senftleben U, Li Z-W, Baud V, Karin M. IKKβ is essential for protecting T cells from TNFα-induced apoptosis. Cell 2001;14: 217–30. Aggarwal S, Gollapudi S, Gupta S. Increased TNF-α-induced apoptosis in lymphocytes from aged humans: changes in TNF-α receptor expression and activation of caspases. J Immunol 1999;162:2154–61. Aggarwal S, Gupta S. Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl2, and Bax. J Immunol 1998;160:1627–37. Gupta S, Bi R, Gollapudi S. Differential sensitivity of naïve and memory subsets of human CD8+ T cells to TNF-α-induced apoptosis. J Clin Immunol 2006;26:193–203. Gupta S, Gollapudi S. Central and effector memory CD4+ and CD8+ T cells display differential sensitivity to TNF-α-induced apoptosis. N Y Acad Sci 2005;1050:108–15. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest 2002;110:1389–98.
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[33] Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, et al. Caspase-12 mediated endoplasmic reticulum-specific apoptosis and cytotoxicity by amyloid β. Nature 2000;403: 98–103. [34] Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, et al. p28BAP31, a Bcl-2/Bcl-xL and procaspase-8 associated protein in the endoplasmic reticulum. J Cell Biol 1997;139:327–8. [35] Lam M, Dubyak G, Chen L, Nunez G, Miesfeld RL, Distelhorst CW. Evidence that Bcl-2 can inhibit apoptosis induced by regulating endoplasmic reticulum-associated fluxes. Proc Natl Acad Sci U S A 1994;91:6569–73. [36] Monteiro J, Baltiwala F, Ostere H, Gregersen PK. Shortened telomere in clonally expanded CD28−CD8+ T cells imply a replicative history that is distinct from there CD28+CD8+ counterparts. J Immunol 1996;162:6572–9. [37] Monteiro J, Baltiwala F, Ostrer H, Gregersen PK. Shortened telomeres in clonally expanded CD28−CD8+ T cells imply a replicative history that is distinct from their CD28+CD8+ counterparts. J Immunol 1996;156:3587. [38] Sprent J. Turnover of memory phenotype CD8+ T cells. Microbes Infect 2003;5:227–31.
Selective dysregulation of the FcgammaIIB receptor on memory B cells in SLE. The inappropriate expansion and activation of autoreactive memory B cells and plasmablasts contributes to loss of self-tolerance in systemic lupus erythematosus (SLE). Defects in the inhibitory Fc receptor, FcgammaRIIB, have been shown to contribute to B cell activation and autoimmunity in several mouse models of SLE. In this paper, Mackay M. et. al. (J Exp Med 2006; 203: 2157-64) demonstrate that expression of FcgammaRIIB is routinely up-regulated on memory B cells in the peripheral blood of healthy controls, whereas up-regulation of FcgammaRIIB is considerably decreased in memory B cells of SLE patients. This directly correlates with decreased FcgammaRIIB-mediated suppression of B cell receptor-induced calcium (Ca2+) response in those B cells. They also found substantial overrepresentation of African-American patients among those who failed to upregulate FcgammaRIIB. These results suggest that the inhibitory receptor, FcgammaRIIB, may be impaired at a critical checkpoint in SLE in the regulation of memory B cells; thus FcgammaRIIB represents a novel target for therapeutic interventions in this disease.
T-bet regulates the fate of Th1 and Th17 lymphocytes in autoimmunity. IL-17-producing T cells (Th17) have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. However, little is known about the transcription factors that regulate these cells. Although it is clear that the transcription factor T-bet plays an essential role in the differentiation of IFN-gamma-producing CD4+Th1 lymphocytes, the potential role of T-bet in the differentiation of Th17 cells is not completely understood. In this study, Gocke AR. et. al. (J Immunol 2007; 178: 1341-8) show that therapeutic administration of a small interfering RNA specific for T-beta significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of myelin basic protein-specific Th1 autoreactive T cells. Moreover, T-bet was found to directly regulate transcription of the IL-23R, and, in doing so, influenced the fate of Th17cells, which depend on optimal IL-23 production for survival. It was shown for the first time that suppression of T-bet ameliorates EAE by limiting the differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of IL-23R.
