Fig 2: LTßR regulates thymic tuft cell development.a Intracellular staining of mTEClow from control Foxn1Cre and LTßRTEC mice for expression of the tuft cell marker DCLK1. Bar graphs indicate absolute cell numbers and percentages within mTEClow in Foxn1Cre mice (closed symbols) and LTßRTEC mice (open symbols) n = 7 biologically independent samples, over three independent experiments. Significant P values using two-tailed unpaired t test as follows: no. of tuft cells p = 0.0001, % tuft cells p = 0.002. b Confocal image of thymus section from Foxn1Cre and LTßRTEC mice, stained for the mTEC marker ERTR5 (magenta) and DCLK1 (cyan), representative of n = 3 independent biological samples. Scale bars denote 20 µm. c qPCR analysis of CD104- mTEClow FACS sorted from Foxn1Cre (closed symbols) and LTßRTEC (open symbols) mice, for expression of the tuft cell markers Pou2f3, Dclk1 and Trpm5, data are representative of three biological sorts. d Alymphoid 2dGuo-treated FTOC cultured for 4 days in the presence or absence of agonistic anti-LTßR (2 µg/ml) were analysed by flow cytometry for intracellular expression of DCLK1 to detect tuft cells. Tuft cells in freshly isolated adult WT mTEClow are shown for comparison, data are representative of three independent experiments. e qPCR analysis of control (closed circles) and anti-LTßR stimulated (open circles) dGuo FTOC for expression of Pou2f3 and Dclk1, with levels of mRNA expression in adult mTEClow (closed squares) for comparison, data are representative of three biological sorts from six FTOC cultured lobes/sort. f qPCR analysis of control (closed cirlces) and anti-LTßR stimulated (open circles) dGuo FTOC for expression of Ccl21a. All data are represented as mean ± SEM. ***P < 0.001 and **P < 0.01. Source data are provided as a Source Data file.

Fig 3: The adult mTEClow compartment is phenotypically diverse.a EpCAM1+Ly51-UEA1+ total mTECs from digested adult C57/BL6 (WT) thymus were subdivided into mTEClow and mTEChi on the basis of MHCII and CD80 expression. Cell surface (CD104) and intracellular (DCLK1 and CCL21) expression within total mTEClow is shown, which identifies three mTEClow subsets: CD104+CCL21+ cells (blue), CD104-CCL21-DCLK1+ thymic tuft cells (magenta) and CD104-CCL21-DCLK1- ‘triple-negative’ cells (green). b qPCR analysis showing mRNA levels of the tuft cell signature genes Pou2f3, Dclk1 and Trpm5 in CD104+ and CD104- mTEClow subsets that were FACS sorted from WT adult thymus, data are representative of three biological sorts. c Confocal microscopy of frozen tissue sections of adult CCL21tdTOM thymus stained with antibodies to DCLK1 (cyan), representative of n = 3 independent biological samples. Counterstaining with DAPI is in blue, CCL21tdTOM is in magenta and scale bars denote 20 µm. d Analysis of the ontogenetic frequency of mTEClow subsets identified in a, WT mice of 1 day, 1 week, 2 week, 4 week and 8 week were analysed, CD104+CCL21+ cells: 1 day n = 10, 1 week n = 10, 2 week n = 7, 4 week n = 7, 8 week n = 6, tuft cells: 1 day n = 9, 1 week n = 9, 2 week n = 6, 4 week n = 7, 8 week n = 10, triple-negative cells: 1 day n = 6, 1 week n = 6, 2 week n = 6, 4 week n = 6, 8 week n = 7, over three independent experiments per age. All data are represented as mean ± SEM. Source data are provided as a Source Data file.

Fig 4: Impaired production Of CD104+CCL21+ mTEClow In LTßRTEC mice.a mTEClow from Foxn1Cre and LTßRTEC mice, analysed for surface expression of CD104 and intracellular expression of CCL21. Bar graphs show cell percentages and absolute cell numbers in Foxn1Cre (n = 6 biologically independent samples, closed symbols) and LTßRTEC mice (n = 6 biologically independent samples, open symbols), over three independent experiments. Significant P values using two-tailed unpaired t test are as follows: no. of cells P = 0.0009. b MFI levels of intracellular CCL21 expression in pre-gated CD104+CCL21+ mTEClow from Foxn1Cre (closed symbols) and LTßRTEC (open symbols) mice, n = 6 biologically independent samples, over three independent experiments. c Levels of Ccl21 mRNA in FACS sorted CD104+ mTEClow from Foxn1Cre (closed symbols) and LTßRTEC mice (open symbols), data representative of three biological sorts. d Alymphoid 2dGuo-treated FTOC cultured for 4 days in the presence or absence of agonistic anti-LTßR (2 µg/ml) were pooled and analysed by flow cytometry for the expression of CCL21 and CD104. Freshly isolated adult WT mTEClow were stained alongside for comparison. Significant P values using two-tailed unpaired t test are as follows: % cells P = 0.0003, no. of cells P = 0.0102. Six FTOC cultured lobes were pooled per data point, n = 3 biologically independent samples, over three independent experiments. All data are represented as mean ± SEM. *P < 0.05 and ***P < 0.001. Source data are provided as a Source Data file.