Additional file 1 of Modulating tumor infiltrating myeloid cells to enhance bispecific antibody-driven T cell infiltration and anti-tumor response
Creators: name: Park, Jeong A.
name: Wang, Linlin
name: Cheung, Nai-Kong V.
Publisher: name: figshare
Year: 2021
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1. Supplementary Table S1. Distribution of CD3, CD4, and CD8 T cells in neuroblastoma patient-derived xenografts by positive pixel count analyses. Abbreviations; CL, clodronate liposome. Supplementary Table S2. Tumor-infiltrating lymphocytes quantified by the area under each BLI curvesSupplementary Table S2. Tumor-infiltrating lymphocytes are quantified by the area under each BLI curve. Abbreviations; SCLC, small cell lung carcinoma; CDX, cell line-derived xenograft; PDX, patient-derived xenograft; CL, clodronate liposome; LD, low-dose; ID, intermediate-dose. Supplementary Fig. S1. (A) Neuroblastoma PDX bearing mice were treated by GD2-BsAb armed T cells (GD2-EATs) or unarmed T cells. (B) Tumors were harvested on day 10 and analyzed by flow cytometry. G-MDSC was gated as anti-mouse CD45+CD11b+Ly6G+; M-MDSC was gated as anti-mouse CD45+CD11b+Ly6G-Ly6Chi; TAM was gated as antimouse CD45+CD11b+Ly6G-Ly6CloF4/80+; Ly6Clo MDSC was gated as anti-mouse CD45+CD11b+Ly6GLy6CloF4/80-. The frequencies of each tumor-infiltrating leukocyte were compared among groups: a, no treatment; b, unarmed T cells; c, GD2-EATs. Supplementary Fig. S2. (A) Peripheral blood mononuclear cells (PBMCs) and GD2-BsAb were administered intravenously to the mice bearing 143B osteosarcoma cell line-derived xenograft (CDX). (B) Tumors were harvested on day 14 and immunohistochemical (IHC) stained with anti-CD11b antibody and anti-CD3 antibody. Supplementary Fig. S3. (A) Tumors were harvested on day 10 and analyzed by flow cytometry. G-MDSC was gated as anti-mouse CD45+CD11b+Ly6G+; M-MDSC was gated as anti-mouse CD45+CD11b+Ly6G-Ly6Chi; TAM was gated as antimouse CD45+CD11b+Ly6G-Ly6CloF4/80+; Ly6Clo MDSC was gated as anti-mouse CD45+CD11b+Ly6GLy6CloF4/80-. a, GD2-EATs; b, GD2-EATs plus anti-Ly6G antibody; c, GD2-EATs plus anti-Ly6C antibody; d, GD2-EATs plus anti-CSF1R antibody; e, GD2-EATs plus clodronate liposome (CL). Supplementary Fig. S4. (A) Osteosarcoma cell line xenografts were treated by GD2-BsAb armed T cells (GD2-EATs) or unarmed T cells with anti-Gr1 antibody or anti-Ly6G antibody. (B) CBC analyses were done on day 5 and compared among groups. (C) Tumors harvested on day 60 were analyzed by flow cytometry, and the frequencies of human CD45(+) or human CD8 (+) tumor-infiltrating lymphocytes were compared among groups. (D) Immunohistochemical (IHC) staining of tumor sections by anti-human CD3 antibody (x10) on day 60 post-treatment. The number of T cells was compared among groups using Q-path analysis. (E) In vivo anti-tumor effect of GD2-EATs with anti-Ly6G or anti-GR1 antibody was tested against 143B osteosarcoma cell line xenografts. Supplementary Fig. S5. (A) Luciferase transduced T cells [Luc(+) T cells] or Luciferase transduced GD2-BsAb armed T cells [Luc(+) GD2-EATs] were administered with anti-Ly6G antibody to the mice bearing neuroblastoma patient-derived xenograft (PDX). (B) Bioluminescence in the lesions of the tumor was monitored. The bioluminescence images on day 8 and quantification of the bioluminescence in the lesions of the tumor. Supplementary Fig. S6. (A) Osteosarcoma cell line xenografts were treated by GD2-BsAb armed T cells (GD2-EATs) with anti-Ly6C antibodies. (B) CBC analyses were done on day 5 and compared among groups. (C) Tumors harvested on day 60 were analyzed by flow cytometry, and the frequencies of human CD45(+) or human CD8 (+) tumor-infiltratinglymphocytes were compared among groups. (D) Immunohistochemical (IHC) staining of tumor sections by antihuman CD3 antibody (x10) on day 60 post-treatment. The number of T cells was compared among groups using Qpath analysis. (E) In vivo anti-tumor effect of GD2-EATs with anti-Ly6C antibody was tested against 143B osteosarcoma cell line xenografts. Supplementary Fig. S7. (A) Clodronate liposome (CL, 100μL) depleted macrophages in the liver. Livers were stained with immunofluorescence antibodies (green, murine CD68; blue, nuclei). (B) Anti-CSF1R antibody and 10μL of CL successfully depleted macrophages in the spleen. Spleens were processed with immunohistochemical (IHC) staining using murine CD68 antibody. (C) IHC staining of human CD3(+) T cells in 143B tumor sections aftertreatment with GD2-EATs plus anti-CSF1R antibody (x20). CD3(+) T cell number was compared with tumors treated with GD2-EATs alone. (D) IHC staining of human CD45(+) T cells in M14 tumor sections after treatment with T cells plus GD2-BsAb and CL (x10), and the number of CD45(+) T cells was compared. Supplementary Fig. S8. (A) Luciferase transduced T cells [Luc(+) T cells] were injected with clodronate liposome (CL) to small cell lung cancer (SCLC) cell line xenografts. The bioluminescence of Luc(+) T cell was monitored. The bioluminescence images on day 4 and quantitation of the bioluminescence in the lesions of the tumor. (B) Luciferase transduced HER2-BsAb armed T cells [Luc(+) HER2-EATs] were administered with anti-CSF1R antibody to HER2(+) osteosarcoma PDXs bearing mice. The bioluminescence of Luc(+) HER2-EATs was monitored. The bioluminescence images on day 4 and quantitation of the bioluminescence in the lesions of the tumor. Supplementary Fig. S9. (A) In vivo anti-tumor effect of GD2-BsAb armed T cells (GD2-EATs) plus clodronate liposome (CL) was studied in 143B osteosarcoma cell line xenograft model. (B) The anti-tumor effect of GD2-EATs plus anti-CSF1R antibody was tested against osteosarcoma patient-derived xenografts (PDXs), and overall survival was analyzed.Supplementary Fig. S10. (A) Increasing doses of dexamethasone with GD2-BsAb armed T cells (GD2-EATs) were administered to mice bearing neuroblastoma patient-derived xenograft (PDX). TH1 cell cytokines were measured at different time points post-GD2-BsAb armed T cell (GD2-EAT) injection. (B) In vivo TH1 cell cytokine release was compared amonggroups. Supplementary Fig. S11. (A) Tumors were harvested on day 25 post-treatment and analyzed by flow cytometry. G-MDSC was gated as antimouse CD45+CD11b+Ly6G+; M-MDSC was gated as anti-mouse CD45+CD11b+Ly6G-Ly6Chi; TAM was gated as anti-mouse CD45+CD11b+Ly6G-Ly6CloF4/80+; Ly6Clo MDSC was gated as anti-mouse CD45+CD11b+Ly6G-Ly6CloF4/80-. G1, no treatment; G2, unarmed T cells; G3, unarmed T cells plus high-dose (HD) dexamethasone;G4, GD2-EATs; G5, GD2-EATs plus low-dose (LD) dexamethasone; G6, GD2-EATs plus intermediate-dose (ID) dexamethasone; G7, GD2-EATs plus high-dose (HD) dexamethasone.Supplementary Fig. S12. (A) Luciferase transduced T cells [Luc(+) T cells] or Luciferase transduced GD2-BsAb armed T cells [Luc(+) GD2-EATs] were administered with increasing doses of dexamethasone to the mice bearing osteosarcoma patientderived xenograft (PDX). (B) Bioluminescence in the lungs and in the lesions of the tumor was monitored. Thebioluminescence images in the lungs on day 1 and in the lesions of tumor on day 5 and quantitation of the bioluminescence in the lesions of the tumor. Supplementary Fig. S13.(A) In vivo anti-tumor effect of dexamethasone premedication on GD2-BsAb directed T cell immunotherapy. 1x107 of T cells and 5μg of GD2-BsAb were administered with increasing doses of dexamethasone to mice bearing neuroblastoma patient-derived xenograft (PDX). (B) Increasing doses of dexamethasone with GD2-BsAb armed T cells (GD2-EATs) were administered to treat osteosarcoma PDXs. Tumor growth, body weight change, and overallsurvival were analyzed.