Programmed Cell Death-1: Programmed Cell Death-Ligand 1 Interaction Protects Human Cardiomyocytes Against T-Cell Mediated Inflammation and Apoptosis Response In Vitro

Abstract

Aim: Immunological checkpoint therapy is considered a powerful method for cancer therapy and acts by re-activating autologous T cells to kill the cancer cell. Myocarditis cases have been reported in cancer patients after immunological therapy; for example, nivolumab treatment is a monoclonal antibody that blocks programmed cell death-1/programmed cell death ligand-1 ligand interaction. This project provided insight into the inflammatory response as a benchmark to investigate the potential cardiotoxic effect of T cell response to the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis in regulating cardiomyocyte injury in vitro.

Methods and results: We investigated cardiomyopathy resulted from the PD-1/PD-L1 axis blockade using the anti-PD-1 antibody in Rockefeller University embryonic stem cells-derived cardiomyocytes (RUES2-CMs) and a melanoma tumor-bearing murine model. We found that nivolumab alone did not induce inflammatory-related proteins, including PD-L1 expression, and did not induce apoptosis, which was contrary to doxorubicin, a cardiotoxic chemotherapy drug. However, nivolumab was able to exacerbate the immune response by increasing cytokine and inflammatory gene expression in RUES2-CMs when co-cultured with CD4⁺ T lymphocytes and induced apoptosis. This effect was not observed when RUES2-CMs were co-cultured with CD8⁺ T lymphocytes. The in vivo model showed that the heart function of tumor-bearing mice was decreased after treatment with anti-PD-1 antibody and demonstrated a dilated left ventricle histological examination. The dilated left ventricle was associated with an infiltration of CD4⁺ and CD8⁺ T lymphocytes into the myocardium. PD-L1 and inflammatory-associated gene expression were significantly increased in anti-PD-1-treated tumor-bearing mice. Cleaved caspase-3 and mouse plasma cardiac troponin I expressions were increased significantly.

Conclusion: PD-L1 expression on cardiomyocytes suppressed T-cell function. Blockade of PD-1 by nivolumab enhanced cardiomyocyte inflammation and apoptosis through the enhancement of T-cell response towards cardiomyocytes.

Keywords: PD-1; PD-L1; T-lymphocytes; human embryonic stem cell-derived cardiomyocytes; immune-related cardiotoxicity; nivolumab.

Figure 1

Establishment and validation of RUES2 and differentiation of cardiomyocytes in vitro. (A) Schematic of the RUES2-CMs differentiation protocol. (B) Flow cytometric analysis of pluripotent stem cell markers expression: Oct-4, SSEA-4, Nanog, and a positively selected cardiac marker (cTnT). (C) Representative fluorescent images of pluripotent stem cell markers Oct-4 and cardiac markers: α-actinin and cTnT. Scale bar: 50 μm (upper panel); 20 μm (lower panel). (D) PD-L1 expression on RUES2-CMs by flow cytometry and (E) immunofluorescence staining, scale bar: 25 μm. The quantitative result as the right panel. *** p < 0.001 versus control (n = 3), one-way ANOVA, posthoc Bonferroni test. RUES2, Rockefeller University embryonic stem cell line 2; cTnT, cardiac troponin T; Oct4, octamer-binding transcription factor 4; PD-L1, programmed death-ligand 1; RUES2-CMs, Rockefeller University embryonic stem cell line 2-cardiomyocytes.

Figure 2

Effects of nivolumab on cell viability in RUES2-CMs. (A) The effect of nivolumab on cell viability was determined by an MTT assay. Positive control (doxorubicin 5 μg/mL) was provided. *** p < 0.001 versus control (n = 3). (B) Representative images of the TUNEL staining of cardiomyocytes. TUNEL staining was used to detect cell apoptosis (green). The nuclei were counterstained with DAPI (blue). Cyan color represents TUNEL-positive nuclei on merged photos. Positive control (doxorubicin 5 μg/mL) was provided. Scale bar: 50 μm. (C) Representative flow cytometry images of the Annexin V. Positive control (doxorubicin 5 μg/mL) was provided. *** p < 0.001 versus control (n = 3), one-way ANOVA, posthoc Bonferroni test. (D) Representative western blot analysis of caspase3 and inflammation markers—STAT1, NFkB—and the quantitative result (E). Positive control (doxorubicin 5 μg/mL) was provided. *** p < 0.001 versus control (n = 3), one-way ANOVA, posthoc Bonferroni test. Data are shown as the mean ± SD. RUES2-CM, Rockefeller University embryonic stem cell line 2- cardiomyocytes. (F) The PD-L1 expression level in RUES2-CMs analyzed by flow cytometry images. The quantitative result is shown in the right panel. *** p < 0.001 versus control (n = 3), one-way ANOVA, posthoc Bonferroni test.

