A role for E2F1 in the induction of apoptosis during thymic negative selection.

Thymic negative selection is the process in which maturing thymocytes that express T-cell receptors recognizing self are eliminated by apoptotic cell death. The molecular mechanism by which this occurs is poorly understood. Notably, genes involved in cell death, even thymocyte death, such as Fas, Fas-ligand, p53, caspase-1, caspase-3, and caspase-9, and Bcl-2 have been found to not be required for normal thymic negative selection. We have demonstrated previously that E2F1-deficient mice have a defect in thymocyte apoptosis. Here we show that E2F1 is required for normal thymic negative selection. Furthermore, we observed an E2F1-dependent increase of p53 protein levels during the process of thymic clonal deletion, which suggests that E2F1 regulates activation-induced apoptosis of self-reactive thymocytes by a p53-dependent mechanism. In contrast, other apoptotic pathways operating on developing thymocytes, such as glucocorticoid-induced cell death, are not mediated by E2F1. The T lymphocytes that escape thymic negative selection migrate to the peripheral immune system but do not appear to be autoreactive, indicating that there may exist E2F1-independent mechanisms of peripheral tolerance, which protect mice from developing an autoimmune response. We expect that E2F1-deficient mice will provide a useful tool for understanding the molecular mechanism of and the immunological importance of thymic negative selection.     

E2F1-directed activation of Bcl-2 is correlated with lactoferrin-induced apoptosis in Jurkat leukemia T lymphocytes

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.     

Central tolerance to self-antigen expressed by cortical epithelial cells

The exposure of developing thymocytes to high-affinity self-Ag results in T cell tolerance. A predominant mechanism for this is clonal deletion; though receptor editing, anergy induction, and positive selection of regulatory T cells have also been described. It is unclear what signals are involved in determining different tolerance mechanisms. In particular, OT-I mice displayed receptor editing when the high-affinity self-Ag was expressed in cortical epithelial cells (cEC) using the human keratin 14 promoter. To test the hypothesis that receptor editing is a consequence of a unique instruction given by cEC presenting self-Ag, we created mice expressing the 2C and HY ligands under control of the keratin 14 promoter. Alternatively, we studied the fate of developing T cells in OT-I mice where Ag was presented by all thymic APC. Surprisingly, we found that the tolerance mechanism was not influenced by the APC subset involved in presentation. Clonal deletion was observed in 2C and HY models even when Ag was presented only by cEC; and receptor editing was observed in OT-I mice even when Ag was presented by all thymic APC. These results suggest that different TCRs show intrinsic differences in thymic tolerance mechanism.     

Cutting edge: contributions of apoptosis and anergy to systemic T cell tolerance

Multiple pathways can induce and maintain peripheral T cell tolerance. The goal of this study was to define the contributions of apoptosis and anergy to the maintenance of self-tolerance to a systemic Ag. Upon transfer into mice expressing OVA systemically, OVA-specific DO11 CD4+ T cells are activated transiently, cease responding, and die. Bim is the essential apoptosis-inducing trigger and apoptosis proceeds despite increased expression of Bcl-2 and Bcl-x. However, preventing apoptosis by eliminating Bim does not restore proliferation or cytokine production by DO11 cells. While Foxp3 is transiently induced, anergy is not associated with the stable development of regulatory T cells. Thus, apoptosis is dispensable for tolerance to a systemic self-Ag and cell-intrinsic anergy is sufficient to tolerize T cells.     

Cutting edge: Enhanced IL-2 signaling can convert self-specific T cell response from tolerance to autoimmunity

Naive and memory T cells show differences in their response to antigenic stimulation. We examined whether this difference extended to the peripheral deletion of T cells reactive to self-Ag or, alternatively, the induction of autoimmunity. Our results show that although both populations where susceptible to deletion, memory T cells, but not naive T cells, also gave rise to autoimmunity after in vivo presentation of skin-derived self-Ags. The same migratory dendritic cells presented self-Ag to both naive and memory T cell populations, but only the latter had significant levels of the effector molecule granzyme B. Memory T cells also expressed increased levels of the high affinity IL-2 receptor chain after self-Ag recognition. Provision of IL-2 signaling using a stimulatory complex of anti-IL-2 Ab and IL-2 drove the otherwise tolerant naive T cells toward an autoimmune response. Therefore, enhanced IL-2 signaling can act as a major selector between tolerance and autoimmunity.     

Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance.

The mechanisms of allograft tolerance have been classified as deletion, anergy, ignorance and suppression/regulation. Deletion has been implicated in central tolerance, whereas peripheral tolerance has generally been ascribed to clonal anergy and/or active immunoregulatory states. Here, we used two distinct systems to assess the requirement for T-cell deletion in peripheral tolerance induction. In mice transgenic for Bcl-xL, T cells were resistant to passive cell death through cytokine withdrawal, whereas T cells from interleukin-2-deficient mice did not undergo activation-induced cell death. Using either agents that block co-stimulatory pathways or the immunosuppressive drug rapamycin, which we have shown here blocks the proliferative component of interleukin-2 signaling but does not inhibit priming for activation-induced cell death, we found that mice with defective passive or active T-cell apoptotic pathways were resistant to induction of transplantation tolerance. Thus, deletion of activated T cells through activation-induced cell death or growth factor withdrawal seems necessary to achieve peripheral tolerance across major histocompatibility complex barriers.     

Defects in the Bcl-2-regulated apoptotic pathway lead to preferential increase of CD25 low Foxp3+ anergic CD4+ T cells

Defects in the Bcl-2-regulated apoptotic pathway inhibit the deletion of self-reactive T cells. What is unresolved, however, is the nature and fate of such self-reactive T cells escaping deletion. In this study, we report that mice with such defects contained increased numbers of CD25(low)Foxp3(+) cells in the thymus and peripheral lymph tissues. The increased CD25(low)Foxp3(+) population contained a large fraction of cells bearing self-reactive TCRs, evident from a prominent increase in self-superantigen-specific Foxp3(+)Vβ5(+)CD4(+) T cells in BALB/c Bim(-/-) mice compared with control animals. The survival rate of the expanded CD25(low)Foxp3(+) cells was similar to that of CD25(high)Foxp3(+) CD4 T cells in vitro and in vivo. IL-2R stimulation, but not TCR ligation, upregulated CD25 on CD25(low)Foxp3(+)CD4(+) T cells in vitro and in vivo. The expanded CD25(low)Foxp3(+)CD4(+) T cells from Bim(-/-) mice were anergic but also had weaker regulatory function than CD25(high)Foxp3(+) CD4(+) T cells from the same mice. Analysis of Bim(-/-) mice that also lacked Fas showed that the peripheral homeostasis of this expanded population was in part regulated by this death receptor. In conclusion, these results show that self-reactive T cell escapes from thymic deletion in mice defective in the Bcl-2-regulated apoptotic pathway upregulate Foxp3 and become unresponsive upon encountering self-Ag without necessarily gaining potent regulatory function. This clonal functional diversion may help to curtail autoaggressiveness of escaped self-reactive CD4(+) T cells and thereby safeguard immunological tolerance.     

Regulating the immune system: the induction of regulatory T cells in the periphery

The immune system has evolved a variety of mechanisms to achieve and maintain tolerance both centrally and in the periphery. Central tolerance is achieved through negative selection of autoreactive T cells, while peripheral tolerance is achieved primarily via three mechanisms: activation-induced cell death, anergy, and the induction of regulatory T cells. Three forms of these regulatory T cells have been described: those that function via the production of the cytokine IL-10 (T regulatory 1 cells), transforming growth factor beta (Th3 cells), and a population of T cells that suppresses proliferation via a cell-contact-dependent mechanism (CD4+CD25+ TR cells). The present review focuses on the third form of peripheral tolerance - the induction of regulatory T cells. The review will address the induction of the three types of regulatory T cells, the mechanisms by which they suppress T-cell responses in the periphery, the role they play in immune homeostasis, and the potential these cells have as therapeutic agents in immune-mediated disease.     

