The path to new discoveries

Transcription

1 Vol 15 1/2013 The path to new discoveries Highly reliable gene expression profiling Isolation of B cells by MACSxpress Technology for sensitive and concordant microarray analysis (p. 10) From basic to clinical research in immunology Multiple myeloma research Automated purification of CD138 + cells from whole bone marrow for FISH analysis (p. 18) Malaria research Rapid, automated purification of Plasmodium falciparum infected erythrocytes for biochemical studies (p. 31)

2 Contents Page News Enjoy our redesigned website 4 Meet our best kits yet 5 As fast as it gets: cell isolation from whole blood 6 with MACSxpress Technology Automated workflows in immunology research 7 using simplicity to control complexity The CliniMACS CD34 Reagent System 8 a landmark in cellular therapy Reports MACSxpress Technology allows isolation of B cells with high purity 10 for sensitive and concordant microarray-based gene expression profiling Angela Mekes, Stephanie Soltenborn, Ines Dischinger, Volker Huppert, and Bernhard Gerstmayer Isolation of monocytes with high purity directly from whole blood 14 for transcriptome analysis in translational research Christelle Foucher, Sébastien Vachenc, Alexandre Meniccaci, Bernhard Gerstmayer, Gwenola Henrion, Uwe Janssen, Darren Wilbraham, Karine Le Malicot, and Jean-Claude Ansquer An automated method for purification of CD138 + cells from whole 18 bone marrow samples for multiple myeloma research Hossain Mossafa and Sabine Defasque Highly purified peripheral blood γ/δ T cells isolated by MACS Technology 20 respond to NOD2 ligand Lothar Marischen, Hans-Heinrich Oberg, Christian Peters, Sandra Ussat, Hoa Ly, Dieter Kabelitz, and Daniela Wesch Efficient and rapid in vitro generation of fully functional 24 multi-virus-specific CD4 + and CD8 + T cells Anna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer, Ju rgen Schmitz, Mario Assenmacher, and Anne Richter Mouse NK cells isolated to high purity 28 by MACS Technology are fully functional Kathrin Meinhardt, Irena Kroeger, Sabine Mueller, and Evelyn Ullrich Measuring prolyl aminopeptidase activity in extracts prepared from 31 magnetically purified malaria parasites Fabio L. da Silva, Donald L. Gardiner, and Katharine R. Trenholme MACS&more (ISSN ) is published by Miltenyi Biotec GmbH. Editorial board: Anthony Allen, Adrian Arechiga, Conrad Beckers, Caroline Blumer Toti, Sven Brosch, Oliver Burlon, Vincent Chu, Steven Dublin, Guy Hewlett, Christoph Hintzen, Jennifer Horner, Volker Huppert, Claudia Loske, Joumana Masri, Simon Mauch, Mariette Mohaupt, Shane Oram, Gerd Steffens, Nanette von Oppen, Ilka Wege, Raif Yu cel Editor: Ralph Schaloske Graphics & Layout: Miltenyi Biotec GmbH, Graphics team Miltenyi Biotec GmbH Friedrich-Ebert-Straße 68, Bergisch Gladbach, Germany Phone macs@miltenyibiotec.de Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. automacs, CliniMACS, gentlemacs, MACS, MACSmix, MACSQuant, MACSxpress, MultiMACS, PepTivator, Vio, and VioBlue are registered trademarks or trademarks of Miltenyi Biotec GmbH or its affiliates in Germany, the United States, and/or other countries. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Copyright 2013 Miltenyi Biotec GmbH. All rights reserved. 2 MACS & more Vol 15 1/2013

3 MACS&more Vol. Vol /2012 1/2013 Dear Researcher, Detailed research on the properties and function of individual immune cell types deepens our understanding of their role in health and disease. With more than twenty years of experience in biomedical research, Miltenyi Biotec provides a multitude of sophisticated tools for immune cell isolation helping advance research towards the development of future therapies. Usually, the immune system effectively protects the body against disease. However, there are myriads of scenarios in which the immune system is not able to cope with certain challenges, for example, the escape of tumor cells, prolonged inflammation, or autoimmune reactions, all of which can lead to debilitating disease. The immune system is based on a highly organized interplay between a multitude of different cell types each playing a distinct role. In order to accurately analyze the function and characteristics of a specific immune cell type in health and disease, it is essential to use a pure cell population. Miltenyi Biotec offers the tools to isolate immune cells and subsets reliably and efficiently. In this MACS&more issue we present a selection of reports featuring our solutions for magnetic cell separation in a broad spectrum of immunology-related applications. The reports by Angela Mekes et al. and Christelle Foucher et al. highlight the intriguing benefits of our tools for cell isolation directly from whole blood for subsequent microarray-based analyses. Using the new MACSxpress Technology, Mekes et al. showed that B cells isolated from whole blood enabled a more conclusive analysis of cell type specific gene expression than unseparated whole blood or PBMCs. MACSxpress Technology thus provides an excellent basis for biomarker research. lipid-lowering drug fenofibrate. The analysis of differentially expressed genes enabled the construction of functional networks. For their research into malignant cells from multiple myeloma, Hossain Mossafa and Sabine Defasque required a reliable, fast, and standardized technique to enrich CD138 + plasma cells from a large number of bone marrow samples. The automacs Pro Separator and Whole Blood CD138 MicroBeads met all these requirements. Using the isolated CD138 + cells for FISH analysis, the authors were able to reliably detect chromosomal abnormalities that are characteristic for malignant plasma cells. Lothar Marischen et al. investigated the function of the innate receptor NOD2 expressed on γ/δ T cells. For their studies, the authors needed γ/δ T cells and monocytes with an exceptionally high level of purity. MACS Technology enabled Marischen et al. to isolate both cell types from PBMCs to purities of greater than 99%. Likewise, they depleted either cell type from PBMCs to residual percentages of less than 0.1%. Their results support the notion that γ/δ T cells play a role in anti-bacterial immunity. Adoptive transfer of antigen-specific T cells holds great potential for the control of infections after stem cell transplantations. To enhance research into virus-specific T cells, Anna Foerster-Marniok et al. developed a fast and straightforward protocol for the generation and enrichment of T cell populations that are specific for three different viruses. The authors used Miltenyi Biotec s PepTivator Peptide Pools for cell stimulation and the MACS Cytokine Secretion Assay technology to isolate the virus-specific T cells. Isolated virus-specific T cells were fully functional and could be expanded easily. spleen turned out to be challenging. As the C57BL/6 strain is an important model in preclinical NK cell research, Meinhardt et al. set out to solve this issue. They further optimized the depletion procedure and developed a new depletion cocktail, which is now part of the NK Cell Isolation Kit II. This kit enables the isolation of NK cells to high purities from both BALB/c and C57BL/6 spleens. The report by Meinhardt et al. is a great example of how our collaboration with scientists using Miltenyi Biotec products can lead to the development of exciting new research tools. In their quest to identify new targets for antimalarial drug development, Fabio da Silva et al. required preparations of Plasmodium falciparum-infected red blood cells (RBCs) with high purities. To this end, the authors magnetically enriched infected RBCs using the automacs Pro Separator. Unlike immunomagnetic cell isolation based on MACS Technology, their approach did not involve a magnetic labeling step. Instead the parasites labeled themselves: During development P. falciparum digests the host cell s hemoglobin. This leads to the formation of insoluble hemozoin, which has paramagnetic properties. Da Silva et al. were able to identify prolyl aminopeptidase activity in extracts prepared from the magnetically enriched parasites a first step towards the identification of a potential target for drug development. We wish you an inspiring read. Your MACS&more team Foucher et al. used the automacs Pro Separator in combination with Whole Blood CD14 MicroBeads for the fast and reliable isolation of monocytes. The authors analyzed the transcriptome of monocytes purified from whole blood obtained from healthy donors before and after treatment with the For their studies on the function of NK cells, Kathrin Meinhardt et al. obtained highly purified cells from BALB/c mouse spleen using two strategies based on MACS Technology: positive selection and depletion of non-nk cells. However, the isolation of MACS&more high-purity NK online: cells from /macs&more C57BL/6 mouse Vol 15 1/2013 MACS & more 3

4 NEWS Enjoy our redesigned website With you, the busy scientist, in mind, we have redesigned our website. Various new features make miltenyibiotec.com faster, easier, and more intuitive. Save time with easy navigation The simple interface and straightforward navigation guides you to the information you need in no time. The revamped miltenyibiotec.com offers you in-depth insight into products and services for research and its translation into clinical applications. You will also find comprehensive information on individual research areas and numerous support options, as well as general company information. Easy navigation is key to finding appropriate information fast. Start your immunology experiment here To keep you up to date on our wide range of products for your immunology research, we offer a special section on immunologyrelated topics, including autoimmunity, tumor immunology, dermatology, allergy, and infection and inflammation. Discover a plethora of smart products for your sample preparation, cell separation, flow cytometry, molecular applications, and pre-clinical imaging. Also benefit from numerous references and downloads. Find the products you need in just a few clicks The easy-to-use product finder helps you find the right solution for your specific needs, be it an instrument, software, reagents, or consumables. Simply select the appropriate parameters and get started. The product finder guides you to the products you need. Welcome to the immunologist s resource. Start enjoying the new features today at 4 MACS & more Vol 15 1/2013

