Fig. 5

Upregulation of CCL2, CCL8 and CCL12 chemokines, specifically in Ltα^−/− mTEC^lo, correlates with the upregulation of classical NF-κB subunits. a Gating strategy and flow cytometry profiles used to analyse total mTECs (CD45^-Ep-CAM⁺BP-1^loUEA-1⁺), mTEC^lo (CD45⁻Ep-CAM⁺BP-1^loUEA-1⁺CD80^lo) and mTEC^hi (CD45⁻Ep-CAM⁺BP-1^loUEA-1⁺CD80^hi) in WT and Ltα^−/− mice. This gating strategy was used to sort and analyse mTECs in Figs. 3 and 5. The histogram shows numbers of mTEC^lo and mTEC^hi cells in both mice. b Ccl2, ccl8 and ccl12 mRNAs were measured by qPCR in purified mTEC^lo and mTEC^hi from WT (n = 8) and Ltα^−/− (n = 8) mice. c, d MCP1–4 (c) and phosphorylation of Ser536 p65 (d) were analysed by flow cytometry in mTEC^lo from WT and Ltα^−/− mice. Histograms show the MFI of MCP1–4 and phospho p65 (Ser536). e Relb, Rel and Rela mRNAs were measured by qPCR in purified mTEC^lo from WT (n = 8) and Ltα ^−/− (n = 8) mice. f, g RelB (f) and p65 (g) protein levels were analysed by flow cytometry in mTEC^lo from WT and Ltα^−/− mice. Histograms show the MFI of RelB and p65 expression. h LTβR expression was analysed by flow cytometry in WT mTEC^lo. The histogram shows the MFI of LTβR. i Relb, Rel and Rela mRNAs were measured by qPCR in mTECs loaded (n = 6) or not (n = 6) with OVA_323–339 peptide co-cultured with CD4⁺ thymocytes from OTII-Rag2^−/− mice or OTII-Rag2^−/−xLtα ^−/− mice. a–i Data are representative of two independent experiments (n = 3-4 mice per group and per experiment). II abs secondary antibodies, FMO fluorescence minus one, MFI mean fluorescence intensity. Error bars show mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using one-tailed Mann–Whitney test for c, f, g, two-tailed Mann–Whitney test for h and unpaired Student’s t-test for b, e and i