Limits...
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.

Show MeSH

Related in: MedlinePlus

Only TCR-CD28–stimulated DP thymocytes die in response to TCR-CD28 coengagement. (a) TCR-CD28 stimulation will  not kill bystander CD28 KO DP thymocytes. Individual populations of  DP thymocytes from wild-type B6 mice and CD28 KO mice were cultured and stimulated independently with platebound antibodies. % Cell  death of each population of DP thymocytes was quantitated and normalized as described in Materials and Methods. (b) Experimental design. DP  thymocytes from wild-type mice (CD28+Ly5.2+) were mixed in a 1:1 ratio  with DP thymocytes from CD28-deficient mice (CD28−/−Ly 5.1+) and  stimulated by platebound anti–TCR-β and anti-CD28. Thymocytes  were harvested after overnight culture and percent cell death in each population was determined. CD28+/+ and CD28−/− DP thymocytes were  distinguished by the presence or absence of Ly5.1 staining. (c) Bystander  CD28 KO DP thymocytes are not killed by TCR-CD28 signals. DP thymocytes were isolated from wild-type and CD28-deficient (CD28 KO)  mice which differed in Ly5 expression such that wild-type DP thymocytes were Ly5.2+ and CD28 KO thymocytes were Ly5.1+. Harvested  cells were stained with both anti-Ly5.1 antibody and EtBr to determine  cell death in each population of cocultured DP thymocytes. Percent cell death was quantitated and normalized as described in Materials and Methods. (d)  Schematic of the mechanism by which TCR-CD28 coengagement kills DP thymocytes. This figure illustrates two possible CD28-dependent mechanisms of TCR-mediated apoptosis of DP thymocytes, both of which result in death exclusively of TCR-CD28–stimulated DP thymocytes. The upper  figure (i) illustrates one scenario in which simultaneous coengagement of TCR and CD28 molecules directly and cell-autonomously induces an apoptotic program. The lower figure (ii) illustrates an alternative scenario in which simultaneous coengagement of TCR and CD28 induces expression of a  death domain containing receptor (Y) that signals apoptosis upon interaction with its ligand (Y-L, Y ligand) that is also expressed on DP thymocytes. In  this latter case, the ligand could conceivably engage the death receptor in either cis or trans.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199155&req=5

Figure 3: Only TCR-CD28–stimulated DP thymocytes die in response to TCR-CD28 coengagement. (a) TCR-CD28 stimulation will not kill bystander CD28 KO DP thymocytes. Individual populations of DP thymocytes from wild-type B6 mice and CD28 KO mice were cultured and stimulated independently with platebound antibodies. % Cell death of each population of DP thymocytes was quantitated and normalized as described in Materials and Methods. (b) Experimental design. DP thymocytes from wild-type mice (CD28+Ly5.2+) were mixed in a 1:1 ratio with DP thymocytes from CD28-deficient mice (CD28−/−Ly 5.1+) and stimulated by platebound anti–TCR-β and anti-CD28. Thymocytes were harvested after overnight culture and percent cell death in each population was determined. CD28+/+ and CD28−/− DP thymocytes were distinguished by the presence or absence of Ly5.1 staining. (c) Bystander CD28 KO DP thymocytes are not killed by TCR-CD28 signals. DP thymocytes were isolated from wild-type and CD28-deficient (CD28 KO) mice which differed in Ly5 expression such that wild-type DP thymocytes were Ly5.2+ and CD28 KO thymocytes were Ly5.1+. Harvested cells were stained with both anti-Ly5.1 antibody and EtBr to determine cell death in each population of cocultured DP thymocytes. Percent cell death was quantitated and normalized as described in Materials and Methods. (d) Schematic of the mechanism by which TCR-CD28 coengagement kills DP thymocytes. This figure illustrates two possible CD28-dependent mechanisms of TCR-mediated apoptosis of DP thymocytes, both of which result in death exclusively of TCR-CD28–stimulated DP thymocytes. The upper figure (i) illustrates one scenario in which simultaneous coengagement of TCR and CD28 molecules directly and cell-autonomously induces an apoptotic program. The lower figure (ii) illustrates an alternative scenario in which simultaneous coengagement of TCR and CD28 induces expression of a death domain containing receptor (Y) that signals apoptosis upon interaction with its ligand (Y-L, Y ligand) that is also expressed on DP thymocytes. In this latter case, the ligand could conceivably engage the death receptor in either cis or trans.

