ALBERT

Description: Background The presence of abundant macrophages is characteristic morphological hallmark for the diagnosis of Burkitt´s lymphoma. They are considered to be the removers of cellular debris which occurs due to the rapid proliferation of such lymphomas. However, although macrophages represent the main non-malignant population within such lymphomas and therefore can be considered a substantial part of the stroma, little is known about how these cells are being recruited and how they influence malignant growth. Since malignant B–cells show high plasticity and have capacity to differentiate into various phenotypes we analysed the origin of macrophages in a murine model of Burkitt´s lymphoma. In this transgenic model a human c-myc gene is overexpressed in a B-cell specific fashion. Results When long term cultured lymphoma cells from transgenic animals were transferred into wild type animals expressing the congenic marker CD45.1 lymphomas grew out within 14 days post transfer. Lymphomas were minced into single cell suspension and macrophages were sorted by flow cytometry based on the expression of CD11b and F4/80. Approximately 6% of the cells expressed F4/80 and CD11b as macrophage markers and displayed an M2 phenotype. Surprisingly approximately 50% of these macrophages also expressed CD45.2 and therefore were not of host origin but derived from the transferred lymphoma cells. These cells were positive for the c-myc transgene, but displayed reduced expression of c-myc as shown by RT PCR in comparsion to the lymphoma cell line in culture. Furthermore, lymphoma derived macrophages displayed reduced PAX5 expression and were negative for CD19 surface expression. When lymphoma cells were retrovirally transduced to express bcl2 to block apoptosis, and were treated with chemotherapy in vivo, we observed a massive increase in macrophage numbers in remaining tissues suggesting transdifferenziation as an alternative exit strategy in case of cellular stress. Conclusion These results demonstrate that BL cells are able to differentiate into a macrophage phenotype to form their own supporting stroma. In addition, differentiation after DNA damage such as chemotherapy is increased suggesting a new exit strategy when classic apoptotic pathways are blocked. Disclosures: Mackensen: Eli Lilly: Consultancy.

Description: B-cell lymphomas, such as Burkitt's lymphoma, are malignant diseases of the hematopoietic system. They arise from transformed B-cells originating from diverse primary and secondary lymphatic tissues. Like in other cancers, the stroma of lymphomas is typically infiltrated by so-called tumor-associated macrophages (TAMs), which execute manifold tumor-specific functions. Interestingly it was shown, that mature B-cells could be efficiently re-programmed into macrophages by the overexpression of myeloid-specific transcription factors. Moreover, other studies observed in vitro that this lymphoid/myeloid plasticity might be also caused by oncogenes in cultured B-cells of murine lymphoma models. Therefore we consider, if lymphoma B-cells themselves might be a source of TAMs, besides the well-known infiltration of monocytic cells into the tumor environment. Based on the system of CD45.1/2 allotypes, a murine model of lymphoma was thus developed, which allows tracking of the conversion of lymphoma B-cells into TAMs. It could be shown, that some lymphoma B-cells of the established model in fact spontaneously switched into a macrophage-like phenotype. Accordingly, they start to express typical macrophage markers, but seem to perform a transition into a myeloid-like expression profile on the level of transcription as well. Furthermore, analysis of the recombined immunoglobulin heavy chain confirmed the clonal identity of lymphoma b-cells and TAMs. Although these cells do not exhibit an overt immunological phenotype, they show elementary functional properties of macrophages, such as phagocytosis as well as a post-mitotic character. Moreover, transdifferentiated B-cells cells were resistant to chemotherapy and persist after treatment, while lymphoma B-cells were eradicated. Which consequences the lymphoid/myeloid plasticity of lymphoma B-cells has for tumor development, progression, as well as for the outcome of therapy, and if these observations could be transferred to human tumors, will be object of future investigations. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear. Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry. Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells. Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1035 Introduction: Distinct macrophage subsets have been linked with either protective or pathogenic roles in cancer. M2 macrophages have been shown to suppress adaptive tumour-specific immune responses and promote tumour growth, while M1 macrophages eliminate neoplastic cells and activate tumour-killing mechanisms. Macrophages isolated from solid tumours (tumor-associated macrophages, TAM) reveal a suppressive and tumor promoting M2-like phenotype. However, it remains unclear which tumoricidal effector mechanisms are compromised in theses immune cells. While infilatration of macrophages is a characteristic morphological hallmark in Burkitts' lymphoma (BL), the phenotype and functional properties of TAM as part of the stroma are poorly understood. In this study we investigated the phenotype of lymphoma associated macrophages (LAM) and the mechanisms by which macrophages are able to modulate the growth of tumor cells. Methods: We included 19 patients diagnosed with BL and 20 patients with benign reactive lymphadenopathy as a control group. Phenotypic characterizations of LAM were evaluated by immunohistochemistry and by qPCR in paraffin embedded tissues. To investigate anti lymphoma cytotoxicity of distinct macrophage subsets we generated macrophages from PBMC either by GM-CSF to create the M1 phenotype, or by M-CSF to obtain the M2 phenotype. M1 and M2 macrophages were coincubated them with several BL cell lines and cytotoxicity was analyzed by flow cytometry, qPCR and immunofluorescence. Results: First, we could demonstrate that BL infiltrating macrophages displayed an anti-inflammatory M2-phenotype characterized by expression of surface markers such as CD68 and CD163. Second, we identified impaired vitamin D metabolism in LAM and M2 macrophages as demonstrated by low expression of vitamin D receptor and Cyp27B1, and increased expression of Cyp24A1. Third, macrophages revealed a reduced cytotoxic potential towards lymphoma cells which was dependent on the expression of the vitamin D dependent peptide cathelicidin. Finally, we could demonstrate that excess supplementation of calcitriol led to increased cytotoxicity of M1- and M2 macrophages towards BL cells. Conclusions: These results suggest a mechanism in which vitamin D is required for innate immunity to overcome the ability of lymphoma cells to evade macrophage-mediated antitumoral responses. The present findings underscore the importance of vitamin D for sustaining innate immunity and imply that the therapeutic activation of the vitamin D pathway may even result in triggering tumoricidal effector mechanisms of LAM. Disclosures: No relevant conflicts of interest to declare.

Description: In high-grade lymphatic malignancies, such as Burkitt’s lymphoma (BL), macrophage infiltration represents a characteristic morphological hallmark. Although macrophages can be directly cytotoxic against tumor cells, tumor associated macrophages (TAMs) regularly fail to kill them. The mechanism responsible for this loss of function remains unknown. Here, we firstly demonstrate that M1 (classically activated) macrophages selectively kill proliferating lymphoma cells by releasing the antimicrobial peptide cathelicidin in a vitamin D dependent fashion. Moreover, we show that cathelicidin leads to cell death by targeting the mitochondria. In contrast, M2 (alternatively activated) macrophages, and M2-like TAMs in BL exhibit an altered vitamin D metabolism that leads to reduced production of cathelicidin and consequently failure to lyse lymphoma cells. However, treating M2 macrophages with 1,25D or a vitamin D receptor agonist efficiently triggers cathelicidin-mediated tumoricidal activity against BL cells. Taken together, these observations indicate that a therapeutic activation of the vitamin D pathway could restore antitumor activity of TAMs, which are an important player in the tumor stroma. Disclosures: Mackensen: Eli Lilly: Consultancy.

Description: Abstract 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.

Description: 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.