Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages

Abstract

Interactions of developing T cells with Aire⁺ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEC^hi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire⁺ mTEC^hi. However, it remains unknown whether thymocytes control the precursors of Aire⁺ mTEC^hi that are contained in mTEC^lo cells or other mTEC^lo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4⁺ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4⁺ thymocytes induce key transcriptional regulators in mTEC^lo and control the composition of mTEC^lo subsets, including Aire⁺ mTEC^hi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEC^lo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4⁺ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4⁺ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.

Keywords: T cells; cellular crosstalk; immunology; inflammation; medullary thymic epithelial cells; mouse; thymus; tolerance.

Figure 1.. The transcriptional profile and IKKα and p38 MAPK signaling pathways are impaired in mTEC^lo of ΔCD4 mice.

(A, B) Total IKKα, p38 MAPK, phospho-IKKα(Ser180)/IKKβ(Ser181), and p38 MAPK (Thr180/Tyr182) (A) and the ratio of phospho/total proteins (B) analyzed by flow cytometry in mTEC^lo from WT and ΔCD4 mice. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). (C) Scatter plot of gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) of mTEC^lo from WT versus ΔCD4 mice. Genes with fold difference ≥2 and p-adj<0.05 were considered as upregulated or downregulated genes (red and blue dots, respectively). RNA-seq was performed on two independent biological replicates with mTEC^lo derived from 3 to 5 mice. (D) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated (left panel) and the proportion of upregulated TRAs compared to those in the all genome (right panel). ND, not determined. (E) Numbers of induced Aire-dependent, Fezf2-dependent, Aire/Fezf2-dependent, and Aire/Fezf2-independent TRAs. (F) The expression of Aire-dependent (Meig1, Nov), Fezf2-dependent (Fcer2a, Kcnj5), Aire/Fezf2-dependent (Krt1, Reig1), and Aire/Fezf2-independent (Crp, Rsph1) TRAs measured by qPCR in WT (n = 3–4) and ΔCD4 (n = 3–4) mTEC^lo. (G) Expression fold change in HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus ΔCD4 mTEC^lo. The color code represents gene expression level. (H) Heatmaps of genes encoding for cell adhesion molecules and cytokines that were significantly downregulated in mTEC^lo from ΔCD4 mice. (I) Hierarchical clustering and heatmap of mean expression of these cell adhesion molecules and cytokines in mTEC subsets identified by scRNA-seq. Error bars show mean ± SEM, *p<0.05, **p<0.01 using two-tailed Mann–Whitney test for (A), (B) and (F) and chi-squared test for (D).

Figure 1—figure supplement 1.. Gating strategy used to purify mTEC^lo cells.

(A) Total thymic epithelial cells (TECs) were defined as EpCAM⁺ in CD45-negative enriched thymic cells by autoMACS and were further divided into medullary TECs (mTECs) (UEA-1⁺Ly51^lo) and cortical TECs (cTECs) (UEA-1⁻Ly51^hi). mTEC^lo were identified and sorted based on low/intermediate levels of the CD80 co-stimulatory molecule. The purity of sorted mTEC^lo was >98%. (B) Gates used to sort mTEC^lo from WT, ΔCD4, mTEC^ΔMHCII, RipmOVAxOTII-Rag2^-/-, and OTII-Rag2^-/- mice.

Figure 1—figure supplement 2.. Normal total and phosphorylated p65, RelB, and Erk1/2 proteins in mTEC^lo from ΔCD4 mice.

Total p65, RelB, and Erk1/2 (A) and phospho-p65 (Ser536), phospho-RelB (Ser552), and phospho-Erk1/2 (Thr202/Tyr204) (B) proteins were analyzed by flow cytometry in mTEC^lo of WT and ΔCD4 mice. Histograms show the MFI. II Abs: secondary antibodies. Data are representative of two independent experiments (n = 2–3 mice per group and experiment). Error bars show mean ± SEM, *p<0.05 using the Mann–Whitney test.

