Bim alleles more than restored normal number, suggesting that other prosurvival proteins, such as Mcl-131 or Bcl-xL,21 are involved as guardians of Bim toxicity. Because maintenance of homeostasis appears to require a delicate balance between BH3-only proteins and their prosurvival counterparts, it would be interesting to know whether loss of Bim alone is sufficient to compensate for Mcl-1 deficiency in hematopoietic progenitor cells. Puma and Noxa, also able to bind Mcl-1, are also important regulators of lymphoid and myeloid cell apopto-

sis.28,29

2.2. Sizing of the T-cell population

2.2.1. Establishing central tolerance

T-cell progenitors emigrate to the thymus from the bone marrow. Their maturation in the thymus consists of several steps, the purpose of which is the production of a functional TCR. Early T-cell progenitors are CD4–8– and can be subdivided into four populations according to expression of CD25 (interleukin [IL] 2 receptor α) and CD44. Pro-T1 cells (CD25–44+ ) are the most immature and develop successively into pro-T2 (CD25+44+ ), proT3 (CD25+44–), and pro-T4 (CD25–44–) cells.32 Signals through the IL-7 receptor (IL-7R)/γc, c-Kit (SCF receptor), and Flk2 are essential for cell proliferation and survival during the pro-T1 to pro-T3 stages of development.33 Rearrangement of TCRβ genes occurs during the transition from the pro-T2 to the pro-T3 stage.34 Successful production of a pre-TCR35 results in progression to the pro-T4 and CD4+8+ pre-T stages.33 Thymocytes that survive the pre-TCR checkpoint proliferate and differentiate to yield the CD4+ 8+ population. The next maturation step is the rearrangement of the TCRα gene, and thymocytes expressing a complete TCRα/β-CD3 complex become subject to immunological selection on the basis of their TCRα/β specificity.36

All in all, approximately 90% of the pro-thymocytes that enter the thymus will fail the selection process and undergo programmed cell death.37 Immature T cells that do not produce a TCR and those whose TCR is not functional die “by neglect”. T cells that produce a functional TCR are then sorted according to the affinity of this receptor for a particular class II–self-peptide complex, in the process of positive and negative selection. T cells that recognize such complexes with moderate affinity are positively selected and differentiate into CD4+ 8– or CD4–8+ SP thymocytes before escaping to the periphery. The purpose of negative selection is to eliminate T cells whose TCRs have a high affinity for self-antigens and whose transformation into mature T cells and

subsequent escape to the periphery might cause autoimmunity. Such cells, however, sometimes reach the periphery, where they can be induced to die by mechanisms ensuring peripheral tolerance (see Section 2.2.2).

Early T-cell progenitors require the continuous presence of IL-7 for survival (pro-T1 to pro-T3 stages). Mice that lack IL-7, IL-7 receptor alpha (IL-7Rα), or the common gamma chain (γc), which is a component of the receptors for IL-2, -4, -7, -9, -15, and -21, have dramatically reduced numbers of T and B cells.38,39 IL-7–induced survival involves proteins of the Bcl-2 family, because the over-expression of Bcl-2 in IL-7Rα-deficient mice restores T-lymphocyte numbers similar to those of wildtype mice.40,41 In vivo, the protection of early thymocyte progenitors by IL-7 seems to be ensured by Mcl-1 rather than Bcl-2 or Bcl-xL. Indeed, genetic models have demonstrated that neither Bcl-2 nor Bcl-xL are required for early lymphoid development,16,18,20,42 whereas mice deficient for Mcl-1 present an increase in apoptosis before antigen-receptor rearrangement strikingly similar to the phenotype of IL-7 or IL-7Rα knockout mice.31 The nature of the proapoptotic proteins involved in early lymphoid development is still unclear. Experiments in mice lacking Bad, Bid, Hrk, Blk, or Noxa have shown that these BH3-only proteins are not crucial for early lymphoid development.11 Bim is probably involved in the killing of immature thymocytes in the absence of IL-7 signaling because removal of Bim rescued near-normal numbers of mature T cells in IL-7Rα-/- mice.43 Because the rescue was not as complete as the rescue afforded by Bcl-2 over-expression, it is highly probable that other BH3-only proteins may be involved in the killing of T cells in the absence of IL-7 signal. Puma is a good candidate for such an activity, because loss of Puma in cultured myeloid cells rendered the cells resistant to growth factor withdrawal,29 and loss of both Bim and Puma additively increased resistance to cytokine withdrawal,44 suggesting that they act in concert in this killing activity.

Pro-thymocytes must rearrange their TCR genes to produce a functional MHC-restricted TCR. The first step in the production of a TCR is the rearrangement of the genes coding for the TCRβ chain, which in combination with the invariant chain pTα and the CD3 protein, forms the pre-TCR. Successful assembly of a pre-TCR complex constitutes the pre-TCR checkpoint and is absolutely required for further differentiation. Indeed, mice lacking either of the recombination-activating genes, Rag- 1 or Rag-2, are defective in antigen receptor gene rearrangement and the development of their thymocytes is blocked at the pro-T3 stage.45,46,47,48 Lack of CD3 or pTα also blocks T-cell development at the pro-T3 stage.49,50 Over-expression of the antiapoptotic protein Bcl-2

rescues pro–T cells from a lack of IL-7R signaling,40,41 but it does not promote survival of pre–TCR-deficient pro-T3 cells in scid51 or Rag-1−/− mice.40 By contrast, expression of a dominant-negative form of the adaptor protein FADD, FADD-DN, restored significant CD4+ 8+ pre–T-cell numbers in Rag-1−/– animals, demonstrating a role for the death receptor pathway in the apoptosis occurring in the absence of pre-TCR signaling.52 Fas has no role in the process, however, because T-cell development in Fas-deficient Faslpr/lpr/Rag-1−/– mice was still arrested at the CD25+44– pro-T3 stage.52 Assembly of a complete pre-TCR signals survival and proliferation of the pro-T4 population, as well as the next step in the production of the functional TCR, rearrangement of the TCRα gene. Bcl-xL has been proposed to protect DN and DP pre–T cells during TCRα gene rearrangement.53 More recently, the prosurvival Bcl-2 family member A1 was identified as a direct transcriptional target of both preTCR and nuclear factor kappa B (NF-κB).54 A1 could be more important than Bcl-2 or Bcl-xL in this step, because mRNA knockdown of A1 impaired the survival of pre– T-cell lines despite the expression of the other prosurvival proteins. Further investigations will be necessary to understand whether the presence of any death receptor is required at this stage and how A1 can antagonize this apoptotic pathway.

Rearrangement of the TCRα gene results either in a nonfunctional α chain or in a chain that can be part of a TCRα/β/CD3 antigen receptor complex exposed on the surface of the pre–T cells. These immature thymocytes travel from the thymic medulla to the cortex and back and on their way meet thymic stromal antigenpresenting cells (APCs), which present on their surface self-peptides associated with MHC molecules. The fate of developing thymocytes is determined by the affinity of their newly assembled TCR for self-peptide–MHC ligands. Progenitors whose TCR has little or no affinity fail to receive a survival signal and undergo “death by neglect,” a process in which Bcl-2 family proteins play an active role. Indeed, over-expression of Bcl-2 inhibits this type of death,55 but over-expression of FADD-DN does not.56 Even if Bcl-2 over-expression impairs cell death of thymocytes bearing TCRs unable to bind MHC molecules,51,55 their differentiation is arrested at the CD4+ CD8+ stage, indicating that TCR-ligation activates signals required not only for cell survival, but also for differentiation.51,57 Bim is a likely candidate to mediate death by neglect because Bim deficiency confers resistance to cytokine withdrawal-induced cell death to DP thymocytes.27 This, however, has not been formally demonstrated in a mouse model, and other BH3-only proteins may be involved in this process.

