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Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.

McCaughtry TM, Baldwin TA, Wilken MS, Hogquist KA - J. Exp. Med. (2008)

Bottom Line: However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation.We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation.Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c(+) cortical dendritic cells, and elimination of such cells impaired deletion.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology, Laboratory Medicine, and Pathology, University of Minnesota, Minneapolis, MN 55454, USA.

ABSTRACT
The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HY(cd4) model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c(+) cortical dendritic cells, and elimination of such cells impaired deletion.

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HYcd4 mixed BM chimeras as a model system to study clonal deletion in vivo. (A) HYcd4 TCRαo female BM was mixed with congenic B6.PL female and male BM and transferred into congenic female and male hosts. Chimeras were harvested at 6 wk and thymi were analyzed by flow cytometry for expression of Thy1.2, T3.70, CD4, CD8, and active Caspase 3. HYcd4 donor-derived cells were identified by Thy1.2 where indicated. Numbers represent frequency of parent gate. (B) The activation of Caspase 3 in male chimeras compared with female chimeras. The fold change in male over female is indicated. Data represent the mean from 15–17 individuals from six different experiments ± SD. P < 0.0001.
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fig3: HYcd4 mixed BM chimeras as a model system to study clonal deletion in vivo. (A) HYcd4 TCRαo female BM was mixed with congenic B6.PL female and male BM and transferred into congenic female and male hosts. Chimeras were harvested at 6 wk and thymi were analyzed by flow cytometry for expression of Thy1.2, T3.70, CD4, CD8, and active Caspase 3. HYcd4 donor-derived cells were identified by Thy1.2 where indicated. Numbers represent frequency of parent gate. (B) The activation of Caspase 3 in male chimeras compared with female chimeras. The fold change in male over female is indicated. Data represent the mean from 15–17 individuals from six different experiments ± SD. P < 0.0001.

Mentions: To further examine the anatomical location of clonal deletion and factors involved, we sought a system that allowed us to easily test various genetic deficiencies and to limit antigen presentation to particular subsets of cells. To do this, we used a mixed BM chimera strategy where HYcd4 female BM was mixed at a low ratio with competitor BM from WT mice and used to reconstitute female or male recipients. This experimental strategy has the additional advantage that the precursor frequency is reduced, thereby reducing clonal competition and correcting thymic architecture defects, which are typical of TCR transgenics (46, 47). In addition, for some experiments we bred HYcd4 mice onto the nonselecting MHC Class I Db-deficient (Dbo) background to eliminate the possibility of antigen presentation by HYcd4 donor-derived cells (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20080866/DC1). For a summary of mixed chimeras used, see Table I. To validate the use of the mixed BM chimera approach, we first examined the phenotype of HYcd4 cells undergoing positive selection and clonal deletion in mixed BM chimeras. When HYcd4 TCRαo female BM was mixed at a low ratio with congenically marked B6.PL female or male BM and transferred into female or male B6.PL hosts, respectively, we saw characteristic positive and negative selection in female and male mice. This included typical CD4 by CD8 profiles and activation of Caspase 3 (compare Figs. 1 and 3).


Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.

McCaughtry TM, Baldwin TA, Wilken MS, Hogquist KA - J. Exp. Med. (2008)

HYcd4 mixed BM chimeras as a model system to study clonal deletion in vivo. (A) HYcd4 TCRαo female BM was mixed with congenic B6.PL female and male BM and transferred into congenic female and male hosts. Chimeras were harvested at 6 wk and thymi were analyzed by flow cytometry for expression of Thy1.2, T3.70, CD4, CD8, and active Caspase 3. HYcd4 donor-derived cells were identified by Thy1.2 where indicated. Numbers represent frequency of parent gate. (B) The activation of Caspase 3 in male chimeras compared with female chimeras. The fold change in male over female is indicated. Data represent the mean from 15–17 individuals from six different experiments ± SD. P < 0.0001.
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fig3: HYcd4 mixed BM chimeras as a model system to study clonal deletion in vivo. (A) HYcd4 TCRαo female BM was mixed with congenic B6.PL female and male BM and transferred into congenic female and male hosts. Chimeras were harvested at 6 wk and thymi were analyzed by flow cytometry for expression of Thy1.2, T3.70, CD4, CD8, and active Caspase 3. HYcd4 donor-derived cells were identified by Thy1.2 where indicated. Numbers represent frequency of parent gate. (B) The activation of Caspase 3 in male chimeras compared with female chimeras. The fold change in male over female is indicated. Data represent the mean from 15–17 individuals from six different experiments ± SD. P < 0.0001.
Mentions: To further examine the anatomical location of clonal deletion and factors involved, we sought a system that allowed us to easily test various genetic deficiencies and to limit antigen presentation to particular subsets of cells. To do this, we used a mixed BM chimera strategy where HYcd4 female BM was mixed at a low ratio with competitor BM from WT mice and used to reconstitute female or male recipients. This experimental strategy has the additional advantage that the precursor frequency is reduced, thereby reducing clonal competition and correcting thymic architecture defects, which are typical of TCR transgenics (46, 47). In addition, for some experiments we bred HYcd4 mice onto the nonselecting MHC Class I Db-deficient (Dbo) background to eliminate the possibility of antigen presentation by HYcd4 donor-derived cells (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20080866/DC1). For a summary of mixed chimeras used, see Table I. To validate the use of the mixed BM chimera approach, we first examined the phenotype of HYcd4 cells undergoing positive selection and clonal deletion in mixed BM chimeras. When HYcd4 TCRαo female BM was mixed at a low ratio with congenically marked B6.PL female or male BM and transferred into female or male B6.PL hosts, respectively, we saw characteristic positive and negative selection in female and male mice. This included typical CD4 by CD8 profiles and activation of Caspase 3 (compare Figs. 1 and 3).

Bottom Line: However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation.We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation.Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c(+) cortical dendritic cells, and elimination of such cells impaired deletion.

View Article: PubMed Central - PubMed

Affiliation: Center for Immunology, Laboratory Medicine, and Pathology, University of Minnesota, Minneapolis, MN 55454, USA.

ABSTRACT
The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HY(cd4) model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c(+) cortical dendritic cells, and elimination of such cells impaired deletion.

Show MeSH
Related in: MedlinePlus