[Frontiers in Bioscience 2, d61-77, February 15, 1997]

	[Frontiers in Bioscience 2, d61-77, February 15, 1997] Reprints PubMed CAVEAT LECTOR
	ANTIGEN-INDUCED DEATH OF T-LYMPHOCYTES Dieter Kabelitz & Ottmar Janssen Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany Received 1/24/97; Accepted 1/31/97 On-line 2/15/97 2. INTRODUCTION 2.1 Features of apoptosis There are essentially two ways how cells can die, necrosis and apoptosis. Necrosis is characterized by irreversible swelling and lysis of the cell, which usually occurs in response to a damage of rather unphysiological nature. Due to the disruption of the cell membrane, the cellular content is released, resulting in an inflammatory reaction in the neighbouring tissue. In contrast, apoptosis proceeds via an ordered sequence of events that culminates in the suicide of the cell. Hence, apoptosis is also referred to as programmed cell death. Apoptosis is associated with characteristic morphological features which have been studied in great detail (1; see ref. 2 for review). Briefly, cells undergoing apoptosis separate from neighbouring cells and display a characteristic condensation of the nucleus, as well as blebbing of the plasma membranes and formation of the apoptotic bodies containing cell organelles and chromatin fragments. The content of the apoptotic bodies is not released into the environment. Therefore, in contrast to necrosis, apoptosis does not provoke an inflammatory reaction in the surrounding tissue. in vivo, apoptotic cells are rapidly removed by macrophages, possibly through vitronectin receptor-mediated phagocytosis (3). Thus, it is difficult to demonstrate the presence of apoptosis in vivo, due to the rapid clearance of such cells. Due to the action of endonucleases during the apoptotic process, the cellular DNA is cut into oligonucleosomal fragments of approximately 200 bp in length, and multiples thereof. Upon electrophoresis in an agarose gel and staining with ethidium bromide, the fragmented DNA gives rise to a characteristic "apoptotic DNA ladder" (4). Probably all cells (except erythrocytes and thrombocytes) have the intrinsic capacity to undergo apoptosis. While it has been obvious for a long time that apoptosis plays a central role in fetal development and organogenesis (2), its significance in the immune system has been recognized only in recent years (5). For some time, the term apoptosis has been used for the morphological description of a specific way of cell death (1). Only recently has it been possible to decipher major pathways of the molecular mechanisms of apoptosis. In this regard, Caenorhabditis elegans has been widely studied. However, major breakthroughs in the identification of molecules involved in apoptosis have been made recently in immunological systems. In the following sections, we will review the recent evidence that programmed cell death plays an important role in the regulation of T-cell mediated immune responses. While programmed cell death of T-cells is frequently associated with the above described typical features of apoptosis including low molecular weight DNA fragmentation, there are well-documented instances where DNA fragmentation is dispensable (6, 7). Moreover, the central role of the apoptosis-inducing interaction between the Fas molecule and its corresponding ligand, Fas-Ligand (see below), is beyond any doubt. Nevertheless, there are examples where signal-induced lymphocyte death occurs in the apparent absence of Fas/Fas-Ligand interaction (8-11). We have coined the term activation-induced cell death (AICD) to describe the signal-induced programmed cell death of T-lymphocytes (12). Apoptosis may also be triggered by the lack of stimuli required for survival or lack of growth factors ("death by neglect"), as is the case with hematopoietic precursor cells in the absence of appropriate colony-stimulating factors (13). In this article, we will concentrate on the induction of AICD via the antigen-specific T-cell receptor (TCR) molecule. 2.2 Role of death molecules Apoptosis can be initiated by triggering of cell surface receptors such as Fas (APO-1) with monoclonal antibodies (mAb) or by interaction with its specific ligand (Fas-L, CD154). Fas is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor gene family. The mature protein has 319 amino acids (aa) and a calculated molecular weight of 36 kD (14). The Fas molecule consists of an extracellular domain of 157 aa, a hydrophobic transmembrane domain containing 17 aa, and a cytoplasmic tail of 145 aa at its carboxyl terminal (14). The Fas apoptosis signaling domain is contained within a stretch of 68 amino acids in the intracellular part. Fas shares this characteristic "death domain" with the TNF receptor type I (15). Conversely, the natural ligand of the Fas molecule, Fas-L, belongs to the TNF gene family (16, 17). Figure 1: Schematic diagram of the structure of Fas and Fas-L. The extracellular domain of Fas contains three cysteine-rich subdomains (shaded areas). The intracellular death domain is shown as bold line. The black symbols indicate N-glycosylation sites. The Fas-Ligand is a type II membrane protein. The shaded area is the extracelluar region which shares significant homolgy with TNFalpha. For comparison, the schematic structure of TNF receptor type I and TNFalpha are also shown. Adapted and modified from ref. 17. Figure 1 shows a diagram indicating the schematic structure of the Fas and Fas-L molecules. In addition to Fas-L and TNFalpha, other recently identified members of the TNF family such as TRAIL (TNF-related apoptosis-inducing ligand) can trigger apoptosis in susceptible cells (18). The induction of apoptosis via the Fas molecule requires the close proximity of the cytoplasmic tails of several Fas molecules, as it is induced by crosslinking anti-Fas antibodies or by Fas-L in its trimeric form. The Fas apoptosis pathway does not depend on extracellular Ca²⁺ (19, 20) and does not require macromolecular synthesis (14, 21-23). Interestingly, cell death displaying all characteristic morphological features of apoptosis can be triggered via Fas in enucleated cells, indicating that apoptosis can proceed in the absence