Figure 3: SP thymocyte differentiation and survival in LUBAC-deficient mice.

(a) Schematic representation of CD4+ T-cell development in the thymus. Earliest thymic progenitor (ETP) cells undergo progressive differentiation from DN to DP to single-positive (CD4 or CD8) cell. The different stages of thymocyte development are also accompanied by changes in CD24, CD62L, CCR7, CD69 and MHC I (H2-K^b) surface marker expression on the differentiating thymocyte. (b) Flow cytometric analysis of the surface expression of MHC I (H2-K^b) versus CD69 on whole thymocytes from 7- to 15-week-old control, Hoip^ΔCd4, Hoil^ΔCd4 and Sharpin^cpdm mice. (c) Quantification of total cell numbers and percentages of Fraction 4 (MHC I^high CD69^high) and Fraction 5 (MHC I^high CD69^low) populations for control, Hoip^ΔCd4, Hoil^ΔCd4 and Sharpin^cpdm mice. Surface expression of CD24 versus CD62L (d), CD24 versus CCR7 (e), Helios versus CCR7 (f) gated on CD4SP from 7- to 15-week-old control, Hoip^ΔCd4, Hoil^ΔCd4 and Sharpin^cpdm mice. Right, cell numbers of the mature CD24^low CD62L^high, CD24^low CCR7^high and HELIOS^high CCR7^high from CD4SP cells. (g) Contribution to different thymic T cell subsets in 50:50 mixed bone marrow chimeras 8 weeks after reconstitution. Columns show percentage of WT CD45.1⁺ (white bar) and CD45.2⁺ control, Hoip^ΔCd4, Hoil^ΔCd4 and Sharpin^cpdm (black bar) cells in individual chimeric mice. DN; CD4⁻CD8⁻, DP; CD4⁺CD8⁺ SP3; CD4⁺CD24^lowCCR7^high, W1; CD4⁺CCR7^low,HELIOS⁺, W2; CD4⁺CCR7^high,HELIOS⁺, Treg; CD4⁺FOXP3⁺. Each bar represents an individual mouse. (h) Surface expression of CD4 and CD8 on whole thymocytes (upper panels) and B220 and TCRβ on splenocytes (lower panels) from control, Bax^ΔCd4Bak^−/−, Hoip^ΔCd4 and Hoip^ΔCd4Bax^ΔCd4Bak^−/− mice. For c,d,e and f, each symbol represents an individual mouse; small horizontal lines indicate mean±s.d.; *P<0.05, **P<0.01, ***P<0.005 and ****P<0.001, respectively. One-way analysis of variance with a Tukey’s post-hoc test for multiple comparisons was used for statistical analysis. Shpn^cpdm refers to Sharpin^cpdm mice. Data are pooled from six independent experiments with two to six mice per group (b–f) or representative of two independent experiments with four to six mice per group (g), or representative of two independent experiments with one to five mice per group (h).