Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3A and Nrp-1 in human lung tumour cell lines and CTL clone. a Sema-3A expression in human lung tumour cell lines. Total protein extracts were analysed by western blot using anti-Sema-3A mAb. The 16HBE cell line was included as a control. Full length and proteolytically processed proteins are indicated. b Co-expression of Nrp-1 and CD25, and Nrp-1 and PD-1 on P62 T cells stimulated with immobilised anti-CD3. c The P62 clone was unstimulated or stimulated with anti-CD3, pre-incubated with Sema-3A-Fc, and then labelled with anti-human IgG-Fc secondary mAb. Results are gMFI mean ± SEM of triplicate samples. d Sema-3A-Fc inhibits CTL migration toward a CXCL12 gradient. The T-cell clone was stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwell plates and exposed to a gradient of CXCL12 loaded in the lower chambers. The number of T cells that had migrated into the lower chambers was determined. Results are mean chemotaxis index ± SEM of triplicate samples. e Sema-3A-Fc inhibits CD8 + TIL migration toward CXCL12. CD8 + TIL isolated from three human NSCLC tumours were stimulated with anti-CD3, pre-incubated with BSA or Sema-3A-Fc, and then seeded in the upper chambers of transwells and exposed to a CXCL12 gradient. Results are mean chemotaxis index ± SEM of triplicates. f Cytotoxicity of the CTL clone toward autologous tumour cells. The P62 clone was stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward the cognate IGR-Pub cell line was determined. g Cytotoxic activity of freshly isolated TIL toward autologous tumour cells. TIL, freshly isolated from a NSCLC tumour, were stimulated with anti-CD3, pre-incubated in medium or with Sema-3A-Fc. Cytotoxicity toward freshly isolated autologous tumour cells was determined. Data shown for cytotoxic assay correspond to one of three independent experiments. Means ± SEM two-tailed Student’s paired t test c , one-way ANOVA test with Bonferroni correction d , e or two-way ANOVA test with Bonferroni correction f , g . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Western Blot, Control, Incubation, Migration, Chemotaxis Assay, Isolation, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: Expression of Sema-3B and Nrp-1 in B16F10 mouse melanoma model. a Expression of Sema-3B in B16F10 tumour cells. Total protein extracts from B16F10 cells cultured in vitro or isolated ex vivo from tumour grafts were analysed by western blot using anti-Sema-3B mAb. b Surface expression of Nrp-1 on CD4 + and CD8 + T cells infiltrating B16F10 melanoma engrafted in C57BL/6 mice. TIL from individual tumours were isolated at day 15 after tumour cell inoculation. T lymphocytes from spleens and TdLN of tumour-bearing mice were analysed in parallel. Percentages of positive cells are included. Right: percentages of Nrp-1 + cells among CD8 + and CD4 + T cells in TIL ( n = 29 and 31), splenocytes ( n = 29 and 31) and TdLN ( n = 11 and 16). c Expression of Nrp-1 and FoxP3 in CD4 + T cells. T lymphocytes from tumours ( n = 20), spleens ( n = 22) and TdLN ( n = 11) of B16F10 melanoma-bearing mice were analysed at day 15 by flow cytometry. Right: percentages of Nrp-1 among FoxP3 + and FoxP3 − CD4 + T lymphocytes from B16F10. d Expression of CD44 and CD62L on Nrp-1 + and Nrp-1 − CD8 + T cells from B16F10 TIL. Right: Distribution of naive, effector and memory T cells populations among Nrp-1 − and Nrp-1 + CD8 + TIL ( n = 10). Results are representative of 3–5 independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction b or two-way ANOVA test with Bonferroni correction c . * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file a , d

