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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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CD28-dependent and CD28-independent mechanisms of DP thymocyte apoptosis are distinct. (a) TCR and CD28 signals must be received  simultaneously to mediate death of DP thymocytes. DP thymocytes from B6 mice were prestimulated by platebound anti-TCR-β for 6 h. They were  then removed from this stimulus and transferred to wells that had been precoated with the antibodies indicated on the x-axis. Cells were harvested and  stained with EtBr. The background cell death observed in TCR pretreated groups is likely due to cell damage inflicted by their physical removal from  platebound anti-TCR antibody. (b) TCR and second signals derived from APCs do not have to be simultaneous to induce CD28-independent DP thymocyte death. DP thymocytes from CD28 KO (Ly 5.11) mice were prestimulated by platebound anti–TCR-β for 6 h. They were then removed from this  stimulus and transferred either into wells that had been precoated with anti-CD28 or into wells containing APCs from B6 Ly5.2 mice (in a 2:1 ratio with  the DP cells). Cells were harvested and stained with both Ly5.1 and EtBr. DP thymocytes were distinguished from APC by expression of Ly5.1. As can  be seen, preengagement of TCR on DP thymocytes made them susceptible to APC-induced cell death. (c) Schematic of the CD28-independent mechanism of DP thymocyte apoptosis. This figure illustrates the proposed mechanism by which TCR and subsequent APC signals induce apoptosis of DP thymocytes in a CD28-independent manner. TCR prestimulation of DP thymocytes (1) induces upregulation of a molecule X which might express death  domains (2). Subsequent engagement of molecule X with a ligand expressed by APCs (X ligand or X-L) induces apoptosis of only prestimulated DP thymocytes (3).
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Figure 7: CD28-dependent and CD28-independent mechanisms of DP thymocyte apoptosis are distinct. (a) TCR and CD28 signals must be received simultaneously to mediate death of DP thymocytes. DP thymocytes from B6 mice were prestimulated by platebound anti-TCR-β for 6 h. They were then removed from this stimulus and transferred to wells that had been precoated with the antibodies indicated on the x-axis. Cells were harvested and stained with EtBr. The background cell death observed in TCR pretreated groups is likely due to cell damage inflicted by their physical removal from platebound anti-TCR antibody. (b) TCR and second signals derived from APCs do not have to be simultaneous to induce CD28-independent DP thymocyte death. DP thymocytes from CD28 KO (Ly 5.11) mice were prestimulated by platebound anti–TCR-β for 6 h. They were then removed from this stimulus and transferred either into wells that had been precoated with anti-CD28 or into wells containing APCs from B6 Ly5.2 mice (in a 2:1 ratio with the DP cells). Cells were harvested and stained with both Ly5.1 and EtBr. DP thymocytes were distinguished from APC by expression of Ly5.1. As can be seen, preengagement of TCR on DP thymocytes made them susceptible to APC-induced cell death. (c) Schematic of the CD28-independent mechanism of DP thymocyte apoptosis. This figure illustrates the proposed mechanism by which TCR and subsequent APC signals induce apoptosis of DP thymocytes in a CD28-independent manner. TCR prestimulation of DP thymocytes (1) induces upregulation of a molecule X which might express death domains (2). Subsequent engagement of molecule X with a ligand expressed by APCs (X ligand or X-L) induces apoptosis of only prestimulated DP thymocytes (3).

Mentions: The present results clearly indicate that CD28 is not the only surface molecule that the TCR can use to kill DP thymocytes. However, it is possible that even though CD28-dependent and CD28-independent mechanisms of TCR-induced DP thymocyte apoptosis are mediated by different surface receptor molecules on DP thymocytes, the second signals induced by these different proteins might be identical. That this is not the case was revealed by experiments in which TCR and second signals were induced sequentially rather than simultaneously. In this experiment, we prestimulated DP thymocytes with immobilized anti-TCR for 6 h and subsequently transferred them to cultures with platebound anti-CD28. We found that death of prestimulated DP thymocytes was not affected by subsequent stimulation through CD28 (Fig. 7 a). In fact, only subsequent coengagement of both TCR and CD28 together increased apoptosis of prestimulated DP thymocytes above background (Fig. 7 a). In contrast, we found in the same experiment that DP thymocytes from CD28 KO mice similarly prestimulated with immobilized anti-TCR and subsequently transferred to APCs were killed efficiently (Fig. 7 b). Thus, to stimulate DP thymocyte apoptosis, TCR and second signals induced by CD28 must be generated simultaneously, whereas TCR and second signals induced by APC can be generated sequentially, as illustrated in Fig. 7 c. Consequently, CD28-dependent and CD28-independent pathways of cell death represent two distinct mechanisms by which TCR can induce DP thymocyte apoptosis.


