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Immune Cell Biology Department, Naval Medical Research Institute, Bethesda, Maryland 20889-5607; and Department of Immunology, Scripps Research Institute, La Jolla, California 92037
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Acentral feature of the immune system is its ability to respond to foreign antigens while tolerating self-antigens. Burnet's model of self-nonself discrimination proposed that receptor engagements that were stimulatory for mature immune cells would induce immature immune cells to die, resulting in the removal of autoreactive receptor specificities from the developing repertoire (1–2). The removal of autoreactivities from the functional lymphocyte repertoire is known as negative selection. For T cells, negative selection can occur at different developmental stages in the thymus by a variety of mechanisms, including (a) clonal deletion, (b) developmental or clonal arrest, or (c) clonal inactivation or anergy (3–4). CD4+CD8+ double positive (DP)1 thymocytes in particular are susceptible to clonal deletion induced in response to TCR signals (5–7). Curiously, while clonal deletion of DP thymocytes must involve TCR engagement, TCR engagement by itself is insufficient to stimulate DP thymocyte apoptosis in vitro (8–12). Rather, DP thymocyte death has been found to require signals in addition to TCR, such as those provided by CD28 (9–11). This requirement for second signals parallels the requirement for second signals in TCR-mediated activation and proliferation of mature single positive (SP) T cells (13). Because CD28 is not a unique costimulatory molecule for mature T cells (14–22), it is likely that CD28 is not the only molecule capable of transducing second signals for TCR dependent DP thymocyte apoptosis (6, 14, 23–24). Indeed, other molecules have been implicated in clonal deletion of DP thymocytes, such as CD30 (21) and fas (25). The role of fas in negative selection is particularly controversial (26–29).
TCR-dependent apoptosis of mature T lymphocytes was not foreseen in Burnet's model of self-nonself discrimination but is now a recognized consequence of mature T cell activation (reviewed by 30–31). In mature T cells, TCR signals do not directly induce an apoptotic program, but rather act indirectly by upregulating surface expression of members of the TNF family, specifically fas ligand (fasL) and TNF (32–34). These proteins bind to fas or TNF receptor (TNFR) which mediate apoptosis through death domains expressed in their cytosolic regions (35). This mechanism of apoptosis necessarily results in death of both TCR-stimulated T cells and neighboring (or bystander) cells expressing fas or TNFR. In contrast, clonal deletion in the thymus is presumed not to involve bystander death since it should be limited to cells directly stimulated by TCR interactions.
In this study we have focused on identifying mechanisms of TCR-dependent clonal deletion of rigorously purified DP thymocytes to avoid confounding signals that can result from contact with other thymic elements. We identify two distinct mechanisms for generating second signals leading to TCR-induced DP thymocyte apoptosis: (a) a CD28-dependent mechanism that requires simultaneous engagement of TCR and CD28 surface molecules, and (b) a CD28-independent mechanism initiated by TCR signals but mediated subsequently by APC signals. While we found that fas signals can induce DP thymocyte death, they were not involved in either TCR-specific apoptotic mechanism. We propose that the two mechanisms of DP apoptosis revealed in this report represent two distinct TCR-specific clonal deletion strategies in the thymus: (a) a CD28-dependent mechanism specific for antigens on B7+ cells, i.e., APC and medullary epithelium, and (b) a CD28-independent mechanism specific for antigens on B7– cells in the thymic cortex.
Cell Preparation.
Antibodies.
Reagents.
Culture and Stimulation Conditions.
Staining and Flow Cytometry.
Measures of Cell Death.
Materials and Methods
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Abstract
Materials and Methods
Results
Discussion
References
Mice.
Young adult female C57BL/6 (B6) mice, gld/gld mice (B6-SMN C3H fasL), and lpr/lpr mice (B6 mrl lpr/lpr) were obtained from The Jackson Laboratory (Bar Harbor, ME) and young adult female B6 Ly5.2 mice were obtained from the Frederick Cancer Research Center (Frederick, MD). CD28-deficient mice (CD28 KO) (14) were bred to the C57BL/6 background and maintained at the Bethesda Naval Medical Research Institute. Mice transgenic for the human bcl-2 gene (driven by the lck proximal promoter [36] were generously provided by Dr. Stanley Korsmeyer and bred in our facility at the NIH. Mice deficient in either the p55 TNFR (p55) (37) or both p55 and p75 TNFR (38 and 38a) were obtained from Immunex (Seattle, WA).
CD4+CD8+ thymocytes were purified from 4–6-wk-old female mice either by panning on anti-CD8 (83-12-5) coated plates (39) or by Percoll density fractionation (40). Similar experimental results were obtained with cells isolated by either procedure. In each case >95% of isolated cells were CD4+CD8+. APC were prepared by treating splenocytes with anti-CD4 (RL-172), anti-CD8 (3-155), anti-Thy-1 (30H12), and rabbit complement as previously described (3). Viable cells were isolated by centrifugation over Lymphocyte-M (Cedarlane Laboratory, Ltd., Ontario, CA). These cell preparations were free of T cells as determined by CD3 staining and FACS® analysis. Lymph node T cells were prepared from B6 and gld/gld mice by treating single-cell suspensions of cells isolated from a pool of popliteal, inguinal, axillary, brachial, and submandibular lymph nodes with a combination of anti-class II (M5114), anti-NK1.1 (PK136), anti-HSA (J11d) culture supernatants, and rabbit complement.
