Journal: Cancer Communications

Article Title: Targeting TREM2 + and TIMD4 + Macrophages Reprograms the Tumor Microenvironment and Enhances PD-1 Blockade Efficacy in Intrahepatic Cholangiocarcinoma

doi: 10.34133/cancomm.0056

Figure Lengend Snippet: TREM2 + macrophages promote POSTN + CAF formation at the tumor boundary to establish an immune barrier restricting CD8 + T cell infiltration in ICC. (A and B) DEGs analysis (A) and GO enrichment (B) of up-regulated genes in fibroblasts from Trem2 −/− versus WT tumors (Kras-p19-induced ICC mice). (C and D) UMAP plot of human fibroblast cell subtypes (C) and POSTN expression levels (D). (E) Spatial transcriptomics shows expression level and distribution of genes in ICC samples. The dotted line demarcates the boundary between the tumor and peritumoral tissue. (F to H) mIF staining showing spatial distribution (F and G) and quantitative analysis (H) of CD8 + T cells in the presence or absence of an immune barrier ( **** P < 0.0001; Mann–Whitney U test). (I) IHC was performed to evaluate the expression levels of Ck19, Postn, and Cd8a, and SR was performed to evaluate collagen abundance, between the anti-Fap treatment group and the control group. For detailed procedures regarding the preparation of the anti-Fap therapeutic agent, please refer to Materials and Methods. (J) α-SMA and POSTN expression detected by mIF staining in LX-2 cocultured with Trem2 + macrophages or Trem2 − macrophages or treated with TGF-β ( * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant; one-way ANOVA with Dunnett’s test). (K and L) Survival (Kaplan–Meier) analysis of POSTN (K) and TREM2-POSTN coexpression (L) in the FuDan-ICC cohort. Abbreviations: ICC, intrahepatic cholangiocarcinoma; CAF, cancer-associated fibroblast; DEGs, differentially expressed genes; UMAP, Uniform Manifold Approximation and Projection; GO, Gene Ontology; WT, wild type; apCAF, antigen-presenting cancer-associated fibroblast; HSC, hepatic stellate cell; iCAF, inflammatory cancer-associated fibroblast; myCAF, myofibroblastic cancer-associated fibroblast; Prolif_Fibro, proliferating fibroblast; IHC, immunohistochemistry; PanCK, pan-cytokeratin; FAP, fibroblast activation protein; OS, overall survival.

Article Snippet: For polarization, naïve CD4 + T cells (1 × 10 6 cells/ml) were cultured with TGF-β (5 ng/ml; catalog no. HY- P70648 , Medchemexpress), IL-2 (100 U/ml), and reduced anti-CD3/CD28 activation beads (0.5 μg/ml) for 3 to 5 days.

Techniques: Analysis, Expressing, Spatial Transcriptomics, Tissue, Staining, MANN-WHITNEY, Control, Immunohistochemistry, Activation Assay

Journal: Cancer Communications

Article Title: Targeting TREM2 + and TIMD4 + Macrophages Reprograms the Tumor Microenvironment and Enhances PD-1 Blockade Efficacy in Intrahepatic Cholangiocarcinoma

doi: 10.34133/cancomm.0056

Figure Lengend Snippet: TREM2 + macrophages promote immunosuppressive microenvironment in ICC by regulating CD8 + T cell exhaustion and Treg development. (A) Schematic diagram of the experimental workflow for macrophage and T cell coculture assays. Macrophage subsets were isolated from ICC tumors, and naive Cd4 + T cells or Cd8 + T cells were isolated from the spleens of healthy mice for subsequent coculture experiments. (B) Flow cytometry analysis of Foxp3 expression in naive Cd4 + T cells cocultured with sorted Trem2 − macrophages or Trem2 + macrophages for 2 days in the presence of IL-2 and TGF-β. Cells were gated sequentially on single cells, live cells, and Cd4 + T cells. Representative flow cytometry plots and quantification of Foxp3 + cells among Cd4 + T cells are shown ( * P < 0.05, **** P < 0.0001; one-way ANOVA by Dunnett’s test). (C) Flow cytometry analysis of Foxp3 expression in naive Cd4 + T cells cocultured with sorted Trem2 − macrophages or Trem2 + macrophages for 9 days in the presence of IL-2 but without exogenous TGF-β. Cells were gated sequentially on single cells, live cells, and Cd4 + T cells. Representative flow cytometry plots and quantification of Foxp3 + cells among Cd4 + T cells are shown ( * P < 0.05, **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). (D) Batf expression in nTreg cells cocultured with macrophages ( *** P < 0.001; ns, not significant; one-way ANOVA by Dunnett’s test). (E) Spearman correlation analysis between Treg signatures and immune-related genes in the FuDan-ICC cohort (Spearman test). (F) Correlation of TNFSF13B with FOXP3 expression in the Xena pan-cancer cohort (Spearman test). (G) Batf expression in nTreg cells in indicated groups. Flow cytometry analysis of Batf expression in Treg cells treated with recombinant TNFSF13B in the presence or absence of a TNFSF13B inhibitor. Representative histogram plots and quantification of Batf + cells among Foxp3 + T cells are shown ( *** P < 0.001, **** P < 0.0001; one-way ANOVA by Dunnett’s test). (H) Pd-1 expression in Cd8 + T cells stimulated by macrophage subtypes ( * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). (I) In vitro cytotoxicity assay assessing the killing activity of OT-1 Cd8 + T cells against OVA-expressing ICC tumor cells after coculture with macrophage subsets. Tumor cells were visualized by crystal violet staining. Representative images of residual OVA-ICC tumor cells are shown for the control, Cd8 + T cell alone, Trem2 + macrophage–Cd8 + T cell coculture, and Timd4 + macrophage–Cd8 + T cell coculture groups. (J) Heatmap of immunosuppressive molecule expression in macrophage subcell types (scaled value). (K) Spatial transcriptomics of TIMD4 expression and localization in tumor tissue of 2 ICC patients. (L and M) Spearman correlation of TIMD4 with immunosuppressive markers [PDCD1 (L), VSIG4 (M)] in tumor and adjacent tissues (EHBH-ICC cohort). (N) Multiplex immunofluorescence staining of ICC tissue showed the spatial distribution of PanCK, CD8A, and PD-1 signals in tumor and adjacent tissues (PanCK marks epithelial tumor cells, CD8A marks CD8 + T cells, and PD-1 marks PD-1 + cells; the white dashed line indicates the tumor-adjacent tissue boundary; Magnified images of the boxed adjacent-tissue region and single-channel images are shown). (O) Ligand–receptor interaction analysis between macrophage subsets and exhausted CD8 + T cells in adjacent tissues based on scRNA-seq data. The chord diagram shows predicted immune checkpoint ligand–receptor pairs. (P) Bulk RNA-seq analysis of Cd8 + T cells cocultured with sorted Trem2 + macrophages or Timd4 + macrophages. Heatmap shows scaled mRNA expression levels of selected inhibitory receptor- and exhaustion-related genes compared with control Cd8 + T cells. (Q) GO enrichment of up-regulated DEGs in Cd8 + T cells cocultured with Trem2 + macrophages. (R) Expression levels of fatty acid metabolism-related genes and Csf1 in Cd8 + T cells under coculture with macrophages (the mRNA expression levels were log2-transformed; ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant; one-way ANOVA by Dunnett’s test). Abbreviations: ICC, intrahepatic cholangiocarcinoma; WT, wild type; TREM2, triggering receptor expressed on myeloid cells 2; TIMD4, T cell immunoglobulin and mucin domain-containing protein 4; TGF-β, transforming growth factor-β; IL-2, interleukin-2; FOXP3, forkhead box P3; BATF, basic leucine zipper ATF-like transcription factor; Treg, regulatory T cell; nTreg, natural regulatory T cell; TNFSF13B, tumor necrosis factor ligand superfamily member 13B; PD-1, programmed cell death protein 1; OVA, ovalbumin; mIF, multiplex immunofluorescence; PanCK, pan-cytokeratin; RNA-seq, RNA sequencing; DEG, differentially expressed gene; GO, Gene Ontology; TPM, transcripts per million; TME, tumor microenvironment.

Article Snippet: For polarization, naïve CD4 + T cells (1 × 10 6 cells/ml) were cultured with TGF-β (5 ng/ml; catalog no. HY- P70648 , Medchemexpress), IL-2 (100 U/ml), and reduced anti-CD3/CD28 activation beads (0.5 μg/ml) for 3 to 5 days.

Techniques: Isolation, Flow Cytometry, Analysis, Expressing, Recombinant, In Vitro, Cytotoxicity Assay, Activity Assay, Staining, Control, Spatial Transcriptomics, Tissue, Multiplex Assay, Immunofluorescence, RNA Sequencing, Transformation Assay

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: The fabrication of Gel@IVNMs, in vivo application and immunomodulatory mechanism for myocardial infarction (MI) therapy. (A) The stepwise fabrication of the PPM@rapamycin (Rapa) inverse vaccine nanomimics (IVNMs), Gel, and Gel@IVNMs. First, PPMal, PPMan, and Rapa are assembled into the IVNMs via the solvent evaporation method. Concurrently, oDex and gelatin are cross-linked to form a hydrogel network, where IVNMs are encapsulated. (B) The hydrogel is injected into the infarcted myocardium. As the Gel swells, cardiac lysate antigens (CLAs) released by necrotic cardiomyocytes diffuse into it and are captured by Mal on the surface of IVNMs to form antigen-loaded IVNMs (AIVNMs). Lactic acid accumulates at the infarcted site due to the activation of inflammation, followed by a decrease in pH, leading to a mild acidic microenvironment. In response to the mildly acidic pH, the Gel gradually degrades and releases AIVNMs. (C) Released AIVNMs can be taken up by CD206-expressing immune cells, including dendritic cells (DCs), and subsequently modulate DC phenotype by suppressing the mechanistic target of rapamycin (mTOR) pathway (via Rapa-mediated inhibition of mTOR–Rictor–FKBP12 signaling), reducing CD80 and CD86 expression, and increasing programmed death ligand 1 (PD-L1) expression and transforming growth factor-β (TGF-β) secretion. These DCs then present antigens and suppress T cell activation through PD-L1 signaling and TGF-β secretion, fostering regulatory T cell (Treg) expansion. Tregs further modulate macrophage polarization, shifting proinflammatory M1 macrophages toward anti-inflammatory M2 macrophages, thereby balancing proinflammatory and anti-inflammatory cytokine secretion. The established immune tolerance memory can rapidly respond and specifically inhibit inflammatory activation upon restimulation with CLAs. This cascade reduces cardiomyocyte apoptosis, promotes neovascularization, ameliorates myocardial fibrosis, and ultimately enhances heart pumping function, contributing to improved cardiac repair after MI.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: In Vivo, Solvent, Evaporation, Injection, Activation Assay, Expressing, Inhibition

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: The effect of mannose functionalization on the uptake of inverse vaccine nanomimics (IVNMs) by dendritic cells (DCs) in vitro, together with DC phenotypes and cytokine secretion after stimulation with cardiac lysate antigens (CLAs). (A) DC isolation and experimental workflow. The process by which IVNMs capture antigens and are internalized by DCs. After antigen-loaded IVNMs (AIVNMs) are internalized by DCs, the antigens activate mechanistic target of rapamycin (mTOR) signaling, whereas Rapa inhibits mTOR activation, which leads to decreased expression of molecules such as CD80 and CD86, and increased expression of programmed death ligand 1 (PD-L1). Additionally, the secretion of anti-inflammatory factors (such as interleukin-10 [IL-10] and transforming growth factor-β [TGF-β]) is enhanced. (B) Flow cytometry results for the uptake of IVNMs by DCs. (C) Quantification of the uptake of IVNMs by DCs. (D) Fluorescence images of the uptake of IVNMs by DCs, with red fluorescence indicating IVNMs and blue fluorescence indicating cell nuclei. (E and F) Quantification of DC phenotypes by flow cytometry. (G) Representative flow cytometry plots of DC phenotypes. (H) Enzyme-linked immunosorbent assay detection results of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β expression levels in DCs after CLA stimulation and drug administration. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: In Vitro, Isolation, Activation Assay, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Phenotypic changes and inflammatory factor secretion of T cells stimulated by cardiac lysate antigens (CLAs). (A) Coculture protocol for dendritic cells (DCs) and T cells. The tolerogenic DCs (tDCs) present antigens to T cells; meanwhile, programmed death ligand 1 binds to programmed cell death protein 1 on T cells and inhibits T cell activation, promoting the phenotypic conversion of T cells to regulatory T cells (Tregs). Furthermore, due to the lack of activation signals such as CD80 and CD86, some T cells exhibit a weak activation state after receiving antigen presentation, and under the regulation of key factors such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), they undergo phenotypic transition to Tregs. Under restimulation, tDCs phagocytose antigens and present them to Tregs. Tregs are rapidly activated, express high levels of Forkhead box P3 (FOXP3), and directly inhibit T cell activation, while secreting large amounts of anti-inflammatory factors. (B) Flow cytometry (FC) results for T cell phenotype. (C) Quantification of T cell phenotypes by FC. (D) T cell phenotype immunofluorescence staining results, where blue represents cell nuclei, red represents CD127, and green represents CD4. (E) Expression of interferon-γ (IFN-γ), IL-2, IL-10, and TGF-β after coculture of DCs and T cells. (F) Western blot analysis of the activation status of the mechanistic target of rapamycin (mTOR)-p70S6K-S6 pathway after treatment with Gel@PPM, Rapa, inverse vaccine nanomimics (IVNMs), and Gel@IVNMs under CLA stimulation. (G) The percentage of phosphorylation of mTOR, p70S6K, and S6 was statistically analyzed based on the gray values of the western blot results (compared to the unphosphorylated proteins). Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: Activation Assay, Immunopeptidomics, Flow Cytometry, Immunofluorescence, Staining, Expressing, Western Blot, Analysis, Phospho-proteomics

Journal: Research

Article Title: Inverse Vaccine Nanomimics: In situ Antigen-Specific Immune Tolerance Induction for Durable Cardiac Repair Post-Myocardial Infarction

doi: 10.34133/research.1464

Figure Lengend Snippet: Changes in myocardial macrophage phenotypes and inflammatory cytokine profiles after myocardial infarction (MI). (A) Representative immunofluorescence images of heart sections from the Control, Gel@PPM, Gel@PP@Rapa, Gel@OVA-IVNMs, and Gel@IVNMs groups at 7 and 56 d after MI. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), macrophages with CD68 (green), proinflammatory macrophage-associated inducible nitric oxide synthase (iNOS) with red fluorescence, and reparative/immunomodulatory macrophage-associated CD206 with purple fluorescence. Scale bars, 200 μm. (B) Quantification of iNOS- and CD206-positive signals in myocardial sections at 7 and 56 d after MI. (C) Enzyme-linked immunosorbent assay quantification of interleukin-2 (IL-2), interferon-γ (IFN-γ), transforming growth factor-β (TGF-β), and IL-10 levels in myocardial tissue at 7 and 56 d after MI. Data are presented as means ± SD, with individual points representing biological replicates. Statistical significance is indicated as ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The supernatants were collected to quantify the cytokines IFN-γ, IL-10, IL-2, and TGF-β (E-EL-M0048, E-EL-M0046, E-EL-M0042, E-EL-0162, all from Elabscience), by ELISA according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Control, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Tissue

Journal: BioMed Research International

Article Title: Compound Astragalus Oral Liquid Attenuates Ventricular Remodeling in Heart Failure After Myocardial Infarction via the AMPK/TGF‐ β /Smad Signaling Pathway

doi: 10.1155/bmri/1276968

Figure Lengend Snippet: CAOL enhances AMPK activity and suppresses the TGF‐ β /Smad pathway in myocardial tissue. (A–D) The mRNA levels of AMPK , TGF-β , SMAD2 , and SMAD3 were detected, respectively, by RT‐qPCR ( n = 6). (E–L) WB analysis and quantification of the protein levels of AMPK, p‐AMPK, TGF‐ β 1, Smad2, p‐Smad2, Smad3, and p‐Smad3 ( n = 6). Data were shown as mean ± SD, ## p < 0.01 compared with the sham group. ∗ p < 0.05, ∗∗ p < 0.01 compared with the model group, ns means nonsignificant.

Article Snippet: After blocking with nonfat milk at room temperature, membranes were incubated overnight at 4°C with primary antibodies against AMPK (CST, United States), p‐AMPK (CST, United States), TGF‐ β 1 (Proteintech, China), Smad2 (Proteintech, China), Smad3 (Proteintech, China), p‐Smad2 (CST, United States), p‐Smad3 (CST, United States), and GAPDH (Proteintech, China).

Techniques: Activity Assay, Tissue, Quantitative RT-PCR, Analysis