Systemic Agonistic Anti-CD40 Treatment of Tumor-Bearing Mice Modulates Hepatic Myeloid-Suppressive Cells and Causes Immune-Mediated Liver Damage

Abstract

Immune-stimulatory mAbs are currently being evaluated as antitumor agents. Although overall toxicity from these agents appears to be moderate, liver toxicities have been reported and are not completely understood. We studied the effect of systemic CD40 antibody treatment on myeloid cells in the spleen and liver. Naïve and tumor-bearing mice were treated systemically with agonistic anti-CD40 antibody. Immune cell subsets in the liver and spleen, serum transaminases, and liver histologies were analyzed after antibody administration. Nox2(-/-), Cd40(-/-), and bone marrow chimeric mice were used to study the mechanism by which agonistic anti-CD40 mediates its effects in vivo. Suppressor function of murine and human tumor-induced myeloid-derived suppressor cells (MDSC) was studied upon CD40 ligation. Agonistic CD40 antibody caused liver damage within 24 hours after injection in two unrelated tumor models and mice strains. Using bone marrow chimeras, we demonstrate that CD40 antibody-induced hepatitis in tumor-bearing mice was dependent on the presence of CD40-expressing hematopoietic cells. Agonistic CD40 ligation-dependent liver damage was induced by the generation of reactive oxygen species. Furthermore, agonistic CD40 antibody resulted in increased CD80-positive and CD40-positive liver CD11b(+)Gr-1(+) immature myeloid cells. CD40 ligation on tumor-induced murine and human CD14(+)HLA-DR(low) peripheral blood mononuclear cells from patients with cancer reduced their immune suppressor function. Collectively, agonistic CD40 antibody treatment activated tumor-induced myeloid cells, caused myeloid-dependent hepatotoxicity, and ameliorated the suppressor function of murine and human MDSC. Collectively, our data suggest that CD40 may mature immunosuppressive myeloid cells and thereby cause liver damage in mice with an accumulation of tumor-induced hepatic MDSC.

Figure 1. Systemic agonistic anti-CD40 induces immune-mediated liver damage in tumor-bearing mice

Tumor-free (TF) and tumor-bearing (TB) mice received i.p. either 100 μg agonistic CD40 antibody (CD40 Ab) or control IgG (IgG). (A) Serum ALT and AST levels in C57BL/6 TF and EL4 TB (n=6–8 mice/group) were determined 24 hours after antibody injection. Cumulative data expressed as mean ± SEM, representative of 3 independent experiments. (B) Serum ALT and AST levels in BALB/c TF (n=4 mice/group) and 4T1 TB (n=8 mice/group) were determined 24 hours after antibody injection. Cumulative data of two independent experiments are expressed as mean ± SEM. Representative hematoxilin and eosin staining of liver sections from EL4 TB mice 24 hours after IgG (C) or CD40 Ab injection (D). Images show a 20× magnification and yellow bar = 0.2mm. TB bone marrow chimeric mice (donor→recipient) received i.p. either CD40 Ab or IgG (n=4 mice/group). Serum ALT and AST (E) levels were measured 24 hours after antibody injection. Cumulative data expressed as mean ± SEM, representative of 2 independent experiments. n.s., non significant, *P<0.05, ** P<0.01, ***P<0.005: Student’s t test, (E) one-way ANOVA. TF: Tumor-free; TB: Tumor-bearing; IgG: control IgG: CD40 Ab: agonistic anti-CD40.

Figure 2. Agonistic CD40 antibody exacerbates liver inflammation via oxidative stress

TB WT and Nox2^−/− mice (n=4 mice/group) were injected either with CD40 Ab or IgG. Serum ALT and AST levels (A) were measured 24 hours after injection. Cumulative data shown as mean ± SEM are representative of 2 independent experiments. Representative hematoxilin and eosin staining of liver sections from WT TB (B) or Nox2^−/− TB (C) 24 hours after systemic CD40 Ab injection. Images show a 60× magnification and yellow bar = 0.1mm. (D) TF and EL4 TB mice received i.p. either 100 μg agonistic CD40 Ab or control IgG (n=2 mice/group). Mean Fluorescence Intensity (MFI) of DCFDA gated on hepatic CD11b⁺Gr-1⁺ cells was used to quantify ROS production 3 hours after treatment. Data shown as mean ± SEM are representative of 3 independent experiments. (E) Luminescence intensity by luciferase-expressing RIL-175 cells cultured with or without hepatic CD11b⁺ cells derived from EL4 TB mice 3 hours after injection of either IgG or anti-CD40 agonist (n=2 mice/group). 100 U/ml catalase was used to block ROS production and 2 mM H₂O₂ was set as positive control. Data presented here are expressed as mean ± SEM, representative of two independent experiments. n.s. non significant; *P<0.05, ** P<0.01, *** P<0.005; (A,D) Student’s t test, (E) one-way ANOVA.