Anti-CD28 (37.51 [41]) and anti–TCR-β (H57-597 [42]) were affinity purified in our laboratory from hybridoma culture supernatant on columns of protein G– and protein A–Sepharose (Pharmacia LKB Nuclear, Gaithersburg, MD), respectively. Anti-CD2 (RM2-5), anti-CD43 (S7), anti-CD27 (LG.3A10), anti-41BB (1AH2), neutralizing anti–TNF-
(G281-2626), anti-CD80 (1G10), anti-FcR (2.4G2), anti-fas (Jo2), and FITC-conjugated anti-HSA, FITC anti-Ly5.2 (CD45.1) and FITC anti-Ly5.1 (CD45.2) were purchased from Pharmingen laboratories. FITC-conjugated anti-CD5 (Ly-1) was purchased from Beckton Dickinson (San Jose, CA). Anti-CD81 was the product of hybridoma 2F7 (43). Anti-CD9 (9D3 [20]), anti-CD24 (20C9 [44]), anti-CD30 (mCD30.1 [45]), anti-CD30L (M15 [46]), and anti-B7-2 (GL1 [47]), were generously provided by Drs. Hiromi Fujiwara (Osaka University, Osaka, Japan), Charles Janeway (VCI, NIH, Bethesda, MD), Eckhardt Podack (University of Miami School of Medicine, FL), Phil Morrissey (Immunex, Seattle, WA), and Richard Hodes (NIH, Bethesda, MD), respectively.
Murine recombinant TNF- (R&D Labs., Minneapolis, MN) was used at a final concentration of 100 ng/ml. CD30-Ig was generously provided by Dr. Eckhardt Podack. Cyclosporine A was purchased from Calbiochem-Novabiochem (La Jolla, CA) and cycloheximide, wortmannin, and GF109203X were purchased from Sigma Chem. Co. (St. Louis, MO). The caspase inhibitor Cbz-Val-Ala-Asp-(Ome)-fluoromethyl ketone (ZVAD-FMK) was purchased from Enzyme Systems Products (Dublin, CA).
Purified cell populations were cultured for 16–20 h in a 7% CO2 humidified incubator in RPMI 1640 supplemented with 5 x 10–5 M 2-ME and 10% FCS at 37°C. Single-cell suspensions of DP thymocytes were plated in 24-well tissue culture plates (Corning Glass, Corning, NY) at a cell density of 2 x 106/ml in a total of 500 µl per well. When mixed culture experiments were performed, DP cells from CD28-deficient mice were incubated with DP cells from Ly5.2 mice at a 1:1 ratio and with LN T from Ly5.2 mice at a 1:2 or 1:3 ratio. APC were mixed with DP thymocytes at a 2:1 or 3:1 ratio and 3 x 106 total cells were plated per well. For stimulation, wells in a 24-well plate were coated with antibody combinations by incubating them overnight at 4°C with 350 µl of a 10 µg/ml (most antibodies) or 50 µg/ml (anti-CD28) of each affinity-purified antibody specified in PBS.
Cell death was assayed as previously described (9, 48). In brief, 5 x 105 cultured cells were incubated with saturating concentrations of the FITC conjugated antibodies specified in staining medium (HBSS, 0.5% BSA, 0.5% NaN3) for 30 min at 4°C, washed, and then incubated with a 1 µg/ml ethidium bromide (EtBr; Sigma) for another 30 minutes at 4°C. Cells were washed again and analyzed using CellQuest software on a FACScan®. In mixed cell culture experiments, DP thymocytes were identified by expression of Ly5.1, Ly5.2, HSA, and Thy-1, as indicated.
Specific cell death (% cell death) was determined by EtBr staining and was calculated as a normalized value as follows: (%EtBr+ (stimulated) – %EtBr+ (unstimulated control))/(100 – %EtBr+ control). Background EtBr staining of cultured DP thymocytes ranged between 18 and 30%. To compare thymocyte apoptosis in response to TCR-CD28 signals among experimental groups with different internal controls (i.e., different mouse strains, different solvents, etc.), individual responses were normalized and expressed as a killing index. The killing index was calculated as follows: (% DP cell death induced by TCR-CD28 under experimental conditions)/(% DP cell death induced by TCR-CD28 under control conditions). Killing index = 1.0 means that the indicated condition did not affect TCR-CD28– induced apoptosis; killing index >1 means that the indicated condition inhibited TCR-CD28–mediated death. All experiments displayed were performed three or more times with similar results.
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Abstract
Materials and Methods
Results
Discussion
References
Surface Molecules Expressed by CD4+CD8+ Thymocytes That Kill Cells by Either TCR-dependent or TCR-independent Mechanisms.
