- Email: [email protected]
The sound of silence: modulating anergy in T lymphocytes
The sound of silence: modulating anergy in T lymphocytes Samuel D Saibil1,2, Elissa K Deenick1 and Pamela S Ohashi1,2 Understanding the intercellular and intracellular mechanisms that maintain anergy and prevent the induction of full effector function is one avenue that may allow us to manipulate immune responses. Recent studies of T cell receptor (TCR)-proximal signaling events in different models of T cell unresponsiveness have suggested that biochemically distinct forms of anergy may exist in vivo. T cell responsiveness can be altered through the control of the intracellular pool of key second messengers, such as diacylglycerol (DAG) or the lipid modification of signaling molecules, such as the Linker for activated T cells (LAT). Studies on the molecule programmed death-1 (PD-1) and its ligands have revealed that tissue-resident signals are essential in the maintenance of T cell unresponsiveness. Thus, the emerging view is that T cell anergy is a dynamic state whose establishment and maintenance can be influenced by numerous different signaling pathways. Addresses 1 The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, Ontario, Canada 2 Department of Immunology, University of Toronto, Toronto, Ontario, Canada Corresponding author: Ohashi, Pamela S ([email protected])
Current Opinion in Immunology 2007, 19:658–664 This review comes from a themed issue on Autoimunnity Edited by Ann Marshak-Rothstein and Pam Ohashi Available online 18th October 2007 0952-7915/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2007.08.005
Introduction Anergy is a functional definition. It denotes a hyporesponsive state induced by antigen in which T cell proliferation and cytokine production are impaired upon subsequent exposures to cognate antigen. The induction of this nonresponsiveness is important in peripheral tolerance in preventing the activation of self-reactive clones that have not undergone thymic deletion [1]. As well, anergy is tied to peripheral tolerance because regulatory T cells (Treg) have been described as being anergic [2]. Understanding the mechanisms which enforce the anergic state is of interest because it may permit us to manipulate T cell function in disease. For example, preventing the induction of anergy may hold the key to eliciting better anti-tumor responses, whereas converting T cell activation to anergy may be relevant for controlling autoimmunity. Current Opinion in Immunology 2007, 19:658–664
Historically, the anergic state was first described in T cell clones as a unresponsive state induced by signaling through the T cell receptor (TCR) in the absence of costimulation [3–6]. Subsequent work by many groups has lead to the development of a two-signal model of T cell activation. Signals through the antigen receptor alone (signal 1) induces anergy while signal 1 in conjunction with proper costimulation (signal 2) results in T cell activation [7,8]. Although the two-signal model is simplistic, and the concepts have evolved over time to reflect the complex interactions that occur between a dendritic cell and a T cell, T cell anergy is often modeled according to the two-signal theory. Investigation of the signaling pathways engaged by signal 1 alone found that sustained calcium signals in the absence of activation of other signaling pathways, such as AP-1 and NF-kB, resulted in the induction of clonal anergy. These calcium signals activated the NFAT transcription factor that was responsible for the expression of genes associated with the establishment of anergy [9,10]. These genes included the transcription factors Egr-2 and Egr-3 as well as the E3 ubiquitin ligases Itch, Cbl-b and gene related to anergy in lymphocytes (GRAIL) [11,12]. Studies have also shown the GRAIL is upregulated in Treg cells, consistent with the anergic phenotype of these cells [13]. The mechanism through which these anergy-associated genes enforce anergy remains elusive, although recent work has identified a potential substrate for GRAIL called Rho guanine dissociation inhibitor (RhoGDI) [14]. Previous evidence has also suggested that the E3 ligases target key signaling molecules such as phospholipase C-g1 (PLCg1) and protein kinase C u (PKCu) for ubiquitinmediated proteasomal degradation [11,15]. Furthermore, studies have also shown that PKCu-deficient T cell display an anergic pheonotype after challenge with an agonistic peptide in vivo. The phenotype of the PKCu/ mice overlaps with the phenotype of CD28/ mice, reinforcing the connection between CD28 and PKCu for promoting T cell activation versus anergy [16]. A major problem for studying anergy induction in vivo is that unlike in vitro systems, all of the stimuli that a T cell receives cannot be as clearly defined or controlled. The dominant paradigm which explains the induction of anergy in vivo is that in a non-inflammatory environment immature antigen presenting cells (APC) present antigen without the proper costimulatory molecules which results in the induction of T cell anergy and tolerance. Conversely, the introduction of pathogen-derived signals results in the maturation of APCs and the presentation of antigen with sufficient costimulatory signals to lead to www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 659
the activation of T cells [17]. Recent work, however, has illustrated that there are multiple different signaling pathways that when perturbed are sufficient to induce or maintain T cell unresponsiveness in vivo. Thus, the emerging view is that T cell anergy is not a uniform state and different signaling inputs can result in T cell becoming unresponsive and enforce tolerance.
Anergy is a consequence of the inhibition of signaling pathways It has long been appreciated that anergic T cells display reduced Ras signaling [18,19]. However, the signaling mechanisms responsible for reduced Ras signaling in anergic cells remained obscure, primarily because of technical constraints on the satisfactory manipulation of Ras signaling in anergic cells. Recent progress has defined new mechanisms that dampen Ras signaling pathways. DGK
Zha et al. [20] utilized the coxsackie and adenovirus receptor (CAR) transgenic system to transfect active Ras into quiescent anergic cells. Transgenic expression of the CAR in T cells allows the cells to be transfected efficiently by adenoviral vectors without requiring the cell to be activated. Using this system to introduce active Ras in T cells, they found that active Ras was sufficient to reverse the anergic phenotype and restore Erk activation and IL-2 secretion in T cells rendered anergic both in vitro and in vivo. Using microarray analysis, these authors identified diacylglycerol kinase-a (DGK-a) as a candidate gene whose induction correlated with the induction of anergy and whose activity impinged upon Ras signaling. DGKs convert diacylglycerol (DAG) to phosphatidic acid; DAG activates Ras via the activation of the Ras guanine nucleotide-exchange factor RasGRP1. Accordingly, these authors investigated the activation of RasGRP1 in anergic cells as well as the effects of DGK-a on T cell anergy. They found diminished RasGRP1 localization to the plasma membrane in T cells anergized in vivo and that DGK-a over-expression in naı¨ve cells was sufficient to induce an anergy-like phenotype. From these results, these authors concluded that induction of DGK-a diminishes the intracellular pool of DAG and subsequently results in T cell anergy via the prevention of Ras signaling. This study, however, did not investigate the effects of DGK-a activity upon the activation of PKCu, another signaling molecule activated by DAG. Given the importance of PKCu-mediated signaling preventing anergy [11,16,21], it is conceivable that in vivo DGK-a promotes T cell unresponsiveness through diminishing signaling through both Ras and PKCu. T cells express two isoforms of DGKs, DGK-a and DGKz [22,23]. Further evidence supporting a role for DGKs in the induction of anergy came from the study of T cells from mice lacking either DGK-a or DGK-z [24]. T cells from mice lacking DGK-a were found to have enhanced www.sciencedirect.com
activation of the Ras signaling pathway. As well, Dgka/ T cells were resistant to the induction of anergy in vivo. Dgkz/ T cells also showed a resistance to anergy induction. Unfortunately, mice lacking both DGK-a or DGK-z displayed a severe thymic phenotype and had few peripheral T cells to study. T cells from mice lacking DGK-z treated with a pharmacological inhibitor of DGK-a, however, displayed an increased resistance to anergy induction as compared to mice lacking DGK-a alone. Thus, by modulating the intracellular pool of DAG, DGKs appear to play a role in promoting T cell anergy. PAG