Susceptibility of naïve and subsets of memory T cells to apoptosis via multiple signaling pathways Sudhir Gupta ⁎, Sastry Gollapudi Division of Basic and Clinical Immunology, Medical Sciences I, C-240, University of California, Irvine, CA 92697, United States Received 5 December 2006; accepted 6 February 2007 Available online 7 March 2007
Abstract Apoptosis is mediated via death receptor, the mitochondrial, and the endoplasmic reticulum pathway. Following activation of naïve T cells with antigens, different subsets of memory T cells are generated. In this review we have discussed relative sensitivity/ resistance of naïve and different subsets of memory T cells to apoptosis via different signaling pathways. Molecular basis for differential sensitivity to apoptosis is discussed. © 2007 Elsevier B.V. All rights reserved. Keywords: TNF-α receptors; Oxidative stress; Endoplasmic reticulum; NF-κB; FLIP; VDAC
Contents 1. Introduction . . . . . . . . . . . . . . . . . . . 2. Death receptor pathway of apoptosis . . . . . . 3. Mitochondrial pathway of apoptosis . . . . . . 4. Endoplasmic reticulum stress-induced apoptosis Acknowledgement . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction There is only one way to conceive but multiple ways to die. Cell death occurs by necrosis, autophagy, and apoptosis. Apoptosis plays an important role in cellular homeostasis and in deletion of self-reactive lymphocytes and removal of effector cells following termination of an immune response. There are three major signaling path⁎ Corresponding author. Tel.: +1 949 824 5818; fax: +1 949 824 4362. E-mail address: [email protected] (S. Gupta). 1568-9972/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2007.02.005
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ways of apoptosis (Fig. 1): the death receptor pathway [1–3], the mitochondrial pathway [3–5], and a recently recognized the endoplasmic reticulum (ER) stress pathway [6,7]. Caspases, which are cysteine proteases, serve as molecular chainsaws to cleave a number of membrane and cytoplasmic substrates, inducing morphological and biochemical features of apoptosis [2]. In all three different apoptosis pathways, common executioner caspases are activated; however, apoptosis via different pathways is associated with activation of distinct initiator caspases [8].
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Fig. 1. Three different pathways of apoptosis. Extrinsic pathway (death receptor pathway) is mediated by interaction between death receptor and death receptor ligand. Mitochondrial pathway is mediated by release of cytochrome c (cyto c) and the endoplasmic reticulum (ER) stress pathway is mediated by calcium release. There is a cross-talk between various pathways of apoptosis.
Following exposure to antigen, naïve T cells (TN) unõdergo clonal expansion followed by a phase of contraction during which antigen-specific T cells undergo apoptosis, and then a small number of antigen-specific T cells are retained as memory T cells. The development of immunological memory provides a long-lasting defense to the host. The memory T cells display differential expression of adhesion molecules (CD62L) and chemokine receptors (CCR-7), which allow them to home into lymph nodes, non-lymphoid tissue and mucosal sites, and to respond to microbes at peripheral tissue sites [9–12]. CCR7+ and CD62high T cells are found in lymph nodes (central memory), whereas CCR7− and CD62Llow (effector memory) are found in extranodal sites such as in the liver and lung. These subpopulations of naïve, central and effector memory T cells are identified by a number of cell surface proteins [12,13]. Recently, we have further characterized these subsets of CD8+ T cells [14]. Naïve CD8+ T cells, in addition to expression of CD45RA and CCR7, also express CD27 and CD28, whereas central memory (TCM) CD8+ T cells retain these cell surface antigens except CD45RA. Effector memory CD8+ T cells are further subdivided into two subsets. One subset of effector memory (TEM) is CCR7−CD45RA−, whereas a second set of effector memory CD8+ T cells reexpress CD45RA (TEMRA). TEMRA CD8+ T cells are CD27− and CD28. Although it is generally considered
