Journal: NPJ Precision Oncology

Article Title: Comprehensive single-cell RNA analysis reveals intertumoral microenvironment heterogeneity and hub niche of carcinogenesis in thyroid cancer

doi: 10.1038/s41698-025-00924-7

Figure Lengend Snippet: A Circle plot summarizing the maximum number of interactions among individual cell types in metastatic lesions. The thickness of the connecting lines represents the interaction strength. B Comparison of the overall information flow, including the number and strength of interactions, within the inferred networks between PTC and metastatic lesions. C Heatmap displaying the potential outgoing and incoming interaction strength of each cellular cluster in PTC and metastatic lesions. D Heatmap illustrating the relative signaling contribution of each cell group based on the number and strength of interactions, comparing PTC with metastatic lesions. E Differential network centrality analysis based on EMT-like cancer-associated fibroblasts (CAFs), comparing PTC and metastatic lesions. F Bubble heatmap showing the cell-cell communication of selected ligand-receptor pairs between EMT-like CAFs and CD8 + PDCD1 + T cells. Dot size indicates the P -value, while color represents the communication probability. G Multiplex immunohistochemistry (mIHC) validation of the cross-talk between SAA1+ CAFs and T cells via the PPIA-BSG ligand-receptor interaction.

Article Snippet: We performed multiplex immunofluorescence staining using the following primary antibodies: THBS1 rabbit anti-human antibody (Affinity Biosciences; catalog no. DF6848), CD47 rabbit anti-human antibody (Affinity Biosciences; catalog no. DF6649), CD68 rabbit anti-human antibody (Affinity Biosciences; catalog no. DF7518), APOE rabbit anti-human antibody (Affinity Biosciences; catalog no. AF5178), CD3 rabbit anti-human antibody (Affinity Biosciences; catalog no. DF6848), THBS1 rabbit anti-human antibody (Affinity Biosciences; catalog no. DF6594), BSG rabbit anti-human antibody (Affinity Biosciences; catalog no. AF5221), COL3A1 rabbit anti-human antibody (Affinity Biosciences; catalog no. AF5457), PPIA rabbit anti-human antibody (Proteintech Group; catalog no. 10720-1-AP), and SAA1 rabbit anti-human antibody (Proteintech Group; catalog no. 16721-1-AP).

Techniques: Comparison, Multiplex Assay, Immunohistochemistry, Biomarker Discovery

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: The indicated mice were sublingually inoculated with cotton ball-saturated PBS (Sham) or C. albicans (OPC). Each symbol represents one mouse. A. Total mRNA from tongue homogenates of infected WT mice was subjected to qPCR for Il22 and Il17a and normalized to Gapdh at each time point. Graphs show mean ± SEM. Data are pooled from 4–9 mice per group. B. Fungal burdens were determined by CFU enumeration on YPD/Amp agar at day 5 p.i. Graphs show geometric mean ± SD. Data were pooled from 3 independent experiments. Dashed line indicates limit of detection (~30 CFU/g). C. Tongue homogenates were prepared on day 2 p.i. Left: A representative FACS plot showing percent of CD11b+Ly6G+ neutrophils (gated on live, CD45+ cells). Right: Data from 3 independent experiments. D-E. Il17a and Il17f in total RNA from tongue on day 2 p.i. was assessed by qPCR relative to Gapdh. Graphs show mean ± SEM relative to Sham-infected WT mice. F. Mice were injected i.p. with anti-IL-22 or isotype control IgG (150 μg) on days −1, 0, 1, 2, and 3 relative to infection. CFU was assessed on day 4, pooled from 2 independent experiments. G. Mice were treated with anti-IL-17A or isotype control (IgG2a) (200 μg) injected i.p. on days −1, 1, and 3 relative to infection. CFU was assessed on day 5, pooled from 3 independent experiments. H. Weight loss was assessed daily, shown relative to day 0 in mice from panel F. Data were analyzed by ANOVA or Student’s t-test, with Mann-Whitney U test for fungal load analysis.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Infection, Injection, Control, MANN-WHITNEY

