3-Methylcholanthrene-induced transforming growth factor-beta-producing carcinomas, but not sarcomas, are refractory to regulatory T cell-depletion therapy

Abstract

Regulatory T cells (Tregs) are major immunosuppressors in tumor-bearing hosts. Although Treg-depletion therapy has been shown to induce a complete cure in tumor-bearing mice, this treatment is not always successful. Using 3-methylcholanthrene-induced primary mouse tumors, we examined the distinct regulation of Treg-mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)-bearing mice compared with sarcoma-bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)-beta, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg-depletion therapy by treatment with anti-CD25 mAb. The refractoriness of SCC against Treg-depletion therapy was due to the rapid recovery of Tregs in SCC-bearing mice compared with sarcoma-bearing mice. However, combination treatment of anti-TGF-beta mAb with anti-CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC-bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg-depletion therapy using anti-CD25 mAb treatment. We also proposed a novel Treg-blocking combination therapy using anti-CD25 mAb and anti-TGF-beta mAb to induce a complete cure of tumor-bearing hosts.

Figure 1

Regulatory T cells (Tregs) were increased in draining lymph node (DLN), but not distal LN or spleen, in squamous cell carcinoma (SCC)‐bearing host mice. (A) CMC‐1 (2 × 10⁶; representative SCC) was inoculated intradermally. The numbers of CD4 ⁺ Foxp3 ⁺ T cells in tumor‐side axillary DLN (filled circle), contralateral axillary LN (distal LN; open circle), and spleen (filled triangle) was analyzed 0, 4, 8, 12, and 16 days after CMC‐1 inoculation. The data shown are mean ± SD of three mice in each experimental group. *P < 0.05 DLN versus distal LN. Similar results were obtained in two separate experiments. (B) Tregs were detected in LN and spleen of normal mice, and DLN, distal LN, and spleen of tumor‐bearing mice by immunohistochemical analysis. Representative data are shown. Green, Foxp3; red, CD4. Magnification, ×200.

Figure 2

The number of regulatory T cells (Tregs) in draining lymph node (DLN) increases in 3‐methylcholanthrene (MCA)‐induced squamous cell carcinoma (SCC)‐bearing mice, but not in sarcoma‐bearing mice. (A) BALB/c mice were inoculated intradermally with primary sarcoma lines (CMS‐G2 and CMS‐G4; 2 × 10⁶) and primary SCC lines (CMC‐1 and CMC‐9; 2 × 10⁶). DLN of the tumor‐bearing mice were immunostained with anti‐CD4 mAb and anti‐Foxp3 mAb. Distributions of CD4 ⁺ Foxp3 ⁺ Tregs in the tumor DLN were evaluated by immunohistochemical observation. Green, Foxp3; red, CD4. Magnification, ×200. (B) The numbers of Foxp3 ⁺ Tregs of the sections were counted. The data shown are mean ± SD of five fields of each DLN. *P < 0.02 sarcoma versus SCC.

Figure 3

Squamous cell carcinomas (SCCs) predominantly induce regulatory T cells (Tregs) by their ability to produce high levels of active transforming growth factor (TGF)‐β. (A) CMC‐1 (SCC; 2 × 10⁶) was inoculated intradermally into BALB/c mice (day 0). Anti‐TGF‐β mAb (500 μg/mL) was i.p. injected at days 0, 3, 6, and 9. At day 10, we evaluated the number of Tregs by FACSCalibur. The number was calculated as follows: total cell number × rate of Tregs among total cells. The data shown are the mean ± SD of three mice. Similar results were obtained in three separate experiments. (B) Draining lymph nodes of mice treated with anti‐TGF‐β or control IgG mAb were immunostained with anti‐CD4 mAb and anti‐Foxp3 mAb 13 days after tumor inoculation. (C) TGF‐β activities in the culture supernatants of six kinds of sarcoma lines and eight kinds of SCC lines were determined by growth inhibition assay of Mv1Lu cells. We defined the inhibition rate (%) as an inhibition index (II) of active TGF‐β. Similar results were obtained in three separate experiments. *P < 0.006 between the two indicated groups.

Figure 4

3‐Methylcholanthrene‐induced carcinomas, not sarcomas, are resistant to anti‐CD25 mAb treatment. Five kinds of primary sarcoma (A) or squamous cell carcinoma (B) cell lines (2 × 10⁶) were intradermally inoculated into BALB/c mice pre‐treated with control mAb or anti‐CD25 mAb (50 μg/mouse). Effects of the regulatory T cell depletion were evaluated by monitoring subsequent tumor growth. Similar results were obtained in two separate experiments.

Figure 5

Number of regulatory T cells rapidly restored in non‐rejected tumor‐bearing mice treated with anti‐CD25 mAb. CMS‐G4 (sarcoma; 2 × 10⁶) and CMC‐1 (carcinoma; 2 × 10⁶ or 2 × 10⁵) cells were intradermally inoculated into mice pre‐treated with anti‐CD25 mAb. Seven days after tumor inoculation, we evaluated absolute numbers of CD4 ⁺ Foxp3 ⁺ T cells in draining lymph nodes (DLNs) of the tumor‐bearing mice by flow cytometric analysis. As controls (Cont), we analyzed tumor‐free mice treated with control IgG or anti‐CD25 mAb. The data shown are the mean ± SD of four mice. Similar results were obtained in three separate experiments. –, tumor free or not applicable. *P < 0.03 versus CMS‐G4‐bearing mice group.

Figure 6

Inhibiting regulatory T cell recovery by treatment with anti‐transforming growth factor (TGF)‐β mAb leads to complete rejection of carcinoma. CMC‐1 (2 × 10⁵) cells were inoculated at day 0 into BALB/c mice pre‐treated with control IgG of PC61 (open circle, open triangle) or anti‐CD25 mAb (PC61; filled circle, filled triangle). Anti‐TGF‐β mAb (1D11; open triangle, filled circle) or control IgG of 1D11 (open circle, filled triangle) was injected i.p. six times at 2‐day intervals. Effects of the anti‐CD25 mAb and anti‐TGF‐β treatment were evaluated by monitoring subsequent tumor growth. The data shown are the mean ± SD of four mice. Photographs are representative at day 14. Similar results were obtained in three separate experiments.