that TEMRA subset is lacking from CD4+ T cells, we have observed a very small subset of TEMRA CD4+ T cells. 2. Death receptor pathway of apoptosis Death receptors belong to a large family of tumor necrosis factor receptor (TNFR), including CD95, TNFR, TRAIL and others [15]. Although we have observed similar differential sensitivity of naïve and memory subsets to CD95-mediated apoptosis (unpublished observations), TNF-α-induced apoptosis has been extensively studied. Therefore, we will review relative sensitivity/resistance of naïve and different memory subsets to TNF-α-induced apoptosis. TNF-α exerts its biological activity by binding to type I and type II receptors (TNFR-I and TNFR-II) and activating several signaling pathways [16]. These receptors share common extracellular domains; however, TNFR-I contains death domain (DD) whereas TNFR-II lacks DD. Although TNFR-II lacks a cytoplasmic DD, several investigators have reported that TNFR-II may induce/potentiate TNF-αinduced apoptosis [17,18]. Both cell survival and cell death signals mediated by TNF-α require distinct sets of adapters and other downstream signaling molecules. Upon ligation with TNF-α, TNFR-I undergoes trimerization of its receptor death domains, which in turn recruits an adapter protein, TNFR-associated death domain
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(TRADD). TRADD then may recruit another adapter molecule, the Fas-associated death domain (FADD). FADD then recruits pro-caspase-8, which becomes active by its dimerization. The remaining downstream signaling steps are similar to those described above for CD95mediated apoptosis. Alternatively, TRADD may recruit distinct sets of adapter proteins, TRAF-2 (TNFR-associated factor-2) and receptor interactive protein (RIP). TRAF-2 and RIP stimulate pathways leading to activation of MAP kinase and NF-κB respectively. MAPK may inhibit [19] or promote apoptosis [20]. NF-κB is a repressor of apoptosis [21,22]. NF-κB exists as either a heterodimer or homodimer of a subfamily of Rel family of proteins. The predominant form of NF-κB is a heterodimer comprising of p50 (NF-κB1) and p65 (RelA). When cells are exposed to TNF-α, IκB is phosphorylated at two specific serine residues, which is a signal for ubiquitination and degradation of IκB by the 26S proteosome. Free NF-κB dimers are released and translocated to the nucleus, where they activate transcription of a number of anti-apoptotic genes including cIAPs (inhibitor of apoptosis proteins), FLIP, TRAF-1, TRAF-2, Bcl-2, and Bcl-xL [23,24]. An inhibition of NF-κB is associated with upregulation of Bax, suggesting that Bax is negatively regulated by NF-κB [25]. The protein kinase complex that phosphorylates IκB in response to TNF-α contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ [21,22]. IKKβ is essential for the activation of IKK by TNF-α. Furthermore, it has been demonstrated that IKKβ is essential for protecting cells from apoptosis, including T cells from TNF-α-induced apoptosis [26,27]. Previously, we have reported TNF-α-induced and CD95-induced apoptosis in CD45RA+ naïve and CD45RA− memory CD4+ and CD8+ T cells [28,29]. However, CD45RA is also present in TEMRA memory T cells and CD45RA− memory cells contain both TCM and TEM. Therefore, we have examined the relative sensitivity of naïve and various memory CD8+ T cell subsets to TNFα-induced apoptosis [30,31]. Our data show that TN and TCM CD8+ T cells were sensitive whereas TEM and TEMRA CD8+ T cells were resistant to TNF-α-induced apoptosis. Apoptosis profile correlated with the profile of activation of caspase-8 and caspase-3. However, no correlation was observed between relative sensitivity of four CD8+ T cell subsets to TNF-α-induced apoptosis and the expression of TNFR-I or TNFR-II, suggesting that downstream signaling molecules may be responsible for differential sensitivity to apoptosis. We have shown that phosphorylation of IKKα/β and IκB and activation of NF-κB following activation with TNF-α, was higher in TEM and TEMRA CD8+ T cells as compared to TN and TCM CD8+ T cells. Since NF-κB
suppresses apoptosis by inducing anti-apoptotic genes, we examined the expression of anti-apoptotic proteins by Western blotting. We observed relatively increased expression of Bcl-2, Bcl-XL and FLIP, and decreased expression of pro-apoptotic Bax (which is negatively regulated by NF-κB) in TEM and TEMRA CD8+ T cells, as compared to TN and TCM CD8+ T cells. No difference in MAPK activation was observed between various subsets. In summary, anti-apoptotic molecules are upregulated, and pro-apoptotic molecules are downregulated in TEM and TEMRA CD8+ T cells that are relatively resistant to apoptosis. 