of a nucleus (6). The signaling through Fas involves the activation of an acidic sphingomyelinase (24) giving rise to the generation of ceramide, a complex lipid which induces rapid apoptosis when added in a membrane-permeable form to cell cultures (25). Ceramide mediates Fas-induced apoptosis through the activation of the Ras signaling pathway (26). In turn, the activation of Ras is associated with the rapid and transient synthesis of reactive oxygen intermediates (ROI), pointing to an important role of ROI in Fas-mediated apoptosis (27, 28). In addition, other components of the Fas-dependent death pathway (FADD, RIP) have been identified by searching for proteins that interact with the intracellular death domain of Fas (29, 30). Other potential regulators of Fas-dependent apoptosis include the FAP-1 phosphatase and the serine/threonine kinase FAST (31, 32). Although it has been reported that the activation of a protein tyrosine kinase is involved in Fas signaling (33), we have established experimental systems where Fas-mediated apoptosis clearly proceeds in the absence of detectable tyrosine phosphorylation (34; and Oberg et al., submitted for publication). Following coimmunoprecipitation with anti-Fas mAb, four cell death-associated proteins (CAP; cytotoxicity-dependent APO-1-associated proteins) have been identified, which form a death-inducing signaling complex (DISC) (35). One of the CAP encodes a 55 kDa protein termed FLICE which shares homology to Interleukin-1ß-converting enzyme (ICE) -like cysteine proteases (36); this molecule has been independently cloned by Boldin et al. and has been termed MACH by this group (37). The cleavage of cellular substrates including poly (ADP-ribose) polymerase (PARP) and by ICE/CED-3 like proteases is a central event in the execution of apoptosis (38-40). Accordingly, apoptosis can be inhibited by peptides that act as competitive substrates for ICE-like proteases (38, 40, 41). The Fas/Fas-L system plays a major role in the induction and regulation of programmed cell death in T-lymphocytes. A commonly considered scenario proceeds as follows (see Figure 2). Upon stimulation of activated T-cells via the CD3/TCR complex, Fas-L mRNA and cell surface expression are rapidly upregulated. Fas-L binds to the surface-expressed Fas molecule on the same or on neighbouring cells and triggers apoptosis (42-44). Since Fas-L is rapidly cleared from the cell surface through the action of matrix metalloproteases (45), both "suicide" and "fratricide" can be initiated (Figure 2). Although the role of the Fas/Fas-L system in the induction of T-cell apoptosis is unambiguous, it is evident that other molecules are perhaps equally important. Under certain circumstances, TNFalpha can act as a mediator of apoptosis in mature T-lymphocytes (46, 47). The potential role of the recently described TRAIL molecule (18) in T-cell apoptosis has not yet been precisely defined. It is unlikely that all types ofT-cell apoptosis involve the Fas/Fas-L system (8, 9, 11). Fas-independent apoptosis triggered through the cell surface CD2 molecule has been described (10). Moreover, immobilized mAb against the CTLA4 costimulatory molecule has been reported to induce apoptosis in antigen-specific human T-lymphocytes (48). It is unknown whether the Fas system is involved in this case. The possibility that AICD can take place in T-lymphocytes without the involvement of Fas/Fas-L, will be further discussed (see below). 2.3 Apoptosis in intrathymic T-cell development Immature precursor cells lacking CD3/TCR enter the thymus. During intrathymic development, TCR gene rearrangement takes place, and cell surface-expressed heterodimeric TCRs are randomly generated. The vast majority (> 95 %) of developing thymocytes do not leave the thymus, and die by apoptosis. Only a minority of thymocytes is rescued from programmed cell death, due to positive selection of cells expressing TCR molecules of appropriate affinity/avidity for self MHC molecules to allow for the differentiation into mature self MHC-restricted T-cells (see ref. 49 for review). The significance of the Fas/Fas-L system in the intrathymic apoptosis is not entirely clear. Although the Fas antigen is expressed on thymocytes (50), there is only low intrathymic expression of the corresponding Fas-L (16). More importantly, negative intrathymic T-lymphocyte selection (by apoptosis) seems to develop to a normal extent in Fas-defective lpr or Fas-L-defective gld mice (51, 52). However, there is evidence that the Fas/Fas-L interaction is involved in the modulation of thymocyte apoptosis (53). In addition, there are synergistic interactions between the Fas molecule and the CD3/TCR complex in the induction of programmed cell death in thymocytes (54). Immature thymocytes readily undergo apoptosis following treatment with glucocorticoids or anti-CD3 mAb, both in vivo and in vitro (4, 55-57). While these stimuli induce apoptosis irrespective of the antigen specificity of the TCR, selective depletion (by apoptosis) of thymocytes expressing specifically reactive TCR can be triggered by exogenous superantigens such as staphylococcus aureus enterotoxins (58) or by endogenous superantigens encoded by e.g. mouse mammary tumor virus (59). Taken together, there is no doubt that apoptosis is an important physiological process that contributes to the ordered development of the immune system and the developmental shaping of the TCR repertoire. More recent evidence indicates, however, that apoptosis also regulates immune responses of peripheral T-lymphocytes in the mature immune system. Figure 2: Role of the Fas/Fas-Ligand system in the induction of programmed cell death in T-lymphocytes and their targets. Activated T-cells stimulated via the CD3/T-cell receptor complex through the recognition of antigen presented by MHC molecules rapidly express Fas-L molecules. In different scenarios, Fas-L molecules cleaved from the T-cell surface by matrix metalloproteases, or Fas-L molecules still anchored in the cell membrane, can induce apoptosis either in the same cell (autocrine suicide), or in a neighbouring T-cell (fratricide), or in a Fas⁺ target cell (paracrine lysis).

