T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection
1997; Springer Nature; Volume: 16; Issue: 9 Linguagem: Inglês
10.1093/emboj/16.9.2282
ISSN1460-2075
AutoresLinda K. Clayton, Yoseph Ghendler, Emiko Mizoguchi, Raymond J. Patch, Timothy D. Ocain, Kim Orth, Atul K. Bhan, Vishva M. Dixit, Ellis L. Reinherz,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle1 May 1997free access T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection Linda K. Clayton Corresponding Author Linda K. Clayton Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Yoseph Ghendler Yoseph Ghendler Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Emiko Mizoguchi Emiko Mizoguchi Department of Pathology, Massachusetts General Hospital, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Raymond J. Patch Raymond J. Patch Department of Medicinal Chemistry, Procept, Inc., 840 Memorial Drive, Cambridge, MA, 02139 USA Search for more papers by this author Timothy D. Ocain Timothy D. Ocain Department of Medicinal Chemistry, Procept, Inc., 840 Memorial Drive, Cambridge, MA, 02139 USA Search for more papers by this author Kim Orth Kim Orth Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Search for more papers by this author Atul K. Bhan Atul K. Bhan Department of Pathology, Massachusetts General Hospital, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Vishva M. Dixit Vishva M. Dixit Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Search for more papers by this author Ellis L. Reinherz Ellis L. Reinherz Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Linda K. Clayton Corresponding Author Linda K. Clayton Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Yoseph Ghendler Yoseph Ghendler Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Emiko Mizoguchi Emiko Mizoguchi Department of Pathology, Massachusetts General Hospital, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Raymond J. Patch Raymond J. Patch Department of Medicinal Chemistry, Procept, Inc., 840 Memorial Drive, Cambridge, MA, 02139 USA Search for more papers by this author Timothy D. Ocain Timothy D. Ocain Department of Medicinal Chemistry, Procept, Inc., 840 Memorial Drive, Cambridge, MA, 02139 USA Search for more papers by this author Kim Orth Kim Orth Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Search for more papers by this author Atul K. Bhan Atul K. Bhan Department of Pathology, Massachusetts General Hospital, Boston, MA, 02115 USA Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Vishva M. Dixit Vishva M. Dixit Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA Search for more papers by this author Ellis L. Reinherz Ellis L. Reinherz Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Department of Medicine, Boston, MA, 02115 USA Search for more papers by this author Author Information Linda K. Clayton 1,3, Yoseph Ghendler1,3, Emiko Mizoguchi2,4, Raymond J. Patch5, Timothy D. Ocain5, Kim Orth6, Atul K. Bhan2,4, Vishva M. Dixit6 and Ellis L. Reinherz1,3 1Laboratory of Immunobiology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA 2Department of Pathology, Massachusetts General Hospital, Boston, MA, 02115 USA 3Department of Medicine, Boston, MA, 02115 USA 4Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA 5Department of Medicinal Chemistry, Procept, Inc., 840 Memorial Drive, Cambridge, MA, 02139 USA 6Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, 48109 USA The EMBO Journal (1997)16:2282-2293https://doi.org/10.1093/emboj/16.9.2282 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3ϵ monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death. Introduction The T-cell repertoire is generated through a tightly regulated developmental program of selection or 'filtering' in which thymocytes bearing immunologically desirable T-cell receptor (TCR) specificities are preserved and those expressing harmful specificities are eliminated (reviewed in Fowlkes and Pardoll, 1989). T-lineage cells expressing autoreactive TCRs are deleted