Co-Targeting PD-1 and IL-33/ST2 Pathways for Enhanced Acquired Anti-Tumor Immunity in Breast Cancer

Abstract

Despite advances in immunotherapy, the treatment of breast cancer still remains a major global problem. In a previous study, we showed that co-blockade of Interleukin-33/ST2 and Programmed death-1/Programmed death-ligand (PD-1/PD-L) signaling pathways strongly slows progression by enhancing the antitumor capacity of natural killer (NK) cells. The main aim of this study is to elucidate the exact effect of co-blockade on the T lymphocyte and macrophage effector cells. 4T1 cells were used to induct breast cancer in female BALB/C and BALB/C ST2^-/- mice. The mice, both BALB/C and BALB/C ST2^-/-, were treated with anti-PD-1 antibody on certain days. After the mice were sacrificed, T cells and macrophages were analyzed using flow cytometry; dual co-blockade increased significantly the percentage of M1 macrophages in the tumor microenvironment, followed by an increase in expression of CD86⁺ and TNFα⁺. T cell accumulation was significantly higher in the spleen and within the tumor microenvironment, with elevation in activation markers such as Interleukin-17, CD69, NKG2D, and FasL and a decrease in Interleukin-10 and FoxP3 expression. Co-blockade of the PD-1/PD-L axes and IL-33/ST2 axes shows promising results in reestablishing an effective immune response and offers a new perspective on improving immune response to breast carcinoma.

Keywords: IL-33; T cells; anti-PD-1; breast cancer; macrophages.

Figure 1

Dual blockade of the PD-1/PD-L and IL-33/ST2 axes does not alter the percentage of spleen-derived macrophages. The graphs and representative plots representing the percentage of F4/80⁺ cells (A) and expression of TNFα in F4/80⁺ cells (B) derived from the spleens of WT (BALB/C), ST2^−/− (BALB/C ST2 knockout), anti-PD-1-treated WT (WT + PD-1) and anti-PD-1-treated ST2^−/− (ST2^−/− + PD-1) mice. Data are presented as average ± SEM (n = 7 mice per group). Statistical significance was tested via the Kruskal–Wallis test and a post hoc Mann–Whitney Rank Sum test.

Figure 2

Dual blockade of the PD-1/PD-L and IL-33/ST2 axes enhances the accumulation of M1 macrophages within the tumor microenvironment. The graphs and representative plots represent the percentage of F4/80⁺ cells (A), CD86⁺F4/80⁺ cells (B) and expression of TNFα in F4/80⁺ cells (C) derived from primary breast tumor of WT (BALB/C), ST2^−/− (BALB/C ST2 knockout), anti-PD-1-treated WT (WT + PD-1), and anti-PD-1-treated ST2^−/− (ST2^−/− + PD-1) mice. The upper panel contains a fact plot with a gating strategy for mononuclear cells (leukocytes). Data are presented as average ± SEM (n = 7 mice per group). Statistical significance was tested via the Kruskal–Wallis test and post hoc Mann–Whitney Rank Sum test. * p < 0.05.

Figure 3

Anti-PD-1 treatment increases the percentage of T cells and the expression of activation molecules in the spleens of ST2^−/− mice. The graphs and representative plots represent the percentage of CD3⁺CD49b⁻ cells (A), NKG2D⁺CD3⁺CD49b⁻ cells (B), CD107a⁺ CD3⁺CD49b⁻ cells (C), and IL-17⁺ CD3⁺CD49b⁻ cells (D) derived from spleens of WT (BALB/C), ST2^−/− (BALB/C ST2 knockout), anti-PD-1-treated WT (WT + PD-1), and anti-PD-1-treated ST2^−/− (ST2^−/− + PD-1) mice. Data are presented as average ± SEM (n = 7 mice per group). Statistical significance was tested by Kruskal–Wallis test and post hoc Mann–Whitney Rank Sum test.* p < 0.05; **p < 0.01.

Figure 4

Pro-tumoricidal phenotypes of T cells in the tumor microenvironment of anti-PD-1-treated ST2^−/− mice. The graphs and representative plots represent the percentage of CD3⁺CD49b⁻ cells (A), IL-10⁺CD3⁺CD49b⁻ cells (B), FoxP3⁺CD3⁺CD49b⁻ cells (C), CD69⁺CD3⁺CD49b⁻ cells (D), NKG2D⁺CD49b⁻ cells (E), and FasL⁺CD3⁺CD49b⁻ cells (F) derived from primary tumors of WT (BALB/C), ST2^−/− (BALB/C ST2 knockout), anti-PD-1-treated WT (WT + PD-1), and anti-PD-1-treated ST2^−/− (ST2^−/− + PD-1) mice. Data are presented as average ± SEM (n = 7 mice per group). Statistical significance was tested by Kruskal–Wallis test and post hoc Mann–Whitney Rank Sum test. * p < 0.05.

Figure 5

Dual blockade stimulates acquired anti-tumor immunity by boosting T cells’ activity and macrophage polarization towards the M1 phenotype, which in turn can also help to stimulate overall T cell activity, leading to more efficient anti-tumor response.