Journal Articles

Cancer Immunobiology

Basic research studies that extend knowledge in the field of cancer immunotherapy

M1 macrophage recruitment correlates with worse outcome in SHH Medulloblastomas

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BACKGROUND: Recent progress in molecular analysis has advanced the understanding of medulloblastoma (MB) and is anticipated to facilitate management of the disease. MB is composed of 4 molecular subgroups: WNT, SHH, Group 3, and Group 4. Macrophages play a crucial role in the tumor microenvironment; however, the functional role of their activated phenotype (M1/M2) remains controversial. Herein, we investigate the correlation between tumor-associated macrophage (TAM) recruitment within the MB subgroups and prognosis. METHODS: Molecular subgrouping was performed by a nanoString-based RNA assay on retrieved snap-frozen tissue samples. Immunohistochemistry (IHC) and immunofluorescence (IF) assays were performed on subgroup identified samples, and the number of polarized macrophages was quantified from IHC. Survival analyses were conducted on collected clinical data and quantified macrophage data. RESULTS: TAM (M1/M2) recruitment in SHH MB was significantly higher compared to that in other subgroups. A Kaplan-Meier survival curve and multivariate Cox regression demonstrated that high M1 expressers showed worse overall survival (OS) and progression-free survival (PFS) than low M1 expressers in SHH MB, with relative risk (RR) values of 11.918 and 6.022, respectively. CONCLUSION: M1 rather than M2 correlates more strongly with worse outcome in SHH medulloblastoma.

Author Info: (1) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. (2) Medical Research Collaborating Center, Seoul National

Author Info: (1) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. (2) Medical Research Collaborating Center, Seoul National University Hospital, Seoul, South Korea. (3) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. (4) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. (5) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. (6) Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea. (7) Department of Pathology, Yonsei University, College of Medicine, Severance Hospital, Seoul, Republic of Korea. (8) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea. (9) Division of Pediatric Neurosurgery, Seoul National University Children's Hospital, 101 Daehakro, Jongno-gu, 110-744, Seoul, Republic of Korea. phi.jihoon@gmail.com.

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A genome-wide survey of mutations in the Jurkat cell line

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BACKGROUND: The Jurkat cell line has an extensive history as a model of T cell signaling. But at the turn of the 21st century, some expression irregularities were observed, raising doubts about how closely the cell line paralleled normal human T cells. While numerous expression deficiencies have been described in Jurkat, genetic explanations have only been provided for a handful of defects. RESULTS: Here, we report a comprehensive catolog of genomic variation in the Jurkat cell line based on whole-genome sequencing. With this list of all detectable, non-reference sequences, we prioritize potentially damaging mutations by mining public databases for functional effects. We confirm documented mutations in Jurkat and propose links from detrimental gene variants to observed expression abnormalities in the cell line. CONCLUSIONS: The Jurkat cell line harbors many mutations that are associated with cancer and contribute to Jurkat's unique characteristics. Genes with damaging mutations in the Jurkat cell line are involved in T-cell receptor signaling (PTEN, INPP5D, CTLA4, and SYK), maintenance of genome stability (TP53, BAX, and MSH2), and O-linked glycosylation (C1GALT1C1). This work ties together decades of molecular experiments and serves as a resource that will streamline both the interpretation of past research and the design of future Jurkat studies.

Author Info: (1) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. lhgioia@scripps.edu. (2) Next Generation Sequencing Core, The Scripps Research Institute, La

Author Info: (1) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. lhgioia@scripps.edu. (2) Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, California, 92037, USA. (3) Next Generation Sequencing Core, The Scripps Research Institute, La Jolla, California, 92037, USA. (4) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA. (5) Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA.

