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Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

Roberts NA, White AJ, Jenkinson WE, Turchinovich G, Nakamura K, Withers DR, McConnell FM, Desanti GE, Benezech C, Parnell SM, Cunningham AF, Paolino M, Penninger JM, Simon AK, Nitta T, Ohigashi I, Takahama Y, Caamano JH, Hayday AC, Lane PJ, Jenkinson EJ, Anderson G - Immunity (2012)

Bottom Line: The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance.In turn, generation of Aire(+) mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire.Hence, our data attributed a functional importance to the temporal development of Vγ5(+) γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Immune Regulation, University of Birmingham, Birmingham, B15 2TT, UK.

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Related in: MedlinePlus

In Fetal Thymus, Vγ5+ Thymocytes Associate with Developing Aire-Expressing Medullary Environments(A) E17 thymus lobes from B6 mouse embryos were stained with antibodies to CD8 (white), Vγ5TCR (green), and EpCAM1 (red). CD8 expression denotes the cortical areas containing CD4+CD8+ thymocytes.(B) Quantitation of the distribution of Vγ5+ thymocytes in cortical and medullary areas of E17 thymic sections. Each point represents an individual thymus lobe, and horizontal lines on the graph represent the mean.(C) An image of a frozen section of E17 thymus stained for CD8 (white), CD45RB (red), and Vγ5TCR (green).(D) Quantitation of the frequency of RORγ+IL-7Rα+ LTi and Vγ5TCR+ thymocytes in E17 thymic medullary regions, with each point representing an individual thymus lobe, and horizontal lines on the graph representing the mean.(E) Image of an E17 frozen thymus section for CD8 (white), Aire (red), and Vγ5TCR (green).(F) Confocal image of a medullary area within an E17 thymus section stained with antibodies to RORγ (green), IL-7Rα (yellow), TR5 (red), γδTCR (blue), and Aire (white). Nuclei are stained with DAPI (gray). For clarity, a series of images with various marker combinations is shown.
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fig1: In Fetal Thymus, Vγ5+ Thymocytes Associate with Developing Aire-Expressing Medullary Environments(A) E17 thymus lobes from B6 mouse embryos were stained with antibodies to CD8 (white), Vγ5TCR (green), and EpCAM1 (red). CD8 expression denotes the cortical areas containing CD4+CD8+ thymocytes.(B) Quantitation of the distribution of Vγ5+ thymocytes in cortical and medullary areas of E17 thymic sections. Each point represents an individual thymus lobe, and horizontal lines on the graph represent the mean.(C) An image of a frozen section of E17 thymus stained for CD8 (white), CD45RB (red), and Vγ5TCR (green).(D) Quantitation of the frequency of RORγ+IL-7Rα+ LTi and Vγ5TCR+ thymocytes in E17 thymic medullary regions, with each point representing an individual thymus lobe, and horizontal lines on the graph representing the mean.(E) Image of an E17 frozen thymus section for CD8 (white), Aire (red), and Vγ5TCR (green).(F) Confocal image of a medullary area within an E17 thymus section stained with antibodies to RORγ (green), IL-7Rα (yellow), TR5 (red), γδTCR (blue), and Aire (white). Nuclei are stained with DAPI (gray). For clarity, a series of images with various marker combinations is shown.