Figure 1—figure supplement 3.. Impaired TRA expression in mTEC^lo from MHCII^-/- mice.

(A) The expression of Aire-dependent (Meig1, Nov), Fezf2-dependent (Fcer2a, Kcnj5), Aire/Fezf2-dependent (Krt1, Reig1), and Aire/Fezf2-independent (Crp, Rsph1) tissue-restricted self-antigens (TRAs) was measured by qPCR in purified mTEC^lo from WT (n = 3), ΔCD4 (n = 3), and MHCII^-/- (n = 3) mice. (B) Itgb6, Cdh2, Il21, and Il5 mRNAs were measured by qPCR in WT (n = 4) and ΔCD4 (n = 4) mTEC^lo.

Figure 1—figure supplement 4.. Identification of thymic epithelial cell (TEC) subsets by single-cell RNA-seq.

(A) UMAP visualization of single-cell RNA-seq data on TECs reanalyzed from Wells et al., 2020. Six clusters were identified corresponding to cortical TECs (cTECs), CCL21⁺ medullary TECs (mTECs) (mTEC I), TAC-TECs (transit-amplifying cells), Aire⁺ (mTEC II), post-Aire (mTEC III), and Tuft-like (mTEC IV) mTECs. (B) Expression of selected marker genes overlaid on UMAP visualization. Scale bars represent the log2 expression of the indicated genes.

Figure 2.. The transcriptional and functional properties of mTEC^lo are impaired in mTEC^ΔMHCII mice.

(A) Percentages of CD69⁺ OTII CD4⁺ T cells cultured or not with variable numbers of OVA_323-339-loaded WT or mTEC^ΔMHCII mTECs derived from two independent experiments (n = 2–3 mice per group and experiment). (B) Scatter plot of gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) of mTEC^lo from WT versus mTEC^ΔMHCII mice. Genes with fold difference ≥2 and p-adj<0.05 were considered as upregulated or downregulated genes (red and blue dots, respectively). RNA-seq was performed on two independent biological replicates with mTEC^lo derived from 3 to 5 mice. (C) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated in mTEC^lo from WT versus mTEC^ΔMHCII mice. ND, not determined. (D) Numbers of induced TRAs regulated or not by Aire and/or Fezf2. (E) Aire-dependent (Crabp1), Fezf2-dependent (Coch, Sult1c2), Aire/Fezf2-dependent (Fabp9), and Aire/Fezf2-independent (Spon2, Upk3b) TRAs were measured by qPCR in mTEC^lo from WT (n = 4) and mTEC^ΔMHCII (n = 4) mice. (F) Scatter plot of gene expression variation in mTEC^lo from WT versus mTEC^ΔMHCII mice and in mTEC^hi from WT versus Aire^-/- mice. The loess fitted curve is shown in blue and the induced Aire-dependent genes (fold change [FC] > 5) in red. (G) Heatmap of significantly downregulated activation factors in mTEC^lo from mTEC^ΔMHCII mice. (H) Aire and Fezf2 mRNAs were measured by qPCR in mTEC^lo from WT (n = 3–4) and mTEC^ΔMHCII (n = 4) mice. (I) FC in the expression of HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus mTEC^ΔMHCII mice. The color code represents gene expression level. (J) Heatmap of significantly downregulated cytokines, chemokines, and cell adhesion molecules in mTEC^lo from mTEC^ΔMHCII mice. (K) Hierarchical clustering and heatmap of mean expression of these activation factors, cell adhesion molecules, chemokines, and cytokines in mTEC subsets identified by scRNA-seq. Error bars show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 using two-tailed Mann–Whitney test for (A), (E) and (H) and chi-squared test for (C) and (F).

Figure 2—figure supplement 1.. Altered expression of some cytokines, cell adhesion molecules, and chemokines in mTEC^lo from mTEC^ΔMHCII mice.

