The thymic medulla: a unique microenvironment for intercellular self-antigen transfer

Abstract

Central tolerance is shaped by the array of self-antigens expressed and presented by various types of thymic antigen-presenting cells (APCs). Depending on the overall signal quality and/or quantity delivered in these interactions, self-reactive thymocytes either apoptose or commit to the T regulatory cell lineage. The cellular and molecular complexity underlying these events has only recently been appreciated. We analyzed the ex vivo presentation of ubiquitous or tissue-restricted self-antigens by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs), the two major APC types present in the medulla. We found that the ubiquitously expressed nuclear neo-self-antigen ovalbumin (OVA) was efficiently presented via major histocompatibility complex class II by mTECs and thymic DCs. However, presentation by DCs was highly dependent on antigen expression by TECs, and hemopoietic cells did not substitute for this antigen source. Accordingly, efficient deletion of OVA-specific T cells correlated with OVA expression by TECs. Notably, OVA was only presented by thymic but not peripheral DCs. We further demonstrate that thymic DCs are constitutively provided in situ with cytosolic as well as membrane-bound mTEC-derived proteins. The subset of DCs displaying transferred proteins was enriched in activated DCs, with these cells being most efficient in presenting TEC-derived antigens. These data provide evidence for a unique, constitutive, and unidirectional transfer of self-antigens within the thymic microenvironment, thus broadening the cellular base for tolerance induction toward promiscuously expressed tissue antigens.

Figure 1.

Efficient presentation of TEC-derived self-antigens by thymic DCs. mTECs and DCs from the thymus (Thy-DC), spleen (Sp-DC), and lymph nodes (LN-DC) of (A) L^d-nOVA, (C) BALB/c, or (E) C57BL/6 mice, respectively, were co-cultured for 3 d with antigen-specific TCs. Presentation of OVA (A), P1A (C), and PLP (E) was assessed by antigen-specific proliferation of naive DO11.10 CD4⁺ TCs, P1A-TCR transgenic CD8⁺ TCs, or the PLP-specific TC line TPLP-15-5-2. Shown are the triplicate mean values ± SD of one representative of at least two experiments performed. Transcription of (B) OVA, (D) P1A, and (F) DM20 by the indicated cell populations was evaluated by quantitative (B and F) or semiquantitative (D) PCR analysis. Expression levels were normalized to HPRT/Ubiquitin (B) or β-actin (D and F). Error bars in B and F indicate the SD of triplicates of the same cDNA preparation.

Figure 2.

Ex vivo presentation of the nuclear neo–self-antigen OVA by thymic DCs and efficient deletion of DO11.10 TCs in vivo depend on antigen transfer from radio-resistant stromal cells. (A) Lethally irradiated BALB/c WT or L^d-nOVA transgenic mice were reconstituted with TC-depleted BM cells of the indicated donor strains. After 5–8 wk, CD11c⁺ DCs of the thymus (Thy-DC) and spleen (Sp-DC) and mTECs, respectively, were isolated and co-cultured with DO11.10 CD4⁺ TCs. OVA presentation was assessed by antigen-specific proliferation of TCs. Bars represent threefold titration steps of the indicated APC population ranging from 3 × 10⁵–10³ cells/well. Shown are the triplicate mean values ± SD of one representative of two or more experiments performed. (B) 5–6 wk after reconstitution, the number of total CD4 single-positive and DO11.10 clonotype-specific CD4 single-positive thymocytes was assessed. Symbols represent values of 7–10 individual thymi in each group. *, P < 0.005. (C) FACS analysis of clonotypic DO11.10 CD4 single-positive spleen TCs of each chimera type. Shown are overlay histograms of three individuals. Arrows indicate DO11.10 clonotype-specific CD4⁺ TCs.

Figure 3.

Cotransfer of membrane-bound and cytoplasmic proteins from epithelial cells marks DCs with efficient cross-presentation. (A and B) Lethally irradiated CB6/F1 mice were reconstituted with either BALB/c (A) or C57BL/6 (B) TC-depleted BM. After 6 wk, low density TSCs were enriched by Nycodenz density gradient centrifugation. Cells were stained with mAb against CD11c, EpCAM, and the parental MHC class II haplotypes I-A^b and I-A^d. Thymic DCs were defined as CD11c^high EpCAM^intermediate cells. Expression of the respective host-specific MHC haplotype by thymic DCs is displayed as shaded histograms, whereas lines represent expression of the indicated haplotypes by C57BL/6 (solid), CB6/F1 (dashed), or BALB/c (dotted) thymic DCs. (C) EGFP-containing cells are detectable within the CD11c⁺ thymic DC population of FoxN1-eGFP mice. Thymic CD11c⁺ cells were preenriched using MACS technology and analyzed by flow cytometry, excluding autofluorescent cells. (D) EGFP-expressing TECs are distinguishable from eGFP-containing thymic DCs on the basis of differential CD11c expression, as shown by flow-cytometric analysis of single-cell suspensions. Cells were isolated and pooled from five FoxN1-eGFP thymi. (E) EpCAM expression of FoxN1-eGFP CD11c⁺ thymic DCs correlates with eGFP fluorescence intensity, as shown for CD45⁻, nonautofluorescent cells. (F) EpCAM-expressing CD11c⁺ thymic DCs (Thy-DC) present TEC-derived OVA most efficiently. CD11c⁺ thymic DCs of L^d-nOVA mice were enriched according to differential CD11c and EpCAM coexpression, resulting in the indicated subpopulations of ≥99% purity. CD11c⁺ splenic DCs (Sp-DC) were enriched by magnetic separation (purity ≥ 90%). OVA presentation was assessed by proliferation of OVA-specific DO11.10 CD4⁺ TCs after a 3-d co-culture period. Shown are the means of triplicates ± SD. Each figure (C–F) is representative of two to six independent experiments.

Figure 4.

EGFP-containing thymic DCs of FoxN1-eGFP mice display a mature phenotype. TSCs of FoxN1-eGFP mice were preenriched for CD11c⁺ cells using MACS technology (purity ≥ 60%). Subsequently, positively selected cells were stained for CD11c and EpCAM (Fig. 3 B) together with one additional marker and analyzed by flow cytometry. Shaded histograms indicate negative controls representing isotype control or secondary antibody stainings. Expression of indicated marker molecules by eGFP⁻ TSCs is displayed in red and expression by eGFP⁺ cells is displayed in blue. Arrows indicate marker profiles correlating with eGFP content. For details see Materials and methods. Each histogram is representative of one to three independent experiments.