Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

Abstract

Cancer cells can evade host immune systems via multiple mechanisms. Transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that induces regulatory T cell (Tregs) differentiation and is involved in immune evasion mechanisms in cancer. The inhibition of the TGF-β1 signaling pathway can suppress cancer progression and metastasis through the modulation of anticancer immune responses. However, to best of our knowledge, no implementation of treatments targeting TGF-β1 has been reported in dog cancers. This study aimed to examine whether TGF-β1 is upregulated in canine cancers. We measured TGF-β1 concentrations in culture supernatants of canine melanoma cell lines and in serum samples from dogs with oral malignant melanoma. TGF-β1 production was observed in several cell lines, and serum TGF-β1 levels were elevated in dogs with oral malignant melanoma. Interestingly, the addition of recombinant TGF-β1 to canine peripheral blood mononuclear cell cultures decreased Th1 cytokine production and increased differentiation of CD4⁺CD25⁺Foxp3⁺ lymphocytes, suggesting that TGF-β1 is immunosuppressive in canine immune systems. We developed a decoy receptor for TGF-β, namely TGF-βRII-Ig, by identifying an open reading frame of the canine TGFBR2 gene. TGF-βRII-Ig was prepared as a recombinant fusion protein of the extracellular region of canine TGF-βRII and the Fc region of canine IgG-B. As expected, TGF-βRII-Ig bound to TGF-β1. In the presence of TGF-β1, the treatment with TGF-βRII-Ig increased Th1 cytokine production and decreased the differentiation of CD4⁺CD25⁺Foxp3⁺ lymphocytes. Our results suggest that TGF-βRII-Ig competitively inhibits the immunosuppressive effects of TGF-β1 and thereby activates immune responses. This study demonstrated the potential of TGF-βRII-Ig as a novel biologic for canine melanoma.

Keywords: TGF-β1; biologic; cancer immunotherapy; canine; immunosuppression; melanoma.

Figure 1

TGF-β1 expression in canine cancers. (A) TGF-β1 production by canine cancer cell lines. TGF-β1 concentration in 10% FBS RPMI 1640 is shown as the background (a dashed line). One-way ANOVA with post-hoc Dunnett's-test was used for statistical analysis under the assumption of equal variance between samples (*p <0.05 vs. 10% FBS RPMI 1640). (B) TGF-β1 serum levels in dogs with oral malignant melanoma. The red bar shows the median value. The Mann-Whitney U-test was used for statistical analysis.

Figure 2

Effect of TGF-β1 on Th1 cytokine production and Tregs differentiation in PBMC cultures. (A) IL-2, (B) IFN-γ, and (C) TNF-α concentrations in cell culture supernatant were measured by ELISA. (D) The percentage of CD25⁺Foxp3⁺ cells among CD4⁺ lymphocytes was measured by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis (n = 6, *p <0.05).

Figure 3

Identification of canine TGFBR2 gene. (A) Multiple alignment of TGF-βRII. The deduced amino acid sequence of canine TGFBR2 gene was aligned with those of other species. (B) The phylogenetic tree of canine TGF-βRII in relation to those of other species. GenBank accession numbers were listed beside the scientific names. The scale bar indicates the branch length.

Figure 4

Preparation of recombinant TGF-βRII-Ig and Control IgG-B. (A) The amino acid sequences of TGF-βRII-Ig. The predicted signal peptide of TGF-βRII, extracellular region, and canine IgG-B Fc region are marked with a dashed line, underline, and double underline, respectively. (B) The amino acid sequences of Control IgG-B. The signal peptide of the canine antibody light chain and canine IgG-B Fc region are marked with a dashed line and double underline, respectively. (C) SDS-PAGE of the purified Ig proteins. Analysis in reducing (left) and non-reducing (right) conditions. Arrowheads indicate the protein bands with expected molecular weights. (D) Binding of TGF-βRII-Ig to TGF-β1. Each dot represents the average of absorbance obtained from three independent experiment. Error bars represent the standard deviation. One-way ANOVA with post-hoc Tukey's-test was used for statistical analysis (*p <0.05).

Figure 5

Effect of TGF-βRII-Ig on Th1 cytokine production and Tregs differentiation of in PBMC cultures in the presence of TGF-β1. (A) IL-2, (B) IFN-γ, and (C) TNF-α in cell culture supernatants were measured by ELISA. (D) The percentage of CD25⁺Foxp3⁺ cells among CD4⁺ lymphocytes was calculated by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis (n = 6, *p <0.05).