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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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Vγ5+ Thymocytes Express Rankl and Induce Aire+ mTEC Development(A) Reaggregate thymus organ cultures were prepared from either 2 dGuo-treated thymic stromal cells alone (left), or with added LTi (middle) or added Vγ5+ fetal thymocytes (right). In some cultures, recombinant OPG was added at a final concentration of 10 μg/ml (bottom). After 5 days, cultures were disaggregated, and FACS analysis is shown for EpCAM1 and nuclear Aire, gated on CD45−Ly51− cells. Numbers indicate percentages of cells.(B and C) Quantitative PCR analysis of Rankl is shown for thymocyte populations and total Vγ5+ thymocytes (B) and Rankl expression in thymocytes and CD45RB subsets of Vγ5+ thymocytes (C). Levels of mRNA were normalized to ACTB (β-actin).(D) Cell numbers of Aire+EpCAM1+Ly51− mTECs within freshly disaggregated E17 thymus lobes of the indicated mouse strains. Each point represents a single thymus lobe, with horizontal lines representing the mean. Asterisks indicate statistically significant differences; ∗∗∗p < 0.001, ∗∗p < 0.006, n.s., not significant.
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fig2: Vγ5+ Thymocytes Express Rankl and Induce Aire+ mTEC Development(A) Reaggregate thymus organ cultures were prepared from either 2 dGuo-treated thymic stromal cells alone (left), or with added LTi (middle) or added Vγ5+ fetal thymocytes (right). In some cultures, recombinant OPG was added at a final concentration of 10 μg/ml (bottom). After 5 days, cultures were disaggregated, and FACS analysis is shown for EpCAM1 and nuclear Aire, gated on CD45−Ly51− cells. Numbers indicate percentages of cells.(B and C) Quantitative PCR analysis of Rankl is shown for thymocyte populations and total Vγ5+ thymocytes (B) and Rankl expression in thymocytes and CD45RB subsets of Vγ5+ thymocytes (C). Levels of mRNA were normalized to ACTB (β-actin).(D) Cell numbers of Aire+EpCAM1+Ly51− mTECs within freshly disaggregated E17 thymus lobes of the indicated mouse strains. Each point represents a single thymus lobe, with horizontal lines representing the mean. Asterisks indicate statistically significant differences; ∗∗∗p < 0.001, ∗∗p < 0.006, n.s., not significant.

Mentions: To investigate the possibility that Vγ5+ DETC thymocyte progenitors influence the formation of embryonic mTEC microenvironments, we first made reaggregate thymus organ cultures (RTOCs) by using 2 dGuo fetal thymus lobes, known to contain the Rank+ progenitors of Aire+ mTECs (Rossi et al., 2007), into which either purified Vγ5+ thymocytes or LTi were added. After 5 days, RTOCs were disaggregated and analyzed by flow cytometry for the appearance of mature EpCAM1+Ly51−Aire+ mTECs. Consistent with our previous observations that mTEC progenitor development depends upon hematopoietic cell crosstalk (Rossi et al., 2007), Aire+ mTECs were absent in RTOCs initiated without added hematopoietic cells (Figure 2A, left) but were found to be present after the addition of LTi (Figure 2A, middle). Strikingly, analysis of RTOCs initiated with Vγ5+ thymocytes (Figure 2A, right) also induced the emergence of a defined cohort of EpCAM1+Ly51−Aire+ mature mTECs, providing direct evidence that DETC progenitors can influence the formation of embryonic medullary thymic microenvironments. Despite an approximate 100-fold difference in Rankl expression in LTi cells and Vγ5+ thymocytes (Figure 2B), both cell types induced a similar proportion of Aire+ mTECs in RTOC experiments (Figure 2A, middle and right). Importantly, RTOC experiments in which Rank-Rankl interactions were inhibited by addition of the soluble decoy receptor OPG completely abrogated Aire+ mTEC development induced by both Vγ5+ thymocytes and LTi cells (Figure 2A). Collectively, these experiments demonstrate the potency of Rank signaling in mTEC development and directly show that Rankl expression by Vγ5+ thymocytes and LTi cells underpins the ability of these cells to induce Aire+ mTEC development.


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)

Vγ5+ Thymocytes Express Rankl and Induce Aire+ mTEC Development(A) Reaggregate thymus organ cultures were prepared from either 2 dGuo-treated thymic stromal cells alone (left), or with added LTi (middle) or added Vγ5+ fetal thymocytes (right). In some cultures, recombinant OPG was added at a final concentration of 10 μg/ml (bottom). After 5 days, cultures were disaggregated, and FACS analysis is shown for EpCAM1 and nuclear Aire, gated on CD45−Ly51− cells. Numbers indicate percentages of cells.(B and C) Quantitative PCR analysis of Rankl is shown for thymocyte populations and total Vγ5+ thymocytes (B) and Rankl expression in thymocytes and CD45RB subsets of Vγ5+ thymocytes (C). Levels of mRNA were normalized to ACTB (β-actin).(D) Cell numbers of Aire+EpCAM1+Ly51− mTECs within freshly disaggregated E17 thymus lobes of the indicated mouse strains. Each point represents a single thymus lobe, with horizontal lines representing the mean. Asterisks indicate statistically significant differences; ∗∗∗p < 0.001, ∗∗p < 0.006, n.s., not significant.
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fig2: Vγ5+ Thymocytes Express Rankl and Induce Aire+ mTEC Development(A) Reaggregate thymus organ cultures were prepared from either 2 dGuo-treated thymic stromal cells alone (left), or with added LTi (middle) or added Vγ5+ fetal thymocytes (right). In some cultures, recombinant OPG was added at a final concentration of 10 μg/ml (bottom). After 5 days, cultures were disaggregated, and FACS analysis is shown for EpCAM1 and nuclear Aire, gated on CD45−Ly51− cells. Numbers indicate percentages of cells.(B and C) Quantitative PCR analysis of Rankl is shown for thymocyte populations and total Vγ5+ thymocytes (B) and Rankl expression in thymocytes and CD45RB subsets of Vγ5+ thymocytes (C). Levels of mRNA were normalized to ACTB (β-actin).(D) Cell numbers of Aire+EpCAM1+Ly51− mTECs within freshly disaggregated E17 thymus lobes of the indicated mouse strains. Each point represents a single thymus lobe, with horizontal lines representing the mean. Asterisks indicate statistically significant differences; ∗∗∗p < 0.001, ∗∗p < 0.006, n.s., not significant.
Mentions: To investigate the possibility that Vγ5+ DETC thymocyte progenitors influence the formation of embryonic mTEC microenvironments, we first made reaggregate thymus organ cultures (RTOCs) by using 2 dGuo fetal thymus lobes, known to contain the Rank+ progenitors of Aire+ mTECs (Rossi et al., 2007), into which either purified Vγ5+ thymocytes or LTi were added. After 5 days, RTOCs were disaggregated and analyzed by flow cytometry for the appearance of mature EpCAM1+Ly51−Aire+ mTECs. Consistent with our previous observations that mTEC progenitor development depends upon hematopoietic cell crosstalk (Rossi et al., 2007), Aire+ mTECs were absent in RTOCs initiated without added hematopoietic cells (Figure 2A, left) but were found to be present after the addition of LTi (Figure 2A, middle). Strikingly, analysis of RTOCs initiated with Vγ5+ thymocytes (Figure 2A, right) also induced the emergence of a defined cohort of EpCAM1+Ly51−Aire+ mature mTECs, providing direct evidence that DETC progenitors can influence the formation of embryonic medullary thymic microenvironments. Despite an approximate 100-fold difference in Rankl expression in LTi cells and Vγ5+ thymocytes (Figure 2B), both cell types induced a similar proportion of Aire+ mTECs in RTOC experiments (Figure 2A, middle and right). Importantly, RTOC experiments in which Rank-Rankl interactions were inhibited by addition of the soluble decoy receptor OPG completely abrogated Aire+ mTEC development induced by both Vγ5+ thymocytes and LTi cells (Figure 2A). Collectively, these experiments demonstrate the potency of Rank signaling in mTEC development and directly show that Rankl expression by Vγ5+ thymocytes and LTi cells underpins the ability of these cells to induce Aire+ mTEC development.

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