Generation of both cortical and Aire(+) medullary thymic epithelial compartments from CD205(+) progenitors

Abstract

In the adult thymus, the development of self-tolerant thymocytes requires interactions with thymic epithelial cells (TECs). Although both cortical and medullary TECs (cTECs/mTECs) are known to arise from common bipotent TEC progenitors, the phenotype of these progenitors and the timing of the emergence of these distinct lineages remain unclear. Here, we have investigated the phenotype and developmental properties of bipotent TEC progenitors during cTEC/mTEC lineage development. We show that TEC progenitors can undergo a stepwise acquisition of first cTEC and then mTEC hallmarks, resulting in the emergence of a progenitor population simultaneously expressing the cTEC marker CD205 and the mTEC regulator Receptor Activator of NF-κB (RANK). In vivo analysis reveals the capacity of CD205(+) TECs to generate functionally competent cortical and medullary microenvironments containing both cTECs and Aire(+) mTECs. Thus, TEC development involves a stage in which bipotent progenitors can co-express hallmarks of the cTEC and mTEC lineages through sequential acquisition, arguing against a simple binary model in which both lineages diverge simultaneously from bipotent lineage negative TEC progenitors. Rather, our data reveal an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage-restricted counterparts.

Figure 1

Emergence of the CD205⁺ TEC compartment during thymus ontogeny. (A) Thymus/parathyroid rudiments from Ly75^−/− embryos were analyzed for CD205 and CD40 after gating on EpCAM1⁺CD45⁻ TECs. (B) FoxN1:eGFP embryos were analyzed for CD205 and CD40 expression, and data are shown after gating on EpCAM1⁺CD45⁻FoxN1:eGFP⁺ cells. (C) WT E13-E17 thymus rudiments were isolated and analyzed as above. (D) E11 and (E) E12 thymus/parathyroid rudiments from FoxN1:eGFP embryos were cultured for 1 day in the presence (bottom) or absence (top) of anti-RANK, and FoxN1:eGFP⁺ and FoxN1:eGFP⁻ EpCAM1⁺ subsets were analyzed for CD40 and MHC class II expression. Gates to discriminate FoxN1:eGFP⁺ and FoxN1:eGFP⁻ TEC subsets were set using WT thymus preparations. Data shown are representative of at least three separate experiments.

Figure 2

A TEC subset expressing the cTEC marker CD205 responds to RANK stimulation. (A, B) FoxN1:eGFP thymus/parathyroid rudiments from (A) E11 and (B) E12 embryos were cultured for 1 day in the absence (top) or presence (bottom) of 10 μg/mL anti-RANK. CD205 expression is shown following gating on EpCAM1⁺CD45⁻FoxN1eGFP⁺ cells, with CD205⁻ CD205^Low and CD205^High subsets analyzed for MHC class II and CD40 expression. (C) Quantitative analysis of CD40 and MHC class II induction in the presence (black bars) or absence (open bars) of RANK stimulation in E12 FoxN1:eGFP⁺ TECs is shown. Data are shown as mean + SEM of n = 4 and are representative of three experiments performed. An unpaired student t-test was performed, **p < 0.005 and ***p < 0.0005.

Figure 3

CD205⁺ TECs generate both cTECs and Aire⁺ mTECs in vivo. (A) E15 thymus lobes stained for CD45, EpCAM1, CD40, and CD205 before (top) and after (bottom) cell sorting are shown. (B) Confocal microscopy of grafts initiated from CD205⁺40⁻ TECs, after transplantation into WT mice is shown. “C” denotes cortex, “M” denotes medulla. Top left panel is a composite tile scan at 250× final magnification, top right and lower panels taken at 250× and 400× final magnification, respectively. (C) Flow cytometric analysis of the EpCAM1⁺CD45⁻ TEC compartment of CD205⁺40⁻ TEC grafts (bottom), with unmanipulated dGuo FTOC grafts as comparison (top) is shown. (D) Analysis of thymocytes from 6-week dGuo FTOC grafts (top) and CD205⁺CD40⁻ E15 TEC grafts (bottom) for the expression of CD4, CD8, FoxP3, and CD25 is shown. Dot plots (right) show FoxP3/CD25 expression in CD4⁺8⁻αβTCR^hi cells. Data shown are representative of at least three experiments.