Claudia Papewalis

IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo.

The antitumor effect of IFN-α is mediated by the activation of CTLs, NK cells, and the generation of highly potent Ag-presenting dendritic cells (IFN-DCs). In this study, we show that IFN-DCs generated in vitro from monocytes express CD56 on their surface, a marker which has been thought to be specific for NK cells. FACS analyses of CD56+ and CD56− IFN-DCs showed a nearly identical pattern for most of the classical DC markers. Importantly, however, only CD56+ IFN-DCs exhibited cytolytic activity up to 24% that could almost completely be blocked (−81%) after coincubation with anti-TRAIL. Intracytoplasmatic cytokine staining revealed that the majority of IFN-DCs independently of their CD56 expression were IFN-γ positive as well. In contrast, CD56+ IFN-DCs showed stronger capacity in stimulating allogenic T cells compared with CD56− IFN-DC. Based on these results, five patients with metastasized medullary thyroid carcinoma were treated for the first time with monocyte-derived tumor Ag-pulsed IFN-DCs. After a long term follow-up (in mean 37 mo) all patients are alive. Immunohistochemical analyses of delayed-type hypersensitivity skin reaction showed a strong infiltration with CD8+ cells. In two patients no substantial change in tumor morphology was detected. Importantly, by analyzing PBMCs, these patients also showed an increase of Ag-specific IFN-γ-secreting T cells. In summary, we here describe for the first time that cytotoxic activity of IFN-DCs is mainly mediated by an IFN-DC subset showing partial phenotypic and functional characteristics of NK cells. These cells represent another mechanism of the antitumor effect induced by IFN-α.

Adipocyte-secreted factors increase osteoblast proliferation and the OPG/RANKL ratio to influence osteoclast formation.

Several studies have reported a positive relationship of the body fat mass and bone density. However, it is not clear whether adipocyte-derived signaling molecules directly act on osteoblasts or osteoclasts. Therefore, we investigated the effect of fat cell-secreted factors on the proliferation and differentiation of preosteoblasts and the molecular mechanisms involved. This stimulation led to an increased proliferation of MC3T3-E1 and primary preosteoblastic cells (2.8-fold and 1.5-fold, respectively; p<0.0001), which could be reduced with inhibitors of protein tyrosine kinases, FGFR1 and PI3K. Concordantly, we found human adipocytes to secrete bFGF and bFGF to mimic the effect of adipocyte-secreted factors. The ratio of OPG/RANKL secretion in primary human preosteoblasts increased 9-fold (mRNA and protein) when stimulated with adipocyte-secreted factors. Moreover, osteoblasts which were prestimulated with adipocyte-secreted factors inhibited the formation of osteoclasts. In conclusion, human adipocytes secrete factors that directly act on preosteoblasts and alter their crosstalk with osteoclasts. These in vitro findings reflect the higher bone mass in obese people and attribute it to effects of adipocyte-secreted factors on bone formation.

Evidence of a Combined Cytotoxic Thyroglobulin and Thyroperoxidase Epitope-Specific Cellular Immunity in Hashimoto's Thyroiditis