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: A. RNASeq was performed on whole tongue mRNA from WT, Il22−/− and Il17ra−/− mice subjected to OPC and harvested on day 1 p.i. Venn diagram of differentially regulated or overlapping genes in infected Il22−/− and Il17ra−/− compared to WT mice. 215 genes were regulated both by IL-22 and IL-17RA, whereas 368 genes are regulated only by IL-22, and 931 genes were regulated only by IL-17RA. B. GSEA enrichment of predicted IL-6/STAT3 gene sets in Il17ra−/− and Il22−/− mice from panel A. C. Ingenuity Pathway Analysis of RNASeq data from panel A, indicating that STAT3 is an upstream regulator integrating Il22- and Il17ra- driven transcriptional networks. D. IF staining of tongue frozen sections with DAPI and anti-pSTAT3 (Tyr705) in WT, Il22−/− and Il17ra−/− mice harvested 2 days p.i. Size bar = 200 μm. E. qPCR of Il22 in tongue mRNA from WT or Il17ra−/− mice at 2 days p.i. normalized to Gapdh F. IF staining of DAPI, pSTAT3 (Tyr705) and K14 in WT or Il22ra1−/− mice at 2 days p.i. Size bar = 200 μm. G. The indicated mice were subjected to OPC and fungal burden quantified at day 5 p.i.. Data are pooled from 3 experiments H. All mice except Il22−/− were administered tamoxifen for 5 d, subjected to OPC and fungal burden assessed on day 5 p.i. Bars show geometric mean ± SD. Data was pooled from 3 experiments. Data analyzed by ANOVA with Mann-Whitney U test.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Infection, Staining, MANN-WHITNEY

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: The indicated mice were subjected to OPC. Each symbol represents one mouse A. Tongues were harvested on day 2 p.i. and Il22 mRNA was assessed by qPCR, normalized to Gapdh. Graphs show mean ± SEM relative to sham-infected WT mice. B. WT mice were infected with the indicated C. albicans strains. Il22 mRNA in tongue on day 2 p.i. was assessed by qPCR, normalized to Gapdh. Data were pooled from 2 independent experiments. C. BM from indicated donors was transferred into irradiated recipients. After 6–9 weeks, mice were subjected to OPC and fungal burdens assessed on day 5 p.i. Data were pooled from 2 experiments. D. On day 2 p.i., tongue homogenates from IL22TdTomato mice were stained for the TCRβ and TCRγδ and gated on the live CD45+TdTomato+ population. Left: representative FACS plots. Right: Pooled results from 3 independent experiments. Each symbol represents data from 2 pooled tongues. Data were analyzed by ANOVA or Student’s t-test, with Mann-Whitney U test for fungal load analysis. E. Comparisons of the relative percentages of IL-22+ cells (left) or IL-17+ cells (data from (29)) isolated from tongues of mice 2 days p.i. and analyzed by flow cytometry.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Infection, Irradiation, Staining, MANN-WHITNEY, Isolation, Flow Cytometry

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: The indicated mice were subjected to OPC. A. Fungal burdens were assessed on day 5 p.i. Bars show geometric mean ± SD. Data were pooled from 2 independent experiments. B. BM from indicated donors was transferred into irradiated recipients. After 6–9 weeks, mice were subjected to OPC and fungal burdens assessed on day 5 p.i. Data were pooled from 2 experiments. C. Frozen sections from WT tongues were co-stained with DAPI and Abs against K13, K14 or IL-22RA1. Suprabasal and basal epithelial layers are indicated. Images are representative of a minimum of 3 sections. Size bar = 200 μm. D. Top: Fungal burdens were assessed on day 5 p.i. Data are pooled from 3 independent experiments. Bottom: IF staining of tongues from the indicated mice were co-stained with DAPI and α-IL-22RA1 Abs. Size bar = 200 μm. E. Top: All mice except Il22−/− were administered tamoxifen for 5 days prior to OPC, and fungal burden assessed on day 5 p.i. Bars show geometric mean ± SD. Bottom: Frozen sections from tongues from the indicated mice were co-stained with DAPI and α-IL-22RA1. Size bar = 200 μm. Data were pooled from 3 independent experiments and analyzed by ANOVA with Mann-Whitney U test.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Irradiation, Staining, MANN-WHITNEY

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: RNASeq was performed on tongue mRNA from WT, Il22−/− or Il17ra−/− mice, isolated 24 h p.i. A. GSEA of mitotic spindle checkpoint pathway and cell death pathway genes. Normalized enrichment score is shown on Y-axis. B. Heatmap of cell cycle pathway genes in global differential gene expression analysis (Partekflow) in WT or Il22−/− mice. C. IF staining of DAPI, Ki67 and K14 in WT and Il22ra1−/− mice at 2 days p.i. Data are representative of images from 2 mice per group. Size bar = 100 μm. D. BrdU was administered 24 h p.i., and tongues harvested on day 2 p.i. Cell cycle/apoptotic status of CD45−EpCAM+ cells was determined by bromodeoxyuridine (BrdU) and 7-aminoactinomycin (7AAD) staining. Data show mean ± SEM. E. Frequency of CD45−EpCAM+ cells staining positive for active (cleaved) caspase-3 in tongue homogenates at day 2 pi measured by flow cytometry. Left: Representative FACS plot. Right: Pooled data from 4 independent samples showing mean fluorescence intensity (MFI) of cleaved Caspase 3 within the CD45−EpCAM+ compartment. Data analyzed by Student’s t-test. F. DAPI and TUNEL staining of tongue sections from the indicated mice at day 2 p.i. Images are representative of 4 mice per group. Size bar = 200 μm. G. Quantification of TUNEL+ cells from panel. F. Data analyzed by ANOVA and post-hoc Tukey’s test.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Isolation, Gene Expression, Staining, Flow Cytometry, Fluorescence, TUNEL Assay

Journal: Science immunology

Article Title: Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

doi: 10.1126/sciimmunol.aba0570

Figure Lengend Snippet: A. Heatmap of genes implicated in tissue repair, wound healing, keratinization and epithelial differentiation. B. Heatmap of IL-17 signature genes in OPC. C. qPCR of Il17ra expression normalized to Gapdh in tongue tissue from the indicated mice subjected to OPC and analyzed on day 2 p.i.. Data show mean ± SEM relative to sham-infected mice. Data analyzed by ANOVA or Student’s t-test. D. IF staining of IL-17RA and DAPI in the indicated mice on day 2 p.i. E. IL-17RA expression in CD45−EpCAM+ oral epithelial cells in WT or Il22−/− mice during OPC. Top: representative FACS histogram. Bottom: Pooled data from 2 independent experiments. Size bar = 200 μm. F. Expression of BD3 (Defb3) mRNA in tongue from WT or Il22−/− mice during OPC, normalized to Gapdh. Data analyzed by ANOVA or student’s t-test. G. Diagram of stratified oral epithelium during a first encounter with C. albicans. Fungal hyphae induces cellular damage and secrete the peptide candidalysin, which facilitates tissue invasion and activates innate IL-17- and IL-22-producing lymphocytes (see Refs (29, 61)). IL-17 was shown previously to act dominantly on K13+ cells of the suprabasal epithelial layer (SEL) (31). In contrast, IL-22/STAT3 promotes proliferation of the K14+ basal epithelial layer (BEL) that serves to restore the IL-17R-expressing SEL, thus maintaining IL-17-induced antifungal signals such as β-defensins and neutrophil responses that are required to mediate clearance of C. albicans. Diagram created on Biorender.com.

Article Snippet: We thank U. Siebenlist (NIH) for Act1 −/− mice, Amgen for Il17ra −/− mice and anti-IL-17RA Abs, Genentech for Il22 −/− mice and anti-IL-22 Abs, Janssen for anti-IL-17A antibodies, X. Lin (MD Anderson) for Card9 −/− mice, and F. De Beer (U. Kentucky) for Saa1/2 −/− mice.

Techniques: Expressing, Infection, Staining