Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Structural and biological characterization of SIS and UBM-SIS meshes and isolated MBVs. A) Surface and cross-sectional morphology of SIS and UBM-SIS meshes by SEM. B) Quantification of mesh thickness (n = 10). C) Pore size and porosity analysis of mesh (n = 10). D) Live/dead staining of fibroblasts on SIS and UBM-SIS at day 1, 4, and 7. (E) CCK-8 assay of fibroblast viability on meshes (n = 5). F) Immunofluorescence staining of fibroblasts (TGF-β1, day 3), SMCs (phalloidin, day 7) and HUVECs (CD31, day 14) on SIS and UBM-SIS meshes, and SEM and DAPI staining of SMCs (day 21) coverage and cellular infiltration. White dashed lines delineate the upper and lower boundaries of the ECM scaffold. The yellow dashed line indicates the infiltration depth, defined as the distance from the scaffold surface to the DAPI-positive nucleus formed as a cellular floor and used for quantitative analysis. G) Quantification of TGF-β1, cytoskeletal area (phalloidin) and CD31 expression (n = 5). H) Quantification of cellular infiltration across mesh thickness (n = 5). I) Schematic of ECM components retained in decellularized ECM mesh. J) H&E and Masson's trichrome staining of mesh. K) Residual DNA quantification. L) Analysis of cytokine and growth factor profiling upon mesh-specific difference (n = 4). M) Workflow for MBV isolation and analysis. N) SEM images of MBV embedded on ECM. O) TEM images of MBV morphology. P) NTA analysis of MBV (n = 4). Q) Western blot detection of exosomal markers in MBV. Data are presented as mean ± SD. Each dot represents an independent sample. Statistical significance was determined using two-tailed unpaired Student's t-test for comparisons between two groups (B, C, and K), or one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (E, G, and H), where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Isolation, Pore Size, Staining, CCK-8 Assay, Immunofluorescence, Expressing, Western Blot, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Bioactivity and immunomodulatory properties of MBVs derived from SIS and UBM-SIS meshes. A) Schematic illustration of MBV-regulated cellular activities during ECM remodeling via their interactions with fibroblasts, SMCs, HUVECs, and macrophages to validate MBVs as bioactive components embedded within parent ECM. Nuclei are labeled with DAPI (blue); PKH26 (red) marks MBVs; phalloidin (green) stains F-actin. B) Immunofluorescence staining of fibroblasts (TGF-β1, collagen I), SMCs (phalloidin), and HUVECs (CD31) after treatment with SIS MBVs or UBM-SIS MBVs. C) Quantification of fluorescence signal area per cell for respective markers (n = 5). D) Western blot analysis of marker proteins in MBV-treated cells. E) Relative protein expression levels normalized to GAPDH (n = 3). F) Schematic of macrophage polarization model with/without MBV treatment. G) Immunostaining of macrophages for F4/80, iNOS (M1-like), and Arg-1 (M2-like) under different stimulations and MBV-treated conditions. H) Quantification of mean fluorescence intensity (MFI) of iNOS and Arg-1 (n = 5). I) Western blot analysis of pro- and anti-inflammatory markers in MBV-treated macrophages and LPS + IFN-γ-treated macrophages (control). J) Quantification of relative protein levels (n = 3). K) Heatmap of RT-qPCR analysis showing cytokine and ECM regulator gene expression in MBV-treated macrophages (n = 3). L) Representative fluorescence images of DCFH staining in macrophages following different treatments. M) Quantification of DCFH fluorescence area per cell (n = 5). Data are presented as mean ± SD. Each dot represents an independent biological replicate. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Derivative Assay, Labeling, Immunofluorescence, Staining, Fluorescence, Western Blot, Marker, Expressing, Immunostaining, Control, Quantitative RT-PCR, Gene Expression

