Cetuximab and IL-15 Promote NK and Dendritic Cell Activation In Vitro in Triple Negative Breast Cancer

Abstract

Triple Negative Breast Cancer (TNBC) treatment is still challenging, and immunotherapy is a potential approach in this tumor subtype. Cetuximab is an IgG1 monoclonal antibody (mAb) directed against Epidermic Growth Factor Receptor (EGFR), a protein overexpressed in a subgroup of TNBC patients and associated with poor prognosis. Previously, we demonstrated in vitro that Cetuximab triggers Ab-dependent cell cytotoxicity against TNBC cells. In this study, using co-cultures including TNBC cells, and NK and Dendritic Cells (DCs) from healthy donors, we studied the effect of Cetuximab-activated NK cells on DC function. Given that we already demonstrated that TNBC has an immunosuppressive effect on NK cells, we also tested Cetuximab combination with IL-15. We determined that Cetuximab opsonization of TNBC cells increased IFN-γ and TNF-α production by NK cells co-cultured with DCs. Moreover, we showed that NK cells activated by TNBC cells opsonized with Cetuximab promoted tumor material uptake and maturation of DCs, as well as their ability to produce IL-12. Furthermore, the stimulation with IL-15 increased the activation of NK cells and the maturation of DCs. These results suggest that IL-15 may enhance the efficacy of Cetuximab in the treatment of TNBC by promoting activation of both NK cells and DCs.

Keywords: ADCC; Cetuximab; NK cells; dendritic cells; triple negative breast cancer.

Figure 1

NK cells promoted DC uptake of TNBC cells opsonized with Cetuximab. Cytotoxicity of IIB-BR-G cells (left) and their uptake by DCs (right) after a co-culture of DCs with IIB-BR-G cells opsonized with 10 µg/mL isotype control (IC) or Cetuximab in the absence (DCs) or presence of NK cells (DCs+NK) at a DCs:NK:IIB-BR-G ratio of 1:1:1 for 4 h at 37 °C. (A) Upper panel: simplified gating strategy for CFSE⁺ IIB-BR-G cells (left) and CD11c⁺ DCs (right). Lower panel: for the different experimental conditions, representative dot plots showing tumor cell lysis (% of 7AAD⁺ cells within CFSE⁺ tumor cells) are presented in the left, and tumor cell uptake by DCs (% of CFSE⁺ cells within the CD11c⁺ DCs) in the right. (B) Bars with different letters are statistically different, p < 0.05 (ANOVA) (n = 3).

Figure 2

NK cell activation and IFN-γ and TNF-α production were enhanced by Cetuximab opsonization of IIB-BR-G cells. NK cells were co-cultured with IIB-BR-G cells opsonized with IC or Cetuximab, in the absence (NK) or presence of DCs (DCs+NK), in a DCs:NK:IIB-BR-G ratio of 1:1:1 for 24 h. (A) Representative histograms showing CD25, CD69 and HLA-DR expression in NK cells after co-cultures in the presence of DCs. (B) Expression of CD25, CD69 and HLA-DR in NK cells (n = 3–6). Bars with different letters are statistically different, p < 0.05 (ANOVA). (C) IFN-γ and TNF-α concentration in the co-culture supernatants (n = 3–4).

Figure 3

NK cells promoted DC maturation when IIB-BR-G cells were coated with Cetuximab. DCs were co-cultured with IIB-BR-G opsonized with IC or Cetuximab, in the absence (DCs) or presence of NK cells (DCs+NK), in a DCs:NK:IIB-BR-G ratio of 1:1:1 for 24 h. (A) Representative histograms showing CD83 and CD86 expression in DCs after co-cultures in the presence of NK cells. (B) Expression of CD83, CD86, CD80 and HLA-DR in DCs. Bars with different letters are statistically different, p < 0.05 (ANOVA) (n = 3–6). nMFI: normalized geometric mean fluorescence intensity.

Figure 4

NK cells promoted IL-12 production by DCs when TNBC cells were coated with Cetuximab. IL-12p70 concentration in the 24-h co-culture supernatant of cells transfected with CD40L and DCs, which were first stimulated with IIB-BR-G cells opsonized with IC or Cetuximab, in the absence (DCs) or presence of NK cells (DCs+NK) for 24 h (n = 2).

Figure 5

IL-15 increased NK cell activation and DC maturation triggered by Cetuximab. DCs were co-cultured with NK cells and IIB-BR-G cells opsonized with IC or Cetuximab in the absence (DCs+NK) or presence of 10 ng/mL IL-15 (DCs+NK+IL-15) for 24 h. (A) Expression of CD25 and CD69 in NK cells (n = 4). (B) Concentration of IFN-γ and TNF-α in the co-culture supernatant (n = 3–4). (C) Expression of CD83 and CD86 in DCs (n = 6). Bars with different letters are statistically different, p < 0.05 (ANOVA).