Keith L. Knutson

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.

Blockade of B7-H1 improves myeloid dendritic cell–mediated antitumor immunity

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)-γ. 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.

Tumor antigen-specific T helper cells in cancer immunity and immunotherapy

Historically, cancer-directed immune-based therapies have focused on eliciting a cytotoxic T cell (CTL) response, primarily due to the fact that CTL can directly kill tumors. In addition, many putative tumor antigens are intracellular proteins, and CTL respond to peptides presented in the context of MHC class I which are most often derived from intracellular proteins. Recently, increasing importance is being given to the stimulation of a CD4+ T helper cell (Th) response in cancer immunotherapy. Th cells are central to the development of an immune response by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages and mast cells. Two predominant Th cell subtypes exist, Th1 and Th2. Th1 cells, characterized by secretion of IFN-γ and TNF-α, are primarily responsible for activating and regulating the development and persistence of CTL. In addition, Th1 cells activate antigen-presenting cells (APC) and induce limited production of the type of antibodies that can enhance the uptake of infected cells or tumor cells into APC. Th2 cells favor a predominantly humoral response. Particularly important during Th differentiation is the cytokine environment at the site of antigen deposition or in the local lymph node. Th1 commitment relies on the local production of IL-12, and Th2 development is promoted by IL-4 in the absence of IL-12. Specifically modulating the Th1 cell response against a tumor antigen may lead to effective immune-based therapies. Th1 cells are already widely implicated in the tissue-specific destruction that occurs during the pathogenesis of autoimmune diseases, such as diabetes mellitus and multiple sclerosis. Th1 cells directly kill tumor cells via release of cytokines that activate death receptors on the tumor cell surface. We now know that cross-priming of the tumor-specific response by potent APC is a major mechanism of the developing endogenous immune response; therefore, even intracellular proteins can be presented in the context of MHC class II. Indeed, recent studies demonstrate the importance of cross-priming in eliciting CTL. Many vaccine strategies aim to stimulate the Th response specific for a tumor antigen. Early clinical trials have shown that focus on the Th effector arm of the immune system can result in significant levels of both antigen-specific Th cells and CTL, the generation of long lasting immunity, and a Th1 phenotype resulting in the development of epitope spreading.

Generation of T-Cell Immunity to the HER-2/neu Protein After Active Immunization With HER-2/neu Peptide–Based Vaccines

PURPOSE The HER-2/neu protein is a nonmutated tumor antigen that is overexpressed in a variety of human malignancies, including breast and ovarian cancer. Many tumor antigens, such as MAGE and gp100, are self-proteins; therefore, effective vaccine strategies must circumvent tolerance. We hypothesized that immunizing patients with subdominant peptide epitopes derived from HER-2/neu, using an adjuvant known to recruit professional antigen-presenting cells, granulocyte-macrophage colony-stimulating factor, would result in the generation of T-cell immunity specific for the HER-2/neu protein. PATIENTS AND METHODS Sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers were enrolled. Vaccines were composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally. Peripheral-blood mononuclear cells were evaluated at baseline, before vaccination, and after vaccination for antigen-specific T-cell immunity. Immunologic response data are presented on the 38 subjects who completed six vaccinations. Toxicity data are presented on all 64 patients enrolled. RESULTS Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides (stimulation index, 2.1 to 59) and 68% to a HER-2/neu protein domain (stimulation index range, 2 to 31). Epitope spreading was observed in 84% of patients and significantly correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P =.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. CONCLUSION The majority of patients with HER-2/neu-overexpressing cancers can develop immunity to both HER-2/neu peptides and protein. In addition, the generation of protein-specific immunity, after peptide immunization, was associated with epitope spreading, reflecting the initiation of an endogenous immune response. Finally, immunity can persist after active immunizations have ended.