Mentions: While fas and TNFR are not involved in TCR-CD28– induced apoptosis, additional members of the TNFR family expressing the “death domain” associated with apoptotic signaling continue to be identified (56–58). To determine if TCR-CD28 coengagement induced apoptosis in trans of bystander thymocytes via ligands (known and unknown), we used thymocytes from CD28 KO mice as bystander cells for they do not die in response to TCR-CD28 stimulation (Fig. 3 a). In this mixed culture experiment, we stimulated cocultures of DP thymocytes from wild-type (B6-Ly5.2) and CD28 KO mice with both anti-TCR and anti-CD28 platebound antibodies (Fig. 3 b). We found that the majority of wild-type DP thymocytes died in cocultures in response to TCR-CD28 coengagement. In contrast, bystander CD28 KO DP thymocytes did not die even though they were present in the same cocultures and even though they were stimulated by the immobilized anti-TCR antibodies (Fig. 3 c). These results demonstrate that TCR-CD28 coengagement only induces death of DP thymocytes that had been stimulated by both TCR and CD28 signals, and suggests one of two scenarios as illustrated in Fig. 3 d. Either TCR-CD28 transduces intracellular signals that directly activate an intrinsic death program in DP thymocytes, or TCR-CD28 coengagement stimulates surface expression of an unknown death domain bearing molecule that signals cell death upon interaction with its ligand which must also be expressed on DP thymocytes. In the latter case, the death receptor-ligand interaction could conceivably operate either on an individual DP thymocyte (i.e., cis) or between interacting DP thymocytes (trans).


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)