in the thymus via a process termed negative selection (reviewed in Nossal, 1994). Multiple lines of evidence utilizing both normal and transgenic mice show that this negative selection process occurs during a restricted stage(s) of thymic differentiation (Fowlkes et al., 1988; Kisielow et al., 1988; Fowlkes and Pardoll, 1989; Murphy et al., 1990; Vasquez et al., 1992; Sebzda et al., 1994). The deletion process requires TCR recognition of antigenic peptides displayed on APCs in complex with MHC class I or class II molecules and generally involves thymocytes at the DP stage of development. Although the mechanism of negative selection is unknown, the targeted thymocytes die via apoptosis (Murphy et al., 1990), a physiologically controlled form of cell death utilized by metazoan organisms during normal development as well as for homeostasis (Vaux, 1993; Steller, 1995; Vaux and Strasser, 1996). Apoptosis occurs as a consequence of extracellular signaling events such as crosslinking of certain receptors including CD95 (Trauth et al., 1989; Yonehara et al., 1989; Itoh and Nagata, 1993; Alderson et al., 1994; Takahashi et al., 1994), TNF receptors (Tartaglia et al., 1993) and a recently identified Death Receptor 3 (DR3) (Chinnaiyan et al., 1996a) or as a result of growth factor withdrawal (reviewed in Yang and Korsmeyer, 1996). Upon induction of apoptosis, cells undergo a morphologically characteristic process of nuclear condensation, blebbing of cellular membranes and disintegration into small fragments which are removed by phagocytes (Vaux, 1993; Vaux and Strasser, 1996; for review see Steller, 1995). Recent analyses of the death pathways in various systems have begun to delineate the biochemical basis of apoptosis. Particularly, activation of ICE-like cysteine proteases now termed caspases has proven to be a hallmark of apoptotic death (reviewed in Nalin, 1995; Henkart, 1996). There are presently 10 human homologues of the ced 3 cysteine protease first defined by genetic analysis of cell death in Caenorhabditis elegans (Alnemri et al., 1996; Duan et al., 1996a; Fernandes-Alnemri et al., 1996; Muzio et al., 1996; reviewed in Henkart, 1996). These enzymes exist as inactive proenzymes which become active when cleaved to subunits of ∼17–20 kDa and ∼10–12 kDa. The molecular structures of ICE (caspase-1) and Yama (caspase-3) have been determined (Walker et al., 1994; Wilson et al., 1994; Rotonda et al., 1996). The active enzymes exist as tetramers made up of two large and two small subunits. Comparison of these structures suggests that the caspase family falls into two major groups: those that most resemble caspase-1 and those that most resemble caspase-3 (and also ced-3). The substrate binding pockets of these two groups have distinct differences pointing to potentially unique substrate specificities. The active sites contain a critical cysteine within the canonical pentapeptide, QACR/QG, and R and Q residues (Arg179, Gln283 and Arg341 for example in caspase-3) conserved among all caspase family members. A unique characteristic of these enzymes is that they cleave after an aspartic acid residue in their substrate (Sleath et al., 1990; Howard et al., 1991; Thornberry et al., 1994). Thus, the fact that activation of these enzymes occurs by cleavage at aspartic acid residues suggests both autocatalytic capabilities and the possibility of a cascade of various cysteine proteases with one family member activating others during apoptosis. To date, the characterization of caspase enzymatic activities has typically been investigated using transfected cell lines. Hence, the physiological roles of most of these various cysteine proteases in vivo are at present unclear. Three cysteine proteases, caspase-3, Mch2 (caspase-6) and ICE-LAP3 (caspase-7), have been shown to be proteolytically activated by apoptotic stimuli, implying a central role for this family of enzymes in cell death in vivo (Chinnaiyan et al., 1996b; Duan et al., 1996b; Orth