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




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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

3. Activation-induced cell death (AICD) of mature T-lymphocytes

AICD can be triggered in activated T-lymphocytes via the CD3/TCR molecular complex and certain other surface molecules involved in T-cell activation such as CD2. Monoclonal anti-CD3/TCR antibodies have been widely used to study AICD in T-cells. It is assumed that this system adequately mimics the activation of T-cells through TCR-mediated recognition of antigen, but the strength (and perhaps the quality) of the signal generated by anti-CD3/TCR mAb versus antigenic peptide presented by appropriate MHC molecules may be quite different. Examples of mAb-and antigen-induced AICD of mature T-cells will be discussed in the following sections.

3.1 Induction via the CD3/T-cell receptor molecular complex

The heterodimeric alphaß (or gammadelta) TCR molecule is closely associated with the CD3 polypeptide complex on the surface of T-lymphocytes. While the clonally distributed alphaß chains of the TCR serve to recognize short antigenic peptides presented by antigen-presenting cells in the context of MHC class I or class II molecules, the signal transduction occurs via the TCR xi chain and the epsilon, gamma and delta subunits of the CD3 molecule (see ref. 60 for review). Anti-CD3 mAb (as well as anti-TCR mAb) have been shown to exert antigen-like effects on T-cells, and thus stimulate Ca2+ influx, cytokine synthesis and interleukin-2 (IL-2)-dependent proliferation (61, 62). As mentioned above, anti-CD3/TCR mAb induce apoptosis in immature thymocytes (55-57, 63). However, T-cells at other stages of differentiation are also susceptible to anti-CD3-induced growth arrest, again associated with the induction of programmed cell death. The spontaneous proliferation of transformed T-cells at various stages of differentiation as well as of T-cell hybridomas was inhibited by anti-CD3 mAb, and cell death associated with DNA fragmentation was observed (64-69). More recently, it was realized that the induction of growth arrest and cell death by anti-CD3/TCR mAb was not restricted to immature thymocytes and transformed T-cells, but could be similarly triggered in mature, non-transformed T-lymphocytes. Early studies indicated that such anti-CD3/TCR mAb inhibited the IL-2-driven in vitro proliferation of activated peripheral murine and human T-lymphocytes (70-73). It was confirmed by several groups that cross-linking of the CD3/TCR complex by mAb triggered AICD in these instances (74-76). The early observation that anti-CD3/TCR mAb can initiate AICD in normal T-lymphocytes raised a great deal of interest in the question of how AICD might contribute to the regulation of cellular immune responses. Moreover, the molecular mechanisms controlling the induction or prevention of AICD have developed into a scientific topic of utmost interest to immunologists. It is obvious that a precise understanding of the underlying rules is a prerequisite for successful attempts to modulate AICD, e.g. by pharmacological intervention (77). As would be expected, resting peripheral T-lymphocytes are generally resistant to AICD triggered by anti-CD3/TCR mAb; instead, these cells respond to CD3/TCR signaling by the induction of a proliferative response. Under in vitro culture conditions, it takes seveal days of stimulation before freshly isolated mature peripheral T-cells acquire sensitivity towards AICD (22, 78-80). In response to anti-TCR mAb, AICD is induced both in T-cells expressing the conventional alphaß TCR as well as in T-cells expressing the gammadelta TCR (81). In contrast to resting mature T-cells of adults, neonatal murine T-cells display enhanced sensitivity to AICD and undergo apoptosis in response to primary TCR-mediated stimulation (82). The induction of AICD sensitivity of mature T-cells is influenced by several parameters. Surprisingly, the T-cell growth factor IL-2 has been implicated in the priming of mature T-lymphocytes for AICD (83, 84). It was suggested that the role of growth factors in the induction of apoptosis is to drive T-cells into the S phase of the cell cycle where they are sensitive to TCR-triggered AICD (85). In addition to cytokines, the ligation of cell surface molecules can modulate AICD. Thus, cross-linking of the CD4 T-cell antigen by immobilized anti-CD4 mAb or by the Human Immunodeficiency Virus (HIV) envelope protein, gp120, has been shown to prime CD4+ T-lymphocytes for subsequent apoptosis triggered via the CD3/TCR complex (86, 87). This mechanism might contribute to the continuous decline of CD4 T-lymphocytes in HIV-infected individuals. Only a minor fraction of circulating CD4+ T-cells is actually infected with HIV; it has been suggested that the disappearance of non-infected CD4 T-cells is due to priming by gp120/anti-gp120 immune complexes and subsequent encounter of antigen through the TCR (88, 89). It appears, however, that the role of CD4 in the regulation of AICD is more complex. While cross-linking of the CD4 molecule primes resting T-cells for apoptosis, we have recently observed opposite effects on activated T-lymphocytes. Thus, ligation of CD4 by anti-CD4 mAb or HIV-1 gp120 drastically inhibited subsequent AICD triggered through CD3/TCR, due to the prevention of upregulation of Fas-L (Oberg et al., submitted for publication). Based on these in vitro observations, we would postulate that gp120 present in the serum of HIV-infected individuals might actually inhibit AICD of circulating activated CD4+ T-cells. Independent of the seemingly different role of the CD4 molecule for the regulation of AICD in resting versus activated T-cells, it is obvious that the sensitivity to AICD is not limited to CD4+ T-cells (90) but is shared by CD8+ T-cells (74). Importantly, functionally distinct subpopulations of CD4+ T-cells are equally susceptible to anti-CD3-stimulated programmed cell death, independent of their capacity to exert anti-CD3 mAb-mediated redirected lysis of Fc-receptor-positive target cells (90). While the molecular mechanisms underlying the differential outcome of CD3 signaling (proliferative response or AICD) are not precisely understood, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAM) that are the principle signaling components revealed a significant role of the TCR xi chain in the signaling of AICD (91). The conserved 18 amino acid ITAM sequence is found three times in the TCR xi chain and once each in the CD3 subunits gamma, delta and epsilon. The induction of efficient AICD required the ITAM motifs of the xi chain, particularly the membrane proximal Z1 ITAM (91). Taken together, there are multiple examples where AICD has been successfully triggered in normal preactivated T-lymphocytes by mAb against CD3 or TCR. In the following sections, we will briefly review the current evidence that AICD can be also initiated by TCR ligands that are naturally encountered by T-lymphocytes.

3.2 AICD triggered by superantigens

In contrast to conventional antigens, superantigens need not be processed by antigen-presenting cells. They activate T-cells by directly bridging the MHC class II molecules on antigen-presenting cells with the variable region of the TCR ß chain (92). Most superantigens identified to date are bacterial proteins such as the staphylococcus aureus enterotoxins (SE) or products of endogenous or exogenous (retro)viruses. The injection of SEB into BALB/c mice leads to a transient increase in SEB-reactive Vß8-expressing T-cells, followed by selective depletion of these cells, due to the induction of programmed cell death (93). Although not all Vß8 cells are deleted following the injection of SEB into mice, the treated animals are tolerant to subsequent challenge with SEB (93, 94). In addition to the deletion of SEB-reactive cells due to apoptosis, other mechanisms such as the induction of anergy in the remaining Vß8 cells contribute to the functional status of tolerance in such mice (95-101). The deletion of Vß8+ CD4+ T-cells in response to SEB occurs in the absence of CD8+ T-cells, in support of the notion that activated CD4+ T-cells undergo suicide rather than being killed by CD8+ T-cells (102). AICD following exposure to SEB can be modulated by additional signals. in vivo, clonal deletion of Vß8+ T-cells is significantly increased when mice are injected with hydrocortisone before the application of SEB (103). Under in vitro conditions, the induction of AICD in sensitized lymph node T-cells by restimulation with SEB is inhibited by mAb directed against lymphocyte function-associated antigen-1 (LFA-1) or intercellular adhesion molecule-2 (ICAM-2), indicating that cell interactions mediated through adhesion molecules can play a role in AICD (104).

Like activated murine T-cells, human CD4 and CD8 T-lymphocytes are susceptible to superantigen-induced AICD (105-107). In contrast to murine T-cells, activated human T-cells express MHC class II antigens. Therefore, AICD of cloned human CD4+ T-cells is triggered by SE superantigens in the absence of additional antigen-presenting cells (105). In the presence of antigen-presenting cells, AICD proceeds to a similar extent, but a proliferative response is simultaneously initiated in the fraction of surviving clone cells (105). Again, the induction of AICD by SE superantigens was inhibited by mAb directed against LFA-1 (CD11a/CD18; ref. 106). While the analysis with human lymphocytes is largely restricted to in vitro experimental systems, clonal deletion of SE-reactive human T-lymphocytes also occurs in vivo in the model of SCID mice reconstituted with human fetal liver and thymus (107).

In addition to bacterial superantigens, endogenous or exogenous retroviral superantigens also delete peripheral T-cells expressing the adequate TCR Vß elements by induction of AICD. Intrathymic deletion of Vß14-bearing thymocytes occurs in mice transgenic for mouse mammary tumor virus (MMTV) (59). Upon transfer of lymphocytes expressing endogenous MMTV, or of lymphocytes maternally infected with MMTV, into recipients lacking this retroviral superantigen, deletion of T-cells expressing the reactive TCR Vß elements occurs in vivo (108, 109). Therefore, superantigens of both bacterial and viral origin are potent inducers of AICD in activated T-lymphocytes from various species.

