KLF2 controls thymic epithelial cell homeostasis, impacting regulatory T cell development and immune tolerance

Figures

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  Figure 1. TEC-specific deficiency in Klf2 disrupts the mTEC compartment (A) Total thymic cellularity was assessed in thymi isolated from littermate control (Klf2fl/fl) and KLF2 cKO (Foxn1Cre:Klf2fl/fl) mice at postnatal day 3 and at 2 and 10 weeks of age. (B and C) (B) Total TECs (CD45−EpCAM+) were analyzed for cTEC (Ly51+) and mTEC (UEA+) composition and (C) cellularity. (D) Frequency and cellularity of mTEClo (MHC IIlowCD80low), mTEChi (MHC IIhighCD80high) and Aire+ mTECs within the total mTEC (UEA+) of 2- and 10-week-old control and KLF2 cKO mice. Cell population frequencies in representative flow cytometry plots are presented as mean ± SD. Statistically significant differences are indicated by ∗ and in blue. Bar graphs represent the mean ± SD, with each dot representing an individual mouse. Data are representative of 2–6 independent experiments per time-point. P, post-natal; WK, weeks. Statistical analysis was carried out using an unpaired two-tailed Student’s t test; ∗p < 0.05; ∗∗p < 0.01.

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  Figure 2. TEC-specific deficiency in Klf2 alters broader and distinct genetic programs in cTEC and mTEC. Thymi from 2-week-old control (Klf2fl/fl) and KLF2 cKO (Foxn1Cre:Klf2fl/fl) mice were isolated, and FACS-sorted cTECs (CD45−EpCAM+Ly51+UEA−) and mTECs (CD45−EpCAM+Ly51−UEA+) were subjected to RNAseq (A) Principal component analysis and hierarchical clustering of the indicated cTEC and mTEC samples. (B) Volcano plot depicting down-regulated (blue) and up-regulated (red) differentially expressed genes (DEGs) in cTECs and mTECs from KLF2 cKO versus control samples. Numbers in the top corners of each volcano plot indicate the respective number of up- and down-regulated genes. Multiple parameter graphs indicate the top five down- and up-regulated gene ontology (GO) terms within the differentially expressed genes of cTECs and mTECs. (C) Venn diagrams depict the number of KLF2 cKO mTEC up-regulated (right) and down-regulated (left) genes classified as Aire-dependent (Aire dep) or Aire-independent (Aire indep) tissue-restricted genes (TRGs). Bar graphs indicate the frequency of identified Aire dep and Aire indep genes in KLF2 cKO mTEC DEGs. (D) Heatmaps representing the deviation to the mean…

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  Figure 3. TEC-specific deficiency in Klf2 limits thymopoiesis and regulatory T cell differentiation. Representative pseudocolor plots and bar graphs depicting the distribution and cellularity of distinct thymocyte cell subsets of 10-week-old control (Klf2fl/fl) and KLF2 cKO (Foxn1Cre:Klf2fl/fl) mice (A) CD4 and CD8 expression on total thymocytes defines DN, DP, SP4, and SP8 subsets. (B) CD25 and Foxp3 expression within TCRβ+ SP4 defines immature (CD25+Foxp3- and CD25-Foxp3+) and mature (CD25+Foxp3+) thymic regulatory T cell subsets. Data are representative of 3–6 independent experiments. (C) Regulatory T cell in vitro suppression assay measuring the proliferation of CFSE-labelled, FACS-sorted splenic responder T cells (T conventional; TCRβ+CD4+CD25−) from control mice, co-cultured with FACS-sorted control or KLF2 cKO thymic regulatory T cells (Treg; TCRβhighCD4+CD25+) at a T_reg:T_conventional ratio of 1:1 and 1:3. The negative control was set in the absence of polyclonal stimulation, and the positive control corresponded to maximal CFSE dilution in stimulated T conventional cells cultured in the absence of regulatory T cells. Histograms are from a representative experiment out of three…

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  Figure 4. KLF2 cKO mice manifest signs of compromised peripheral immunological tolerance (A) Representative H&E staining of the different degrees of infiltration in the salivary glands. Score 0 corresponds to the absence of detectable tissue infiltration Scale bars, 50 μm. (B) Severity scores for lymphocytic infiltration in salivary (SG) and lacrimal (LG) glands, and liver (LV) of 13 months-old of control (Klf2fl/fl) and KLF2 cKO(Foxn1Cre:Klf2fl/fl) mice. (C) ELISA quantification of anti-nuclear autoantibodies in the serum of 13 months-old control and KLF2 cKO mice. Serum from 6-month-old RELB knockout mice was used as a positive control for the detection of anti-nuclear antibodies. Bar graphs represent the mean ± SD, with each dot representing an individual mouse. ∗∗p < 0.01. (D) severity scores for lymphocytic infiltration in SG, LG, and LV of and 6–8 months-old AireWT, Klf2fl/flAire−/− and KLF2 cKOAire−/− mice. Pie charts represent absent (0, white), light (1, light gray), moderate (2, dark gray) and severe lesions (3, black) and each segment of the charts represents one mouse. Statistical analysis was carried out using an unpaired two-tailed Student’s t test; ∗∗p < 0.01.