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Expressing, Cell Culture, In Vitro, Isolation, Ex Vivo, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Regulation of antitumour CD8 T-cell immunity and checkpoint blockade immunotherapy by Neuropilin-1

doi: 10.1038/s41467-019-11280-z

Figure Lengend Snippet: The Nrp-1 + PD-1 hi TIL subset is enriched with activated antigen-specific CD8 + T cells. a Staining of CD8 + TIL with dextramers. C57BL/6 mice were engrafted with B16F10 and then vaccinated with Trp2 and gp100 peptides. On day 15, TIL were isolated from tumours. Right: Percentages of Trp2 ( n = 8) and gp100 ( n = 5) dextramer-positive T cells among Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + TIL. b Expression of IFNγ ( n =20) and TNF ( n =18) in Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − CD8 + T cells. TIL were stimulated for 4 h with autologous tumour cells. c Degranulation of CD8 + TIL. TIL were stimulated with autologous tumour cells; then, T-cell subsets were analysed for expression of CD107a ( n = 18). d Cytotoxicity of freshly isolated CD8 + TIL. CD8 + TIL were pre-incubated in medium or with anti-Nrp-1, anti-PD-1 or a combination of both mAb; then, cytotoxicity toward autologous tumour cells was determined. e Increase in MHC-I and PD-L1 expression on tumour cells co-cultured with autologous CD8 + TIL. Kinetic studies of H-2-K b /-D b and PD-L1 expression on B16F10 co-cultured with CD8 + TIL. Expression profiles (left), percentages of positive cells (middle) and gMFI (right) of MHC-I (upper panels) and PD-L1 (lower panels) are shown. f Expression of perforin in CD8 + T cells. TIL were stimulated with autologous tumour cells in the absence or presence of neutralising anti-Nrp-1, anti-PD-1 or anti-Nrp-1 plus anti-PD-1 then, T-cell subsets were analysed for expression of perforin ( n = 5). g Anti-Nrp-1 re-establishes migration of Nrp-1 + PD-1 hi T cells toward B16F10. TIL were pre-incubated in medium or with neutralising anti-Nrp-1, -PD-1, a combination of both mAb or an isotype control. Cells were seeded in the upper chambers of transwells and then exposed to a gradient of B16F10 supernatant, enriched in Sema-3B, loaded in the lower chambers. The numbers of Nrp-1 + PD-1 hi , Nrp-1 − PD-1 + and Nrp-1 − PD-1 − T cells that had migrated were determined by flow cytometry. Results are representative of three independent experiments. Means ± SEM one-way ANOVA test with Bonferroni correction a – g . * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Blots were then probed with anti-Sema-3B mAb (clone 904201, R&D systems MAB5440, 1 μg/ml), anti-Sema-3A (clone 215803, R&D systems MAB1250, 1 μg/ml) or anti-β-actin-peroxidase (clone AC-15, Merck A3854, 1/50 000), followed by secondary anti-mouse (Santa cruz Biotechnology sc-2031) or anti-rat (R&D systems HAF005) horseradish peroxidase-conjugated Ab.

Techniques: Staining, Isolation, Expressing, Incubation, Cell Culture, Migration, Control, Flow Cytometry

Journal: American Journal of Physiology - Cell Physiology

Article Title: SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF

doi: 10.1152/ajpcell.00681.2023

Figure Lengend Snippet: Reduced semaphorin class 3B (SEMA3B) and neuropilin 1 (NRP1) levels associate with lung function loss in idiopathic pulmonary fibrosis (IPF). A : SEMA3B transcript levels were lower in patients with IPF ( n = 160) compared to healthy subjects ( n = 108) based on the RNA microarray dataset GSE47460. **** P < 0.0001. B and C : GSE47460 data show a positive correlation between SEMA3B transcript levels and both percent forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DL CO ) in both IPF (red) and healthy subjects (blue). The blue line represents the linear correlation. D : RNA-sequencing data from GSE150910 confirms decreased SEMA3B transcript levels in IPF lungs compared to healthy controls (CTRL; n = 40–60/group). **** P < 0.0001. E and F : similar to GSE47460, GSE150910 data show a positive correlation between SEMA3B transcript levels and percent FVC or DL CO in IPF (red) and healthy subjects (blue). G : NRP1 transcript levels were also reduced in IPF lungs compared to healthy lungs according to the RNA-sequencing data from GSE150910. **** P < 0.0001. H and I : a positive correlation between NRP1 transcript levels and both percent FVC and DL CO is observed in IPF (red) and healthy subjects (blue) from the GSE150910 dataset. The correlation coefficient ( r ) and P value are displayed within the scatter plots.