3.3 AICD triggered by conventional antigens

It has been known for some time that specific antigen can inhibit the proliferative response of murine mature T-lymphocytes (110-112). The recognition of the specific antigenic peptide presented by appropriate MHC class I molecules triggers the self-destruction of cytotoxic T-cells, due to the induction of AICD (113-117). in vivo, such a mechanism seems to contribute to the termination of an ongoing cellular immune response. Systemic infection with lymphocytic choriomeningitis virus (LCMV) is accompanied by cellular expansion of virus-specific CD8+ T-cells. Following virus control, the number of CD8+ T-cells decreases, due to the induction of apoptosis (118). In transgenic mice expressing a transgenic TCR specific for LCMV glycoprotein peptide 33-41, the injection of this peptide induced deletion of transgenic T-cells by AICD (119). Similarly, the exposure to specific antigen induces peripheral CD8+ T-cell deletion also in other TCR transgenic mice with different TCR specificity, e.g. for the male-specific H-Y antigen (120-122) or the nucleoprotein of influenza virus (123). Transgenic mice where the majority of peripheral T-cells express one single TCR molecule with known antigen specificity, provide convenient models to study antigen-triggered AICD in vivo. However, there are clear-cut examples that antigen can trigger AICD also in activated non-transgenic, normal T-lymphocytes. Both CD8 and CD4 T-cells are susceptible to AICD induced through TCR-mediated recognition of allogeneic MHC class I (124-127) or MHC class II molecules (128, 129), respectively. In addition, AICD is triggered in CD4+ T-cells by the specific antigenic peptide, e.g. tetanus toxoid or myelin basic protein, presented by the appropriate MHC class II molecules (130-132). Thus, a large body of evidence supports the notion that AICD can be triggered in activated cells through the TCR-mediated recognition of antigen. The extent of AICD is in part controlled by the antigen dosage. Early studies have demonstrated that the antigen-induced suppression of the in vitro growth of antigen-specific T-lymphocytes requires high concentrations of antigen (110, 111). More recently, the antigen dosage has been correlated with the TCR avidity and the extent of apoptosis. The studies of Alexander-Miller and coworkers revealed that in CD8+ T-lymphocytes, AICD is preferentially triggered by high concentrations of peptide antigen in cytotoxic T-cells displaying high avidity TCR (133). Similarly, the induction of apoptosis in antigen-specific CD4 T-cells occurs preferentially at high antigen concentrations (134, 135). Taken together, it appears that antigen dosage is a major factor controlling the induction of AICD in activated mature T-lymphocytes.

3.4 Role of Fas/Fas-Ligand interaction in antigen-induced AICD

AICD triggered in transformed T-cells and T-cell hybridomas by stimulation of the CD3/TCR complex is mediated via the induction of Fas-L expression and subsequent interaction of Fas-L with the Fas/CD95 antigen (42-44). Similarly, the superantigen-induced AICD of activated normal T-lymphocytes seems to depend on Fas/Fas-L interactions and can be inhibited by reagents that interfere with this interaction, e.g. some anti-Fas mAb or Fas-Fc fusion proteins (42, 136, 137). Several lines of evidence indicate that the Fas/Fas-L system is also operational in vivo in the course of deletion of peripheral T-lymphocytes. Thus, apoptotic T-cells isolated from the peripheral blood or the draining lymph nodes of mice injected with bacterial superantigen express high levels of Fas and Fas-L (138, 139). Moreover, lpr mice carrying a genetic defect in the Fas gene (140) are unable to normally delete peripheral T-cells following the injection of bacterial superantigen (138, 141) or conventional peptide antigen (51), and have a defect in AICD triggered in vitro by superantigen or anti-CD3 mAb (141-143). There is evidence, however, that AICD can also take place in vivo in the absence of a functional Fas molecule. Thus, it was observed by Tucek-Szabo and coworkers that the injection of high concentrations of anti-CD3 mAb triggered apoptosis to a comparable degree in lymph node T-cells from wild-type and lpr mice (144). In addition, the in vivo elimination of male-antigen (H-Y)-specific T-cells occurs normally when lymph node cells from female H-Y TCR transgenic lpr/lpr mice are injected into B6 nu/nu male mice (145). Moreover, the peripheral CD4+ T-cell deletion following the injection of influenza hemagglutinin into TCR transgenic mice was similar in mice of normal or Fas defective lpr background (47). In the latter case, the peripheral deletion of transgenic T-cells following the in vivo administration of the specific antigenic peptide was prevented by a neutralizing anti-TNFalpha mAb (47), suggesting that TNFalpha might be involved as an effector molecule in AICD of mature T-lymphocytes (46, 47). Despite the clear-cut evidence for the important role of the Fas/Fas-L system in AICD triggered via the CD3/TCR complex, it is obvious that, at least under certain conditions, AICD can proceed in the absence of a functional Fas molecule. It is conceivable that TNFalpha, the related TRAIL molecule (18), or other ligand-receptor interactions might transduce death signals in these situations.