Interestingly, in human T cells anergized in vitro, Ras signaling was found to be inhibited via a mechanism involving the phosphoprotein associated with glycosphingolipid-enriched mircodomains (PAG) and its Fynmediated recruitment of the C-terminal Src kinase (CSK) [25]. In these human T cells, PAG was able to block Ras and promote anergy without affecting DGK expression. Thus, there are potentially other signaling mechanisms that can block Ras to promote anergy in T cells. A recent report has also implicated the Fyn- PAG complex in the establishment of anergy in murine T cells [26]. Although this report did not directly examine the effect of Fyn-PAG on Ras activation, the finding that the Fyn-PAG complex is involved suggests that a similar mechanism may exist in murine and human anergic T cells. Further investigation is required to assess the roles of Fyn and PAG in silencing Ras signaling and promoting anergy in vivo in both mice and men. Anergy in vivo: beyond Ras
Most studies on the induction of anergy in vivo utilize T cells bearing a transgenic T cell receptor. Administration of soluble cognate peptide antigen for the transgenic TCR can induce an unresponsive state cells bearing the transgenic TCR [27]. The signaling pathways responsible for the anergic state induced by soluble peptide administration are believed to be similar to those engaged in vitro. Mice deficient for many of the molecules identified as important for imposing anergy in vitro, such as Cbl-b [28,29], or Egr-3 [30] as well as mice expressing a dominant negative GRAIL molecule [12], are resistant the induction of peptide-induced anergy in vivo [11,15]. Another model of in vivo anergy induction, however, involves the transfer of TCR transgenic T cells into a T cell-depleted host that ubiquitously expresses cognate antigen for the transferred cells. These transferred T cells also enter into an unresponsive state, called ‘adaptive tolerance’ by Schwartz and colleagues [31]. ‘Adaptively tolerant’ T cells are functionally anergic but differ from clonally anergized cells in their requirements for antigen persistence to maintain unresponsiveness and by the effect IL-2 has on unresponsiveness ([32] and see Table 1 one for comparison of adaptive tolerance and Current Opinion in Immunology 2007, 19:658–664
660 Autoimunnity
Table 1 A comparison of different models of T cell anergy Type of anergy Clonal anergy Adaptive tolerance Peptide-induced anergy
In vitro or In vivo model
Antigen exposure required to maintain anergy
In vitro In vivo In vivo
Transient Persistent Transient
Anergy reversed by IL-2 Yes No Yes
TCR-proximal signaling defects
# # # #
clonal anergy). Given these functional distinctions, Chiodetti et al. [33] characterized the signaling defects in cells made anergic in vitro versus cells made adaptively tolerant in vivo. They found that the signaling defects between these two unresponsive states were indeed different, as cells made anergic in vitro had impaired activation of the Ras signaling pathway. In adaptively tolerant cells, however, the signal defect was more proximal to the TCR, as these cells displayed impaired activation of Zap70 kinase activity. These studies did not, however, provide a mechanistic explanation for the differences observed. As well, it would be of interest to compare the alterations in signaling molecules found in adaptively tolerant cells versus cells rendered anergic in vivo via peptide administration. The absence of Cbl-b was reported not to impart resistance to anergy induction in the adaptive tolerance model [33]. This is in stark contrast to the findings in peptide-induced anergy models and implies that there are most likely different signaling molecules enforcing unresponsiveness in these two in vivo models. Collectively, these data suggest that in vivo there may be multiple different signaling programs which are responsible for enforcing an anergic state. These various anergy programs may be differentially engaged depending upon the context in which the antigen is encountered, such as in a transient manner in a peptide-induced model versus the persistent antigen used in the adaptive tolerance model. LAT
The linker for activation of T cells (LAT) is a scaffold protein that is a central signaling nexus for the activation of T cells. Phosphorylation of LAT by ZAP70 initiates signaling events that results in the recruitment and activation of phospholipase C-g1 (PLC-g1), PI3K and Ras signaling pathways. Recently, it was discovered that upon restimulation, cells rendered anergic in vitro or in vivo displayed decreased phosphorylation of LAT [34]. Surprisingly, this defect in LAT phosphorylation was not because of decreased ZAP70 activity or degradation of phosphorylated LAT. Rather, a decrease in the palmitoylation of LAT led to the defective recruitment of LAT to the lipid rafts. Surprisingly, the palmitoylation defect of LAT in anergic cells was not global, as Fyn was palmitoylated normally in anergic cells. This suggests that one of the mechanisms through which the anergic state is maintained is through the alteration of the Current Opinion in Immunology 2007, 19:658–664
Ras/MAPK activation Zap70 activity Ras/MAPK activation, LAT palmitolylation
palmitoylation of key signaling molecules. In mice, there are at least 23 potential palmitoyl acyl transferases (PATs), which have been identified to date [35]. It appears that each PAT may display some degree of specificity. Thus, it is of interest to identify the PAT responsible for palmitoylating LAT. Moreover, given that other signaling molecules, such as CD4 and PAG, are palmitoylated, it is possible that they are also differentially palmitoylated in anergic T cells. p27Kip1 and cell cycle
One controversial issue is whether the process of anergy induction is fundamentally linked with cell cycle arrest during the primary encounter with antigen, or if the two processes can be dissociated. Previous studies on the cell cycle inhibitor p27Kip1 have suggested that cell cycle arrest and the establishment of the unresponsive state are inseparable, as forced expression of p27Kip1 in T cells induces an anergic-like state [36] and p27 expression correlates with anergy induction [37,38]. Conversely, other studies have indicated that anergy induction can be uncoupled from cell cycle arrest [39,40]. Recent studies in p27Kip1 knockin mice, which express a mutant form of p27 that lacks its cyclin-dependent kinase (CDK)binding domain (called p27D mice [41]), and thus are unable to inhibit CDKs, have helped reconcile this confusion in the literature. It was found that T cells from these p27Dmice were indeed resistant to the induction of anergy in vivo [42]. Interestingly, however, these p27D T cells underwent the same number of cell divisions in vivo as wildtype cells during the process of anergy induction. Thus, it was not proliferation per se which prevented the induction of anergy in the p27D T cells. These cells had sustained CDK activity that was found to phosphorylate and inhibit Smad3 transcriptional activity. Therefore, p27Kip1 is important for the induction of anergy but blocking the entry into cell cycle was not the critical event for preventing anergy. Recent studies comparing the effects of rapamycin to those of the cell cycle inhibiting drug sanglifehrin A (SFA) on the induction of anergy also demonstrated that proliferation and the imposition of anergy can be uncoupled [43]. It was found that whilst both rapamycin and SFA inhibited proliferation, only rapamycin treatment caused the T cells to become anergic. The inhibition of mTOR activation by rapamycin, and not cell www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 661
cycle progression, was shown to be important for the establishment of unresponsiveness. These authors further demonstrated that this decrease in mTOR activity resulted in decreased phosphorylation of S6K-1 (S6 Kinase-1) and that this decreased in S6K-1 signaling was associated with anergy. Intriguingly, signaling downstream of the PI3K-PKB axis, which is activated by costimulation [44,45], has been reported to both suppress p27 expression [46,47] and activate mTOR [48]. Thus, it is possible that via sustained PKB activation T cells avoid the induction of anergy via the combined suppression of Smad3 activity and the activation of mTOR. This possibility is consistent with previous studies that have demonstrated that T cells with hyperactivation of the PI3K-PKB axis are resistant to becoming anergic ([49] and SDS, EKD unpublished observation).
Intercellular interactions maintain anergy
periphery in the maintenance of the unresponsive state. Collectively, these studies establish a role for PD-L1 in the establishment of the anergic state in models of both CD4+ and CD8+ T cell anergy. Complicating the interpretation of these studies, however, is the recent discovery that PDL1 was able to bind to B7-1 [60]. In vitro ligation of PDL1 expressed on T cells lacking CD28 and CTLA-4 by a B7-1 fusion protein was able to deliver an inhibitory signal to T cells and inhibit proliferation. Interestingly, ligation of B7-1 on T cells lacking PD-1 by PD-L1 fusion protein was also able to inhibit proliferation. Thus, it appeared that the PD-L1:B7-1 interaction signaled bi-directionally, and that expression of either B7-1 or PD-L1 on T cells was sufficient to delivery an inhibitory signal when the B7-1:PD-L1 interaction was engaged. In light of this finding, further study is required to determine whether the effects of PDL1 on T cell anergy are mediated through its interactions with PD-1, B7-1 or both.
