Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla - PubMed (original) (raw)

Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla

Guillaume E Desanti et al. J Immunol. 2012.

Abstract

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.

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Figures

Figure 1. RANKL and CD40L Are Differentially Expressed In Innate Cells and αβTCR+ Thymocytes

Lymphoid Tissue inducer (LTi) cells (CD4+IL-7Rα+CD8α−B220−CD11c−TcRγδ−CD3ε−TCRβ−) and Vγ5+TCR (Vγ5+CD3ε+CD4−CD8α−) thymocytes from 7d FTOC (A), together with adult thymocyte subsets defined by expression of CD4, CD8 and TCRβ (B) were analysed for expression of RANKL and CD40L by flow cytometry. Note that gates are set using isotype control antibodies for RANKL, and cells obtained from _Cd40lg_−/− mice for CD40L expression. Panel C shows frequencies of CD40L+ and RANKL+ cells in adult thymocytes. Error bars represent S.E.M. Open bars, RANKL+CD40L− cells; hatched bars, RANKL+CD40L+ cells, grey bars RANKL−CD40L+ cells. Analysis of Vγ5+TCR and LTi cells was performed on pooled batches of approximately 60 and 100 FTOC, respectively. The dot plots shown are representative of two independent experiments. Dot plots and histograms representing RANKL and CD40L expressions on adult RAG2-GFP thymocytes are representative of two independent experiments with 4 mice analysed in each of these experiments.

Figure 2. RANKL and CD40L Define Early And Late Stages of αβT-cell Maturation

Panel A shows CD25 expression and intracellular FoxP3 expression in total CD4+8−TCRbhi adult thymocytes. In panel B, CD4+8−TCRβhi adult thymocytes from adult Rag2GFP mice were divided into CD25− and CD25+ subsets shown in the right dotplot of Panel A, and subsequently analysed for Rag2GFP in conjunction with CD69 expression. Panels C-D show frequencies of RANKL and CD40L expressing cells in the indicated CD25/RagGFP/CD69 subsets of CD4+8−TCRβhi thymocytes. Error bars represent S.E.M. Open bars, RANKL+CD40L− cells; hatched bars, RANKL+CD40L+ cells, grey bars RANKL−CD40L+ cells. The dot plot showing FoxP3 expression is representative of four independent experiments with one mouse analysed in each of these experiments. Dot plots and histograms showing RANKL and CD40L expression alongside CD25, GFP and CD69 on adult RAG2-GFP thymocytes are representative of two independent experiments with 4 mice analysed in each of these experiments.

Figure 3. CD40 Expression By mTEC Is Reduced In The Absence of The TNF-R Superfamily Member RANK

Panel A shows expression of MHC class II and CD80, used to define mTEClow and mTEChigh compartments, in total CD45−EpCAM+Ly51low mTEC within freshly digested thymuses from 3-4 week old WT and _Tnfrsf11a_−/− mice. mTEClow and mTEChigh fractions in WT and _Tnfrsf11a_−/− mice were compared for expression of CD40 (panel B), and Panel C shows Mean Fluorescence Intensity analysis of CD40 expression on the indicated mTEC subsets. Statistical analysis performed used a Mann-Whitney test (unpaired, two-tailed, 95% of confidence) with * indicating P<0.05 and N.S.: non-significant. Dot plots are representative of two independent experiments on 3 to 6 weeks old mice. In total, five mice of each group were studied.

Figure 4. RANK Stimulation of mTEC Induces CD40 Expression Prior to Aire

2-dGuo treated FTOC were cultured in either the absence (A) or presence (B and C) of 5mg/mL of agonistic anti-RANK antibody for the indicated time. Panel B shows FACS analysis of disaggregated lobes for CD40/CD80 (upper panels) and CD40/Aire expression (lower panels), after gating on CD45−EpCAM+Ly51− mTEC. Note the early induction of CD40 expression. Panel C shows qPCR analysis of Csna and Spt1 mRNA expression during the timecourse of RANK stimulation. Dot plots and qPCR are representative of two independent experiments, with PCR reactions performed in replicate. Three to four pooled 2-dGuo treated FTOC were required for each condition.

Figure 5. CD40-CD40L Interactions Control The Balance of Proliferation Within The mTEC Compartment

Panel A shows quantatitive analysis of Ki67+ cells within the total mTEC population, obtained by flow cytometry analysis, in WT and _Cd40lg_−/− mice. Panel B shows flow cyometric analysis of digested thymuses from 7-8 week old WT (upper panels) and _Cd40lg_−/− (lower panels) mice, gated on CD80− MHCIIlow and CD80+ MHCIIhigh subsets of CD45−EpCAM1+Ly51low mTEC. Staining is shown for levels for MHC class II and the proliferation marker Ki67. Panel C shows quantitative analysis of Ki67+ cells within the mTEClow and mTEChigh subsets in WT and _Cd40lg_−/− mice. Statistical analysis performed used a Mann-Whitney test (unpaired, two-tailed, 95% of confidence) with ** indicating P<0.005 and N.S.: non-significant. The histogram in panel A is a summary of two independent experiments. In total, five mice of each group were studied. The dot plots are representative of three independent experiments on 6-9 week old male mice. In total, seven WT and six _Cd40lg_−/− mice were studied.

Figure 6. CD40-CD40L Interactions Do Not Control The Rate Of Apoptosis Within The mTEC Compartment

Panel A shows flow cytometric analysis of DAPI/AnnexinV staining in digested thymuses from 11-15 week old WT (upper panels) and _Cd40lg_−/− (lower panels) mice, gated on MHC-IIlow and MHC-IIhigh subsets of CD45−EpCAM1+Ly51low mTEC. Panel B shows quantitative analysis of AnnexinV+ DAPI−/Int. cells within mTEClow and mTEChigh subsets in WT and _Cd40lg_−/− mice. Panel C shows quantitative analysis of AnnexinV+ DAPIhigh cells within mTEClow and mTEChigh subsets in WT and _Cd40lg_−/− mice. Statistical analysis performed used a Mann-Whitney test (unpaired, two-tailed, 95% of confidence) with N.S.: non-significant. The dot plots are representative of two independent experiments. The histograms are the summary of these two independent experiments. Three WT and three _Cd40lg_−/− mice were studied in each experiment.

Figure 7. The Involvement of RANK and CD40 During Fetal and Adult mTEC Development

A model of mTEC development involving the TNF-Receptor superfamily members RANK and CD40 is presented. During the fetal program of thymus development, the generation of the first cohorts of Aire+ mTEC involves RANK but not CD40 signalling, via interactions with RANKL+CD40L− innate like cells. In contrast, in an adult program of mTEC development, RANK signalling is followed by CD40 upregulation and signalling, a two-step process involving interactions with distinct CD4 thymocyte subsets.

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Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla - PubMed (original) (raw)