[Frontiers in Bioscience 2, d61-77, February 15, 1997]
Reprints
PubMed
CAVEAT LECTOR

ANTIGEN-INDUCED DEATH OF T-LYMPHOCYTES

Dieter Kabelitz & Ottmar Janssen

Department of Immunology, Paul-Ehrlich-Institute, Langen, Germany

Received 1/24/97; Accepted 1/31/97 On-line 2/15/97

2. INTRODUCTION

2.1 Features of apoptosis

There are essentially two ways how cells can die, necrosis and apoptosis. Necrosis is characterized by irreversible swelling and lysis of the cell, which usually occurs in response to a damage of rather unphysiological nature. Due to the disruption of the cell membrane, the cellular content is released, resulting in an inflammatory reaction in the neighbouring tissue. In contrast, apoptosis proceeds via an ordered sequence of events that culminates in the suicide of the cell. Hence, apoptosis is also referred to as programmed cell death. Apoptosis is associated with characteristic morphological features which have been studied in great detail (1; see ref. 2 for review). Briefly, cells undergoing apoptosis separate from neighbouring cells and display a characteristic condensation of the nucleus, as well as blebbing of the plasma membranes and formation of the apoptotic bodies containing cell organelles and chromatin fragments. The content of the apoptotic bodies is not released into the environment. Therefore, in contrast to necrosis, apoptosis does not provoke an inflammatory reaction in the surrounding tissue. in vivo, apoptotic cells are rapidly removed by macrophages, possibly through vitronectin receptor-mediated phagocytosis (3). Thus, it is difficult to demonstrate the presence of apoptosis in vivo, due to the rapid clearance of such cells. Due to the action of endonucleases during the apoptotic process, the cellular DNA is cut into oligonucleosomal fragments of approximately 200 bp in length, and multiples thereof. Upon electrophoresis in an agarose gel and staining with ethidium bromide, the fragmented DNA gives rise to a characteristic "apoptotic DNA ladder" (4). Probably all cells (except erythrocytes and thrombocytes) have the intrinsic capacity to undergo apoptosis. While it has been obvious for a long time that apoptosis plays a central role in fetal development and organogenesis (2), its significance in the immune system has been recognized only in recent years (5). For some time, the term apoptosis has been used for the morphological description of a specific way of cell death (1). Only recently has it been possible to decipher major pathways of the molecular mechanisms of apoptosis. In this regard, Caenorhabditis elegans has been widely studied. However, major breakthroughs in the identification of molecules involved in apoptosis have been made recently in immunological systems. In the following sections, we will review the recent evidence that programmed cell death plays an important role in the regulation of T-cell mediated immune responses. While programmed cell death of T-cells is frequently associated with the above described typical features of apoptosis including low molecular weight DNA fragmentation, there are well-documented instances where DNA fragmentation is dispensable (6, 7). Moreover, the central role of the apoptosis-inducing interaction between the Fas molecule and its corresponding ligand, Fas-Ligand (see below), is beyond any doubt. Nevertheless, there are examples where signal-induced lymphocyte death occurs in the apparent absence of Fas/Fas-Ligand interaction (8-11). We have coined the term activation-induced cell death (AICD) to describe the signal-induced programmed cell death of T-lymphocytes (12). Apoptosis may also be triggered by the lack of stimuli required for survival or lack of growth factors ("death by neglect"), as is the case with hematopoietic precursor cells in the absence of appropriate colony-stimulating factors (13). In this article, we will concentrate on the induction of AICD via the antigen-specific T-cell receptor (TCR) molecule.

2.2 Role of death molecules

Apoptosis can be initiated by triggering of cell surface receptors such as Fas (APO-1) with monoclonal antibodies (mAb) or by interaction with its specific ligand (Fas-L, CD154). Fas is a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor gene family. The mature protein has 319 amino acids (aa) and a calculated molecular weight of 36 kD (14). The Fas molecule consists of an extracellular domain of 157 aa, a hydrophobic transmembrane domain containing 17 aa, and a cytoplasmic tail of 145 aa at its carboxyl terminal (14). The Fas apoptosis signaling domain is contained within a stretch of 68 amino acids in the intracellular part. Fas shares this characteristic "death domain" with the TNF receptor type I (15). Conversely, the natural ligand of the Fas molecule, Fas-L, belongs to the TNF gene family (16, 17).

Figure 1: Schematic diagram of the structure of Fas and Fas-L. The extracellular domain of Fas contains three cysteine-rich subdomains (shaded areas). The intracellular death domain is shown as bold line. The black symbols indicate N-glycosylation sites. The Fas-Ligand is a type II membrane protein. The shaded area is the extracelluar region which shares significant homolgy with TNFalpha. For comparison, the schematic structure of TNF receptor type I and TNFalpha are also shown. Adapted and modified from ref. 17.

Figure 1 shows a diagram indicating the schematic structure of the Fas and Fas-L molecules. In addition to Fas-L and TNFalpha, other recently identified members of the TNF family such as TRAIL (TNF-related apoptosis-inducing ligand) can trigger apoptosis in susceptible cells (18). The induction of apoptosis via the Fas molecule requires the close proximity of the cytoplasmic tails of several Fas molecules, as it is induced by crosslinking anti-Fas antibodies or by Fas-L in its trimeric form. The Fas apoptosis pathway does not depend on extracellular Ca²⁺ (19, 20) and does not require macromolecular synthesis (14, 21-23). Interestingly, cell death displaying all characteristic morphological features of apoptosis can be triggered via Fas in enucleated cells, indicating that apoptosis can proceed in the absence of a nucleus (6). The signaling through Fas involves the activation of an acidic sphingomyelinase (24) giving rise to the generation of ceramide, a complex lipid which induces rapid apoptosis when added in a membrane-permeable form to cell cultures (25). Ceramide mediates Fas-induced apoptosis through the activation of the Ras signaling pathway (26). In turn, the activation of Ras is associated with the rapid and transient synthesis of reactive oxygen intermediates (ROI), pointing to an important role of ROI in Fas-mediated apoptosis (27, 28). In addition, other components of the Fas-dependent death pathway (FADD, RIP) have been identified by searching for proteins that interact with the intracellular death domain of Fas (29, 30). Other potential regulators of Fas-dependent apoptosis include the FAP-1 phosphatase and the serine/threonine kinase FAST (31, 32). Although it has been reported that the activation of a protein tyrosine kinase is involved in Fas signaling (33), we have established experimental systems where Fas-mediated apoptosis clearly proceeds in the absence of detectable tyrosine phosphorylation (34; and Oberg et al., submitted for publication). Following coimmunoprecipitation with anti-Fas mAb, four cell death-associated proteins (CAP; cytotoxicity-dependent APO-1-associated proteins) have been identified, which form a death-inducing signaling complex (DISC) (35). One of the CAP encodes a 55 kDa protein termed FLICE which shares homology to Interleukin-1ß-converting enzyme (ICE) -like cysteine proteases (36); this molecule has been independently cloned by Boldin et al. and has been termed MACH by this group (37). The cleavage of cellular substrates including poly (ADP-ribose) polymerase (PARP) and by ICE/CED-3 like proteases is a central event in the execution of apoptosis (38-40). Accordingly, apoptosis can be inhibited by peptides that act as competitive substrates for ICE-like proteases (38, 40, 41).