5 NEWS Meet our best kits yet Miltenyi Biotec introduces the next generation of immune cell isolation kits. Exciting new updates have been made to some of our most popular kits, resulting in new kits that offer unmatched purity, greater recovery, and faster cell isolation. Miltenyi Biotec s continuous quest for product refinement has led to the next generation of kits for the isolation of plasmacytoid dendritic cells (PDCs), monocytes, NK cells, pan T cells, and T cell subsets. Updates were made on various levels. Two kits for the isolation of human PDCs were revamped with depletion cocktails that were redeveloped from scratch, allowing for unmatched PDC purity and recovery. Regular communication with users of our products allowed us to optimize the kits according to the users specific needs. The Pan Monocyte Isolation Kit, for example, was enhanced for excellent recovery of human classical, non-classical, and intermediate monocyte subsets. Besides ensuring outstanding separation performance, the protocols of various kits were modified to save valuable time. Systematic optimization of the reagents allowed us to minimize, for example, the time used for cell labeling, and in many cases it became possible to omit washing steps. Mouse NK cells, for example, can now be isolated in less time with unrivaled purity using the new NK Cell Isolation Kit II. Pure pan monocytes, pan T cells, as well as CD4 + and CD8 + T cells can be obtained in as little as 25 minutes and the isolation of human Treg cells or NK cells can be accomplished up to 20 minutes faster than before. The original kits were already very effective and immensely popular. However, when we realized that there was a way to tweak them, we didn t hesitate to go the extra mile, points out Dr. Claudia Loske, Product Manager, Immunology. Miltenyi Biotec is dedicated to continuously advancing products that provide researchers with the best possible tools stay tuned for the next updates. If you have questions about the individual kits and how they can enhance and accelerate your research, contact our technical support team. Get quick, helpful, local support from a support team in your specific country. Contact information is available at com/support. To get immediate support online, use the Live Chat feature on the starting page of our website. Vol 15 1/2013 MACS & more 5

6 NEWS As fast as it gets: cell isolation from whole blood with MACSxpress Technology Isolating leukocytes from large whole blood samples with no time to lose a challenge numerous labs face every day. Miltenyi Biotec provides the solution: the new MACSxpress Technology. The MACSxpress CD4 T Cell Isolation Kit, human enables isolation of untouched cells directly from whole blood. Fast, safe, and convenient With the MACSxpress Cell Isolation Kits, Miltenyi Biotec is breaking new ground. The technology enables the fastest isolation of cells from whole blood. MACSxpress Technology was specifically developed for cell isolation from large whole blood volumes, in a short time, and with great convenience, explains Volker Huppert, project leader at Miltenyi Biotec s R&D department. Using MACSxpress Kits it takes only 20 minutes to prepare pure leukocyte subsets from whole blood. This means that the entire MACSxpress Cell Isolation procedure is shorter than the sample preparation step in traditional cell isolation protocols. The short process opens up valuable time for users to accomplish other tasks. As the procedure does not involve any centrifugation step or erythrocyte lysis, less pipetting is required, and aerosol formation is minimized. This high level of safety is particularly relevant when processing untested blood samples, which potentially contain infectious particles. Minimal sample handling also means maintenance of sample integrity and a reduced risk for the user. Large-scale isolation of leukocyte subsets Studies often require numerous tests to be conducted with the same sample. Such multiparameter assays require large numbers of isolated cells, which are obtained using MACSxpress Cell Isolation Kits. Using these How it works MACSxpress Cell Isolation is based on a simple procedure: Erythrocytes are aggregated and sedimented without centrifugation. Non-target cells are removed by immunomagnetic depletion with MACSxpress Beads, yielding target cells of high purity. kits up to 30 ml of whole blood can be processed in a single run. Especially when isolating cells with low frequencies, such as NK cells, it is crucial to start with a large sample volume in order to end up with a sufficient number of cells for downstream analysis. MACSxpress Kits meet these requirements perfectly, says Volker Huppert. Simple yet powerful MACSxpress Kits are the perfect solutions for a wide range of applications, whether in basic or translational immunology research. The procedure involves only minimal sample handling, which makes MACSxpress Technology the ideal choice for omics studies, biomarker research, or for drug discovery research. Multicenter research studies can also benefit from the new technology: Users don t need any special expertise, equipment requirements are minimal, and experiments can be carried out in any lab in no time. The simplicity of MACSxpress Technology is compelling, Volker Huppert points out. MACSxpress Cell Isolation Kits are currently available for B cells, naive B cells, NK cells, pan T cells, CD4 + T cells, and CD8 + T cells. Cell isolation with MACSxpress Technology Labeling Separation 20 min Density gradient centrifugation Centrifugation without brake 35 min Also read the report on page 10 of this MACS&more issue: Mekes et al. used MACSxpress Technology to isolate B cells for sensitive gene expression analysis. For general information on MACSxpress Kits visit Time (min) MACSxpress Cell Isolation versus density gradient centrifugation. A leukocyte subset isolation with MACSxpress Technology takes less time than a preparative density gradient centrifugation. With MACSxpress Kits whole blood samples of up to 30 ml can be processed, whereas density gradient centrifugation has a maximum capacity of 15 ml per sample. 6 MACS & more Vol 15 1/2013

7 NEWS Automated workflows in immunology research using simplicity to control complexity Many research projects focusing on immune cell biology require a high sample throughput enabling complex, large-scale experiments with the option to evaluate a high number of parameters simultaneously. Miltenyi Biotec and major liquid handling system providers are working together to provide platforms for sophisticated automated workflows, including cell separation, cell culture, and functional assays. Facing the challenge With the growing complexity of experiments in today s immunology research, the need for high-throughput automated workflow solutions increases. Workflow automation ensures consistent and reliable results through highly standardized, operator-independent procedures. This is of particular relevance in translational research, where multiple centers work together towards a common goal. The minimization of laborious manual handling steps has many advantages and amenities. Productivity increases, as experiments can continue over night and on weekends, and, instead of performing tedious pipetting steps, researchers can focus on other tasks. Automation is key Miltenyi Biotec collaborates closely with major providers of liquid handling systems (LHS), including Tecan and Hamilton Robotics, towards the development of automated workflows supporting most demanding research applications. An example of a project in the planning is a platform for automated monitoring of immune cell activity in the context of tumor formation. The platform is based on the compelling synergy of LHS and Miltenyi Biotec instruments, including the MultiMACS Cell24 Separator for cell separation and the MACSQuant Analyzer for flow cytometric quality control and endpoint analysis. Complex experiment workflow walk-away processes The automated workflow for monitoring immune responses starts with the isolation of monocytes and T cells from PBMCs by the MultiMACS Cell24 Separator. The MACSQuant Analyzer then automatically assesses purity and yield of the separated cell populations. Isolated monocytes are differentiated into monocyte-derived dendritic cells (Mo-DCs) and T cells are cultured for later use. The MACSQuant Analyzer monitors the Mo-DC differentiation status utilizing the reagents of the Mo-DC Differentiation Inspector. As complex the workflow may be, this platform simplifies matters the operator just starts the process and walks away, points out Dr. Raif Yu cel, Product Manager Automation at Miltenyi Biotec. Automation not only saves a lot of time, it also makes for excellent reproducibility. In the next step of the workflow, differentiated Mo-DCs are turned into tumor antigen presenting cells by incubation with tumorspecific peptides. Finally, the antigenpresenting Mo-DCs are cocultured with the T cells, while the MACSQuant Analyzer, in combination with MACS Cytokine Secretion Assays, determines the T cells cytokine response. The magnitude of cytokine production allows valuable conclusions on the repertoire of tumor antigen-specific T cells in the PBMCs. The huge versatility of the LHS and our instruments, cell separation reagents, antibodies, and cell culture products opens up vast possibilities for basic and translational research, Dr. Yu cel says. Workflows can be adapted to meet specific cell isolation requirements, culture conditions, and assays. For more information visit The MultiMACS Cell24 Separator integrated into the LHS for automated high-throughput cell separation. Vol 15 1/2013 MACS & more 7

8 NEWS The CliniMACS CD34 Reagent System a landmark in cellular therapy Miltenyi Biotec strives for improvement of scientific understanding and medical progress by providing products and services that advance biomedical research and cellular therapy. Working together with many internationally recognized professionals in centers throughout the world, we design, support, and coordinate clinical studies in areas such as hematological malignancies and stem cell therapies. AML is characterized by the rapid growth of myeloid bone marrow cells. Transplantation of allogeneic hematopoietic stem cells (HSCT) is regarded as the single most effective treatment for the prevention of reoccurrence of the disease in patients in complete remission after induction therapy. 1,2 However, major complications are associated with allogeneic stem cell transplantation, such as acute and chronic graft-versus-host disease (GvHD). Depending on the severity, GvHD can be associated with increased morbidity and mortality following transplantation. Since both acute and chronic GvHD are caused by the presence of donor T cells in the graft, depletion of graft T cells (TCD) before transplantation seemed to be a suitable preventative strategy. However this strategy was not universally accepted by clinicians because of doubts about the efficacy of the engineered graft. The uncertainty associated with treatment with HSCT was addressed by a recent study carried out by researchers from the Blood and Marrow Transplant Clinical Trials Network (Study BMT CTN 0303). They reported that TCD and HSCT, following intensive chemotherapy of AML patients, can be performed reproducibly in a multicenter setting using the CliniMACS CD34 Reagent System.³,⁴ The data from the BMT CTN 0303 study were compared to that of a similar study in which patients had received a hematopoietic stem cell graft that was not T cell depleted (BMT CTN 0101). The data from this comparison demonstrated that TCD with the CliniMACS CD34 Reagent System, as the sole method for preventing GvHD, resulted in a low incidence of acute and chronic GvHD in patients. This method of TCD did not have any negative impact on engraftment success, patient survival, or relapse rate.⁵ The effect on the occurrence of chronic GvHD was especially notable: at the 2-year time point, chronic GvHD occurred with a frequency of only 19% in BMT CTN 0303 compared to 50% in the study with the non-engineered graft, a statistically significant difference. Due to the highly efficient TCD there was no need for any immunosuppressive prophylaxis post transplantation, a feature that may influence quality of life in a positive way. Furthermore, the transplantation of a T cell depleted graft in the absence of an immunosuppressive agent may provide an ideal platform for further adoptive cell therapy post transplantation. References 1. Cornelissen, J.J. et al. (2007) Blood 109: Koreth, J. et al. (2009) JAMA 301: Devine, S.M. et al. (2011) Biol. Blood Marrow Transplant. 17: Keever-Taylor, C.A. et al. (2012) Biol. Blood Marrow Transplant. 18: Pasquini, M.C. et al. (2012) J. Clin. Oncol. 30: The CliniMACS CD34 Reagent System components, Instrument, CD34 Reagent, Tubing Sets, and PBS/ EDTA Buffer, are manufactured and controlled under an ISO certified quality system. In Europe, the CliniMACS System components are available as CE-marked medical devices. In the USA, the CliniMACS System components, including the CliniMACS Reagents, are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). CliniMACS MicroBeads are for research use only and not for use in humans. 8 MACS & more Vol 15 1/2013

