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

  1. Noella Lopes
  2. Nicolas Boucherit
  3. Jérémy C Santamaria
  4. Nathan Provin
  5. Jonathan Charaix
  6. Pierre Ferrier
  7. Matthieu Giraud
  8. Magali Irla  Is a corresponding author
  1. Aix-Marseille University, CNRS, INSERM, Centre d'Immunologie de Marseille-Luminy, France
  2. Nantes Université, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064, France
8 figures, 1 table and 5 additional files

Figures

Figure 1 with 4 supplements
The transcriptional profile and IKKα and p38 MAPK signaling pathways are impaired in mTEClo 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 mTEClo 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 mTEClo 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 mTEClo 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) mTEClo. (G) Expression fold change in HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus ΔCD4 mTEClo. The color code represents gene expression level. (H) Heatmaps of genes encoding for cell adhesion molecules and cytokines that were significantly downregulated in mTEClo 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 mTEClo 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+Ly51lo) and cortical TECs (cTECs) (UEA-1Ly51hi). mTEClo were identified and sorted based on low/intermediate levels of the CD80 co-stimulatory molecule. The purity of sorted mTEClo was >98%. (B) Gates used to sort mTEClo from WT, ΔCD4, mTECΔMHCII, RipmOVAxOTII-Rag2-/-, and OTII-Rag2-/- mice.

Figure 1—figure supplement 2
Normal total and phosphorylated p65, RelB, and Erk1/2 proteins in mTEClo 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 mTEClo 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 mTEClo 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 mTEClo 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) mTEClo.

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 with 1 supplement
The transcriptional and functional properties of mTEClo are impaired in mTECΔMHCII mice.

(A) Percentages of CD69+ OTII CD4+ T cells cultured or not with variable numbers of OVA323-339-loaded WT or mTECΔMHCII 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 mTEClo from WT versus mTECΔMHCII 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 mTEClo derived from 3 to 5 mice. (C) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated in mTEClo from WT versus mTECΔMHCII 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 mTEClo from WT (n = 4) and mTECΔMHCII (n = 4) mice. (F) Scatter plot of gene expression variation in mTEClo from WT versus mTECΔMHCII mice and in mTEChi 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 mTEClo from mTECΔMHCII mice. (H) Aire and Fezf2 mRNAs were measured by qPCR in mTEClo from WT (n = 3–4) and mTECΔMHCII (n = 4) mice. (I) FC in the expression of HDAC3-induced transcriptional regulators and other transcription factors significantly upregulated in WT versus mTECΔMHCII mice. The color code represents gene expression level. (J) Heatmap of significantly downregulated cytokines, chemokines, and cell adhesion molecules in mTEClo from mTECΔMHCII 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 mTEClo from mTECΔMHCII mice.

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

Figure 3 with 3 supplements
The composition in medullary thymic epithelial cell (mTEC) subsets is altered in ΔCD4 and mTECΔMHCII mice.

(A) Flow cytometry profiles, frequencies, and numbers of mTEClo and mTEChi in WT, ΔCD4, and mTECΔMHCII 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 mTECΔMHCII mice stained for Aire (green) and Fezf2 (red). 12 and 20 sections derived from two WT, two ΔCD4, and two mTECΔMHCII 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, mTEClo, and mTEChi (C), of CCL21+ cells in mTEClo (D) and of DCKL1+ cells in Aire- mTEClo (E) from WT, ΔCD4, and mTECΔMHCII 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 mTECΔMHCII mice.

(A, B) Flow cytometry profiles and frequencies of Ki-67+ proliferating Aire-Fezf2+ and Aire+Fezf2+ cells in mTEClo (A) and mTEChi (B) from WT, ΔCD4, and mTECΔMHCII 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 mTECΔMHCII 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 mTECΔMHCII 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- mTEClo cells in WT, ΔCD4, and mTECΔMHCII 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, mTEClo, and mTEChi 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 mTEClo from WT, ΔCD4, and MHCII-/- mice. (C) Flow cytometry profiles, frequencies, and numbers of DCKL1+ cells in Aire- mTEClo. 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 with 2 supplements
Highly self-reactive CD4+ thymocytes control the transcriptional and functional properties of mTEClo.

