Journal: Nature Communications

Article Title: SARS-CoV-2 in severe COVID-19 induces a TGF-β-dominated chronic immune response that does not target itself

doi: 10.1038/s41467-021-22210-3

Figure Lengend Snippet: a Titration of SARS-Cov-2-S protein-specific IgG, IgA, and IgM antibodies in the serum of three COVID-19 ICU patients. ΔMFI calculation as described in Fig. . Dotted line indicates a ΔMFI of 1500. b Workflow for the isolation and analysis of SARS-CoV-2-and measles-reactive CD4 + T lymphocytes. c Cells for single cell sequencing were sorted using FACS as PI - CD19 - CD14 - CD3 + CD4 + CD154 + or CD137 + (gating strategy as used for the analysis shown in Supplementary Fig. ). UMAP representation of 3727 CD4 + T cells from three COVID-19 ICU patients and two healthy controls (SARS-CoV-2-stimulated cells from one control and measles-stimulated cells from another control). Four clusters were identified based on transcriptional similarity using shared nearest-neighbor (SNN) modularity optimization (left). An additional CD14 + cluster (124 cells) and six outlying T cells with considerably less UMI-counts than the average were also identified but excluded from further analysis. Heatmap of signature genes of the four clusters. Depicted are genes with an absolute log2 fold change > log2(2) and a p -value < 0.01 after Bonferroni correction (two-sided Wilcoxon rank sum test). Colors corresponds to z -scores of the average expression. d Expression levels of selected signature genes and cytokines.

Article Snippet: Cells were also stained for 15 min with the following anti-human antibodies: CD3 (OKT3, BV785, Biolegend, Cat. No. 317330, 1:100), CD4 (SK3, PE-Cy5.5, Biolegend, Cat. No. 35-0047-42, 1:200), CD19 (H1B19, V500, BD Biosciences, Cat. No. 561121, 1:100), CD14 (TM1, Pacific Orange, DRFZ in-house conjugation, 1:100), CD137 (4B4-1, PE, Miltenyi Biotec, Cat. No. 130-093-476, 1:25), and CD154 (5C8, biotin, Miltenyi Biotec, Cat. No. 5190204135, 1:10).