9 The CliniMACS Prodigy Next generation of integrated cell processing Automated cell processing from cell sample to final product Cell washing Density gradient separation MACS Cell Separation Cell culture Fully automated Closed system At the time of publication, the components of the CliniMACS Prodigy line are for research use only and not for human therapeutic or diagnostic use. For an updated regulatory status in your country, please ask your local representative. clinimacs-prodigy.com Vol 15 1/2013 MACS & more 9

10 MACSxpress Technology allows isolation of B cells with high purity for sensitive and concordant microarray-based gene expression profiling Angela Mekes, Stephanie Soltenborn, Ines Dischinger, Volker Huppert, and Bernhard Gerstmayer Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Introduction Microarray-based gene expression profiling is an excellent tool for the identification of biomarkers in blood cells. However, whole blood and PBMCs, which are frequently used for biomarker research, are complex mixtures of many different cell types and subsets. Therefore, subtle changes in gene expression within a particular subpopulation can escape detection, if these complex mixtures are used for analysis. There is also a large variability in the proportions of blood cell subsets among different donors, which can further mask expression changes within a particular cell type. The use of isolated blood cell subsets has greatly enhanced biomarker research. Lyons et al.¹ used purified leukocyte subsets isolated by MACS Technology for their biomarker research in systemic lupus erythematosus. The authors identified disease-associated differentially expressed genes in purified CD4 + cells and monocytes that were not detectable in PBMCs. Similarly, Lee et al.² identified signatures in magnetically purified CD8 + T cells in their biomarker research on Crohn s disease and ulcerative colitis, which they otherwise would not have detected in unseparated PBMCs. For omics research, in general, it is desirable to have simple and short procedures that are highly reproducible and require minimal handling. The novel MACSxpress Technology for the fast isolation of cells directly from whole blood meets all these requirements. Using this technology, we isolated B cells from different donors to high purity. The obtained gene expression patterns were distinct from the corresponding whole blood and PBMC samples and showed enrichment of B cell specific gene transcripts as well as depletion of non-b cell related gene transcripts. Materials and methods B cell isolation For the isolation of B cells, 30 ml of EDTAanticoagulated whole blood were incubated with the MACSxpress B Cell Isolation Cocktail for 5 min in a 50-mL tube. During the incubation step, the tube was gently rotated using the MACSmix Tube Rotator. The B Cell Isolation Cocktail contains antibodyconjugated MACSxpress Beads magnetically labeling the non-target cells. Following the labeling step, the tube was placed in the magnetic field of a MACSxpress Separator. The labeled non-target cells adhered to the tube wall and the aggregated erythrocytes sedimented to the bottom, whereas the supernatant contained pure unlabeled B cells, which could immediately be used for further experiments. Samples obtained from four healthy donors were processed. Flow cytometry To assess the purity of B cells after isolation with MACSxpress Technology, cells were labeled with CD45-VioBlue, CD19-APC, and CD20-PE, before and after separation. Flow cytometry was performed on the MACSQuant Analyzer. For comparison, PBMCs prepared by density gradient centrifugation from the same whole blood sample were analyzed. Sample preparation for microarray analysis RNA was extracted from whole blood (stabilized with PAXgene Blood RNA Tubes) using the PAXgene Blood RNA Kit (Qiagen), and from PBMCs and isolated B cells using the NucleoSpin RNA II system (Macherey-Nagel). RNA quality, i.e., RNA integrity number (RIN), was assessed using the Agilent 2100 Bioanalyzer platform and the integrated software. For linear T7-based amplification, 50 ng of total RNA were used. Cy 3-labeled crna was prepared 10 MACS & more Vol 15 1/2013

11 Whole blood PBMCs Isolated B cells 7.43% 12.77% 97.98% CD19-APC CD19-APC CD19-APC CD20-PE CD20-PE Donor % B cells in whole blood sample % B cells in PBMCs A C D E means±sd 5.63± ± ±3.35 % B cells after MACSxpress Separation Figure 1 Flow cytometric analysis of whole blood, PBMCs, and isolated B cells. Cells were stained as indicated in the materials and methods section, and analyzed on the MACSQuant Analyzer. Data were gated on leukocytes. Dot plots are shown for one representative donor. The table summarizes the results for samples from four different donors. by using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer s protocol. Hybridization of Agilent Whole Genome Oligo Microarrays Hybridization was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 1.65 μg of Cy3-labeled fragmented crna in hybridization buffer were hybridized overnight (17 hours, 65 C) to Agilent Whole Human Genome Oligo Microarrays 4 44K V1 using Agilent s recommended hybridization chamber and oven. Subsequently, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with pre-heated Agilent Gene Expression Wash Buffer 2 for 1 min at 37 C. The last washing step was performed with acetonitrile for 30 s at room temperature. Microarray analysis Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers, and calculates statistical confidences. Results and discussion Isolation of B cells with MACSxpress Technology MACSxpress Technology allows the isolation of B cells to excellent purities up to 98% (fig. 1). The average purity was about 95% (n=4). The recovery of B cells isolated from whole blood ranged between 68% and 83%. RNA quality A RIN value of greater than 5 is considered to indicate that RNA quality is sufficient for gene expression profiling experiments³. In our experiments, RNA prepared from whole blood, PBMCs, and purified B cells consistently showed RIN values of approximately 8. RNA yields were sufficient for gene expression profiling experiments (table 1). Whole blood PBMCs Isolated B cells RIN 7.93± ± ±0.51 RNA yield (µg/ml whole blood) 2.6 ± ± ±0.012 Table 1 RIN values and yields for RNA extracted from different sample materials. RNA was extracted as described in the materials and methods section. Values represent samples from four different donors (means±sd). Microarray analysis Expression analysis of various cell markers in whole blood vs. the purified B cell fraction allowed us to further validate the purity of the isolated B cells (fig. 2). As anticipated CD19 + cells were efficiently enriched, as indicated by a 20-fold increase in light units ( 3,000 LU in whole blood vs. 60,000 LU in purified B cells). Vol 15 1/2013 MACS & more 11

12 qn-normalized raw data qn-normalized raw data qn-normalized raw data qn-normalized raw data qn-normalized raw data qn-normalized raw data 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2, ,000 70,000 60,000 50,000 40,000 30,000 20,000 10, , , , , , , , , ,000 50, ,600 1,400 1,200 1, , ,323 4,049 10,770 3, , , , , ,865 1, ,234 7,208 3, , ,269 12,168 A C D E 14,472 7, ,853 1, ,567 12,057 5,673 28, ,798 1,733 10,203 5,120 4,054 Figure 2 Microarray-based analysis of cell type specific gene expression. Gene expression analysis was performed as indicated in the materials and methods section. Data indicate quantile-normalized raw data (light units, LU) for whole blood samples, PBMCs, and isolated B cells, from four different donors (A,C,D,E) each. CD14 CD19 HBA1 MPO PF4 14,472 4, , TCL1A 15,584 13,312 4,864 16, ,849 A C D E 41 57, , , , , , , ,744 A C D E Whole blood PBMCs Isolated B cells Likewise, the lymphocyte-associated marker TCL1A showed an increase from 10,000 LU to 100,000 LU, whereas CD14 expression was almost absent ( 10,000 LU vs. 50 LU). The reticulocyte/erythrocyte associated marker HBA1 showed high values of 450,000 LU in whole blood samples, which was in the range of saturation, while the purified B cells showed a reduced value of 5,000 LU. The values for the neutrophil marker MPO were reduced from 250 LU to 30 LU, and the platelet marker PF4 from 400 LU to 2 LU. These results confirm that MACSxpress Technology allowed the efficient removal of non-b cells from whole blood. We compared expression profiles of whole blood samples, PBMCs, and purified B cells in an unsupervised cluster analysis with 4,100 genes as input. Genes not associated with A_Whole_blood D_Whole_blood C_Whole_blood E_Whole_blood A_PBMCs D_PBMCs C_PBMCs E_PBMCs A_Isolated_B_cells D_Isolated_B_cells C_Isolated_B_cells E_Isolated_B_cells SLA2 KLRD1 MAF LEF1 SRGN ENST SIRPG TRA@ ICOS CD3E UBASH3A LAT A_32_P TXK S1PR2 CD8B FLJ22662 CD2 CD3G C4orf18 CSTA GIMAP7 PTGER2 GIMAP8 DUSP6 FCN2 FCN1 CD33 USP6NL EBF1 MS4A1 FCRL2 IL7 COBLL1 BANK1 BLNK CD19 L0C AIM2 FCRLA PNOC CPNE5 WDR34 Figure 3 Gene expression profiling of cells isolated by MACSxpress Technology. Gene expression analysis was performed as indicated in the materials and methods section. Data were extracted from an unsupervised heatmap (input 4,100 genes) and are shown for whole blood samples, PBMCs, and isolated B cells, from four different donors each. 12 MACS & more Vol 15 1/2013

13 B cells, such as CD3, CD8, and CD33, were not represented in the purified B cell fraction. In contrast, the B cell marker CD19 was highly abundant in this fraction (fig. 3). Overall, the heat map reveals considerable differences between purified B cells and the unpurified fractions (whole blood and PBMCs). A twoway analysis of variance showed that 2,111 of 4,100 genes were significantly differentially expressed in purified B cells vs. unpurified cells. This suggests that subtle yet significant gene expression changes within the B cell fraction might escape detection if unpurified cells are used for analysis. Conclusion Gene expression analysis of isolated cell populations is more sensitive and conclusive than analysis of whole blood or PBMCs. MACSxpress Technology is a fast method (as little as 20 min) for the efficient isolation of cells from large whole blood volumes (up to 30 ml) requiring minimal handling steps. Isolated B cells show high purities. The isolation procedure is gentle to cells. Cells can be immediately used for downstream applications. RNA extracted from isolated cells is of high quality and suitable for gene expression analysis. MACSxpress Technology is an ideal method for the isolation of cells prior to gene expression analysis. References 1. Lyons, P.A. et al. (2010) Ann. Rheum. Dis. 69: Lee, J.C. et al. (2011) J. Clin. Invest. 121: Fleige, S. and Pfaffl, M.W. (2006) Mol. Aspects Med. 27: MACS Product or Services* MACSxpress B Cell Isolation Kit, human Order no MACSxpress Separator MACSmix Tube Rotator MACSQuant Analyzer Genomic services * Products and services are for research use only. MACSxpress Cell Isolation Kits are also available for naive B cells, NK cells, pan T cells, CD4 + T cells, and CD8 + T cells. For further information visit For more information on Miltenyi Biotec's comprehensive genomic and bioinformatic services visit New Effective activation of pdcs and B cells High-quality oligonucleotides for TLR9 stimulation DNA oligonucleotides (ODNs) containing the CpG motif represent specific toll-like receptor 9 (TLR9) ligands, enabling studies on the role of pdcs and B cells in innate immunity and inflammation. Miltenyi Biotec offers four different classes of CpG ODNs of outstanding quality for best possible activation of your specific cell type. High reproducibility: activity tested for each batch Ultrapure, endotoxin-free Fully compatible with MACS Cell Separation Products Easy to handle: reconstitution protocol included Unique ODNs for superior cell activation miltenyibiotec.com Vol 15 1/2013 MACS & more 13

14 Isolation of monocytes with high purity directly from whole blood for transcriptome analysis in translational research Christelle Foucher 1, Sébastien Vachenc 1, Alexandre Meniccaci 1, Bernhard Gerstmayer 2, Gwenola Henrion 1, Uwe Janssen 2, Darren Wilbraham 3, Karine Le Malicot 1, and Jean-Claude Ansquer 1 1 Laboratoires Fournier SA, formely an Abbott Company, Daix, France 2 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 3 Quintiles Limited, London, UK Introduction Fenofibrate is a lipid-lowering drug used in the treatment of dyslipidemia. Fenofibrate effects have been attributed to the activation of the nuclear transcription factor peroxisome proliferator activated receptor-α (PPARα). PPARα plays a role in the regulation of tissue factor expression in human monocytes and might thus influence atherothrombosis¹,². In this study, we assessed the effects of fenofibrate on gene expression in peripheral blood monocytes of healthy donors. Microarray-based transcriptome analysis is a powerful tool in translational research for evaluating drug effects on particular blood cell types. However, for the reliable detection of subtle changes in gene expression in a certain cell type, it is crucial to use isolated cell populations of high purity for analysis. In particular for translational research projects involving multiple clinical centers and operators, it is vital to use cell isolation protocols that are short, simple, and highly reproducible, and avoid steps that are prone to variability, such as the preparation of peripheral blood mononuclear cells (PBMCs). Positive selection of CD14 + monocytes by MACS Technology is a well-established and reliable procedure. Here we used the automacs Pro Separator and Whole Blood CD14 MicroBeads to magnetically isolate monocytes directly from whole blood and achieved high cell purities and yields. This protocol avoids both PBMC preparation and erythrocyte lysis, which are both laborious and can lead to variation in cell separation results. Microarray experiments and comparison of gene expression at three different time points of fenofibrate treatment allowed for the identification of differentially expressed sequences (DES) and modulated biological functions. The experiment workflow is summarized in figure 1. Subjects, materials, and methods Subjects and study design Twenty six healthy males or post menopausal (natural or chirurgical) females not receiving hormone replacement therapy (HRT) or having stopped HRT for at least 1 month, aged years inclusive, were recruited in this openlabel, single-center research study to receive a standard dose of fenofibrate as one 145 mg tablet daily treatment (Lipanthyl, Laboratoires Whole blood samples from healthy donors Isolation of monocytes with automacs Pro Separator and Whole Blood CD14 MicroBeads Microarray analysis of purified CD14 + monocytes Figure 1 Workflow for the isolation of CD14 + monocytes directly from whole blood and subsequent transcriptome analysis. Fournier SA, Dijon, France). Subjects with a body mass index (BMI) 30 kg/m 2 or <18 kg/ m 2 or with known hypersensitivity to fibrates, or females with child bearing potential without a reliable method of contraception, having received an investigational drug in the last 90 days before date of inclusion were not included in the study. All subjects had normal folate levels (mean±sd: 15.2±11.1 ng/ml) and vitamin B12 levels (404.1±165.2 pg/ml) at inclusion in the study. The study included a screening phase from a few days up to 3 weeks (wks) and a treatment phase of 6 wks. Blood was drawn and monocytes were separated at baseline, after 7 to 10 days, and after 6 wks of treatment. Ethic approval was obtained from the Guy s Hospital Research Ethics Committee, London, UK. Freely given informed consent was obtained from each subject before enrollment. Isolation of monocytes from whole blood Freshly drawn whole blood (40 ml) was anticoagulated using EDTA. Two aliquots (15 ml each) were magnetically labeled with Whole Blood CD14 MicroBeads. Labeled CD14 + monocytes were automatically isolated in two runs using the automacs Pro Separator according to the manufacturer s protocol. Isolated monocytes from both runs were combined and analyzed by flow cytometry to determine viability, yield, and purity. For subsequent RNA extraction, isolated cells were centrifuged and flash-frozen. 14 MACS & more Vol 15 1/2013

15 Flow cytometry Cells were labeled with CD14-PE and CD45- FITC antibodies before and after separation, and analyzed by flow cytometry. CD15 antibodies were used to evaluate the frequency of granulocytes. All antibodies were obtained from Miltenyi Biotec. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. RNA extraction and microarray analysis Total RNA was extracted from flash-frozen cell samples using the NucleoSpin RNA II system (Macherey-Nagel). RNA was quantitated and RIN values were assessed using the Agilent 2100 Bioanalyzer platform and the integrated software. RNA was amplified and labeled with Cy 3 using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). RNA was hybridized to Agilent Whole Human Genome Oligo Microarrays (4 44K). Statistical analysis A global effect of the treatment on the 78 (26 3) intensity profiles was determined using a 2-way ANOVA considering donors and times of sample collection, adjusted by using the Benjamini-Hochberg false-discovery rate (FDR) method to adjust p values and control for the first species error. Pairwise comparisons of the DES between study visits were performed using the Student-Newman-Keuls (NK) test. A significant effect of the treatment on the sequences was concluded for p For functional analysis, pathways and networks were constructed based on the classification of all the DES modulated in at least one pairwise comparison (p 0.01 for NK test) via a k-means clustering approach (user-defined number of clusters = 11, cosine correlation and centroid-based search). Functional networks were constructed using the Ingenuity Pathway Analysis software (IPA version 8.6 build 93815). Results and discussion Magnetic isolation of monocytes directly from whole blood CD14 + monocytes were enriched directly from whole blood in an automated fashion using the automacs Pro Separator and Whole Blood CD14 MicroBeads. In the example shown in figure 2 the frequency of monocytes in the whole blood sample amounted to 6%. MACS Technology allowed us to separate monocytes to purities greater than 97% (fig. 2, dot plots). The mean purity of monocytes in 78 samples from 26 donors amounted to 94.1±6.17%. The mean cell yield was ± Purified monocytes showed consistently high viabilities of 96%. For details see the table in figure 2. Quality and yield of RNA extracted from isolated monocytes The yield of monocytes magnetically isolated from 30 ml of whole blood was high, which allowed us to extract large amounts of RNA for microarray analysis. The yield of RNA extracted from isolated monocytes (n=78) amounted to 2.15±0.73 µg (mean±sd). RIN values reached 9.57±0.47 (mean±sd) indicating consistently high RNA quality for sensitive, reliable microarray experiments. Microarray analysis of monocytes isolated with MACS Technology We compared gene expression in monocytes isolated from peripheral blood of healthy donors at 0, 1, and 6 wks of fenofibrate CD14-PE Before enrichment CD45-FITC treatment. We identified 5,187 sequences that were differentially modulated in at least one of the pairwise comparisons. The distribution was as follows: 3,924 DES between 0 and 1 wk, 1,973 DES between 0 and 6 wks, and 2,904 DES between 1 and 6 wks of fenofibrate treatment (fig. 3). K-means clustering led to the identification of 11 clusters showing different trends over time in gene expression after fenofibrate treatment. One of the k-means clusters, which included 476 DES, showed a trend to down-regulation after 1 wk and a trend to up-regulation between 1 and 6 wks of treatment. Out of these 476 DES, 324 had mapped gene identities, 231 were eligible for networks, and 220 were eligible for function pathway analysis around immunological disease, cell-mediated immune response, cellular assembly and organization, cellular movement, antigen presentation, cardiovascular disease, cellular growth and proliferation, lipid metabolism, molecular transport, inflammatory response etc. (fig. 4). Parameter Mean sd Frequency of monocytes in whole blood sample (% among CD45 + cells) Purity of monocytes after enrichment (% among CD45 + cells) Yield of enriched cells (number of cells per ml) Viability of enriched cells (% live cells) Figure 2 Isolation of CD14 + monocytes from whole blood using the automacs Pro Separator and Whole Blood CD14 MicroBeads. Cells were enriched as indicated in the subjects, materials, and methods section. Before and after enrichment, cells were labeled with CD14-PE and CD45-FITC and analyzed by flow cytometry. CD14-PE After enrichment 6.15% 97.6% CD45-FITC Vol 15 1/2013 MACS & more 15

16 Best-in-class flow cytometry The MACSQuant Family of flow cytometers Powerful and compact 3 lasers and 10 optical parameters Discover new answers Powerful four-laser functionality in a compact three-laser layout Achieve more Automated labeling and analysis of up to 96 samples in a single run Real-time remote support At just the touch of a button Search deeper Enhanced rare cell analysis with proven MACS Technology 16 MACS & more Vol 15 1/2013 macsquant.com