CONTEXT Hashimotos thyroiditis (HT) is a common autoimmune disease leading to thyroid destruction due to lymphocytic infiltration. Only rare data are available regarding the recognition of specific cellular antigens, e.g. of thyroperoxidase (TPO) and thyroglobulin (Tg). OBJECTIVE The aim of this study was to quantify and characterize TPO- and Tg-epitope-specific CD8-positive T cells of HT patients. DESIGN Six different human leukocyte antigen (HLA)-A2 restricted, TPO- or Tg-specific tetramers were synthesized and used for measuring CD8-positive T cells in HT patients and controls. RESULTS The frequency of peripheral TPO- and Tg-specific CD8-positive T cells was significantly higher in HLA-A2-positive HT patients (2.8 ± 9.5%) compared with HLA-A2-negative HT patients (0.5 ± 0.7%), HLA-A2-positive nonautoimmune goiter patients (0.2 ± 0.4%), and HLA-A2-positive healthy controls (0.1 ± 0.2%). The frequency of Tg-specific T cells (3.0%) was very similar to those of TPO-specific CD8-positive T cells (2.9%). Subgroup analyses revealed a steady increase of the number of epitope-specific CD8-positive T cells from 0.6 ± 1.0% at initial diagnosis up to 9.4 ± 18.3% in patients with long-lasting disease. Analyses of the number of thyroid-infiltrating cells as well as the cytotoxic capacity revealed a similar picture for TPO- and Tg-specific T cells. CONCLUSION We here report for the first time that both antigens, TPO and Tg, are recognized by CD8-positive T cells and are involved in the thyroid destruction process leading to clinical disease manifestation.

Dendritic Cell Vaccination with Xenogenic Polypeptide Hormone Induces Tumor Rejection in Neuroendocrine Cancer

Purpose: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). Experimental Design: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid–modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. Results: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8+ T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-γ-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8+ cells. Immunohistochemistry revealed strong CD8+ tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (−74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. Conclusion: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.

Immunoregulatory Natural Killer Cells Suppress Autoimmunity by Down-Regulating Antigen-Specific CD8+ T Cells in Mice

Natural killer (NK) cells belong to the innate immune system. Besides their role in antitumor immunity, NK cells also regulate the activity of other cells of the immune system, including dendritic cells, macrophages, and T cells, and may, therefore, be involved in autoimmune processes. The aim of the present study was to clarify the role of NK cells within this context. Using two mouse models for type 1 diabetes mellitus, a new subset of NK cells with regulatory function was identified. These cells were generated from conventional NK cells by incubation with IL-18 and are characterized by the expression of the surface markers CD117 (also known as c-Kit, stem cell factor receptor) and programmed death (PD)-ligand 1. In vitro analyses demonstrated a direct lysis activity of IL-18-stimulated NK cells against activated insulin-specific CD8(+) T cells in a PD-1/PD-ligand 1-dependent manner. Flow cytometry analyses revealed a large increase of splenic and lymphatic NK1.1(+)/c-Kit(+) NK cells in nonobese diabetic mice at 8 wk of age, the time point of acceleration of adaptive cytotoxic immunity. Adoptive transfer of unstimulated and IL-18-stimulated NK cells into streptozotocin-treated mice led to a delayed diabetes development and partial disease prevention in the group treated with IL-18-stimulated NK cells. Consistent with these data, mild diabetes was associated with increased numbers of NK1.1(+)/c-Kit(+) NK cells within the islets. Our results demonstrate a direct link between innate and adaptive immunity in autoimmunity with newly identified immunoregulatory NK cells displaying a potential role as immunosuppressors.

Dendritic cells as potential adjuvant for immunotherapy in adrenocortical carcinoma

Objective  Adrenocortical carcinoma (ACC) is a rare malignancy associated with a dismal prognosis. Dendritic cells (DCs) are professional antigen‐presenting cells leading to an antitumour immune response. The aim of this study was to elaborate two methods of antigen delivery to DCs and to evaluate an immunotherapy protocol in ACC patients.

Amino Acid-Modified Calcitonin Immunization Induces Tumor Epitope-Specific Immunity in a Transgenic Mouse Model for Medullary Thyroid Carcinoma