Il21, Il5, Il15, Ccl22, and Icam2 mRNAs were measured by qPCR in mTEC^lo from WT (n = 4) and mTEC^ΔMHCII (n = 3–4) mice. Error bars show mean ± SEM, *p<0.05 using two-tailed Mann–Whitney test.

Figure 3.. The composition in medullary thymic epithelial cell (mTEC) subsets is altered in ΔCD4 and mTEC^ΔMHCII mice.

(A) Flow cytometry profiles, frequencies, and numbers of mTEC^lo and mTEC^hi in WT, ΔCD4, and mTEC^ΔMHCII mice. Data are representative of 2–3 independent experiments (n = 2–5 mice per group and experiment). (B) Confocal images of thymic sections from WT, ΔCD4, and mTEC^ΔMHCII mice stained for Aire (green) and Fezf2 (red). 12 and 20 sections derived from two WT, two ΔCD4, and two mTEC^ΔMHCII mice were quantified. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire⁺Fezf2^-, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells, respectively. The histogram shows the density of Aire⁺Fezf2^-, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells. (C–E) Flow cytometry profiles, frequencies, and numbers of Aire^-Fezf2^-, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells in total mTECs, mTEC^lo, and mTEC^hi (C), of CCL21⁺ cells in mTEC^lo (D) and of DCKL1⁺ cells in Aire^- mTEC^lo (E) from WT, ΔCD4, and mTEC^ΔMHCII mice. II Abs: secondary antibodies. Data are representative of 2–3 independent experiments (n = 2–5 mice per group and experiment). Error bars show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using unpaired Student’s t-test for (B) and two-tailed Mann–Whitney test for (A) and (C-E).

Figure 3—figure supplement 1.. Normal proliferation of Aire^-Fezf2⁺ and Aire ⁺Fezf2⁺ medullary thymic epithelial cell (mTECs) in ΔCD4 and mTEC^ΔMHCII mice.

(A, B) Flow cytometry profiles and frequencies of Ki-67⁺ proliferating Aire^-Fezf2⁺ and Aire⁺Fezf2⁺ cells in mTEC^lo (A) and mTEC^hi (B) from WT, ΔCD4, and mTEC^ΔMHCII mice. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). Error bars show mean ± SEM.

Figure 3—figure supplement 2.. Reduced post-Aire medullary thymic epithelial cells (mTECs) in ΔCD4 and mTEC^ΔMHCII mice.

(A) Representative thymic sections stained with antibodies against involucrin (red), TPA (green), and Aire (magenta). c and m denote the cortex and the medulla, respectively. The graph shows the number of involucrin⁺TPA⁺Aire^- cells per medulla. 15 medullas derived from two WT, two ΔCD4, and two mTEC^ΔMHCII mice were quantified, respectively. Scale bar: 200 μm. Error bars show mean ± SEM, **p<0.01 using unpaired Student’s t-test. (B) Gating strategy used to identify Aire^- mTEC^lo cells in WT, ΔCD4, and mTEC^ΔMHCII mice.

Figure 3—figure supplement 3.. Analysis of medullary thymic epithelial cell (mTEC) subsets in MHCII^-/- mice.

(A) Flow cytometry profiles and numbers of Aire^-Fezf2^-, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells in total mTECs, mTEC^lo, and mTEC^hi from WT, ΔCD4, and MHCII^-/- mice. II Abs: secondary antibodies. Data are representative of two independent experiments (n = 2–4 mice per group). (B) Flow cytometry profiles, frequencies, and numbers of CCL21⁺ cells in mTEC^lo from WT, ΔCD4, and MHCII^-/- mice. (C) Flow cytometry profiles, frequencies, and numbers of DCKL1⁺ cells in Aire^- mTEC^lo. Data are representative of two independent experiments (n = 2–3 mice per group). Error bars show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using the Mann–Whitney test.

Figure 4.. Highly self-reactive CD4⁺ thymocytes control the transcriptional and functional properties of mTEC^lo.