PD-L1
Programmed death 1 (PD-1) is an inhibitory member of the CD28 receptor superfamily that is expressed upon T cells after antigen encounter [50]. To date, two ligands for PD-1 have been identified PD-L1/B7-H1 and PD-L2/B7DC. These ligands display a different pattern of expression, as PD-L1 is expressed upon haematopoetic cells as well as upon cells of multiple different lineages, including cells of the endothelium, cardiomyocytes, pancreatic islets cells and in the placenta [51]. Conversely, PD-L2 is expressed exclusively upon macrophages and dendritic cells [52]. Studies using knockout mice have implicated PD-1 in the maintenance of T cell tolerance, as mice lacking PD-1 develop an autoimmune disease [53,54]. Furthermore, genetic ablation of PD-1 on T cells in an adoptive transfer model was able to convert tolerizing stimuli to T cell priming [55]. From these studies, however, it was unclear as to which ligand for PD-1 was responsible for mediating tolerance. As well, it was also unclear as to which cells had to express PD-1 ligands to mediate tolerance.
Much work is also required to elucidate the signaling machinery recruited by PD-L1 ligation to enforce anergy. Almost nothing is known about the signaling molecules activated by the intracellular tail of B7-1 or PD-L1. In fact, little is known about the signaling initiated downstream of PD-1. The cytoplasmic domain of PD-1 contains an ITIM motif, which has been reported to recruit the tyrosine phosphatases SHP-2 and SHP-1 [61,62]. Whether other inhibitory molecules must be recruited to initiate and maintain anergy is presently unclear. Of note, PD-1 has also recently been reported as an important mediator of the immune dysfunction in chronic viral infection known as ‘clonal exhaustion’ [63–65]. Therapeutic strategies aimed at blocking PD-1 to restore immune function against a chronic viral infection now must bear the caveat that this strategy could result in reversal of T cell anergy and the initiation of autoimmunity. In addition, similar strategies are being explored to promote anti-tumor immunity in vivo.
Concluding remarks Experiments in non-obese diabetic (NOD) mice found that PD-L1 expression on the cells of the pancreas was essential for delaying the onset of autoimmunity [56]. Similarly, in another model of autoimmune diabetes, blocking antibodies to PD-L1, but not PD-L2, converted tolerance to autoimmunity [57]. From these studies, it was clear that tissue expression of PD-L1 was important for mediating tolerance. What was not clear, however, was through which mechanism this tolerance was maintained. Two recent studies have determined that PD-L1 plays a key role in maintaining T cell anergy. Blocking antibodies to PD-L1 or genetic ablation of PD-L1 was sufficient to reverse established anergy and promote autoimmunity in two different models of autoimmune diabetes [58,59]. Moreover, in one study [59] it was found that PD-L1 plays a role in the lymphoid organs in the establishment of T cell anergy as well as in the www.sciencedirect.com
Many strides have been made in unraveling the complex signaling networks which underlie the anergic state in T cells. On the basis of these current findings, it is apparent that alterations to multiple different signaling pathways are able to result in state of T cell hyporesponsiveness (See Figure 1). Similar defects in signaling have also been reported in Treg cell populations that have an anergic phenotype [66]. However, gene profiling studies have shown that T cells rendered anergic have a different gene expression profile than Treg cells, in at least one model of induced anergy [67]. The challenge remains to understand the relative importance of each pathway to the induction of anergy and the significance of the different anergic states that exist in vivo. As well, with the discovery of the contribution of tissue-resident PD-L1-mediated signals for the maintenance of the anergic state, the paradigm that anergy is the outcome of the dialogue Current Opinion in Immunology 2007, 19:658–664
662 Autoimunnity
Figure 1
A comparison of signaling in T cell activation vs. anergy. Signaling schematic depicting the molecular differences in signaling tolerance versus activation. Solid arrows represent strong signals, dotted arrows represent weak signals. Suggested pathways are demarcated with a question mark. The size of the circles for LAT represent the relative size of the palmitoylated vs. unpalmitoylated pools.
planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness. J Immunol 1987, 138:3704-3712.
solely between the T cell and the APC is no longer valid. Interactions with cells other than DCs are also required for anergy. It is becoming clear that the maintenance of the T cell anergy is an active process in which continuous signals are required to preserve tolerance and keep T cells ‘silent’.
7.
Bretscher P, Cohn M: A theory of self-nonself discrimination. Science 1970, 169:1042-1049.
8.
Schwartz RH: Costimulation of T lymphocytes: The role of CD28, CTLA-4 and B7/BB1 in interleukin-2 production and immunotherapy. Cell 1992, 71:1065-1068.
References and recommended reading
9.
Vanier LE, Prud’homme GJ: Cyclosporin A markedly enhances superantigen-induced peripheral T cell deletion and inhibits anergy induction. J Exp Med 1992, 176:37-46.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Walker LS, Abbas AK: The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol 2002, 2:11-19.
2.
Sakaguchi S: Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004, 22:531-562.
3.
Lamb JR, Skidmore BJ, Green N, Chiller JM, Feldmann M: Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J Exp Med 1983, 157:1434-1447.
10. Macian F, Garcia-Cozar F, Im SH, Horton HF, Byrne MC, Rao A: Transcriptional mechanisms underlying lymphocyte tolerance. Cell 2002, 109:719-731. 11. Heissmeyer V, Macian F, Im SH, Varma R, Feske S, Venuprasad K, Gu H, Liu YC, Dustin ML, Rao A: Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat Immunol 2004, 5:255-265. 12. Seroogy CM, Soares L, Ranheim EA, Su L, Holness C, Bloom D, Fathman CG: The gene related to anergy in lymphocytes, an E3 ubiquitin ligase, is necessary for anergy induction in CD4 T cells. J Immunol 2004, 173:79-85.
4.
Nossal GJV: Cellular mechanisms of immunological tolerance. Annu Rev Immunol 1983, 1:33-62.
13. Mackenzie DA, Schartner J, Lin J, Timmel A, Jennens-Clough M, Fathman CG, Seroogy CM: GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype. J Biol Chem 2007, 282:9696-9702.
5.
Jenkins MK, Schwartz RH: Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 1987, 165:302-319.
14. Su L, Lineberry N, Huh Y, Soares L, Fathman CG: A novel E3 ubiquitin ligase substrate screen identifies Rho guanine dissociation inhibitor as a substrate of gene related to anergy in lymphocytes. J Immunol 2006, 177:7559-7566.
6.
Quill H, Schwartz RH: Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in
15. Jeon M-S, Atfield A, Venuprasad K, Krawczyk C, Sarao R, Elly C, Yang C, Arya S, Bachmaier K, Su L et al.: Essential role of the E3
Current Opinion in Immunology 2007, 19:658–664
www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 663
ubiquitin ligase Cbl-b in T cell anergy induction. Immunity 2004, 21:167-177. 16. Berg-Brown NN, Gronski MA, Jones RG, Elford AR, Deenick EK, Littman DR, Ohashi PS: PKCu signals activation versus tolerance in vivo. J Exp Med 2004, 199:743-752.
34. Hundt M, Tabata H, Jeon MS, Hayashi K, Tanaka Y, Krishna R, De Giorgio L, Liu YC, Fukata M, Altman A: Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect. Immunity 2006, 24:513-522. This study demonstrates that lipid modification of LAT, via palmitoylation, is important for the maintenance of the anergic state.
17. Reis e Sousa C: Dendritic cells in a mature age. Nat Rev Immunol 2006, 6:476-483.
35. Fukata M, Fukata Y, Adesnik H, Nicoll RA, Bredt DS: Identification of PSD-95 palmitoylating enzymes. Neuron 2004, 44:987-996.
18. Li W, Whaley CD, Mondino A, Mueller DL: Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Science 1996, 271:1272-1276.
36. Boussiotis VA, Freeman GJ, Taylor PA, Berezovskaya A, Grass I, Blazar BR, Nadler LM: p27kip1 functions as an anergy factor inhibiting interleukin 2 transcription and clonal expansion of alloreactive human and mouse helper T lymphocytes. Nat Med 2000, 6:290-297.
