Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: Binding properties of PBA-0111 to GARP and GARP/TGF-β1. (A) Increasing concentrations of PBA-0111 were titrated on HEK293T cells overexpressing GARP, GARP/TGF-β1 or empty vector (EV) control, and the binding potency was analyzed by flow cytometry. (B) Increasing concentrations of PBA-0111, an ABBV-151-like antibody, a DS-1055a-like antibody, or an afucosylated isotype control were titrated on L-428 lymphoma cell line, which endogenously expresses GARP/TGF-β1 and the binding potency was analyzed by flow cytometry. Data in (A) and (B) are shown as mean ± standard deviation (SD) and are representative of at least three independent experiments performed in duplicates. (C) Flow cytometry analysis of binding of PBA-0111, DS-1055a-like, or ABBV-151-like antibody to human platelets at 10 µg/mL. Data are expressed as fold change in median fluorescence intensity (MdFI) relative to the afucosylated isotype control, (n=6 independent donors, **p=0.0062, n.s, p=0.06 comparing to PBA-0111, Kruskal-Wallis test). (D) Biolayer interferometry (BLI) binding analysis of PBA-0111 against recombinant extracellular GARP, GARP/TGF-β1, latency associated peptide (LAP), TGF-β1, latent TGF-β1, latent TGF-β2, latent TGF-β3, or GARP paralog leucine-rich repeat-containing protein 15 (LRRC15) at 500 nM. All samples were tested in at least three independent experiments in duplicate. (E) HEK-Blue™ TGF-β reporter assay. Raji GARP/TGF-β1 cells were pre-incubated with the indicated antibodies for 30 minutes and then co-cultured with HEK-Blue™ TGF-β cells. After 48h at 37 °C, SEAP activity was assessed using QUANTI-Blue™ Solution and optical density (OD) measured at 630 nm. Data shown as mean ± SD, n= 9, from 3 independent experiments. Statistical differences were assessed by Kruskal-Wallis test with Dunn´s multiple comparisons versus vehicle or afucosylated isotype control, **p<0.001. (F) Flow cytometry analysis of GARP/TGF-β1 complex formation on Raji-GARP cells. Cells were preincubated with 0.2 ng/mL to 50 µg/mL of PBA-0111, ABBV-151-like antibody, DS-1055a-like antibody, or afucosylated isotype control prior to addition of recombinant latent TGF-β1 at final concentration of 1.5 µg/mL. Complex formation was detected using an anti-LAP antibody. The graph is representative of at least three independent experiments. (G) TGF-β1 signalling was assessed in a Jurkat cell line overexpressing β8-GARP, stimulated with plate-bound anti-CD3 beads and soluble anti-CD28 in the presence of indicated antibodies (20 μg/mL) for 24 h at 37 °C. Cell lysates were analyzed by western blot and densitometric analysis of pSMAD2 bands was performed. (H) pSMAD2 band intensities from WB were normalized to the loading control (GAPDH) and then to isotype control,(n= 6 gels, ABBV-151 n=8 samples, 4 MHG-8 and 4 LGH.10.6). Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparisons to IgG1.aF control, ***p=0.0002 (ABBV-151, n=8), **p=0.0037 (ADWA11). Statistical significance of PBA-0111 in single comparison to IgG1. aF was performed with Mann-Whitney analysis, p=0.002.

Article Snippet: Protein lysates were run in 4-20% Mini-Protean TGX Gel (BioRad), transferred to membranes followed by overnight incubation with anti-pSMAD2 antibody (Cell signalling technology, 3108L) and subsequent anti-rabbit IgG-HRP (R&D Systems, AF008) for one hour at RT.