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Functional comparison of MBVs derived from SIS and UBM in modulating inflammation, angiogenesis, and matrix remodeling. A) Schematic of macrophage polarization model under LPS + IFN-γ stimulation with or without MBV treatment. B) Immunofluorescence staining of iNOS (red) and Arg-1 (green) in macrophages treated with SIS MBVs, UBM MBVs, or UBM-SIS MBVs. C) Quantification of mean fluorescence intensity (MFI) of iNOS and Arg-1 (n = 5). D–G) RT-qPCR analysis of pro- (( TNF-α, IL-6 ) and anti-inflammatory ( IL-10, TGF-β1 ) cytokine gene expression in MBV-treated macrophages (n = 4). H) Schematic of analysis of MBV-treated HUVECs and fibroblasts cultured in Matrigel. I) 3D immunostaining of CD31 + tube-like structures in HUVECs after MBV treatment. J–K) Quantification of tube-like area percentage and number of tube-like structures per field (n = 6). L) 3D two-photo images of TGF-β1 expression in fibroblasts cultured with different MBVs. M) Quantification of TGF-β1-positive volume percentage in fibroblasts (n = 4). N-P) Western blot analysis of NF-κB and STAT3 pathway proteins in MBV-treated macrophages, angiogenic signaling proteins (AKT, ERK1/2) in MBV-treated HUVECs, TGF-β/Smad signaling pathway in MBV-treated fibroblasts. Quantification of respective signaling molecules (n = 3). Data are presented as mean ± SD. Each dot represents an independent biological replicate. Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Functional Assay, Comparison, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Quantitative RT-PCR, Gene Expression, Cell Culture, Immunostaining, Expressing, Western Blot

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Inflammatory immune responses following mesh implantation. A) Schematic illustration and representative macroscopic images of seroma tissues collected from the explants at 1 week. B) ELISA analysis of cytokines in the seroma fluid after 1 week (n = 4). C) Representative immunofluorescence images of CD11b + cell infiltration in mesh at 1 and 4 weeks. Scale bars: left, 1000 μm; right, 50 μm. D–E) Quantification of CD11b + cell density (n = 5, 4 samples per rat). F) Representative immunofluorescence images of CD68 (yellow), CD86 (green), and CD206 (red) staining of SIS and UBM-SIS at 1 and 4 weeks. Scale bars: top, 1000 μm; bottom, 50 μm. G–H) Quantification of CD68 + macrophage infiltration and M2-like/M1-like phenotypic distribution at 1 week (n = 5). I) Statistical comparison of M2-like/M1-like ratios between SIS and UBM-SIS groups (n = 5). J–K) CD68 + macrophage infiltration and CD206 + /CD86 + phenotypic distribution at 4 weeks (n = 5, 4 samples per rat). L) Quantification of M2-like/M1-like ratios at 4 weeks (n = 5, 4 samples per rat). M−O) Representative immunofluorescence images of iNOS and Arg-1 at tissue-mesh interfaces at 1 and 4 weeks, with quantitative analysis of positive area (n = 5, with 4 samples per rat). Scale bars: 50 μm. P) RT-qPCR analysis of pro- (( TNF-α, IL-6 ) and anti-inflammatory ( IL-10, TGF-β1 ) cytokine gene expression in SIS and UBM-SIS explants. Q) Schematic summary of immune response transition induced by SIS versus UBM-SIS MBV-containing meshes over 4 weeks. The asterisk indicates the implanted mesh. Data are presented as mean ± SD. For (B, I, L, and P), mean value of each rat (n = 5) was used for statistical comparisons. For (D, E, G, H, J, K, N, and O), each dot represents one section-level sample, where the value of each animal for statistical comparisons was obtained by averaging measurements from 4 samples. Statistical comparisons were performed within each time point using two-tailed unpaired Student's t-test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Comparison, Quantitative RT-PCR, Gene Expression, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: ECM remodeling and mechanical evaluation of meshes and explants. A) Representative immunofluorescence images showing collagen I (red) and collagen III (green) deposition in the center and interface regions of SIS and UBM–SIS explants at 8 weeks. Scale bars: overview = 1000 μm, magnified images = 100 μm. B–E) Quantification of total collagen (I + III), collagen I, and collagen III positive expression and collagen I/III ratio in the center and interface regions (n = 5, 4 samples per rat). F–G) Polar plot of collagen fiber orientation in the center and interface regions analyzed by orientation distribution. H) Orientation coherency of collagen fibers in the center and interface regions (n = 5, 4 samples per rat). I) Aspect ratio analysis indicating collagen fibril anisotropy (n = 4). J–K) Representative immunofluorescence staining of TGF-β1 and α-SMA at 8 weeks and corresponding quantification of positive area (n = 5, 4 samples per rat). L, M) Mechanical characterization of meshes and explants showing ultimate tensile strength and elongation at break across different time points (n = 4). N) Comparison of tensile strength and elongation of explants with native abdominal wall components, including posterior and anterior rectus sheath, linea alba, peritoneum, and transversalis fascia. The asterisk indicates the implanted mesh. Data are presented as mean ± SD. For (B-E, H, and K), mean value of each rat (n = 5) was used for statistical comparisons. Each dot represents one section-level sample, where the value for each animal was obtained by averaging measurements from 4 samples. For (I, L, and M), mean value of each rat (n = 4) was used for statistical comparisons. Statistical significance was determined using two-tailed unpaired Student's t-test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Expressing, Staining, Comparison, Two Tailed Test

