Evidences of CTLA-4 and PD-1 Blocking Agents-Induced Cardiotoxicity in Cellular and Preclinical Models

Figures

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  Figure 1. Effects of Nivolumab and Ipilimumab mAbs on SK-BR-3 tumor cells co-cultured with hPBMCs as well as on human cardiomyocytes co-cultured with hPBMCs. (A) Cell viability assay on SKBR-3 tumor cells, treated with Nivolumab or Ipilimumab mAb for 24 h, in the presence of hPBMCs. Untreated cells (white bars) or cells treated with an unrelated control Immunoglobulin G (IgG) (grey bars) were used as negative controls. Cells were counted by Trypan blue after the removal of lymphocytes. (B) LDH assay on the supernatant of co-cultures treated as indicated. Cell lysis was measured as described in the Materials and Methods section. (C) Representative images of SK-BR-3 tumor cells co-cultured with lymphocytes and treated as indicated. (D) Cell viability assay on HFC cells, treated with Nivolumab or Ipilimumab mAb for 24 h, in the presence of lymphocytes. Untreated cells (white bars) or cells treated with an unrelated control IgG (grey bars) were used as negative controls. (E) LDH assay on the supernatant of co-cultures treated as indicated. Cell lysis was measured as described in the Materials and Methods section. (F) Representative images of HFC treated in the presence of lymphocytes. Error…

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  Figure 2. Pro-inflammatory effects of Nivolumab and Ipilimumab mAbs (alone or combined) on human cardiomyocytes co-cultured with hPBMCs. (A) Leukotriene type B4 production by HFC cells, treated with Nivolumab or Ipilimumab mAb alone (or combined) for 24 h, in the presence of lymphocytes. Untreated or treated cells with an unrelated control IgG (grey bars for both) were used as negative controls. (B) p65/NF-kB expression (fold of control) of cardiomyocytes treated as indicated before. (C) NLRP3 expression (fold of untreated cells) of cardiomyocytes treated with ICIs. (D) MyD88 expression (fold of control) of cardiomyocytes treated with ICIs. For each experiment, cell lysates were analyzed as described in the Materials and Methods section. Error bars depict means ± SD. *** p < 0.001; ** p < 0.01; * p < 0.05. (E) NLRP3 imaging through confocal laser scanning microscope (CLSM) method in cardiomyocytes unexposed or exposed to 200 nM of Nivolumab (F) or Ipilimumab (G) or Nivolumab and Ipilimumab in combination (H) for 24 h. Red: actin staining; Green: NLRP3 staining. Scale bar: 50 µm.

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  Figure 3. Pro-inflammatory cytokines secreted by cardiomyocytes after Nivolumab and Ipilimumab mAbs treatments. Interleukin-1 β (A) Interleukin-6 (B) Interleukin-8 (C), and TGF-β (D) production by HFC cells, treated with Nivolumab or Ipilimumab mAb for 24 h, in the presence of lymphocytes. Untreated or treated cells with an unrelated control IgG (grey bars for both) were used as negative controls. Details of the experiments are described in the Materials and Methods section. Error bars depict means ± SD. ** p < 0.01; * p < 0.05.; ns = not significant.

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  Figure 4. (A) Binding assays of Nivolumab and Ipilimumab on mouse or human purified recombinant proteins; ELISA assays of the indicated antibodies used at increasing concentrations on purified human (black bars) or mouse (grey bars) PD-1 or CTLA-4 proteins. graphic of fractional shortening (FS) (B) ejection fraction (EF), (C) radial strain (D), and longitudinal strain (E) in mice untreated (sham) or treated with Ipilimumab. Images of strain in untreated (F) or Ipilimumab-treated (G) mice. Error bars depict means ± SD. ** p < 0.01; * p < 0.05.; ns: not significant.

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  Figure 5. Inflammation pathways in the heart tissue of mice untreated (sham) or treated with Ipilimumab. Mice were treated as described in the Materials and Methods section; after their appropriate sacrifice, cardiac tissues were lysed and treated for quantitative analysis of leukotrienes B4 (A), nuclear factor NF-kappa-B p65 subunit (B), NLRP3 inflammasome (C), MYD88 (D), and 12 pro and anti-inflammatory cytokines (E). Error bars depict means ± SD. ** p < 0.01; * p < 0.05.; ns = not significant.