(A) Relb mRNA was measured by qPCR in mTEClo from RipmOVAxOTII-Rag2-/- (n = 4) and OTII-Rag2-/- (n = 5) mice. (B) Total and phospho-RelB (Ser552) were analyzed by flow cytometry in mTEClo 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 mTEClo 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 mTEClo derived from 5 to 8 mice. (D) Numbers of tissue-restricted self-antigens (TRAs) and non-TRAs in genes up- and downregulated in mTEClo 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 mTEClo from RipmOVAxOTII-Rag2-/- (n = 4) and OTII-Rag2-/- (n = 4) mice. (G) Scatter plot of gene expression variation in mTEClo from RipmOVAxOTII-Rag2-/- versus OTII-Rag2-/- mice and in mTEChi 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 mTEClo 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 mTEClo 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 mTEClo 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 mTEClo 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 mTEClo 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 mTEClo from RipmOVAxOTII-Rag2-/- and OTII-Rag2-/- mice.

(A) Mean expression of Relb in mTEClo 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 mTEClo from RipmOVAxOTII-Rag2-/- (n = 3–4) and OTII-Rag2-/- (n = 3–4) mice.

Figure 5 with 3 supplements
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-Ly51hi), mTECs (UEA-1+Ly51lo) (B), TEClo (MHCIIloUEA-1lo), cTEChi (MHCIIhiUEA-1lo), mTEClo (MHCIIloUEA-1hi), and mTEChi (MHCIIhiUEA-1hi) (C), α6-integrinhiSca-1hi TEPC-enriched cells in TEClo (D) in CD45neg-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+Fezf2lo, Aire-Fezf2+, and Aire+Fezf2+ cells, respectively. The histogram shows the density of Aire+Fezf2lo, 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, mTEClo, and mTEChi (F), of CCL21+ cells in mTEClo (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
mTEClo and mTEChi cells are increased in RipmOVAxOTII-Rag2-/- compared to OTII-Rag2-/- mice.

Flow cytometry profiles, frequencies, and numbers of mTEClo and mTEChi 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 mTEClo (A) and mTEChi (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- mTEClo cells in RipmOVAxOTII-Rag2-/- and OTII-Rag2-/- mice.

H3K27me3 and H3K4me3 landscape in tissue-restricted self-antigen (TRA) genes of mTEClo 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 mTEClo. 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 mTEClo 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 mTEClo 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 mTEClo 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 with 1 supplement
The adoptive transfer of T cells from mTECΔMHCII mice into Rag2-/- recipients induces autoimmunity.

(A) Tissue-restricted self-antigens (TRAs) underexpressed in mTEClo from mTECΔMHCII 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 mTECΔMHCII mice. (C) Body weight of Rag2-/- recipients transferred with splenic T cells from WT or mTECΔMHCII 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 (CD44loCD62Lhi), effector memory (EM; CD44hiCD62Llo) and central memory (CM; CD44hiCD62Lhi) 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 mTECΔMHCII 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 mTEClo that drive their differentiation and function.

Antigen-specific interactions between CD4+ thymocytes and mTEClo 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