19. Fields PE, Gajewski TF, Fitch FW: Blocked Ras activation in anergic CD4+ T cells. Science 1996, 271:1276-1278. 20. Zha Y, Marks R, Ho AW, Peterson AC, Janardhan S, Brown I, Praveen K, Stang S, Stone JC, Gajewski TF: T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-alpha. Nat Immunol 2006, 7:1166-1173. This report and ref. [24] establish DGKs and DAG metabolism as key molecules in blocking Ras signaling and establishing anergy. 21. Marsland BJ, Nembrini C, Schmitz N, Abel B, Krautwald S, Bachmann MF, Kopf M: Innate signals compensate for the absence of PKC-{theta} during in vivo CD8(+) T cell effector and memory responses. Proc Natl Acad Sci U S A 2005, 102:14374-14379. 22. Zhong XP, Hainey EA, Olenchock BA, Zhao H, Topham MK, Koretzky GA: Regulation of T cell receptor-induced activation of the Ras-ERK pathway by diacylglycerol kinase zeta. J Biol Chem 2002, 277:31089-31098. 23. Sanjuan MA, Jones DR, Izquierdo M, Merida I: Role of diacylglycerol kinase alpha in the attenuation of receptor signaling. J Cell Biol 2001, 153:207-220. 24. Olenchock BA, Guo R, Carpenter JH, Jordan M, Topham MK, Koretzky GA, Zhong XP: Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat Immunol 2006, 7:1174-1181. See annotation to ref. [20] 25. Smida M, Posevitz-Fejfar A, Horejsi V, Schraven B, Lindquist JA: A novel negative regulatory function of PAG: blocking Ras activation. Blood 2007. This report shows that PAG-Fyn complex can promote anergy in human T cells by blocking Ras activation independent of DGKs. 26. Davidson D, Schraven B, Veillette A: PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes. Mol Cell Biol 2007, 27:1960-1973. 27. Kearney ER, Pape KA, Loh DY, Jenkins MK: Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1994, 1:327-339. 28. Bachmaier K, Krawczyk C, Kozieradzki I, Kong Y-Y, Sasaki T, Oliveira-dos-Santos AJ, Mariathasan S, Bouchard D, Wakeham A, Itie A et al.: Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 2000, 403:211-216. 29. Chiang YJ, Kole HK, Brown K, Naramura M, Fukuhara S, Hu R-J, Jang IK, Gutkind JS, Shevach E, Gu H: Cbl-b regulates the CD28 dependence of T-cell activation. Nature 2000, 403:216-220. 30. Safford M, Collins S, Lutz MA, Allen A, Huang CT, Kowalski J, Blackford A, Horton MR, Drake C, Schwartz RH et al.: Egr-2 and Egr-3 are negative regulators of T cell activation. Nat Immunol 2005, 6:472-480.
37. Wells AD, Walsh MC, Sankaran D, Turka LA: T cell effector function and anergy avoidance are quantitatively linked to cell division. J Immunol 2000, 165:2432-2443. 38. Rowell EA, Walsh MC, Wells AD: Opposing roles for the cyclindependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function. J Immunol 2005, 174:3359-3368. 39. Colombetti S, Benigni F, Basso V, Mondino A: Clonal anergy is maintained independently of T cell proliferation. J Immunol 2002, 169:6178-6186. 40. Allen A, Zheng Y, Gardner L, Safford M, Horton MR, Powell JD: The novel cyclophilin binding compound, sanglifehrin A, disassociates G1 cell cycle arrest from tolerance induction. J Immunol 2004, 172:4797-4803. 41. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A: Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 1996, 85:721-732. 42. Li L, Iwamoto Y, Berezovskaya A, Boussiotis VA: A pathway regulated by cell cycle inhibitor p27Kip1 and checkpoint inhibitor Smad3 is involved in the induction of T cell tolerance. Nat Immunol 2006, 7:1157-1165. Study shows that p27 is important in establishing anergy by allowing Smad3 transcriptional activity and not by blocking cell cycle entry. 43. Zheng Y, Collins SL, Lutz MA, Allen AN, Kole TP, Zarek PE, Powell JD: A role for mammalian target of rapamycin in regulating T cell activation versus anergy. J Immunol 2007, 178:2163-2170. This study demonstrates mTOR activation and not cell cycle progression is important in determining T cell activation versus anergy. 44. Parry RV, Reif K, Smith G, Sanson DM, Hemmings BA, Ward SG: Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B. Eur J Immunol 1997, 27:2495-2501. 45. Song J, Salek-Ardakani S, Rogers PR, Cheng M, van Parijs L, Croft M: The costimulation-regulated duration of PKB activation controls T cell longevity. Nat Immunol 2004:1-9. 46. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL: PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 2002, 8:1145-1152. 47. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R et al.: PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 2002, 8:1153-1160. 48. Inoki K, Li Y, Zhu T, Wu J, Guan KL: TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002, 4:648-657.
31. Schwartz RH: T cell anergy. Annu Rev Immunol 2003, 21:305-334. 32. Rocha B, Tanchot C, von Boehmer H: Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen. J Exp Med 1993, 177:1517-1521. 33. Chiodetti L, Choi S, Barber DL, Schwartz RH: Adaptive tolerance and clonal anergy are distinct biochemical states. J Immunol 2006, 176:2279-2291. This study demonstrates the signaling differences between clonal anergy and adaptive tolerance. www.sciencedirect.com
49. Suzuki A, Yamaguchi MT, Ohteki T, Sasaki T, Kaisho T, Kimura Y, Yoshida R, Wakeham A, Higuchi T, Fukumoto M et al.: T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity 2001, 14:523-534. 50. Okazaki T, Honjo T: The PD-1-PD-L pathway in immunological tolerance. Trends Immunol 2006, 27:195-201. 51. Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, Freeman GJ, Sharpe AH: Regulation of PD-1, PD-L1, and PD-L2 Current Opinion in Immunology 2007, 19:658–664
664 Autoimunnity
expression during normal and autoimmune responses. Eur J Immunol 2003, 33:2706-2716. 52. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, Shin T, Tsuchiya H, Pardoll DM, Okumura K et al.: Expression of programmed death 1 ligands by murine T cells and APC. J Immunol 2002, 169:5538-5545. 53. Nishimura H, Nose M, Hiai H, Minator N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999, 11:141-151. 54. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N et al.: Autoimmune dilated cardiomyopathy in PD-1 receptor deficient mice. Science 2001, 291:319-322. 55. Probst HC, McCoy K, Okazaki T, Honjo T, van den Broek M: Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4. Nat Immunol 2005, 6:280-286. 56. Keir ME, Liang SC, Guleria I, Latchman YE, Qipo A, Albacker LA, Koulmanda M, Freeman GJ, Sayegh MH, Sharpe AH: Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 2006, 203:883-895. Refs. [56–59] establish tissue-resident PD-L1 signals important to the imposition and maintenance of the anergic state. 57. Martin-Orozco N, Wang YH, Yagita H, Dong C: Cutting Edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens. J Immunol 2006, 177:8291-8295. See annotation to ref. [56]. 58. Fife BT, Guleria I, Gubbels BM, Eagar TN, Tang Q, Bour-Jordan H, Yagita H, Azuma M, Sayegh MH, Bluestone JA: Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway. J Exp Med 2006, 203:2737-2747. See annotation to ref. [56]. 59. Tsushima F, Yao S, Shin T, Flies A, Flies S, Xu H, Tamada K, Pardoll DM, Chen L: Interaction between B7-H1 and PD-1
Current Opinion in Immunology 2007, 19:658–664
determines initiation and reversal of T-cell anergy. Blood 2007, 110:180-185. See annotation to ref. [56]. 60. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ: Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 2007, 27:111-122. Reference demonstrates that PD-L1 and B7-1 can ligate each other. 61. Parry RV, Chemnitz JM, Frauwirth KA, Lanfranco AR, Braunstein I, Kobayashi SV, Linsley PS, Thompson CB, Riley JL: CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol 2005, 25:9543-9553. 62. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R et al.: PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2001, 2:261-268. 63. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R: Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 2006, 439:682-687. 64. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS et al.: Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 2006, 12:1198-1202. 65. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C et al.: PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006, 443:350-354. 66. Hickman SP, Yang J, Thomas RM, Wells AD, Turka LA: Defective activation of protein kinase C and Ras-ERK pathways limits IL2 production and proliferation by CD4+CD25+ regulatory T cells. J Immunol 2006, 177:2186-2194. 67. Knoechel B, Lohr J, Zhu S, Wong L, Hu D, Ausubel L, Abbas AK: Functional and molecular comparison of anergic and regulatory T lymphocytes. J Immunol 2006, 176:6473-6483.
www.sciencedirect.com
Current Opinion in Immunology 2007, 19:658–664 This review comes from a themed issue on Autoimunnity Edited by Ann Marshak-Rothstein and Pam Ohashi Available online 18th October 2007 0952-7915/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2007.08.005
Introduction Anergy is a functional definition. It denotes a hyporesponsive state induced by antigen in which T cell proliferation and cytokine production are impaired upon subsequent exposures to cognate antigen. The induction of this nonresponsiveness is important in peripheral tolerance in preventing the activation of self-reactive clones that have not undergone thymic deletion [1]. As well, anergy is tied to peripheral tolerance because regulatory T cells (Treg) have been described as being anergic [2]. Understanding the mechanisms which enforce the anergic state is of interest because it may permit us to manipulate T cell function in disease. For example, preventing the induction of anergy may hold the key to eliciting better anti-tumor responses, whereas converting T cell activation to anergy may be relevant for controlling autoimmunity. Current Opinion in Immunology 2007, 19:658–664
Historically, the anergic state was first described in T cell clones as a unresponsive state induced by signaling through the T cell receptor (TCR) in the absence of costimulation [3–6]. Subsequent work by many groups has lead to the development of a two-signal model of T cell activation. Signals through the antigen receptor alone (signal 1) induces anergy while signal 1 in conjunction with proper costimulation (signal 2) results in T cell activation [7,8]. Although the two-signal model is simplistic, and the concepts have evolved over time to reflect the complex interactions that occur between a dendritic cell and a T cell, T cell anergy is often modeled according to the two-signal theory. Investigation of the signaling pathways engaged by signal 1 alone found that sustained calcium signals in the absence of activation of other signaling pathways, such as AP-1 and NF-kB, resulted in the induction of clonal anergy. These calcium signals activated the NFAT transcription factor that was responsible for the expression of genes associated with the establishment of anergy [9,10]. These genes included the transcription factors Egr-2 and Egr-3 as well as the E3 ubiquitin ligases Itch, Cbl-b and gene related to anergy in lymphocytes (GRAIL) [11,12]. Studies have also shown the GRAIL is upregulated in Treg cells, consistent with the anergic phenotype of these cells [13]. The mechanism through which these anergy-associated genes enforce anergy remains elusive, although recent work has identified a potential substrate for GRAIL called Rho guanine dissociation inhibitor (RhoGDI) [14]. Previous evidence has also suggested that the E3 ligases target key signaling molecules such as phospholipase C-g1 (PLCg1) and protein kinase C u (PKCu) for ubiquitinmediated proteasomal degradation [11,15]. Furthermore, studies have also shown that PKCu-deficient T cell display an anergic pheonotype after challenge with an agonistic peptide in vivo. The phenotype of the PKCu/ mice overlaps with the phenotype of CD28/ mice, reinforcing the connection between CD28 and PKCu for promoting T cell activation versus anergy [16]. A major problem for studying anergy induction in vivo is that unlike in vitro systems, all of the stimuli that a T cell receives cannot be as clearly defined or controlled. The dominant paradigm which explains the induction of anergy in vivo is that in a non-inflammatory environment immature antigen presenting cells (APC) present antigen without the proper costimulatory molecules which results in the induction of T cell anergy and tolerance. Conversely, the introduction of pathogen-derived signals results in the maturation of APCs and the presentation of antigen with sufficient costimulatory signals to lead to www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 659
the activation of T cells [17]. Recent work, however, has illustrated that there are multiple different signaling pathways that when perturbed are sufficient to induce or maintain T cell unresponsiveness in vivo. Thus, the emerging view is that T cell anergy is not a uniform state and different signaling inputs can result in T cell becoming unresponsive and enforce tolerance.
Anergy is a consequence of the inhibition of signaling pathways It has long been appreciated that anergic T cells display reduced Ras signaling [18,19]. However, the signaling mechanisms responsible for reduced Ras signaling in anergic cells remained obscure, primarily because of technical constraints on the satisfactory manipulation of Ras signaling in anergic cells. Recent progress has defined new mechanisms that dampen Ras signaling pathways. DGK
Zha et al. [20] utilized the coxsackie and adenovirus receptor (CAR) transgenic system to transfect active Ras into quiescent anergic cells. Transgenic expression of the CAR in T cells allows the cells to be transfected efficiently by adenoviral vectors without requiring the cell to be activated. Using this system to introduce active Ras in T cells, they found that active Ras was sufficient to reverse the anergic phenotype and restore Erk activation and IL-2 secretion in T cells rendered anergic both in vitro and in vivo. Using microarray analysis, these authors identified diacylglycerol kinase-a (DGK-a) as a candidate gene whose induction correlated with the induction of anergy and whose activity impinged upon Ras signaling. DGKs convert diacylglycerol (DAG) to phosphatidic acid; DAG activates Ras via the activation of the Ras guanine nucleotide-exchange factor RasGRP1. Accordingly, these authors investigated the activation of RasGRP1 in anergic cells as well as the effects of DGK-a on T cell anergy. They found diminished RasGRP1 localization to the plasma membrane in T cells anergized in vivo and that DGK-a over-expression in naı¨ve cells was sufficient to induce an anergy-like phenotype. From these results, these authors concluded that induction of DGK-a diminishes the intracellular pool of DAG and subsequently results in T cell anergy via the prevention of Ras signaling. This study, however, did not investigate the effects of DGK-a activity upon the activation of PKCu, another signaling molecule activated by DAG. Given the importance of PKCu-mediated signaling preventing anergy [11,16,21], it is conceivable that in vivo DGK-a promotes T cell unresponsiveness through diminishing signaling through both Ras and PKCu. T cells express two isoforms of DGKs, DGK-a and DGKz [22,23]. Further evidence supporting a role for DGKs in the induction of anergy came from the study of T cells from mice lacking either DGK-a or DGK-z [24]. T cells from mice lacking DGK-a were found to have enhanced www.sciencedirect.com
activation of the Ras signaling pathway. As well, Dgka/ T cells were resistant to the induction of anergy in vivo. Dgkz/ T cells also showed a resistance to anergy induction. Unfortunately, mice lacking both DGK-a or DGK-z displayed a severe thymic phenotype and had few peripheral T cells to study. T cells from mice lacking DGK-z treated with a pharmacological inhibitor of DGK-a, however, displayed an increased resistance to anergy induction as compared to mice lacking DGK-a alone. Thus, by modulating the intracellular pool of DAG, DGKs appear to play a role in promoting T cell anergy. PAG
Interestingly, in human T cells anergized in vitro, Ras signaling was found to be inhibited via a mechanism involving the phosphoprotein associated with glycosphingolipid-enriched mircodomains (PAG) and its Fynmediated recruitment of the C-terminal Src kinase (CSK) [25]. In these human T cells, PAG was able to block Ras and promote anergy without affecting DGK expression. Thus, there are potentially other signaling mechanisms that can block Ras to promote anergy in T cells. A recent report has also implicated the Fyn- PAG complex in the establishment of anergy in murine T cells [26]. Although this report did not directly examine the effect of Fyn-PAG on Ras activation, the finding that the Fyn-PAG complex is involved suggests that a similar mechanism may exist in murine and human anergic T cells. Further investigation is required to assess the roles of Fyn and PAG in silencing Ras signaling and promoting anergy in vivo in both mice and men. Anergy in vivo: beyond Ras
Most studies on the induction of anergy in vivo utilize T cells bearing a transgenic T cell receptor. Administration of soluble cognate peptide antigen for the transgenic TCR can induce an unresponsive state cells bearing the transgenic TCR [27]. The signaling pathways responsible for the anergic state induced by soluble peptide administration are believed to be similar to those engaged in vitro. Mice deficient for many of the molecules identified as important for imposing anergy in vitro, such as Cbl-b [28,29], or Egr-3 [30] as well as mice expressing a dominant negative GRAIL molecule [12], are resistant the induction of peptide-induced anergy in vivo [11,15]. Another model of in vivo anergy induction, however, involves the transfer of TCR transgenic T cells into a T cell-depleted host that ubiquitously expresses cognate antigen for the transferred cells. These transferred T cells also enter into an unresponsive state, called ‘adaptive tolerance’ by Schwartz and colleagues [31]. ‘Adaptively tolerant’ T cells are functionally anergic but differ from clonally anergized cells in their requirements for antigen persistence to maintain unresponsiveness and by the effect IL-2 has on unresponsiveness ([32] and see Table 1 one for comparison of adaptive tolerance and Current Opinion in Immunology 2007, 19:658–664
660 Autoimunnity
Table 1 A comparison of different models of T cell anergy Type of anergy Clonal anergy Adaptive tolerance Peptide-induced anergy
In vitro or In vivo model
Antigen exposure required to maintain anergy
In vitro In vivo In vivo
Transient Persistent Transient
Anergy reversed by IL-2 Yes No Yes
TCR-proximal signaling defects
# # # #
clonal anergy). Given these functional distinctions, Chiodetti et al. [33] characterized the signaling defects in cells made anergic in vitro versus cells made adaptively tolerant in vivo. They found that the signaling defects between these two unresponsive states were indeed different, as cells made anergic in vitro had impaired activation of the Ras signaling pathway. In adaptively tolerant cells, however, the signal defect was more proximal to the TCR, as these cells displayed impaired activation of Zap70 kinase activity. These studies did not, however, provide a mechanistic explanation for the differences observed. As well, it would be of interest to compare the alterations in signaling molecules found in adaptively tolerant cells versus cells rendered anergic in vivo via peptide administration. The absence of Cbl-b was reported not to impart resistance to anergy induction in the adaptive tolerance model [33]. This is in stark contrast to the findings in peptide-induced anergy models and implies that there are most likely different signaling molecules enforcing unresponsiveness in these two in vivo models. Collectively, these data suggest that in vivo there may be multiple different signaling programs which are responsible for enforcing an anergic state. These various anergy programs may be differentially engaged depending upon the context in which the antigen is encountered, such as in a transient manner in a peptide-induced model versus the persistent antigen used in the adaptive tolerance model. LAT
The linker for activation of T cells (LAT) is a scaffold protein that is a central signaling nexus for the activation of T cells. Phosphorylation of LAT by ZAP70 initiates signaling events that results in the recruitment and activation of phospholipase C-g1 (PLC-g1), PI3K and Ras signaling pathways. Recently, it was discovered that upon restimulation, cells rendered anergic in vitro or in vivo displayed decreased phosphorylation of LAT [34]. Surprisingly, this defect in LAT phosphorylation was not because of decreased ZAP70 activity or degradation of phosphorylated LAT. Rather, a decrease in the palmitoylation of LAT led to the defective recruitment of LAT to the lipid rafts. Surprisingly, the palmitoylation defect of LAT in anergic cells was not global, as Fyn was palmitoylated normally in anergic cells. This suggests that one of the mechanisms through which the anergic state is maintained is through the alteration of the Current Opinion in Immunology 2007, 19:658–664
Ras/MAPK activation Zap70 activity Ras/MAPK activation, LAT palmitolylation
palmitoylation of key signaling molecules. In mice, there are at least 23 potential palmitoyl acyl transferases (PATs), which have been identified to date [35]. It appears that each PAT may display some degree of specificity. Thus, it is of interest to identify the PAT responsible for palmitoylating LAT. Moreover, given that other signaling molecules, such as CD4 and PAG, are palmitoylated, it is possible that they are also differentially palmitoylated in anergic T cells. p27Kip1 and cell cycle
One controversial issue is whether the process of anergy induction is fundamentally linked with cell cycle arrest during the primary encounter with antigen, or if the two processes can be dissociated. Previous studies on the cell cycle inhibitor p27Kip1 have suggested that cell cycle arrest and the establishment of the unresponsive state are inseparable, as forced expression of p27Kip1 in T cells induces an anergic-like state [36] and p27 expression correlates with anergy induction [37,38]. Conversely, other studies have indicated that anergy induction can be uncoupled from cell cycle arrest [39,40]. Recent studies in p27Kip1 knockin mice, which express a mutant form of p27 that lacks its cyclin-dependent kinase (CDK)binding domain (called p27D mice [41]), and thus are unable to inhibit CDKs, have helped reconcile this confusion in the literature. It was found that T cells from these p27Dmice were indeed resistant to the induction of anergy in vivo [42]. Interestingly, however, these p27D T cells underwent the same number of cell divisions in vivo as wildtype cells during the process of anergy induction. Thus, it was not proliferation per se which prevented the induction of anergy in the p27D T cells. These cells had sustained CDK activity that was found to phosphorylate and inhibit Smad3 transcriptional activity. Therefore, p27Kip1 is important for the induction of anergy but blocking the entry into cell cycle was not the critical event for preventing anergy. Recent studies comparing the effects of rapamycin to those of the cell cycle inhibiting drug sanglifehrin A (SFA) on the induction of anergy also demonstrated that proliferation and the imposition of anergy can be uncoupled [43]. It was found that whilst both rapamycin and SFA inhibited proliferation, only rapamycin treatment caused the T cells to become anergic. The inhibition of mTOR activation by rapamycin, and not cell www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 661
cycle progression, was shown to be important for the establishment of unresponsiveness. These authors further demonstrated that this decrease in mTOR activity resulted in decreased phosphorylation of S6K-1 (S6 Kinase-1) and that this decreased in S6K-1 signaling was associated with anergy. Intriguingly, signaling downstream of the PI3K-PKB axis, which is activated by costimulation [44,45], has been reported to both suppress p27 expression [46,47] and activate mTOR [48]. Thus, it is possible that via sustained PKB activation T cells avoid the induction of anergy via the combined suppression of Smad3 activity and the activation of mTOR. This possibility is consistent with previous studies that have demonstrated that T cells with hyperactivation of the PI3K-PKB axis are resistant to becoming anergic ([49] and SDS, EKD unpublished observation).
Intercellular interactions maintain anergy
periphery in the maintenance of the unresponsive state. Collectively, these studies establish a role for PD-L1 in the establishment of the anergic state in models of both CD4+ and CD8+ T cell anergy. Complicating the interpretation of these studies, however, is the recent discovery that PDL1 was able to bind to B7-1 [60]. In vitro ligation of PDL1 expressed on T cells lacking CD28 and CTLA-4 by a B7-1 fusion protein was able to deliver an inhibitory signal to T cells and inhibit proliferation. Interestingly, ligation of B7-1 on T cells lacking PD-1 by PD-L1 fusion protein was also able to inhibit proliferation. Thus, it appeared that the PD-L1:B7-1 interaction signaled bi-directionally, and that expression of either B7-1 or PD-L1 on T cells was sufficient to delivery an inhibitory signal when the B7-1:PD-L1 interaction was engaged. In light of this finding, further study is required to determine whether the effects of PDL1 on T cell anergy are mediated through its interactions with PD-1, B7-1 or both.
PD-L1
Programmed death 1 (PD-1) is an inhibitory member of the CD28 receptor superfamily that is expressed upon T cells after antigen encounter [50]. To date, two ligands for PD-1 have been identified PD-L1/B7-H1 and PD-L2/B7DC. These ligands display a different pattern of expression, as PD-L1 is expressed upon haematopoetic cells as well as upon cells of multiple different lineages, including cells of the endothelium, cardiomyocytes, pancreatic islets cells and in the placenta [51]. Conversely, PD-L2 is expressed exclusively upon macrophages and dendritic cells [52]. Studies using knockout mice have implicated PD-1 in the maintenance of T cell tolerance, as mice lacking PD-1 develop an autoimmune disease [53,54]. Furthermore, genetic ablation of PD-1 on T cells in an adoptive transfer model was able to convert tolerizing stimuli to T cell priming [55]. From these studies, however, it was unclear as to which ligand for PD-1 was responsible for mediating tolerance. As well, it was also unclear as to which cells had to express PD-1 ligands to mediate tolerance.
Much work is also required to elucidate the signaling machinery recruited by PD-L1 ligation to enforce anergy. Almost nothing is known about the signaling molecules activated by the intracellular tail of B7-1 or PD-L1. In fact, little is known about the signaling initiated downstream of PD-1. The cytoplasmic domain of PD-1 contains an ITIM motif, which has been reported to recruit the tyrosine phosphatases SHP-2 and SHP-1 [61,62]. Whether other inhibitory molecules must be recruited to initiate and maintain anergy is presently unclear. Of note, PD-1 has also recently been reported as an important mediator of the immune dysfunction in chronic viral infection known as ‘clonal exhaustion’ [63–65]. Therapeutic strategies aimed at blocking PD-1 to restore immune function against a chronic viral infection now must bear the caveat that this strategy could result in reversal of T cell anergy and the initiation of autoimmunity. In addition, similar strategies are being explored to promote anti-tumor immunity in vivo.
Concluding remarks Experiments in non-obese diabetic (NOD) mice found that PD-L1 expression on the cells of the pancreas was essential for delaying the onset of autoimmunity [56]. Similarly, in another model of autoimmune diabetes, blocking antibodies to PD-L1, but not PD-L2, converted tolerance to autoimmunity [57]. From these studies, it was clear that tissue expression of PD-L1 was important for mediating tolerance. What was not clear, however, was through which mechanism this tolerance was maintained. Two recent studies have determined that PD-L1 plays a key role in maintaining T cell anergy. Blocking antibodies to PD-L1 or genetic ablation of PD-L1 was sufficient to reverse established anergy and promote autoimmunity in two different models of autoimmune diabetes [58,59]. Moreover, in one study [59] it was found that PD-L1 plays a role in the lymphoid organs in the establishment of T cell anergy as well as in the www.sciencedirect.com
Many strides have been made in unraveling the complex signaling networks which underlie the anergic state in T cells. On the basis of these current findings, it is apparent that alterations to multiple different signaling pathways are able to result in state of T cell hyporesponsiveness (See Figure 1). Similar defects in signaling have also been reported in Treg cell populations that have an anergic phenotype [66]. However, gene profiling studies have shown that T cells rendered anergic have a different gene expression profile than Treg cells, in at least one model of induced anergy [67]. The challenge remains to understand the relative importance of each pathway to the induction of anergy and the significance of the different anergic states that exist in vivo. As well, with the discovery of the contribution of tissue-resident PD-L1-mediated signals for the maintenance of the anergic state, the paradigm that anergy is the outcome of the dialogue Current Opinion in Immunology 2007, 19:658–664
662 Autoimunnity
Figure 1
A comparison of signaling in T cell activation vs. anergy. Signaling schematic depicting the molecular differences in signaling tolerance versus activation. Solid arrows represent strong signals, dotted arrows represent weak signals. Suggested pathways are demarcated with a question mark. The size of the circles for LAT represent the relative size of the palmitoylated vs. unpalmitoylated pools.
planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness. J Immunol 1987, 138:3704-3712.
solely between the T cell and the APC is no longer valid. Interactions with cells other than DCs are also required for anergy. It is becoming clear that the maintenance of the T cell anergy is an active process in which continuous signals are required to preserve tolerance and keep T cells ‘silent’.
7.
Bretscher P, Cohn M: A theory of self-nonself discrimination. Science 1970, 169:1042-1049.
8.
Schwartz RH: Costimulation of T lymphocytes: The role of CD28, CTLA-4 and B7/BB1 in interleukin-2 production and immunotherapy. Cell 1992, 71:1065-1068.
References and recommended reading
9.
Vanier LE, Prud’homme GJ: Cyclosporin A markedly enhances superantigen-induced peripheral T cell deletion and inhibits anergy induction. J Exp Med 1992, 176:37-46.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Walker LS, Abbas AK: The enemy within: keeping self-reactive T cells at bay in the periphery. Nat Rev Immunol 2002, 2:11-19.
2.
Sakaguchi S: Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 2004, 22:531-562.
3.
Lamb JR, Skidmore BJ, Green N, Chiller JM, Feldmann M: Induction of tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza hemagglutinin. J Exp Med 1983, 157:1434-1447.
10. Macian F, Garcia-Cozar F, Im SH, Horton HF, Byrne MC, Rao A: Transcriptional mechanisms underlying lymphocyte tolerance. Cell 2002, 109:719-731. 11. Heissmeyer V, Macian F, Im SH, Varma R, Feske S, Venuprasad K, Gu H, Liu YC, Dustin ML, Rao A: Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat Immunol 2004, 5:255-265. 12. Seroogy CM, Soares L, Ranheim EA, Su L, Holness C, Bloom D, Fathman CG: The gene related to anergy in lymphocytes, an E3 ubiquitin ligase, is necessary for anergy induction in CD4 T cells. J Immunol 2004, 173:79-85.
4.
Nossal GJV: Cellular mechanisms of immunological tolerance. Annu Rev Immunol 1983, 1:33-62.
13. Mackenzie DA, Schartner J, Lin J, Timmel A, Jennens-Clough M, Fathman CG, Seroogy CM: GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype. J Biol Chem 2007, 282:9696-9702.
5.
Jenkins MK, Schwartz RH: Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 1987, 165:302-319.
14. Su L, Lineberry N, Huh Y, Soares L, Fathman CG: A novel E3 ubiquitin ligase substrate screen identifies Rho guanine dissociation inhibitor as a substrate of gene related to anergy in lymphocytes. J Immunol 2006, 177:7559-7566.
6.
Quill H, Schwartz RH: Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in
15. Jeon M-S, Atfield A, Venuprasad K, Krawczyk C, Sarao R, Elly C, Yang C, Arya S, Bachmaier K, Su L et al.: Essential role of the E3
Current Opinion in Immunology 2007, 19:658–664
www.sciencedirect.com
Sound of silence: modulating anergy in T lymphocytes Saibil, Deenick and Ohashi 663
ubiquitin ligase Cbl-b in T cell anergy induction. Immunity 2004, 21:167-177. 16. Berg-Brown NN, Gronski MA, Jones RG, Elford AR, Deenick EK, Littman DR, Ohashi PS: PKCu signals activation versus tolerance in vivo. J Exp Med 2004, 199:743-752.
34. Hundt M, Tabata H, Jeon MS, Hayashi K, Tanaka Y, Krishna R, De Giorgio L, Liu YC, Fukata M, Altman A: Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect. Immunity 2006, 24:513-522. This study demonstrates that lipid modification of LAT, via palmitoylation, is important for the maintenance of the anergic state.
17. Reis e Sousa C: Dendritic cells in a mature age. Nat Rev Immunol 2006, 6:476-483.
35. Fukata M, Fukata Y, Adesnik H, Nicoll RA, Bredt DS: Identification of PSD-95 palmitoylating enzymes. Neuron 2004, 44:987-996.
18. Li W, Whaley CD, Mondino A, Mueller DL: Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T cells. Science 1996, 271:1272-1276.
36. Boussiotis VA, Freeman GJ, Taylor PA, Berezovskaya A, Grass I, Blazar BR, Nadler LM: p27kip1 functions as an anergy factor inhibiting interleukin 2 transcription and clonal expansion of alloreactive human and mouse helper T lymphocytes. Nat Med 2000, 6:290-297.
19. Fields PE, Gajewski TF, Fitch FW: Blocked Ras activation in anergic CD4+ T cells. Science 1996, 271:1276-1278. 20. Zha Y, Marks R, Ho AW, Peterson AC, Janardhan S, Brown I, Praveen K, Stang S, Stone JC, Gajewski TF: T cell anergy is reversed by active Ras and is regulated by diacylglycerol kinase-alpha. Nat Immunol 2006, 7:1166-1173. This report and ref. [24] establish DGKs and DAG metabolism as key molecules in blocking Ras signaling and establishing anergy. 21. Marsland BJ, Nembrini C, Schmitz N, Abel B, Krautwald S, Bachmann MF, Kopf M: Innate signals compensate for the absence of PKC-{theta} during in vivo CD8(+) T cell effector and memory responses. Proc Natl Acad Sci U S A 2005, 102:14374-14379. 22. Zhong XP, Hainey EA, Olenchock BA, Zhao H, Topham MK, Koretzky GA: Regulation of T cell receptor-induced activation of the Ras-ERK pathway by diacylglycerol kinase zeta. J Biol Chem 2002, 277:31089-31098. 23. Sanjuan MA, Jones DR, Izquierdo M, Merida I: Role of diacylglycerol kinase alpha in the attenuation of receptor signaling. J Cell Biol 2001, 153:207-220. 24. Olenchock BA, Guo R, Carpenter JH, Jordan M, Topham MK, Koretzky GA, Zhong XP: Disruption of diacylglycerol metabolism impairs the induction of T cell anergy. Nat Immunol 2006, 7:1174-1181. See annotation to ref. [20] 25. Smida M, Posevitz-Fejfar A, Horejsi V, Schraven B, Lindquist JA: A novel negative regulatory function of PAG: blocking Ras activation. Blood 2007. This report shows that PAG-Fyn complex can promote anergy in human T cells by blocking Ras activation independent of DGKs. 26. Davidson D, Schraven B, Veillette A: PAG-associated FynT regulates calcium signaling and promotes anergy in T lymphocytes. Mol Cell Biol 2007, 27:1960-1973. 27. Kearney ER, Pape KA, Loh DY, Jenkins MK: Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1994, 1:327-339. 28. Bachmaier K, Krawczyk C, Kozieradzki I, Kong Y-Y, Sasaki T, Oliveira-dos-Santos AJ, Mariathasan S, Bouchard D, Wakeham A, Itie A et al.: Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 2000, 403:211-216. 29. Chiang YJ, Kole HK, Brown K, Naramura M, Fukuhara S, Hu R-J, Jang IK, Gutkind JS, Shevach E, Gu H: Cbl-b regulates the CD28 dependence of T-cell activation. Nature 2000, 403:216-220. 30. Safford M, Collins S, Lutz MA, Allen A, Huang CT, Kowalski J, Blackford A, Horton MR, Drake C, Schwartz RH et al.: Egr-2 and Egr-3 are negative regulators of T cell activation. Nat Immunol 2005, 6:472-480.
37. Wells AD, Walsh MC, Sankaran D, Turka LA: T cell effector function and anergy avoidance are quantitatively linked to cell division. J Immunol 2000, 165:2432-2443. 38. Rowell EA, Walsh MC, Wells AD: Opposing roles for the cyclindependent kinase inhibitor p27kip1 in the control of CD4+ T cell proliferation and effector function. J Immunol 2005, 174:3359-3368. 39. Colombetti S, Benigni F, Basso V, Mondino A: Clonal anergy is maintained independently of T cell proliferation. J Immunol 2002, 169:6178-6186. 40. Allen A, Zheng Y, Gardner L, Safford M, Horton MR, Powell JD: The novel cyclophilin binding compound, sanglifehrin A, disassociates G1 cell cycle arrest from tolerance induction. J Immunol 2004, 172:4797-4803. 41. Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A: Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kip1). Cell 1996, 85:721-732. 42. Li L, Iwamoto Y, Berezovskaya A, Boussiotis VA: A pathway regulated by cell cycle inhibitor p27Kip1 and checkpoint inhibitor Smad3 is involved in the induction of T cell tolerance. Nat Immunol 2006, 7:1157-1165. Study shows that p27 is important in establishing anergy by allowing Smad3 transcriptional activity and not by blocking cell cycle entry. 43. Zheng Y, Collins SL, Lutz MA, Allen AN, Kole TP, Zarek PE, Powell JD: A role for mammalian target of rapamycin in regulating T cell activation versus anergy. J Immunol 2007, 178:2163-2170. This study demonstrates mTOR activation and not cell cycle progression is important in determining T cell activation versus anergy. 44. Parry RV, Reif K, Smith G, Sanson DM, Hemmings BA, Ward SG: Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B. Eur J Immunol 1997, 27:2495-2501. 45. Song J, Salek-Ardakani S, Rogers PR, Cheng M, van Parijs L, Croft M: The costimulation-regulated duration of PKB activation controls T cell longevity. Nat Immunol 2004:1-9. 46. Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL: PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization. Nat Med 2002, 8:1145-1152. 47. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R et al.: PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 2002, 8:1153-1160. 48. Inoki K, Li Y, Zhu T, Wu J, Guan KL: TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 2002, 4:648-657.
31. Schwartz RH: T cell anergy. Annu Rev Immunol 2003, 21:305-334. 32. Rocha B, Tanchot C, von Boehmer H: Clonal anergy blocks in vivo growth of mature T cells and can be reversed in the absence of antigen. J Exp Med 1993, 177:1517-1521. 33. Chiodetti L, Choi S, Barber DL, Schwartz RH: Adaptive tolerance and clonal anergy are distinct biochemical states. J Immunol 2006, 176:2279-2291. This study demonstrates the signaling differences between clonal anergy and adaptive tolerance. www.sciencedirect.com
49. Suzuki A, Yamaguchi MT, Ohteki T, Sasaki T, Kaisho T, Kimura Y, Yoshida R, Wakeham A, Higuchi T, Fukumoto M et al.: T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity 2001, 14:523-534. 50. Okazaki T, Honjo T: The PD-1-PD-L pathway in immunological tolerance. Trends Immunol 2006, 27:195-201. 51. Liang SC, Latchman YE, Buhlmann JE, Tomczak MF, Horwitz BH, Freeman GJ, Sharpe AH: Regulation of PD-1, PD-L1, and PD-L2 Current Opinion in Immunology 2007, 19:658–664
664 Autoimunnity
expression during normal and autoimmune responses. Eur J Immunol 2003, 33:2706-2716. 52. Yamazaki T, Akiba H, Iwai H, Matsuda H, Aoki M, Tanno Y, Shin T, Tsuchiya H, Pardoll DM, Okumura K et al.: Expression of programmed death 1 ligands by murine T cells and APC. J Immunol 2002, 169:5538-5545. 53. Nishimura H, Nose M, Hiai H, Minator N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 1999, 11:141-151. 54. Nishimura H, Okazaki T, Tanaka Y, Nakatani K, Hara M, Matsumori A, Sasayama S, Mizoguchi A, Hiai H, Minato N et al.: Autoimmune dilated cardiomyopathy in PD-1 receptor deficient mice. Science 2001, 291:319-322. 55. Probst HC, McCoy K, Okazaki T, Honjo T, van den Broek M: Resting dendritic cells induce peripheral CD8+ T cell tolerance through PD-1 and CTLA-4. Nat Immunol 2005, 6:280-286. 56. Keir ME, Liang SC, Guleria I, Latchman YE, Qipo A, Albacker LA, Koulmanda M, Freeman GJ, Sayegh MH, Sharpe AH: Tissue expression of PD-L1 mediates peripheral T cell tolerance. J Exp Med 2006, 203:883-895. Refs. [56–59] establish tissue-resident PD-L1 signals important to the imposition and maintenance of the anergic state. 57. Martin-Orozco N, Wang YH, Yagita H, Dong C: Cutting Edge: Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens. J Immunol 2006, 177:8291-8295. See annotation to ref. [56]. 58. Fife BT, Guleria I, Gubbels BM, Eagar TN, Tang Q, Bour-Jordan H, Yagita H, Azuma M, Sayegh MH, Bluestone JA: Insulin-induced remission in new-onset NOD mice is maintained by the PD-1-PD-L1 pathway. J Exp Med 2006, 203:2737-2747. See annotation to ref. [56]. 59. Tsushima F, Yao S, Shin T, Flies A, Flies S, Xu H, Tamada K, Pardoll DM, Chen L: Interaction between B7-H1 and PD-1
Current Opinion in Immunology 2007, 19:658–664
determines initiation and reversal of T-cell anergy. Blood 2007, 110:180-185. See annotation to ref. [56]. 60. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ: Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 2007, 27:111-122. Reference demonstrates that PD-L1 and B7-1 can ligate each other. 61. Parry RV, Chemnitz JM, Frauwirth KA, Lanfranco AR, Braunstein I, Kobayashi SV, Linsley PS, Thompson CB, Riley JL: CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol Cell Biol 2005, 25:9543-9553. 62. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R et al.: PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol 2001, 2:261-268. 63. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH, Freeman GJ, Ahmed R: Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 2006, 439:682-687. 64. Trautmann L, Janbazian L, Chomont N, Said EA, Gimmig S, Bessette B, Boulassel MR, Delwart E, Sepulveda H, Balderas RS et al.: Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 2006, 12:1198-1202. 65. Day CL, Kaufmann DE, Kiepiela P, Brown JA, Moodley ES, Reddy S, Mackey EW, Miller JD, Leslie AJ, DePierres C et al.: PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 2006, 443:350-354. 66. Hickman SP, Yang J, Thomas RM, Wells AD, Turka LA: Defective activation of protein kinase C and Ras-ERK pathways limits IL2 production and proliferation by CD4+CD25+ regulatory T cells. J Immunol 2006, 177:2186-2194. 67. Knoechel B, Lohr J, Zhu S, Wong L, Hu D, Ausubel L, Abbas AK: Functional and molecular comparison of anergic and regulatory T lymphocytes. J Immunol 2006, 176:6473-6483.
www.sciencedirect.com