Immune-induced epithelial to mesenchymal transition in vivo generates breast cancer stem cells

The breast cancer stem cell (BCSC) hypotheses suggest that breast cancer is derived from a single tumor-initiating cell with stem-like properties, but the source of these cells is unclear. We previously observed that induction of an immune response against an epithelial breast cancer led in vivo to the T-cell-dependent outgrowth of a tumor, the cells of which had undergone epithelial to mesenchymal transition (EMT). The resulting mesenchymal tumor cells had a CD24(-/lo)CD44(+) phenotype, consistent with BCSCs. In the present study, we found that EMT was induced by CD8 T cells and the resulting tumors had characteristics of BCSCs, including potent tumorigenicity, ability to reestablish an epithelial tumor, and enhanced resistance to drugs and radiation. In contrast to the hierarchal cancer stem cell hypothesis, which suggests that breast cancer arises from the transformation of a resident tissue stem cell, our results show that EMT can produce the BCSC phenotype. These findings have several important implications related to disease progression and relapse.

Folate receptor alpha as a tumor target in epithelial ovarian cancer.

OBJECTIVES Folate receptor alpha (FRalpha) is a folate-binding protein overexpressed on ovarian and several other epithelial malignancies that can be used as a target for imaging and therapeutic strategies. The goal of this study is to improve historical data that lack specific information about FRalpha expression in rare histological subtypes, primary disease versus metastatic foci, and recurrent disease. METHODS FRalpha expression was analyzed by immunohistochemistry on 186 primary and 27 recurrent ovarian tumors, including 24 pairs of samples obtained from the same individuals at diagnosis and at secondary debulking surgery. For 20 of the 186 primaries, simultaneous metastatic foci were also analyzed. FRalpha staining was analyzed in light of disease morphology, stage, grade, debulking status, and time from diagnosis to recurrence and death. RESULTS FRalpha expression was apparent in 134 of 186 (72%) primary and 22 of 27 (81.5%) recurrent ovarian tumors. In 21 of 24 (87.5%) matched specimens, recurrent tumors reflected the FRalpha status detected at diagnosis. Metastatic foci were similar to primary tumors in FRalpha staining. FRalpha status was not associated with time to recurrence or overall survival in either univariate or multivariable analyses. CONCLUSION FRalpha expression occurs frequently, especially in the common high-grade, high-stage serous tumors that are most likely to recur. New findings from this study show that FRalpha expression is maintained on metastatic foci and recurrent tumors, suggesting that novel folate-targeted therapies may hold promise for the majority of women with either newly diagnosed or recurrent ovarian cancer.

Intraepithelial Effector (CD3+)/Regulatory (FoxP3+) T-Cell Ratio Predicts a Clinical Outcome of Human Colon Carcinoma

BACKGROUND & AIMS Regulatory T cells (Tregs) express the forkhead box transcription factor (FoxP3) and suppress the antitumor immune response. We investigated whether the intratumoral densities of FoxP3(+) and effector CD3(+) lymphocytes are associated with prognosis of patients with colon cancer. METHODS FoxP3 and CD3 expression and location were determined in stage II and III colon carcinomas (n = 160) and normal mucosa (n = 25) by immunohistochemistry; CD4 and FoxP3 were localized by dual immunofluorescence microscopy. T-cell markers were compared with pathological variables, DNA mismatch repair status, and patient survival using Cox proportional hazards models. RESULTS FoxP3(+) and CD3(+) T-cell densities were increased in carcinomas compared with autologous normal mucosa (P < .0001). An increase in intraepithelial FoxP3(+) cells was associated with poor tumor differentiation (P = .038), female sex (P = .028), and advanced patient age (P = .042). FoxP3(+) cell density was not prognostic, yet patients with tumors with reduced intraepithelial CD3(+) T-cell densities had reduced disease-free survival (DFS) rates (hazard ratio [HR], 1.87 [95% confidence interval, 1.10-3.16]; P = .018). A low intraepithelial CD3(+)/FoxP3(+) cell ratio predicted reduced DFS (46.2% vs 66.7% survival at 5 years; HR, 2.17 [95% confidence interval, 1.11-4.23]; P = .0205). The prognostic impact of these markers was maintained when tumors were stratified by mismatch repair status. By multivariate analysis, a low CD3(+)/FoxP3(+) cell ratio (P= .0318) and low numbers of CD3(+) T cells (P = .0397) predicted shorter DFS times and were stronger prognostic variables than tumor stage or number of lymph node metastases. CONCLUSIONS A low intraepithelial CD3(+)/FoxP3(+) cell ratio and reduced numbers of CD3(+) T cells were associated with shorter patient survival time, indicating the importance of an effector to Treg cell ratio in colon cancer prognosis.

Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer

Immunomodulatory strategies, such as antibody therapy and cancer vaccines, are increasingly being considered as potential adjuvant therapies in patients with advanced stage breast cancer to either treat minimal residual disease or prevent relapse. However, little is known concerning the incidence and magnitude of the pre-existent breast cancer specific immune response in this patient population. Using the HER-2/neu oncogenic protein as a model, a well-defined tumor antigen in breast cancer, we questioned whether patients with advanced stage HER-2/neu overexpressing breast and ovarian cancers (III/IV) had evidence of pre-existent immunity to HER-2/neu. Forty-five patients with stage III or IV HER-2/neu overexpressing breast or ovarian cancer were evaluated for HER-2/neu specific T cell and antibody immunity. Patients enrolled had not received immunosuppressive chemotherapy for at least 30 days (median 5 months, range 1–75 months). All patients were documented to be immune competent prior to entry by DTH testing using a skin test anergy battery. Five of 45 patients (11%) were found to have a significant HER-2/neu specific T cell response as defined by a stimulation index ≥ 2.0 (range 2.0–7.9). None of eight patients who were HLA-A2 had a detectable IFNγ secreting T-cell precursor frequency to a well-defined HER-2/neu HLA-A2 T cell epitope, p369-377. Three of 45 patients (7%) had detectable HER-2/neu specific IgG antibodies, range 1.2–8.9 μg/ml. These findings suggest that patients with advanced stage HER-2/neu overexpressing breast and ovarian cancer can mount a T cell and/or antibody immune response to their tumor. However, in the case of the HER-2/neu antigen, the pre-existent tumor specific immune response is found only in a minority of patients.

TGFβ/TNFα-Mediated Epithelial–Mesenchymal Transition Generates Breast Cancer Stem Cells with a Claudin-Low Phenotype

Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial-mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer.

Augmented HER-2–Specific Immunity during Treatment with Trastuzumab and Chemotherapy

Purpose: Passive immunotherapy with antitumor antibodies has the potential to induce active tumor immunity via the opsonic enhancement of immunogenicity of tumor antigen. We have assessed whether immune sensitization to the HER-2/neu tumor antigen occurs during treatment with the anti-HER-2/neu monoclonal antibody trastuzumab. Experimental Design: Twenty-seven patients treated with trastuzumab and chemotherapy were assessed for the induction of HER-2/neu–specific immunity. Sera and peripheral blood mononuclear cells obtained before and after trastuzumab therapy were compared for the presence of anti-HER-2/neu endogenous Igλ antibodies and HER-2/neu–specific CD4 responses by ELISA and enzyme-linked immunospot, respectively. Results: Anti-HER-2/neu antibodies were detectable in 8 of 27 (29%) patients before trastuzumab treatment and in 15 of 27 (56%) patients during trastuzumab treatment. In the overall study population, anti-HER-2/neu humoral responses significantly increased during therapy (P < 0.001) and were not associated with development of an anti-idiotypic response. In 10 evaluable individuals, 6 showed augmented HER-2/neu–specific CD4 T-cell responses during therapy. Of the 22 individuals treated for metastatic disease, those patients showing objective clinical responses exhibited more frequent (P = 0.004) and larger (P = 0.006) treatment-associated anti-HER-2/neu humoral responses. Conclusion: Humoral immune sensitization occurs during treatment with chemotherapy and trastuzumab. Further studies are warranted to investigate whether augmented anti-HER-2/neu humoral and cellular immunity contributes mechanistically to clinical outcome.