To identify the molecular requirements for TCR-dependent DP thymocyte apoptosis, we stimulated purified DP thymocytes in vitro and assessed them for EtBr uptake, which identifies cells undergoing apoptosis (9, 48). We initially examined surface molecules for their ability to induce apoptosis of DP thymocytes in the absence of TCR engagement (Fig. 1 a). In fact, we identified two stimuli that induced DP thymocyte apoptosis in a TCR-independent manner, namely (a) fas engagement by immobilized platebound antibody and (b) TNF engagement of TNFR. Antibody engagement of surface fas molecules induced apoptosis of most DP thymocytes, consistent with others' observations and the high expression of fas on DP thymocytes (49); and soluble TNF- engagement of surface receptors induced apoptosis of 20% of DP thymocytes, which may represent a distinct TNF- responsive subpopulation (50) (Fig. 1 a). Our present finding that immobilized anti-fas antibody efficiently induces DP thymocyte death differs from that of Ogasawara et al. (51) who found that metabolic inhibitors were necessary to enhance DP thymocyte death induced by soluble anti-fas antibodies. We think that our different observations may be due to differences in the efficiency by which fas is crosslinked by soluble versus immobilized antibody.
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We next assessed molecules that could induce DP thymocyte apoptosis in the presence of TCR engagement (Fig. 1 b). As expected (9), TCR engagement alone failed to induce significant DP thymocyte apoptosis (Fig. 1 b). We examined three specific sets of surface proteins for their ability to cooperate with the TCR to kill DP thymocytes (Fig. 1 b and data not shown), namely (a) molecules thought to be costimulatory (CD9 [20]), CD28 [reviewed in 53], CD43 [19], and CD81 [54], (b) coactivating molecules that enhance TCR signaling (CD2, CD4, CD5, CD8, CD24, CD69, and LFA-1), and (c) selected TNFR family members (CD27, CD30, 41BB), which may also exhibit costimulatory activity (15, 17–18, 21–22, 52). Importantly, antibody engagement of each of these molecules cooperated with TCR signaling to upregulate CD5 expression on DP thymocytes (data not shown). However, only CD28 cooperated with TCR to induce DP thymocyte apoptosis (Fig. 1 b).
Thus, there exist both TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis: TCR-independent mechanisms can be mediated by either fas or TNFR engagement, whereas the TCR-dependent mechanisms can be mediated by coengagement of TCR and CD28.
TCR/CD28–induced Apoptosis Is Independent of fas and TNFR Interactions, and Is Strictly Confined to DP Thymocytes That Have Received both TCR and CD28 Signals.
The ability of fas and TNF- to mediate DP thymocyte apoptosis raised the possibility that both mechanisms of DP thymocyte apoptosis (TCR-independent and TCR-dependent) may ultimately result from engagement of fas or TNFR. To address this possibility, we examined the ability of TCR-CD28 coengagement to kill purified DP thymocytes from mice deficient in (a) fas (lpr, reviewed in reference 55), (b) fas ligand (gld), (c) the p55 murine TNFR, or (d) both the p55 and p75 murine TNFRs (38, 38a) (Fig. 2). None of the mutations significantly affected the ability of TCR-CD28 coengagement to induce apoptosis of DP thymocytes, indicating that neither fas nor the TNF receptors p55 or p75 were required (Fig. 2). In addition, neutralizing anti–TNF- antibodies had no effect on TCR-CD28– induced apoptosis of wild-type DP thymocytes (data not shown). We also found that TCR-CD28–induced death was not blocked by CD30 Ig, a fusion protein that blocks CD30-CD30L interactions (data not shown). We conclude that TCR-CD28 coengagement induces DP thymocyte apoptosis by a mechanism that is independent of fas/fasL, TNF-TNFR, and CD30-CD30L interactions.
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(PKC (61, 62), (b) the protein synthesis inhibitor, cycloheximide, and (c) the caspase (ICE-family protease) inhibitor, ZVAD-FMK (63, 64) (Fig. 4). Consistent with the participation of caspases, TCR-CD28 coengagement failed to induce significant apoptosis in DP thymocytes from bcl-2 transgenic mice which constitutively overexpress the anti-apoptotic bcl-2 protein (Fig. 4).
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, require protein synthesis, and result in the activation of intracellular caspases that are regulated by bcl-2.
Identification of a TCR-dependent but CD28-independent Mechanism of DP Thymocyte Apoptosis.
Our studies using an array of antibodies to known molecules on the surface of DP thymocytes did not identify any proteins other than CD28 that could cooperate with TCR to induce DP thymocyte apoptosis. To determine if any such molecule existed, we asked whether APC expressed ligands, known or unknown, for surface molecules that would cooperate with TCR to induce DP thymocyte apoptosis. In this coculture experiment, we stimulated DP thymocytes from wild-type and CD28 KO mice with platebound anti-TCR in the presence of APC. We confined our assessment of EtBr staining to DP thymocytes by excluding APCs from the analysis using an allelic marker, Ly5. DP thymocytes from wild-type mice died in response to TCR stimulation in the presence of APC. More importantly, DP thymocytes from CD28 KO mice also died in response to TCR stimulation in the presence of APC, indicating that APC possess ligands that engage molecules other than CD28 that can induce death of TCR-stimulated thymocytes. It is important to note that this CD28-independent mechanism of DP thymocyte death strictly requires engagement of TCR on DP thymocytes, as APCs did not kill unstimulated CD28 KO DP thymocytes (Fig. 6). From these data we can conclude that APC express or secrete ligands for surface molecules on DP thymocytes that are capable of cooperating with the TCR to induce TCR-dependent but CD28-independent apoptosis.