17 Conclusion The automacs Pro Separator in combination with Whole Blood CD14 MicroBeads allows for rapid and robust magnetic isolation of monocytes with high yields. Isolated CD14 + monocytes showed purities of 94% on average. The use of isolated monocytes enables sensitive and accurate microarray-based transcriptome analysis of research samples. Significant short-term (1 wk) and middleterm (6 wks) effects of fenofibrate on gene expression in monocytes were observed. Identification of a large number of DES within a k-means cluster allowed for the construction of functional networks with eligible gene identities involved in particular in metabolic and inflammatory pathways wks vs. 1 wk (3,924 DES) 0 wks vs. 6 wks (1,973 DES) 1 wk vs. 6 wks (2,904 DES) References 1. Marx, N. et al. (2001) Circulation 103: Neve, B.P. et al. (2001) Circulation 103: Figure 3 Venn diagram for comparisons of DES at three time points of fenofibrate treatment. The Student-Newman-Keuls algorithm was applied for all pairwise comparisons. For details see the subjects, materials, and methods section. 0 vs. 1 wk 0 vs. 6 wks 1 vs. 6 wks Figure 4 Functional networks constructed from a k-means cluster. Network-eligible genes from a cluster showing a trend to down-regulation after 1 wk and a trend to up-regulation between 1 and 6 wks of fenofibrate treatment were used as seeds for in silico network generation. Down-regulation is indicated in green and up-regulation in red. For details see the subjects, materials, and methods section. MACS Product or Services* Order no. automacs Pro Separator Starter Kit Whole Blood CD14 MicroBeads, human CD14, CD15, and CD45 antibodies Visit Genomic services Visit *Products and services are for research use only. Vol 15 1/2013 MACS & more 17

18 REPORT Report An automated method for purification of CD138 + cells from whole bone marrow samples for multiple myeloma research Hossain Mossafa and Sabine Defasque Laboratoire Cerba, Département Génétique Cergy Pontoise, Saint Ouen L Aumone, France Introduction Multiple myeloma (MM), the second most common hematological malignancy, is a plasma cell (PC) disorder in the bone marrow (BM). MM is characterized by a large clinical heterogeneity despite the homogeneous morphological appearance of malignant PCs. Chromosomal aberrations are a hallmark of MM, and distinct genetic abnormalities characterize the major subtypes of the disease. As an addition to interphase fluorescence in situ hybridization (FISH), the more global assessment of the underlying cytogenetics by a genome-wide analysis of malignant PCs, using high-density, single-nucleotide polymorphism (SNP) arrays for molecular karyotyping, has significantly improved the detection and identification of genetic lesions. FISH and/or molecular karyotyping experiments with unseparated BM samples have a 30 to 50% probability of showing false results or failure due to technical reasons. This is especially true for samples from BM aspirates that were affected by very low PC infiltration in the BM. Purified CD138 + cells are a prerequisite to increasing the sensitivity of FISH analysis or SNP arrays. The quality of FISH and molecular karyotyping results depends on the degree of PC purity and DNA integrity after isolation. Given the daily demands of laboratories there is a need for an automated platform for the preparation of pure CD138 + cell populations. Here we used Whole Blood CD138 MicroBeads and the automacs Pro Separator for the fast and reliable automated isolation of CD138 + PCs from whole BM samples for MM research. Materials and methods Isolation of CD138 + cells For the isolation of CD138 + cells directly from whole BM samples (1.5 to 3 ml), cells were magnetically labeled with Whole Blood CD138 MicroBeads (Miltenyi Biotec). Program posselwb in combination with the Clean process was used for separation of CD138 + cells on the automacs Pro Separator (Miltenyi Biotec) according to the manufacturer s protocol. Purities of isolated cell populations were determined by flow cytometry using CD138 and CD38 antibodies. The purity of DNA was analyzed using a NanoDrop Instrument (Thermo Fisher Scientific). FISH analysis After separation CD138 + PCs were hybridized with the LSI D13S25 (13q14.3) Single Color and/or the LSI IGH/FGFR3 Dual Color, Dual Fusion Translocation Probe (t(4;14)(p16;q32)), and/or the LSI P53 (17p13.1) Single Color Probe and analyzed by fluorescence microscopy. Results Isolation of CD138 + cells directly from BM samples In a study of 100 MM samples from whole BM, we evaluated the efficiency and performance of separation, cell purity, and the quality of DNA after purification of CD138 + cells. Purities of CD138 + PCs after isolation with Whole Blood CD138 MicroBeads and the automacs Pro Separator amounted to 95% in average (table 1). For the vast majority of MM samples (95%) we obtained enough cells for the performance of the recommended panel of FISH analyses and genome-wide analysis. FISH analysis of CD138 + cells CD138 + cell enrichment prior to FISH analysis more than doubled the detection rate of abnormalities (83% vs. 40% in unseparated cells), and frequencies of abnormalities reached significant levels. Figure 1 shows examples of FISH analyses of isolated CD138 + cells, comparing normal cells (left) with malignant cells (right). The normal cell in figure 1A Sample Detectable PCs in bone marrow samples (%) CD138 + PCs following isolation (%) Table 1 Frequency of CD138 + PCs prior to and after enrichment from BM samples using Whole Blood CD138 MicroBeads and the automacs Pro Separator. PC purity was determined by flow cytometry using CD138 and CD38 antibodies. 18 MACS & more Vol 15 1/2013

19 shows two orange signals representing the two alleles of the D13S319 locus. The malignant cell shows only one orange signal, due to a deletion affecting locus D13S319 or monosomy of chromosome 13. The normal cell in figure 1B shows two orange (FGFR3) and two green (IgH) signals. The malignant cell (fig. 1B, right) shows an abnormal signal pattern with one orange (FGFR), one green (IGH), and three fusion signals, resulting from the chromosomal translocation (t(4;14) FGFR3/IgH). NanoDrop Analysis of DNA extracted from isolated CD138 + PCs revealed high purity (data not shown). Conclusion As a routine MM research laboratory, we receive numerous BM samples every day. Isolation of CD138 + cells from these samples is necessary to increase the sensitivity of downstream assays, such as FISH analysis or molecular karyotyping by SNP arrays. Since 2007, we have performed more than 6,000 PC isolations. This large number necessitates a reliable, rapid, and standardized method, A B CD138 + plasma cells allowing us to isolate CD138 + cells from multiple samples in a convenient way, while maintaining sample integrity. Whole Blood CD138 MicroBeads in combination with the automacs Pro Separator meet all of our lab s requirements. The automacs Pro Separator allows the standardization of cell separation processes and ensures a rapid handling of MM samples. Whole Blood CD138 MicroBeads enable fast isolation of CD138 + cells directly from BM samples, thus minimizing hands-on time and maximizing the yield of target cells. No sample preparation is required, such as density gradient centrifugation or red blood cell lysis. Purified CD138 + cells can be immediately subjected to FISH or molecular analyses. The detection rate of chromosomal abnormalities per sample in MM and PC dyscrasia significantly improves when analysis is performed on purified populations of CD138 + PCs. The platform allows the generation of reproducible and consistent results even in multi-user settings. The trusted resource for your cell research Miltenyi Biotec product catalog 2013/2014 B del(13): locus D13S319 Hot off the press, our new catalog features more products than ever before. Get a comprehensive overview of our entire portfolio of products and services. From broad background information to details on specific products, you will easily find the information you need. Myeloma: t(4;14) FGFR3/ lgh Figure 1 FISH analysis of CD138 + cells. PCs were isolated from whole BM using Whole Blood CD138 MicroBeads. Isolated cells were subjected to FISH analysis with (A) LSI D13S25 Single Color Probe, or (B) LSI IGH/FGFR3 Dual Color, Dual Fusion Translocation Probe. In both pictures a normal cell is shown on the left and a malignant, mutated cell on the right. MACS Product* automacs Pro Separator Starter Kit Order no Whole Blood CD138 MicroBeads CD138-PE CD38-APC CD19-FITC * Products are for research use only. Order your copy today! miltenyibiotec.com/catalog2013 Vol 15 1/2013 MACS & more 19

20 Highly purified peripheral blood γ/δ T cells isolated by MACS Technology respond to NOD2 ligand Lothar Marischen, Hans-Heinrich Oberg, Christian Peters, Sandra Ussat, Hoa Ly, Dieter Kabelitz, and Daniela Wesch Institute of Immunology, University of Kiel, Kiel, Germany Introduction CD3 + γ/δ T cells display characteristics of the adaptive and the innate immune system¹. The dominant subset of γ/δ T cells expresses a Vγ9Vδ2 T cell receptor (TCR), which recognizes phosphorylated intermediates of the bacterial non-mevalonate isoprenoid biosynthesis pathway. Synthetic phosphoantigens such as bromohydrin pyrophosphate (BrHPP) are capable of inducing large-scale Vγ9Vδ2 T cell expansion, which is independent of processing and presentation of such molecules by classical MHC molecules¹,². After TCR stimulation, γ/δ T cells rapidly release cytokines, such as IFN-γ and MIP-1α (CCL3), thereby activating other cells of the immune system³. Additionally, γ/δ T cells possess features of innate immune cells, such as antigen-presenting capacity and express pattern recognition receptors (PRR), including Toll-like receptors (TLR)⁴,⁵. TLR recognize a broad variety of structurally conserved molecules derived from microbes. TLR ligands have been shown to costimulate TCRactivated γ/δ T cells by enhancing production of cytokines and chemokines⁶,⁷. Besides TLR, another class of PRR, the nucleotide-binding leucine-rich repeat receptors (NLR), was detected in γ/δ T cell lines. However, these studies did not address whether the NLR nucleotide-binding oligomerization domain (NOD) 2 was expressed in freshly isolated γ/δ T cells⁸. Our studies revealed that highly purified γ/δ T cells express NOD2 mrna and NOD2 protein⁹. Furthermore, we investigated a possible function of NOD2 in freshly isolated γ/δ T cells by stimulating the cells with the minimally bioactive motif muramyl dipeptide (MDP) that mimics bacterial peptidoglycan. Cytokine IL-1β GRO cross-linked TCR medium medium MDP-LD Materials and methods Cell isolation Human γ/δ T cells were depleted from PBMCs using the Anti-TCRγ/δ MicroBead Kit (Miltenyi Biotec). For depletion of human CD14 + monocytes, CD14 MicroBeads (Miltenyi Biotec) were used. Cell separation was performed using LS Columns (Miltenyi Biotec). After depletion, the proportion of residual γ/δ T cells or monocytes was <0.1%. control (+) (-) MDP-DD Figure 1 Production of IL-1β and GRO in response to TCR cross-linking and MDP-LD. γ/δ T cells were purified by positive selection with MACS Technology. 4 10⁶ cells per well (24-well plate) were cultured in the absence or presence of rabbit anti-mouse Ig (cross-linked TCR) and MDP-LD or MDP-DD as indicated. Culture supernatants were analyzed for cytokines after 24 h. Positive and negative controls were included in the array. 20 MACS & more Vol 15 1/2013

