Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc and treated with VV and/or α-PD-L1 as described. Single cells were made from primary tumours collected from tumour-bearing mice at day 5 post first treatment, blocked with α-CD16/32 Ab and then stained with antibodies against CD45, CD8, CD4, PD-1, ICOS, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and Foxp3 to determine the quantities of CD8 + T cells ( a ), CD8 + T-cell activation ( b – d ), CD8 + T-cell exhaustion ( e – h ), Treg cells ( i ), CD8 + /CD4 + Foxp3 + T cells ( j ) and CD4 + Foxp3 − T cells ( k , l ) in the TME. Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent phenotypic analysis. Data were analysed using Student's t- test (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Staining, Activation Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: ( a ) B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Splenic CD8 + T cells (4 × 10 5 ) were isolated from naive and MC38-luc-bearing mice that received different treatments 18 days post tumour cell injection and restimulated with mitomycin C-treated MC38-luc or B16 cancer cells (4 × 10 4 cells each) in the presence of 4000-rad-irradiated CD8-depleted naive B6 splenocytes (2 × 10 6 ) in 200 μl RPMI-1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 for 2 days. The concentration of IFN-γ in the culture supernatants was tested by ELISA. The statistical analyses were performed with t -test. ( b ) Naive or MC38-luc-bearing B6 mice with dual treatments, which survived for more than 60 days, were s.c. rechallenged with 1 × 10 6 MC38-luc cancer cells. The primary tumour size was measured and presented here. ( c ) In a separate experiment, B6 mice were inoculated with 5 × 10 5 MC38-luc cells i.p. and treated with VV plus α-PD-L1 or PBS at day 5 post tumour inoculation, α-PD-L1 Ab was injected every 2 days for a total of four times. α-CD8 Ab (250 μg per injection), α-CD4 Ab (150 μg per injection) or α-IFN-γ Ab (200 μg per injection) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells or neutralize circulating IFN-γ as scheduled in c , and the overall survival was monitored by Kaplan–Meier analysis and analysed using log rank test ( d ).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Isolation, Injection, Irradiation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc and treated with VV and/or α-PD-L1 as described. Single cells were made from primary tumours collected from tumour-bearing mice at day 5 post first treatment, blocked with α-CD16/32 Ab and then stained with antibodies against CD45, CD8, CD4, PD-1, ICOS, PD-1, CTLA-4, TIM-3, LAG-3, TIGIT and Foxp3 to determine the quantities of CD8 + T cells ( a ), CD8 + T-cell activation ( b – d ), CD8 + T-cell exhaustion ( e – h ), Treg cells ( i ), CD8 + /CD4 + Foxp3 + T cells ( j ) and CD4 + Foxp3 − T cells ( k , l ) in the TME. Of note, the anti-PD-L1 antibody clone 10F.9G2 was used for therapy while clone MHI5 was used for subsequent phenotypic analysis. Data were analysed using Student's t- test (* P <0.05; ** P <0.01; *** P <0.001).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Staining, Activation Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: ( a ) B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Splenic CD8 + T cells (4 × 10 5 ) were isolated from naive and MC38-luc-bearing mice that received different treatments 18 days post tumour cell injection and restimulated with mitomycin C-treated MC38-luc or B16 cancer cells (4 × 10 4 cells each) in the presence of 4000-rad-irradiated CD8-depleted naive B6 splenocytes (2 × 10 6 ) in 200 μl RPMI-1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 for 2 days. The concentration of IFN-γ in the culture supernatants was tested by ELISA. The statistical analyses were performed with t -test. ( b ) Naive or MC38-luc-bearing B6 mice with dual treatments, which survived for more than 60 days, were s.c. rechallenged with 1 × 10 6 MC38-luc cancer cells. The primary tumour size was measured and presented here. ( c ) In a separate experiment, B6 mice were inoculated with 5 × 10 5 MC38-luc cells i.p. and treated with VV plus α-PD-L1 or PBS at day 5 post tumour inoculation, α-PD-L1 Ab was injected every 2 days for a total of four times. α-CD8 Ab (250 μg per injection), α-CD4 Ab (150 μg per injection) or α-IFN-γ Ab (200 μg per injection) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells or neutralize circulating IFN-γ as scheduled in c , and the overall survival was monitored by Kaplan–Meier analysis and analysed using log rank test ( d ).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Isolation, Injection, Irradiation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: In one experiment, the i.p. MC38-luc tumour model was treated with VV and/or α-PD-L1 as described. Tumour tissues were collected at day 5 post first treatment and were used for extraction of total RNA. RT–qPCR assays were performed to determine the levels of IFN-γ ( a ), granzyme B ( b ) and perforin ( c ) in the TME. Data were analysed using Student's t- test (* P <0.05; ** P <0.01).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Extraction, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Rational combination of oncolytic vaccinia virus and PD-L1 blockade works synergistically to enhance therapeutic efficacy

doi: 10.1038/ncomms14754

Figure Lengend Snippet: ( a ) B6 mice were intraperitoneally inoculated with 5 × 10 5 MC38-luc cancer cells and treated with VV and/or α-PD-L1 as described. Splenic CD8 + T cells (4 × 10 5 ) were isolated from naive and MC38-luc-bearing mice that received different treatments 18 days post tumour cell injection and restimulated with mitomycin C-treated MC38-luc or B16 cancer cells (4 × 10 4 cells each) in the presence of 4000-rad-irradiated CD8-depleted naive B6 splenocytes (2 × 10 6 ) in 200 μl RPMI-1640 medium supplemented with 10% FBS at 37 °C, 5% CO 2 for 2 days. The concentration of IFN-γ in the culture supernatants was tested by ELISA. The statistical analyses were performed with t -test. ( b ) Naive or MC38-luc-bearing B6 mice with dual treatments, which survived for more than 60 days, were s.c. rechallenged with 1 × 10 6 MC38-luc cancer cells. The primary tumour size was measured and presented here. ( c ) In a separate experiment, B6 mice were inoculated with 5 × 10 5 MC38-luc cells i.p. and treated with VV plus α-PD-L1 or PBS at day 5 post tumour inoculation, α-PD-L1 Ab was injected every 2 days for a total of four times. α-CD8 Ab (250 μg per injection), α-CD4 Ab (150 μg per injection) or α-IFN-γ Ab (200 μg per injection) were intraperitoneally injected into mice to deplete CD8+ T cells, CD4+ T cells or neutralize circulating IFN-γ as scheduled in c , and the overall survival was monitored by Kaplan–Meier analysis and analysed using log rank test ( d ).

Article Snippet: Anti-mouse PD-L1 Ab (clone 10F.9G2), α-mouse CD8 Ab (clone 53-6.7), α-mouse CD4 Ab (clone GK1.5) and α-mouse IFN-γ Ab (clone XMG1.2) were purchased from Bio X Cell (West Lebanon, NH, USA).

Techniques: Isolation, Injection, Irradiation, Concentration Assay, Enzyme-linked Immunosorbent Assay