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Cytomegalovirus: an unlikely ally in the fight against blood cancers

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CMV infection is a potentially fatal complication in patients receiving HSCT, but recent evidence indicates that CMV has strong anti-leukemia effects due in part to shifts in the composition of NK-cell subsets. NK-cells are the primary mediators of the anti-leukemia effect of allogeneic HSCT and infusion of allogeneic NK-cells has shown promise as a means of inducing remission and preventing relapse of several different hematologic malignancies. The effectiveness of these treatments is limited, however, when tumors express HLA-E, a ligand for the inhibitory receptor NKG2A which is expressed by the vast majority of post-transplant reconstituted and ex vivo expanded NK-cells. It is possible to enhance NK-cell cytotoxicity against HLA-E(pos) malignancies by increasing the proportion of NK-cells expressing NKG2C (the activating receptor for HLA-E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2C(pos) /NKG2A(neg) NK-cells is typically low in healthy adults, but it can be increased by CMV infection or ex vivo expansion of NK-cells using HLA-E transfected feeder cells and IL-15. In this review, we will discuss the role of CMV-driven NKG2C(pos) /NKG2A(neg) NK-cell expansion on anti-tumor cytotoxicity and disease progression in the context of hematologic malignancies, and explore the possibility of harnessing NKG2C(pos) /NKG2A(neg) NK-cells for cancer immunotherapy. This article is protected by copyright. All rights reserved.

Author Info: (1) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences

Author Info: (1) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA. (2) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA. (3) Laboratory of Integrated Physiology, Department of Health and Human Performance, University of Houston, 3875 Holman Street, Houston, Texas, 77204, USA. Department of Nutritional Sciences, The University of Arizona, 1177 E. Fourth Street, Tucson, Arizona, 85721, USA.

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hIL-15-gene modified human natural killer cells (NKL-IL15) exhibit anti-human leukemia functions

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PURPOSE: Natural killer (NK) cells can kill transformed cells and represent anti-tumor activities for improving the immunotherapy of cancer. In previous works, we established human interleukin-15 (hIL-15) gene-modified NKL cells (NKL-IL15) and demonstrated their efficiency against human hepatocarcinoma cells (HCCs) in vitro and in vivo. To further assess the applicability of NKL-IL15 cells in adoptive cellular immunotherapy for human leukemia, here we report their natural cytotoxicity against leukemia in vitro and in vivo. METHODS: Flow cytometry, ELISA and MTT methods were performed for molecular expression, cell proliferation and cytotoxicity assays. Leukemia xenograft NOD/SCID mice were established by subcutaneous injection with K562 cells, and then treated with irradiated NKL cells. RESULTS: We found NKL-IL15 cells displayed a significant high cytolysis activity against both human leukemia cell lines and primary leukemia cells from patients, accompanied with up-regulated expression of molecules related to NK cell cytotoxicity such as perforin, granzyme B and NKp80. Moreover, cytokines secreted by NKL-IL15 cells, including TNF-alpha and IFN-gamma, could induce the expression of NKG2D ligands on target cells, which increased the susceptibility of leukemia cells to NK cell-mediated cytolysis. Encouragingly, NKL-IL15 cells significantly inhibited the growth of leukemia cells in xenografted NOD/SCID mice and prolonged the survival of tumor-bearing mice dramatically. Furthermore, NKL-IL15 cells displayed stimulatory effects on hPBMCs, indicating the immunesuppressive status of leukemia patients could be improved by NKL-IL15 cell treatment. CONCLUSIONS: These results provided evidence that IL-15 gene-modification could augment NK cell-mediated anti-human leukemia function, which would improve primary NK cell-based immunotherapy for leukemia in future.

Author Info: (1) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (2) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical

Author Info: (1) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (2) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (3) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. (4) Institute of Immunopharmaceutical Sciences, School of Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan, China. zhangj65@sdu.edu.cn.