The central tolerance response to male antigen in normal mice is deletion and not receptor editing

It is widely accepted that developing T cells can undergo clonal deletion in the thymus in response to a high affinity self-Ag. This is largely based on studies of TCR transgenics. However, encounter with high affinity self-Ag can also result in receptor editing in TCR transgenic models. Because all TCR transgenics display ectopic receptor expression, the tolerance mechanism that predominates in normal mice remains an open question. When self-Ag drives receptor editing during T cell development, one expects to find in-frame, self-reactive TCRalpha joins on TCR excision circles (TRECs), which are the products of secondary V/J recombination in the TCRalpha locus. Such joins are not expected if clonal deletion occurs, because the progenitor cell would be eliminated by apoptosis. To test the relative utilization of receptor editing vs clonal deletion, we determined the frequency of in-frame, male-specific joins on TRECs in male and female HYbeta transgenic mice. In comparison with female HYbeta transgenic mice, our analysis showed a lower frequency of TRECs with male-reactive V17J57 joins in male mice. Thus, it would appear that receptor editing is not a predominant tolerance mechanism for this self-Ag.     

Regulation of apoptosis in mature alphabeta+CD4-CD8- antigen-specific suppressor T cell clones

The regulation of apoptosis in mature CD4+ or CD8+ alphabeta+ T cells has been well studied. How the survival and death is regulated in peripheral CD4-CD8- (double negative, DN) alphabeta+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of alphabeta+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-alpha/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.     

The role of the common cytokine receptor gamma-chain in regulating IL-2-dependent, activation-induced CD8+ T cell death.

IL-2-dependent, activation-induced T cell death (AICD) plays an important role in peripheral tolerance. Using CD8+ TCR-transgenic lymphocytes (2C), we investigated the mechanisms by which IL-2 prepares CD8+ T cells for AICD. We found that both Fas and TNFR death pathways mediate the AICD of 2C cells. Neutralizing IL-2, IL-2R alpha, or IL-2R beta inhibited AICD. In contrast, blocking the common cytokine receptor gamma-chain (gamma c) prevented Bcl-2 induction and augmented AICD. IL-2 up-regulated Fas ligand (FasL) and down-regulated gamma c expression on activated 2C cells in vitro and in vivo. Adult IL-2 gene-knockout mice displayed exaggerated gamma c expression on their CD8+, but not on their CD4+, T cells. IL-4, IL-7, and IL-15, which do not promote AICD, did not influence FasL or gamma c expression. These data provide evidence that IL-2 prepares CD8+ T lymphocytes for AICD by at least two mechanisms: 1) by up-regulating a pro-apoptotic molecule, FasL, and 2) by down-regulating a survival molecule, gamma c.     

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.     

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.     

Immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4(+) T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8(+) T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8(+) tolerance in SM-Ova mice. We show herein that Ova-specific CD8(+) T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8(+) T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8(+) T cells.     

Survivin reduces activation-induced T cell death in G1 phase

A process termed activation-induced cell death (AICD) is responsible for peripheral T cell tolerance after negative selection of self-reactive T cells, and deletion of hyperactivated T cells following the immune response. Cells in G1 phase of the cell cycle are most susceptible to AICD. We have investigated the relationship between the induction of AICD by phorbol 12-myristate 13-acetate plus ionomycin during the cell cycle and the expression of survivin, an inhibitor of the apoptosis protein (LAP) family. AICD was highly induced in cells of the human T cell line Jurkat E6.1 arrested in G1 phase, whereas survivin was hardly expressed in G1 and instead it was highly expressed in G2/M. Moreover, transient over-expression of survivin in G1 partially blocked the induction of AICD. These results suggest that survivin inhibits the induction of AICD, especially in G1 phase.     

Resistance of Crohn's disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.

Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-alpha and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-alpha failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.