et al., 1996a; Schlegel et al., 1996). Given that apoptosis is linked to negative selection in the thymus, we herein examine the potential role of caspase activity in antigen-triggered deletion of CD4+CD8+ double-positive (DP) fetal and adult thymocytes. Using peptide-based enzyme substrates we show that TCR ligation by peptide/MHC activates a procaspase in DP thymocytes which then causes cell death; specific enzyme inhibitors block this death process. These findings provide the molecular basis for negative selection. Results Caspases are irreversibly inhibited by tri- or tetrapeptide sequences (VAD, YVAD and/or DEVD) linked to a chemical moiety such as fluoromethyl ketone (fmk) or various acyloxymethyl ketones (amk) which covalently modify the enzyme, thereby inactivating its catalytic function (Chapman, 1992; Thornberry et al., 1992, 1994; Rotonda et al., 1996). The peptide-based inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVADfmk), blocks the induction of apoptosis induced by expression of REAPER protein when added to Drosophila Schneider cells (Pronk et al., 1996); such experiments demonstrate that REAPER-induced apoptosis requires activation of a caspase. In order to analyze the effect of inhibition of members of the caspase family of cysteine proteases on negative selection in the thymus, we have utilized a fetal thymic organ culture (FTOC) system. FTOC was chosen over either in vivo whole-animal studies or in vitro cell suspension cultures for several reasons. First, FTOC allows analysis of the effects of pharmaceutical agents on thymocyte development in a system which more closely mimics in vivo conditions than does thymocyte suspension culture. Second, pharmacophores which cannot be utilized in whole mice either due to systemic toxicity or limitations in their bioavailability can be assayed easily in FTOC. Furthermore, the relatively small volumes required minimize the amount of chemical required. Caspase inhibition in FTOC prevents DP thymocyte deletion mediated by anti-CD3ϵ mAb Prior studies utilizing the anti-CD3ϵ-specific hamster anti-mouse mAb 145-2C11 (2C11) have shown that DP thymocytes are susceptible to undergo apoptotic cell death upon exposure to 2C11 in FTOC (Smith et al., 1989) or upon parenteral in vivo administration (Shi et al., 1991). We therefore first analyzed the effect of a caspase inhibitor on anti-CD3ϵ-induced deletion of the CD4+CD8+ DP thymocytes using C57BL/6 animals. Fetal thymocytes were dissected on day 14.5 of pregnancy and cultured in transwells for 4 days. On day 4, additions were made to the FTOCs including 2C11 mAb in the presence or absence of 100 μM zVADfmk, an irreversible inhibitor of cysteine proteases, and thymocytes harvested 18 h later. Figure 1A shows the FACS analysis of C57BL/6 fetal thymuses after such a treatment. Upon 2C11 mAb addition, the percent of DP thymocytes is reduced from 66% in the control culture to 37% in the culture treated for 18 h with 200 μg/ml 2C11 mAb (Figure 1A, compare panels a and b). Moreover, the deletion of these DP thymocytes is inhibited by a 2 h preincubation with zVADfmk (Figure 1A, panel d). Note that zVADfmk alone does not alter the percentage of DP thymocytes (Figure 1A, panel c) nor does dimethyl sulfoxide (DMSO), the additive used to solubilize zVADfmk, affect the deletion of DP thymocytes (Figure 1A, panel e). A control hamster anti-mouse mAb, H28 (Becker et al., 1989), directed against a segment of the TCR α chain which is inaccessible on intact thymocytes, has no effect on the FTOC (data not shown). Figure 1.A caspase inhibitor blocks deletion of DP thymocytes induced by anti-CD3ϵ mAb and glucocorticoids. (A) FACS analysis is presented for FTOC from C57BL/6 mice following 18 h of treatment with: (a) no addition; (b) 200 μg/ml 145-2C11 mAb; (c) 100 μm zVADfmk; (d) 2 h treatment with 100 μm zVADfmk followed by 18 h with 200 μg/ml 145-2C11 mAb; (e) 2 h treatment with 0.25% DMSO followed by 18 h with 200 μg/ml 145-2C11 mAb. (B) FACS analysis following 18 h of treatment with: (a) no addition; (b) 0.1 μM dexamethasone; (c) 2 h with 100 μM zVADfmk followed by 18 h with 0.1 μM dexamethasone; (d) 2 h pretreatment with 0.25% DMSO followed by 18 h with 0.1 μM dexamethasone. Fetal lobes (three to five per group) were cultured for 4 days, treated as indicated and harvested on day 5. Lobes were dissociated and thymocytes stained with directly conjugated anti-CD8-Red613 and anti-CD4-PE. 10 000 cells were analyzed per dot plot. The numbers within the quadrants represent the percent of cells in that quadrant. Download figure Download PowerPoint zVADfmk inhibits glucocorticoid-induced DP thymocyte death in FTOC DP thymocytes are exquisitely sensitive to pharmacological doses of glucocorticoids in vivo (Wyllie et al., 1980). The rapid thymic involution known to result from stress is a consequence, in large part, also of endogenous release of steroids. To determine whether caspase inhibition blocks DP thymocyte apoptosis induced by corticosteroids, the effects of dexamethasone on DP thymocyte survival in FTOC were examined in the presence or absence of zVADfmk. As shown in Figure 1B, dexamethasone at 0.1 μM reduces the percentage of DP thymocytes from 74% to 19% (Figure 1B, compare panels a and b). A 2 h pretreatment with 100 μM zVADfmk protects the CD4+CD8+ population from dexamethasone-induced death (Figure 1B, panel c). The inhibition of deletion is specific to the zVADfmk reagent and not due to the DMSO solvent, as shown in Figure 1B, panel d. These results demonstrate that two well-known inducers of CD4+CD8+ DP thymocyte death, anti-CD3ϵ mAb and glucocorticoids, are blocked by inhibition of cysteine protease activity. zVADfmk protects DP thymocytes from antigen-induced deletion in N15 TCR tg RAG-2−/− H-2b mice Given that negative selection of thymocytes in vivo is antigen-driven, we next tested the effect of zVADfmk on specific peptide-induced deletion of DP thymocytes in FTOC derived from TCR tg mice. To this end, we employed the N15 TCR tg mouse which bears a class I MHC-restricted TCR and recognizes the vesicular stomatitis virus nuclear protein octapeptide VSV8 in the context of H-2 Kb (Y.Ghendler, R.E.Hussey, T.Witte, E.Mizoguchi, L.K. Clayton, A.K.Bhan, S.Koyasu, H.-C.Chang and E.L. Reinherz, manuscript submitted). The VSV8 peptide interacts with Kb with high affinity such that a single in vitro or in vivo exposure efficiently loads the Kb molecules in the thymus of these animals. We additionally constructed the animals on a RAG-2−/− background (Shinkai et al., 1992), thereby guaranteeing exclusive expression of this TCR on the surface of T-lineage cells. In these experiments, fetuses were dissected at day 15.5 since we observed that the development of N15 tg RAG-2−/− animals was -slightly slower than that of the corresponding wild-type C57BL/6 animals. Figure 2a shows that after 5 additional days of FTOC, 60% of N15 TCR tg RAG-2−/− H-2b thymocytes are DP and 24% are CD8+ SP thymocytes. Addition of 10−5 M VSV8 peptide to the N15 FTOCs 18 h before analysis results in massive depletion of CD4+CD8+ DP thymocytes with a reduction from 60% to 2% (Figure 2b). zVADfmk completely blocks this antigen-induced depletion (Figure 2c). Importantly, zVADmk, a compound identical to zVADfmk (Figure 3) except for the absence of the fluoride atom which is required for the irreversible inhibition of caspases, has no effect on the antigen-induced depletion of DP thymocytes as shown in Figure 2d. Hence, inhibition of enzymatic function is a prerequisite for protecting DP thymocytes from depletion by specific antigen. Exposure of the FTOC to the DMSO solvent before VSV8 treatment has no effect (Figure 2e). Figure 2 also shows that the depletion of DP thymocytes is specifically induced by the VSV8 peptide: addition to the FTOC of an equivalent amount of the unrelated SEV9 Sendai virus-derived peptide, which binds to Kb with a comparable affinity with that of VSV8 but which is not recognized by the N15 TCR, does not result in thymocyte death (Figure 2, panel f). Figure 2.A caspase inhibitor blocks peptide/MHC induced deletion of DP thymocytes. FACS analysis of FTOC from N15 TCRtg RAG-2−/− H-2b mice following 18 h treatment with: (a) no addition; (b) 10 μM VSV8 peptide; (c) 2 h with 100 μM zVADfmk followed by 18 h with 10 μM VSV8; (d) 2 h with 100 μM zVADmk followed by 18 h with 10 μM VSV8; (e) 2 h with 0.25% DMSO followed by 18 h with 10 μM VSV8; (f) 18 h with 10 μM SEV9. Fetal lobes (five per group) were cultured 4 days, treated as indicated and harvested on day 5. Lobes were dissociated and stained with directly conjugated anti-CD8-Red613 and anti-CD4-PE. 10 000 cells were analyzed per dot plot. The numbers within the quadrants represent the percent of cells in that quadrant. Download figure Download PowerPoint Figure 3.Structures of the peptide-based caspase inhibitors and related compounds. The structures of the peptide-based inhibitor analogues used in these studies are shown along with their molecular weight as verified by mass spectrometry. The trivial names of the compounds are also given. Note that the cell-permeable inhibitors (zVADfmk and zVADmk) have the P1 aspartate protected as the methyl ester. On the other hand, the P1 aspartate of the biotinylated peptides has a free acid at the β position. Download figure Download PowerPoint Histological analysis of antigen-induced apoptosis in FTOC of N15 TCR tg RAG-2−/− H-2b mice: blockade of cell death by zVADfmk Negative selection results in apoptosis of DP thymocytes as judged by morphological criteria or induction of DNA fragmentation as analyzed by gel electrophoresis or TUNEL assay (Murphy et al., 1990; Surh and Sprent, 1994). We confirmed that apoptosis in this TCR tg system is induced by the VSV8 peptide in N15 FTOC using a terminal deoxynucleotidyl transferase (TdT) assay on histological sections as demonstrated in Figure 4. This method exploits the fact that apoptosis results in DNA cleavage, the ends of which serve as a substrate for the TdT enzyme, allowing cells undergoing death to be labeled with biotinylated dUTP. Sections were prepared from fetal thymic lobes cultured as described above and treated for 4 h with 10−5 M VSV8 peptide with either no pretreatment or a 1 h pretreatment with zVADfmk or zVADmk. This early time point was chosen for the characterization as we had previously observed that, after overnight exposure to VSV8, dying DP thymocytes from adult animals were already removed by an efficient process involving the non-lymphoid stromal components of the thymus (Y.Ghendler, R.E.Hussey, T.Witte, E.Mizoguchi, L.K.Clayton, A.K. Bhan, S.Koyasu, H.-C.Chang and E.L.Reinherz, manuscript submitted). The effective removal of corpses has also been previously described in both tg and non-tg thymuses (Surh and Sprent, 1994). Figure 4.Inhibition of caspase activity blocks antigen-induced apoptosis of thymocytes. TUNEL analysis was performed on histological sections of thymuses from N15 tg RAG−/− H-2b FTOC. Thymuses were: untreated (control); treated with 10 μM VSV8 peptide for 4 h (VSV8); 1 h with 100 μM zVADfmk and 4 h with 10 μM VSV8 peptide (VSV8 + zVADfmk); 1 h with 100 μM zVADmk and 4 h with 10 μM VSV8 peptide (VSV8 + zVADmk). A 40× objective was used. Download figure Download PowerPoint As shown in Figure 4, VSV8 treatment resulted in an obvious increase in the number of TdT-positive cells as compared with the control culture which was not exposed to the VSV8 peptide. Pretreatment with zVADfmk before VSV8 addition reduces the number of TdT-positive cells to that of the control. In contrast, zVADmk-pretreated cultures which were exposed to VSV8 had levels of TdT-positive cells similar to cultures treated with VSV8 alone. Table I shows quantitative immunohistological results for three N15 tg RAG-2−/− H-2b FTOC and a littermate control non-tg RAG-2−/− FTOC. Thus, the depletion of DP thymocytes in the N15 TCR tg RAG-2−/− H-2b FTOC is blocked specifically with a cysteine protease inhibitor and this inhibition correlates with a reduction in the number of apoptotic cells as determined by TdT assays. To our knowledge, this is the first demonstration that inhibition of a caspase(s) prevents apoptotic cell death induced by antigen triggering of the TCR on immature DP thymocytes. Table 1. Quantitation of cell death in FTOC by TUNEL assay Treatment TdT-positive cells (mm2) N15tg #1 N15tg #2 N15tg #3 non-tg Control 7a 28 7 1 VSV8 429 456 441 9 zVADfmk + VSV8 6 1 2 6 zVADmk +VSV8 360 525 461 ND In the above experiments (n = 3) of which these results are representative, fetuses were offspring of N15 tg+/− Rag-2−/− H-2b males and RAG-2−/− H-2b females and derived from the same litters. a Mean number of TdT-positive cells/mm2 in six fields from Figure 4. ND, not determined. TCR-triggered activation of caspase(s): a biochemical analysis Although the above results clearly demonstrate that the zVADfmk cysteine protease inhibitor can block antigen-triggered cell death, it remained to be determined whether TCR ligation specifically activates a cysteine protease. Members of the caspase family exist as proenzymes which are cleaved by an activation step to give rise to ∼20 kDa and ∼12 kDa subunits (reviewed in Henkart, 1996). The heterodimeric subunits then associate to form a tetramer. Because the tetramer—but not the inactive proenzyme—binds the substrate, we developed a biochemical assay to examine the state of caspases in thymuses of unstimulated or in vivo-VSV8-triggered N15 tg mice. To this end, we tested several possible substrates including biotin–YVADamk and biotin–DEVDamk (Figure 3). Because the biotin–DEVDamk substrate appears to have the highest affinity for the thymic caspase, only these data will be presented. The significance of this substrate selectivity will be discussed below. N15 transgenic RAG-2−/− H-2b mice were injected in the tail vein with PBS, 24 μg VSV8 or control SEV9 peptide and extracts prepared from the thymocytes of these adult mice 2 h after injection. Lysate equivalent to 2×106 thymocytes was incubated with or without 2 μM biotin–DEVDamk, which binds irreversibly to caspases at varying rates depending on the specific protease. Subsequently, the treated lysates were analyzed by 12.5% SDS–PAGE and blotted onto nitrocellulose. The membranes were then incubated with streptavidin horseradish peroxidase and developed by ECL. A band of ∼17 kDa is induced in the 2 h VSV8-treated thymocytes (Figure 5a). This band is not found in lysates from thymuses of PBS- or SEV9-injected animals, and is not detected in thymic lysates without prior incubation with biotin–DEVDamk (Figure 5a). As expected, purified, recombinant Yama/caspase-3 and Mch2α (caspase-6) proteases bind the biotin–DEVDamk substrate in this assay (Figure 5b). Figure 5.Western blot analysis reveals antigen-induced activation of a thymic caspase. (A) Thymic extracts prepared from PBS, VSV8 or SEV9 injected N15 tg RAG-2−/− H-2b mice were incubated 15 min with (+) or without (−) 2 μM biotin-DEVDamk, run on 12.5% SDS–PAGE under non-reducing conditions and blotted onto nitrocellulose. The membrane was incubated with horseradish peroxidase-conjugated streptavidin and developed by ECL. Each lane contained thymic extract equivalent to 2×106 thymocytes. VSV8 peptide injections were performed 1.5 h before. (B) 140 ng of purified, recombinant Mch2α and 3.5 ng of purified, recombinant Yama were incubated 15 min with 2 μM biotin–DEVDamk and analyzed as described in (a). (C) Thymic extracts prepared from mice 2 h after VSV8 peptide injection were incubated 15 min with 2 μM biotin–DEVDamk alone, 2 μM zVADfmk alone, or preincubated 15 min with an excess of 1000×, 500×, 100×, 10× or an equal concentration of zVADfmk relative to biotin–DEVDamk. Biotin–DEVDamk was then added to 2 μM and the incubation continued for 15 min. Lysates were then run on 12.5% SDS–PAGE and analyzed as described in (a). (D) Thymic extracts isolated from mice 2 h after VSV8 peptide injection were incubated 15 min with 2 μM biotin–DEVDamk alone (−), or, alternatively, incubated 15 min with 200 μM zVADfmk or zVADmk and then biotin–DEVDamk added to 2 μM for another 15 min. Lysates were then analyzed as described in (a). Arrows indicate the position of the activated thymic caspase subunit which binds the biotin–DEVDamk. Download figure Download PowerPoint The above functional studies utilizing zVADfmk and the biochemical analysis with biotin–DEVDamk collectively show that cysteine proteases are activated during antigen-induced negative selection in the thymus. Whether the same caspase(s) interacts with both of these inhibitors remained to be determined. This possibility was tested by competitive inhibition analysis wherein the above thymic lysates were first preincubated with up to 1000-fold molar excess of zVADfmk before addition of the biotin–DEVDamk. If both substrate inhibitors bound to the same enzyme, then the non-biotin-labeled zVADfmk would block the ability of biotin–DEVDamk to bind to the activated thymic caspase and hence, eliminate detection of the ∼17 kDa subunit by streptavidin horseradish peroxidase. Alternatively, if these inhibitors bound to different enzymes, then there would be no change in the appearance of the caspase band as detected by biotin–DEVDamk. Figure 5c shows that, if the thymic lysates are incubated first with zVADfmk at molar concentrations ranging from 1000- to 100-fold that of the biotin–DEVDamk substrate, the ∼17 kDa band is no longer detectable by Western blot analysis. In contrast, at lower zVADfmk:biotin–DEVDamk ratios (10:1 or 1:1), the biotin–DEVDamk substrate is bound by the TCR-triggered cysteine protease and therefore detected as a ∼17 kDa biotinylated inhibitor–enzyme complex. Thus, the irreversible binding of zVADfmk which blocks VSV8-induced depletion of the DP thymocytes in FTOC competes with the biotin–DEVDamk substrate for binding to the same ∼17 kDa band. This result links the antigen-induced negative selection of thymocytes as measured by depletion of DP thymocytes in FTOC to the appearance of an activated cysteine protease detectable in the thymuses of mice undergoing negative selection. Figure 5d shows that, while a 100-fold molar excess of zVADfmk blocks binding of the biotin–DEVDamk substrate to the antigen-activated cysteine protease, the same excess of zVADmk does not block this interaction. This finding is consistent with our observation that zVADmk fails to block antigen-induced depletion of the DP thymocytes in FTOC. Localization of activated caspase to TCR-triggered cortical thymocytes in N15 tg RAG-2−/− H-2b fetal and adult mice To localize the activated caspase within the thymus of fetal and adult N15 tg RAG−/− H-2b mice, we performed two-color immunohistological analysis using biotin–DEVDamk and anti-CD4 mAb. In the N15 RAG−/− H-2b mice, CD4 staining is essentially limited to the DP thymocytes because the N15 TCR is class I MHC-restricted, resulting in positive selection of CD8+ SP cells exclusively. In Figure 6A, we show a control FTOC section stained with biotin–DEVDamk and anti-CD4. The majority of thymocytes demonstrate CD4 staining (red/brown), consistent with the large percentage of DP thymocytes present in the thymus of these animals. Staining with biotin–DEVDamk (blue) is negative. In contrast, 4 h after treatment with VSV8, the CD4 staining has become patchy and the morphology of the CD4 cells is altered with less discrete cell boundaries. In addition, there is widespread biotin–DEVDamk staining throughout the cortex, indicative of antigen-induced activation of a thymic caspase. Consistent with the biochemical analysis, this activation is blocked by pretreatment with zVADfmk but not with zVADmk. Figure 6.Histological analysis of thymic caspase expression in fetal and adult thymuses. (A) Sections of thymic lobe
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