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Finally, these data suggest that TCR prestimulation (in the absence of CD28 coengagement) "prepares" DP thymocytes to undergo apoptosis upon subsequent interaction with APC, possibly by inducing expression of surface molecules containing death domains that bind APC-derived ligands. Consistent with such a possibility, cycloheximide reduced by over 70% the number of TCR prestimulated DP thymocytes that died upon subsequent exposure to APC (data not shown), indicating that new protein synthesis is required to make TCR prestimulated DP thymocytes vulnerable to death. TNFR family members are attractive candidate molecules whose synthesis could be induced on DP thymocytes by TCR prestimulation. Indeed, CD30 has been reported to play a role in thymocyte negative selection (21). Nevertheless, CD30 does not appear to be involved as neither soluble CD30-Ig fusion protein (45) (Fig. 8) nor antibodies specific for the CD30 ligand (data not shown) blocked the ability of APCs to mediate CD28-independent apoptosis of prestimulated DP thymocytes.
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Discussion |
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The CD28-dependent mechanism of apoptosis is a consequence of simultaneous TCR and CD28 coengagement on DP thymocytes and results exclusively in death of DP thymocytes that have simulataneously received both TCR and CD28 signals. Such apoptotic signals do not require the activity of PI 3-kinase, a signaling molecule associated with CD28 (69), but are resistant to the effects of cyclosporine A, a hallmark of CD28 involvement in mature T cells (70). Apoptosis induced by TCR-CD28 coengagement requires PKC and employs a common effector pathway involving caspases that is inhibitible by bcl-2. The present results are consistent with either of two possibilities: simultaneous TCR-CD28 signals induce DP thymocyte death either by directly initiating a novel intracellular apoptotic program or by inducing surface expression of an uncharacterized death receptor (e.g., a TNFR family member) that is engaged by surface ligands. In either case, the CD28-dependent mechanism of death requires that both TCR and CD28 signals be delivered simultaneously and requires that the death ligand, if it exists, be expressed on DP thymocytes for these were the only cells present in our cultures. We favor the possibility that TCR-CD28 directly signals an apoptotic program because we have found that TCR-CD28–induced apoptosis of DP thymocytes occurs efficiently at very low cell densities at which cell-cell interactions are unlikely to occur (data not shown). However, we cannot exclude the possibility that TCR/CD28 stimulation results in lethal death receptor/ligand interactions occurring in cis on the surface of individual DP thymocytes (Fig. 3 d).
The mechanism responsible for CD28-independent DP apoptosis is distinct from that of TCR-CD28–induced apoptosis. CD28-independent DP thymocyte apoptosis does not require costimulatory signals. Rather, TCR engagement alone on DP thymocytes appears sufficient to induce surface expression of a molecule which triggers death upon subsequent engagement by ligands expressed by APCs. This proposed surface molecule has features of death domain containing receptors, such as some members of the TNFR family. Thus, TCR signals may stimulate expression of a TNFR family member which is neither fas, nor CD30, nor 41BB, but which generates apoptotic signals when engaged by an appropriate ligand expressed or secreted by APC.
It is conceivable that all programmed cell death is initiated by signals transduced by death domains on specialized surface receptors. From this perspective, surface death receptors would be responsible for death of DP thymocytes stimulated either by TCR-CD28 costimulatory signals or by CD28-independent TCR signals alone. However, it is important to appreciate that the identity and specificity of the death receptors induced by TCR-CD28 signals must be different than those induced by TCR signals alone. This conclusion is based on our data showing that TCR-CD28– stimulated DP thymocytes kill themselves or each other, but do not kill TCR-stimulated DP bystander cells (Fig. 3 c). Rather, TCR-stimulated DP thymocytes are induced to die upon subsequent interaction with APCs (Fig. 7 b). Therefore, from the perspective that all cell death is mediated by death domain containing receptors, the ligand for TCR-CD28–induced death receptors must be expressed on DP thymocytes themselves, whereas the ligand for TCR induced death receptors is not expressed on DP thymocytes but is expressed on APCs.
The importance of APCs in inducing clonal deletion in the thymus has long been appreciated (8, 71–73). However, the mechanism by which APCs induce DP thymocyte apoptosis has not been fully understood. Here we show for the first time that APCs can induce death of TCR stimulated DP thymocytes in two distinct ways via two distinct sets of ligands. Our observation that TCR signals can induce DP thymocyte apoptosis by two different mechanisms is relevant to an understanding of clonal deletion of DP thymocytes during normal in vivo development. In Fig. 9 we illustrate a model of in vivo clonal deletion that is based on our present data. We propose that the two mechanisms of TCR-mediated DP apoptosis identified in this report represent two distinct strategies to rid the developing DP thymocyte repertoire of autoreactive specificities during thymocyte development (Fig. 9). Only DP thymocytes whose TCRs bind MHC–peptide complexes on cells expressing the CD28 ligands B7-1 or B7-2 will be killed as a consequence of TCR and CD28 coengagement. Because B7-1 and B7-2 are not expressed in the thymic cortex but are only expressed on APCs and medullary epithelial cells, this form of clonal deletion would be confined to DP thymocytes autoreactive to self-antigens encountered either at the corticomedullary junction, where most APCs are located, or on medullary epithelium. In contrast, high-affinity interactions of TCR on DP thymocytes with self-antigens in the cortex would induce expression of a surface molecule that "tags" DP thymocytes for future disposal (i.e., a "death tag"). Such DP thymocytes would not die in response to TCR engagement per se, but would travel through the cortex and enter the corticomedullary junction where they would encounter a "screen" of APCs constitutively expressing or secreting a ligand that bind the putative death tag and induce apoptosis.