Techniques: Binding Assay, Plasmid Preparation, Control, Flow Cytometry, Standard Deviation, Analysis, Fluorescence, Recombinant, Reporter Assay, Incubation, Cell Culture, Activity Assay, Concentration Assay, Western Blot, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: TGFβ determines epithelial tissue spacing by regulating mesenchymal condensation

doi: 10.64898/2026.03.16.712215

Figure Lengend Snippet: A. Schematic showing two possible mechanisms to spatially pattern proliferation in the embryonic lung. B. Representative immunoblots for pSMAD2 and GAPDH in lungs cultured with RepSox or vehicle control, and graph showing the normalized pSMAD2 band intensity from 3 replicates. Shown are the average and s.d. of normalized intensity of the pSMAD2 band. C. Fluorescence images of EdU incorporation in the epithelium and mesenchyme of lung explants cultured with RepSox or vehicle control. White arrows denote closely apposed branch stalks. Solid and dotted boxes denote the regions between b1 and b2, respectively. Scale bars, 100 µm. D. Graph showing the percentage of EdU-positive epithelial cells in lung explants cultured with RepSox or vehicle control. Shown are the average and s.d. of 6 lungs per condition. n.s. stands for not significant. *** p <0.001. E. Graph showing the percentage of EdU-positive mesenchymal cells in lung explants cultured with RepSox or vehicle control. Shown are the average and s.d. of 3 lungs per condition. ** p <0.005. F. Graph showing the density of mesenchymal cells near the tip in lung explants cultured with RepSox or vehicle control. Shown are the average and s.d. of at least 4 lungs per condition. * p <0.05.

Article Snippet: Samples were then incubated with primary antibodies against E-cadherin (1:100; BD Biosciences, 610182), pSMAD1/5/9 (1:100; Invitrogen, PA5-64711), pan-laminin (1:100; Invitrogen, PA1-16730), perlecan (1:100; Invitrogen, MA1-06821), or pSMAD2 (1:100; Cell Signaling, 18338).

Techniques: Western Blot, Cell Culture, Control, Fluorescence

Journal: bioRxiv

Article Title: TGFβ determines epithelial tissue spacing by regulating mesenchymal condensation

doi: 10.64898/2026.03.16.712215

Figure Lengend Snippet: ( A ) Fluorescence images of nuclei (blue) and EdU incorporation (red) in lung explants inserted with TGFβ-containing bead or vehicle control. The beads were inserted proximal to b1. In contrast to control, few EdU-positive mesenchymal cells are observed in TGFβ-containing bead-inserted lung. White arrows denote proximal stalk of b1. ( B ) Fluorescence images of epithelium in branch-implanted lung explants treated with RepSox or vehicle control. The distance between implanted and native branches is small in RepSox-treated lungs. Scale bars, 100 µm and 10 µm (inset). ( C ) Fluorescence images of nuclei in lung explants cultured for 12- or 24-hours after implantation of beads with different TGFβ concentrations. ( D ) Graph showing the sizes of condensations in bead-implanted lung explants with different initial TGFβ concentration and culture time. ( E ) Fluorescence images of nuclei (blue) and pSMAD2 (red) counterstained for E-cadherin (green). Strong circular pSMAD2 staining is observed near TGFβ-containing beads. ( F ) Fluorescence images of nuclei (blue) and perlecan (red) in lung explants inserted with TGFβ-containing bead or vehicle control. Strong perlecan staining is observed in mesenchymal condensations near TGFβ-containing beads. ( G ) Fluorescence images of nuclei in lung explants cultured with or without RepSox after being inserted with TGFβ-containing beads or vehicle control. Mesenchymal condensations are only observed in lungs inserted with TGFβ-containing beads and cultured without RepSox. ( H ) pSMAD2 staining and nuclear-masked pSMAD2 staining intensity near the branch. Scale bar, 50 µm. Subepithelial mesenchymal cells exhibit strong pSMAD2 staining. Cyan dotted lines denote condensations ( D, F, G ). Scale bars, 100 µm ( A, C, E, F, G ).

Article Snippet: Samples were then incubated with primary antibodies against E-cadherin (1:100; BD Biosciences, 610182), pSMAD1/5/9 (1:100; Invitrogen, PA5-64711), pan-laminin (1:100; Invitrogen, PA1-16730), perlecan (1:100; Invitrogen, MA1-06821), or pSMAD2 (1:100; Cell Signaling, 18338).