Mentions: We previously showed that generation of the first cohorts of Aire+ mTECs in the embryonic thymus involves signals from Rankl+ LTi cells (Rossi et al., 2007), a population originally identified as a key player in the development of stromal microenvironments within secondary lymphoid tissues such as lymph node (Mebius, 2003). However, when we analyzed thymus medulla formation in Rorc−/− embryonic mice lacking LTi, we found Aire+ mTECs still present, albeit at reduced numbers (White et al., 2008). This nonessential role for LTi cannot be explained by Rankl provision by positively selected αβTCR+ thymocytes, because Aire+ mTECs are present in Rorc−/− embryonic mice prior to αβ T cell selection (White et al., 2008). To investigate the possibility that previously unidentified cell types influence initial thymus medulla formation in the developing embryonic thymus, we screened the cellular makeup of medullary areas in E17 thymus tissue sections. Of the cell types analyzed, and consistent with an earlier report (Farr et al., 1990), a pan-γδTCR antibody defined a striking concentration of γδTCR+ thymocytes within developing embryonic EpCAM1+ medullary areas (not shown). Moreover, by using a Vγ5TCR-specific antibody, we found that essentially all of the medullary-resident γδTCR+ thymocytes at this stage expressed the Vγ5TCR (Figures 1A and 1B), representing DETC progenitors. Further analysis showed that some medullary-resident Vγ5+ T cells expressed high levels of CD45RB (Figure 1C), a maturational marker of intrathymic DETC progenitors (Lewis et al., 2006), suggestive of a link between thymus medulla development and DETC progenitor maturation. Confocal analysis of embryonic thymus sections stained to reveal the localization and frequency of Vγ5+ thymocytes and RORγ+CD4+CD3−IL-7Rα+ LTi showed an abundance of Vγ5+ thymocytes relative to LTi cells, with quantitative analysis indicating an approximate 100:1 ratio for Vγ5+ thymocytes:LTi within medullary areas (Figure 1D). Importantly, dual staining with antibodies to Aire and Vγ5TCR revealed individual medullary areas containing both Aire+ mTECs and Vγ5+ thymocytes (Figure 1E), whereas staining with Aire and pan-γδTCR antibodies together with IL-7Rα and RORγ antibodies to reveal IL-7Rα+RORγ+ LTi demonstrated the presence of both γδTCR+ thymocytes and LTi within individual Aire-expressing medullary areas (Figure 1F). Analysis of the anatomical distribution of LTi and Vγ5 thymocytes in relation to Aire+ mTECs in thymus tissue sections failed to reveal a defined pattern to the distribution of these cells within multiple medullary areas (not shown). However, although there may be no difference in the topological positioning of these cells in the thymic medulla, the combined presence of Vγ5+ thymocytes and LTi cells within individual medullary areas suggests that they act collectively to influence mTEC development. Taken together, these results indicate that in the fetal thymus, in addition to the presence of LTi cells, there is an anatomical association between fetal-specific Vγ5 thymocytes and thymic medullary epithelial cells.


Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

Roberts NA, White AJ, Jenkinson WE, Turchinovich G, Nakamura K, Withers DR, McConnell FM, Desanti GE, Benezech C, Parnell SM, Cunningham AF, Paolino M, Penninger JM, Simon AK, Nitta T, Ohigashi I, Takahama Y, Caamano JH, Hayday AC, Lane PJ, Jenkinson EJ, Anderson G - Immunity (2012)

In Fetal Thymus, Vγ5+ Thymocytes Associate with Developing Aire-Expressing Medullary Environments(A) E17 thymus lobes from B6 mouse embryos were stained with antibodies to CD8 (white), Vγ5TCR (green), and EpCAM1 (red). CD8 expression denotes the cortical areas containing CD4+CD8+ thymocytes.(B) Quantitation of the distribution of Vγ5+ thymocytes in cortical and medullary areas of E17 thymic sections. Each point represents an individual thymus lobe, and horizontal lines on the graph represent the mean.(C) An image of a frozen section of E17 thymus stained for CD8 (white), CD45RB (red), and Vγ5TCR (green).(D) Quantitation of the frequency of RORγ+IL-7Rα+ LTi and Vγ5TCR+ thymocytes in E17 thymic medullary regions, with each point representing an individual thymus lobe, and horizontal lines on the graph representing the mean.(E) Image of an E17 frozen thymus section for CD8 (white), Aire (red), and Vγ5TCR (green).(F) Confocal image of a medullary area within an E17 thymus section stained with antibodies to RORγ (green), IL-7Rα (yellow), TR5 (red), γδTCR (blue), and Aire (white). Nuclei are stained with DAPI (gray). For clarity, a series of images with various marker combinations is shown.
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fig1: In Fetal Thymus, Vγ5+ Thymocytes Associate with Developing Aire-Expressing Medullary Environments(A) E17 thymus lobes from B6 mouse embryos were stained with antibodies to CD8 (white), Vγ5TCR (green), and EpCAM1 (red). CD8 expression denotes the cortical areas containing CD4+CD8+ thymocytes.(B) Quantitation of the distribution of Vγ5+ thymocytes in cortical and medullary areas of E17 thymic sections. Each point represents an individual thymus lobe, and horizontal lines on the graph represent the mean.(C) An image of a frozen section of E17 thymus stained for CD8 (white), CD45RB (red), and Vγ5TCR (green).(D) Quantitation of the frequency of RORγ+IL-7Rα+ LTi and Vγ5TCR+ thymocytes in E17 thymic medullary regions, with each point representing an individual thymus lobe, and horizontal lines on the graph representing the mean.(E) Image of an E17 frozen thymus section for CD8 (white), Aire (red), and Vγ5TCR (green).(F) Confocal image of a medullary area within an E17 thymus section stained with antibodies to RORγ (green), IL-7Rα (yellow), TR5 (red), γδTCR (blue), and Aire (white). Nuclei are stained with DAPI (gray). For clarity, a series of images with various marker combinations is shown.
Mentions: We previously showed that generation of the first cohorts of Aire+ mTECs in the embryonic thymus involves signals from Rankl+ LTi cells (Rossi et al., 2007), a population originally identified as a key player in the development of stromal microenvironments within secondary lymphoid tissues such as lymph node (Mebius, 2003). However, when we analyzed thymus medulla formation in Rorc−/− embryonic mice lacking LTi, we found Aire+ mTECs still present, albeit at reduced numbers (White et al., 2008). This nonessential role for LTi cannot be explained by Rankl provision by positively selected αβTCR+ thymocytes, because Aire+ mTECs are present in Rorc−/− embryonic mice prior to αβ T cell selection (White et al., 2008). To investigate the possibility that previously unidentified cell types influence initial thymus medulla formation in the developing embryonic thymus, we screened the cellular makeup of medullary areas in E17 thymus tissue sections. Of the cell types analyzed, and consistent with an earlier report (Farr et al., 1990), a pan-γδTCR antibody defined a striking concentration of γδTCR+ thymocytes within developing embryonic EpCAM1+ medullary areas (not shown). Moreover, by using a Vγ5TCR-specific antibody, we found that essentially all of the medullary-resident γδTCR+ thymocytes at this stage expressed the Vγ5TCR (Figures 1A and 1B), representing DETC progenitors. Further analysis showed that some medullary-resident Vγ5+ T cells expressed high levels of CD45RB (Figure 1C), a maturational marker of intrathymic DETC progenitors (Lewis et al., 2006), suggestive of a link between thymus medulla development and DETC progenitor maturation. Confocal analysis of embryonic thymus sections stained to reveal the localization and frequency of Vγ5+ thymocytes and RORγ+CD4+CD3−IL-7Rα+ LTi showed an abundance of Vγ5+ thymocytes relative to LTi cells, with quantitative analysis indicating an approximate 100:1 ratio for Vγ5+ thymocytes:LTi within medullary areas (Figure 1D). Importantly, dual staining with antibodies to Aire and Vγ5TCR revealed individual medullary areas containing both Aire+ mTECs and Vγ5+ thymocytes (Figure 1E), whereas staining with Aire and pan-γδTCR antibodies together with IL-7Rα and RORγ antibodies to reveal IL-7Rα+RORγ+ LTi demonstrated the presence of both γδTCR+ thymocytes and LTi within individual Aire-expressing medullary areas (Figure 1F). Analysis of the anatomical distribution of LTi and Vγ5 thymocytes in relation to Aire+ mTECs in thymus tissue sections failed to reveal a defined pattern to the distribution of these cells within multiple medullary areas (not shown). However, although there may be no difference in the topological positioning of these cells in the thymic medulla, the combined presence of Vγ5+ thymocytes and LTi cells within individual medullary areas suggests that they act collectively to influence mTEC development. Taken together, these results indicate that in the fetal thymus, in addition to the presence of LTi cells, there is an anatomical association between fetal-specific Vγ5 thymocytes and thymic medullary epithelial cells.

Bottom Line: The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance.In turn, generation of Aire(+) mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire.Hence, our data attributed a functional importance to the temporal development of Vγ5(+) γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Immune Regulation, University of Birmingham, Birmingham, B15 2TT, UK.

Show MeSH
Related in: MedlinePlus