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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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Inhibitors of TCR-CD28–mediated death of DP thymocytes. DP thymocytes from wild-type (B6) mice were stimulated by  platebound anti–TCR-β and anti-CD28 in the presence or absence of  the following pharmacological agents: the calcineurin inhibitor, cyclosporine A (1 μg/ml); the PI-3-kinase inhibitor, wortmannin (800 ng/ml); the  PKCγ inhibitor, GF109203x (800 ng/ml); the protein synthesis inhibitor,  cycloheximide (10 μg/ml); and the caspase inhibitor, ZVAD-FMK (100  μM). To compare the effects of various reagents on TCR-CD28–mediated DP thymocyte apoptosis in experiments performed with different  solvent controls, individual responses were normalized to their respective  controls (killing index). As positive controls for the pharmacologic agents  used: cyclosporine A and GF109203x used in this experiment inhibited  TCR-mediated CD5 upregulation, and wortmannin used in this experiment blocked NK-mediated target cell lysis (data not shown). Also displayed in the same format are the results of anti–TCR-CD28 stimulation  of DP thymocytes isolated from bcl-2 transgenic mice.
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Figure 4: Inhibitors of TCR-CD28–mediated death of DP thymocytes. DP thymocytes from wild-type (B6) mice were stimulated by platebound anti–TCR-β and anti-CD28 in the presence or absence of the following pharmacological agents: the calcineurin inhibitor, cyclosporine A (1 μg/ml); the PI-3-kinase inhibitor, wortmannin (800 ng/ml); the PKCγ inhibitor, GF109203x (800 ng/ml); the protein synthesis inhibitor, cycloheximide (10 μg/ml); and the caspase inhibitor, ZVAD-FMK (100 μM). To compare the effects of various reagents on TCR-CD28–mediated DP thymocyte apoptosis in experiments performed with different solvent controls, individual responses were normalized to their respective controls (killing index). As positive controls for the pharmacologic agents used: cyclosporine A and GF109203x used in this experiment inhibited TCR-mediated CD5 upregulation, and wortmannin used in this experiment blocked NK-mediated target cell lysis (data not shown). Also displayed in the same format are the results of anti–TCR-CD28 stimulation of DP thymocytes isolated from bcl-2 transgenic mice.

Mentions: To characterize the molecular basis for TCR-CD28–induced apoptosis of DP thymocytes, we assessed specific inhibitors for their ability to abrograte DP thymocyte death (Fig. 4). We found that neither cyclosporine A (reviewed in reference 59) nor wortmannin (60, 61) inhibited TCR-CD28–mediated death (Fig. 4), indicating that calcineurin and PI 3-kinase activity are not required. In contrast, DP thymocyte apoptosis induced by TCR-CD28 coengagement was inhibited by (a) GF109203X, a specific inhibitor of protein kinase Cγ (PKCγ (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).


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)

Inhibitors of TCR-CD28–mediated death of DP thymocytes. DP thymocytes from wild-type (B6) mice were stimulated by  platebound anti–TCR-β and anti-CD28 in the presence or absence of  the following pharmacological agents: the calcineurin inhibitor, cyclosporine A (1 μg/ml); the PI-3-kinase inhibitor, wortmannin (800 ng/ml); the  PKCγ inhibitor, GF109203x (800 ng/ml); the protein synthesis inhibitor,  cycloheximide (10 μg/ml); and the caspase inhibitor, ZVAD-FMK (100  μM). To compare the effects of various reagents on TCR-CD28–mediated DP thymocyte apoptosis in experiments performed with different  solvent controls, individual responses were normalized to their respective  controls (killing index). As positive controls for the pharmacologic agents  used: cyclosporine A and GF109203x used in this experiment inhibited  TCR-mediated CD5 upregulation, and wortmannin used in this experiment blocked NK-mediated target cell lysis (data not shown). Also displayed in the same format are the results of anti–TCR-CD28 stimulation  of DP thymocytes isolated from bcl-2 transgenic mice.
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Related In: Results  -  Collection

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

Figure 4: Inhibitors of TCR-CD28–mediated death of DP thymocytes. DP thymocytes from wild-type (B6) mice were stimulated by platebound anti–TCR-β and anti-CD28 in the presence or absence of the following pharmacological agents: the calcineurin inhibitor, cyclosporine A (1 μg/ml); the PI-3-kinase inhibitor, wortmannin (800 ng/ml); the PKCγ inhibitor, GF109203x (800 ng/ml); the protein synthesis inhibitor, cycloheximide (10 μg/ml); and the caspase inhibitor, ZVAD-FMK (100 μM). To compare the effects of various reagents on TCR-CD28–mediated DP thymocyte apoptosis in experiments performed with different solvent controls, individual responses were normalized to their respective controls (killing index). As positive controls for the pharmacologic agents used: cyclosporine A and GF109203x used in this experiment inhibited TCR-mediated CD5 upregulation, and wortmannin used in this experiment blocked NK-mediated target cell lysis (data not shown). Also displayed in the same format are the results of anti–TCR-CD28 stimulation of DP thymocytes isolated from bcl-2 transgenic mice.
Mentions: To characterize the molecular basis for TCR-CD28–induced apoptosis of DP thymocytes, we assessed specific inhibitors for their ability to abrograte DP thymocyte death (Fig. 4). We found that neither cyclosporine A (reviewed in reference 59) nor wortmannin (60, 61) inhibited TCR-CD28–mediated death (Fig. 4), indicating that calcineurin and PI 3-kinase activity are not required. In contrast, DP thymocyte apoptosis induced by TCR-CD28 coengagement was inhibited by (a) GF109203X, a specific inhibitor of protein kinase Cγ (PKCγ (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).

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