Techniques: Fluorescence, Control, Cell Culture, Concentration Assay, Staining

Journal: Research

Article Title: Mapping Immune-Inflammatory Niches on Zirconia Bone Implants: Single-Cell Transcriptomic Profiling

doi: 10.34133/research.1162

Figure Lengend Snippet: Ti and ZrO 2 implants induce early osseointegration and immune inflammation. (A) Transcriptional levels of osteogenesis-related and immune-inflammatory genes. Osteogenic genes: Runx2 , Osterix , and Tgfb1 . Immune-inflammatory genes: Il1b , Mmp9 , and Il23a . (B) Receiver operating characteristic (ROC) analysis based on bulk RNA-seq data. AUC, area under the curve. (C) Protein expression levels of osteogenesis-related and immune-inflammatory markers. Osteogenic proteins: RUNX2, OSX, TGFB1, pSMAD2/SMAD2, and pSMAD3/SMAD3. Immune-inflammatory proteins: IL1B, MMP9, and IL23A. (D) Statistical analysis of protein expression levels. Statistical significance was determined using one-way analysis of variance. RUNX2, RUNX family transcription factor 2; OSX, osterix; TGFB1, transforming growth factor beta 1; SMAD2, SMAD family member 2; SMAD3, SMAD family member 3; IL1B, interleukin 1 beta; MMP9, matrix metallopeptidase 9; IL23A, interleukin 23 subunit alpha.

Article Snippet: The membranes were blocked with 5% nonfat milk for 1 h, followed by overnight incubation at 4 °C with the following primary antibodies: RUNX2 (Bioss, bs-1134R, 1:1,000, China), OSX (Bioss, bs-25532R, 1:1,000, China), pSMAD2 (Bioss, bs-3419R, 1:1,000, China), pSMAD3 (Bioss, bs-3425R, 1:1,000, China), IL1B (Bioss, bs-0812R, 1:1,000, China), MMP9 (Bioss, bsm-54040R, 1:3,000, China), IL23A (Bioss, bs-1193R, 1:1,000, China), NOS2 (Bioss, bs-0162R, 1:2,000, China), CD206 (Bioss, bsm-55604R, 1:1,000, China), TGFB1 (Proteintech, 81746-2-RR, 1:1,000, China), SMAD2 (Proteintech, 12570-1-AP, 1:3,000, China), and SMAD3 (Proteintech, 30130-1-AP, 1:2,000, China).

Techniques: RNA Sequencing, Expressing

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Conditional knockout of Tgfβ1 in macrophage lineage cells reduces pSmad2 levels and promotes functional recovery in SCI mice. a – d ELISA analysis showing the concentration of TGF-β in the spinal cord and the serum after SCI in each mouse for the groups indicated (* P < 0.05, ** P < 0.01, n = 4). e Representative Western blots showing the activation of phosphorylated Smad (pSmad) signaling. f Representative images of immunofluorescent staining of pSmad2 + (red), platelet-derived growth factor receptor-β + (PDGFR-β + ) (green) pericytes , and DAPI (blue) staining of nuclei at 7 days after SCI. Scale bar, 10 µm. g Representative immunofluorescence images of PDGFR-β + (green) pericytes and DAPI (blue) staining of nuclei at 2 weeks after SCI. Scale bars, 200 µm. h Quantitative analysis of the number of pSmad2 + cells (**** P < 0.000 1, n = 6). i Quantitative analysis of the number of PDGFR-β + cells (** P < 0.01, n = 6). j , k Illustrations of the hot plate test and the von Frey test. l Quantitative analysis of hindpaw withdrawal time responding to temperature (hot plate test, ** P < 0.01, n = 8). m , n Quantitative analysis of hindpaw withdrawal frequency responding to mechanical stimulation (von Frey test, 0.7 mN and 3.9 mN, * P < 0.05, ** P < 0.01, n = 8). o Quantitative analysis of Basso Mouse Scale (BMS) score between Tgfb1 flox / flox control mice after SCI, Tgfb1 LysM-cre −/− mice after SCI, and Tgfb1 flox/flox control mice without SCI (* P < 0.05, n = 8). PWF paw withdrawal frequency. LF left forepaw, RF right forepaw, RH right hindpaw. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques: Knock-Out, Functional Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Activation Assay, Staining, Derivative Assay, Immunofluorescence, Hot Plate Test, Control

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Conditional knockout of TGF-β type 2 receptor ( Tgfbr2 ) in pericytes reduces fibrotic scar formation in SCI mice. a Schematic diagram of Tgfbr2 knockout in Glast-Cre + pericytes. b Representative images of immunofluorescent analysis of PGP9.5 + (red) nerve fibers, fibronectin + (green) fibrotic scar, and DAPI (blue) staining of nuclei at 4 weeks after SCI. Scale bar, 200 µm. c , d Quantitative analysis of the intensity value of PGP9.5 and fibronectin (* P < 0.05, n = 6). e Representative images of immunofluorescent analysis of nerve axon–specific β-III-tubulin + (green), collagen III + (red) fibrotic scar, and DAPI (blue) staining of nuclei at 4 weeks after SCI. Scale bar, 200 µm. Right images are high resolution versions of the boxed regions in the left images. Scale bar, 50 µm. f Quantitative analysis of the intensity value of collagen III (* P < 0.05, n = 6). g Representative images of immunofluorescent analysis of neurotransmitter marker 5-HT + (red), nerve axon–specific β-III-tubulin + (green), and DAPI (blue) staining of nuclei in spinal cord lesion site of T10 in Tgfbr2 flox/flox sham group mice, Tgfbr2 flox/flox control mice, and Tgfbr2 Glast-creER −/− mice at 4 weeks after SCI. Scale bar, 200 µm. Right images are high-resolution versions of the boxed regions in the left images. Scale bar, 50 µm. h , i Quantitative analysis of the intensity value of 5-HT and β-III-tubulin (* P < 0.05, n = 6). j Representative images of immunofluorescent analysis of pSmad2 + (red), PDGFR-β + (green) pericytes and DAPI (blue) staining of nuclei at 7 days after SCI. Scale bar, 10 µm. k , l Quantitative analysis of the intensity mean value of PDGFR-β and the number of pSmad2 + cells (*** P < 0.001, **** P < 0.000 1, n = 6). m Representative Western blots showing the activation of pSmad signaling. Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques: Knock-Out, Staining, Marker, Control, Western Blot, Activation Assay

Journal: Bone Research

Article Title: TGF-β-induced fibrotic scar formation limits recovery of spinal cord injury

doi: 10.1038/s41413-026-00507-7

Figure Lengend Snippet: Neonatal mice completely recover from SCI without scarring. a Schematic diagram illustrating the timeline of the experimental procedures. b Representative images of immunofluorescent analysis of pSmad2 (red), PDGFR-β (green), and DAPI (blue) in the spinal cord lesion site in neonatal mice on days 2, 7, and 12 with or without SCI. Scale bar, 50 µm. Right images are high-resolution versions of the boxed regions in the left images. Scale bar, 50 µm. c , d Quantitative analysis of the number of pSmad2 + cells and PDGFR-β + cells (** P < 0.01, *** P < 0.001, **** P < 0.000 1, n = 6). e Representative images of immunofluorescent analysis of β-III-tubulin (green), collagen III (red), and DAPI (blue) in the spinal cord lesion site in neonatal mice on days 2 and 12, and adult mice with or without SCI. Scale bar, 200 µm. f , g Quantitative analysis of the intensity mean value of collagen III + fibrotic scar and β-III-tubulin + nerves (* P < 0.05, **** P < 0.000 1, n = 6). Statistical significance was determined by multifactorial ANOVA, and all data are shown as means ± standard deviations. Source data are provided as a Source Data file

Article Snippet: We used antibodies recognizing mouse pSmad2 (1:1 000, Cell Signaling Technology, Inc., Danvers, MA, USA) and Smad2 (1:1 000, Cell Signaling Technology, Inc.) to examine the protein concentrations in the lysates.

Techniques: