Review

Mechanisms for safeguarding tolerance to self

Under normal (healthy) conditions the immune system does not attack and destroy the tissues of its host—a state called self-tolerance. A number of mechanisms have evolved to safeguard animals against autoimmune attack, including deletion of autoreactive lymphocytes, sequestration of lymphocytes from sites where certain self-antigens are expressed (immunological ignorance), developmental arrest, de-sensitization of autoreactive lymphocytes (anergy) and finally immunological suppression by so-called regulatory T cells (T regs) and regulatory factors.¹ Of these mechanisms only deletion of autoreactive lymphocytes constitutes an irreversible process—all others are potentially reversible. Upon activation and exposure to inflammatory signals, T and B lymphocytes can alter expression of surface molecules that regulate their migration and thereby gain access to tissues, which are not visited by their naive counterparts, and this may cause a breakdown in 'immunological ignorance'. Furthermore, developmentally arrested or so-called 'anergic' lymphocytes may recuperate responsiveness to stimulation by their cognate antigens upon cessation of sustained antigen receptor triggering or when they are exposed to certain cytokines. Finally, the repertoire and/or activity of T regulatory cells may change in response to immunological challenge and thereby liberate the 'regulated' T lymphocytes from their shackles. Thus, deletion of autoreactive lymphocytes plays a central role in immunological tolerance to self but it is by no means the only mechanism for protection against autoimmune attack. In fact, we^{2, 3} and others⁴ speculate that development of severe autoimmune pathology may well require combined subversion of more than one, perhaps all, of these protective mechanisms.

Deletion of autoreactive lymphocytes for self-tolerance—prediction and experimental confirmation

Burnet⁵ and Talmage⁶ were the first to predict that lymphocytes expressing antigen receptors specific for self-antigens will be deleted during their development. This hypothesis was first tested and confirmed by analysing the fate of immature thymocytes expressing TCR/ antigen receptors specific for so-called endogenous superantigens, which are encoded in the germline by retrovirus-like sequences.^{7, 8, 9, 10} In contrast to 'conventional' antigens, which are presented to T cells as peptide fragments embedded into class I or class II MHC molecules and stimulate a small fraction of the T cell repertoire (generally <1/10⁵), superantigens are presented to T cells as whole polypeptides associated with class II MHC molecules and stimulate a readily identifiable portion of the TCR repertoire (generally 2–10%). Specificity for superantigens bound to class II MHC chains is conferred to a large extent by the variable region of the TCR chain; for example, most TCRv3 expressing T cells respond to Mls-2^a presented by I-E class II MHC proteins.¹⁰ Using panels of TCRv-specific monoclonal antibodies and collections of mouse strains it was shown by several laboratories that T cells that respond to endogenous superantigens are deleted during their development in the thymus.^{7, 8, 9, 10} Experiments using transgenic mice expressing on all of their T lymphocytes TCR/ antigen receptors of defined specificity demonstrated that not only superantigen specific T cells but also T cells specific for conventional antigens presented by either class I or class II MHC are deleted during their differentiation in the thymus.^{11, 12, 13, 14} For example, expression of a TCR/ specific for a peptide derived from the male HY antigen presented by class I^b MHC molecules caused deletion of thymocytes in transgenic males 11 but positive selection in females.¹⁵ Depending on the nature of the self-antigen and/or timing of expression of the transgene-encoded TCR/, deletion was found to occur at either the early CD4⁺8⁺ (DP: double positive) stage or at the transition from the late CD4⁺8⁺ to the mature (SP: single positive) CD4⁺8^- or CD4^-8⁺ stage. Depending on the experimental system that was examined, self-antigens were reportedly presented by either epithelial cells, haemopoietic (that is, dendritic) cells or even both cell types (reviewed in Goodnow et al.¹). With respect to anatomical location, it is widely believed that deletion of autoreactive thymocytes occurs predominantly at the boundary between the cortex and medulla, the so-called corticomedullary junction, which is rich in antigen presenting dendritic cells (reviewed in Goodnow et al.¹).

Experiments in which naive TCR/ transgenic T cells were injected into mice expressing their cognate antigen (either endogenously or encoded by a transgene) plus the MHC molecules required for presentation, demonstrated that deletion of autoreactive T cells can also occur at the mature stage in peripheral lymphoid organs and non-lymphoid tissues.¹⁶ In addition, studies using B cell antigen receptor (BCR) transgenic mice and transgenic mice expressing the cognate antigen revealed that autoreactive B lymphocytes, like T lymphoid cells, can be deleted at an immature stage during development in the bone marrow or at a mature stage in the spleen or lymph nodes, provided that no T cell help is available.^{17, 18, 19} Collectively, these studies demonstrate that signalling through antigen receptors can cause deletion of autoreactive T and B lymphocytes both during their development in the thymus or bone marrow, respectively, or at the mature stage in peripheral lymphoid organs, such as the spleen or lymph nodes.

