Infiltration by macrophages represents a characteristic morphological hallmark in high-grade lymphatic malignancies such as Burkitt's lymphoma (BL). Although macrophages can, in principle, target neoplastic cells and mediate antibody-dependent cellular cytotoxicity (ADCC), tumor-associated macrophages (TAMs) regularly fail to exert direct cytotoxic functions. The underlying mechanisms responsible for this observation remain unclear. We demonstrate that inflammatory M1 macrophages kill proliferating high-grade B cell lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D-dependent fashion. We show that cathelicidin directly induces cell death by targeting mitochondria of BL cells. In contrast, anti-inflammatory M2 macrophages and M2-like TAMs in BL exhibit an altered vitamin D metabolism, resulting in a reduced production of cathelicidin and consequently in inability to lyse BL cells. However, treatment of M2 macrophages with the bioactive form of vitamin D, 1,25D3, or a vitamin D receptor agonist effectively induces cathelicidin production and triggers tumoricidal activity against BL cells. Furthermore, rituximab-mediated cytotoxicity of vitamin D-treated M2 macrophages is cathelicidin-dependent. Finally, vitamin D treatment of 25-hydroxyvitamin D (25D)-deficient volunteers in vivo or primary TAMs in vitro improves rituximab-mediated ADCC against B cell lymphoma cells. These data indicate that activation of the vitamin D signaling pathway activates antitumor activity of TAMs and improves the efficacy of ADCC.

Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B‐cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR‐induced Ca2+ signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B‐cell‐specific Grb2‐deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B‐cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR‐induced Ca2+ signalling, alterations in mitogen‐activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B‐cell‐specific Grb2‐deficient mice showed impaired IgG and B‐cell memory responses, and impaired germinal centre formation. Thus, Grb2‐dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

Infiltration by macrophages represents a characteristic morphological hallmark in high grade lymphatic malignancies such as Burkitt’s lymphoma (BL). Although macrophages can in principle target neoplastic cells and mediate antibody-dependent cytotoxicity (ADCC), tumor associated macrophages (TAMs) regularly fail to exert direct cytotoxic functions. The underlying mechanisms responsible for this observation remain unclear. Here we demonstrate that inflammatory M1 macrophages kill proliferating high grade B-cell lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D-dependent fashion. Moreover, we show that cathelicidin directly induces cell death by targeting mitochondria of BL-cells. In contrast, anti-inflammatory M2 macrophages and M2-like TAMs in BL exhibit an altered vitamin D metabolism resulting in a reduced production of cathelicidin and consequently in inability to lyse BL cells. However, treatment of M2 macrophages with the bioactive form of vitamin D, 1,25D, or a vitamin D receptor agonist effectively induces cathelicidin production and triggers tumoricidal activity against BL cells. Furthermore, rituximab-mediated cytotoxicity of M2 macrophages is cathelicidin dependent and vitamin D treatment of 25D deficient volunteers improves rituximab-mediated ADCC against BL cells. These data indicate that activation of the vitamin D signaling pathway may activate antitumor activity of TAMs and improve the efficacy of ADCC. Disclosures No relevant conflicts of interest to declare.

BackgroundA given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC.Principal FindingsWe generated C57BL/6-derived embryonic stem cells that are transgenic for a humanized c-Myc gene and established a mouse strain (hc-Myc) that expresses human c-MYC instead of the murine ortholog. These transgenic animals harbor the humanized c-Myc gene integrated into the endogenous murine c-Myc locus. Despite the lack of the endogenous murine c-Myc gene, homozygous mice show a normal phenotype indicating that human c-MYC can replace its murine ortholog.ConclusionsThe newly established hc-Myc mouse strain provides a model system to study in detail the adverse effects of therapies that target the human c-MYC protein. To mimic the clinical situation, hc-Myc mice may be cross-bred to mice that develop tumors due to overexpression of human c-MYC. With these double transgenic mice it will be possible to study simultaneously the therapeutic efficiency and adverse side effects of MYC-specific therapies in the same mouse.

1098 Macrophages make up the bulk of the leukocyte infiltrate in most solid tumors. These so-called tumor associated macrophages (TAMs) are thought to be recruited by tumor-derived chemokines and converted to a tumor-permissive state. TAMs are called M2, or alternatively-activated macrophages, to distinguish them from M1, or classically-activated macrophages. Of interest, these TAMs are not able to fight the tumor; instead, they play a tumor-supporting role via their ability to promote angiogenesis and metastases. In Burkitt's lymphoma infiltration of the tumor masses by macrophages is a characteristic morphological hallmark, while the phenotype and the relevance of TAMs as part of the stroma are poorly understood. Using a murine model of spontaneous Burkitt's lymphoma (λ-hu-c-myc transgenic mice), we therefore characterized macrophages infiltrating lymphoma masses and show here that infiltrating macrophages display an anti-inflammatory M2-phenotype characterized by the expression of surface markers, such as CD206. In addition, they secrete TGFβ and IL-10, which are known not only to support B-cell growth, but suppress T-cell immunity at the tumor site. In vitro analysis of human macrophages generated from PBMC either by GM-CSF to generate the M1 phenotype, or by M-CSF to obtain the M2 phenotype, revealed that both types display a distinct cytotoxic potential. When incubated together with different Burkitt's lymphoma cells, M2 macrophages did not efficiently lyse lymphoma cells. In contrast, M1 macrophages revealed a strong cytotoxic activity towards lymphoma cells, in a TNF, TRAIL, FasL and NO independent manner, as shown by antibody blocking studies. Investigating this mechanism further, we found that M1 macrophages, in contrast to M2 macrophages, express high levels of the Vitamin D inducible Cathelicidin antimicrobial peptide. Strikingly, we demonstrate that cathelicidin has a cytotoxic effect on lymphoma cells, but not on non-malignant B-cells. Of note, excess supplementation of Vitamin-D increased the cytotoxicity of M2 macrophages against lymphoma cells. We conclude that Vitamin D may therefore point into a therapeutic option to influence a tumor stromal component that greatly impacts on survival and immunogenicity of malignant B-cells. Disclosures: Mackensen: Micromet Inc.: Research Funding.