Regulatory T (T(reg)) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Failure of host antitumor immunity may be caused by exaggerated suppression of tumor-associated antigen-reactive lymphocytes mediated by T(reg) cells; however, definitive evidence that T(reg) cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4(+)CD25(+)FOXP3(+) T(reg) cells in 104 individuals affected with ovarian carcinoma, that human tumor T(reg) cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor T(reg) cells are associated with a high death hazard and reduced survival. Human T(reg) cells preferentially move to and accumulate in tumors and ascites, but rarely enter draining lymph nodes in later cancer stages. Tumor cells and microenvironmental macrophages produce the chemokine CCL22, which mediates trafficking of T(reg) cells to the tumor. This specific recruitment of T(reg) cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking T(reg) cell migration or function may help to defeat human cancer.

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.

IntroductionAdaptive immunity plays a crucial role in tumor immunosurveillance. [1][2][3] It has been shown that tumor-infiltrating effector T cells are associated with improved prognoses in multiple human cancers, 4-6 whereas tumor-infiltrating regulatory T (Treg) cells are negatively associated with patient outcome. 6,7 Th17 cells are newly identified effector CD4 ϩ T cells. Th17 cells and interleukin-17 (IL-17) play an active role in inflammation and autoimmune diseases. [8][9][10][11][12][13][14][15] Th17 cells are found in both mouse and human tumors. 16,17 However, the biologic role of Th17 cells is poorly understood in the tumor microenvironment. In this report, we examined the phenotype, cytokine profile, generation, functional relevance, and immunologic and clinical predictive values of Th17 cells in 201 patients with ovarian cancers. We provide novel insight into the nature of Th17 cells in the tumor microenvironment in patients with cancer. This information may be useful for designing more effective cancer immunotherapies. Methods Human subjectsWe studied previously untreated patients with 201 ovarian carcinomas. Survival data were available for 85 patients (supplemental Table 1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Patients gave written, informed consent in accordance with the Declaration of Helsinki. The study was approved by the University of Michigan Institutional Review Board. Cells and tissuesCells and tissues were obtained from ascites, blood, lymph nodes, and tumors as we described. 16,18,19 Immune cells, including monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells, and T-cell subsets, were enriched using paramagnetic beads (StemCell Technologies) and sorted with FACSAria (Becton Dickinson) as we described. 16,18,19 Cell purity was more than 98% as confirmed by flow cytometry (LSR II; Becton Dickinson). FACSFor cytokine detection, the cells were stimulated with phorbol myristate acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1 M; Sigma-Aldrich) for 4 hours before staining. Cells were first stained extracellularly with specific antibodies against human CD3, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD25, CD39, CD45, CD45RO, CD49a, CD49c, CD49d, CD49e, CD56, CD123, CD161, PD-1, CCR4, CCR6, CCR7, CXCR4, HLA-DR, and annexin V (BD Biosciences), CCR2, CXCR3, and CCR5 (R&D Systems), EpCam (StemCell Technologies), then were fixed and permeabilized with Perm/Fix solution (eBioscience), and finally were stained intracellularly with anti-IL-2, anti-IL-10, anti-IL-17, anti-tumor necrosis factor-␣, anti-interferon-␥ (IFN-␥), anti-Granzyme A, anti-Ki-67, and anti-FOXP3 (all from BD Biosciences, except anti-IL-17, eBioscience). Samples were acquired on a LSR II (BD Biosciences), and data were analyzed with DIVA software (BD Biosciences). Th17 induction and suppressionFresh peripheral blood and tumor-associated CD14 ϩ macrophages were sorted 19 and cocultured with T cells as indicated for 3 to 5 days in the An In...

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.

B7-H4 is a recently identified B7 family member. We previously showed that ovarian tumor and associated macrophages expressed B7-H4; tumor B7-H4 + macrophages and CD4 + CD25 + FOXP3 + regulatory T cells (Treg cells) suppressed tumor-associated antigen-specific T-cell immunity. To determine the pathologic relationship between B7-H4, macrophages, and Treg cells in the tumor environment, in addition to Treg cell numbers, we quantified B7-H4 expression in the tumor and tumor-associated macrophages in 103 patients with ovarian carcinoma. We observed that the intensity of B7-H4 expression in macrophages was significantly correlated with Treg cell numbers in the tumor. Further, both Treg cells and macrophage B7-H4, but not tumor B7-

To directly dissect the role of each immune component in human tumor immunopathogenesis, we have studied the interaction between dendritic cells and T cells in the tumor environment of patients with ovarian carcinoma. We previously reported that functional plasmacytoid dendritic cells, but not functionally mature myeloid dendritic cells, accumulated in tumor microenvironments. We now show that tumor ascites macrophage-derived dendritic cells induced tumor-associated antigen-specific CD8+ T cells with effector functions. Strikingly, tumor ascites plasmacytoid dendritic cells induced interleukin-10+ CCR7+ CD45RO+ CD8+ regulatory T cells. Four characteristics have been identified in tumor plasmacytoid dendritic cell-induced CD8+ regulatory T cells: (a) induction of CD8+ regulatory T cells is independent of CD4+ CD25+ T cells; (b) CD8+ regulatory T cells significantly suppress myeloid dendritic cell-mediated tumor-associated antigen-specific T cell effector functions through interleukin-10; (c) repetitive myeloid dendritic cell stimulation can recover CD8+ regulatory T cell-mediated poor T cell proliferation, but not T cell effector function; (d) CD8+ regulatory T cells express functional CCR7, and efficiently migrate with lymphoid homing chemokine MIP-3beta. Primary suppressive CCR7+ CD45RO+ CD8+ T cells are found in the tumor environment of patients with ovarian cancers. Thus, tumor-associated plasmacytoid dendritic cells contribute to the tumor environmental immunosuppressive network. Collectively, tumors manipulate tumor microenvironmental dendritic cell subset distribution and function to subvert tumor immunity. The data are relevant to understanding tumor immunopathology as well as reevaluating tumor immunotherapeutic strategies.

Angiogenesis is essential for both primary and metastatic tumor growth. Tumor blood vessel formation is complex and regulated by many factors. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites of patients with untreated ovarian carcinoma. We observed high numbers of plasmacytoid dendritic cells (PDCs) and significant stromal-derived factor (CXCL-12/SDF)-1 in their malignant ascites, attracting PDCs into the tumor environment. We now show that tumor-associated PDCs induced angiogenesis in vivo through production of tumor necrosis factor ␣ and interleukin 8. By contrast, myeloid dendritic cells (MDCs) were absent from malignant ascites. MDCs derived in vitro suppressed angiogenesis in vivo through production of interleukin 12. Thus, the tumor may attract PDCs to augment angiogenesis while excluding MDCs to prevent angiogenesis inhibition, demonstrating a novel mechanism for modulating tumor neovascularization. Because dendritic cells (DCs) have long been known to affect tumor immunity, our data also implicate DCs in regulation of tumor neoangiogenesis, suggesting a novel role of DCs in tumor pathology.

Tumors express tumor-associated antigens (TAA) and thus should be the object of immune attack. Nonetheless, spontaneous clearance of established tumors is rare. Much work has demonstrated that tumors have numerous strategies either to prevent presentation of TAA, or to prevent TAA presentation in the context of T-cell co-signaling molecules. Thus, it was thought that lack of TAA-specific immunity was largely a passive process: tumors simply did not present enough TAA, or antigen-presenting cells did not have sufficient stimulatory capacity. On this basis, attempts were made to bolster TAA-specific immunity by using optimal antigen-presenting cells or by growing TAA-specific effector T cells ex vivo followed by adoptive transfer. These approaches met with some success in mouse models of human tumors, and showed some early clinical efficacy in human trials, although long-term efficacy remains to be established, and logistical problems are considerable. These studies established the concept that experimentally induced TAA-specific immunity is a rational and potentially efficacious means to treat cancer, including ovarian cancer. Nonetheless, recent work demonstrates that lack of naturally induced TAA-specific immunity is not simply a passive process. We discuss recent data clearly demonstrating that 'tumors actively prevent induction of TAA-specific immunity through induction of TAA-specific tolerance'. This tolerance is mediated in part by regulatory T cells (Tregs). Means to revert these tolerizing conditions represent a novel anticancer therapeutic stratagem. We discuss Tregs in this regard in human ovarian cancer and present evidence that depleting Treg in human cancer, including ovarian cancer, using denileukin diftitox (Ontak), improves immunity and may be therapeutic.