Introduction into apoptosis signalling

Morphological analysis of TCR/ transgenic thymocytes undergoing negative selection in culture and studies of immortalized cell lines (that is, B lymphoma lines or T hybridoma lines) stimulated with agonistic antibodies to their antigen receptors indicated that deletion of autoreactive lymphocytes occurs through apoptotic cell death.^{20, 21, 22}

Apoptosis is a genetically programmed process for killing unwanted and potentially dangerous cells that is evolutionarily highly conserved.^{23, 24} Demolition of the cells is mediated by aspartate-specific cysteine proteases, called caspases, which cleave and thereby destroy a large number of structural proteins (for example, lamins, gelsolin) and also activate latent enzymes that dismantle nucleic acids (caspase activated DNAse called CAD) or other cellular constituents.²⁵ Caspases are present in healthy cells in an inactive (zymogen) state and according to their structure, function and mode of activation, they can be divided into two groups: 'initiator caspases' (for example, caspase-8 and -9 in mammals and Caenorhabditis elegans CED-3) and 'effector caspases' (for example, caspase-3, -6 and -7 in mammals). The 'effector caspases' are mainly responsible for the proteolysis of structural proteins and activation of CAD. They are synthesized as zymogens of 35–40 kDa with short prodomains and are activated by proteolytic cleavage by 'initiator caspases'. This liberates fragments of 20 and 10 kDa, which are assembled into the fully active tetrameric (p20₂p10₂) enzyme. 'Initiator caspases' are mainly responsible for proteolytic activation of the 'effector caspases' and are synthesized as zymogens with long prodomains. These prodomains mediate interaction with so-called adaptor proteins (FADD/Mort1 binds to procaspase-8 whereas Apaf-1 binds to caspase-9), which promote activation of the 'initiator caspases' by inducing a conformational change.

Mammals have two distinct although ultimately converging apoptosis signalling pathways (Figure 1).^{24, 26} Activation of so-called 'death receptors', members of the tumour necrosis factor receptor (TNF-R) family with an intracellular 'death domain' (for example, Fas/APO-1/CD95, TNF-R1) by their corresponding ligands (FasL, TNF) or agonistic antibodies triggers formation of an intracellular 'death inducing signalling complex' (DISC) in which FADD promotes activation of caspase-8. Experiments with gene-targeted as well as transgenic mice have shown that FADD^{27, 28} and caspase-8^{29, 30, 31} are essential for 'death receptor'-induced apoptosis signalling. Conversely, apoptosis induced by a range of developmentally programmed cues, growth factor deprivation or treatment with many cytotoxic drugs does not require 'death receptors', caspase-8 or its activator FADD.^{26, 27, 29, 32, 33} Instead, these stimuli trigger apoptosis by a signalling cascade that is regulated by the Bcl-2 protein family and involves permeabilization of the outer mitochondrial membrane (MOMP) leading to the activation of caspase-9 by its adaptor Apaf-1 in conjunction with cytochrome c and dATP.^{24, 34, 35} This pathway is referred to as the 'intrinsic', 'mitochondrial' or 'Bcl-2-regulated' apoptotic pathway. We prefer the last nomenclature, because many death stimuli that activate this pathway are actually applied from the outside (for example, TGF, loss of cell attachment, cytotoxic drugs) and because mitochondria may not be absolutely essential for this pathway. For example, although cytochrome c/Apaf-1-mediated caspase-9 activation features prominently in this pathway, cells lacking either of these proteins still die and lose clonogenic potential and some may even exhibit classical features of apoptosis when exposed to death stimuli (for example, cytokine deprivation or treatment with certain cytotoxic drugs) that can be efficiently antagonized by Bcl-2 overexpression or loss of proapoptotic Bim or combined Bax/Bak deficiency.^{36, 37, 38, 39}

Figure 1.

Two distinct but ultimately converging pathways to apoptosis in mammalian cells. Engagement of 'death receptors', such as Fas, by their ligands leads to the activation of 'initiator caspase', caspase-8, with the help of adaptor proteins, such as FADD and TRADD. Caspase-8 proteolytically activates 'effector caspases' (caspases-3, -6, -7), which cause cell demolition by cleaving vital structural proteins and through proteolytic activation of the DNAse CAD. In the 'Bcl-2-regulated' (also called 'mitochondrial' or 'intrinsic') pathway, developmental cues, cytokine deprivation or cytotoxic stimuli cause activation of BH3-only proteins (a proapoptotic subgroup of the Bcl-2 protein family). The BH3-only proteins cause activation of Bax/Bak (the second proapoptotic subgroup within the Bcl-2 protein family) either directly and/or indirectly by interaction with the prosurvival Bcl-2 family members (the two prevalent opposing models are shown in Figure 2). Upon activation, Bax/Bak cause permeabilization of the mitochondrial outer membrane (through a presently still unresolved process), thereby eliciting release of cytochrome c (and other mitochondrial apoptogenic proteins) into the cytoplasm. The interaction between cytochrome c, Apaf-1 and procaspase-9 generates active initiator caspase-9, which can then proteolytically activate the 'effector caspases', the point where the two pathways converge. In certain cell types (for example, hepatocytes) caspase-8-mediated proteolytic activation of the BH3-only protein Bid is critical for Fas-induced apoptosis, providing a link between the two apoptosis signalling pathways.

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The Bcl-2 protein family, the critical regulators of this pathway, can be subdivided into three subgroups. The prosurvival family members, including Bcl-2, Bcl-x_L, Bcl-w, A1/Bfl1 and Mcl-1, all contain up to four regions of homology called Bcl-2 Homology (BH) domains. Experiments with gene-targeted mice have shown that different prosurvival proteins are critical for the survival of different cell types. Bcl-2-deficient mice develop fatal polycystic kidney disease, lymphopoenia and turn prematurely grey due to abnormally increased apoptosis of renal epithelial cell progenitors, mature lymphocytes and melanocyte progenitors.^{40, 41} Mice lacking Bcl-x die around embryonic day 14 (E14) due to widespread apoptosis of erythroid progenitors and neuronal cells.⁴² Bcl-w-deficient mice are largely normal but males are sterile due to abnormally increased apoptosis of developing sperm cells.^{43, 44} Embryonic mice lacking Mcl-1 in all tissues die prior to implantation and experiments with gene-targeted mice in which mcl-1 was deleted in specific tissues demonstrated that it is also critical for survival of immature as well as mature lymphocytes⁴⁵ and hemopoietic stem cells.⁴⁶ Loss of A1a, one of the four A1 genes in mice (three expressed and one pseudo-gene) causes abnormally accelerated apoptosis of granulocytes⁴⁷ and mast cells⁴⁸ in culture, but due to possible redundancy amongst the a1 genes additional functions might have been obscured.

Bax, Bak and Bok/Mtd are proapoptotic but, curiously, they contain extensive sequence similarity (three BH regions: BH1, BH2 and BH3) and at least in the case of Bax also remarkable structural similarity to the prosurvival Bcl-2 family members.⁴⁹ Bax/Bak proteins are thought to be kept in check by binding to prosurvival Bcl-2 family members in healthy cells but have been shown to change their conformation and to undergo homo-dimerization and even oligomerization during apoptosis induction.⁵⁰ Gene-targeting experiments have shown that Bax and Bak have largely redundant functions but are essential for the death of many cell types triggered by a large range of apoptotic stimuli. Although mice lacking either Bax or Bak are normal with the exception of defective spermatogenesis seen in bax^-/- males, animals lacking both (bax^-/-bak^-/- mice) mostly die during development and those that survive for a few weeks all have webbed feet and develop progressive lymphadenopathy and splenomegaly due to defective programmed death of interdigitating epithelial cells and mature lymphocytes, respectively.^{51, 52} Remarkably, fibroblasts, lymphocytes and certain other cell types from bax^-/-bak^-/- mice were found to be dramatically resistant to cytokine deprivation and a broad range of cytotoxic drugs with the exception that lymphoid cells were normally sensitive to FasL.^{51, 52}

The second proapoptotic subgroup of the Bcl-2 family contains Bad, Bik/Blk/Nbk, Hrk/DP5, Bid, Bim/Bod, Bmf Puma/Bbc3 and Noxa, and because these proteins share with each other and the remainder of the family only the BH3 domain they are called BH3-only proteins.⁵³ All BH3-only proteins can bind to prosurvival Bcl-2 family members but they differ substantially in their specificity. For example, Bim and Puma can bind with high affinity (low or sub-nM range) to all prosurvival members of the Bcl-2 family, while active, truncated Bid (tBid) binds to nearly all with the exception of Bcl-2. In contrast, Bad binds only to Bcl-2, Bcl-x_L and Bcl-w and, conversely, Noxa only to Mcl-1 and A1.^{54, 55} Direct binding of at least certain BH3-only proteins (Bim, Bid and Puma) to Bax/Bak has also been reported^{56, 57, 58} but the affinities of these interactions are not clear. What is widely accepted is that killing by activation or overexpression of all BH3-only proteins requires Bax/Bak.^{59, 60} Based on these observations, two models for apoptosis induction have been proposed (Figure 2). According to one, apoptosis initiation requires direct activation of Bax/Bak by BH3-only proteins and prosurvival family members function as a sink to bind BH3-only proteins.⁵⁷ The other model postulates that apoptosis induction requires binding and antagonism of all prosurvival family members present in a particular cell by BH3-only proteins⁶¹ and that this then liberates Bax/Bak to cause permeabilization of the outer mitochondrial membrane and activation of the caspase cascade. It remains possible that these two models are not fully mutually exclusive and it appears likely that modifications will have to be made to these models to better explain all the currently available and newly emerging data.

Figure 2.

Two opposing models for Bax/Bak activation. The 'direct activation model' stipulates that certain BH3-only proteins, the so-called 'activator BH3-only proteins', including Bim, Bid and possibly also Puma, bind and thereby activate Bax and/or Bak directly, whereas so-called 'sensitiser BH3-only proteins' (Bad, Hrk, Bmf, Noxa, Bik) bind to prosurvival Bcl-2 family members, thereby causing release of the 'activator BH3-only proteins'. In the 'indirect model', Bax/Bak are kept in check by prosurvival Bcl-2 family members until BH3-only proteins bind to the prosurvival Bcl-2 family members thereby causing release and activation of Bax/Bak.

