Benedikt Jacobs

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.

Trans-Presentation of IL-15 Dictates IFN-Producing Killer Dendritic Cells Effector Functions

IFN-producing killer dendritic cells (IKDC) were initially described as B220+CD11c+CD3−NK1.1+ tumor-infiltrating cells that mediated part of the antitumor effects of the combination therapy with imatinib mesylate and IL-2. In this study, we show their functional dependency on IL-15 during homeostasis and inflammatory processes. Trans-presentation of IL-15 by IL-15Rα allows dramatic expansion of IKDC in vitro and in vivo, licenses IKDC for TRAIL-dependent killing and endows IKDC with immunizing potential, all three biological attributes not shared by B220−NK cells. However, IL-15 down-regulates the capacity of IKDC to induce MHC class I- or II-restricted T cell activation in vitro. Trans-presentation of IL-15 by IL-15Rα allows IKDC to respond to TLR3 and TLR4 ligands for the production of CCL2, a chemokine that is critical for IKDC trafficking into tumor beds (as described recently). We conclude that IKDC represent a unique subset of innate effectors functionally distinguishable from conventional NK cells in their ability to promptly respond to IL-15-driven inflammatory processes.

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.

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.

Nilotinib combined with interleukin-2 mediates antitumor and immunological effects in a B16 melanoma model.

The immune system contributes to tumor cell killing which can be enhanced by cancer chemotherapeutics and immune modulatory pharmaceuticals such as tyrosine kinase inhibitors (TKIs). Recently, the beneficial effect of natural killer (NK) cells was demonstrated when combining interleukin-2 (IL-2) with the TKI imatinib. The aim of the present study was to address the antitumor and immunological effects of recently approved TKIs. Therefore, we focused on the comparison of the efficacy between imatinib and nilotinib in combination with IL-2 in a murine B16F10 melanoma model. Both TKIs possessed antitumor activity in vivo. However, the combination of nilotinib and IL-2 showed a superior outcome. Importantly, both the use of immunodeficient Rag2γc-/- mice, which lack T-lymphocytes, B-lymphocytes and NK cells, as well as NK cell-depletion in C57Bl/6 mice reduced the therapeutic effect of nilotinib. Flow cytometry revealed a significant increase in the IFN-γ-producing CD27+ NK cell subpopulation following treatment with nilotinib and IL-2. Furthermore, the therapeutic antitumor effect of nilotinib/IL-2 was completely lost in IFN-γ-/- mice. In summary, we suggest that nilotinib combined with IL-2 confers high antitumor activity involving the subset of IFN-γ-producing CD27+ NK cells. These new insights are of high importance for the understanding and development of immunotherapeutic protocols using TKIs.

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.

NK Cell Subgroups, Phenotype, and Functions After Autologous Stem Cell Transplantation

High-dose chemotherapy with consecutive autologous stem cell transplantation (autoSCT) is a well-established treatment option for patients suffering from malignant lymphoma or multiple myeloma. Natural killer (NK) cells are an important part of the immune surveillance, and their cell number after autoSCT is predictive for progression-free and overall survival. To improve knowledge about the role of NK cells after autoSCT, we investigated different NK cell subgroups, their phenotype, and their functions in patients treated with autoSCT. Directly after leukocyte regeneration (>1000 leukocytes/μl) following autoSCT, CD56++ NK cells were the major NK cell subset. Surprisingly, these cells showed unusually high surface expression levels of CD57 and killer Ig-like receptors (KIRs) compared to expression levels before or at later time points after autoSCT. Moreover, these NK cells strongly upregulated KIR2DL2/3/S2 and KIR3DL1, whereas KIR2DL1/S1 remained constant, indicating that this cell population arose from more immature NK cells instead of from activated mature ones. Remarkably, NK cells were already able to degranulate and produce IFN-γ and MIP-1β upon tumor interaction early after leukocyte regeneration. In conclusion, we describe an unusual upregulation of CD57 and KIRs on CD56++ NK cells shortly after autoSCT. Importantly, these NK cells were functionally competent upon tumor interaction at this early time point.