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)C57BL/6J backgroundCharles RiverRRID:IMSR_JAX:000664
Genetic reagent (M. musculus)Ciitatm2Wrth/Ciitatm2WrthLeibundGut-Landmann et al., 2004RRID:MGI:3052466C57BL/6 background, ΔCD4 mice
Genetic reagent (M. musculus)H2dlAb1-Ea/H2dlAb1-EaMadsen et al., 1999RRID:MGI:4436873C57BL/6 background, MHCII-/- mice
Genetic reagent (M. musculus)K14xCiitaIII+IV-/-Irla et al., 2008C57BL/6 background, mTECΔMHCII mice
Genetic reagent (M. musculus)Tg(TcraTcrb)425CbnBarnden et al., 1998RRID:MGI:3762632C57BL/6 background, OTII mice
Genetic reagent (M. musculus)Tg(Ins2-TFRC/OVA)296WehiKurts et al., 1996RRID:MGI:3623748C57BL/6 background, Rip-mOVA mice
Genetic reagent (M. musculus)Rag2tm1Fwa/Rag2tm1FwaShinkai et al., 1992RRID:MGI:2174910C57BL/6 background, Rag2-/- mice
AntibodyAnti-IKKα (rabbit polyclonal)Cell Signaling TechnologyCat# 2682; RRID:AB_331626FACS (1:500)
AntibodyAnti-phospho IKKα (Ser180)/IKKβ(Ser181) (rabbit polyclonal)Cell Signaling TechnologyCat# 2681S; RRID:AB_331624FACS (1:500)
AntibodyAnti-p38 MAPK (rabbit polyclonal)Cell Signaling TechnologyCat# 9212; RRID:AB_330713FACS (1:500)
AntibodyAnti-phospho p38 MAPK (Thr180/Tyr182) (rabbit polyclonal)Cell Signaling TechnologyCat# 9211S; RRID:AB_331641FACS (1:500)
AntibodyAnti-Erk1/2(rabbit polyclonal)Cell Signaling TechnologyCat# 9102; RRID:AB_330744FACS (1:500)
AntibodyAnti-phospho Erk1/2 (Thr202/Tyr204) (rabbit polyclonal)Cell Signaling TechnologyCat# 9101S;RRID:AB_331646FACS (1:500)
AntibodyAnti-NF-κB p65 (clone D14E12, rabbit monoclonal)Cell Signaling TechnologyCat# 8242S; RRID:AB_10859369FACS (1:500)
AntibodyPhospho-NF-κB p65 (Ser536) (clone 93H1, rabbit monoclonal)Cell Signaling TechnologyCat# 3033S; RRID:AB_331284FACS (1:3000)
AntibodyAnti-RelB (clone C-19, rabbit polyclonal)Santa Cruz BiotechnologyCat# sc-226; RRID:AB_632341FACS (1:200)
AntibodyAnti-phospho RelB (ser552) (clone D41B9, rabbit monoclonal)Cell Signaling TechnologyCat# 5025S; RRID:AB_10622001FACS (1:1000)
AntibodyAnti-DCLK1 (clone D2U3L, rabbit monoclonal)Cell Signaling TechnologyCat# 62257; RRID:AB_2799622FACS (1:200)
AntibodyAnti-H3K4me3 (rabbit polyclonal)AbcamCat# ab8580; RRID:AB_306649FACS(1:1000)ChIP-seq (2 µg:25 µg chromatin)
AntibodyAnti-H3K27me3 (clone C36B11, rabbit monoclonal)Cell Signaling TechnologyCat# 9733; RRID:AB_2616029FACS(1:1000)ChIP-seq(1:50)
AntibodyPE-Cy7 anti-CD326 (EpCAM) (clone G8.8, rat monoclonal)eBioscienceCat# 25-5791-80; RRID:AB_1724047FACS(1:3000)
AntibodyAlexa Fluor 488 anti-Aire (clone 5H12, rat monoclonal)eBioscienceCat# 53-5934-82; RRID:AB_10854132FACS, IF(1:200)
AntibodyPE anti-Ly51 (clone BP-1, mouse monoclonal)BD BiosciencesCat# 553735; RRID:AB_395018FACS(1:3000)
AntibodyPerCP-Cy5.5 anti-CD80 (clone 16-10A1, Armenian hamster monoclonal)BioLegendCat# 104722; RRID:AB_2291392FACS(1:200)
AntibodyeFluor 450 anti-Ki-67 (clone SolA15, rat monoclonal)eBioscienceCat# 48-5698-82; RRID:AB_11149124FACS(1:200)
AntibodyAnti-Fezf2 (clone F441, rabbit polyclonal)IBL TecanCat# JP18997; RRID:AB_2341444FACS, IF(1:200)
AntibodyAnti-Involucrin (clone Poly19244, rabbit polyclonal)BioLegendCat# 924401; RRID:AB_2565452IF(1:100)