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IL-35-producing B cells in gastric cancer patients

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A significant characteristic of advanced gastric cancer (GC) is immune suppression, which can promote the progression of GC. Interleukin 35 (IL-35) is an immune-suppressing cytokine, and it is generally recognized that this cytokine is secreted by regulatory T (Treg) cells. Recently, studies have found that IL-35 can also be produced by B cells in mice. However, scientific studies reporting that IL-35 is secreted by B cells in humans, specifically in cancer patients, are very rare.Blood samples were collected from 30 healthy controls (HCs) and 50 untreated GC patients, and IL-35-producing B cells in the peripheral blood were investigated. Moreover, Treg cells (CD4CD25CD127), myeloid-derived suppressor cells (MDSCs) (CD14HLA-DR) and other lymphocyte subsets (CD3, CD4, CD8 T cells, activated and memory CD4 T cells, activated CD8 T cells, CD14 monocytes, and IL-10-producing B cells) were also examined.IL-35-producing B cells were significantly upregulated in patients with advanced GC. Furthermore, the frequency of IL-35-producing B cells was positively correlated with the frequencies of Treg cells (CD4CD25CD127), MDSCs (CD14HLA-DR), IL-10-producing B cells, and CD14 monocytes in these GC patients.In summary, the frequency of IL-35-producing B cells is significantly elevated in advanced GC; this outcome implies that this group of B cells may participate in GC progression.

Author Info: (1) Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou. (2) Department of Pharmacology, JiangXi Medical College, Shangrao, China. (3) Department of Gastroenterology

Author Info: (1) Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou. (2) Department of Pharmacology, JiangXi Medical College, Shangrao, China. (3) Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou.

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Single-Molecule Light-Sheet Imaging of Suspended T Cells

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Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

Author Info: (1) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (2) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (3) Department of Chemistry, University

Author Info: (1) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (2) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (3) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (4) Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom. (5) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (6) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. (7) Radcliffe Department of Medicine and MRC Human Immunology Unit, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom. Electronic address: simon.davis@imm.ox.ac.uk. (8) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: dk10012@cam.ac.uk. (9) Department of Chemistry, University of Cambridge, Cambridge, United Kingdom. Electronic address: sl591@cam.ac.uk.

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Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function

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When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8(+) T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8(+) T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8(+) T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8(+) T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8(+) T cell activation, mediated by the binding of empty HLA-I to CD8.

Author Info: (1) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States. (2) Department of Microbiology and Immunology, Yerkes National Primate Research Center, Emory

Author Info: (1) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States. (2) Department of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University School of Medicine, Atlanta, United States. (3) Research and Development, Sirona Genomics, Immucor, Inc, Mountain View, United States. (4) Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States.

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A Novel Three-Dimensional Immune Oncology Model for High-Throughput Testing of Tumoricidal Activity

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The latest advancements in oncology research are focused on autologous immune cell therapy. However, the effectiveness of this type of immunotherapy for cancer remediation is not equivalent for all patients or cancer types. This suggests the need for better preclinical screening models that more closely recapitulate in vivo tumor biology. The established method for investigating tumoricidal activity of immunotherapies has been study of two-dimensional (2D) monolayer cultures of immortalized cancer cell lines or primary tumor cells in standard tissue culture vessels. Indeed, a proven means to examine immune cell migration and invasion are 2D chemotaxis assays in permeabilized supports or Boyden chambers. Nevertheless, the more in vivo-like three-dimensional (3D) multicellular tumor spheroids are quickly becoming the favored model to examine immune cell invasion and tumor cell cytotoxicity. Accordingly, we have developed a 3D immune oncology model by combining 96-well permeable support systems and 96-well low-attachment microplates. The use of the permeable support system enables assessment of immune cell migration, which was tested in this study as chemotactic response of natural killer NK-92MI cells to human stromal-cell derived factor-1 (SDF-1alpha). Immune invasion was assessed by measuring NK-92MI infiltration into lung carcinoma A549 cell spheroids that were formed in low-attachment microplates. The novel pairing of the permeable support system with low-attachment microplates permitted simultaneous investigation of immune cell homing, immune invasion of tumor spheroids, and spheroid cytotoxicity. In effect, the system represents a more comprehensive and in vivo-like immune oncology model that can be utilized for high-throughput study of tumoricidal activity.

Author Info: (1) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (2) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (3) Life Sciences Division, Corning

Author Info: (1) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (2) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States. (3) Life Sciences Division, Corning Incorporated, Kennebunk, ME, United States.

