Tumor rejection by modulation of tumor stromal fibroblasts

Abstract

Interleukin (IL)-4-secreting tumors are rejected in mice, an effect that is thought to be immune mediated. However, solid tumors are embedded in a stroma that often contains tumor-promoting fibroblasts, a cell population whose function is also affected by IL-4. Here we show that IL-4-secreting tumors grew undiminished in IL-4 receptor (R)-deficient (IL-4R-/-) mice. In IL-4R+/+ mice they were long-term suppressed in the absence of T cells but complete rejection required T cells, compatible with the assumption that hematopoietic cells needed to respond to IL-4. Surprisingly, bone marrow (BM) chimeric mice revealed that IL-4R expression exclusively on non-BM-derived cells was sufficient for tumor rejection. Fibroblasts in the tumor stroma were identified as a target cell type for IL-4 because they accumulated in IL-4-secreting tumors and displayed an activated phenotype. Additionally, coinjection of IL-4R+/+ but not IL-4R-/- fibroblasts was sufficient for the rejection of IL-4-secreting tumors in IL-4R-/- mice. Our data demonstrate a novel mechanism by which IL-4 contributes to tumor rejection and show that the targeted modulation of tumor-associated fibroblasts can be sufficient for tumor rejection.

Figure 1.

Biphasic rejection of IL-4–producing tumor cells requires host IL-4R but not IL-4. BALB/c WT (wt; ▵), Rag2^−/− (•), IL-4^−/− (⋄), IL-4R^−/− (▪), and RAG2^−/−/IL-4R^−/− mice (□) were injected with 2 × 10⁶ J558-IL-4 cells and tumor growth was measured. One representative experiment per experimental group (five to six mice per group) is shown. In total, 30 BALB/c (six experiments), 11 RAG2^−/− (two experiments), 17 IL-4^−/− (two experiments), 33 IL-4R^−/− (six experiments), and 11 RAG2^−/−/IL-4R^−/− mice (two experiments) were analyzed. In three of the experiments with J558-IL-4 cells in IL-4R^−/− mice, a group with parental J558L cells in WT mice (n = 13) was included showing no significant difference in growth kinetics between the two groups. All experiments are presented individually in Table SI, available at http://www.jem.org/cgi/content/full/jem.20030849/DC1.

Figure 2.

IL-4R expression on non-BM–derived cells is sufficient for the rejection of J558-IL-4 cells. For the generation of BM chimeras, irradiated (9 Gy) BALB/c WT (wt) mice were reconstituted with BM cells from (C) WT (WT > WT) or (F) IL-4R^−/− mice (IL-4R^−/− > WT). Irradiated IL-4R^−/− mice were reconstituted with BM cells from (D) IL-4R^−/− (IL-4R^−/− > IL-4R^−/−) or (E) WT (WT > IL-4R^−/−) mice. (C–F) 2 × 10⁶ J558-IL-4 cells were injected s.c. in the indicated BM chimeras, (A) untreated WT, and (B) IL-4R^−/− mice. Pooled results from two independent experiments performed 10 and 17 wk after reconstitution are shown. In total, 9–10 mice per group were analyzed. Numbers in the graph indicate the number of mice that rejected the tumor per number of analyzed mice. (A–F, histograms) To analyze whether the peripheral lymphocytes were derived from donor or recipient BM, the up-regulation of MHC class II on splenic B cells in response to IL-4 was tested. Spleen cells from untreated mice of each experimental group were stimulated with 2 ng/ml recombinant IL-4 for 16 h or were left untreated. The expression of MHC class II on B220⁺ cells was determined by flow cytometry. Red lines show MHC II expression on untreated B220⁺ cells and blue lines show MHC II expression after incubation with IL-4. An isotype-matched control antibody was used to verify staining specificity (unpublished data). One representative experiment out of two is shown.

Figure 3.

Fibroblasts in J558-IL-4 tumors of WT mice have an altered phenotype. WT (A and C) and IL-4R^−/− mice (B and D) were injected s.c. with 10⁷ J558-IL-4 (A and B) or J558L cells (C and D) and tumors were removed 6 d later. Tumor tissue sections were stained with a mAb that stains fibroblasts/extracellular matrix (ER-TR7). Arrows indicate staining around lumen-containing structures typical for blood vessels. (E and F) 2 × 10⁶ J558-IL-4 cells were injected s.c. into (E) RAG2^−/− and (F) RAG2^−/−/IL-4R^−/− mice and tumors were removed at 1 cm in diameter. Because the tumors grew with different kinetics in IL-4R–deficient and IL-4R–competent mice (see Fig. 1), they were removed from RAG2^−/−/IL-4R^−/− mice at day 8 and from RAG2^−/− mice at day 30. Tumor tissue sections were stained with a mAb specific for collagen I. Representative sections for four tumors analyzed per group are shown. ×100.

Figure 4.

IL-4 responsiveness of fibroblasts in the tumor is sufficient for rejection of J558-IL-4 cells. 10⁶ lung fibroblasts from (A) WT or (B) IL-4R^−/− mice were mixed with 10⁶ J558-IL-4 cells, injected s.c. into IL-4R^−/− mice, and tumor growth was measured. Combined data from two independent experiments with two to three mice/group are shown.