Article Figures & Data
Figures
- Figure 1.
Anti-HER2/CD3 TDB increased intratumoral lymphocyte infiltration in autochthonous HER2 overexpressing mammary tumors. MMTV-huHER2 TG tumors treated for 6 days with 0.5 mg/kg anti-HER2/CD3 TDB (4D5/2C11;red) or vehicle (blue) as indicated. A, Representative images of anti-CD3 and anti-HER2 immunostaining. Scale bar, 100 μm. B, Flow cytometry analysis of lymphocytes and indicated cell types. Each dot represents one tumor. Welch t test was used for the statistical analysis. Error bars, mean ± SEM.
- Figure 2.
CD4+ T cells were dispensable for antitumor efficacy of anti-HER2/CD3 TDB. Tumor-bearing MMTV-huHER2/huCD3e TG mice were dosed weekly with 0.5 mg/kg anti-HER2/CD3 TDB (4D5/2C11). CD4+ T cells were depleted by treatment with 20 mg/kg anti-CD4 on D-2, D3 and D7. A, Flow cytometry analysis of peripheral blood at one day prior TDB treatment (D-1, blue), 1 (D1; red), 7 (D7; brown) or 14 (D14; purple) days after HER2-TDB treatment. B, Flow cytometry analysis on CD4+ T cell or C, Treg cells in tumors 20 days after anti-HER2/CD3 TDB treatment. D, Tumor growth of individual animals over time. E, Waterfall plot of maximum percentage change in tumor volume from D0. D and E, Vehicle (blue), anti-HER2/CD3 TDB (red), anti-CD4 (magenta) and combination treatment of anti-CD4 and anti-HER2/CD3 TDB (green). Error bars, mean ± SEM.
- Figure 3.
Both T-cell recruitment and proliferation contributed to the accumulation of tumor-infiltrating CD8+ T cells. A, Flow cytometry analysis of CD45+ lymphocytes and CD8+ T cells in blood of tumor-bearing MMTV-huHER2 TG mice 1 day after treatment with vehicle (blue) or FTY720 (red). B, Flow cytometry analysis of CD8+ T cells and CD4+ T cells in MMTV-huHER2/huCD3e TG tumors 6 days of indicated treatment. Vehicle (blue), 0.5 mg/kg anti-HER2/CD3 TDB (4D5/40G5;red), FTY720 (brown) and combination of FTY720 and anti-HER2/CD3 TDB (purple). Parametric unpaired t test was used for the statistical analysis. C, Cell Trace Violet (CTV) labeled CD8+ T cells were adoptively transferred to mice with Fo5 tumor allografts 1 day before TDB treatment. Flow cytometry analysis of intratumoral CTV-labeled CD8+ T cells in the tumors 4 days after TDB treatment (0.5 mg/kg). D, Left, representative flow cytometry dot plots of CTV-labeled CD8+ T cells. The red box is the gate for proliferating CD8+ T cells and the blue box is the gate for nonproliferating CD8+ T cells. Right: summary data of proliferating CD8+ cells in Fo5 tumors treated with control-TDB (blue) or anti-HER2/CD3 TDB for 4 days. E, Ki67 stain of CTV-positive CD8+ from parallel samples. Welch t test was used for the statistical analysis in A, C, D, and E. Error bars, mean ±SEM.
- Figure 4.
Anti-HER2/CD3 TDB induced acute release of proinflammatory cytokines in mammary tumors. Tumor-bearing MMTV-huHER2/huCD3e mice were treated with either vehicle (blue) or 0.5 mg/kg anti-HER2/CD3 (4D5/2C11) TDB (red) for 2 hours. Tumor lysate protein concentration was normalized to 20 mg/mL. Cytokines (A) and chemokines (B) were analyzed using Luminex. The levels of cytokine or chemokine are presented as pg/mg, that is, pg of cytokine/chemokine as mg of tumor protein. Welch t test was used for the statistical analysis. Error bars, mean ± SEM.
- Figure 5.
Anti-HER2/CD3 TDB induced expression of CXCR3 ligands in tumors and CXCR3 on T cells. A, CXCL9 and CXCL11 in MMTV-huHER2/huCD3e TG tumor lysate was analyzed using ELISA 2 hours after treatment with 0.5 mg/kg anti-HER2/CD3 TDB (4D5/2C11). The levels of CXCL9 and CXCL11 are presented as pg/mL, that is, pg of cytokine per mL of tumor lysate which is in protein concentration of 20 mg/mL. B, CXCR3 on CD4+, CD8+ T cells and T-cell subsets in peripheral blood was analyzed using flow cytometry. Naïve CD8+ T cells (CD8+CD62L+CD44−, blue), central memory CD8+ T cells (CD8+CD62L+CD44+, red), effective memory CD8+ T cells (CD8+CD62L−CD44+, brown), and effector CD8+ T cells (CD8+CD62L−CD44−, purple). C and D, MMTV-huHER2 TG mice treated with anti-HER2/CD3 TDB and blood was collected at indicated time points. Flow cytometry analysis for expression of CXCR3 on CD8+ (C) and CD4+ T cells (D) in blood. Welch t test was used for the statistical analysis. Error bars, mean ± SEM.
- Figure 6.
IFNγ-induced CXRC3 signaling is required for CD8+ T-cell recruitment and antitumor efficacy of anti-HER2/CD3 TDB. A, Treatment schedule of Fo5 mice as indicated. Black arrows were the days for anti-IFNγ, or anti-CXCR3 or the control Ig. Red arrows depict days for control-TDB or 0.5 mg/kg anti-HER2/CD3 TDB treatment. Serum was collected 1 day after TDB treatment (brown arrow) for cytokine analysis. Tumor cell flow cytometry was performed 4 days after TDB treatment (purple arrow). B, Serum CXCL9 and CXCL10 levels at 24 hours post-anti-HER2/CD3 TDB administration. C, Flow cytometric analysis of tumor-infiltrating CD8+ T cells 4 days after treatment. Welch t test was used for the statistical analysis. D, Effect of anti-IFNγ and anti-CXCR3 treatment on anti-HER2/CD3 antitumor activity in Fo5 tumors. Data reported as mean ± SEM.
Additional Files
Supplementary Data
- Supplemental Figure 1-5 - Fig. S1. Subset analysis of tumor infiltrating and splenic CD8+ T cells after treatment with anti-HER2/CD3 TDB; Fig. S2. Anti-HER2/CD3 TDB increases intra-tumoral lymphocyte infiltration in Fo5 allograft model of HER2 overexpressing breast cancer within immunocompetent mice; Fig. S3. Anti-HER2/CD3 treatment did not induce peripheral CD8+ T cell proliferation; Fig. S4. Anti-HER2/CD3 TDB induced acute release of pro-inflammatory cytokines in serum; Fig. S5. Induction of CXCR3 ligands within Fo5 tumors in response to anti-HER2/CD3.
Volume 24, Issue 24
