Ilona Kryczek

Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival

Regulatory T (Treg) 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 Treg cells; however, definitive evidence that Treg cells have an immunopathological role in human cancer is lacking. Here we show, in detailed studies of CD4+CD25+FOXP3+ Treg cells in 104 individuals affected with ovarian carcinoma, that human tumor Treg cells suppress tumor-specific T cell immunity and contribute to growth of human tumors in vivo. We also show that tumor Treg cells are associated with a high death hazard and reduced survival. Human Treg 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 Treg cells to the tumor. This specific recruitment of Treg cells represents a mechanism by which tumors may foster immune privilege. Thus, blocking Treg cell migration or function may help to defeat human cancer.

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

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.

Plasmacytoid dendritic cells induce CD8+ regulatory T cells in human ovarian carcinoma.

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.

Regulatory T cells in ovarian cancer: biology and therapeutic potential.

Tumors express tumor‐associated antigens (TAA) and thus should be the object of immune attack. Nonetheless, spontaneous clearance of established tumors is rare. 1 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. 1,2 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. 3 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. 4 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. 5–12 We discuss recent data clearly demonstrating that ‘tumors actively prevent induction of TAA‐specific immunity through induction of TAA‐specific tolerance’. 13 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.

Dendritic Cell Subsets Differentially Regulate Angiogenesis in Human Ovarian Cancer

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 α 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.

290 DEPLETING REGULATORY T CELLS IS ASSOCIATED WITH IMPROVED IMMUNITY AND TUMOR CLEARANCE IN HUMAN CANCER

Despite a compelling logic, tumor immunotherapy has generally failed. Work by us and others suggests that this failure owes in part to tumor-mediated immune dysfunction. CD4+CD25+ regulatory T cells (Tregs) are thought to contribute to this dysfunction. In support, we show that in ovarian cancer patients, Tregs block tumor-specific immunity, promote tumor growth and predict poor survival. We hypothesize that depleting Tregs will improve immunity and improve therapeutic outcomes in cancer patients. Ontak is a recombinant protein fusing the CD25-binding domain of IL-2 to the active domain of diphtheria toxin. It was designed to kill CD4+CD25+ leukemia cells. We tested the hypothesis that Ontak kills CD4+CD25+ Tregs and thus improves immunity in cancer patients. Patients 1-3 (advanced-stage cancer of the ovary, breast or lung) received a single intravenous infusion of 9 μg/kg Ontak. Patient 4 had relapsed stage IIIC ovarian cancer and received a single dose of Ontak at 12 μg/kg. Blood immune cells from before and after treatment were enumerated and cytokines were studied by flow cytometry. T cell suppression was also tested. Prior to infusion, the mean blood CD4+CD25+ cells/mm3 was 126, which was 26.8% of CD4+ T cells. This decreased to a mean of 78 cells/mm3 blood and mean of 19.0% of CD4+ T cells 3-5 days after Ontak. Also, interferon-γ-expressing T cells increased approximately 50% and FOXP3 (a functional marker for Tregs) was reduced after Ontak. In patient 4, CD4+CD25+ cells inhibited T cell proliferation in vitro prior to but not after treatment. Further, a single Ontak-mediated Treg depletion reduced blood CA-125 from 121 to 38 U/mL four weeks later in patient 4, suggesting clinical efficacy. Patient 4 then received 6 additional doses of Ontak at 12 μg/kg with a further decrease in blood CA-125 level to 16 U/mL four weeks after the final dose. Additionally, a PET/CT fusion demonstrated dramatic reduction of metastatic disease. The CA-125 level remains normal two months after the final Ontak dose. Our data demonstrate for the first time that Ontak efficiently depletes Tregs in patients with cancer, and Treg depletion is associated with improved T cell immunity, normalized CA-125 and reduced tumor metastasis. The data may help explain failures of current tumor immunotherapy protocols. It further suggests that Treg depletion or a combination with current immunotherapeutic regimens will be a novel strategy for treating patients with cancer.