AntibodyPE anti-Ly-6A/E (Sca-1) (clone D7, rat monoclonal)BD BiosciencesCat# 553108; RRID:AB_394629FACS(1:600)
AntibodyBiotin anti-CD49f (α6-integrin) (clone GoH3, rat monoclonal)BioLegendCat# 313604; RRID:AB_345298FACS(1:200)
AntibodyAlexa Fluor 647 anti-I-Ab (MHCII) (clone AF6-120.1, mouse monoclonal)BioLegendCat# 116412; RRID:AB_493141FACS(1:200)
AntibodyBrilliant Violet 421 anti-CD4 (clone RM4-5, rat monoclonal)BioLegendCat# 100544; RRID:AB_11219790FACS(1:200)
AntibodyPerCP-Cy5.5 anti-CD4 (clone RM4-5, rat monoclonal)BD BiosciencesCat# 550954; RRID:AB_393977FACS(1:200)
AntibodyPacific Blue anti-CD8α (clone 53-6.7, rat monoclonal)BD BiosciencesCat# 558106; RRID:AB_397029FACS(1:200)
AntibodyPE/Cy7 anti-CD8α (clone 53-6.7, rat monoclonal)BioLegendCat# 100722; RRID:AB_312761FACS(1:600)
AntibodyAlexa Fluor 488 anti-CD44 (clone IM7, rat monoclonal)BioLegendCat# 103016; RRID:AB_493679FACS(1:200)
AntibodyPE anti-CD69 (clone H1.2F3, rat monoclonal)BioLegendCat# 104508; RRID:AB_313111FACS(1:400)
AntibodyPE anti-CD62L (clone MEL-14, rat monoclonal)BD BiosciencesCat# 553151; RRID:AB_394666FACS(1:300)
AntibodyPerCP-Cy5.5 anti-CD3ε (clone 17A2, rat monoclonal)BD BiosciencesCat# 560527;RRID:AB_1727463FACS(1:200)
AntibodyAlexa Fluor 405 anti-CCL21 (clone 59106, rat monoclonal)R&D SystemsCat# IC457VFACS(1:100)
AntibodyCD45 MicroBeads, mouse (clone 30F11.1, rat monoclonal)MiltenyiCat# 130052301; RRID:AB_2877061
AntibodyCy5 anti-rabbit IgG (goat polyclonal)InvitrogenCat# A10523; RRID:AB_2534032FACS(1:500)
AntibodyCyanine 3 anti-rabbit IgG (goat polyclonal)InvitrogenCat# A10520; RRID:AB_2534029IF(1:500)
AntibodyFITC anti-TCR Vβ2 (clone B20.6, rat monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ3 (clone KJ25, Armenian hamster monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ4 (clone KT4, rat monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ5.1, 5.2 (clone MR9-4, mouse monoclonal)BD BiosciencesCat# 553189; RRID:AB_394697FACS(1:100)
AntibodyFITC anti-TCR Vβ6 (clone RR4-7, rat monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyPE anti-TCR Vβ8.1, 8.2 (clone MR5-2, mouse monoclonal)BioLegendCat# 140103; RRID:AB_10641144FACS(1:300)
AntibodyFITC anti-TCR Vβ9 (clone MR10-2, mouse monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyPE anti-TCR Vβ10b (clone B21.5, rat monoclonal)BD BiosciencesCat# 553285; RRID:AB_394757FACS(1:300)
AntibodyBiotin anti-TCR Vβ11 (clone RR3-15, rat monoclonal)BD BiosciencesCat# 553196; RRID:AB_394702FACS(1:300)
AntibodyFITC anti-TCR Vβ12 (clone MR11-1, mouse monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ13 (clone MR12-3, mouse monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ14 (clone 14-2, rat monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyFITC anti-TCR Vβ17a (clone KJ23, rat monoclonal)BD BiosciencesCat# 557004; RRID:AB_647180FACS(20 µl per 106 cells)
AntibodyCD4+ T cell isolation kit, mouseMiltenyi BiotecCat# 130-104-454
Peptide, recombinant proteinPerCP-Cy5.5 StreptavidinBioLegendCat# 405214; RRID:AB_2716577FACS(1:400)
Peptide, recombinant proteinAlexa Fluor 488 StreptavidinInvitrogenCat# S11223IF(1:1000)
Peptide, recombinant proteinOvalbumin (323–339)PolyPeptideCat# SC13035 μM
