human dpp4 cd26 antibody  (R&D Systems)

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: PDGFRα + DPP4 + fibroblasts are progenitors with enhanced fibrogenic capacity in TED. a) UMAP plot shows five diverse fibroblast subgroups labeled as G1–G5 after unsupervised clustering of fibroblasts from both control‐derived and TED‐derived orbital adipose SVF. All scRNA‐seq analyses presented in the figure are based on a total of five SVF samples, which include two control and three TED‐associated SVF samples. b) Dot plot showing the expression of representative markers of progenitor cells ( CD24 and DPP4 ), fibrosis ( ELN and FN1 ), and adipogenesis ( PPARG and FABP4 ) in the G1 to G3 subgroups. Dot size represents the proportion of cells expressing the denoted gene. Color indicates the average normalized expression level within the cluster. c) The left panel of the feature plot and violin plot shows the distribution and expression levels of representative markers of progenitor cells ( DPP4 ), adipogenesis ( APOD ), and fibrosis ( CTGF ) in the G1 to G5 subgroups. The right panel shows three highly expressed marker genes detected in G1 ( CADM3 ), G2 ( Glypican 3 (GPC3) ), and G3 ( TAGLN ) on a UMAP diagram and a violin plot. The y ‐axis of the violin plot represents the normalized read count. d) Biological process results of gene ontology enrichment analysis in G1 to G3 sorted by p ‐adjust < 0.05. e) Pseudotime trajectories of fibroblast subgroups G1 to G5 (upper), colored by Seurat clusters (middle) and split into individual groups (bottom) using Monocle 2. The pseudotime trajectory map is shown from dark to light blue.

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Labeling, Control, Derivative Assay, Expressing, Marker

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: PDGFRα + DPP4 + fibroblasts within OAT display significant fibrogenic capacity in TED. a) Hematoxylin and eosin (H&E), Masson's trichrome (Masson), and immunofluorescence staining of DPP4 (green) and DAPI (blue) in control and TED serial section tissue samples (scale bar = 100 µm, n = 3 patients). b) Schematic of experimental design for flow cytometry. DPP4 + cells indicate PDGFRα + DPP4 + fibroblasts, and GPC3 + cells indicate PDGFRα + GPC3 + fibroblasts in all the figures. c,d) Isolation results of c) PDGFRα + DPP4 + fibroblasts and d) PDGFRα + GPC3 + fibroblasts from SVF by flow cytometry. e) qRT‐PCR assay results of fibrosis‐ (red) and adipogenesis‐ (green) associated marker gene expression in PDGFRα + DPP4 + and PDGFRα + GPC3 + fibroblasts isolated via flow cytometry. Data are presented as mean ± standard deviation (SD) ( n = 3, representing three biologically independent experiments conducted using three different patient‐derived cells). Two‐sided unpaired t ‐test, * p < 0.05, ** p < 0.01, *** p < 0.001. f) Single‐cell RNA sequencing showing similar expression patterns of fibrosis‐ (red) and adipogenesis‐ (green) marker genes in the SVF of OAT from three patients with TED.

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Control, Flow Cytometry, Isolation, Quantitative RT-PCR, Marker, Gene Expression, Standard Deviation, Derivative Assay, RNA Sequencing, Expressing

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: PDGFRα + DPP4 + fibroblasts accumulate in fibrotic zones of the TED orbital adipose with macrophages. a) CellChat analysis infers cell–cell interactions and cross‐talk strength within the TED‐derived SVF subtypes. The left panel predicts overall interactions; the right panel focuses on PDGFRα + DPP4 + cells (labeled DPP4 + in the figure). Line width indicates the strength of interaction between subclusters. Significant interactions (L–R pairs) with a value > 10 and p < 0.05 are shown. b) Representative images showing H&E, Masson, and immunofluorescence staining for CD68 (red), DPP4 (green), and DAPI (blue) in serial sections from OAT from control and TED samples (scale bar = 100 µm, n = 3 patients). c–e) Characteristics of macrophages from OAT in patients with TED. (c) UMAP of SVF cells from the OAT of three patients with TED, colored by subgroup identity. Each dot represents a single cell. d) Proportions of M1/M2 macrophage subgroups from TED samples. e) Feature plot and violin plot showing the representative marker expression in M1/M2 macrophage subgroups. f) Representative images of H&E, Masson, and immunofluorescence staining of CD206 (red), DPP4 (green), and DAPI (blue) in serial sections of OAT from control and TED samples (scale bar = 100 µm, n = 3 patients).

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Derivative Assay, Labeling, Immunofluorescence, Staining, Control, Marker, Expressing

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: M2 macrophage‐induced PDGFRα + DPP4 + cell fibrosis in vitro. a) Schematic representation of an in vitro experimental investigating the interaction between PDGFRα + DPP4 + cells (isolated from patients' SVF, labeled DPP4 + in the figure) and THP‐1 induced M2 macrophage. Utilizing PDGFRα + GPC3 + cells (isolated from patients SVF, labeled GPC3 + in figure) as control. b) qRT‐PCR analysis results of fibrosis‐associated gene expression of PDGFRα + DPP4 + fibroblasts and PDGFRα + GPC3 + cells after treatment with the supernatant of M2 or M0 macrophages for 48 h. M0 macrophage‐conditioned medium was used as control. Data are presented as mean values ± standard deviation (SD) ( n = 6, representing six biologically independent experiments conducted using six different patient‐derived cells). Two‐sided unpaired t ‐test, ** p < 0.01, *** p < 0.001.

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: In Vitro, Isolation, Labeling, Control, Quantitative RT-PCR, Gene Expression, Standard Deviation, Derivative Assay

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: Signaling interactions reveal that macrophages may activate PDGFRα + DPP4 + fibroblasts via the GAS6‐AXL pathway. a) Cell‐Chat analysis results of potential interactions between PDGFRα + DPP4 + or PDGFRα + GPC3 + fibroblasts and M1/M2 macrophage subgroups. Heatmap shows the inferred outgoing communication patterns of secreting cells (left) and the incoming communication patterns of receiving cells (right), revealing corresponding inferred latent patterns and cell groups, as well as signaling pathways. b) Jointly projecting and clustering signaling pathways from fibroblasts and macrophages into a shared two‐dimensional manifold according to their functional similarity. Circles represent the signaling networks from fibroblast and macrophage subgroups, and each circle represents the communication network of one signaling pathway. Circle size is proportional to the total communication probability (Commun. Prob). Different colors represent different groups of signaling pathways. c) Bubble plot shows selected L–R pairs of PDGFRα + DPP4 + cells and M2 macrophages. The colors range from blue to red, representing low to high communication probability. The bubble size represents the corresponding p ‐values. d) Overall pathways involved in cross‐talk between PDGFRα + DPP4 + fibroblasts and M2 macrophages. The line width indicates the interaction strength. e,f) Heatmap showing the importance of each cluster, based on four network centrality measures, of e) SEMA3 and (f) GAS signaling. g) Representative serial‐section images of H&E staining (Slice 1), immunofluorescence staining of SEMA3C/NRP‐1 (Slice 2), and GAS6/AXL (Slice 3) in OAT from patients with TED (scale bar = 100 µm, n = 3 patients). SEMA3C and GAS6 are stained red; NRP‐1 and AXL are green, and DAPI is blue.

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Protein-Protein interactions, Functional Assay, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: PDGFRα + DPP4 + Fibroblasts‐Macrophage Crosstalk Induces Orbital Fibrosis in Treatment‐Resistant Thyroid Eye Disease via the GAS6‐AXL Pathway

doi: 10.1002/advs.202511404

Figure Lengend Snippet: AXL inhibitor TP0903 inhibits fibrosis in PDGFRα + DPP4 + fibroblasts induced by M2 macrophages derived from THP‐1 cells and from TED patients. a) Comparison of relative GAS6 mRNA expression in M0 macrophages, LPS‐induced M1 macrophages, and IL‐4 and IL‐13‐induced M2 macrophages derived from THP‐1 cells. Data are presented as mean ± standard deviation (SD) ( n = 3). Two‐sided unpaired t ‐test, ** p < 0.01. b) ELISA results of GAS6 concentration in the supernatant of M0 and M2 macrophages derived from THP‐1 cells. Data are presented as mean ± SD ( n = 3). Two‐sided unpaired t ‐test, ** p < 0.01. c) Schematic representation of in vitro experiment verifying our hypothesis that blocking GAS6‐AXL signaling with TP‐0903 pre‐treatment reduces M2 macrophage‐mediated fibrosis. d) qRT‐PCR analysis of mRNA expression of fibrosis‐associated genes COL1A1, COL3A1, ELN, FN1, ACTA2 , and TIMP1 in PDGFRα + DPP4 + fibroblasts after treatment with M2 or M0 macrophage conditioned medium, with or without TP0903. M0 macrophage‐conditioned medium was used as a control. Data are presented as mean ± SD ( n = 6, representing six biologically independent experiments conducted using six different patient‐derived cells). Two‐sided unpaired t ‐test, * p < 0.05, ** p < 0.01, *** p < 0.001. e) Schematic representation of the experimental design to investigate the interaction between PDGFRα + DPP4 + fibroblasts and monocyte‐derived M2 macrophages in vitro. f) ELISA results of GAS6 concentration in the supernatant of M0 and M2 macrophages, derived from monocytes of patients with TED. Data are presented as mean ± SD ( n ≥ 3, representing three biologically independent experiments conducted using three different patient‐derived cells). Two‐sided unpaired t‐ test, ** p < 0.01. g) qRT‐PCR analysis of mRNA expression of fibrosis‐associated genes COL1A1, COL3A1, ELN, FN1, ACTA2 , and TIMP1 in PDGFRα + DPP4 + cells after treatment with monocyte‐derived M2 or M0 macrophage conditioned medium with or without TP0903. M0 macrophage‐conditioned medium was used as control. Data are presented as mean ± SD ( n = 6, representing six biologically independent experiments conducted using six different patient‐derived cells). Two‐sided unpaired t‐ test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After overnight incubation with primary antibodies, including DPP4 (R&D Systems, Minneapolis, MN, USA, catalogue no. AF1180, 1:250), CD68 (Invitrogen, Waltham, MA, USA, catalogue no. PA5‐89134, 1:100), CD206 (Abcam, China, catalogue no. ab64693, 1:100), SEMA3C (Abclonal, China, catalogue no. A15386, 1:100), NRP‐1 (Santa, China, catalogue no. sc‐5307, 1:100), GAS6 (Santa, catalogue no. sc‐376087, 1:100), and AXL (Abclonal, catalogue no. A17874, 1:100) at 4 °C, the sections were washed three times with PBS and incubated with secondary antibodies for 1 h at room temperature.

Techniques: Derivative Assay, Comparison, Expressing, Standard Deviation, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vitro, Blocking Assay, Quantitative RT-PCR, Control