Apoptosis shapes T cell development and immune responses. The pro-apoptotic molecules Bax and Bak have overlapping essential functions in T cell development and homeostasis.
Apoptotic cell death is critical in the development of T cells because it "weeds-out" those cells that lack useful antigen receptors or are self-reactive and therefore dangerous. In addition, T cell death is involved in limiting the magnitude and duration of immune responses to infection. Mammals have two distinct apoptosis signaling pathways: one initiated by "death receptors"—members of the tumor necrosis factor receptor family with an intracellular "death domain"—and the other initiated by cell stress that is regulated by pro- and anti-apoptotic members of the Bcl-2 protein family1. Previous in vitro experiments have shown that lymphocytes lacking both Bax and Bak are resistant to apoptotic stimuli that can be countered by Bcl-2—such as cytokine deprivation, glucocorticoids and DNA damage—but remain sensitive to Fas activation2. In this issue of Nature Immunology, Rathmell et al. now demonstrate that the two closely related pro-apoptotic Bcl-2 family members Bax and Bak have overlapping and essential functions in developmentally programmed T cell death in vivo3.
The combined loss of Bax and Bak causes developmental abnormalities in many tissues2. Thus, to exclude indirect effects on lymphocytes, recombination-activating gene 1−deficient (RAG-1-/-) mice were reconstituted with Bax-/-Bak-/- or control (wild-type) hematopoietic stem cells. Using two experimental models—T cell receptor (TCR)-CD3 antibody treatment in vitro and endogenous superantigen-mediated TCR-CD3 stimulation in vivo—the authors demonstrate that Bax and Bak are required for negative selection of autoreactive thymocytes. The role played by Bax and Bak in the apoptosis of thymocytes that failed positive selection was not examined, but these proteins are likely important in this death program because it can be inhibited by their antagonist Bcl-24,
5. Closer inspection of the T cell phenotype in mice reconstituted with Bax-/-Bak-/- stem cells revealed the accumulation of large numbers of TCR+CD4-CD8- thymocytes3. Because strong TCR stimulation, as occurs during negative selection, down-regulates CD4 and CD8 coreceptor expression, these cells probably represent abnormally surviving autoreactive thymocytes, rather than cells that failed to die despite failing positive selection. These TCR+CD4-CD8- T cells do not appear to emigrate into peripheral lymphoid organs, and this form of developmental arrest may therefore serve as a safeguard against autoimmune tissue destruction.
Regulation of immune responses by apoptosis of activated T lymphocytes may occur via two distinct mechanisms. Repeated ligation of TCRs up-regulates Fas ligand (FasL) expression, resulting in FasL-Fas−mediated fratricide (or suicide), and activated T cells undergo apoptosis when essential survival factors (cytokines) become limiting as a consequence of waning inflammation. In response to in vivo stimulation with an exogenous superantigen, T cells lacking Bax and Bak undergo normal activation and proliferation but persist for prolonged periods3. Protracted survival of T cells activated by environmental antigens is probably responsible for the progressive accumulation of quiescent T lymphocytes with a "memory" phenotype in nonimmunized mice reconstituted with a Bax-/-Bak-/- immune system. This accumulation of T cells is most likely due to escape from cytokine withdrawal−induced apoptosis, as Bax-/-Bak-/- lymphocytes are normally sensitive to Fas-induced apoptosis2. It will be interesting (although admittedly difficult) to generate mice lacking Bax, Bak and FasL (or Fas) to understand what happens if both mechanisms for killing activated T cells are absent. Such analyses will help to provide insights into the development of autoimmune diseases or lymphoid malignancies.
The T lymphocyte abnormalities caused by Bax and Bak deficiency are strikingly similar to those found in mice lacking the pro-apoptotic BH3-only Bcl-2 family member Bim6. Bim-deficient mice have defects in negative selection of autoreactive thymocytes7 and deletion of activated T cells at the termination of immune responses8. Collectively, these data indicate that cell killing requires an interplay between the two subclasses of pro-apoptotic Bcl-2 family members: the Bax-Bak−like proteins—which have three Bcl-2 homology regions (BH1, BH2 and BH3) and significant structural similarity compared to their pro-survival relatives—and the distantly related BH3-only proteins—which share only a short BH3 region with the family9. Transfection experiments have shown that Bax and Bak are required for cell death induced by enforced expression of BH3-only proteins10,
11. Three scenarios are therefore possible. First, BH3-only proteins might initiate apoptosis by neutralizing the ability of Bcl-2−like pro-survival proteins to keep Bax and Bak−like proteins and (indirectly) caspase adaptor proteins in check (Fig. 1a). Second, BH3-only proteins might directly activate Bax and Bak−like proteins (Fig. 1b), although binding with physiologically meaningful affinity has not yet been documented. Third, BH3-only proteins and Bax and Bak act in parallel to neutralize the ability of Bcl-2−like proteins to keep caspase adaptors in check (Fig. 1c). There is much controversy concerning caspase activation9: does it occur only after the release of apoptogenic factors (such as cytochrome c and Diablo) from the mitochondria or is apoptosis initiated by caspase activation upstream of the mitochondria, with disruption of mitochondrial membrane triggering an amplification mechanism via the cytochrome c−Apaf-1−caspase-9 apoptosome?
Figure 1. Possible mechanisms for the function of Bax and Bak−like proteins and BH3-only proteins in T cell apoptosis.
The Bcl-2 family−regulated apoptosis signaling pathway can be initiated by strong stimulation of the TCR-CD3 complex, cytokine deprivation and many other physiological or experimentally applied forms of cells stress. Different death signals activate distinct BH3-only proteins to initiate apoptosis, but all require Bax and/or Bak−like proteins (which act downstream of the BH3-only proteins) for cell killing. Three scenarios are therefore possible. (a) BH3-only proteins initiate apoptosis by neutralizing the ability of Bcl-2−like pro-survival proteins to keep Bax and Bak−like proteins and (indirectly) caspase adaptor proteins in check. (b) BH3-only proteins directly activate Bax and Bak−like proteins. (c) BH3-only proteins and Bax and Bak act in parallel to neutralize the ability of Bcl-2−like proteins to keep caspase adaptors in check.
Programmed cell death occurs not only during T cell differentiation but also during the development of cells of the B lymphoid, myeloid and erythroid lineages. So, what are the consequences of the combined loss of Bax and Bak in other hematopoietic cell types? Mice reconstituted with Bax-/-Bak-/- cells develop splenomegaly, but the proportions of CD4+ and CD8+ T cells are normal, indicating that other cell populations must also be elevated in these animals. It would be interesting to investigate the role played by Bax and Bak and BH3-only proteins, such as Bim, in the negative selection of autoreactive B cells, termination of humoral immune responses and programmed death of myeloid cells. This could be done by crossing animals deficient in pro-apoptotic Bcl-2 family members with transgenic mice expressing autoreactive B cell antigen receptors or with mutant mice lacking cytokines such as BAFF (B cell activating factor), G-CSF (granulocyte-macrophage colony-stimulating factor), M-CSF or their receptors, which are essential for the development of B cells or myeloid cells. These investigations might identify the cell death mechanisms that contribute to lymphoid or myeloid immunodeficiencies.
The study by Rathmell et al. has produced a surprising finding3. Despite what might have been expected, lymphocyte accumulation was not unlimited in Bax-/-Bak-/- mice; instead, it was curtailed by reduced progenitor proliferation in the thymus3. What could be the nature of this negative feedback mechanism? There appears to be no general role for Bax and Bak in cell cycling because progenitor proliferation and thymic T lymphocyte output were normal early after lethally irradiated RAG-1-/- mice were reconstituted with Bax-/-Bak-/- stem cells, when peripheral T cell numbers were not yet excessive3. It is noteworthy that reduced progenitor cell proliferation is only found in mutant mice that accumulate resting "memory" T cells (Bax-/-Bak-/--, Bim-/-- and Bcl-2−transgenic), but not in animals that accumulate even larger numbers of "effector" T cells that are cycling (for example, in CTLA-4-/- mice) or post-mitotic (for example, lpr or gld mice). This suggests that the negative feedback results from competition for limiting growth factors that are consumed by progenitors and "memory" T cells but not by "effector" T cells. Alternatively, "memory" T cells, but not "effector" T cells, might produce inhibitory cytokines (such as transforming growth factor-−like factors) that inhibit progenitor cell proliferation. It might be interesting to determine whether negative feedback of thymic T cell output in Bax-/-Bak-/- mice can be overcome by injecting growth factors or neutralizing antibodies to growth-inhibitory cytokines or by introducing null mutations in genes encoding cell cycle inhibitors such as p21, p27 and Rb. Because abnormalities in cell death control synergize with dysregulated cell cycle in tumorigenesis12, these studies may provide interesting insights into the process of neoplastic transformation.
Thus, together Bax and Bak are critical for programmed suicide during T lymphopoiesis and probably also in the development of other cell lineages. Abnormalities in these proteins (or their regulators) likely play a role in cancer and autoimmune disease.
−like factors) that inhibit progenitor cell proliferation. It might be interesting to determine whether negative feedback of thymic T cell output in Bax-/-Bak-/- mice can be overcome by injecting growth factors or neutralizing antibodies to growth-inhibitory cytokines or by introducing null mutations in genes encoding cell cycle inhibitors such as p21, p27 and Rb. Because abnormalities in cell death control synergize with dysregulated cell cycle in tumorigenesis