21 To enrich γ/δ T cells or monocytes from PBMCs, the Anti-TCRγ/δ MicroBead Kit or CD14 MicroBeads were used. PBMCs were pre-treated with FcR Blocking Reagent (Miltenyi Biotec) to avoid non-specific binding of antibodies (Abs) to FcR-bearing cells. The purity of the positively selected A SSC CD3 Before depletion After depletion of γ/δ T cells FSC cells ranged between 83 and 97% when only one LS Column was used for positive selection. This pre-enrichment of γ/δ T cells by MACS Technology was necessary to avoid time-consuming flow sorting of PBMCs. To definitely ensure the depletion of residual monocytes in the enriched γ/δ T cell fraction B SSC no stain Before depletion After depletion of monocytes 2.14 % 0 % % 0 % FSC and vice versa, a further purification step was done by sorting with a FACSAria cell sorter (BD Biosciences) or by using another LS Column. The purity of the cells was then >99%. For further details, please refer to Marischen et al.⁹. Activation of cells PBMCs and γ/δ T cell- or monocyte-depleted PBMCs were stimulated with the γ/δ T cell specific antigen BrHPP (200 nm, Innate Pharma ) in the presence of ril-2 (Novartis). Positively selected γ/δ T cells or monocytes were cultured in uncoated wells or wells coated with 1 µg/ml rabbit anti-mouse Ig, which binds to anti-tcrγ/δ mab present on the γ/δ T cells or CD14 mab present on monocytes after labeling with MACS MicroBeads. Additionally, all cell cultures were incubated with 10 µg/ml MDP-LD isomer (tlrl-mdp, Invivogen ) or as a control with inactive MDP-DD isomer (tlrlmdpc; Invivogen) for 24 h without ril-2 in serum-free X-VIVO 15 medium (Lonza ). For detailed culture conditions see reference 9. C MIP-1α (pg/ml) γ/δ incl. γ/δ w/o γ/δ w/o γ/δ & CD14 D IFN-γ (pg/ml) medium MDP-LD MDP-DD 0 CD14 incl. γ/δ w/o γ/δ w/o γ/δ & CD14 Cytokine analysis Cytokines were analyzed in cell culture supernatants by using the RayBio Human Cytokine Antibody Array VI & 6.1 Map and VII & 7.1 Map (Hoelzel Diagnostic), which allows simultaneous detection of 2 60 cytokines and chemokines. Signals were detected by chemiluminescence, followed by semiquantitative analysis with the AIDA software (Raytest). To determine the intensity, local background was subtracted from each value and normalized against the positive controls of each membrane. IFN-γ and MIP- 1α were detected by DuoSet sandwich ELISA (R&D Systems) according to the manufacturer s instructions. Figure 2 MIP-1α and IFN-γ production by PBMCs depleted or not of γ/δ T cells and monocytes. (A, B) Gates were set on lymphocytes (A, upper dot plots) or monocytes (B, upper dot plots) to determine γ/δ T cells or monocytes among PBMCs, respectively, before and after depletion of the appropriate cell population. The frequencies of CD3 + TCRγ/δ + T cells (A, lower dot plots) or CD14 + monocytes (B, lower dot plots) among PBMCs of one representative donor before and after depletion of γ/δ T cells or monocytes, respectively, are shown. (C, D) Undepleted PBMCs (incl. γ/δ), PBMCs depleted of γ/δ T cells (w/o γ/δ), and PBMCs depleted of both γ/δ T cells and monocytes (w/o γ/δ & CD14) were stimulated with BrHPP and ril-2 in the absence (medium) or presence of active MDP-LD or inactive MDP-DD as indicated ⁵ cells per well (96-well plates) were used throughout the experiment. Concentrations of MIP-1α (C) and IFN-γ (D) in the supernatants were determined by ELISA after 24 h. Results of fig. 2D were reproduced from Marischen et al. (ref. 9) with the permission of John Wiley and Sons. Results of one representative experiment out of four are shown in figure 2 C and D. Flow cytometry To determine the proportion of γ/δ T cells among PBMCs, cells were labeled with anti- CD3 mab and anti-tcrγ/δ mab. Monocytes among PBMCs were analyzed after staining with anti-cd14 mab. Antibodies were obtained from BD Biosciences. Vol 15 1/2013 MACS & more 21

22 A no stain 83.1 % C MIP-1α (pg/ml) B no stain 99.9 % 89.1 % 99.7 % γ/δ CD14 D 600 IFN-γ (pg/ml) MDP-LD or MDP-DD for 24 h (fig. 2C, D). In these experiments, we analyzed the cytokine MIP-1α, which can be secreted by both γ/δ T cells and monocytes, and IFN-γ, which is exclusively produced by T cells. We observed that MDP-LP, but not inactive MDP-DD, enhanced MIP-1α production in undepleted PBMCs, but also in PBMCs depleted of γ/δ T cells. The depletion of monocytes almost abolished the secretion of MIP-1α suggesting that MDP-LP reactive monocytes were the major producers of MIP-1α. Moreover, MDP- LD, but not MDP-DD, enhanced IFN-γ secretion in BrHPP-stimulated PBMCs, which was abrogated after γ/δ T cell depletion (fig. 2D). These experiments indicate a moderate direct costimulatory effect of MDP-LD on γ/δ T cells, which was investigated in further experiments using highly purified γ/δ T cells from 18 healthy donors (see ref. 9). 0 γ/δ monocytes medium MDP-LD MDP-DD Figure 3 Effects of MDP-LD on highly purified monocytes and γ/δ T cells. (A, B) The purities of γ/δ T cells (A) or CD14 + monocytes (B) were determined after MACS Separation with one LS Column (A and B, left dot plots) and after subsequent flow sorting (A and B, right dot plots), shown for one representative donor. (C, D) Highly purified γ/δ T cells (1.5 10⁵) were stimulated through the TCR via immobilized rabbit antimouse Ab in the presence or absence of MDP-LD or MDP-DD, whereas monocytes (1.5 10⁵) were cultured in medium or activated by MDP-LD or MDP-DD as indicated. Concentrations of MIP-1α (C) and IFN-γ (D) in the supernatants were analyzed by ELISA after 24 h. Results of figure 3D were reproduced from Marischen et al. (ref. 9) with the permission of John Wiley and Sons. Results and discussion MDP enhanced cytokine secretion in purified γ/δ T cells γ/δ T cells were isolated from PBMCs from three healthy donors by positive selection using an LS Column. The purity of the cells was between 83 and 97%. Purified γ/δ T cells were cultured in medium or stimulated by TCR cross-linking (via rabbit anti-mouse Ig) in the absence or presence of MDP-LD or MDP-DD for 24 h. IL-1β and GRO production were measured by a human cytokine Ab array. Figure 1 shows that MDP-LD induced secretion of IL-1β and GRO by the purified TCR-stimulated γ/δ T cells, whereas MDP-DD had no effect. However, IL-1β and GRO are produced mainly by monocytes, and not by γ/δ T cells. This suggests that residual monocytes were present in the γ/δ T cell population, 0 γ/δ monocytes which could be responsible for production of these cytokines. Similar to our results, Lancioni and colleagues observed that CD4 + T cells with a purity of 97% differ from highly purified CD4 + T cells with regard to their responses to LPS.¹⁰ Therefore, we examined in more detail γ/δ T cell or monocyte-depleted PBMCs and highly purified, freshly isolated γ/δ T cells or monocytes. Abrogation of TCR/MDP-LP induced IFN-γ production after depletion of γ/δ T cells from PBMCs PBMCs were completely depleted of γ/δ T cells (fig. 2A) or monocytes (fig. 2B) by MACS Technology. Undepleted PBMCs or PBMCs depleted of γ/δ T cells or PBMCs depleted of both γ/δ T cells and monocytes were stimulated with BrHPP in the absence or presence of MDP-LD directly increases IFN-γ production in highly purified γ/δ T cells From the same PBMCs shown in figure 2 as well as from 3 additional healthy donors (see ref. 9), γ/δ T cells and CD14 + monocytes were isolated by a combination of MACS Separation and flow sorting or by two consecutive MACS Separations. We obtained highly purified cells from both separation procedures. The data for cells purified by the combination of MACS Separation and flow sorting from one representative donor are shown in figure 3A and B. Highly purified monocytes stimulated with MDP-LD produced higher levels of MIP- 1α than the control or cells incubated with inactive MDP-DD (fig. 3C). Freshly isolated, highly purified γ/δ T cells from the same donor secreted IFN-γ in response to TCR crosslinking. IFN-γ secretion was slightly enhanced in the presence of MDP-LD, but not with MDP- DD (fig. 3D). Purified monocytes did not produce IFN-γ under these culture conditions (fig. 3D). Similar results were obtained after two consecutive MACS Separations as shown in figure 6 of reference 9. Conclusion MACS Separation followed by flow sorting as well as two consecutive MACS Separations permit the isolation of highly purified γ/δ T cells or CD14 + monocytes from PBMCs. The highly purified cell populations allow functional studies on innate receptors, such 22 MACS & more Vol 15 1/2013

