ALBERT

Beschreibung: Key Points Lack of KLRG1 and T-bet expression is a unique feature of DNT and subsets of single positive T cells in ALPS patients. Genetic, phenotypic, and transcriptional evidence indicates that DNT in ALPS patients derive from both CD4+ and CD8+ T cells.

Beschreibung: Abstract 584 Specific T-cell responses are initiated by T-cell receptor (TCR) recognition of peptide-MHC-complexes on antigen presenting cells (APCs). Upon specific interaction of T cells with APCs T cells capture membrane fragments and surface molecules of APCs in a process termed trogocytosis. Exchange of membrane molecules/antigens between immune cells has been observed for a long time, but the mechanisms and functional consequences of these transfers remain unclear. Here, we demonstrate that human antigen-specific CD8+ T cells do acquire the co-inhibitory molecule programmed death ligand 1 (PD-L1) from mature monocyte-derived dendritic cells (mDC) and tumor cells in an antigen-specific manner. The kinetics of PD-L1 transfer revealed a maximal PD-L1 expression on antigen-specific T cells within 3–4 hours after co-incubation with antigen-pulsed APCs, being detectable up to 72 hours. Antigen-pulsed immature DCs were less effective in transfering surface molecules such as PD-L1 onto CD8+ T cells after antigen-specific recognition. Using a transwell system we could show that the acquisition of PD-L1 requires cell-cell contact. Furthermore, PD-L1 cannot be acquired by T cells from a lysate of mDCs. The transfer process is impaired after pretreatment of T cells with concanamycin A, a specific inhibitor of vacuolar ATPases, playing an important role in membrane trafficking. Moreover, fixation of DCs with glutaraldehyde completely abrogated the acquisition of PD-L1 on T cells suggesting that an active interaction between APCs and T cells is required for trogocytosis. Of importance, CD8+ T cells which acquired PD-L1 complexes, were able to induce apoptosis of neighbouring PD-1 expressing CD8+ T cells, that could be completely blocked by an anti-PD-L1 antibody. In summary our data demonstrate for the first time that human antigen-specific CD8+ T cells take up functionally active PD-L1 from APCs in an antigen-specific fashion, leading to apoptosis of PD-1 expressing T cells. The transfer of functionally active co-inhibitory molecules from APCs onto human CD8+ T cells may serve to limit clonal expansion of antigen-specific T-cell responses but may also play a major role for T-cell exhaustion in chronic infection and tumor immunosurveillance. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3491 Novel methods to quantify chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) based on single-nucleotide polymorphism (SNP) specific real-time quantitative polymerase chain reaction (qPCR) require high amounts of input DNA. The applicability of these methods in cases where DNA quantity is limiting, however, is currently unclear. Here, we demonstrate that SNP typing performed with just 5ng of input DNA still allowed for the distinction of positive (mean ct 28.05) and negative (ct 〉 36) signals. In addition, we confirm the high informative value of a set of 19 SNP markers, with 12 markers exceeding 20% informativity in our population (n=74). Of interest, a four-fold reduction of input DNA for qPCR standard curves did not alter PCR efficiencies. Most importantly, in 7 out of 16 allogeneic HSCT patients who experienced disease relapse, a retrospective analysis using the SNP-qPCR method revealed re-appearance of recipient chimerism earlier (mean 95 days) compared to previous results obtained by a short tandem repeat (STR) specific PCR. Taken together our data clearly demonstrate that SNP-qPCR is more sensitive compared to the widely used STR-PCR even with reduced amounts of input DNA. We therefore recommend that SNP-qPCR is preferable to STR-PCR for chimerism analysis in cases where DNA quantity is a limiting factor. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3004 Clinical studies exploiting the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation (HSCT) have provided promising results. It is known that NK cells are a heterogeneous population and can be divided into functionally distinct NK cell subpopulations. Murine NK cells can be separated along their expression of CD27 and CD11b and CD117 (c-kit). However, the functional relevance of distinct NK cell subsets in graft-versus-host-disease (GVHD) has not been investigated in detail so far. We have established different protocols for ex vivo isolation and expansion of murine NK cell subpopulations. These NK subsets were further analyzed in vitro and in vivo in an allogeneic murine GVHD model. Here we report on different genomic, phenotypic and functional properties of 4 NK cell subsets. Our data clearly demonstrate that CD27+ NK cells revealed the highest IFN-g production upon coculture with tumor cells and/or IL-2. Interestingly, the CD11b+ NK cells express multiple genes of cytotoxic pathways and develop the highest cytotoxic capacity towards tumor cells. We observed up to 60% tumor lysis by CD27- CD11b+ NK cells compared to 40–45% by CD27+ CD11b+, about 25% by CD27+ CD11b- and 10% by c-kit+ CD11b- NK cells at an effector-target ratio of 5:1, respectively. Furthermore, the CD11b+ NK cell subset significantly reduced T cell proliferation induced by allogeneic dendritic cells in mixed lymphocytes reactions. Next, we analyzed the migratory capacity and tissue-specific homing of FACS-sorted NK cell subsets by adoptive transfer of congeneic CD45.1+ and Luc+ NK cell subpopulations in autologous and allogeneic bone marrow transplantation. Of interest, FACS analysis and in vivo imaging showed that CD11b+ NK cells migrated to peripheral GVHD target organs, whereas CD27+ NK cells preferentially homed to the bone marrow. Finally, this study addressed for the first time the role of distinct NK cell subpopulations in the development of GVHD in a fully MHC mismatched HSCT mouse model. Importantly, we identified the CD11b+ NK cell population as the NK cell subset that significantly diminished GVHD. In vivo imaging of Luc+CD11b+ NK cells revealed that this subset migrates to the colonic tissue to prevent development of GVHD colitis as shown by colonoscopy. In summary, our comparative study outlines that only CD11b+ NK cells, migrating to the peripheral GVHD target organs and providing the most efficient cytolytic capacity directed against allogeneic dendritic cells, protect against GVHD. These new insights are highly relevant for the selection of optimal NK cell subsets in the field of cellular immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). Recently, a novel subset of TCRαβ+ CD4- CD8- (double negative, DN) T cells has been described to possess a very potent suppressive activity on allogeneic T-cell responses in both mice and humans. Of interest, clinical studies in patients after allogeneic stem cell transplantation (SCT) revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. Interleukin (IL)-7 is required for survival and homeostasis of mature T-cells and has been shown to modulate the functional activity of Treg cells. Here we asked whether IL-7 affects the suppressive activity of human DN T cells. We found that human DN T cells isolated from peripheral blood lymphocytes do express the IL-7 receptor and are able to transduce IL-7 downstream signaling. Intriguingly, IL-7 significantly diminished the suppressive activity of DN T cells towards allogeneic CD4+ and CD8+ T cells. Thereby, IL-7 did not render the responder T cells insensitive to suppression. Instead, blocking of key signaling molecules revealed that IL-7 has a direct impact on the suppressive activity of DN T cells. Further analyses demonstrated that IL-7 activates Akt/mTOR signaling in DN T cells. Importantly, selective inhibition of the Akt or mTOR protein kinase was able to reverse the IL-7 effect thereby restoring the suppressive function of DN T cells, whereas inhibition of other central T-cell signaling pathways did not. Thus, DN T-cell mediated suppression is impaired by IL-7 due to crucial activation of the Akt/mTOR signaling cascade. These findings indicate that IL-7 and Akt/mTOR signaling are critical factors for the suppressive capacity of DN T cells. Targeting of these pathways by pharmacological agents may restore and/or enhance DN T-cell functionality in GvHD. Disclosures No relevant conflicts of interest to declare.