Techniques: Titration, Isolation, Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Splenocytes (A-D) and tumor-infiltrating lymphocytes (TIL) (E-H) were isolated from HIS mice 16-18 days after tumor implantation and analyzed by flow cytometry. A, frequency of splenic T cells in tumor-bearing or naïve HIS mice; parent population refers to frequency (%) of CD3 + T cells within human CD45 + cells and CD4 + and CD8 + T cells within CD3 + T cells. B , CD8 + T cell differentiation defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ) in tumor-bearing or naïve HIS mice. C-D , expression of indicated markers on CD8 + T cells from spleen of tumor-bearing or naïve HIS mice. E, frequency of CD4 + and CD8 + T cells within TILs, gated on total CD3 + T cells. F, CD8 + T cell differentiation within TILs. G-H, expression of indicated markers on CD8 + T cells within TILs. I, expression of CD137 on splenic CD8 + and CD4 + T cells of tumor-bearing or naïve HIS mice. J, expression of PD-1 and CD137 on splenic CD8 + T cells of tumor-bearing mice. K , expression of PD-1 on splenic CD8 + T cells based on CD137 in tumor-bearing HIS mice or bulk CD8 + T cells from naïve HIS mice. L, CD8 + T cell differentiation in spleen of tumor-bearing HIS mice. M-O , expression of indicated markers on splenocyte-derived CD8 + T cell populations based on expression of CD137 and PD-1. Data are pooled from at least 3 independent experiments, n=10-23 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by paired t-test, one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Isolation, Tumor Implantation, Flow Cytometry, Cell Differentiation, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation, expansion and characterization of T cells from HIS mice bearing autologous LCL tumors. B , fold expansion of FACS sorted splenic CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). C , CD8 + T cell differentiation after ex vivo expansion defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ), T EMRA (CD45RA + CD62L - ). D, IFN-ψ ELISpot of expanded T cells in 5:1 (E:T) co-culture with autologous LCL tumor cells for 24 hours. Spot count is normalized to the spots produced by expanded CD8 + bulk T cells from naïve HIS mice. E , TNF⍺ ELISA of supernatant of expanded T cells in co-culture (5:1, E:T) with autologous LCL tumor cells for 24 hours. TNF⍺ concentration is normalized to the TNF⍺ secretion from expanded CD8 + bulk T cells derived from naïve mice. Data are pooled from at least 3 independent experiments, n=6-19 mice per group. For each experiment, a different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by mixed-effects analysis (paired), RM one-way ANOVA or 2way ANOVA, as appropriate. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Cell Differentiation, Ex Vivo, Enzyme-linked Immunospot, Co-Culture Assay, Produced, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: Transcriptome analysis and pathway analysis of expanded CD8 + T cells from tumor-bearing HIS mice (CD137 + , CD137 - and CD137 - PD1 - ) or naïve HIS mice (bulk). A, Upset plot (intersect) showing number of differentially expressed genes between groups in bulk RNAseq. p(FDR) < 0.05, log2 FC > 1.5. B , PCA plot of RNAseq showing PC1 and PC2. C, Top 50 upregulated and D, downregulated genes of T cell subsets based on the DEG between CD137 + vs. CD137 - PD1 - CD8 + T cells. p(FDR) < 0.05, log2 FC > 1.5. E, Volcano plot showing DEG between CD137 + and CD137 - PD1 - CD8 + T cells with genes of interest highlighted in yellow. F , differential expression of genes of interest between groups. G , Overrepresentation analysis (ORA) of upregulated pathways in CD137 + CD8 + T cells. H , Gene set enrichment analysis (GSEA) of signatures described on the y-axis. Gene ratio (# genes related to GO term / total number of sig genes) is displayed on the x-axis. Signatures with an adjusted p-value <0.05 are highlighted with a red box. Shown are data from 5 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , total number of individual clonotypes found per population. Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. B, TCR (CDR3 of TRA , TRB , TRG and TRD ) sequence sample diversity estimation using Hill numbers method, with Q=1 describing the Shannon diversity. C, rare clonal proportion showing the occupied repertoire space by clonotypes with defined counts (1, 2-3, 4-10, etc.). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. D, relative abundance of clonotypes with defined frequencies (size). Left: data from individual experiments, right: pooled data for group analysis. Wilcoxon test. E, repertoire overlap analysis cross-comparing every population from every experiment (each with different donor). F , repertoire overlap comparing the repertoire of bulk CD8 + T cells from naïve HIS mice to the populations from tumor-bearing HIS mice from individual experiments (each with different donor; data from individual experiments are indicated by different symbols). Mixed effects analysis with Tukey’s multiple comparisons test. G , tracking of clonotypes over populations. The top 10 most abundant clonotypes of the TCR repertoire of CD137 + CD8 + T cells from one representative experiment are shown. H , proportion of the top 10 most abundant clonotypes (from repertoires of CD137 + CD8 + T cells) in the repertoire of all populations, correlated with the spot count of IFN-ψ ELISpot. Shown are data from 4 individual experiments, each experiment with different human HPC donor for reconstitution of HIS mice and autologous tumor.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Sequencing, Enzyme-linked Immunospot

Journal: bioRxiv

Article Title: Human effector CD8 + T cells with an exhausted-like phenotype control tumor growth in vivo