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LMP2 immunoproteasome promotes lymphocyte survival by degrading apoptotic BH3-only proteins

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The role of the immunoproteasome is perceived as confined to adaptive immune responses given its ability to produce peptides ideal for MHC Class-I binding. Here, we demonstrate that the immunoproteasome subunit, LMP2, has functions beyond its immunomodulatory role. Using LMP2-deficient mice, we demonstrate that LMP2 is crucial for lymphocyte development and survival in the periphery. Moreover, LMP2-deficient lymphocytes show impaired degradation of key BH3-only proteins, resulting in elevated levels of pro-apoptotic BIM and increased cell death. Interestingly, LMP2 is the sole immunoproteasome subunit required for BIM degradation. Together, our results suggest LMP2 has important housekeeping functions and represents a viable therapeutic target for cancer. This article is protected by copyright. All rights reserved.

Author Info: (1) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (2) The Walter, Eliza Hall Institute of Medical Research, 1G Royal

Author Info: (1) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (2) The Walter, Eliza Hall Institute of Medical Research, 1G Royal Melbourne Hospital, Parkville, VIC, 3010, Australia. Department of Paediatrics, University of Melbourne, Parkville, Australia. Department of Psychiatry, University of Melbourne, Parkville, Australia. Murdoch Children's Research Institute, Parkville, Australia. (3) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (4) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (5) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (6) Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, VIC, 3004, Australia. (7) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (8) The Walter, Eliza Hall Institute of Medical Research, 1G Royal Melbourne Hospital, Parkville, VIC, 3010, Australia. (9) Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, VIC, 3004, Australia. (10) Australian National University, Canberra, Australia. (11) Department of Immunology, Monash University, AMREP Melbourne, 3004, Victoria, Australia. (12) The Walter, Eliza Hall Institute of Medical Research, 1G Royal Melbourne Hospital, Parkville, VIC, 3010, Australia. (13) Peter MacCallum Cancer Centre, East Melbourne, VIC, 3002, Australia. (14) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia. (15) La Trobe Institute of Molecular Science, La Trobe University, Bundoora, VIC, 3086, Australia.

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The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

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Cytotoxic necrotizing factor 1 (CNF1) is a bacterial protein toxin primarily expressed by pathogenic Escherichia coli strains, causing extraintestinal infections. The toxin is believed to enhance the invasiveness of E. coli by modulating the activity of Rho GTPases in host cells, but it has interestingly also been shown to promote inflammation, stimulate host immunity and function as a potent immunoadjuvant. The mechanisms underlying the immunostimulatory properties of CNF1 are, however, poorly characterized, and little is known about the direct effects of the toxin on immune cells. Here, we show that CNF1 induces expression of maturation markers on human immature monocyte-derived dendritic cells (moDCs) without compromising cell viability. Consistent with the phenotypic maturation, CNF1 further triggered secretion of proinflammatory cytokines and increased the capacity of moDCs to stimulate proliferation of allogenic naive CD4+ T cells. A catalytically inactive form of the toxin did not induce moDC maturation, indicating that the enzymatic activity of CNF1 triggers immature moDCs to undergo phenotypic and functional maturation. As the maturation of dendritic cells plays a central role in initiating inflammation and activating the adaptive immune response, the present findings shed new light on the immunostimulatory properties of CNF1 and may explain why the toxin functions as an immunoadjuvant.

Author Info: (1) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. lgmas@sund.ku.dk. (2) Italian Center for Global Health, Istituto Superiore di

Author Info: (1) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. lgmas@sund.ku.dk. (2) Italian Center for Global Health, Istituto Superiore di Sanita; Viale Regina Elena 299, 00161 Rome, Italy. alessia.fabbri@iss.it. (3) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. mnamini@sund.ku.dk. (4) Costerton Biofilm Center, Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. mgivskov@sund.ku.dk. (5) Italian Center for Global Health, Istituto Superiore di Sanita; Viale Regina Elena 299, 00161 Rome, Italy. carla.fiorentini@iss.it. (6) Department of Immunology and Microbiology, University of Copenhagen, Norre Alle 14, 2200 Copenhagen, Denmark. thorkr@sund.ku.dk.

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