A TCR with intermediate affinity for self-peptide– MHC ligands mediates positive selection, and cells with such a TCR receive a survival signal, upregulate Bcl-2,58 and differentiate into SP mature T cells that emigrate to the periphery. By contrast, immature thymocytes that harbor a high-affinity TCR are normally deleted by apoptosis in a process referred to as negative selection. This process seems to happen independently of the death receptor pathway because mice lacking Fas59 or caspase- 85 or mice over-expressing FADD-DN56 show no impairment in the death of autoreactive T cells. Negative selection has been reported to be partially impaired in TRAIL-deficient mice and in the presence of blocking soluble TRAIL receptor (DR5),60,61 but this result has been contradicted.62,63

In the Bcl-2 family, Bim was shown to have a prominent role in the process of negative selection. Bimdeficient mice have a twoto four-fold increase in their numbers of single positive thymocytes and peripheral mature T cells.27 Bim-/- DP thymocytes were almost completely resistant to anti-CD3 antibody stimulation, whereas their wild-type counterparts were extremely sensitive to this treatment.64 In the transgenic TCR HY model,65 loss of Bim prevented the death of DP thymocytes in male mice in which they are normally deleted. Loss of Bim also protected thymocytes against apoptosis induced by superantigens.64 TCR ligation induces accumulation of Bim and its association with Bcl-xL.64 It has been reported that Bim accumulation is mediated by protein kinase C and Ca2+-dependent transcriptional activation.66 In Rag-1–deficient mice reconstituted with Bax/Bak doubly deficient hematopoietic cells, thymocyte development is disrupted, with an alteration in thymocytes subsets similar to that observed in vav-Bcl-2 transgenic and Bim−/− mice. Characteristically, these mice show an increase in the DN and SP numbers and a decrease in the DP population.13,27,67,68 Bax−/−/Bak−/− thymocytes were resistant to both death by neglect and antigen receptor-induced apoptosis.68 This is not really surprising because loss of both Bax and Bax prevents the mitochondrial damage that triggers the caspase activation regulated by the Bcl-2 family. Interestingly, thymocytes from mice in which the immune system has been reconstituted with Apaf-1 and caspase- 9–deficient fetal liver cells underwent normal negative selection.69,70 Similarly, Rag-deficient mice reconstituted with fetal liver cells deficient for both caspases-3 and -7 developed thymi with normal distribution of the SP and DP populations, although DKO thymocytes were highly resistant to apoptosis mediated by the mitochondrial pathway, at least at a short 24-hour time point. This data shows that Apaf-1 and caspases -9, -3, and -7 are not

absolutely required for the death of negatively selected thymocytes. This does not mean, however, that they are not normally used in this death process in wild-type mice. Indeed, cells devoid of Apaf-1 or caspase-9, -3, or -7 still have all the machinery necessary to damage mitochondria and release the contents of the mitochondrial inter-membrane space. It is possible that this mitochondrial damage is sufficient to cause the death of these cells even if the downstream apoptotic machinery is not functional. And it is possible that this death might not be apoptotic in these mutant cells, but rather necrotic.71 Further studies with these mutant mice will be necessary to test these hypotheses.

Another surprising observation is that, although loss of Bim induces a defect in thymic negative selection, over-expression of Bcl-2 does not protect autoreactive cells as well as loss of Bim does.64,72 This suggests that Bim has a function that is not inhibited by Bcl-2, but the nature of this function has not been discovered yet.

Nur77, Nor-1, and Nurr1 are a family of nuclear orphan receptors implicated in the death of autoreactive cells. Expression of both Nur-77 and Nor-1 is rapidly induced in thymocytes upon TCR stimulation, and their upregulation triggers a massive cell death in mature T cells,73,74,75 a process that seems dependent on their transcriptional activity.76 Deficiency in either Nur-77 or Nor-1 does not lead to any impairment of negative selection,77,78 but their inhibition by a dominant-negative mutant inhibited negative selection.74,79,80 Although both Bim and Nur-77 are upregulated upon TCR engagement, there is no evidence that Bim may be a direct target of Nur-77 transcriptional activity.81 The function of Bim is affected in many ways by phosphorylation, and mitogen-activated protein (MAP) kinases ERK1/2, p38, and c-Jun N-terminal kinase (JNK) have been shown to phosphorylate Bim on selected serine and threonine residues.82,83,84 Interestingly, these kinases were also shown to have a role in negative and/or positive selection.66,85,86,87 A mutation of BimEL (T112A) affecting its phosphorylation by JNK was recently shown to impair negative selection in vivo in the HY-TCR transgenic animal model.88 The authors concluded that the rescue of autoreactive thymocytes was due to the fact that the T112A mutation of Bim significantly decreases its binding to Bcl-2. A recently described and nonconventional MAP kinase, MEK5-ERK5, was also recently shown to regulate the level of Nur77 family members by transcriptional activation89 and participate in the apoptosis of developing thymocytes. No direct connection between Bim expression and ERK5 activity was uncovered in this study. Studies on the role of Bcl-2 in autoreactive T cells may still hold

some surprises, because recent reports have indicated that Nur77 can translocate to mitochondria and interact with Bcl-2 family members (Bcl-2, Bcl-B, or A1).90,91 Upon TCR activation in DP lymphocytes, Nur77 and Nor- 1 were shown to translocate to mitochondria and render Bcl-2 proapoptotic by inducing a conformational change that exposes its BH3 domain.92 It is thus possible that Bim and Nur77 may belong to two distinct pathways that converge at the mitochondria.

2.2.2. Peripheral tolerance

Mature CD4+ and CD8+ T cells that emigrate to the periphery should not be able to recognize self-antigens. Some do, however, because not all self-antigens are expressed in the thymus at a sufficient level to induce central tolerance. Mechanisms of peripheral tolerance have evolved to prevent autoimmunity that could result from the presence of autoreactive T cells in the periphery. This has been studied in a model of antigen crosspresentation in which naive T cells from TCR transgenic mice (OVA-specific CD8+ T cells from OTI mice, which do not express the antigen) are transferred into mice that express the antigen (OVA) transgenically. In a first report, deletion of Fas appeared to protect autoreactive CD8+ T cells from peripheral deletion in this system,93 but a subsequent study by the same group refuted this finding and found that transgenic expression of Bcl-2 as well as genetic deletion of Bim could prevent the death of these cells.93

These results were corroborated recently in a similar study using the clone 4 TCR transgenic cell line, which is specific for an H-2 Kd-restricted peptide epitope of the influenza hemagglutinin (HA).95 The adoptive transfer of clone 4 CD8+ T cell into InsHA mice, which express the viral HA on their pancreatic islet β cells, results in T-cell activation by cross-presenting APCs in the pancreatic lymph nodes.96 After adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. This deletion, however, could be inhibited by over-expression of Bcl-2 or targeted deletion of Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis, whereas Bim−/− clone 4 cells were not autoaggressive. This data showed that initiation of clone 4 T-cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl- 2–sensitive and at least partially Bim-dependent mechanism. Additional experiments are required to determine whether another BH3-only protein such as Puma could

be a partner to Bim in the death of autoreactive CD8+ T cells in the periphery, as suggested.44

By contrast, the mitochondrial pathway may have no role in the death of autoreactive CD4+ T cells. Bcl- 2 over-expression failed in inhibiting autoreactive CD4+ T-cell deletion in transgenic mice after adoptive transfer, whereas deletion is significantly impaired in Faslpr/lpr or gld (FasL mutant) mice.97 However, the nature of the pathway involved in this process may depend on the quantity, the nature, and the expression pattern of the self-antigen.