The Fas/Fas-L system plays a major role in the induction and regulation of programmed cell death in T-lymphocytes. A commonly considered scenario proceeds as follows (see Figure 2). Upon stimulation of activated T-cells via the CD3/TCR complex, Fas-L mRNA and cell surface expression are rapidly upregulated. Fas-L binds to the surface-expressed Fas molecule on the same or on neighbouring cells and triggers apoptosis (42-44). Since Fas-L is rapidly cleared from the cell surface through the action of matrix metalloproteases (45), both "suicide" and "fratricide" can be initiated (Figure 2). Although the role of the Fas/Fas-L system in the induction of T-cell apoptosis is unambiguous, it is evident that other molecules are perhaps equally important. Under certain circumstances, TNFalpha can act as a mediator of apoptosis in mature T-lymphocytes (46, 47). The potential role of the recently described TRAIL molecule (18) in T-cell apoptosis has not yet been precisely defined. It is unlikely that all types ofT-cell apoptosis involve the Fas/Fas-L system (8, 9, 11). Fas-independent apoptosis triggered through the cell surface CD2 molecule has been described (10). Moreover, immobilized mAb against the CTLA4 costimulatory molecule has been reported to induce apoptosis in antigen-specific human T-lymphocytes (48). It is unknown whether the Fas system is involved in this case. The possibility that AICD can take place in T-lymphocytes without the involvement of Fas/Fas-L, will be further discussed (see below).

2.3 Apoptosis in intrathymic T-cell development

Immature precursor cells lacking CD3/TCR enter the thymus. During intrathymic development, TCR gene rearrangement takes place, and cell surface-expressed heterodimeric TCRs are randomly generated. The vast majority (> 95 %) of developing thymocytes do not leave the thymus, and die by apoptosis. Only a minority of thymocytes is rescued from programmed cell death, due to positive selection of cells expressing TCR molecules of appropriate affinity/avidity for self MHC molecules to allow for the differentiation into mature self MHC-restricted T-cells (see ref. 49 for review). The significance of the Fas/Fas-L system in the intrathymic apoptosis is not entirely clear. Although the Fas antigen is expressed on thymocytes (50), there is only low intrathymic expression of the corresponding Fas-L (16). More importantly, negative intrathymic T-lymphocyte selection (by apoptosis) seems to develop to a normal extent in Fas-defective lpr or Fas-L-defective gld mice (51, 52). However, there is evidence that the Fas/Fas-L interaction is involved in the modulation of thymocyte apoptosis (53). In addition, there are synergistic interactions between the Fas molecule and the CD3/TCR complex in the induction of programmed cell death in thymocytes (54). Immature thymocytes readily undergo apoptosis following treatment with glucocorticoids or anti-CD3 mAb, both in vivo and in vitro (4, 55-57). While these stimuli induce apoptosis irrespective of the antigen specificity of the TCR, selective depletion (by apoptosis) of thymocytes expressing specifically reactive TCR can be triggered by exogenous superantigens such as staphylococcus aureus enterotoxins (58) or by endogenous superantigens encoded by e.g. mouse mammary tumor virus (59). Taken together, there is no doubt that apoptosis is an important physiological process that contributes to the ordered development of the immune system and the developmental shaping of the TCR repertoire. More recent evidence indicates, however, that apoptosis also regulates immune responses of peripheral T-lymphocytes in the mature immune system.

Figure 2: Role of the Fas/Fas-Ligand system in the induction of programmed cell death in T-lymphocytes and their targets. Activated T-cells stimulated via the CD3/T-cell receptor complex through the recognition of antigen presented by MHC molecules rapidly express Fas-L molecules. In different scenarios, Fas-L molecules cleaved from the T-cell surface by matrix metalloproteases, or Fas-L molecules still anchored in the cell membrane, can induce apoptosis either in the same cell (autocrine suicide), or in a neighbouring T-cell (fratricide), or in a Fas⁺ target cell (paracrine lysis).