Chemical compound, drugLiberase TMRocheCat# 0540112700150 μg/ml
Chemical compound, drugDNase IRocheCat# 10104159001100 μg/ml
Chemical compound, drugTRIzolThermo Fisher ScientificCat# 15596018
Software, algorithmGraphPad PrismGraphPad SoftwareRRID:SCR_002798
Software, algorithmFlowJoFlowJohttps://www.flowjo.com/RRID:SCR_008520
Software, algorithmFiji/ImageJ softwareFiji-ImageJhttps://imagej.nih.gov/ij/RRID:SCR_003070
Software, algorithm7500 Real-Time PCR SoftwareThermo Fisherhttps://www.thermofisher.com/us/en/home/technical-resources/software-downloads/applied-biosystems-7500-real-time-pcr-system.htmlRRID:SCR_014596
Software, algorithmPheatmap 0.2https://github.com/raivokolde/pheatmap (Kolde, 2018)RRID:SCR_016418
Software, algorithmSeuratHao et al., 2021RRID:SCR_016341
Sequence-based reagentActin-FWSigma-AldrichPCR primersCAGAAGGAGATTACTGCTCTGGCT
Sequence-based reagentActin-RVSigma-AldrichPCR primersGGAGCCACCGATCCACACA
Sequence-based reagentAire-FWSigma-AldrichPCR primersGCATAGCATCCTGGACGGCTTCC
Sequence-based reagentAire-RVSigma-AldrichPCR primersCTGGGCTGGAGACGCTCTTTGAG
Sequence-based reagentCcl19-FWSigma-AldrichPCR primersGCTAATGATGCGGAAGACTG
Sequence-based reagentCcl19-RVSigma-AldrichPCR primersACTCACATCGACTCTCTAGG
Sequence-based reagentCcl2-FWSigma-AldrichPCR primersTGGAGCATCCACGTGTTG
Sequence-based reagentCcl2-RVSigma-AldrichPCR primersACTCATTGGGATCATCTTGCT
Sequence-based reagentCcl22-FWSigma-AldrichPCR primersCTGATGCAGGTCCCTATGGT
Sequence-based reagentCcl22-RVSigma-AldrichPCR primersGGAGTAGCTTCTTCACCCAG
Sequence-based reagentCcl25-FWSigma-AldrichPCR primersGCCTGGTTGCCTGTTTTGTT
Sequence-based reagentCcl25-RVSigma-AldrichPCR primersACCCAGGCAGCAGTCTTCAA
Sequence-based reagentCdh2-FWSigma-AldrichPCR primersAGCGCAGTCTTACCGAAGG
Sequence-based reagentCdh2-RVSigma-AldrichPCR primersTCGCTGCTTTCATACTGAACTTT
Sequence-based reagentCoch-FWSigma-AldrichPCR primersGTGCAGCAAAACCTGCTACAA
Sequence-based reagentCoch -RVSigma-AldrichPCR primersAGCTAGGACGTTCTCTTTGGT
Sequence-based reagentCrabp1-FWSigma-AldrichPCR primersCAGCAGCGAGAATTTCGACGA
Sequence-based reagentCrabp1-RVSigma-AldrichPCR primersCGCACAGTAGTGGATGTCTTGA
Sequence-based reagentCrp-FWSigma-AldrichPCR primersCATAGCCATGGAGAAGCTAC
Sequence-based reagentCrp-RVSigma-AldrichPCR primersCAGTGGCTTCTTTGACTCTG
Sequence-based reagentCsn2-FWSigma-AldrichPCR primersCTCCACTAAAGGACTTGACAG
Sequence-based reagentCsn2-RVSigma-AldrichPCR primersACCTTCTGAAGTTTCTGCTC
Sequence-based reagentFabp9-FWSigma-AldrichPCR primersCACTGCAGACAACCGAAAAG
Sequence-based reagentFabp9-RVSigma-AldrichPCR primersTCTGTTTGCCAAGCCATTTT
Sequence-based reagentFam183b-FWSigma-AldrichPCR primersCGTGTGGGGCAGATGAAGAAT
Sequence-based reagentFam183b-RVSigma-AldrichPCR primersGGTGAATGAGGTTCAGGAACTTG
Sequence-based reagentFcer2a-FWSigma-AldrichPCR primersCCAGGAGGATCTAAGGAACGC
Sequence-based reagentFcer2a-RVSigma-AldrichPCR primersTCGTCTTGGAGTCTGTTCAGG
Sequence-based reagentFezf2-FWSigma-AldrichPCR primersCAGCACTCTCTGCAGACACAA
Sequence-based reagentFezf2-RVSigma-AldrichPCR primersTGCCGCACTGGTTACACTTA
Sequence-based reagentGrap-FWSigma-AldrichPCR primersGATCAGGGAGAGTGAGAGTTCC
Sequence-based reagentGrap-RVSigma-AldrichPCR primersCAGCTCGTTGAGGGAGTTGA
Sequence-based reagentIcam2-FWSigma-AldrichPCR primersATCAACTGCAGCACCAACTG
Sequence-based reagentIcam2-RVSigma-AldrichPCR primersACTTGAGCTGGAGGCTGGTA
Sequence-based reagentIl15-FWSigma-AldrichPCR primersAGCAGATAACCAGCCTACAGGA
Sequence-based reagentIl15-RVSigma-AldrichPCR primersTGTTGAAGATGAGCTGGCTATGG
Sequence-based reagentIl21-FWSigma-AldrichPCR primersCGCCTCCTGATTAGACTTCG
Sequence-based reagentIl21-RVSigma-AldrichPCR primersTGGAGCTGATAGAAGTTCAGGA
Sequence-based reagentIl5-FWSigma-AldrichPCR primersCCGCCAAAAAGAGAAGTGTGGCGA
Sequence-based reagentIl5-RVSigma-AldrichPCR primersGCCTCAGCCTTCCATTGCCCA
Sequence-based reagentIl7-FWSigma-AldrichPCR primersGGGTCCTGGGAGTGATTATGG
Sequence-based reagentIl7-RVSigma-AldrichPCR primersCGGGAGGTGGGTGTAGTCAT
Sequence-based reagentItgad-FWSigma-AldrichPCR primersCGAAAGGGTTCAGACTTTGC
Sequence-based reagentItgad-RVSigma-AldrichPCR primersACACCTCCACGGATAGAAGTC
Sequence-based reagentItgb6-FWSigma-AldrichPCR primersGCTGGTCTGCCTGTTTCTGC
Sequence-based reagentItgb6-RVSigma-AldrichPCR primersTGAGCAGCTTTCTGCACCAC
Sequence-based reagentKcnj5-FWSigma-AldrichPCR primersAAAACCTTAGCGGCTTTGTATCT
Sequence-based reagentKcnj5-RVSigma-AldrichPCR primersAAGGCATTAACAATCGAGCCC
Sequence-based reagentKrt1-FWSigma-AldrichPCR primersTGGGAGATTTTCAGGAGGAGG
Sequence-based reagentKrt1-RVSigma-AldrichPCR primersGCCACACTCTTGGAGATGCTC
Sequence-based reagentMeig1-FWSigma-AldrichPCR primersCTTCAGCGGAGGGACAATAC
Sequence-based reagentMeig1-RVSigma-AldrichPCR primersCAAGGTTTCAAGGTGGGTGT
Sequence-based reagentNov-FWSigma-AldrichPCR primersAGACCCCAACAACCAGACTG
Sequence-based reagentNov-RVSigma-AldrichPCR primersCGGTAAATGACCCCATCGAAC
Sequence-based reagentNts-FWSigma-AldrichPCR primersGCAAGTCCTCCGTCTTGGAAA
Sequence-based reagentNts-RVSigma-AldrichPCR primersTGCCAACAAGGTCGTCATCAT
Sequence-based reagentReig1-FWSigma-AldrichPCR primersATGGCTAGGAACGCCTACTTC
Sequence-based reagentReig1-RVSigma-AldrichPCR primersCCCAAGTTAAACGGTCTTCAGT
Sequence-based reagentResp18-FWSigma-AldrichPCR primersCCAGCCAAGATGCAGAGTTCGTTAAAG
Sequence-based reagentResp18-RVSigma-AldrichPCR primersTCAGTCAGCAACAAGGTTGAGGCCCAC
Sequence-based reagentRsph1-FWSigma-AldrichPCR primersACGGGGACACATATGAAGGA
Sequence-based reagentRsph1-RVSigma-AldrichPCR primersGGCCGTGCTTTTTATTTTTG
Sequence-based reagentSpon2-FWSigma-AldrichPCR primersATGGAAAACGTGAGTCTTGCC
Sequence-based reagentSpon2-RVSigma-AldrichPCR primersTGATGCTGTATCTAGCCAGAGG
Sequence-based reagentSult1c2-FWSigma-AldrichPCR primersATGGCCTTGACCCCAGAAC
Sequence-based reagentSult1c2-RVSigma-AldrichPCR primersTCGAAGGTCTGAATCTGCCTC
Sequence-based reagentUpk3b-FWSigma-AldrichPCR primersCATCTGGCTAGTGGTGGCTTT
Sequence-based reagentUpk3b-RVSigma-AldrichPCR primersGGTAATGTCATATAGTGGCCGTC
OtherBiotinylated Lotus Tetragonolobus Lectin (LTL)Vector LaboratoriesCat# B-1325; RRID:AB_2336558IF(1:500)
OtherFITC Ulex Europaeus Agglutinin I (UEA I)Vector LaboratoriesCat# FL-1061; RRID:AB_2336767FACS(1:600)
OtherSuperScript II Reverse TranscriptaseThermo FisherCat# 18064022
OtherSYBR Premix Ex Taq master mixTakaraCat# RR390A
OthermiRNeasy Micro KitQIAGENCat# 217084
OtherTruSeq ChIP Library Preparation KitIlluminaCat# IP-202-2012

Additional files

Supplementary file 1

List of tissue-restricted self-antigens (TRAs) differentially expressed in mTEClo from WT and ΔCD4 mice.

https://cdn.elifesciences.org/articles/69982/elife-69982-supp1-v1.pdf
Supplementary file 2

List of tissue-restricted self-antigens (TRAs) differentially expressed in mTEClo from WT and mTECΔMHCII mice.

https://cdn.elifesciences.org/articles/69982/elife-69982-supp2-v1.pdf
Supplementary file 3

List of tissue-restricted self-antigens (TRAs) differentially expressed in mTEClo from OTII-Rag2-/- and RipmOVAxOTII-Rag2-/- mice.

https://cdn.elifesciences.org/articles/69982/elife-69982-supp3-v1.pdf
Supplementary file 4

Main target organs and fold change associated with the expression of tissue-restricted self-antigens (TRAs) differentially expressed in mTEClo from WT and mTECΔMHCII mice.

https://cdn.elifesciences.org/articles/69982/elife-69982-supp4-v1.pdf
Transparent reporting form
https://cdn.elifesciences.org/articles/69982/elife-69982-transrepform1-v1.pdf

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  1. Noella Lopes
  2. Nicolas Boucherit
  3. Jérémy C Santamaria
  4. Nathan Provin
  5. Jonathan Charaix
  6. Pierre Ferrier
  7. Matthieu Giraud
  8. Magali Irla
(2022)
Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages
eLife 11:e69982.
https://doi.org/10.7554/eLife.69982