B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma

Abstract

Tumor-associated macrophages are a prominent component of ovarian cancer stroma and contribute to tumor progression. B7-H4 is a recently identified B7 family molecule. We show that primary ovarian tumor cells express intracellular B7-H4, whereas a fraction of tumor macrophages expresses surface B7-H4. B7-H4+ tumor macrophages, but not primary ovarian tumor cells, suppress tumor-associated antigen-specific T cell immunity. Blocking B7-H4-, but not arginase-, inducible nitric oxide synthase or B7-H1 restored the T cell stimulating capacity of the macrophages and contributes to tumor regression in vivo. Interleukin (IL)-6 and IL-10 are found in high concentrations in the tumor microenvironment. These cytokines stimulate macrophage B7-H4 expression. In contrast, granulocyte/macrophage colony-stimulating factor and IL-4, which are limited in the tumor microenvironment, inhibit B7-H4 expression. Ectopic expression of B7-H4 makes normal macrophages suppressive. Thus, B7-H4+ tumor macrophages constitute a novel suppressor cell population in ovarian cancer. B7-H4 expression represents a critical checkpoint in determining host responses to dysfunctional cytokines in ovarian cancer. Blocking B7-H4 or depleting B7-H4+ tumor macrophages may represent novel strategies to enhance T cell tumor immunity in cancer.

Figure 1.

Ovarian tumor cells express intracellular B7-H4. B7-H4 expression was analyzed by RT-PCR and FACS in (a) fresh primary ovarian tumor cells and (b) ovarian tumor cell lines. FACS results expressed the mean percentage of B7-H4–expressing cells. OC8, OC21, and OC38 are three established ovarian tumor cell lines. Fresh primary ovarian tumor cells were isolated and sorted from ascites and tumor tissues with a phenotype of lin⁻EpCam⁺CD45⁻CD14⁻. Two of six representative patient samples are shown for panel a. Three of eight cell lines are shown for panel b. Filled histogram, B7-H4 expression; open histogram, isotype control.

Figure 2.

Tumor-associated macrophages express B7-H4. (a and b) Tumor ascites macrophages express B7-H4. FACS analysis showed that ascites macrophages, but not control nonmalignant ascites macrophages, or fresh normal blood monocytes expressed B7-H4. Results are expressed as the mean of the percentage of B7-H4⁺ cells ± SEM in total macrophages. Filled histogram, B7-H4 staining; open histogram, isotype control. (c) High prevalence of B7-H4⁺ tumor macrophages in tumor ascites. FACS analysis was used to determine the prevalence of B7-H4⁺ tumor macrophages and CD3⁺CD4⁺CD25⁺ cells in tumor ascites (*, P < 0.001). Results are expressed as the mean of percentage ± SEM in tumor ascites mononuclear cells. (d and e) Tumor tissues were stained with anti–human B7-H4, anti–human CD3, anti–human-Ham56, and control antibody as described in Materials and methods, and analyzed with confocal microscope. (d) Tumor mass macrophages and tumor cells express B7-H4. B7-H4, green; Ham56, red. Tumor macrophages are identified as Ham56⁺ cells (red). Ham56⁺B7-H4⁺ cells are yellowish. Large numbers of tumor macrophages form a barrier surrounding tumor islets. (e) Tumor-infiltrating T cells are B7-H4⁻. Tumor cells, but not CD3⁺ T cells (red), expressed B7-H4 (green). 1 of 60 representative patient samples is shown for panels d and e.

Figure 3.

Tumor ascites IL-6 and IL-10 stimulate macrophage B7-H4 expression. (a) Tumor ascites stimulated macrophage B7-H4 expression. Fresh normal blood monocytes were cultured 72 h with ovarian tumor ascites in the presence or absence of anti–human IL-6 or anti–human IL-10 antibodies. B7-H4 expression was analyzed by FACS. Results are expressed as the mean of B7-H4⁺ cell percentage ± SEM in total macrophages. (b) High concentrations of IL-6 and IL-10 in tumor ascites were detected by ELISA. (c) Recombinant IL-6 or IL-10 stimulated macrophage B7-H4 protein in a dose-dependent manner after a 72 h culture. B7-H4 expression was analyzed by FACS. Results are expressed as the mean of B7-H4⁺ cell percentage ± SEM in total macrophages. (d and e) Recombinant IL-4 and GM-CSF reduced macrophage B7-H4 mRNA and protein induction. Fresh blood monocytes were cultured with different concentrations of cytokines. (d) B7-H4 mRNA was detected by RT-PCR at 24 h. (e) B7-H4 surface protein was detected by FACS at 72 h. One of seven independent experiments is shown.

Figure 4.

Tumor macrophages suppress TAA-specific T cell immunity in vitro. (a–e) Autologous tumor ascites CD3⁺CD25⁻ T cells were stimulated with Her-2/neu peptide-loaded MDC (TAA-MDC) as described in Materials and methods. (a) Tumor T cell proliferation was detected by [³H]thymidine incorporation (CPM). Results are expressed as the mean of cpm ± SEM. TAA-specific T cell IFN-γ (b and c) and IL-2 (b and d) production was detected by intracellular staining (FACS) and ELISA. (e) Tumor macrophages inhibited Her-2/neu–specific T cell cytotoxicity. MDC-activated Her-2/neu–specific T cells were effector cells, and Her-2/neu peptide-loaded T2 cells were target cells. Her-2/neu–specific cytotoxicity was determined by FACS (see Materials and methods). Tumor macrophage to TAA-MDC ratio was 1:1 for panels b–e. (f) B7-H4⁺ tumor macrophages significantly suppress T cell proliferation. B7-H4⁺ or B7-H4⁻ tumor macrophages were sorted from malignant ascites using a FACSaria. Normal macrophages were from M-CSF–treated normal peripheral blood monocytes. Tumor ascites CD3⁺ T cells were stimulated with anti-CD3 antibody and blood monocytes for 3 d in the presence of different concentrations of tumor macrophages or normal macrophages. T cell proliferation was detected by [³H]thymidine incorporation. Results are expressed as the mean of cpm ± SEM. TAM, tumor macrophages; macrophage, MΦ.

Figure 5.

Blockade of B7-H4 improved macrophage-mediated T cell activation. (a and b) B7-H4–blocking oligos reduced macrophage B7-H4 expression. Fresh blood monocytes were exposed to B7-H4–blocking oligos and control oligos with medium, tumor ascites, and IL-10. (a) B7-H4 mRNA was detected at 24 h by RT-PCR with serial dilutions of cDNA. (b) B7-H4 protein was detected at 72 h by FACS. One of nine independent experiments is shown. (c and d) Blockade of B7-H4 improved tumor ascites-conditioned macrophage-mediated T cell activation in vitro. Tumor ascites-conditioned macrophages were previously treated with B7-H4–blocking oligos or control oligos and were subsequently subject to activating T cells for 72 h. (c) T cell proliferation was measured by [³H]thymidine incorporation. Results are expressed as the mean of cpm ± SEM. (d) Cell cycle in T cells was analyzed by FACS. Results are expressed as the mean of the percentage of T cells ± SEM in each cycle phase. The ratio between macrophages and T cells was 1:1 for panel d. n = 4–8; *, P < 0.05 compared with control oligos.

Figure 6.

B7-H4⁺ macrophages mediate an immunosuppression independent of B7-H1, arginase, and iNOS. (a) B7-H1, arginase, and iNOS expression on tumor ascites-associated macrophages. B7-H1 expression was analyzed by FACS. Arginase I and iNOS expression was detected by Western blot. A, B, C, D, and E represent 5 individual patients with ovarian cancer. (b) Tumor ascites-conditioned macrophages mediated T cell suppression through B7-H4. T cells were stimulated with tumor ascites-conditioned macrophages for 72 h in the presence of the indicated conditions. T cell proliferation was measured by [³H]thymidine incorporation in the last 16 h. The results were expressed as the percentage of suppression (mean ± SEM). The ratio between macrophages and T cells was 1:2 for panel d. n = 5; *, P < 0.05.

Figure 7.

Forced B7-H4 expression confers suppressive capacity of normal macrophages. Fresh blood monocytes were transfected using a plasmid encoding human B7-H4 or EGFP cDNA or control plasmid carried the reversed cDNA sequence of B7-H4. (a) 20 h after transfection, B7-H4 and GFP expression were detected by FACS. One of five experiments is shown. (b) Different concentrations of the transfected cells were added into T cell culture in the presence of anti–human CD3 and normal monocytes. T cell proliferation was detected by [³H]thymidine incorporation on day 3. One of five experiments is shown.

Figure 8.

Blockade of B7-H4 improved macrophage-mediated T cell activation in vivo. (a) Blocking tumor ascites-conditioned macrophage B7-H4 results in tumor regression in vivo. Mice were injected with human primary ovarian tumors as described in Materials and methods. Controls received no additional injection (▴, group 1). Treatments were tumor-specific T cells plus B7-H4–blocking oligo-treated macrophages (◯, group 2), and tumor-specific T cells plus control oligo-treated macrophages (▪, group 3). Mean ± SD of tumor volumes is shown (n = 5–7 mice per group). The T cell injection day was counted as day 0. Macrophage, MΦ. (b) Macrophages and T cell were colocalized in tumor tissue. Similar results were observed in group 2 and 3. Macrophages were identified as Ham56⁺ cells (green) and T cells as CD3⁺ cells (red). n = 5.