23 as NOD2, expressed by freshly isolated γ/δ T cells. In order to obtain PBMCs devoid of γ/δ T cells and CD14 + monocytes, these cells were completely removed by MACS Separation. The biologically active MDP-LD isomer, but not the inactive MDP-DD isomer, enhanced IFN-γ production by TCR-stimulated, freshly isolated, highly purified γ/δ T cells, further underscoring a role of γ/δ T cells in anti-bacterial immunity. Acknowledgment We thank John Wiley and Sons for the kind permission to reproduce figures from Marischen et al. (ref. 9). References 1. Hayday, A.C. (2000) Annu. Rev. Immunol. 18: Espinosa, E. et al. (2001) J. Biol. Chem. 276: Wesch, D. et al. (2005) Curr. Med. Chem. Anti- Inflammatory & Anti-Allergy Agents 4: Brandes, M. et al. (2005) Science 309: Wesch, D. et al. (2006) J. Immunol. 176: Pietschmann, K. et al. (2009) Scand. J. Immunol. 70: Wesch, D. et al. (2011) Cell. Mol. Life Sci. 68: Hedges, J.F. et al. (2005) J. Immunol. 174: Marischen, L. et al. (2011) Scand. J. Immunol. 74: Lancioni, C.L. et al. (2009) J. Immunol. Methods 344: MACS Product* Order no. Anti-TCRγ/δ MicroBead Kit CD14 MicroBeads FcR Blocking Reagent, human LS Column Pre-Separation Filter * Products are for research use only. Flexible High yields of viable single cells from virtually any tissue, including tumors, lung, spleen, and intestine Time-saving Run up to eight programs in parallel or independently Dissociate and discover gentlemacs Dissociators Get a good start with automated tissue dissociation Reproducible Standardized tissue dissociation and homogenization using optimized procedures Safe Closed system, sterile sample handling Walk-away processing Automated heaters enable enzymatic dissociation at 37 C gentlemacs.com Vol 15 1/2013 MACS & more 23

24 Efficient and rapid in vitro generation of fully functional multi-virus-specific CD4 + and CD8 + T cells Anna Foerster-Marniok, Verena Traska, Olaf Brauns, Sven Kramer, Ju rgen Schmitz, Mario Assenmacher, and Anne Richter Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Introduction Antigen-specific T cells play a critical role in the regulation of immune responses and the elimination of virus-infected or malignant cells in the human body. T cells are essential, e.g., for the control of the outgrowth of Epstein- Barr virus (EBV)-infected B cells. CD4 + and CD8 + T cells specific for the EBV antigens BZLF1¹-⁴ and EBNA-1⁵-¹¹ have been found in EBV-infected individuals. Virus-specific T cells also hold great potential for clinical use. The adoptive transfer of clinical-grade CD4 + and CD8 + T cells specific for adenovirus (AdV) hexon, cytomegalovirus (CMV) pp65, and EBV antigens is a valuable tool for the treatment of AdV¹², CMV¹³-¹⁶, and EBV¹⁷,¹⁸ infections after hematopoietic stem cell transplantation. To further enhance research on virus-specific T cells, we have established a protocol for the efficient and rapid generation of EBV-, AdV-, and CMV-specific (tri-virus-specific) T cells. The virus-specific CD4 + and CD8 + T cells were stimulated in vitro using peptide pools, and subsequently magnetically enriched according to their IFN-γ secretion. The tri-virus-specific T cell population could be easily expanded without major alterations in the proportions of the respective specificities. Materials and methods Generation of multi-virus-specific T cells For the generation of multi-virus-specific T cells we stimulated 10⁹ PBMCs from leukapheresis products of several healthy PBMCs Mixed antigen loading PBMCs Stimulation with a mix of antigens Enrichment of IFN γ secreting T cells T cell expansion donors with a combination of four PepTivator Peptide Pools (Miltenyi Biotec) covering CMV pp65 or IE-1, AdV hexon, or EBV EBNA-1 or BZLF-1 for four hours. The simultaneous addition of four peptide pools to a single Generation of multi-virus-specific T cells 1/4 Loading antigen 1 Separate antigen loading 1/4 Loading antigen 2 PBMCs Stimulation 1/4 Loading antigen 3 Enrichment of IFN-γ secreting T cells T cell expansion Analysis for specificities Intracellular cytokine staining after in vitro restimulation with a mixture and individual peptide pools 1/4 Loading antigen 4 Figure 1 Experimental procedure for the generation of multi-virus-specific T cells. For details see the materials and methods section. 24 MACS & more Vol 15 1/2013

25 A Mixed antigen loading Separate antigen loading B CD4 CD8 Before enrichment After enrichment 0.42 %* 98.6 %* 1.45 %* 99.8 %* CD4 CD8 Before enrichment After enrichment 0.46 %* 98.4 %* 1.80 %* 99.5 %* Number of enriched IFN-γ + T cells per 10⁹ lymphocytes 1 10⁷ 1 10⁶ 1 10⁵ LP 3/1 mix LP 3/1 separate LP 19/2 mix LP 19/2 separate LP 6/2 mix LP 6/2 separate LP 11/2 mix LP 11/2 separate 1 10⁴ secreted IFN-γ * among CD4 + /CD8 + T cells secreted IFN-γ CD4 + T cells CD8 + T cells Figure 2 Enrichment of multi-virus-specific T cells after stimulation with individual peptide pools or a combination thereof. PBMCs from four different donors were stimulated in two different ways as described in the materials and methods section: i) Cells were stimulated with a combination of pp65, IE-1, hexon, and BZLF1 peptide pools in a single batch (mixed antigen loading), or ii) cells were loaded with the individual peptide pools in four aliquots, and aliquots were subsequently combined for further stimulation (separate antigen loading). Subsequently, virus-specific T cells were enriched according to IFN-γ secretion. Before and after enrichment IFN-γ secreting CD4 + and CD8 + T cells were analyzed by flow cytometry. (A) Results from one representative donor are shown. Numbers indicate frequencies among CD4 + or CD8 + cells. (B) Numbers of IFN-γ positive CD4 + and CD8 + T cells enriched from PBMCs of four different donors. sample might decrease the activation efficacy for each peptide pool due to competition of peptides for MHC binding. As a control, we divided the PBMC samples into four aliquots, incubated each aliquot with a single peptide pool for two hours, and recombined the four aliquots for T cell stimulation for another four hours. Afterwards, we used the Large Scale IFN-γ Secretion Assay Enrichment Kit, human, from Miltenyi Biotec to magnetically select antigen-activated IFN-γ secreting CD4 + and CD8 + T cells. Purities of enriched cells were determined by flow cytometry using the MACSQuant Analyzer (Miltenyi Biotec). Enriched multi-virus-specific CD4 + and CD8 + T cells were expanded in the presence of IL 2 and feeder cells for 9 13 days. A flowchart illustrating the experimental procedure is shown in figure 1. Analysis of PBMCs and multi-virus-specific T cells for antigen specificity For the analysis of antigen specificity and functionality of the enriched, expanded multivirus-specific T cells, we examined the IFN-γ response after short-term in vitro restimulation with individual peptide pools or a mixture thereof. For comparison, we also restimulated PBMCs. The IFN-γ response was determined by intracellular cytokine staining using an A Mixed antigen loading Separate antigen loading B CD4 Unstimulated sample Stimulated sample 0.11%* 31.39%* 0.03%* 33.99%* CD4 Unstimulated sample Stimulated sample 0.04%* 51.40%* 0.08%* 61.74%* % IFN-γ + T cells LP 3/1 mix LP 3/1 separate LP 19/2 mix LP 19/2 separate LP 6/2 mix LP 6/2 separate LP 11/2 mix LP 11/2 separate CD8 CD intracellular IFN-γ * among CD4 + /CD8 + T cells 0 CD4 + T cells CD8 + T cells Figure 3 Restimulation of enriched and expanded multi-virus-specific T cells. Enriched IFN-γ secreting cells were expanded for 9 to 13 days. Subsequently, cells were restimulated with a mixture of peptide pools or were left unstimulated. Cells were analyzed for IFN-γ production by intracellular staining and flow cytometry. (A) Results from one representative donor are shown. Numbers indicate frequencies among CD4 + and CD8 + T cells, respectively. (B) Frequencies of IFN-γ positive CD4 + and CD8 + T cells. Results from four different donors are shown. Vol 15 1/2013 MACS & more 25

26 A Relative frequency of specific CD4 + T cells (%) donor 3/1 donor 19/2 donor 11/2 PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate Relative frequency of specific CD8 + T cells (%) donor 3/1 donor 19/2 donor 6/2 donor 11/2 PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate PBMC mix separate BZLF1 EBNA-1 AdV hexon IE-1 pp65 B Number of IFNγ + CD4 + T cells 1 10⁸ 1 10⁷ 1 10⁶ 1 10⁵ 1 10⁴ CMV pp65 PBMC after expansion 1 10⁷ 1 10⁶ 1 10⁵ 1 10⁴ 1 10³ CMV IE-1 AdV hexon EBV EBNA-1 EBV BZLF1 PBMC after expansion 1 10⁸ 1 10⁷ 1 10⁶ 1 10⁵ 1 10⁴ PBMC after expansion 1 10⁶ 1 10⁵ 1 10⁴ 1 10³ PBMC after expansion 1 10⁶ 1 10⁵ 1 10⁴ 1 10³ PBMC after expansion LP 11/2 mix LP 11/2 separate LP 6/2 mix LP 6/2 separate LP 19/2 mix LP 19/2 separate LP 3/1mix LP 3/1 separate 1 10⁸ 1 10⁷ 1 10⁷ 1 10⁷ 1 10⁷ Number of IFNγ + CD8 + T cells 1 10⁷ 1 10⁶ 1 10⁵ 1 10⁶ 1 10⁵ 1 10⁴ 1 10⁶ 1 10⁵ 1 10⁴ 1 10⁶ 1 10⁵ 1 10⁴ 1 10⁶ 1 10⁵ 1 10⁴ 1 10⁴ PBMC after expansion 1 10³ PBMC after expansion 1 10³ PBMC after expansion 1 10³ PBMC after expansion 1 10³ PBMC after expansion Figure 4 Analysis of individual virus-specific T cell populations. Multi-virus-specific T cells were enriched as indicated in figure 2 and expanded. PBMCs and multi-virus-specific T cell lines were restimulated with individual peptide pools or a mixture thereof. The relative frequencies and absolute cell numbers of T cells with a single antigen specificity were calculated based on total cell numbers and the frequencies of IFN-γ positive T cells among PBMCs and multi-virus-specific T cells upon restimulation with individual peptide pools. Data from different donors are shown as indicated. Anti-IFN-γ-PE antibody (Miltenyi Biotec). CD4 + and CD8 + cells were then analyzed by flow cytometry using the MACSQuant Analyzer. Based on total cell numbers and the frequencies of IFN-γ positive T cells among PBMCs and multi-virus-specific T cells upon restimulation with individual peptide pools, we calculated the relative frequencies and absolute cell numbers of T cells with a single antigen specificity. Results and discussion Magnetic enrichment of multi-virusspecific CD4 + and CD8 + T cells Using the Large Scale IFN-γ Secretion Assay Enrichment Kit, we were able to consistently enrich virus-specific CD4 + and CD8 + T cells to purities higher than 90%. Flow cytometric analyses of IFN-γ secreting CD4 + and CD8 + T cells in PBMCs before and after magnetic enrichment are shown in figure 2A for one representative donor. Prior to enrichment, cells were stimulated in two different ways: Cells were either stimulated with a combination of pp65, IE-1, hexon, and BZLF1 peptide pools in a single batch, or cells were loaded with the individual peptide pools in four aliquots, and aliquots were subsequently combined for further stimulation. Both stimulation procedures resulted in comparable frequencies of IFN-γ secreting T cells before enrichment 26 MACS & more Vol 15 1/2013

