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T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Punt JA, Havran W, Abe R, Sarin A, Singer A - J. Exp. Med. (1997)

Bottom Line: Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities.One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28.We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

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TCR-independent  and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface  molecules that possess costimulatory/coactivating activity and  antibodies against TNFR family  members were assessed for their  ability to induce apoptosis of rigorously purified DP thymocytes.  Control EtBr percentages ranged  between 18 and 30% in all experiments presented in this report.  (a) Only TNF-α and fas induce  death of DP thymocytes in the  absence of TCR stimulation. DP  thymocytes isolated from young  adult female B6 mice were stimulated by platebound antibodies or  by 100 ng/ml recombinant murine TNF-α. Cells were harvested  after overnight incubation and  percent cell death was quantitated  by EtBr staining (see Materials  and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as  indicated. Anti–TCR-β (H5-597) was plated at a concentration of 10 μg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell  death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than  anti-CD28) was plated at both 10 and 50 μg/ml concentrations. Results from the 10 μg/ml plating preparations are shown and were indistinguishable  from results with 50 μg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).
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Figure 1: TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface molecules that possess costimulatory/coactivating activity and antibodies against TNFR family members were assessed for their ability to induce apoptosis of rigorously purified DP thymocytes. Control EtBr percentages ranged between 18 and 30% in all experiments presented in this report. (a) Only TNF-α and fas induce death of DP thymocytes in the absence of TCR stimulation. DP thymocytes isolated from young adult female B6 mice were stimulated by platebound antibodies or by 100 ng/ml recombinant murine TNF-α. Cells were harvested after overnight incubation and percent cell death was quantitated by EtBr staining (see Materials and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as indicated. Anti–TCR-β (H5-597) was plated at a concentration of 10 μg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than anti-CD28) was plated at both 10 and 50 μg/ml concentrations. Results from the 10 μg/ml plating preparations are shown and were indistinguishable from results with 50 μg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).

Mentions: 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) TNFα 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-α engagement of surface receptors induced apoptosis of 20% of DP thymocytes, which may represent a distinct TNF-α 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.


T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Punt JA, Havran W, Abe R, Sarin A, Singer A - J. Exp. Med. (1997)

TCR-independent  and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface  molecules that possess costimulatory/coactivating activity and  antibodies against TNFR family  members were assessed for their  ability to induce apoptosis of rigorously purified DP thymocytes.  Control EtBr percentages ranged  between 18 and 30% in all experiments presented in this report.  (a) Only TNF-α and fas induce  death of DP thymocytes in the  absence of TCR stimulation. DP  thymocytes isolated from young  adult female B6 mice were stimulated by platebound antibodies or  by 100 ng/ml recombinant murine TNF-α. Cells were harvested  after overnight incubation and  percent cell death was quantitated  by EtBr staining (see Materials  and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as  indicated. Anti–TCR-β (H5-597) was plated at a concentration of 10 μg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell  death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than  anti-CD28) was plated at both 10 and 50 μg/ml concentrations. Results from the 10 μg/ml plating preparations are shown and were indistinguishable  from results with 50 μg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199155&req=5

Figure 1: TCR-independent and TCR-dependent mechanisms of DP thymocyte apoptosis. Antibodies against cell surface molecules that possess costimulatory/coactivating activity and antibodies against TNFR family members were assessed for their ability to induce apoptosis of rigorously purified DP thymocytes. Control EtBr percentages ranged between 18 and 30% in all experiments presented in this report. (a) Only TNF-α and fas induce death of DP thymocytes in the absence of TCR stimulation. DP thymocytes isolated from young adult female B6 mice were stimulated by platebound antibodies or by 100 ng/ml recombinant murine TNF-α. Cells were harvested after overnight incubation and percent cell death was quantitated by EtBr staining (see Materials and Methods). (b) Only CD28 cooperates with TCR to induce death of DP thymocytes. Cells were stimulated by platebound antibody combinations as indicated. Anti–TCR-β (H5-597) was plated at a concentration of 10 μg/ml. Even though anti-CD28 antibody was unique in its ability to induce cell death, every experimental antibody enhanced TCR-mediated upregulation of CD5 (data not shown). In a and b, each experimental antibody (other than anti-CD28) was plated at both 10 and 50 μg/ml concentrations. Results from the 10 μg/ml plating preparations are shown and were indistinguishable from results with 50 μg/ml concentrations. Cells were harvested and percent cell death was quantitated by EtBr staining (Materials and Methods).
Mentions: 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) TNFα 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-α engagement of surface receptors induced apoptosis of 20% of DP thymocytes, which may represent a distinct TNF-α 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.

Bottom Line: Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities.One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28.We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

Show MeSH
Related in: MedlinePlus