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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-CD28 killing of DP thymocytes is not mediated by  fas-fasL or TNF-TNFR interactions. DP thymocytes were isolated from  the mice indicated (wild-type B6, gld/gld (fasL deficient), lpr/lpr (fas deficient), TNFR (p55)–deficient, and TNFR (p55 and p75)–deficient mice  strains. Single-cell suspensions were stimulated overnight by platebound  anti–TCR-β and anti-CD28 antibodies, and then were harvested and  stained with EtBr. To compare thymocyte apoptosis from different mouse  strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as  a killing index.
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Figure 2: TCR-CD28 killing of DP thymocytes is not mediated by fas-fasL or TNF-TNFR interactions. DP thymocytes were isolated from the mice indicated (wild-type B6, gld/gld (fasL deficient), lpr/lpr (fas deficient), TNFR (p55)–deficient, and TNFR (p55 and p75)–deficient mice strains. Single-cell suspensions were stimulated overnight by platebound anti–TCR-β and anti-CD28 antibodies, and then were harvested and stained with EtBr. To compare thymocyte apoptosis from different mouse strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as a killing index.

Mentions: The ability of fas and TNF-α to mediate DP thymocyte apoptosis raised the possibility that both mechanisms of DP thymocyte apoptosis (TCR-independent and TCR-dependent) may ultimately result from engagement of fas or TNFR. To address this possibility, we examined the ability of TCR-CD28 coengagement to kill purified DP thymocytes from mice deficient in (a) fas (lpr, reviewed in reference 55), (b) fas ligand (gld), (c) the p55 murine TNFR, or (d) both the p55 and p75 murine TNFRs (38, 38a) (Fig. 2). None of the mutations significantly affected the ability of TCR-CD28 coengagement to induce apoptosis of DP thymocytes, indicating that neither fas nor the TNF receptors p55 or p75 were required (Fig. 2). In addition, neutralizing anti–TNF-α antibodies had no effect on TCR-CD28– induced apoptosis of wild-type DP thymocytes (data not shown). We also found that TCR-CD28–induced death was not blocked by CD30 Ig, a fusion protein that blocks CD30-CD30L interactions (data not shown). We conclude that TCR-CD28 coengagement induces DP thymocyte apoptosis by a mechanism that is independent of fas/fasL, TNF-TNFR, and CD30-CD30L interactions.


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-CD28 killing of DP thymocytes is not mediated by  fas-fasL or TNF-TNFR interactions. DP thymocytes were isolated from  the mice indicated (wild-type B6, gld/gld (fasL deficient), lpr/lpr (fas deficient), TNFR (p55)–deficient, and TNFR (p55 and p75)–deficient mice  strains. Single-cell suspensions were stimulated overnight by platebound  anti–TCR-β and anti-CD28 antibodies, and then were harvested and  stained with EtBr. To compare thymocyte apoptosis from different mouse  strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as  a killing index.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: TCR-CD28 killing of DP thymocytes is not mediated by fas-fasL or TNF-TNFR interactions. DP thymocytes were isolated from the mice indicated (wild-type B6, gld/gld (fasL deficient), lpr/lpr (fas deficient), TNFR (p55)–deficient, and TNFR (p55 and p75)–deficient mice strains. Single-cell suspensions were stimulated overnight by platebound anti–TCR-β and anti-CD28 antibodies, and then were harvested and stained with EtBr. To compare thymocyte apoptosis from different mouse strains with different internal controls, we have normalized individual responses to their respective controls. The normalized value is referred to as a killing index.
Mentions: The ability of fas and TNF-α to mediate DP thymocyte apoptosis raised the possibility that both mechanisms of DP thymocyte apoptosis (TCR-independent and TCR-dependent) may ultimately result from engagement of fas or TNFR. To address this possibility, we examined the ability of TCR-CD28 coengagement to kill purified DP thymocytes from mice deficient in (a) fas (lpr, reviewed in reference 55), (b) fas ligand (gld), (c) the p55 murine TNFR, or (d) both the p55 and p75 murine TNFRs (38, 38a) (Fig. 2). None of the mutations significantly affected the ability of TCR-CD28 coengagement to induce apoptosis of DP thymocytes, indicating that neither fas nor the TNF receptors p55 or p75 were required (Fig. 2). In addition, neutralizing anti–TNF-α antibodies had no effect on TCR-CD28– induced apoptosis of wild-type DP thymocytes (data not shown). We also found that TCR-CD28–induced death was not blocked by CD30 Ig, a fusion protein that blocks CD30-CD30L interactions (data not shown). We conclude that TCR-CD28 coengagement induces DP thymocyte apoptosis by a mechanism that is independent of fas/fasL, TNF-TNFR, and CD30-CD30L interactions.

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