Fig 1: Expression of the MHC class I-like molecule CD1d and the chemokine CXCL16. (a) Representative flow cytometry for CD1d expression in hepatic immune cells and splenocytes. Hepatic MNCs and splenocytes were isolated from WT and LXRa/ß-KO mice, and stained with APC-conjugated anti-CD1d antibody together with FITC-conjugated anti-F4/80 antibody and Pacific Blue-conjugated anti-CD11b antibody. Expression of CD1d was analyzed in hepatic MNCs, splenocytes, neutrophils (F4/80-CD11b+), BMDMs (F4/80loCD11b+) and resident Kupffer cells (F4/80hiCD11b-). Similar results were obtained in repeated experiments (n = 4 for each analysis). (b) Cd1d1 mRNA expression in hepatic MNCs, whole liver samples and splenocytes. (c) Cd1d1 mRNA expression in F4/80+ Kupffer cells/macrophages, CD146+ liver sinusoidal endothelial cells and CD11c+ dendritic cells isolated from hepatic MNCs. (d) Cxcl16 mRNA expression in hepatic MNCs. (e) Cxcl16 mRNA expression in F4/80+ Kupffer cells/macrophages, CD146+ liver sinusoidal endothelial cells and CD11c+ dendritic cells isolated from hepatic MNCs. Hepatic MNCs with collagenase digestion, whole liver samples and splenocytes were obtained from WT and LXRa/ß-KO mice (n = 5) (a, b and d). Hepatic MNCs were isolated after collagenase perfusion, and F4/80+ cells, CD146+ cells and CD11c+ cells were sorted with magnetic beads. *P < 0.05 (Student’s t test).
Fig 2: Decreased population of iNKT cells in the thymus of LXRa/ß-KO mice. (a) Numbers of iNKT cells in thymocytes. (b) Cd1d1 and Zbtb16 mRNA expression and (c) Nr1h3 and Nr1h2 mRNA expression in the thymus. (d) mRNA expression of Nr1h3 and Nr1h2 in iNKT cells, TECs, CD4+ T cells and CD8+ T cells from the thymus. Thymocytes were stained with PE-conjugated CD1d tetramer, FITC-conjugated anti-ßTCR, PE-Cy7-conjugated anti-EpCAM and APC-conjugated anti-CD45 to separate iNKT cells (CD45+ßTCR+CD1d-tetramer+ cells) and TECs (EpCAM+ cells), or with FITC-conjugated anti-ßTCR, PE-conjugated anti-CD4 and PE-Cy7-conjugated anti-CD8a to separate CD4+ T cells (CD45+ßTCR+CD4+ cells) and CD8+ T cells (CD45+ßTCR+CD8a+ cells). (e) The populations of thymic iNKT cells of stage 0, stage 1, stage 2 and stage 3. Thymocytes were isolated from WT and LXRa/ß-KO mice, and stained with FITC-conjugated anti-NK1.1, PE-conjugated CD1d tetramer, PE-Cy5-conjugated anti-ßTCR, Brilliant Violet-510-conjugated anti-CD24 and APC-conjugated anti-CD44. iNKT cell stages were determined as stage 0 (CD24+CD44-), stage 1 (CD24-CD44-NK1.1-), stage 2 (CD24-CD44+NK1.1-) and stage 3 (CD24-CD44+NK1.1+) in the population of ßTCR+CD1d-tetramer+-gated cells (iNKT cells). Stg, stage. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test). n.d., not detected (around or below detection limits).
Fig 3: Decreased population of iNKT cells in the liver of LXRa/ß-KO mice. (a) Representative flow cytometry for NKT cells (NK1.1+ßTCR+) and NK cells (NK1.1+ßTCR-) in hepatic MNCs. (b) Numbers of NKT cells and NK cells. (c) Representative flow cytometry for iNKT cells (ßTCR+CD1d-tetramer+). (d) Numbers of iNKT cells. (e) Representative flow cytometry for CD69 expression in NK1.1+ßTCR+-gated NKT cells and NK1.1+ßTCR--gated NK cells. (d) Numbers of CD69-positive cells in NKT cells and NK cells. (f) Percentages of NK1.1-CD44- cells (stage 1), NK1.1-CD44+ cells (stage 2) and NK1.1+CD44+ cells (stage 3) in iNKT cells. Hepatic MNCs were isolated from WT, LXRa-KO, LXRß-KO and LXRa/ß-KO mice and stained with fluorescein isothiocyanate-conjugated (FITC-conjugated) anti-ßTCR, phycoerythrin-conjugated (PE-conjugated) anti-NK1.1, biotin-conjugated anti-CD69 and PE-Cy5-streptavidin (a, b, d and e), with PE-conjugated CD1d tetramer and PE-Cy5-conjugated anti-ßTCR (c and d) or with FITC-conjugated anti-NK1.1, PE-conjugated CD1d tetramer, PE-Cy5-conjugated anti-ßTCR and allophycocyanin-conjugated (APC-conjugated) anti-CD44 (g) (n = 4). (a-f) **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). (g) Stg, stage. *P < 0.05 (Student’s t test).
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