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Gene targeting experiments have identified the essential roles of the eight known BH3-only proteins (Figure 3). Consistent with the observation that Bim, Puma and Bid bind to all or nearly all (Bid does not appear to bind with high affinity to Bcl-2) prosurvival Bcl-2 family members, their loss causes substantial phenotypic abnormalities, whereas loss of BH3-only proteins that bind only to select Bcl-2-like proteins (for example, Bad or Bik) has only minor impact.^{3, 53} Mice lacking Bim accumulate three- to five-fold excess numbers of mature lymphoid and myeloid cells, up to 200-fold increased numbers of immunoglobulin (Ig)-secreting plasma cells, antinuclear autoantibodies and on a mixed C57BL/6x129SV genetic background even fatal SLE-like autoimmune glomerulonephritis.⁶² Lymphoid, myeloid, neuronal and other cell types from bim^-/- mice were shown to be abnormally resistant to cytokine deprivation, deregulated calcium flux, endoplasmic reticulum (ER) stress and certain other apoptotic stimuli.^{62, 63, 64, 65, 66, 67} Curiously, although the bim gene does not contain a binding site for the tumour suppressor p53,⁶⁸ which is essential for DNA damage-induced apoptosis,^{69, 70, 71} bim^-/- thymocytes were found to have significant albeit minor resistance to -irradiation and etoposide.^{62, 72} Bim-deficient cells are, however, normally sensitive to certain other apoptotic stimuli, such as treatment with phorbol ester.⁶²

Figure 3.

Different developmental cues and cytotoxic stimuli activate distinct BH3-only proteins. Different developmentally programmed death stimuli or cytotoxic stress signals (for example, activated by certain chemotherapeutic drugs) activate through transcriptional and/or post-translational mechanisms distinct BH3-only proteins. Some stimuli (for example, cytokine deprivation) activate several BH3-only proteins (Bim, Puma, Bad and Hrk). It is also possible that a given death stimulus will activate different BH3-only proteins in different cell types. For example, growth factor deprivation causes Hrk activation only in neuronal but not in haematopoietic cells.

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Loss of Puma, which is transcriptionally regulated by the tumour suppressor p53,^{73, 74} renders a broad range of cell types resistant to DNA damage-induced apoptosis^{75, 76} (which depends largely on p53). Consistent with the observation that Puma expression can also be induced by certain p53-independent apoptotic stimuli,⁷⁷ puma^-/- lymphoid and myeloid cells were also found to be abnormally resistant to cytokine deprivation and treatment with glucocorticoids or phorbol ester^{72, 75, 76} (which trigger apoptosis through p53-independent pathways). Surprisingly, although - and UV-irradiation induced apoptosis are both p53-dependent,^{69, 70} loss of Puma renders cells resistant to the former but not the latter, whereas Noxa plays a major role in UVR-induced apoptosis of fibroblasts in culture and keratinocytes in the skin of live mice.⁷⁸ This indicates that different forms of DNA damage may activate p53 in different ways thereby affecting its ability to regulate different target genes (puma versus noxa). Alternatively, different DNA lesions may activate in addition to p53 a second (specific) signalling pathway, which together with p53 determines which target genes are activated.

Loss of Bid renders hepatocytes resistant to Fas-induced apoptosis but thymocytes and T lymphocytes remain normally sensitive.^{79, 80} This indicates that 'death receptor' induced caspase-8 activation on its own is sufficient for killing of certain cell types (for example, lymphoid cells) but amplification of caspase activation through tBid>Bax/Bak>Apaf-1>caspase-9 signalling is required in others (for example, hepatocytes).

Mice lacking Bad,⁸¹ Bik⁸² or Hrk^{83, 84} appear largely normal, although loss of Bad or Hrk affords fibroblasts or neuronal cells, respectively, with minor protection from growth factor deprivation in culture.

Many apoptotic stimuli induce expression of more than one BH3-only protein in a given cell type and apoptosis triggered by certain stimuli can be partially inhibited by loss of more than one BH3-only protein. For example, loss of either Bim⁶² or Puma^{75, 76} protect lymphoid and myeloid cells from cytokine deprivation. This indicates significant functional overlap between individual BH3-only proteins and this has indeed been formally demonstrated through analysis of mice lacking two of these apoptosis initiators. For example, although bim^-/- and bik^-/- males are normally fertile those lacking both Bim and Bik (bim^-/-bik^-/-) are infertile due to abnormal accumulation of early sperm cell progenitors, which precludes normal differentiation.⁸⁵ Moreover, combined loss of Bim and Puma renders lymphoid and myeloid cells more resistant to cytokine deprivation, treatment with glucocorticoids and certain other apoptotic stimuli than loss of either BH3-only protein alone.⁸⁶ In fact these bim^-/-puma^-/- cells are as (or nearly as) resistant as bax^-/-bak^-/- or Bcl-2 overexpressing cells, indicating that these two BH3-only proteins account for most of the apoptosis inducing activity in this setting.

