Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance

Abstract

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21⁺ mTECs and Aire⁺ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

Keywords: Aire; CCL21; CP: Immunology; Fezf2; autoimmunity; central tolerance; medullary thymic epithelial cell; negative selection; regulatory T cell; thymic tuft cell; thymus medulla.

Figure 1.. Tissue inflammation in mice deficient in Aire and/or CCL21

(A) Hematoxylin and eosin (H&E)-stained sections of the indicated tissues from female 20-week-old wild-type (WT), Aire knockout (KO), CCL21-KO, and Aire/ CCL21 double KO (DKO) mice. Representative data from at least 5 independent measurements (n = 5–8) are shown. Scale bars, 100 μm. (B) Pie chart showing the inflammation score of the indicated tissues from the indicated mice at 20 weeks of age. Each pie chart shows the scores for one mouse. Open and closed circles in the Aire-KO group indicate the GH strain and CB stain, respectively, as described in the STAR Methods. (C) Inflammation score of the indicated tissues from the indicated mice(n = 6–13) in at least 5 independent measurements at 10 weeks of age (lacrimal glands and salivary glands) and 20 weeks of age (retina, lungs, and liver). Means ± SEMs are plotted. **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figures S1 and S2.

Figure 2.. Tissue damage in mice deficient in Aire and/or CCL21

(A) Tear volume measured in the indicated mice. The length of cotton threads with absorbed tears was normalized to body weight (n = 6–8, 10–11 weeks old, 6 independent measurements). Plotted is mean ± SEM. ***p < 0.001, ****p < 0.0001. (B and C) Numbers of inflammation foci in lung (B) and liver (C) sections. (D) AST concentrations in serum of the indicated mice (n = 7–8, 20 weeks old, 2 independent measurements, mean ± SEM). ***p < 0.001. (E) Representative macroscopic images of spleens from the indicated mice. Scale intervals indicate 1 mm.

Figure 3.. Tissues in athymic mice transplanted with a thymus deficient in Aire and/ or CCL21

(A and B) Kidney of a B6-nude mouse 6 weeks after thymus transplantation. Shown are a macroscopic image (A) and H&E-stained sections (B) of the transplanted thymus under the kidney capsule in B6-nude mice. c, cortex. m, medulla. *, kidney. Scale bar, 100 μm. (C and D) H&E-stained liver sections from B6-nude mice transplanted with the thymus from the indicated mice. Sham operation mice received no thymus transplantation. Yellow arrowheads indicate foci, and green arrowheads indicate fibrosis (C). Representative data (n = 3–5) from 6 independent experiments are shown. Scale bars, 500 μm. Mean ± SEM of the number of foci per area (D) in the liver. *p < 0.05, ***p < 0.001; ns, not significant. See also Figures S3 and S4.

Figure 4.. Negative selection of thymocytes in mice deficient in Aire and/or CCL21

(A) Representative flow cytometry profiles for the detection of pMOG-tetramer-binding cells (top) and pApoB-tetramer-binding cells (bottom) in TCRβ^high CD4 single-positive (SP) thymocytes (PI⁻TCRβ^highCD4⁺CD8−CD90.2 ⁺CD11b⁻CD11c⁻B220⁻NK1.1−) from indicated mice. Numbers indicate the frequency of cells in the boxes. (B–D) Numbers (means ± SEs) of TCRβ^high CD4 SP thymocytes (B), pMOG-tetramer-binding TCRβ^high CD4 SP thymocytes (n = 6–11 in 9 independent measurements) (C), and pApoB-tetramer-binding TCRβ^high CD4 SP thymocytes (n = 4–11 in 7 independent measurements) (D). Shown are data obtained from more than 5.0 × 10⁷ total thymocytes. *p < 0.05, **p < 0.01.

Figure 5.. CD25⁺ Foxp3⁺ Treg cells in a thymus of mice deficient in Aire and/or CCL21

(A) Representative profiles for the detection of CD25⁺Foxp3⁺ Treg cells in TCRβ^high CD4 SP thymocytes from the indicated mice. Numbers indicate the frequency of cells in the boxes. (B and C) Number (mean ± SE) of CD25⁻Foxp3⁻ TCRβ^high CD4 SP thymocytes (B) and CD25⁺Foxp3⁺TCRβ^high CD4 SP Treg cells (C) in the indicated mice (n = 5–13, 8–11 weeks, 6 independent experiments). (D) Representative CD73 expression profiles of CD25⁺Foxp3⁺TCRβ^high CD4 SP Treg cells in the thymus and the spleen of the indicated mice. Numbers in boxes indicate frequencies of cells. (E and F) Number (mean ± SE) of CD73⁺CD25⁺Foxp3⁺TCRβ^high CD4 Treg cells in the thymus (n = 7–8,8–12 weeks, 4 independent experiments, E) and the spleen (n = 5–7, 8–12 weeks, 4 independent experiments, F). (G) Number (mean ± SE) of CD25⁻Foxp3⁺ TCRβ^high CD4 SP Treg cell precursor thymocytes in the indicated mice (n = 5–13, 8–11 week, 6 independent experiments). Ratios of cell numbers in comparison with the numbers in control WT mice are also shown. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 6.. Single-cell RNA sequencing analysis in Fezf2-deficient TECs

(A and B) Uniform manifold approximation and projection (UMAP) profiles of Fezf2 (A) and Aire (B) expression (red) in Epcam⁺Cd45⁻ TECs (gray) isolated from the indicated mice. (C and D) UMAP profile of Epcam⁺Cd45⁻ TECs (C) reveals cell clusters as indicated based on gene expression profiles shown in violin plots (D). Equivalent expression of Epcam and Foxn1 indicates that all 4 clusters are TECs (D). Selective expression of Psmb11, Prss16, Aire, Ccl21a, and Pou2f3 (D) identifies the cTEC cluster, Aire⁺ mTEC cluster, CCL21⁺ mTEC cluster, and mimetic mTEC cluster (C). Mice analyzed were Fezf2^flox/flox homozygotes in the absence or presence of the Foxn1-Cre-transgene and in the absence or presence of the β5t^iCre/+ heterozygous knockin allele (D). Psmb11 encodes β5t, and the β5t-iCre allele carries the iCre sequence knocked in in place of the Psmb11 sequence in the mouse genome. Consequently, Psmb11 transcripts in β5t^iCre/+ TECs were reduced to 51.6% of those in β5t^+/+ control mice (p < 0.001), whereas Psmb11 transcripts in Foxn1-Cre⁺ TECs were 89.3% of those in Foxn1-Cre⁻ control mice (not statistically significant) (D). β5t^iCre/+ mice, in which Psmb11 mRNA in cTECs is reduced to approximately half, did not show any reduction in β5t protein or its function. (E) Pie charts showing the proportions of the indicated clusters in Epcam⁺Cd45⁻ TECs from the indicated mice. Numbers of total Epcam⁺Cd45⁻ TECs in the indicated mice are shown in parentheses. (F-H) UMAP profiles of the indicated genes (red). (I) Immunofluorescence analysis of the thymus sections from Fezf2^flox/flox control mice, β5t^iCre/+ Fezf2^flox/flox TEC-specific Fezf2-deficient mice, and Pou2f3-deficient mice. DCLK1-expressing thymic tuft cells are detected in the Ly51⁻ medullary region in the thymus from control mice. Representative data from 3 individual measurements are shown. Scale bars, 250 μm. Plotted are the numbers of DCLK1⁺ cells in 1-mm² areas in the thymus medulla, counted in 3 areas per mouse (n = 9). See also Figures S5 and S6

Figure 7.. Lymphocyte infiltration in mice deficient in Aire and Fezf2

(A) H&E-stained sections of the indicated tissues from female 20-week-old WT; Aire KO; TEC-specific, Fezf2-deficient (Fezf2-TEC-KO); and Aire-KO/Fezf2-TEC-KO mice. Representative data from at least 3 independent measurements (n = 5–6) are shown. Scale bars, 100 μm. (B) Pie chart showing the inflammation score of the indicated tissues from the indicated mice at 20 weeks of age. Closed and open circles in Fezf2-TEC-KO and Aire-KO/Fezf2-TEC-KO groups indicate β5t^iCre/+ mice and Foxn1-Cre-transgenic mice, respectively. (C and D) Inflammation scores (C) and numbers of inflammation foci (D) of the indicated tissues from the indicated mice (n = 7–11) in at least 5 independent measurements at 20 weeks of age. Mean ± SEM is plotted. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S7.