Whereas Bcl-xL prevails over Bcl-2 for the survival of immature DP thymocytes, Bcl-2 expression is required for the survival of mature naive T cells in the periphery. The increase of Bcl-2 in resting cells could result from IL-4, -6, and -7 stimulation,98 as well as IL-2 stimulation after T-cell activation.99,100 If Bcl-2 expression is required for survival, it is not essential for cytokinemediated proliferation, because Bcl-2 transgenic T cells are able to survive, but do not proliferate, for instance, in the absence of Il-2.101 The role of Bcl-2 in the survival of T cells in blood and peripheral lymphoid organs has been highlighted by the fact that Bcl-2–deficient mice present a deficit in mature T cells.16,18,42 This deficiency can be compensated by the concomitant loss of Bim, showing that Bim plays an essential role in the apoptosis of mature T cells.27,30

In response to infection or immunization, T cells that express antigen-specific TCRs become activated and proliferate, and some differentiate into effector cells.102 Activated T cells then produce cytokines that help coordinate the immune response aimed at eliminating the pathogen. Clearance of the antigen is accompanied by the shutdown of T-cell immune responses and involves apoptosis of a large fraction of antigen-activated T cells. This prevents accumulation of no longer needed and potentially dangerous effector cells, thereby maintaining homeostasis and precluding immunopathology.

The relative contributions of the two distinct apoptotic pathways in the termination of T-cell immune responses has been a matter of controversy for a long time. FasL and Fas have been implicated because activation-induced cell death, an in vitro model in which mitogen-activated T-cell blasts are killed by TCR restimulation, which causes FasL upregulation, is inhibited by FasL or Fas inactivation.103,104,105 Moreover, clearance of staphylococcus enterotoxin B (SEB)–activated TCR Vβ8+ T cells was reported to depend partially on Fas.106,107 The mitochondrial pathway has been implicated in immune response shutdown, because Bcl-2 over-expression,108 loss of Bim,43,109 or Bax and Bak deficiency68 inhibited death of T cells stimulated in vitro or

in vivo by a single dose of SEB or in vivo after infection with human herpes simplex virus (HSV-1). Involvement of the death receptor pathway in clonal contraction was supported by the accumulation, in mice as in humans defective for either Fas (Faslpr/lpr) or its ligand (FasL),110,111 of excess mature T and B cells as well as “unusual” αβTCR+ CD4− CD8−B220+ T cells, resulting in progressive lymphadenopathy and splenomegaly. Involvement of the mitochondrial pathway in the same process was supported by the observations that overexpression of Bcl-2 or loss of Bim also led to the accumulation of mature T and B cells in the periphery.13,27 However, the “unusual” αβTCR+ CD4−CD8− B220+ T cells were not observed in Bcl-2 transgenic and Bim-deficient animals, strongly suggesting that these cells are normally eliminated by a system relying on Fas/FasL interaction, that is, the death receptor pathway.

Part of the controversy was solved recently, as a result of the observation that mice lacking both Fas and Bim (Faslpr/lprBim−/−) accumulate extraordinary amounts of mature T, B, and the trademark “unusual” αβTCR+ CD4−CD8− B220+ T cells, resulting in spectacular lymphadenopathy, splenomegaly, and an autoimmune pathology that develops very early.112,113,114 Clonal contraction was found to depend only on Bim in a model of acute viral infection (HSV-1), but to be the result of a cooperation between Fas and Bim in a model of chronic viral infection (MHV-68).112 It thus appears that the relative contribution of the two main apoptotic pathways may be determined by the nature of the immune response (acute vs. chronic). In acute immune responses, the drop in cytokine amounts after clearance of the pathogen or injected immunogen triggers apoptosis.102,109,115 A single administration of SEB, which is known to be eliminated quickly from the body,116 would therefore mimic an acute infection. In contrast, TCR re-stimulation of activated T cells in vitro or repeated administration of SEB to mice is more likely to imitate the repeated TCR activation that is thought to occur during chronic immune responses, such as persistent infections or stimulation with self-antigens.102,115 Cooperation of both pathways is particularly obvious when considering the αβTCR+CD4− CD8− B220+ T cells. These cells indeed exist because of the loss of a functional death receptor pathway, because they do not accumulate in Bim-/- or Bcl-2–transgenic mice. But their accumulation in Faslpr/lprBim-/- mice exceeding by far the amounts observed in mice lacking Fas-only clearly shows the importance of Bim to limit this cell population in “normal” circumstances. The catastrophic consequences of the failure to properly eliminate activated B and T cells at the end of an immune response are