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)

CD28-dependent and CD28-independent mechanisms of DP thymocyte apoptosis are distinct. (a) TCR and CD28 signals must be received  simultaneously to mediate death of DP thymocytes. DP thymocytes from B6 mice were prestimulated by platebound anti-TCR-β for 6 h. They were  then removed from this stimulus and transferred to wells that had been precoated with the antibodies indicated on the x-axis. Cells were harvested and  stained with EtBr. The background cell death observed in TCR pretreated groups is likely due to cell damage inflicted by their physical removal from  platebound anti-TCR antibody. (b) TCR and second signals derived from APCs do not have to be simultaneous to induce CD28-independent DP thymocyte death. DP thymocytes from CD28 KO (Ly 5.11) mice were prestimulated by platebound anti–TCR-β for 6 h. They were then removed from this  stimulus and transferred either into wells that had been precoated with anti-CD28 or into wells containing APCs from B6 Ly5.2 mice (in a 2:1 ratio with  the DP cells). Cells were harvested and stained with both Ly5.1 and EtBr. DP thymocytes were distinguished from APC by expression of Ly5.1. As can  be seen, preengagement of TCR on DP thymocytes made them susceptible to APC-induced cell death. (c) Schematic of the CD28-independent mechanism of DP thymocyte apoptosis. This figure illustrates the proposed mechanism by which TCR and subsequent APC signals induce apoptosis of DP thymocytes in a CD28-independent manner. TCR prestimulation of DP thymocytes (1) induces upregulation of a molecule X which might express death  domains (2). Subsequent engagement of molecule X with a ligand expressed by APCs (X ligand or X-L) induces apoptosis of only prestimulated DP thymocytes (3).
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Related In: Results  -  Collection

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Figure 7: CD28-dependent and CD28-independent mechanisms of DP thymocyte apoptosis are distinct. (a) TCR and CD28 signals must be received simultaneously to mediate death of DP thymocytes. DP thymocytes from B6 mice were prestimulated by platebound anti-TCR-β for 6 h. They were then removed from this stimulus and transferred to wells that had been precoated with the antibodies indicated on the x-axis. Cells were harvested and stained with EtBr. The background cell death observed in TCR pretreated groups is likely due to cell damage inflicted by their physical removal from platebound anti-TCR antibody. (b) TCR and second signals derived from APCs do not have to be simultaneous to induce CD28-independent DP thymocyte death. DP thymocytes from CD28 KO (Ly 5.11) mice were prestimulated by platebound anti–TCR-β for 6 h. They were then removed from this stimulus and transferred either into wells that had been precoated with anti-CD28 or into wells containing APCs from B6 Ly5.2 mice (in a 2:1 ratio with the DP cells). Cells were harvested and stained with both Ly5.1 and EtBr. DP thymocytes were distinguished from APC by expression of Ly5.1. As can be seen, preengagement of TCR on DP thymocytes made them susceptible to APC-induced cell death. (c) Schematic of the CD28-independent mechanism of DP thymocyte apoptosis. This figure illustrates the proposed mechanism by which TCR and subsequent APC signals induce apoptosis of DP thymocytes in a CD28-independent manner. TCR prestimulation of DP thymocytes (1) induces upregulation of a molecule X which might express death domains (2). Subsequent engagement of molecule X with a ligand expressed by APCs (X ligand or X-L) induces apoptosis of only prestimulated DP thymocytes (3).
Mentions: The present results clearly indicate that CD28 is not the only surface molecule that the TCR can use to kill DP thymocytes. However, it is possible that even though CD28-dependent and CD28-independent mechanisms of TCR-induced DP thymocyte apoptosis are mediated by different surface receptor molecules on DP thymocytes, the second signals induced by these different proteins might be identical. That this is not the case was revealed by experiments in which TCR and second signals were induced sequentially rather than simultaneously. In this experiment, we prestimulated DP thymocytes with immobilized anti-TCR for 6 h and subsequently transferred them to cultures with platebound anti-CD28. We found that death of prestimulated DP thymocytes was not affected by subsequent stimulation through CD28 (Fig. 7 a). In fact, only subsequent coengagement of both TCR and CD28 together increased apoptosis of prestimulated DP thymocytes above background (Fig. 7 a). In contrast, we found in the same experiment that DP thymocytes from CD28 KO mice similarly prestimulated with immobilized anti-TCR and subsequently transferred to APCs were killed efficiently (Fig. 7 b). Thus, to stimulate DP thymocyte apoptosis, TCR and second signals induced by CD28 must be generated simultaneously, whereas TCR and second signals induced by APC can be generated sequentially, as illustrated in Fig. 7 c. Consequently, CD28-dependent and CD28-independent pathways of cell death represent two distinct mechanisms by which TCR can induce DP thymocyte apoptosis.

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