3. Mitochondrial pathway of apoptosis Several stimuli, including chemotherapeutic agents, UV radiation, oxidative stress and others, appear to mediate apoptosis via the mitochondrial pathway. Mitochondrial pathway of apoptosis and structure of mitochondrial have been reviewed elsewhere [3–5]. Mitochondria contain two well-defined compartments: the matrix, surrounded by the inner membrane (IM), and the intermembrane space, which is surrounded by the outer membrane (OM). The IM contains various molecules, including ATP synthase, electron transport chain, and adenine nucleotide translocator. Under physiological conditions these molecules allow the respiratory chain to create an electrochemical gradient (membrane potential). The OM contains a voltage-dependent anion channel (VDAC). Bcl-2 is located on the IM and appears to play an important role in the maintenance of mitochondrial membrane potential (ΔΨm). The intermembrane space contains holocytochrome c, certain pro-caspases, adenylate kinase 2, Endo G, Daiblo/Smac, and apoptosis-inducing factor. The permeabilization of the OM, therefore, results in the release of these molecules into the cytoplasm. IM permeabilization leads to changes in ΔΨm. Once released from the mitochondria, cytochrome c binds to an adapter molecule Apaf-1 (Apoptotic protease-activating factor) in the presence of ATP/dATP, and recruits pro-caspase 9 to form apoptosome. Pro-caspase-9 is dimerized and activated without undergoing cleavage, and active caspases-9 activate executioner caspases to orchestrate apoptosis. The mitochondrial membrane permeabilization, and the release of cytochrome c, are controlled by members of the Bcl-2 family. We have recently examined the relative sensitivity of TN, TCM, TEM, and TEMRA CD4+ and CD8+ T cells to H2O2-induced apoptosis (manuscript in preparation). We observed that both TN and TCM CD4+ and CD8+ T cells were sensitive to H2O2-induced apoptosis, which was associated with an activation of caspase-9 and caspase-3, release of cytochrome c, and reduction in intracellular glutathione (GSH) levels. Furthermore, TCM CD4+ and
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CD8+ T cells showed greater sensitivity to H2O2-induced apoptosis than TN CD4+ and CD8+ T cells. In contrast, TEM and TEMRA CD4+ and CD8+ T cells were resistant to H2O2-induced apoptosis and to caspase activation. In vitro addition of GSH inhibited H2O2-induced apoptosis in both TN and TCM CD4+ and CD8+ T cells. These data strongly suggest that, similar to death receptor signaling, TN and TCM are sensitive to death signaling via mitochondrial pathway, whereas TEM and TEMRA are relatively resistant to apoptosis. Furthermore, we have investigated various molecular mechanisms of differential sensitivity of naïve and memory CD4+ and CD8+ T cells to oxidative stress. Bcl-XL expression and VDAC expression were overexpressed in TEM and TEMRA CD4+ and CD8+ T cells, whereas Bax overexpression was observed in TN and TCM CD4+ and CD8+ T cells. 4. Endoplasmic reticulum stress-induced apoptosis Ca++ storage and signaling, as well as folding, modification, and sorting of newly synthesized proteins, are among the main functions of the ER [32]. Disturbance of any of these functions can lead to ER stress, which in turn may induce apoptosis [6,7]. Both overload and depletion of the ER Ca++ pool can result in changes in protein folding and in ER stress. Prolonged ER stress stimulates the activation of pro-caspase-12 [33]. Pro-caspase 12 is localized in the ER membrane and is activated and
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cleaved by m-calpain during ER stress, or in response to mobilization of intracellular Ca++ stores. Activated caspase-12 then activates caspase-3. Several ER member proteins interact with Bcl-2 family members and influence apoptosis [34,35]. ER stress can be induced by changes in Ca++ contents within the ER [6]. Although a role of intracellular Ca++ in apoptosis is well known, a role of ER stress has not been investigated. Recently, we have used a number of approaches to study the effect of ER stress on apoptosis of various subsets of CD8+ T cells (unpublished data). Thapsigargin, which increases intracellular calcium, is an ER stressor. We examined the effect of thapsigargin on TN, TCM, TEM, and TEMRA CD8+ T cell apoptosis. Thapsigargin, in a concentration-dependent manner, induced apoptosis in all four subsets of CD8+ T cells; however, TN and TCM showed significantly greater sensitivity to thapsigargin-induced apoptosis, as compared to TEM and TEMRA; albeit TEMRA was almost completely resistant to thapsigargin-induced apoptosis. Furthermore, we observed that thapsigargin enhanced TNF-α-induced apoptosis in TN and TCM to a greater extent than in TEM and none in TEMRA. This correlated with an increase in intracellular Ca+, which was higher in TN and TCM, as compared to TEM and TEMRA CD8+ T cells (unpublished observations). It is unclear why TN and TCM T cells are sensitive to apoptosis, whereas TEM and TEMRA T cells are resistant to
Fig. 2. Phenotypes and changes in apoptotic pathways in naïve (TN), central memory (TCM), and two types of effector memory (TEM and TEMRA) T cells/VDAC=voltage-dependent anion channel.