Since BH3-only proteins are essential initiators of apoptosis, their activity is subject to stringent transcriptional as well as post-translational control.^{53, 87} For example, Puma and Noxa can both be regulated transcriptionally by the tumour suppressor p53,^{73, 74} and Puma also by FOXO3a⁸⁸ and most likely also by certain other transcription factors.⁷⁷ Bim is transcriptionally regulated by Foxo3a in response to growth factor deprivation⁸⁹ and by CHOP in response to ER stress.⁶⁷ Bim can also be regulated post-translationally through ERK-mediated phosphorylation which is thought to affect its binding to prosurvival Bcl-2 family members and targets it for ubiquitination and proteasomal degradation.^{90, 91, 92, 93} Bim⁹⁴ and Bmf⁹⁵ can apparently also be regulated by binding to dynein light chains, which causes their localization to the dynein- or actin-based motor complexes, respectively. The proapoptotic activity of Bid is controlled predominantly by proteolysis by caspase-8 and certain other caspases,^{96, 97} which facilitates its myristoylation and translocation to the outer mitochondrial membrane.⁹⁸

In conclusion, BH3-only proteins are essential for apoptosis initiation and activated in a stimulus- and cell type-specific manner. Bax/Bak are also essential for cell killing, functioning downstream of the BH3-only proteins and they promote by a presently still poorly understood mechanism permeabilization of the outer mitochondrial membrane and activation of the caspase cascade. In contrast, the prosurvival Bcl-2 family members are essential for cell survival and they interact with both the BH3-only proteins as well as the Bax/Bak-like proteins.

Mechanisms for apoptotic death of autoreactive T and B lymphocytes

Initial morphological and biochemical studies with thymocytes from TCR transgenic mice²⁰ and T hybridoma cells stimulated with TCR/CD3 crosslinking antibodies²² indicated that negative selection of autoreactive T cells is mediated by apoptotic cell death. This raised the question of which mechanism of apoptosis signalling—'death receptor' signalling or the 'Bcl-2-regulated' pathway is activated and which pro- and antiapoptotic regulators are critical. Studies with transgenic mice showed that Bcl-2 overexpression can inhibit the developmental death of autoreactive thymocytes specific for either Mls superantigens⁹⁹ or conventional antigens (as examined in anti-HY TCR/bcl-2 doubly transgenic mice).¹⁰⁰ In contrast, null mutations in Fas¹⁰¹ or loss of FADD^{27, 102} or caspase-8^{30, 32} function, which are required for apoptosis induction by all 'death receptors', did not affect deletion of autoreactive thymocytes (both Mls- or conventional antigen specific ones). Collectively, these results show that the death of autoreactive thymocytes is mediated by the 'Bcl-2-regulated' pathway and that 'death receptor' signalling plays no role in this process. This indicated also that TCR/CD3 stimulation must activate one or several BH3-only proteins in autoreactive thymocytes, which then initiate apoptosis. Bim appeared to be a good candidate, since calcium flux has been implicated as a signal critical for deletion of autoreactive thymocytes¹⁰³ and since loss of Bim renders thymocytes and immature cells resistant to apoptosis triggered by deregulated calcium flux.^{62, 104} Indeed, crosses between bim^-/- and three different TCR transgenic strains of mice as well as analysis of fetal thymic organ cultures stimulated with superantigens demonstrated that loss of Bim inhibited the deletion of autoreactive thymocytes both at the immature CD4⁺8⁺ and the semimature CD4⁺8⁺HSA⁺ stage.^{62, 105} Loss of Bim-protected autoreactive thymocytes regardless of whether they expressed class I or class II MHC-restricted TCR/.⁶² The effects of loss of Bim were specific because negative selection of autoreactive thymocytes was found to occur normally in mice lacking Puma, Bid, Bik, Bad, Hrk, Noxa or Bmf (E Michalak, A Villunger, T Kaufmann, L Coultas, P Bouillet and A Strasser, unpublished observations).

Not only immature thymocytes but also mature, naive T cells can be deleted when they are stimulated by self-antigen. This can for example be demonstrated experimentally by transferring naive T cells from TCR transgenic mice (in which the cognate antigen is not expressed) into mice in which the cognate antigen is present (either endogenously or transgene encoded).¹⁶ Another model system that is used for analysis of TCR/CD3 stimulation induced T cell killing involves treatment of activated (mature, proliferating) T cells or immortalized T hybridoma cells with agonistic antibodies to the TCR/CD3 complex. This model is often referred to as 'activation induced cell death' (AICD).¹⁰⁶ The problem with this model is that proliferating T cell blasts and T hybridoma cells may not be akin to naive T cells—in fact in the case of T hybridoma cells it is unclear what they best represent: immature thymocytes (the fusion partners are thymic lymphoma-derived cell lines), naive, mature T cells or proliferating, activated T cells¹⁰⁷? Perhaps not surprisingly, initial analyses of the mechanism of this T cell deletion using these model systems produced conflicting results. TCR/CD3 stimulation was found to trigger FasL production and consequently autocrine or paracrine FasL-Fas mediated apoptosis in both activated (normal) T cells as well as (immortalized) T hybridoma cells.^{108, 109, 110, 111} However, subsequent studies showed that the killing of naive T cells from OT-1 TCR transgenic mice (receptor specific for a peptide derived from ovalbumin presented by class I MHC H2-K^b) upon transfer into ovalbumin expressing transgenic recipients can be inhibited by Bcl-2 overexpression or loss of Bim.¹¹² Collectively, these results indicate that Bim-dependent 'Bcl-2-regulated' apoptosis signalling is critical for negative selection of autoreactive naive, mature T cells whereas Fas-mediated 'death receptor' signalling contributes to TCR/CD3 stimulation induced killing of activated, proliferating (mature) T cells.

It appears likely that FasL>Fas signalling does play a role in the death of mature, activated T cells, since mice lacking either of these proteins (Fas-deficient lpr or lpr^cg mice or FasL-deficient gld mice) develop progressive lymphadenopathy and splenomegaly and on certain genetic backgrounds (for example, MRL) also fatal SLE-like autoimmune disease.^{113, 114} Many of the T cells accumulating in these mutant mice have an unusual cell surface marker expression profile (Thy1⁺TCR/⁺CD3⁺CD4^-CD8^-B220⁺) reminiscent of chronically activated cells. Moreover, Fas was shown to be critical for the deletion of T cells that had been stimulated repeatedly by their cognate (foreign) antigen in vivo,¹¹⁵ a condition that appears to be mimicked by the AICD model in vitro with normal T cells or immortalized T hybridoma cells.¹¹⁶ In contrast, the killing of antigen-activated T cells during shutdown of an immune response to an acute infection (or single challenge with an injected antigen) is independent of Fas and other 'death receptors' but instead requires the BH3-only protein Bim.^{117, 118} In this setting activated T cells are not killed because of repeated stimulation through their TCR/CD3 complex since all antigen (and hence all TCR stimulatory capacity) has vanished by the time they are dying.^{3, 116} Instead, we^{3, 116} and others¹¹⁹ believe that a drop in the levels of survival sustaining cytokines, as a consequence of pathogen clearance, initiates the death of antigen-activated T cells during termination of an acute immune response. Since loss of Bim renders T cells refractory to cytokine deprivation induced apoptosis,⁶² it therefore makes sense that Bim is critical for the death of T cells during shutdown of an acute immune response. Although Fas-mediated 'death receptor' signalling and 'Bcl-2-regulated' apoptosis signalling are mechanistically distinct, these pathways are coordinated and cooperate in the killing of T lymphocytes chronically stimulated by self-antigens in vivo, as demonstrated by the massively increased lymphadenopathy and splenomegaly seen in bcl-2 transgenic lpr mice compared to lpr littermates.²⁶ Moreover, Bcl-2 overexpression and loss of Fas (bcl-2 transgenic lpr mice) synergise in protecting T cells from the death induced by repeated in vivo stimulation with the superantigen staphylococcus enterotoxin B (SEB).²⁶

B lymphoid cells are also subject to stringent selection on the basis of the specificity of their antigen receptors. This was first demonstrated by using transgenic mice expressing on their B cells a BCR specific for hen egg lysozyme (HEL) or H-2K^b class I MHC and then crossing these animals with animals expressing a HEL transgene (either systemically or in specific tissues as a pseudo self-antigen)¹²⁰ or with mice naturally expressing the H-2K^b class I MHC self-antigen.¹⁹ This experimental setup revealed that immature B cells in the bone marrow are subject to negative selection if they express an autoreactive BCR. Moreover, experiments in which mature, naive B cells from BCR transgenic mice (developing in the absence of the cognate self- or pseudo self-antigen) were transferred into mice that express the cognate antigen showed that B cells are also subject to negative selection at this later stage of development.^{17, 18} Indeed, it was found that BCR stimulation can even cause deletion of B lymphocytes as late in differentiation as the germinal centre stage.^{121, 122} The mechanisms for deletion of autoreactive B cells were initially probed by crossing the above-mentioned BCR transgenic mice with Fas-deficient mutant lpr mice or transgenic mice overexpressing Bcl-2 in their B lymphocytes. These experiments showed that the 'Bcl-2-regulated' apoptotic pathway is required for the killing of both autoreactive immature B cells during their development in the bone marrow¹²³ as well as the deletion of autoreactive mature B cells in peripheral lymphoid organs.¹²⁴ In contrast Fas-mediated 'death receptor' signalling appeared to be critical for BCR stimulation induced apoptosis of germinal centre B cells.¹²⁵ Since Bim was found to be critical for the deletion of autoreactive T cells, its role in negative selection of B cells was examined by crossing bim^-/- mice with anti-HEL BCR transgenic mice. These studies demonstrated that Bim is also essential for deletion of autoreactive B lymphocytes, both during their differentiation in the bone marrow and as mature B cells in peripheral lymphoid organs.¹²⁶ Loss of Bim afforded B cells less protection against BCR crosslinking-induced apoptosis in culture (a widely used in vitro model for negative selection of B cells) than Bcl-2 overexpression.¹²⁶ This indicated that BH3-only proteins in addition to Bim may be in involved in the deletion of autoreactive B cells. Bik was a possible candidate, since its expression was shown to be upregulated by BCR crosslinking in transformed human B lymphoma derived cell lines and in primary B cells.¹²⁷ However, B cells from bik^-/- mice were normally sensitive to BCR crosslinking-induced apoptosis⁸² and those from bim^-/-bik^-/- double knock-out mice responded like those from bim^-/- animals.⁸⁵ Since Bim/Puma doubly deficient accumulate greater numbers of B cells in their spleens and lymph nodes than bim^-/- mice,⁸⁶ it is possible that Puma contributes to BCR crosslinking-induced killing of B cells.