Figure 3

Validation of PD-1 expression on CD4⁺ and CD8⁺ T lymphocytes. (A) Representative flow cytometry results of PD-1 and activation marker CD25 expression on CD4+ and CD8+ T lymphocytes after anti-CD3/CD28 stimulation. PD-1, programmed death-1. (B) Representative micrographs showing immunostained PD-1⁺ cells (green) on the CD4⁺ and CD8⁺ T lymphocytes. Scale bar: 50 μm. (C) Quantification of cTnT⁺/PD-L1 expression by flow cytometry analysis of levels of cTnT⁺/PD-L1 expression on RUES2-CMs co-cultured with CD4⁺ and CD8⁺ T lymphocytes with nivolumab treatment at different doses. ** p < 0.01, *** p < 0.001 versus RUES2-CMs alone group; # p < 0.05, ### p < 0.001 versus CD4/RUES2-CMs human IgG4 isotype control (w/o co-culture) group (n = 3). (D) Representative western blot analysis on RUES2-CMs co-cultured with CD4⁺ and CD8⁺ T lymphocytes containing nivolumab treatment with different doses and quantitative results (E–G). * p < 0.05, ** p < 0.01, *** p < 0.001 versus RUES2-CMs alone group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus CD4/RUES2-CMs human IgG4 isotype control (w/o co-culture) group (n=3; & p < 0.05 versus CD8/RUES2-CMs human IgG4 isotype control (w/o co-culture) group; & p < 0.05, versus CD8/RUES2-CMs human IgG4 isotype control (w/o co-culture) group (n = 3), one-way ANOVA, posthoc Bonferroni test. (H) Quantitative result of ELISA assay, showing production of IFN-γ in CD4⁺ T lymphocytes/RUES2-CMs and CD8⁺ T lymphocytes/RUES2-CMs co-culture medium. ** p < 0.01, *** p < 0.001 versus CD4+ T lymphocytes alone group; # p < 0.05, ### p < 0.001 versus CD4/RUES2-CMs human IgG4 isotype control (w/o co-culture) group (n = 3).

Figure 4

Effects of anti-PD-1 immunotherapy on tumor growth in tumor-bearing mice. (A) Representative micrographs showing immunostained PD-L1⁺ cells (green) on the B16-F10 cells. Scale bar: 50 μm. Representative immunohistochemistry staining for IgG (B,C) and PD-L1 (D,E) on mouse heart. The inset box, showing the enlarged pictures for the indicated square area. Scale bars in (B) and (D), 100 μm; scale bars in (C) and (E), 50 μm. Red arrow: PD-L1⁺ cell. (F) Representative PCR analysis of PD-L1 in B16-F10 cells and mouse heart. PD-L1, programmed death-ligand 1. (G) Representative photograph of xenograft tumors at the end of the experiment. (H) Tumor growth curves for each experiment group. ** p < 0.01, *** p < 0.001 versus rat IgG group (n = 7), two-way ANOVA, posthoc Bonferroni test. Effects of anti-PD-1 immunotherapy on mouse heart function. The quantitative data of fractional shortening (I), and ejection fraction (J) of rat IgG and anti-PD-1-treated group. # p < 0.05 versus rat IgG-treated tumor group (n = 7); # p < 0.05 versus anti-PD-1-treated non-tumor group (n = 7). (K) Representative micrographs showing immunostained CD4⁺ and CD8⁺ cells (green) in the myocardium. Scale bar: 1000 μm. (L) Representative western blot analysis and quantitative results (M). *** p < 0.001 versus anti-PD-1-treated non-tumor group; ### p < 0.001 versus rat IgG-treated tumor group (n = 4), two-way ANOVA, posthoc Bonferroni test. Data are shown as the mean ± SD.

Figure 5

Effects of anti-PD-1 immunotherapy on the expression of PD-L1 and inflammation-related markers in tumor-bearing mice. (A) Representative western blot analysis and quantitative results (B). && p < 0.01, ### p < 0.001 versus rat IgG-treated tumor group; *** p < 0.001 versus anti-PD-1-treated non-tumor group; && p < 0.01 versus rat IgG-treated non-tumor group (n = 4), two-way ANOVA, posthoc Bonferroni test. (C) A representative of RT-PCR analysis of IFN-γ and TNF-α in the anti-PD-1-treated non-tumor group and anti-PD-1-treated tumor group. *** p < 0.001 versus anti-PD-1-treated non-tumor group (n = 4), unpaired Student’s t-test. Data are shown as the mean ± SD. (D) Representative western blot analysis of apoptosis markers and quantitative results (E). ## p < 0.01, ### p < 0.001 versus rat IgG-treated tumor group; *** p < 0.001 versus anti-PD-1-treated non-tumor group (n = 4), two-way ANOVA, posthoc Bonferroni test.