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apoptosis. Furthermore, it is not known whether TEM and TEMRA T cells can be rendered sensitive to apoptosis. The relative sensitivity/resistance to apoptosis may be due to differential replicative capacity of these subsets; TN and TCM T cells display high replicative properties, whereas TEM and TEMRA show features of replicative senescence, i.e. short telomere length and poor replicative capacity [36–38]. Therefore, to maintain cellular homeostasis TN and TCM T cells must undergo apoptosis at a much greater rate than poorly replicating TEM and TEMRA T cells. In summary, TN and TCM are sensitive, whereas TEM and TEMRA T cell subsets are relatively resistant to apoptosis by signals provided by multiple distinct pathways of apoptosis. This would suggest that the balance between anti-apoptotic and pro-apoptotic molecules, including Bcl-2 family proteins, FLIP, and IAP may be critical in conferring sensitivity, or resistance, to apoptosis in naïve and different memory subsets of T cells. Data are summarized in Fig. 2. Acknowledgement The work cited was in part supported by grant AG18313. Take-home messages • Naïve and central memory subsets of CD4+ and CD8+ T cells are sensitive to apoptosis by signals provided by death receptor, the mitochondrial, and the endoplasmic reticulum pathways. Sensitivity of naïve and central memory cells to apoptosis via TNF receptor (TNFR) appears to be due to decreased signaling downstream of TNFR, including decreased phosphorylation of IKKα/β and IκB, which results in decreased activation of NF-κB (a survival signal). • Naïve and central memory CD4+ and CD8+ T cells are also sensitive to H2O2-induced apoptosis (the mitochondrial pathway) and the calcium stress (the endoplasmic reticulum pathway)-induced apoptosis. • Effector CD4+ and CD8+ T cells are resistant to apoptosis by multiple pathways, which is associated with increased activation of NF-κB and increased expression of anti-apoptotic proteins (Bcl-2/Bcl-xL, FLIP and IAPs). References [1] Krammer PH. CD95's deadly mission in the immune system. Nature 2000;407:789–95. [2] Gupta S. Decision between life and death during TNF-induced signaling. J Clin Immunol 2002;22:270–8.
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Selective dysregulation of the FcgammaIIB receptor on memory B cells in SLE. The inappropriate expansion and activation of autoreactive memory B cells and plasmablasts contributes to loss of self-tolerance in systemic lupus erythematosus (SLE). Defects in the inhibitory Fc receptor, FcgammaRIIB, have been shown to contribute to B cell activation and autoimmunity in several mouse models of SLE. In this paper, Mackay M. et. al. (J Exp Med 2006; 203: 2157-64) demonstrate that expression of FcgammaRIIB is routinely up-regulated on memory B cells in the peripheral blood of healthy controls, whereas up-regulation of FcgammaRIIB is considerably decreased in memory B cells of SLE patients. This directly correlates with decreased FcgammaRIIB-mediated suppression of B cell receptor-induced calcium (Ca2+) response in those B cells. They also found substantial overrepresentation of African-American patients among those who failed to upregulate FcgammaRIIB. These results suggest that the inhibitory receptor, FcgammaRIIB, may be impaired at a critical checkpoint in SLE in the regulation of memory B cells; thus FcgammaRIIB represents a novel target for therapeutic interventions in this disease.
T-bet regulates the fate of Th1 and Th17 lymphocytes in autoimmunity. IL-17-producing T cells (Th17) have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis. However, little is known about the transcription factors that regulate these cells. Although it is clear that the transcription factor T-bet plays an essential role in the differentiation of IFN-gamma-producing CD4+Th1 lymphocytes, the potential role of T-bet in the differentiation of Th17 cells is not completely understood. In this study, Gocke AR. et. al. (J Immunol 2007; 178: 1341-8) show that therapeutic administration of a small interfering RNA specific for T-beta significantly improved the clinical course of established EAE. The improved clinical course was associated with suppression of newly differentiated T cells that express IL-17 in the CNS as well as suppression of myelin basic protein-specific Th1 autoreactive T cells. Moreover, T-bet was found to directly regulate transcription of the IL-23R, and, in doing so, influenced the fate of Th17cells, which depend on optimal IL-23 production for survival. It was shown for the first time that suppression of T-bet ameliorates EAE by limiting the differentiation of autoreactive Th1 cells, as well as inhibiting pathogenic Th17 cells via regulation of IL-23R.