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Second, the model proposes that CD28-dependent and CD28-independent mechanisms of TCR-mediated DP apoptosis eliminate clones bearing TCR specific for distinct sets of antigens: the CD28-dependent mechanism disposes of TCR reactivities to APC and medullary antigens, whereas the indirect, CD28-independent mechanism of TCR-mediated DP apoptosis disposes of TCR reactivities to cortical antigens. Recently, experimental mice were generated in which expression of MHC class II was confined to cortical epithelium (76). Our model predicts that these experimental mice would lack the CD28-dependent mechanism of death because medullary cells expressing B7 (thymic APC or medullary epithelium) would lack MHC class II expression and fail to engage TCR on DP thymocytes. Indeed, T cells in these animals were not tolerant to self-antigens on Class II+ APCs. Importantly, while CD28-dependent clonal deletion is absent in these mice, our model predicts that the CD28-independent mechanism of clonal deletion should be intact, so that their CD4+ T cells would be tolerant to self-antigens expressed on class II+ cortical epithelium.
Third, the model predicts that there can be a delay between the receipt of a negative selecting TCR signal and the death of the cell by the CD28-independent mechanism. Thus, the presence of autoreactive TCR specificities among DP thymocytes is not necessarily indicative of a failure of negative selection.
Our efforts to define the molecular basis for CD28-dependent mechanisms of TCR-mediated DP apoptosis revealed that CD28 is surprisingly unique in its capacity to cooperate with TCR to directly produce apoptotic signals. Even molecules that have been described as augmenting proliferation of mature T cells, such as CD9, CD43, CD81, CD27, 41BB, and CD30, were not able to stimulate TCR-mediated death of DP thymocytes, so DP thymocyte apoptosis is not the result of simultaneous engagement of TCR with any costimulatory molecule. Rather, CD28 appears to generate unique second signals whose identities have not yet been elucidated. Interestingly, TCR-CD28–induced thymocyte apoptosis does not require the activity of PI 3-kinase, one molecule known to associate with CD28.
The identity of the APC-derived signals responsible for CD28-independent thymocyte apoptosis is not known. While such signals may well involve molecules containing death domains, this study has ruled out two attractive candidates, CD30 and 41BB (Fig. 1 b). However, proteins expressing death domains continue to be identified. In fact, three newly identified molecules containing death domains (DR-3, DR-4, and TRAMP) are expressed in lymphopoietic tissues (56–58).
Finally, it is important to draw a distinction between TCR-mediated apoptosis of DP thymocytes and the phenomenon of negative selection. Negative selection refers to any process that rids a developing T cell repertoire of an autospecificity. Although TCR-mediated clonal deletion of DP thymocytes is thought to be a major component of negative selection, it is clearly not the only mechanism responsible for negative selection. Indeed, we have previously shown that thymocytes can be developmentally arrested before the DP stage in response to TCR signals (3) and others have shown that negative selection can occur after the DP stage of development (77–79). In all likelihood the molecular mechanisms that operate at these other developmental stages (both pre- and post-DP) are distinct from those responsible for TCR-dependent apoptosis of DP thymocytes. In support of this possibility, it is interesting to note that (a) transgenic bcl-2 expression does not always affect negative selection (36) even though it abrogates clonal deletion of DP thymocytes and (b) fas appears to play a role in the TCR-mediated death of semimature T cells in the thymus (79) but clearly does not play a role in the TCR-mediated death of rigorously purified DP thymocytes.
In conclusion, this study identified both TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis and reveals that TCR-dependent mechanisms of DP thymocyte death occur by two mechanisms: (a) a CD28-dependent mechanism in which TCR and CD28 costimulatory signals must be received simultaneously to generate apoptotic signals and (b) a CD28-independent mechanism in which TCR signals are indirectly responsible for apoptosis by upregulating molecules which, when subsequently engaged by APCs, will induce cell death. We propose that these two mechanisms represent two distinct strategies used by the thymus to dispose of autoreactive DP thymocytes and that the strategy used depends on where the antigen is encountered. Hence, DP thymocytes autoreactive to cortical antigens will be removed from the T cell repertoire by the indirect CD28-independent mechanism, whereas DP thymocytes autoreactive to APC or medullary cell antigens will be removed from the repertoire by CD28-dependent mechanisms (Fig. 9). Thus, the present study reveals an unexpected diversity of molecular mechanisms responsible for TCR-specific clonal deletion.
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Submitted: 21 July 1997
Revised: 12 September 1997
1 Abbreviations used in this paper: CMJ, cortico medullary junction; DP, double positive; EtBr, ethidium bromide; fasL, fas ligand; FcR, Fc receptor; SP, single positive; TNFR, TNF receptor.
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References |
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1 Burnet FM. A modification of Jerne's theory of antibody production using the concept of clonal selection, Aust J Sci, 1957, 20, 67–69.
2 Lederberg J. Genes and antibodies: do antigens bear instructions for antibody specificity or do they select cell lines that arise by mutation? , Science, 1959, 129, 1649–1653.
3 Takahama Y, Shores EW & Singer A. Negative selection of precursor thymocytes before their differentiation into CD4+CD8+cells, Science, 1992, 258, 653–656.
