PMC

(A) Stage III melanoma TGFBR3 expression levels were determined by microarray analysis. Complete response rates to ILI therapy by patients with the highest 20% TGFBR3 expression (TGFBR3^hi) and lowest 20% expression (TGFBR3^lo) are shown. 103 patients. (B) sTGFBR3 plasma levels in nonresponding (NR) versus responding (R) stage III melanoma patients. 52 patients. (C) Multivariate survival analysis of stage III melanoma patients according to sTGFBR3 plasma levels. Kaplan-Meier survival curve. Data are mean ± SEM. *P < 0.05, ***P < 0.0005, Spearman correlation calculation (A), Mann-Whitney U test (B), multivariate analysis (C).

(A) In vivo growth of 4T1-NEO and 4T1-TGFBR3 primary tumors in NSG and WT hosts. 15 mice/group. Red, 4T1-NEO WT; blue, 4T1-TGFBR3 WT; green, 4T1-NEO NSG; purple, 4T1-TGFBR3 NSG. (B) 4T1-NEO/4T1-TGFBR3 tumor burden ratios, based on in vivo bioluminescence monitoring. Representative images are also shown. (C) 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 tumor incidence and volume in NSG and WT hosts. 6 mice/group. Representative of 3 individual experiments. (D) CD3 IHC of 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 tumors. 10 random ×40 fields/condition (representative ×40 images are also shown). Representative of 2 independent experiments. (E) TUNEL analysis of 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 tumors resected from WT and NSG hosts. 10 random ×40 fields/condition. Representative of 2 independent experiments. (F) Annexin-V/PI flow cytometry analysis of resected 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 tumors from WT hosts. Data are mean ± SEM. *P < 0.05, **P < 0.005, 2-tailed Student’s t test.

(A) Ccl22 qRT-PCR in the indicated tumor and TDLN tissues. 3–5 tumors/group. Representative of 3 independent experiments. (B) Ccl22 qRT-PCR in 4T1-sTGFBR3_Tet tumors in the presence and absence of doxycycline. Representative of 2 independent experiments. (C) Ccl22 qRT-PCR in 66CL4-TGFBR3-KD and 66CL4-NTC tumors. Representative of 3 independent experiments. (D) CCL22 IHC of 4T1-NEO and 4T1-TGFBR3 TDLNs. 10 ×40 fields over 3 TDLNs/condition. Representative of 2 independent experiments. (E) Ccl22 qRT-PCR of TGF-β–treated bone marrow–derived DCs. IL-4 served as a positive control. Representative of 2 independent experiments. (F) CCL22 immunoprecipitation and Western blot of TGF-β–treated bone marrow–derived DCs. UT, untreated. Representative of 3 independent experiments. (G) CCL22 ELISA of CM after TGF-β treatment of bone marrow–derived DCs. Performed in duplicate. Representative of 2 independent experiments. Data are mean ± SEM. *P < 0.05, **P < 0.005, 2-tailed Student’s t test.

(A) SMAD2 and CCL22 Western blot analysis of TGF-β–treated bone marrow–derived DCs isolated from WT and Cd11c^dnTGFBR2 mice. IL-4 served as a positive control. t-, total. (B) pSMAD2 and IDO Western blot of TGF-β–treated (T; 20 pM) and untreated (U) splenic PDCA-1⁺ pDCs, splenic CD11c⁺PDCA-1^– DCs, and CD8⁺ T cells isolated from WT and Cd11c^dnTGFBR2 mice. Representative of 2 independent experiments. (C) B16-mOVA tumor volume in WT and Cd11c^dnTGFBR2 mice. 8–9 mice/group. Representative of 2 independent experiments. (D) Myc and Ido qRT-PCR of TDLN pDCs isolated from WT and Cd11c^dnTGFBR2 mice. 3 mice/group. Pooled from 2 independent experiments. (E) Foxp3 and Cd8 qRT-PCR of B16-mOVA tumors resected from WT and Cd11c^dnTGFBR2 mice. 4 tumors/group. Representative of 2 independent experiments. (F) CD8 and FOXP3 IHC of B16-mOVA tumors resected from WT and Cd11c^dnTGFBR2 mice. 20 random ×40 fields/group (representative ×40 images are also shown). Representative of 2 independent experiments. (G) K^b-OVA_257–264–specific CD8⁺ T cell tetramer analysis of splenocytes in B16-mOVA tumor-bearing WT and Cd11c^dnTGFBR2 mice. 5 mice/group. Data are mean ± SEM. *P < 0.05, **P < 0.005, 1-way ANOVA (C), 2-tailed Student’s t test (D–G).

(A) Ido qRT-PCR analysis of 4T1-NEO and 4T1-TGFBR3 tumors and TDLNs. (B) IDO immunofluorescence of TDLNs. Representative ×20 images are also shown. (C) TDLN pDC Ido qRT-PCR. Pooled from 3 independent experiments. (D) TDLN pDC IDO immunofluorescence. Representative ×100 images of pDCs isolated in 2 independent experiments are also shown. Isotype controls showed no staining. (E) Ido qRT-PCR analysis of 4T1-NEO and 4T1-TGFBR3 TDLNs after 2G7 treatment. 6 mice/group. (F) In-cell Western blot (left; pooled from 3 independent experiments) and traditional Western blot (right; representative of 2 independent experiments) of pDC IDO expression in the presence or absence of 4T1-NEO or 4T1-TGFBR3 CM. IFN-γ served as a positive control. (G) pDC-derived IDO enzymatic activity, measured after coincubation with 4T1-NEO or 4T1-TGFBR3 CM. sTGFBR3 served as a positive control. Pooled from 3 independent experiments. (H) Whole 4T1-sTGFBR3_Tet TDLN IDO enzymatic assay with or without doxycycline. (I) 4T1-NEO and 4T1-TGFBR3 TDLN-derived pDC mixed lymphocyte proliferation assay. Representative of 2 independent assays. Data are mean ± SEM. *P < 0.05, **P < 0.005, 2-tailed Student’s t test.

(A) Cd8 qRT-PCR and CD3⁺CD8⁺ T cell flow analysis of 4T1-NEO and 4T1-TGFBR3 tumors. (B) Foxp3 qRT-PCR and CD4⁺FOXP3⁺ Treg flow analysis of 4T1-NEO and 4T1-TGFBR3 tumors. (A and B) qRT-PCR, 5 tumors/group, data representative of 3 independent experiments; flow analysis, 9 tumors/group, data pooled from 3 independent experiments. (C) Cd8 qRT-PCR and CD3⁺CD8⁺ T cell flow analysis of 4T1-NEO and 4T1-TGFBR3 TDLNs. 8 mice/group. Pooled from 2 independent experiments. (D) Foxp3 qRT-PCR and FOXP3⁺ IHC of 4T1-NEO and 4T1-TGFBR3 TDLNs. qRT-PCR, 8 mice/group, data pooled from 2 independent experiments; IHC, 5 random ×40 fields/condition, data representative of 2 independent experiments. (E) Allogeneic T cell proliferation assays using distant LN and TDLN tissues resected from 4T1-NEO and 4T1-TGFBR3 tumor-bearing mice. 5 mice/group. Representative of 2 independent experiments. (F) HER2/NEU-specific autologous T cell proliferation assays using 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 TDLNs. 5 mice/group. Representative of 2 independent experiments. (G) HER2/NEU-specific IFN-γ ELISPOT assay of 4T1-NEO-HER2 and 4T1-TGFBR3-HER2 TDLNs. 5–10 mice/group. Performed in triplicate. Flow cytometry data are expressed as a percentage of viable total tumor or TDLN cell number. Data are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, 2-tailed Student’s t test.