(A) Relb mRNA was measured by qPCR in mTEC^lo from RipmOVAxOTII-Rag2^-/- (n = 4) and OTII-Rag2^-/- (n = 5) mice. (B) Total and phospho-RelB (Ser552) were analyzed by flow cytometry in mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). (C) Scatter plot of gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) in mTEC^lo from RipmOVAxOTII-Rag2^-/- versus OTII-Rag2^-/- mice. Genes with fold difference ≥2 and p-adj<0.05 were considered as upregulated or downregulated genes (red and blue dots, respectively). RNA-seq was performed on two independent biological replicates with mTEC^lo derived from 5 to 8 mice. (D) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated in mTEC^lo from RipmOVAxOTII-Rag2^-/- versus OTII-Rag2^-/- mice. ND, not determined. (E) Numbers of induced Aire-dependent, Fezf2-dependent, Aire/Fezf2-dependent, and Aire/Fezf2-independent TRAs. (F) Aire-dependent (Fam183b, Nts), Fezf2-dependent (Resp18, Grap), Aire/Fezf2-dependent (Fabp9), Aire/Fezf2-independent (Csn2, Crp) TRAs, Aire and Fezf2 mRNAs were measured by qPCR in mTEC^lo from RipmOVAxOTII-Rag2^-/- (n = 4) and OTII-Rag2^-/- (n = 4) mice. (G) Scatter plot of gene expression variation in mTEC^lo from RipmOVAxOTII-Rag2^-/- versus OTII-Rag2^-/- mice and in mTEC^hi from WT versus Aire^-/- mice. The loess fitted curve is shown in blue and induced Aire-dependent genes (fold change [FC] > 5) in red. (H) Heatmap of significantly upregulated activation factors in mTEC^lo from RipmOVAxOTII-Rag2^-/- compared to OTII-Rag2^-/- mice. (I, J) Expression FC in HDAC3-induced transcriptional regulators and other transcription factors (I) and in Foxn1 targets (J) in mTEC^lo from RipmOVAxOTII-Rag2^-/- versus OTII-Rag2^-/- mice. The color code represents gene expression level. (K) Heatmap of significantly upregulated cytokines, chemokines, and cell adhesion molecules in mTEC^lo from RipmOVAxOTII-Rag2^-/- mice. (L) Hierarchical clustering and heatmap of mean expression of these activation factors, cell adhesion molecules, chemokines, and cytokines in mTEC subsets identified by scRNA-seq. Error bars show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 using two-tailed Mann–Whitney test for (A), (B) and (F) and chi-squared test for (D) and (G).

Figure 4—figure supplement 1.. Similar levels of total and phosphorylated p65, Erk1/2, p38, and IKKα proteins in mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice.

(A, B) Total p65, Erk1/2, p38, and IKKα (A) and phospho-p65 (Ser536), phospho-Erk1/2 MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), and phospho-IKKα(Ser180)/IKKβ(Ser181) (B) proteins were analyzed by flow cytometry in mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. Histograms show the MFI. (C) Histograms represent the ratio of phospho-p65 (Ser536) to p65, phospho-Erk1/2 MAPK (Thr202/Tyr204) to Erk1/2, phospho-p38 MAPK (Thr180/Tyr182) to p38, and phospho-IKKα (Ser180)/IKKβ(Ser181) to IKKα. II Abs: secondary antibodies. Data are representative of two independent experiments (n = 2–4 mice per group and experiment). Error bars show mean ± SEM.

Figure 4—figure supplement 2.. Expression of Relb, cytokines, chemokines, and cell adhesion molecules that was altered in mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice.

(A) Mean expression of Relb in mTEC^lo subsets identified by scRNA-seq data derived from Wells et al., 2020. (B) Il5, Il15, Il7, Ccl19, Ccl2, Ccl25, Cdh2, and Itgad mRNAs were measured by qPCR in mTEC^lo from RipmOVAxOTII-Rag2^-/- (n = 3–4) and OTII-Rag2^-/- (n = 3–4) mice.