Journal: Bioactive Materials

Article Title: Tissue-specific matrix-bound nanovesicles regulate the immunoregulatory progress of biological mesh-aided abdominal hernia repair

doi: 10.1016/j.bioactmat.2026.03.004

Figure Lengend Snippet: Transcriptomic analysis of explants from different groups after 1 week and comparative analysis of MBV and ECM properties. A) Schematic illustration of tissues collected from the explants at 1 week for distinct signaling pathway analysis. B) Volcano plot showing differentially expressed genes (DEGs) between UBM-SIS and SIS groups (n = 3 per group). C) Heatmap of DEGs between SIS and UBM-SIS groups (red: upregulated, blue: downregulated; cutoff >1.0; n = 3). D) KEGG pathway enrichment analysis of downregulated genes in UBM-SIS compared to SIS. E) Reactome pathway enrichment analysis of downregulated genes in UBM-SIS compared to SIS. F–H) GSEA demonstrating altered gene signatures related to NET formation, NF-κB pathway and cytokine-cytokine receptor interaction. I) Radar plot comparing SIS- and UBM-derived MBVs. The five axes represent key pathways involved in angiogenesis ( ERK ), vascularization ( AKT ), inflammation ( p65 ), immunomodulation ( STAT3 ), and remodeling ( TGF-β/Smad ). J) Radar plot summarizing ECM in vivo performance at 1 and 4 weeks. The five axes represent essential features in ECM remodeling, including adhesion, angiogenesis, inflammation, immunomodulation, and collagen deposition.

Article Snippet: Additional antibodies, such as mouse polyclonal anti -TGF-β1, anti-elastin, CD11b, CD68, CD86, and CD206, along with the BCA Protein Assay Kit, were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Derivative Assay, In Vivo

Journal: Marine Drugs

Article Title: Orally Administered Rhamnan Sulfate from Monostroma nitidum Significantly Inhibits Melanoma Metastasis in Lungs and Aorta of Mice Implanted with B16 Cells

doi: 10.3390/md24040126

Figure Lengend Snippet: ( A ) Changes in the expression of melanoma cell metastasis-related factors in the M + W group mice. The expression levels of melanoma cell metastasis-related factors in the blood and tissues of M + W group mice implanted with B16 cells were compared to those in the control group. Factors that significantly increased are indicated by upward arrows, factors that remained unchanged are indicated by horizontal dotted lines, and factors that significantly decreased are indicated by downward arrows. The combined effects of changes in the expression of each factor are thought to promote melanoma cell metastasis. ( B ) Changes in the expression of melanoma cell metastasis-related factors in the M + RS group mice. The expression levels of melanoma cell metastasis-related factors in the blood and tissues of the M + RS group mice implanted with B16 cells were compared to those in the M + W group. Factors that significantly increased are indicated by upward arrows, factors that remained unchanged are indicated by horizontal dotted lines, and factors that significantly decreased are indicated by downward arrows. The combined effects of changes in the expression of each factor are thought to suppress melanoma cell metastasis. Abbreviations: PAR, protease-activated receptor; TGFβ1, transforming growth factor β1; IL-6, interleukin 6; TF, tissue factor; TNFα, tumor necrosis factor α; F4/80, macrophage marker F4/80; Ly6G, lymphocyte antigen 6 complex locus G6D; PAD4, peptidylarginine deiminase 4; cisH3, citrullinated Histone H3; TM, thrombomodulin; uPA, urokinase-type plasminogen activator; TAFI, thrombin-activatable fibrinolysis inhibitor; TAFIa, activated TAFI; MMP, matrix metalloproteinase; Ang-2, angiopoietin-2; bFGF, basic fibroblast growth factor; Robo4, Roundabout homolog 4; E-cadherin, epithelial cadherin; Snail-1, small family zinc finger 1; Wnt, wingless and int-1; Wnt3a, Wnt family member 3a; LRP, low-density lipoprotein receptor-related protein; Frizzled, Frizzled class receptor.

Article Snippet: The levels of PAR1, PAR2, TF, TNF-α, TGF-β1, Ang-2, bFGF, β-catenin, vimentin, fibronectin, and Snail-1 in tissues were determined using commercially available ELISA kits: PAR1 (MBS753326, MyBioSource, San Diego, CA, USA), PAR2 (MBS4501658; MyBioSource), TF (ab214091; Abcam), TNF-α (KE10002, Proteintech), TGFβ1 (E-EL-M0051, Elabscience, Houston, TX, USA), Ang-2 (MANG20, R&D Systems), bFGF (bs-0217R, Bioss Antibodies), β-catenin (ADI-900-135; Enzo Life Sciences, Executive Blvd Farmingdale, NY, USA), fibronectin (OKCD05702, Aviva Systems Biology, San Diego, CA, USA), vimentin (ELK3731, ELK Biotechnology, Denver, CO, USA), and Snail-1 (LS-F2317-1, LS Bio, Shirley, MA, USA).

Techniques: Expressing, Control, Marker

Journal: Signal Transduction and Targeted Therapy

Article Title: Rationale for multi-epitope TGFβ vaccination in pancreatic cancer: evidence from immunologic and clinical correlates

doi: 10.1038/s41392-026-02626-3

Figure Lengend Snippet: In vitro T cell responses to TGFβ-15, -33, and -38 are detected in healthy donors and pancreatic cancer patients. a Amino acid sequences of TGFβ-02, TGFβ-15, TGFβ-33, and TGFβ-38, with positions mapped within the TGFβ protein. The schematic shows the structural domains of TGFβ, including the signal peptide (SP). The figure was generated with BioRender. b Representative IFNγ ELISPOT wells for TGFβ-33 and TGFβ-38 under unstimulated (Unstim.) and peptide-stimulated conditions in healthy donors (HD) and PDAC patients. c , d PBMCs from HDs ( n = 31; c ) and PDAC patients ( n = 14; d ) were stimulated with TGFβ-15, -33, or -38 peptides. After 12-14 days, cells were restimulated and analyzed by IFNγ ELISPOT. Responses are shown as normalized IFNγ amplitudes in scatter plots (left) with the median indicated and heatmaps (right), normalized to unstimulated controls. Each dot represents a single donor or patient. HD and patient IDs are indicated on the heatmap x -axis. A one-way ANOVA revealed a significant difference in responses to the three peptides for HDs ( p = 0.0272) but not for PDAC patients ( p = 0.0542). p -Values from the post-hoc analysis (Turkey’s multiple comparisons test) are stated in the scatter plots. e , f ICS detection of CD4⁺ ( e ) and CD8⁺ ( f ) T cell responses to TGFβ-33 in a PDAC patient and a healthy donor, respectively. Abbreviations: TGFβ transforming growth factor beta, AA amino acid, SP signal peptide, Unstim. unstimulated, HD healthy donor, PDAC pancreatic ductal adenocarcinoma, IFNγ interferon gamma, TNFα tumor necrosis factor alpha

Article Snippet: Reactions were run on a thermal cycler under standard conditions. qPCR was performed using LightCycler TaqMan Master Mix (Roche Diagnostics, Copenhagen, Denmark) and TaqMan Gene Expression Assays (ThermoFisher Scientific), with TGFβ1 (Hs07289533_m1) normalized to RPLP0 (Hs99999902_m1).

Techniques: In Vitro, Generated, Enzyme-linked Immunospot

Journal: Signal Transduction and Targeted Therapy

Article Title: Rationale for multi-epitope TGFβ vaccination in pancreatic cancer: evidence from immunologic and clinical correlates

doi: 10.1038/s41392-026-02626-3

Figure Lengend Snippet: High-purity cultures of CD4 + CD45RO + memory T cells, isolated from healthy donors and pancreatic cancer patients, respond to stimulation with TGFβ-33. a Flow cytometry confirming the purity of CD4⁺CD45RO⁺ memory T cells co-cultured with peptide-pulsed autologous CD14⁺ monocytes. Gating on live CD3⁺CD4⁺ cells was based on FMO controls. b IFNγ ELISPOT showing T cell responses to TGFβ-33. Left: summary of spot counts with data points indicating technical triplicate spot counts, and bars indicating the mean with SD depicted. Right: representative ELISPOT wells. Cells (3 × 10⁵/well) were stimulated with TGFβ-33 peptide or DMSO. Asterisks (**) indicate responses meeting the DFR(2×) significance threshold. c ICS analysis of CD4⁺ T cells after DMSO (upper panel) or TGFβ-33 peptide (lower panel) stimulation. Abbreviations: SD standard deviation, Unstim. unstimulated, TGFβ transforming growth factor beta, IFNγ interferon gamma, TNFα tumor necrosis factor alpha, DFR distribution-free resampling

Article Snippet: Reactions were run on a thermal cycler under standard conditions. qPCR was performed using LightCycler TaqMan Master Mix (Roche Diagnostics, Copenhagen, Denmark) and TaqMan Gene Expression Assays (ThermoFisher Scientific), with TGFβ1 (Hs07289533_m1) normalized to RPLP0 (Hs99999902_m1).

Techniques: Isolation, Flow Cytometry, Cell Culture, Enzyme-linked Immunospot, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Rationale for multi-epitope TGFβ vaccination in pancreatic cancer: evidence from immunologic and clinical correlates

doi: 10.1038/s41392-026-02626-3

Figure Lengend Snippet: TGFβ-33-specific responses in PBMCs from patients with pancreatic cancer are associated with improved survival. a PBMCs from pancreatic cancer patients ( n = 27) were stimulated in vitro with TGFβ-33 peptide and IL-2 for 14 days, then plated in triplicate IFNγ ELISPOT assays (2 × 10⁵ cells/well). Negative control wells were left unstimulated. Responses at baseline and after four treatment series are shown. Data were normalized by subtracting control well means. Data are represented as mean ± SD, and each data point represents one patient. b Patients were stratified by clinical benefit ( n = 15) or progressive disease (PD, n = 12). Normalized counts from a compare response amplitudes at baseline (left) and on treatment (right). Statistics were performed using the non-parametric Mann-Whitney test. The error bar depicts SD, and each data point represents one patient. c , d Kaplan-Meier curves displaying overall survival ( c ) and progression-free survival ( d ) in patients with baseline TGFβ-33 responses above ( n = 14) or below ( n = 13) the median response to TGFβ-33. Time-to-event analyses were performed using the log-rank (Mantel-Cox) test. e , f Kaplan-Meier curves for overall survival ( e ) and progression-free survival ( f ) in patients stratified by responses above median to TGFβ-15 and TGFβ-33: neither ( n = 9), one epitope ( n = 6), or both ( n = 12). Time-to-event analyses were performed using the log-rank (Mantel-Cox) test. The p -value in green indicates the comparison between the black and green lines, and the p -values in purple refer to the comparison between the black and purple lines. Abbreviations: SD standard deviation, PBMC peripheral blood mononuclear cell, TGFβ transforming growth factor-β

Article Snippet: Reactions were run on a thermal cycler under standard conditions. qPCR was performed using LightCycler TaqMan Master Mix (Roche Diagnostics, Copenhagen, Denmark) and TaqMan Gene Expression Assays (ThermoFisher Scientific), with TGFβ1 (Hs07289533_m1) normalized to RPLP0 (Hs99999902_m1).

Techniques: In Vitro, Enzyme-linked Immunospot, Negative Control, Control, MANN-WHITNEY, Comparison, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Rationale for multi-epitope TGFβ vaccination in pancreatic cancer: evidence from immunologic and clinical correlates

doi: 10.1038/s41392-026-02626-3

Figure Lengend Snippet: TGFβ-specific T cells recognize autologous dendritic cells and cancer cells in a peptide- and protein-dependent manner. a , b Flow cytometry of CD4⁺ T cells from three TGFβ-33-specific cultures ( a ) and CD8⁺ T cells from a TGFβ-33-specific culture ( b ) unstimulated (upper panel) or TGFβ-33 peptide-stimulated (lower panel). c ICS of CD8⁺ (left) and CD4⁺ (right) T cell responses from TGFβ-33 REP cultures co-cultured with autologous DCs, either unpulsed or TGFβ-33 peptide-pulsed (T:DC = 5:1). The ICS for the CD8 + T cells was performed in monoplicates, while the assay for CD4 + T cells was performed using three technical replicates as indicated by the data points. The data is presented as the mean, with error bars indicating SD. d ICS of CD4⁺ T cells from TGFβ-33- and TGFβ-38-specific cultures, co-cultured with mock- or TGFβ-siRNA-transfected THP-1 cells, with or without TGFβ treatment 48 h prior. The experiment was performed with six technical replicates, as indicated by the data points, and presented as the mean ± SD. ns, not significant, and asterisk (**) indicate significance ( p < 0.01) according to a Mann-Whitney non-parametric unpaired test. e IFNγ ELISPOT from the same T cell cultures as seen in ( d ) performed in triplicate, as indicated by the data points. Error bars show SD, and asterisks (*) and (**) indicate significance according to the DFR(eq) or DFR(2×) tests, respectively. f Flow cytometry of the THP-1 cells used in ( d ) and ( e ). HLA-II expression is shown as normalized histograms gated on live cells. The dotted line indicates the gate determined from the FMO control. Percent HLA-II⁺ cells and median fluorescence intensity (MFI) are indicated. Abbreviations: IFNγ interferon gamma, TNFα tumor necrosis factor alpha, SD standard deviation, T T cell, DC dendritic cell, TGFβ transforming growth factor beta, DP double positive

Article Snippet: Reactions were run on a thermal cycler under standard conditions. qPCR was performed using LightCycler TaqMan Master Mix (Roche Diagnostics, Copenhagen, Denmark) and TaqMan Gene Expression Assays (ThermoFisher Scientific), with TGFβ1 (Hs07289533_m1) normalized to RPLP0 (Hs99999902_m1).

Techniques: Flow Cytometry, Cell Culture, Transfection, MANN-WHITNEY, Enzyme-linked Immunospot, Expressing, Control, Fluorescence, Standard Deviation

Journal: Signal Transduction and Targeted Therapy

Article Title: Rationale for multi-epitope TGFβ vaccination in pancreatic cancer: evidence from immunologic and clinical correlates

doi: 10.1038/s41392-026-02626-3

Figure Lengend Snippet: Dendritic cells transfected with mRNA encoding multiple TGFβ-derived epitopes activate T cells with distinct TGFβ specificities. a Schematic of the TGFβ-ALL plasmid for in vitro transcription of TGFβ-ALL-encoding mRNA. To the left is shown the vector map of the plasmid, and to the right is illustrated how dendritic cells transfected with the mRNA would activate anti-Tregs with distinct TGFβ-derived epitope specificities. The figure was generated with BioRender. b ICS analysis of TGFβ-02-specific (left) and TGFβ-15-specific (right) CD4 + T cell cultures responding to their respective peptides. c ICS of four CD4 + T cell cultures cocultured with autologous dendritic cells, either mock-transfected or transfected with TGFβ-ALL mRNA. The experiment was performed using six technical replicates, as indicated by the data points. Bars represent the mean, and the error bars indicate SD. Asterisks (* and **) indicate significance ( p < 0.05 and p < 0.01, respectively) according to an unpaired non-parametric Mann-Whitney test. Abbreviations: TGFβ transforming growth factor beta, Treg regulatory T cell, IFNγ interferon gamma, TNFα tumor necrosis factor alpha, SD standard deviation

Article Snippet: Reactions were run on a thermal cycler under standard conditions. qPCR was performed using LightCycler TaqMan Master Mix (Roche Diagnostics, Copenhagen, Denmark) and TaqMan Gene Expression Assays (ThermoFisher Scientific), with TGFβ1 (Hs07289533_m1) normalized to RPLP0 (Hs99999902_m1).

Techniques: Transfection, Derivative Assay, Plasmid Preparation, In Vitro, Generated, MANN-WHITNEY, Standard Deviation