(A) Oncomine microarray expression analysis of TGFBR3, IDO, CCL22, and FOXP3 in human normal mammary tissues (N) relative to human breast cancers (T). Fold changes in gene expression relative to normal mammary tissues are indicated. See Supplemental Table 2. (B) Loss of TGFBR3 by tumor cells enhances TGF-β–mediated upregulation of IDO and CCL22 by local pDC and mDC populations, respectively, resulting in enhanced Treg activation and recruitment. (C) TGF-β signaling inhibition in combination with HER2/NEU vaccination in a HER2/NEU-expressing 4T1 mammary carcinoma model. AdLacZ was used as control vector. 8 mice/group. Representative of 2 independent experiments. (D) IFN-γ ELISPOT analysis of HER2/NEU-specific T cell responses. PMA/ionomycin served as a positive control; HIV peptide mix served as an irrelevant peptide negative control. 3 mice/group. Representative of 2 independent experiments. **P < 0.005, ***P < 0.0005, 2-tailed Student’s t test.

(A) Oncomine microarray TGFBR3 expression analysis in human benign nevi and primary melanoma tissues. See Supplemental Table 2. (B) DNA hybridization blot analysis of TGFBR3 mRNA levels in normal human skin (N) and melanoma tumor tissues (T). (C) TGFBR3 IHC of human benign nevi, primary melanoma, and metastatic melanoma tumor tissues. 100 core tissues were evaluated. Representative ×20 fields are also shown. (D) B16-mOVA-TGFBR3 tumor growth relative to B16-mOVA control (ctrl) and B16/F10 (B16) tumors in syngeneic hosts. 5–6 tumors/condition. Representative of 3 independent experiments. (E) qRT-PCR of Cd8 and Foxp3 in B16-mOVA-TGFBR3 versus B16-mOVA tumors. 3 tumors/condition. Representative of 2 independent experiments. (F) CD8 and FOXP3 IHC of B16-mOVA-TGFBR3, B16-mOVA, and B16/F10 tumors. 10 fields/tumor, 3 tumors/condition. (G) K^b-OVA_257–264–specific CD8⁺ T cell tetramer analysis of resected splenic and TDLN tissues from B16-mOVA-TGFBR3, B16-mOVA, and B16/F10 tumor-bearing mice. 3–6 mice/group. Representative of 2 independent experiments. Data are mean ± SEM. *P < 0.05, 2-tailed Student’s t test (A, E, and F), 1-way ANOVA (C, D, and G).

(A) Left: sTGFBR3 inhibited downstream TGF-β–mediated signaling in tumor cells in an autocrine manner. Right: sTGFBR3 also inhibited TGF-β signaling in local stromal cells in a paracrine manner. (B) Tumor growth measurements with and without TGF-β–targeted blockade. 2G7, anti–pan–TGF-β mAb. 8 mice/group. (C) Immunofluorescence analysis of pSMAD2 in TDLN tissues. 5 random ×10 fields across 3–4 LNs/condition (representative ×10 and ×50 images are also shown). Representative of 2 independent experiments. (D) CD4⁺FOXP3⁺ Treg flow cytometry of TDLN tissues. 4 mice/group. (E) Doxycycline (Dox) treatment was initiated at different times after 4T1-sTGFBR3_Tet tumor implantation, and mice were monitored for tumor progression. (F) Cd8 qRT-PCR analysis of primary 4T1-sTGFBR3_Tet tumors with or without doxycycline. Flow cytometry analysis of activated CD8⁺ T cells in 4T1-sTGFBR3_Tet TDLNs is also shown. (G) Foxp3 qRT-PCR analysis of primary 4T1-sTGFBR3_Tet tumors with or without doxycycline. Flow cytometry analysis of CD4⁺FOXP3⁺ Tregs in 4T1-sTGFBR3_Tet TDLNs is also shown. (H) CD8 and FOXP3 IHC of 4T1-sTGFBR3_Tet tumors. 20 ×40 fields/tumor, 2–3 tumors/group. Flow cytometry data are expressed as a percentage of viable total tumor cell number. Data are mean ± SEM. *P < 0.05, **P < 0.005, 2-tailed Student’s t test (B and E–G), 1-way ANOVA (C and D), Mann-Whitney U test (H).