doi: 10.1101/2023.10.11.561856

Figure Lengend Snippet: A , schematic of generation of tumor-reactive T cells and subsequent ACT. NSG mice were injected with 2 x 10 LCL s.c. in the flank and after three days, 10 x 10 ex vivo expanded T cells were adoptively transferred intravenously. Transferred T cells and LCL tumors were autologous to each other. B , tumor volume on the day of sacrifice in NSG recipient mice after ACT of the indicated cell populations. C, waterfall plot of tumor size in NSG recipient mice of ACT on the day of sacrifice relative to the tumor volume of control mice (no ACT). Bars depict individual mice. D , frequency of CD3 + T cells (% of human CD45 + cells) in TIL from NSG mice after ACT of the indicated cell populations, measured by flow cytometry. E , frequency of CD8 + T cells (% of total cells) in tumors of NSG mice after ACT of the indicated cell populations, measured by immunohistochemistry. F , differentiation of CD8 + T cells in TIL of NSG mice after ACT of the indicated cell populations defined as T naïve (CD45RA + CD62L + ), T CM (CD45RA - CD62L + ), T EM (CD45RA - CD62L - ) and T EMRA (CD45RA + CD62L - ). G, correlation between tumor volume and infiltration of CD8 + T cells (measured by IHC) in tumors of NSG mice after adoptive transfer of CD137 + CD8 + T cells. H , schematic of ACT. CD137 + CD8 + T cells, CD137 - CD8 + T cells and CD137 - PD-1 - CD8 + T cells were isolated from spleen of tumor-bearing HIS mice or bulk CD8 + T cells from spleen of naïve HIS mice and expanded ex vivo . Recipient HIS mice were injected with 2 x 10 LCL s.c. in the flank and after three days, ex vivo expanded T cells were adoptively transferred intravenously. Tumor-bearing HIS recipient mice received 2 x 10 T cells without prior conditioning/lymphodepletion. Donor and recipient HIS mice as well as LCL were autologous to each other. I, tumor volume on the day of sacrifice in HIS recipient mice after ACT of the indicated cell populations. J, waterfall plot of tumor size on the day of sacrifice of HIS mice receiving ACT relative to the tumor volume of control HIS mice (no ACT). Bars depict individual mice. B-G: Data are pooled from 2-3 independent experiments, n=6-13 per group. I, data are pooled from 2-3 independent experiments (n=6-13 per group); J, data are from 1-3 independent experiments (n=3-13 per group). For each experiment, a with different HPC donor was used for HIS mouse reconstitution and generation of autologous tumor. Significance by one-way ANOVA. Data from individual experiments are indicated by different symbols.

Article Snippet: Antibodies for flow cytometry-based sorting of T cells: CD3-FITC (Biolegend, UCHT1, Cat. 300406), CD4-APC-Cy7 (Biolegend, RPA-T4, Cat. 300518), CD8-BV650 (Biolegend, SK1, Cat. 344730), CD45-Pacific Blue (Biolegend, HI30, Cat. 304029), CD137-APC (Miltenyi, REA765, Cat. 130-110-901), PD-1-PE-Dazzle (Biolegend, EH12.2H7, Cat. 329940), Zombie AquaTM Fixable Viability Kit (Biolegend, Cat. 423101).

Techniques: Injection, Ex Vivo, Control, Flow Cytometry, Immunohistochemistry, Adoptive Transfer Assay, Isolation

Journal: iScience

Article Title: Ad26.COV2.S priming provided a solid immunological base for mRNA-based COVID-19 booster vaccination

doi: 10.1016/j.isci.2022.105753

Figure Lengend Snippet:

Article Snippet: Anti-human CD137 PE clone 4B4-1 , Miltenyi Biotec , Cat#130-119-885; RRID: AB_2783944.

Techniques: Staining, Virus, Clinical Proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Analysis, Software, Imaging

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet:

Article Snippet: InVivoPlus anti-mouse CD137 (4-1BB) , Bio X Cell , Cat# BE0239; RRID: AB_2687721.

Techniques: Purification, Virus, Recombinant, Staining, Saline, Fluorsave, Sequencing, DNA Labeling, Cell Isolation, Sample Prep, Reverse Transcription, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Sequences.

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques:

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: 4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Expressing, Saline, Western Blot, Isolation

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Inhibition, Suspension, Saline, Control, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

doi: 10.3389/fimmu.2018.01848

Figure Lengend Snippet: A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

Article Snippet: Agonist 4-1BB mAb (10 μg/mL; cat: MAB9371, R&D Systems, Minneapolis, MN, USA) ( ) was used to detect the effect of 4-1BB signaling on macrophages 2 h before CS exposure.

Techniques: Expressing