Article Snippet: Formalin-fixed and paraffin-embedded human lung tissue sections of IPF ( n = 6) and healthy ( n = 5) controls were probed with antibodies against SEMA3B (Novus Biologicals) or NRP1 (Proteintech) for immunohistochemistry staining as previously described ( , ).

Techniques: Microarray, RNA Sequencing

Journal: American Journal of Physiology - Cell Physiology

Article Title: SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF

doi: 10.1152/ajpcell.00681.2023

Figure Lengend Snippet: Downregulation of semaphorin class 3B (SEMA3B) and neuropilin 1 (NRP1) in idiopathic pulmonary fibrosis (IPF) lungs. A : violin plot showing the expression levels of SEMA3B and NRP1 across various cell types in non-IPF and IPF lungs. Data are from a publicly available dataset (GSE136831) for non-IPF and IPF lung scRNA-seq. B and C : representative immunohistochemical images of distal lung biopsies from normal and IPF lungs stained with antibodies against SEMA3B ( B ) and NRP1 ( C ). Scale bar = 50 μm. Arrows highlight spindle-shaped mesenchymal cells positive for SEMA3B or NRP1 ( n = 5 per group). D and E : transcript levels of SEMA3B and NRP1 were quantified in fibroblasts isolated from the lungs of IPF patients and healthy controls using RT-PCR. Student’s t test was performed with n = 10–14 per group. *** P < 0.001, ** P < 0.01.

Article Snippet: Formalin-fixed and paraffin-embedded human lung tissue sections of IPF ( n = 6) and healthy ( n = 5) controls were probed with antibodies against SEMA3B (Novus Biologicals) or NRP1 (Proteintech) for immunohistochemistry staining as previously described ( , ).

Techniques: Expressing, Immunohistochemical staining, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF

doi: 10.1152/ajpcell.00681.2023

Figure Lengend Snippet: Semaphorin class 3B (SEMA3B) inhibits TGFβ1-driven extracellular matrix (ECM) gene expression in fibroblasts. A : upregulation of the transcripts of ECM genes [collagen 1A1 (COL1A1), elastin (ELN), and α-smooth muscle actin (αSMA)] in normal lung fibroblasts treated with media, transforming growth factor-β (TGFβ), SEMA3B, or both TGFβ and SEMA3B for 16 h. B : upregulation of the transcripts of ECM genes (COL1A1, ELN, and αSMA) in IPF fibroblasts treated with media, TGFβ, SEMA3B, or both TGFβ and SEMA3B for 16 h. Data are shown as the means ± SE. Student’s t test was performed with n = 4/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Formalin-fixed and paraffin-embedded human lung tissue sections of IPF ( n = 6) and healthy ( n = 5) controls were probed with antibodies against SEMA3B (Novus Biologicals) or NRP1 (Proteintech) for immunohistochemistry staining as previously described ( , ).

Techniques: Gene Expression

Journal: American Journal of Physiology - Cell Physiology

Article Title: SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF

doi: 10.1152/ajpcell.00681.2023

Figure Lengend Snippet: Semaphorin class 3B (SEMA3B) inhibits the transforming growth factor-β1 (TGFβ1)-driven extracellular matrix (ECM) protein production in Idiopathic pulmonary fibrosis (IPF) lung fibroblasts. A : IPF lung fibroblasts were treated with media, SEMA3B (400 ng/mL), and/or TGFβ1 (20 ng/mL) for 72 hours, and cell lysates were immunoblotted with antibodies against collagen 1 (COL1), fibronectin 1 (FN1), α-smooth muscle actin (αSMA), and GAPDH. B : IPF lung fibroblasts were treated with media, SEMA3B (400 ng/mL), and/or TGFβ1 (20 ng/mL) for 72 h, and cell lysates were immunoblotted with antibodies against elastin (ELN) and GAPDH. C : densitometric quantification of COL1, FN1, αSMA, and ELN protein levels normalized to GAPDH. Data are shown as the means ± SE. A one-way ANOVA test was performed with n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Formalin-fixed and paraffin-embedded human lung tissue sections of IPF ( n = 6) and healthy ( n = 5) controls were probed with antibodies against SEMA3B (Novus Biologicals) or NRP1 (Proteintech) for immunohistochemistry staining as previously described ( , ).

Techniques: