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: Medical Science Monitor

Article Title: Expression of Semaphorin 3B (SEMA3B) in Various Grades of Endometrial Cancer

doi: 10.12659/msm.916762

Figure Lengend Snippet: Figure 1. Immunohistochemical localization of SEMA3B in different grades of endometrial cancer. C – control; G – grade of endometrial cancer. 200× magnification.

Article Snippet: The analysis of SEMA3B level changes was performed based on the immunohistochemical reaction with rabbit polyclonal anti-SEMA3B antibody (Novus Biological).

Techniques: Immunohistochemical staining, Control