Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: Tuning of CD73 blockade is required to improve the response of low CD73-expressing MC38 tumors to IR. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 day post-IR. (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–12, *p<0.05 ****p<0.0001 (two-way ANOVA). (D, right panel) Representative histograms of CD73 expression in MC38 and TS/A cells 24-hour post-IR at 6 and 12 Gy compared with non-irradiated (NIR) cells. (D, left panel) cultured cells were analyzed by flow cytometry for their CD73 expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA). (E) TS/A tumor cells were injected subcutaneously in BALB/c mice and when tumors reached 60–70 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 days post-IR. (F) Tumor growth was monitored in treated mice. (G) The Kaplan-Meier survival curves for the treated mice are shown. (I) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=12–13, **p<0.01 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Injection, Irradiation, Cell Culture, Flow Cytometry, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 expression level controls the MC38 tumor response to CD73 blockade treatment. (A) Representative histograms of transfected and non-transfected MC38 cell with CD73 gene analyzed by flow cytometry. (B) Cultured MC38 control (Ctrl) and CD73 high MC38 cells were analyzed by flow cytometry 24-hour post-IR at 12 Gy for their CD73 membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, ***p<0.001, ****p<0.0001 (one-way ANOVA). (C) CD73 high MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 starting 1 day before IR then 2, 6 and 9 days post-IR. (D, F) Tumor growth was monitored in treated mice. (E, G) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=13–14, *p<0.05, ***p<0.001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation; NIR, non-irradiated; ssc-a, side scatter-a.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Control, Membrane, Fluorescence, Staining, Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 blockade treatment regimen affects the expression level of iCOS in tumor infiltrating CD4 + T lymphocytes. C57BL/6 mice with the subcutaneous MC38 tumors and BALB/c mice with subcutaneous TS/A tumors were irradiated and treated with either one dose or four doses of anti-CD73 starting 1 day before IR, then 2, 6 and 9 days post-IR. At day 10 post-IR, tumors were harvested and analyzed for immune infiltrating tumor cells by flow cytometry. CD4 + T lymphocytes infiltrating MC38 tumor were analyzed by flow cytometry for iCOS (A, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (A, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 tumors. (B, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. (B, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=8–10, *p<0.05 (two-way ANOVA). CD4 + T lymphocytes infiltrating MC38 CD73 high tumors were analyzed by flow cytometry for iCOS (C, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (C, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 CD73 high tumors. (D) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 CD73 high tumors are presented for each treatment group. CD4 + T lymphocytes infiltrating TS/A tumor were analyzed by flow cytometry for their iCOS (E, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (E, right panel) Representative histograms of iCOS expression in CD4 + T lymphocytes infiltrating TS/A tumor. (F, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating TS/A tumor are presented for each treatment group. (F, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=4–8, *p<0.05 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Irradiation, Flow Cytometry, Membrane, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: iCOS signaling is involved in CD73 blockade-mediated antitumor effect in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 (starting 1 day before IR then 2, 6 and 9 days post-IR) and anti-iCOS (starting 2 days post IR, then 6 and 9 days post-IR). (B and D) Tumor growth was monitored in treated mice. (C and E) The Kaplan-Meier survival curves for the treated mice are shown. (F) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=6–7, *p<0.05, ****p<0.001, ***p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: One dose of aCD73 improves the antitumor effect of aPD-L1 and IR treatment in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with one dose of anti-CD73 (starting 1 day before IR) and anti-PD-L1 (starting the same day as IR, then 3, 6 and 9 days post-IR). (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. (D) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–14, *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: Anti-IL-9 antibody treatment decreased mast cell infiltration of the CNS. Purified cells (mainly composed of mast cells) were harvested from the CNS on days 0, and 5, 15, and 20 after EAE immunization. CD45 + CD117 + mast cells were detected by flow cytometry. CNS mast cells maintained a high level throughout the disease course in EAE group. However, mast cells were significantly reduced from day 5, and remained low in anti-IL-9 Abs group, compared with IgG group. Data are shown as mean ± SE. * P < 0.01, anti-IL-9 Abs group versus IgG group. CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; SE: Standard error; IL: Interleukin.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Purification, Flow Cytometry

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: IL-9 blockade reduced production of chemokine recruiting mast cells in the CNS. Five days after immunization, mRNA expressions of Pecam1, SCF, Vcam-1, CCL2, and CCL5 in CNS tissue of EAE mice were detected by RT-PCR. After IL-9 neutralization, mRNA expressions of CCL5 and Vcam-1 were significantly decreased in anti-IL-9 Abs group, compared with IgG group. * P < 0.01. Pecam1: Platelet and endothelial cell adhesion molecule 1; SCF: Supercoiling factor; Vcam-1: Vascular cell adhesion molecule 1; CCL2: C-C motif chemokine ligand 2; CCL5: C-C motif chemokine ligand 5; CNS: Central nervous system; EAE: Experimental autoimmune encephalomyelitis; IL: Interleukin; RT-PCR: Reverse transcription-polymerase chain reaction; mRNA: Messenger RNA.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Neutralization

Journal: Chinese Medical Journal

Article Title: Neutralization of Interleukin-9 Decreasing Mast Cells Infiltration in Experimental Autoimmune Encephalomyelitis

doi: 10.4103/0366-6999.204110

Figure Lengend Snippet: In vitro , the effect of anti-IL-9 antibody on splenic mast cells. Splenocytes were harvested from experimental autoimmune encephalomyelitis mice 5 days after MOG immunization. After co-culture with anti-IL-9 antibody or anti-mouse IgG for 7 h, mast cell number was counted by flow cytometry. Splenic mast cells cultured with anti-IL-9 antibody showed significantly lower levels in a dose-dependent manner. This trend was particularly evident with anti-IL-9 antibody concentrations up to 20 μg/ml. * P < 0.05; † P < 0.01. IL: Interleukin; MOG: Myelin oligodendrocyte glycoprotein.

Article Snippet: The following antibodies were used in this study: FITC-anti-mouse CD45 (eBioscience, USA), PE-Cyanine5-anti-mouse CD117 (eBioscience), anti-mouse IL-9 (BE0181; BioXCell, USA), anti-mouse IgG2a isotype control (BE0085; BioXCell), anti-mouse IL-9 receptor (IL-9R) (SC699; Santa Cruz, USA), anti-mouse IL-2Rγ (SC668; Santa Cruz), anti-mouse IgG isotype control (GTX35009; Santa Cruz), and donkey pAb to Rb IgG Alexa Flour 555 (Ab150074; Abcam, USA).

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Cell Culture