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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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Apoptosis occurs asynchronously in vivo, with some cells surviving up to 4 d. BrdU was injected i.p. at the indicated number of hours before harvest. (A) The expression of CD4 and CD8 on BrdU+ T3.70+ thymocytes from representative mice from the indicated time point. (B) Expression of CD5, CD69, and PD-1 on BrdU+ T3.70+ thymocytes over time. (C) The total number of BrdU+ T3.70+ thymocytes versus time after BrdU injection. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0118 at 48 h, P = 0.0070 at 72 h, and P = 0.0037 at 96 h. (D) The frequency of active Caspase 3+ cells as a percentage of BrdU+ T3.70+ thymocytes. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0002 at 12 h, P < 0.0001 at 24 h, P = 0.0004 at 48 h, and P = 0.0061 at 72 h. Activation of Caspase 3 in vitro was determined by stimulating female HYcd4 thymocytes with HYp peptide plus spleen APC for 8 h. Data were normalized for nonspecific death caused by in vitro culture and represent the mean ± SD from triplicate wells.
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fig2: Apoptosis occurs asynchronously in vivo, with some cells surviving up to 4 d. BrdU was injected i.p. at the indicated number of hours before harvest. (A) The expression of CD4 and CD8 on BrdU+ T3.70+ thymocytes from representative mice from the indicated time point. (B) Expression of CD5, CD69, and PD-1 on BrdU+ T3.70+ thymocytes over time. (C) The total number of BrdU+ T3.70+ thymocytes versus time after BrdU injection. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0118 at 48 h, P = 0.0070 at 72 h, and P = 0.0037 at 96 h. (D) The frequency of active Caspase 3+ cells as a percentage of BrdU+ T3.70+ thymocytes. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0002 at 12 h, P < 0.0001 at 24 h, P = 0.0004 at 48 h, and P = 0.0061 at 72 h. Activation of Caspase 3 in vitro was determined by stimulating female HYcd4 thymocytes with HYp peptide plus spleen APC for 8 h. Data were normalized for nonspecific death caused by in vitro culture and represent the mean ± SD from triplicate wells.

Mentions: 2 h after BrdU injection, the majority of labeled thymocytes in both female and male HYcd4 mice were DP (Fig. 2 A), as in WT mice (44). Approximately half of these were T3.70+ in both female and male mice (unpublished data). Although the T3.70+ BrdU+ DPs in female mice did not express CD69 at the 2-h time point, we were surprised to observe that ∼60% of the T3.70+ BrdU+ DPs were already CD69+ in male mice (Fig. 2 B). This indicates a very rapid and efficient response to male antigen very early in the lifespan of the DP thymocyte. Indeed, by 12 h, most labeled T3.70+ DPs expressed a high level of CD69 in male mice. Because there was not a substantial lag in either the expression of HY TCRα or response to male antigen in labeled cells, this was an effective means to measure the kinetics of clonal deletion in vivo. Importantly, we also noted the presence of T3.70+ BrdU+ DPs that were CD69− in male mice at the earliest time points, which suggests that the cd4-mediated expression of the TCRα transgene is in fact delayed until the transition of thymocytes to the DP stage.


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)

Apoptosis occurs asynchronously in vivo, with some cells surviving up to 4 d. BrdU was injected i.p. at the indicated number of hours before harvest. (A) The expression of CD4 and CD8 on BrdU+ T3.70+ thymocytes from representative mice from the indicated time point. (B) Expression of CD5, CD69, and PD-1 on BrdU+ T3.70+ thymocytes over time. (C) The total number of BrdU+ T3.70+ thymocytes versus time after BrdU injection. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0118 at 48 h, P = 0.0070 at 72 h, and P = 0.0037 at 96 h. (D) The frequency of active Caspase 3+ cells as a percentage of BrdU+ T3.70+ thymocytes. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0002 at 12 h, P < 0.0001 at 24 h, P = 0.0004 at 48 h, and P = 0.0061 at 72 h. Activation of Caspase 3 in vitro was determined by stimulating female HYcd4 thymocytes with HYp peptide plus spleen APC for 8 h. Data were normalized for nonspecific death caused by in vitro culture and represent the mean ± SD from triplicate wells.
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Related In: Results  -  Collection

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fig2: Apoptosis occurs asynchronously in vivo, with some cells surviving up to 4 d. BrdU was injected i.p. at the indicated number of hours before harvest. (A) The expression of CD4 and CD8 on BrdU+ T3.70+ thymocytes from representative mice from the indicated time point. (B) Expression of CD5, CD69, and PD-1 on BrdU+ T3.70+ thymocytes over time. (C) The total number of BrdU+ T3.70+ thymocytes versus time after BrdU injection. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0118 at 48 h, P = 0.0070 at 72 h, and P = 0.0037 at 96 h. (D) The frequency of active Caspase 3+ cells as a percentage of BrdU+ T3.70+ thymocytes. Data represent the mean ± SD from four separate time courses including three to seven individual mice. *, P = 0.0002 at 12 h, P < 0.0001 at 24 h, P = 0.0004 at 48 h, and P = 0.0061 at 72 h. Activation of Caspase 3 in vitro was determined by stimulating female HYcd4 thymocytes with HYp peptide plus spleen APC for 8 h. Data were normalized for nonspecific death caused by in vitro culture and represent the mean ± SD from triplicate wells.
Mentions: 2 h after BrdU injection, the majority of labeled thymocytes in both female and male HYcd4 mice were DP (Fig. 2 A), as in WT mice (44). Approximately half of these were T3.70+ in both female and male mice (unpublished data). Although the T3.70+ BrdU+ DPs in female mice did not express CD69 at the 2-h time point, we were surprised to observe that ∼60% of the T3.70+ BrdU+ DPs were already CD69+ in male mice (Fig. 2 B). This indicates a very rapid and efficient response to male antigen very early in the lifespan of the DP thymocyte. Indeed, by 12 h, most labeled T3.70+ DPs expressed a high level of CD69 in male mice. Because there was not a substantial lag in either the expression of HY TCRα or response to male antigen in labeled cells, this was an effective means to measure the kinetics of clonal deletion in vivo. Importantly, we also noted the presence of T3.70+ BrdU+ DPs that were CD69− in male mice at the earliest time points, which suggests that the cd4-mediated expression of the TCRα transgene is in fact delayed until the transition of thymocytes to the DP stage.

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