4 Fowlkes BJ & Ramsdell F. T Cell tolerance, Curr Opin Immunol, 1993, 5, 873–879.[Medline]
5 Kappler JW, Roehm N & Marrack P. T cell tolerance by clonal elimination in the thymus, Cell, 1987, 49, 273–280.[Medline]
6 Fowlkes BJ, Schwartz R & Pardoll DM. Deletion of self-reactive thymocytes occurs at a CD46+CD8+precursor stage, Nature, 1988, 334, 620–623.[Medline]
7 Robey E & Fowlkes BJ. Selective events in T cell development, Annu Rev Immunol, 1994, 12, 675–705.[Medline]
8 Page DM, Kane LP, Allison JP & Hedrick SM. Two signals are required for negative selection of CD4+CD8+thymocytes, J Immunol, 1993, 151, 1868–1880.[Abstract]
9 Punt JA, Osborne BA, Takahama Y, Sharrow SO & Singer A. Negative selection of CD4+CD8+thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28, J Exp Med, 1994, 179, 709–713.
10 Amsen D & Kruisbeek AM. CD28-B7 interactions function to co-stimulate clonal deletion of double-positive thymocytes, Int Immunol, 1996, 8, 1927–1936.
11 Kishimoto H, Cai Z, Brunmark A, Jackson MR, Peterson PA & Sprent J. Differing roles for B7 and intercellular adhesion molecule-1 in negative selection of thymocytes, J Exp Med, 1996, 184, 531–537.
12 Groves T, Parsons M, Miyamoto NG & Guidos CJ. TCR engagement of CD4+CD8+thymocytes in vitro induces early aspects of positive selection, but not apoptosis, J Immunol, 1997, 158, 65–75.[Abstract]
13 Lenschow DJ, Walunas TL & Bluestone JA. CD28/B7 system of T cell costimulation, Annu Rev Immunol, 1996, 14, 233–258.[Medline]
14 Shahinian A, Pfeffer K, Lee KP, Kundig TM, Kishihara K, Wakeman A, Kawai K, Ohashi PS, Thompson CB & Mak TW. Differential T cell costimulatory requirements in CD28-deficient mice, Science, 1993, 261, 609–612.
15 Hubbe M & Altevogt P. Heat-stable antigen/CD24 on mouse T lymphocytes: evidence for a costimulatory function, Eur J Immunol, 1994, 24, 731–737.[Medline]
16 Cocks BG, Chang CC, Carballido JM, Yseel H, de Vries JE & Aversa G. A novel receptor involved in T-cell activation, Nature, 1995, 376, 260–263.[Medline]
17 Del Prete G, De Carli M, D'Elios MM, Daniel KC, Almerigogna F, Alderson M, Smith CA, Thomas E & Romagnani S. CD30-mediated signaling promote the development of human T helper type 2-like T cells, J Exp Med, 1995, 182, 1655–1661.
18 Gravestein LA, Nieland JD, Kruisbeek AM & Borst J. Novel mAbs reveal potent co-stimulatory activity of murine CD27, Int Immunol, 1995, 7, 551–557.
19 Sperling AI, Green JM, Mosley RL, Smith PL, DiPaolo RJ, Klein JR, Bluestone JA & Thompson CB. CD43is a murine T cell costimulatory receptor that functions independently of CD28, J Exp Med, 1995, 182, 139–146.
20 Tai X-G, Yashiro Y, Abe R, Kazuhito T, Wood CR, Morris J, Long A, Ono S, Kobayashi M, Hamaoka T et al.. A role for CD9 molecules in T cell activation, J Exp Med, 1996, 184, 753–758.
21 Amakawa R, Hakem A, Kundig TM, Matsuyama T, Simard JJ, Timms E, Wakeham A, Mittruecker HW, Griesser H, Takimot H et al.. Impaired negative selection of T cells in Hodgkin's disease antigen in CD30-deficient mice, Cell, 1996, 84, 551–562.[Medline]
22 DeBenedette MA, Shahinian A, Mak TW & Watts TH. Costimulation of CD28– T lymphocytes by 4-1BB ligand, J Immunol, 1997, 158, 551–559.[Abstract]
23 Jones LA, Izon DJ, Nieland JD, Linsley PS & Kruisbeek AM. CD28-B7 interactions are not required for intrathymic clonal deletion, Int Immunol, 1993, 5, 503–512.
24 Walunas TL, Sperling AI, Khattri R, Thompson CB & Bluestone JA. CD28 expression is not essential for positive or negative selection of thymocytes or peripheral T cell tolerance, J Immunol, 1996, 153, 1006–1013.
25 Castro JE, Listman JA, Jacobson BA, Wang Y, Lopez PA, Ju S, Finn PW & Perkins DL. Fas modulation of apoptosis during negative selection of thymocytes, Immunity, 1996, 5, 617–627.[Medline]
26 Herron LR, Eisenberg RA, Roper RE, Kakkanaiah VN, Cohen PL & Kotzin BL. Selection of the T cell receptor repertoire in lpr mice, J Immunol, 1993, 151, 3450–3459.[Abstract]
27 Singer GG & Abbas A. The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice, Immunity, 1994, 1, 365–371.[Medline]
28 Suda T & Nagata S. Purification and characterization of the Fas-ligand that induced apoptosis, J Exp Med, 1994, 179, 873–879.
29 Sytwu HK, Liblau RS & McDevitt HO. The roles of Fas/APO-1 (CD95) and TNF in antigen-induced programmed cell death in T cell receptor transgenic mice, Immunity, 1996, 5, 17–30.[Medline]
30 Lenardo MJ. Fas and the art of lymphocyte maintenance, J Exp Med, 1996, 183, 721–724.
31 Russell JH. Activation-induced death of mature T cells in the regulation of immune responses, Curr Opin Immunol, 1995, 7, 382–388.[Medline]
32 Brunner T, Mogil RJ, LaFace D, Yoo NJ, Mahboubi A, Echeverri F, Martin SJ, Force WR, Lynch DH, Ware CF & Green DR. Cell-autonomous Fas (CD95) Fas-ligand interaction mediates activation induced apoptosis in T-cell hybridomas, Nature, 1995, 373, 441–444.[Medline]
33 Dhein J, Walczak H, Baumler C, Debatin KM & Krammer PH. Autocrine T-cell suicide mediated by APO-1 (Fas/CD95), Nature, 1995, 373, 438–441.[Medline]
34 Ju ST, Panka DJ, Cui H, Ettinger R, el-Khatib M, Sherr DH, Stanger BZ & Marshak RA. Fas (CD95)/FasL interactions required for programmed cell death after T-cell activation, Nature, 1995, 373, 444–448.[Medline]
35 Golstein P, Marguet D & Depraetere V. Fas bridging cell death and cytotoxicity: the reaper connection, Immunol Rev, 1995, 146, 45–56.[Medline]
36 Sentman CL, Shutter JR, Hockenbery D, Kanagawa O & Korsmeyer SJ. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes, Cell, 1991, 67, 879–888.[Medline]
37 Rothe J, Lesslauer W, Lotscher H, Lang Y, Koebel P, Kontgen F, Althage A, Zinkernagel R, Steinmetz M & Bluethmann H. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxicity but highly susceptible to infection by Listeria monocytogenes, Nature, 1993, 364, 798–802.[Medline]
38 Leon LR, Kozak W, Peschon J & Kluger MJ. Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice, Am J Physiol, 1997, 272, R563–R569.[Medline]
38 Peschon, J.J., D.S. Torrance, K. Stocking, C.O. Evans, C. Willis, K. Charrier, O.P.J. Morrissey, C.B. Ware, R.G. Goodwin, C.A. Smith, et al. 1998. Pulmonary and systemic inflammatory responses in mice genetically deficient in p55 and p75 tumor necrosis factor receptors. J. Immunol. In press.
39 Nakayama T, June CH, Munitz TI, Sheard M, McCarthy SA, Sharrow SO, Samelson LE & Singer A. Inhibition of T cell receptor expression and function in immature CD4+CD8+cells by CD4, Science, 1990, 249, 1558–1561.
40 Ernst B, Surh CD & Sprent J. Thymic selection and cell division, J Exp Med, 1995, 182, 961–971.
41 Gross JA, Callas E & Allison JP. Identification and distribution of the costimulatory receptor CD28 in the mouse, J Immunol, 1992, 149, 380–388.[Abstract]
42 Kubo RT, Born W, Kappler JW, Marrack P & Pigeon M. Characterisation of a monoclonal antibody which detects all mature β T cell receptors, J Immunol, 1989, 142, 2736–2742.[Abstract]
43 Boismenu R, Rhein M, Fisher WH & Havran WL. A role for CD81 in early T cell development, Science, 1996, 271, 198–200.[Abstract]
44 Liu Y, Jones B, Brady W, Janeway CA Jr & Linsley PS. Co-stimulation of murine CD4 T cell growth: cooperation between B7 and heat-stable antigen, Eur J Immunol, 1992, 22, 2855–2859.[Medline]
45 Bowen MA, Lee RK, Miragliotta G, Nam SY & Podack ER. Structure and expression of murine CD30 and its role in cytokine production, J Immunol, 1996, 156, 442–449.[Abstract]
46 Wiley SR, Goodwin RG & Smith CA. Reverse signaling via CD30 ligand, J Immunol, 1996, 157, 3635–3639.[Abstract]
47 Hathcock KS, Laszlo G, Pucillo C, Linsley P & Hodes RJ. Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function, J Exp Med, 1994, 180, 631–640.
48 Lyons AB, Samuel K, Sanderson A & Maddy AH. Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry, Cytometry, 1992, 13, 1809–1814.
49 Nishimura Y, Ishii A, Kobayashi Y, Yamaski Y & Yonehara S. Expression and function of mouse Fas antigen on immature and mature T cells, J Immunol, 1995, 154, 4395–4403.[Abstract]
50 Hernandez-Caselles T & Stutman O. Immune functions of tumor necrosis factor. I. Tumor necrosis factor induces apoptosis of mouse thymocytes and can also stimulate or inhibit IL-6-induced proliferation depending on the concentration of mitogenic constimulation, J Immunol, 1993, 151, 3999–4012.[Abstract]
51 Ogasawara J, Suda T & Nagata S. Selective apoptosis of CD4+CD8+thymocytes by the anti-fas antibody, J Exp Med, 1995, 181, 485–491.
52 Smith CA, Rarrah T & Goodwin RG. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death, Cell, 1994, 76, 959–962.[Medline]
53 Rudd CE. Upstream-downstream: CD28 cosignaling pathways and T cell function, Immunity, 1996, 4, 527–534.[Medline]
54 Todd SC, Lipps SG, Crisa L, Salomon DR & Tsoukas CD. CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation, J Exp Med, 1996, 184, 2055–2060.
55 Nagata S & Suda T. Fas and Fas ligand: lpr and gld mutations, Immunol Today, 1995, 16, 39–43.[Medline]
56 Bodmer J-L, Burns K, Schneider P, Hofmann K, Steiner V, Thorne M, Bornand T, Hahne M, Schroter M, Becker K et al.. TRAMP, a novel apoptosis-mediating receptor with sequence homology to tumor necrosis factor receptor 1 and Fas (Apo-1/CD95), Immunity, 1997, 6, 79–88.[Medline]
57 Chinnaiyan AM, O'Rourke K, Yu G-L, Lyons RH, Garg M, Duan DR, Xing L, Gentz R, Ni J & Dixit VM. Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95, Science, 1996, 274, 990–992.
58 Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J & Dixit VM. The receptor for the cytotoxic ligand TRAIL, Science, 1997, 276, 111–113.
59 Schreiber SL & Crabtree GR. The mechanism of action of cyclosporin A and FK506, Immunol Today, 1992, 13, 136–142.[Medline]
60 Yano H, Nakanishi S, Kimura K, Hanai N, Saitoh Y, Fukui Y, Nonomura Y & Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol-3 kinase in RBL-2H3 cells, J Biol Chem, 1993, 268, 25846–25856.
61 Bonnema JD, Karnitz LM, Schoon RA, Abraham RT & Leibson PJ. Fc receptor stimulation of phosphatidylinositol 3-kinase in natural killer cells is associated with protein kinase C-independent granule release and cell-mediated cytotoxicity, J Exp Med, 1994, 180, 142–1435.
62 Paya CV, Virelizier JL & Michelson S. Modulation of T-cell activation through protein kinase C- or A-dependent signalling pathways synergistically increases human immunodeficiency virus long terminal repeat induction by cytomegalovirus immediate-early proteins, J Virol, 1991, 65, 5477–5484.
63 Shaw E. Cysteinyl proteinases and their selective inactivation, Adv Enzymol Relat Areas Mol Biol, 1990, 63, 271–346.[Medline]
64 Sarin A, Wu M-L & Henkart P. Different interleukin-1β converting enzyme (ICE) family protease requirements for the apoptotic death of T lymphocytes triggered by diverse stimuli, J Exp Med, 1996, 184, 2445–2450.
65 Ogasawara J, Suda T & Nagata S. Selective apoptosis in CD4+CD8+thymocytes by the anti-fas antibody, J Exp Med, 1995, 181, 485–491.
66 Anderson KL, Anderson G, Michell RH, Jenkinson EJ & Owen JJT. Intracellular signaling pathways involved in the induction of apoptosis in immature thymic T lymphocytes, J Immunol, 1996, 156, 4083–4091.[Abstract]
67 Strasser A, Harris AW, Huang DC, Krammer PH & Cory S. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis, EMBO (Eur Mol Biol Organ) J, 1995, 14, 6136–6147.[Medline]
68 Linsley PS, Brady W, Urnes M, Grosmaire LS, Damle NK & Ledbetter JA. CTLA-4 is a second receptor for the B cell activation antigen B7, J Exp Med, 1991, 174, 561–569.
69 Truitt KE, Hicks CM & Imboden JB. Stimulation of CD28 triggers an association between CD28 and phosphatidylinositol 3-kinase in Jurkat T cells, J Exp Med, 1994, 179, 1071–1076.
70 June CH, Ledbetter JA, Gillespie MM, Lindsten T & Thompson CB. T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression, Mol Cell Biol, 1987, 7, 4472–4481.
71 Lo D & Sprent J. Identity of cells that imprint H-2-restricted T-cell specificity in the thymus, Nature, 1986, 319, 672–675.[Medline]
72 Swat W, Ignatowicz L, von Boehmer H & Kisielow P. Clonal deletion of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen-presenting cells, Nature, 1991, 351, 150–153.[Medline]
73 Matzinger P & Guerder S. Does T-cell tolerance require a dedicated antigen-presenting cell? , Nature, 1989, 338, 74–76.[Medline]
74 Degermann S, Surh CD, Glimcher LH, Sprent J & Lo D. B7 expression on thymic medullary epithelium correlates with epithelium-mediated deletion of V beta 5+ thymocytes, J Immunol, 1994, 152, 3254–3263.[Abstract]
75 Surh CD & Sprent J. T-cell apoptosis detected in situ during positive and negative selection in the thymus, Nature, 1994, 372, 100–103.[Medline]
76 Laufer TM, DeKoning J, Markowitz JS, Lo D & Glimcher LH. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex, Nature, 1996, 383, 81–85.[Medline]
77 MacDonald HR & Lees RK. Programmed death of autoreactive thymocytes, Nature, 1990, 343, 642–644.[Medline]
78 Guidos CJ, Danska JS, Fathman CG & Weissman IL. T cell receptor-mediated negative selection of autoreactive T lymphocyte precursors occurs after commitment to the CD4 or CD8 lineages, J Exp Med, 1990, 12, 835–845.[Medline]
79 Kishimoto H & Sprent J. Negative selection in the thymus includes semimature T cells, J Exp Med, 1997, 185, 263–271.
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