Beschreibung: 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.

Beschreibung: Reactive oxygen species (ROS) such as hydrogen peroxide or superoxide are highly reactive and hyperpermeable. They are continuously produced by mainly the mitochondria during oxidative phosphorylation. Immune cells of especially myeloid origin contribute additionally to the ROS production via the membrane-bound NADPH-oxidase. During inflammation or “inflammatory-like” conditions as often seen in neoplasias ROS production is highly increased. The physiological redox-buffers are overwhelmed leading to the metabolic condition termed oxidative stress. Immune cells, especially NK- and T-cells are very sensitive towards ROS-induced cell death resulting from mitochondrial depolarization. Furthermore, ROS can interfere with nuclear NF-kB translocation thus leading to reduced activation, cytokine production, and cytotoxic activity of immune cells. Functional immune defects at several levels as well as significant shifts in the balance of immune populations have been related to oxidative stress in patients suffering from mainly solid malignant diseases. In this study we sought out to investigate the presence of oxidative stress in patients with chronic lymphocytic leukemia (CLL) and its potential effects on their cellular immune compartment. Peripheral blood and serum from 65 patients with untreated CLL was collected upon informed consent. Oxidative stress was measured based on the concentration of so-called ROS-surrogate markers in the patients' sera. Therefore oxidized lipids, proteins, and DNA bases were quantified by ELISA. The immune compartment was evaluated using multicolor flow cytometry and the analyses included T-, NK-, myeloid-, and dendritic-cell subsets. ROS levels were increased in CLL patients. Based on the concentration of ROS-modified molecules we were able to group our patient cohort into a ROShi and ROSlo group. The ROShi group exhibited lower levels of T-cell activation, while there was a shift of the CD56bright/CD56dim NK-cell balance towards the CD56bright subset. We could not detect significant differences in the myeloid- and dendritic-cell compartment or alterations of T- and NK-cell maturation. Our results implicate that ROS belongs to the tumor immune escape mechanism present in CLL. The use of redox modifying agents in combination with the steadily emerging immune-based approaches could potentially boost their therapeutic efficacy. Disclosures: No relevant conflicts of interest to declare.