Up to now, no relevant tumor antigen has been identified in medullary thyroid carcinoma (MTC). The aim of the present study was to prove the concept of an immunization with an amino acid-modified calcitonin (CT) for the treatment of MTC in a transgenic mouse model. Amino acid-modified (human) CT has been chosen for vaccination because of its higher binding affinity to the murine H2-Kb-MHC molecule. Mice were immunized over 6 months with monthly injections of amino acid-modified CT-pulsed dendritic cells. For enumeration of tumor epitope-specific CD8+ cytotoxic T cells, tetramer analyses were performed. CT peptide-treated mice revealed a mean 0.73 +/- 0.45 and 0.91 +/- 0.59% positive cells, depending on the two tetramers tested, whereas no increase was seen in control protein-immunized mice (0.08-0.12% tetramer-positive cells). Importantly, the subset of CT-specific CD8+ T cells also showed a high expression of interferon-gamma. In line with these results, CT-immunized mice also showed an intratumor infiltration with CD8+ T lymphocytes. Importantly, we also found a diminished tumor outgrowth of -57% and a decrease of the serum CT levels (2.0 +/- 0.1 pg/ml) compared with control protein-immunized Ret/Cal mice (3.0 +/- 0.4 pg/ml). In summary, we show that amino acid-modified CT is recognized from the immune system leading to a specific antitumor immune response and a diminished tumor outgrowth in transgenic MTC mice. The results are of potential importance because they might be applicable to patients with metastatic spread of a MTC.

Increased numbers of tumor-lysing monocytes in cancer patients.

Lymphatic infiltration is a well known phenomenon in different tumors including endocrine malignancies. However, little is known about the role of antigen-presenting cells and T cell activation in this context. The aim of our study was to investigate the quantity and function of CD14+/CD56+ monocytes in tumor patients including endocrine malignancies. First, these cells were characterized in peripheral blood of endocrine and non-endocrine cancer patients as well as in tumor tissue samples. Cancer patients had in mean 3.7 times more CD14+/CD56+ monocytes in the peripheral blood compared to healthy controls (p≤0.0001), while the highest frequencies were seen in patients with heavy tumor load. Importantly, these cells additionally expressed several NK cell markers. A proof of CD14+/CD56+ infiltrations into papillary thyroid carcinoma was shown by immunohistochemical analyses. Functional analyses revealed an apoptosis inducing capacity in vitro after IFN-α re-stimulation. Our data indicate the importance of tumor-lysing monocytes in antitumor immunity.

Chromogranin A as potential target for immunotherapy of malignant pheochromocytoma.

Currently, no effective treatment for malignant pheochromocytoma exists. The aim of our study was to investigate the role of chromogranin A (CgA) as a specific target molecule for immunotherapy in a murine model for pheochromocytoma. Six amino acid-modified and non-modified CgA peptides were used for dendritic cell vaccination. Altogether, 50 mice received two different CgA vaccination protocols; another 20 animals served as controls. In vitro tetramer analyses revealed large increases of CgA-specific cytotoxic T cells (CTL) in CgA-treated mice. Tumors of exogenous applied pheochromocytoma cells showed an extensive infiltration by CD8+ T cells. In vitro, CTL of CgA-treated mice exhibited strong MHC I restricted lysis capacities towards pheochromocytoma cells. Importantly, these mice showed strongly diminished outgrowth of liver tumors of applied pheochromocytoma cells. Our data clearly demonstrate that CgA peptide-based immunotherapy induces a cytotoxic immune response in experimental pheochromocytoma, indicating potential for therapeutic applications in patients with malignant pheochromocytoma.

Identifying tumor antigens in endocrine malignancies

Tumor antigens are surface molecules that are mostly cancer specific, often overexpressed and recognized by the immune system. Therefore, identifying tumor antigens is of key importance for developing new immunotherapies for incurable cancers. For endocrine malignancies, several different tumor-associated antigens have been described, including polypeptide hormones and/or vesicle-associated antigens in Th1-mediated autoimmune diseases. Other antigens have been identified by screening tumor DNA libraries. Furthermore, vaccination studies in humans and animal models have revealed a tumor-antigen-specific immunity and clinical responses with reduced tumor size. Here, we provide an overview of the recent progress achieved in identifying tumor antigens and predict how this knowledge can be used in the future for developing anti-tumor vaccinations.