Figure 3. Systemic anti-CD40 modulates tumor-induced suppressive myeloid cells

C57BL/6 TF and EL4 TB mice (n=4–8 mice/group) injected i.p. either with CD40 Ab or IgG. (A–C) Absolute number of hepatic CD11b⁺Gr-1⁺ cells (A) expressing CD40 (B) and CD80 (C) 24 hours after injection is shown. (D–F) Absolute number of splenic CD11b⁺Gr-1⁺ cells (D) expressing CD40 (E) and CD80 (F). (A–F) Data expressed as mean ± SEM, representative of 3 independent experiments. 1×10⁶ EL4- induced liver CD11b⁺ cells (n=3/mice) were cultured in the presence or absence of 0.1μg CD40 Ab. Arginase activity (G) determined by urea release in the cell lysates was measured with a colorimetric assay 16 hours after incubation. (H) 1×10⁵ EL4- induced liver CD11b⁺ cells (n=3/mice) were cultured in the presence or absence of 0.1μg CD40 Ab. After 24 hours cells were washed with PBS and incubated overnight with 1×10⁵ sorted CD8⁺ OT-I OVA-specific T cells in the presence of 0.1 μg/ml OVA. Intracellular IFN-γ production by CD8⁺ T cells is shown. Data shown as mean ± SEM are representative of 2 independent experiments. 1×10⁶ EL4- induced liver CD11b⁺ cells (n=3/mice) were cultured in the presence or absence of 0.1 μg CD40 Ab. (I) TNF-α from the cell culture supernatants was determined by ELISA. Data shown as mean ± SEM are representative of 2 independent experiments. n.s. non significant, *P<0.05, ** P<0.01, *** P<0.005: (A–G, I) Student’s t test, (H) one-way ANOVA.

Figure 4. Tumor-induced myeloid cells enhance liver inflammation in vivo upon systemic agonistic anti-CD40 treatment

5×10⁷ B16 GMCSF-induced WT liver CD45.1⁺CD11b⁺ cells (n=3 mice) were injected i.v. into TF CD45.2⁺ Cd40^−/− mice (n=6–8 mice/group). Then either CD40 Ab or IgG were injected i.p. TF WT and Cd40^−/− mice received CD40 Ab as control (n= 3mice/group). Serum ALT and AST (A) levels as well as serum TNF-α (B) were measured 16 hours after injection. (A) Cumulative data shown as mean ± SEM are representative of 2 independent experiments. (B) Data shown as mean ± SEM are representative of 2 independent experiments. (C) 5×10⁷ B16 GMCSF-induced WT or Nox2^−/− liver CD11b⁺ cells (n=2 mice/group) were injected i.v. into TF CD45.2⁺ Cd40^−/− mice (n=6–7 mice/group). Then either CD40 Ab or IgG were injected i.p. TF Cd40^−/− mice received CD40 Ab as control (n= 2 mice). Serum ALT levels were measured 16 hours after injection. Cumulative data shown as mean ± SEM are representative of 2 independent experiments. n.s. non significant, *P<0.05: one-way ANOVA.

Figure 5. CD40 ligation impairs suppressor function in human CD14⁺HLA-DR^low MDSC

1×10⁵ sorted CD14⁺HLA-DR^high or CD14⁺HLA-DR^low cells either from healthy (A) or cancer patients (B) were incubated overnight in the presence or absence of human CD40L trimer. Results show the fold change induction in Arginase-1 (ARG1) mRNA expression. Data expressed as mean ± SEM are representative of 2 independent experiments. (C) CD14⁺HLA-DR^low MDSC were incubated at different ratios with CD8⁺ T cells isolated from the remaining CD14⁻ fraction, stimulated with anti-CD3/CD28 and in the presence or absence of human CD40L trimer for 48 hours. Proliferation was assessed by thymidine incorporation. Data expressed as mean ± SEM, representative of 2 independent experiments. (D) 1×10⁷ isolated PBMC from healthy donors (n=5) were cultured in complete RPMI medium in the presence or absence of megaCD40L for 2 hours. ROS production was quantified by measuring DCFDA mean fluorescence intensity (MFI) either on CD14⁺HLA-DR^high or CD14⁺HLA-DR^low cells. Cumulative data are representative of two independent experiments. n.s. non significant, *P<0.05, ** P<0.01, *** P<0.005; (A–C) Student’s t test, (D) two-way ANOVA.