Only TCR-CD28–stimulated DP thymocytes die in response to TCR-CD28 coengagement. (a) TCR-CD28 stimulation will  not kill bystander CD28 KO DP thymocytes. Individual populations of  DP thymocytes from wild-type B6 mice and CD28 KO mice were cultured and stimulated independently with platebound antibodies. % Cell  death of each population of DP thymocytes was quantitated and normalized as described in Materials and Methods. (b) Experimental design. DP  thymocytes from wild-type mice (CD28+Ly5.2+) were mixed in a 1:1 ratio  with DP thymocytes from CD28-deficient mice (CD28−/−Ly 5.1+) and  stimulated by platebound anti–TCR-β and anti-CD28. Thymocytes  were harvested after overnight culture and percent cell death in each population was determined. CD28+/+ and CD28−/− DP thymocytes were  distinguished by the presence or absence of Ly5.1 staining. (c) Bystander  CD28 KO DP thymocytes are not killed by TCR-CD28 signals. DP thymocytes were isolated from wild-type and CD28-deficient (CD28 KO)  mice which differed in Ly5 expression such that wild-type DP thymocytes were Ly5.2+ and CD28 KO thymocytes were Ly5.1+. Harvested  cells were stained with both anti-Ly5.1 antibody and EtBr to determine  cell death in each population of cocultured DP thymocytes. Percent cell death was quantitated and normalized as described in Materials and Methods. (d)  Schematic of the mechanism by which TCR-CD28 coengagement kills DP thymocytes. This figure illustrates two possible CD28-dependent mechanisms of TCR-mediated apoptosis of DP thymocytes, both of which result in death exclusively of TCR-CD28–stimulated DP thymocytes. The upper  figure (i) illustrates one scenario in which simultaneous coengagement of TCR and CD28 molecules directly and cell-autonomously induces an apoptotic program. The lower figure (ii) illustrates an alternative scenario in which simultaneous coengagement of TCR and CD28 induces expression of a  death domain containing receptor (Y) that signals apoptosis upon interaction with its ligand (Y-L, Y ligand) that is also expressed on DP thymocytes. In  this latter case, the ligand could conceivably engage the death receptor in either cis or trans.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Only TCR-CD28–stimulated DP thymocytes die in response to TCR-CD28 coengagement. (a) TCR-CD28 stimulation will not kill bystander CD28 KO DP thymocytes. Individual populations of DP thymocytes from wild-type B6 mice and CD28 KO mice were cultured and stimulated independently with platebound antibodies. % Cell death of each population of DP thymocytes was quantitated and normalized as described in Materials and Methods. (b) Experimental design. DP thymocytes from wild-type mice (CD28+Ly5.2+) were mixed in a 1:1 ratio with DP thymocytes from CD28-deficient mice (CD28−/−Ly 5.1+) and stimulated by platebound anti–TCR-β and anti-CD28. Thymocytes were harvested after overnight culture and percent cell death in each population was determined. CD28+/+ and CD28−/− DP thymocytes were distinguished by the presence or absence of Ly5.1 staining. (c) Bystander CD28 KO DP thymocytes are not killed by TCR-CD28 signals. DP thymocytes were isolated from wild-type and CD28-deficient (CD28 KO) mice which differed in Ly5 expression such that wild-type DP thymocytes were Ly5.2+ and CD28 KO thymocytes were Ly5.1+. Harvested cells were stained with both anti-Ly5.1 antibody and EtBr to determine cell death in each population of cocultured DP thymocytes. Percent cell death was quantitated and normalized as described in Materials and Methods. (d) Schematic of the mechanism by which TCR-CD28 coengagement kills DP thymocytes. This figure illustrates two possible CD28-dependent mechanisms of TCR-mediated apoptosis of DP thymocytes, both of which result in death exclusively of TCR-CD28–stimulated DP thymocytes. The upper figure (i) illustrates one scenario in which simultaneous coengagement of TCR and CD28 molecules directly and cell-autonomously induces an apoptotic program. The lower figure (ii) illustrates an alternative scenario in which simultaneous coengagement of TCR and CD28 induces expression of a death domain containing receptor (Y) that signals apoptosis upon interaction with its ligand (Y-L, Y ligand) that is also expressed on DP thymocytes. In this latter case, the ligand could conceivably engage the death receptor in either cis or trans.
Mentions: While fas and TNFR are not involved in TCR-CD28– induced apoptosis, additional members of the TNFR family expressing the “death domain” associated with apoptotic signaling continue to be identified (56–58). To determine if TCR-CD28 coengagement induced apoptosis in trans of bystander thymocytes via ligands (known and unknown), we used thymocytes from CD28 KO mice as bystander cells for they do not die in response to TCR-CD28 stimulation (Fig. 3 a). In this mixed culture experiment, we stimulated cocultures of DP thymocytes from wild-type (B6-Ly5.2) and CD28 KO mice with both anti-TCR and anti-CD28 platebound antibodies (Fig. 3 b). We found that the majority of wild-type DP thymocytes died in cocultures in response to TCR-CD28 coengagement. In contrast, bystander CD28 KO DP thymocytes did not die even though they were present in the same cocultures and even though they were stimulated by the immobilized anti-TCR antibodies (Fig. 3 c). These results demonstrate that TCR-CD28 coengagement only induces death of DP thymocytes that had been stimulated by both TCR and CD28 signals, and suggests one of two scenarios as illustrated in Fig. 3 d. Either TCR-CD28 transduces intracellular signals that directly activate an intrinsic death program in DP thymocytes, or TCR-CD28 coengagement stimulates surface expression of an unknown death domain bearing molecule that signals cell death upon interaction with its ligand which must also be expressed on DP thymocytes. In the latter case, the death receptor-ligand interaction could conceivably operate either on an individual DP thymocyte (i.e., cis) or between interacting DP thymocytes (trans).

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