Figure 5.. Highly self-reactive CD4⁺ thymocytes control medullary thymic epithelial cell (mTEC) development from an early progenitor stage.

(A–D) Flow cytometry profiles and numbers of total thymic epithelial cells (TECs) (EpCAM⁺) (A), cortical thymic epithelial cell (cTECs) (UEA-1^-Ly51^hi), mTECs (UEA-1⁺Ly51^lo) (B), TEC^lo (MHCII^loUEA-1^lo), cTEC^hi (MHCII^hiUEA-1^lo), mTEC^lo (MHCII^loUEA-1^hi), and mTEC^hi (MHCII^hiUEA-1^hi) (C), α6-integrin^hiSca-1^hi TEPC-enriched cells in TEC^lo (D) in CD45^neg-enriched cells from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. Data are representative of four experiments (n = 3 mice per group and experiment). (E) Confocal images of thymic sections from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice stained for Aire (green) and Fezf2 (red). 11 and 22 sections derived from two RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice were quantified, respectively. Scale bar, 50 μm. Unfilled, dashed and solid arrowheads indicate Aire⁺Fezf2^lo, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells, respectively. The histogram shows the density of Aire⁺Fezf2^lo, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells. (F–H) Flow cytometry profiles, frequencies, and numbers of Aire^-Fezf2^-, Aire^-Fezf2⁺, and Aire⁺Fezf2⁺ cells in total mTECs, mTEC^lo, and mTEC^hi (F), of CCL21⁺ cells in mTEC^lo (G) and of DCKL1⁺ cells in Aire^- mTECs (H) from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. II Abs: secondary antibodies. Data are representative of two independent experiments (n = 3–4 mice per group and experiment). Error bars show mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using unpaired Student’s t-test for (A–E) and two-tailed Mann–Whitney test for (F–H).

Figure 5—figure supplement 1.. mTEC^lo and mTEC^hi cells are increased in RipmOVAxOTII-Rag2^-/- compared to OTII-Rag2^-/- mice.

Flow cytometry profiles, frequencies, and numbers of mTEC^lo and mTEC^hi from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. Data are representative of two independent experiments (n = 3 mice per group and experiment). Error bars show mean ± SEM. **p<0.01, ***p<0.001, ****p<0.0001 using the Mann–Whitney test.

Figure 5—figure supplement 2.. The proliferation of Aire^-Fezf2⁺ and Aire⁺Fezf2⁺ medullary thymic epithelial cells (mTECs) is similar in RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice.

(A, B) Flow cytometry profiles and frequencies of proliferating Ki-67⁺ Aire^-Fezf2⁺ and Aire⁺Fezf2⁺ in mTEC^lo (A) and mTEC^hi (B) from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. Data are representative of two independent experiments (n = 3 mice per group and experiment). Error bars show mean ± SEM.

Figure 5—figure supplement 3.. Post-Aire medullary thymic epithelial cells (mTECs) are increased in RipmOVAxOTII-Rag2^-/- compared to OTII-Rag2^-/- mice.

(A) Representative thymic sections from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice stained with antibodies against involucrin (red), TPA (green), and Aire (magenta). c and m denote the cortex and the medulla, respectively. The graph shows the number of involucrin⁺TPA⁺Aire^- cells per medulla. 15 medullas derived from two RipmOVAxOTII-Rag2^-/- and two OTII-Rag2^-/- mice were quantified, respectively. Scale bar: 200 μm. Error bars show mean ± SEM, ****p<0.0001 using unpaired Student’s t-test. (B) Gating strategy used to identify Aire^- mTEC^lo cells in RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice.

Figure 6.. H3K27me3 and H3K4me3 landscape in tissue-restricted self-antigen (TRA) genes of mTEC^lo from WT, RipmOVAxOTII-Rag2^-/-, and OTII-Rag2^-/- mice.

(A, B) Metagene profiles of the average normalized enrichment of H3K27me3 (A) and H3K4me3 (B) against input for Aire-dependent, Fezf2-dependent, and Aire/Fezf2-independent TRAs as well as for all genes of WT mTEC^lo. Boxplots represent the median enrichment, the 95% CI of the median (notches), and the 75th and 25th percentiles of H3K27me3 and H3K4me3. (C) H3K27me3 and H3K4me3 levels were analyzed by flow cytometry in mTEC^lo from RipmOVAxOTII-Rag2^-/- (n = 4) and OTII-Rag2^-/- (n = 5) mice. Histograms show the MFI. (D) Boxplots represent the median enrichment of H3K27me3 and H3K4me3 of Aire-dependent, Fezf2-dependent, and Aire/Fezf2-independent TRAs and in all genes of mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. (E) Expression (RNA-seq) and H3K27me3 and H3K4me3 chromatin state (ChIP-seq) of the Aire/Fezf2-independent TRA, E2f2, in mTEC^lo from RipmOVAxOTII-Rag2^-/- and OTII-Rag2^-/- mice. *p<0.05, **p<10^–3, ***p<10^–7, using the Mann–Whitney test for (A) and (B) and unpaired Student’s t-test for (C).

Figure 7.. The adoptive transfer of T cells from mTEC^ΔMHCII mice into Rag2^-/- recipients induces autoimmunity.

(A) Tissue-restricted self-antigens (TRAs) underexpressed in mTEC^lo from mTEC^ΔMHCII mice were assigned to their peripheral expression. (B) TCRVβ usage by CD69^- mature CD4⁺ and CD8⁺ thymocytes (left panel) and CD4⁺Foxp3^- and CD8⁺ splenic T cells (right panel) from WT and mTEC^ΔMHCII mice. (C) Body weight of Rag2^-/- recipients transferred with splenic T cells from WT or mTEC^ΔMHCII was monitored during 6 weeks, and tissue infiltration was examined. (D) Weight loss relative to the initial weight. (E, F) Representative spleen pictures and their weights (E) and hematoxylin/eosin counterstained splenic sections (F). Scale bar, 1 mm. The histogram shows follicle areas. (G) Numbers of splenic CD3⁺, CD4⁺, and CD8⁺ T cells and of naive (CD44^loCD62L^hi), effector memory (EM; CD44^hiCD62L^lo) and central memory (CM; CD44^hiCD62L^hi) phenotype. (H) Lung and salivary gland (SG) immune infiltrates detected by hematoxylin/eosin counterstaining. Scale bar, 1 mm. (I, J) Numbers of T cells (I) and of naive, effector and central memory phenotype as well as CD44⁺CD69⁺ and CD44⁺CD69^- T cells (J) in lungs and SG. (K) Schematic of T-cell infiltrates in mice transferred with mTEC^ΔMHCII T cells relative to those transferred with WT T cells. Each circle and black triangles represent an individual mouse and T-cell infiltration in a specific tissue, respectively. Data are representative of two independent experiments (n = 5–7 mice per group and experiment). Error bars show mean ± SEM, ****p<0.0001 using two-way ANOVA for (D) and unpaired Student’s t-test for (B) and (E-J). *p<0.05, **p<0.01, ***p<0.001.

Figure 7—figure supplement 1.. CD4⁺ thymocytes through MHCII/TCR-mediated interactions control transcriptional programs of mTEC^lo that drive their differentiation and function.

Antigen-specific interactions between CD4⁺ thymocytes and mTEC^lo lead to the upregulation of tissue-restricted self-antigen (TRA) regulators, TRAs, key medullary thymic epithelial cell (mTEC)-specific transcription factors, adhesion molecules, cytokines, and chemokines, which correlates with an increased level of the active H3K4me3 histone mark. These interactions enhance the transcriptional activity of TAC-TECs accompanying the transition to Aire⁺Fezf2⁺, as well as post-Aire cells and tuft-like mTECs. They are thus essential to generate a self-tolerant T-cell repertoire and prevent the development of autoimmunity.

Author response image 1.