Collectively, the results described here demonstrate that Bim plays a critical role in the apoptosis of autoreactive T and B cells during their negative selection in either primary lymphoid organs (thymus or bone marrow, respectively) or in the periphery. Because the protection afforded by loss of Bim is incomplete in at least some of these settings, it appears likely that additional proapoptotic factors can also contribute to deletion of autoreactive T and B cells.

Speculating on the mechanisms that lead to Bim activation during deletion of autoreactive T and B cells

Since Bim is essential for the killing of autoreactive T and B lymphocytes during their negative selection, it is important to understand how its expression and function are activated during this process (Figure 4). Experiments with thymocytes isolated from TCR transgenic mice expressing an autoreactive antigen receptor and with thymocytes or B lymphoid cells that had been stimulated in vitro with crosslinking antibodies to the TCR/CD3 complex or the BCR, respectively, indicated that bim is transcriptionally induced during negative selection.^{126, 128, 129, 130, 131} Many regulators of bim transcription have been identified; although not all of these have been examined by analysing mice lacking these regulators, so far none of these have been found to be essential but several have been convincingly excluded as critical activators of bim transcription during T and B cell negative selection. Although calcium signalling and protein kinase c (PKC) are essential for transcriptional induction of bim and thymocyte negative selection, calcineurin and NFAT transcription factors are not required.¹³⁰ Since the calcium-dependent phosphatase calcineurin is essential for positive selection during T cell development, the decision point between positive versus negative selection must be localized somewhere downstream of calcium signalling.¹³⁰ The defects in thymocyte negative selection caused by the specific loss of PTEN in these cells is most likely due to the effect of this phosphatase on regulating TCR/CD3 stimulation activated calcium flux.¹³²

Figure 4.

Several apoptosis regulating pathways activated by TCR/BCR stimulation. TCR/BCR ligation stimulates calcium flux, which then activates bim transcription (via a presently unknown mechanism) and also contributes to the activation of the Nur77/Nurr1/Nor-1 transcription factors. Antigen receptor stimulation is also thought to contribute to the activation of Nur77/Nurr1/Nor-1 through stimulation of ERK5. TCR/BCR ligation is thought to cause post-translational activation of Bim via JNK-mediated phosphorylation. Curiously, phosphorylation of Bim on the same residue (S69) by ERK, which is activated in response to TCR/BCR ligation, inhibits the proapoptotic activity of Bim by causing its dissociation from Mcl-1 and Bcl-x_L and by targeting it for ubiquitination and proteasomal degradation. The two opposing outcomes on Bim activity elicited by JNK- versus ERK-mediated phosphorylation may be explained by differences in recruitment of different adaptor proteins (for example, Pin-1 recruited only by JNK) that may affect Bim conformation and activity.

Full figure and legend (12K)

Nur77 and its close relatives Nurr1 and Nor-1 constitute a family of transcription factors, which are regulated by calcium and are implicated in the death of autoreactive thymocytes during negative selection (reviewed in Sohn et al.¹³³). Initially, two independent screens found that a dominant-negative mutant of Nur77 (later shown to block the activity of all three family members) inhibits TCR/CD3 activation induced apoptosis in T hybridoma cells.^{134, 135} As mentioned above, it is unclear whether this model systems mimics negative selection of autoreactive thymocytes, mature, naive T cells or the AICD of proliferating, mature T cells.¹⁰⁷ It was therefore highly informative when it was reported that transgenic mice expressing this dominant negative mutant of Nur77 in their T lymphoid cells exhibited a pronounced defect in negative selection of autoreactive thymocytes.^{136, 137} Since Nurr1-deficient mice have no defect in deletion of autoreactive thymocytes or mature T cells,¹³⁸ it appears that Nur77, Nurr1 and Nor-1 have overlapping functions in this process and redundancy between Nur77 and Nor-1 has been formally demonstrated in transgenic mice.¹³⁹ The mechanisms by which Nur77, Nurr1 and Nor-1 trigger apoptosis signalling are not clear, but it has been shown that FasL>Fas signalling is not essential.^{139, 140} Since the level of transcriptional activity of Nur77 correlated with the extent of thymocyte apoptosis elicited in transgenic mice¹⁴¹ and since transcriptional induction of bim is critical for apoptosis of autoreactive thymocytes,^{128, 130} it was tempting to speculate that Nur77 and its close relatives transcriptionally activate bim during thymocyte negative selection. However, no conserved Nur77/Nurr1/Nor-1 binding site could be identified within the bim locus (PB, unpublished observations) and transcriptional profiling revealed that bim levels were not abnormally increased in transgenic thymocytes with abnormally increased Nur77 transcriptional activity.¹⁴² Although it has been reported that Nur77 can translocate from the nucleus to the outer mitochondrial membrane where it triggers apoptosis by binding to prosurvival Bcl-2 family members thereby converting them into apoptosis inducers,¹⁴³ this is difficult to reconcile with the observation that the proapoptotic potency of Nur77 correlates with its transcriptional activity.¹⁴¹ Regardless, the available data indicate that Bim activation and Nur77/Nurr1/Nor-1 activation function in parallel pathways in negative selection of autoreactive thymocytes (and possibly also T cells and B lymphoid cells). It will therefore be interesting to generate mice in which both of these pathways are disabled (for example, bim^-/- mice expressing in their T cells a transgene encoding a dominant negative mutant of Nur77) to examine whether these animals have a more severe defect in thymocyte negative selection than mice in which only one of the pathways is blocked. Finally, since apoptosis can be elicited not only by increasing the levels or activity of proapoptotic BH3-only proteins (for example, Bim) but also by a drop in the amount of prosurvival Bcl-2 family members, we wonder whether Nur77/Nu331/Nor-1 might not function as transcriptional repressors of some of these genes.

Foxo3a,⁸⁹ c-Jun⁶³ and CHOP/c-EBP⁶⁷ are other transcriptional regulators that have been shown to control bim transcription either in response to cytokine deprivation (Foxo3a and AP-1) or ER stress (CHOP/c-EBP). The role of Foxo3a and CHOP/c-EBP in thymocyte negative selection has not yet been examined in mice lacking these regulators although such animals are now available. Moreover, although expression of a dominant negative mutant of JNK was reported to inhibit thymocyte negative selection,¹⁴⁴ it is not clear whether this is a consequence of blocking c-Jun activation or due to loss of another activity of JNK.

It has also been found that in response to TCR or BCR stimulation Bim undergoes a post-translational modification in thymocytes or B cells, respectively.^{126, 128, 145} The post-translational modification in Bim appears to be caused by a change in its phosphorylation and these changes were observed in both Bim_EL and Bim_L, the two most abundantly expressed isoforms of Bim.^{146, 147} In thymocytes TCR/CD3 stimulation was also shown to cause increased binding of Bim_EL, Bim_L and possibly also Bim_S (another Bim isoform) to Bcl-x_L, the most highly expressed prosurvival Bcl-2 family in this cell subset.^{128, 145} Although the increased association of Bim with Bcl-x_L is most likely involved in apoptosis initiation, it is presently unclear whether this requires the aforementioned post-translational modification in Bim or is simply a consequence of increased bim transcription. It is, however, noteworthy that changes in Bim phosphorylation have been reported to control the proapoptotic activity of this BH3-only protein.⁹³ ERK-mediated phosphorylation, activated by growth factor receptors or oncogenic kinases (for example, BCR-ABL in CML cells) was shown to target Bim_EL and Bim_L for polyubiquitination and proteasomal degradation, thereby reducing its levels and proapoptotic activity in cells.^{90, 91, 92, 148, 149} Moreover, ERK-mediated phosphorylation is also thought to attenuate the proapoptotic activity of Bim by causing its dissociation from prosurvival Mcl-1 and Bcl-x_L.¹⁵⁰ Curiously, phosphorylation of Bim on the same residue (S69) by a different kinase, JNK, was reported to increase the proapoptotic activity of Bim.^{151, 152} The reported opposing effects of ERK- versus JNK-mediated phosphorylation of the same residue in Bim may be explained by the observation that only JNK recruits the prolyl-isomerase Pin1 and thereby facilitates a conformational change in Bim that is thought to increase its proapoptotic potency.¹⁵³

Changes in the activity of both ERK^{154, 155} as well JNK¹⁴⁴ have been observed during positive and negative selection of thymocytes. Interestingly, ERK, which is thought to inhibit Bim is essential for positive selection,^{154, 155} whereas JNK, implicated in the activation of Bim, is believed to be critical for negative selection¹⁴⁴ during T cell development in the thymus. Thus, the relative strengths of ERK versus JNK activity may determine whether thymocytes undergo positive or negative selection. Biochemical and genetic studies have indicated that the MINK kinase might couple TCR/CD3 stimulation to the activation of JNK and subsequently Bim in negative selection of autoreactive thymocytes.¹⁵⁶ Moreover, it was found that in B cells the protein phosphatase subunit G5PR normally attenuates BCR ligation induced activation of JNK and Bim.¹⁵⁷ Finally, the proapoptotic activity of Bim_EL and Bim_L (but not Bim_S) can also be controlled by regulating its subcellular localization,⁹⁴ but whether this process affects Bim function during positive or negative selection of T or B cells is presently unclear.

Collectively, these investigations reveal that although we know that Bim is essential for TCR- or BCR-stimulation-induced apoptosis in autoreactive T and B lymphoid cells, we still do not understand how it is activated transcriptionally or post-translationally. In fact we do not even know whether the transcriptional induction, the post-translational modification or both are critical for the activation of Bim during deletion of autoreactive lymphocytes and which isoforms of Bim (BimEL, BimL, BimS or perhaps some of the other less abundantly expressed ones¹⁵⁸) is/are essential for cell killing. Further biochemical studies and experiments with genetically modified mice in which subtle mutations have been introduced into the bim gene, which destroy transcription factor binding sites or prevent phosphorylation and/or ubiquitination of Bim, are expected to resolve these questions.

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