27 (fig. 2A). Likewise, purities of enriched cells (fig. 2A and data not shown) and numbers of enriched cells (fig. 2B) were similar in the respective samples from all tested donors, regardless of whether the samples underwent mixed or separate antigen loading. Specificity and functionality of the enriched and expanded multi-virus-specific T cells Within 9 to 13 days cell populations that were generated by either mixed or separate antigen loading expanded between 25- and 496-fold (data not shown). Both cell lines were restimulated with a mixture of peptide pools and analyzed for intracellular IFN-γ production. The percentage of IFN-γ reexpressing CD4 + T cells derived from mixed antigen loading and separate antigen loading amounted to % and %, respectively. Similarly, the percentage of CD8 + T cells amounted to % and %, respectively (fig. 3A,B). These results confirm the high specificity and functionality of the T cell lines. T cell expansion rates and frequencies of T cells re-expressing IFN-γ were similar, regardless of whether the cell lines were originally generated by mixed or separate antigen loading (fig. 3B). original PBMCs were loaded with a mixture of peptide pools or separately with single peptide pools, the obtained T cell lines were comparable with respect to the expansion rate of the individual specificities. Conclusion A combination of several PepTivator Peptide Pools enables the simultaneous, effective activation of CMV-, EBV-, and AdV-specific (tri-virus-specific) CD4 + and CD8 + T cells. Activated tri-virus-specific T cells can be co-enriched using the Large Scale IFN-γ Secretion Assay Enrichment Kit. Enriched tri-virus-specific T cells can be expanded without significantly altering the proportion of the individual specificities. Enriched tri-virus-specific T cells are fully functional and re-express IFN-γ upon restimulation. References 1. Bogedain, C. et al. (1995) J. Virol. 69: Saulquin, X. et al. (2000) Eur. J. Immunol. 30: Precopio, M.L. et al. (2003) J. Immunol. 170: Tynan, F.E. et al. (2005) J. Exp. Med. 202: Blake, N. et al. (2000) J. Immunol. 165: Khanna, R. et al. (1995) Virology 214: Paludan, C. et al. (2002) J. Immunol. 169: Leen, A. et al. (2001) J. Virol. 75: Voo, K.S. et al. (2004) J. Exp. Med. 199: Voo, K.S. et al. (2002) Cancer Res. 62: Long, H.M. et al. (2005) J. Virol. 79: Feuchtinger, T. et al. (2006) Br. J. Haematol. 134: Feuchtinger, T. et al. (2010) Blood 116: Einsele, H. et al. (2002) Blood 99: Mackinnon, S. et al. (2008) Blood Cells Mol. Dis. 40: Peggs, K.S. et al. (2011) Clin. Infect. Dis. 52: Moosmann, A. et al. (2010) Blood 115: Icheva, V. et al. (2013) J. Clin. Oncol. 31: Individual virus-specific T cell populations show similar enrichment and expansion rates The strategy for the enrichment of virusspecific T cells from PBMCs is based on IFN-γ expression induced by stimulation with the peptide pools. Therefore, differences between stimulation efficacies of individual peptide pools would result in differences in IFN-γ responses of the respective virus-specific T cells, and ultimately in a biased proportion of specificities within the enriched multivirus-specific T cell population. To ensure that the proportions of the different specificities are similar before and after enrichment, we compared the relative frequencies (fig. 4A) and absolute numbers (fig. 4B) of T cells specific for each single antigen in PBMCs and in the T cell lines. The relative frequencies of T cells specific for the individual CMV, EBV, and AdV antigens were about the same in PBMCs and in both T cell lines. This demonstrates that all individual virus-specific T cell populations within PBMCs were effectively stimulated and enriched. Moreover, regardless of whether the MACS Product* PepTivator CMV pp65, human PepTivator CMV IE-1, human PepTivator AdV5 Hexon, human PepTivator EBV EBNA-1, human PepTivator EBV BZLF1, human Large Scale IFN-γ Secretion Assay Enrichment Kit, human IFN-γ Secretion Assay Cell Enrichment and Detection Kit (PE), human Order no. For further specificities visit MACSQuant Analyzer Anti-IFN-γ, CD4, and CD8 antibodies Human IL-2 and other cytokines * Products are for research use only. Visit Visit Vol 15 1/2013 MACS & more 27

28 Mouse NK cells isolated to high purity by MACS Technology are fully functional Kathrin Meinhardt 1, Irena Kroeger 1, Sabine Mueller 2, and Evelyn Ullrich 1,3,4 1 Department of Internal Medicine 5 Hematology/Oncology, University of Erlangen, Germany 2 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany 3 Laboratory for Cellular Immunology, Pediatric Hematology & Oncology, J. W. Goethe University, Frankfurt am Main, Germany 4 Center for Cell and Gene Therapy, Frankfurt am Main, Germany Introduction NK cells play a crucial role in both innate and adaptive immunity. Due to their capacity to lyse tumor cells without pre-activation, they hold great potential for use in cellular therapies¹. To investigate the function of NK cells in detail, it is crucial to work with cell populations of high purity. In this study, we compared two different strategies for NK cell isolation based on MACS Technology: i) positive selection of NK cells according to the expression of CD49b (DX5) and ii) depletion of non-nk cells. Both approaches yielded NK cells with high purities from BALB/c mouse spleen. We further optimized the procedure for the depletion of non-nk cells, allowing for the isolation of highly pure NK cells also from C57BL/6 mouse spleen. Isolated NK cells were fully functional in terms of IFN-γ secretion and tumor lysis capacity. Materials and methods Sample preparation Spleens from female, 7 9-week-old C57BL/6 and BALB/c mice were dissociated into singlecell suspensions as described². MicroBeads, mouse (Miltenyi Biotec) in combination with the automacs Pro Separator (Miltenyi Biotec) and the program PosselD according to the standard protocol. Untouched NK cells were isolated using the NK Cell Isolation Kit, mouse (Miltenyi Biotec) and the automacs Pro Separator with program DepleteS. We further improved the depletion cocktail included in this kit to allow % of NK cells Spleen *** 0 0 BALB/c C57BL/6 for a more effective depletion of residual T cells, B cells, and macrophages. This optimized cocktail is now part of the protocol of the new NK Cell Isolation Kit II from Miltenyi Biotec. For NK cell isolation from C57BL/6 spleens, we recommend incubation periods of minutes with antibodies and MicroBeads. Purified cell populations were subsequently analyzed by flow cytometry BALB/c Blood * C57BL/6 Isolation of NK cells CD49b (DX5) + NK cells were isolated by positive selection using CD49b (DX5) Figure 1 Frequencies of NK cells in spleen and peripheral blood from two different mouse strains. NK cells from spleens (n=7) and blood (n=4) from BALB/c and C57BL/6 mice were analyzed by flow cytometry with gating on viable CD49b + NKp46 + cells. ***p=0.0006, *p= Data were adapted from reference MACS & more Vol 15 1/2013

29 Analysis of cytokine production by isolated NK cells To assess the function of isolated NK cells, we determined their capacity to secrete IFN-γ. To this end, the cells were stimulated overnight with IL-2 (50,000 U/mL). The next day, cell culture supernatants were analyzed using the BD OptEIA Mouse IFN-γ ELISA Set (BD Biosciences). BALB/c CD49b CD49b (DX5) MicroBeads NK Cell Isolation Kit NK Cell Isolation Kit II 81% +/ 2 90% +/ % +/ 1.3 Tumor lysis assay The capacity of isolated NK cells to lyse tumor cells was analyzed by using the crystal violet assay as described previously¹. Briefly, 5,000 tumor cells were plated in 96-well plates and cocultured with isolated NK cells at an effector:target ratio of 10:1. To avoid MHC mismatch effects, we used B16F10 melanoma cells together with NK cells from C57BL/6 mice, and CT26 colon carcinoma cells with NK cells from BALB/c mice. The tumor cells were originally derived from the same background as the corresponding NK cells. Results and discussion NK cell frequencies in spleen and peripheral blood from different mouse strains To gain insight into the overall NK cell content of mouse spleen and peripheral blood from the mouse strains C57BL/6 and BALB/c, we analyzed the frequency of CD3 CD49b + NKp46 + NK cells by flow cytometry. Figure 1 indicates that the percentage of NK cells in both spleen and blood from BALB/c mice was approximately twice as high as from C57BL/6 mice. There were no significant differences in the proportions of CD27 + CD11b, CD27 + CD11b +, and CD27 CD11b + NK cell subsets.² Magnetic isolation of splenic NK cells For detailed characterization of NK cells, in particular functional analysis, it is crucial to use cell separation methods that allow for high cell purities. Here we compared different strategies based on MACS Technology for the isolation of NK cells from BALB/c and C57BL/6 mice. The first strategy involved positive selection of NK cells using CD49b (DX5) MicroBeads, based on the expression of the CD49b antigen. The second approach utilized depletion of non-nk cells using the NK Cell Isolation Kit, mouse, resulting in the isolation of untouched NK cells. Figure 2 shows that both approaches allowed C57BL/6 65% +/ 5 77% +/ % +/ 3.1 NKp46 Figure 2 Isolation of splenic NK cells from BALB/c and C57BL/6 mice using different strategies based on MACS Technology. Cells were isolated as described in the materials and methods section. Subsequently, CD49b (DX5) + NKp46 + cells were analyzed by flow cytometry. Numbers indicate mean purities and standard deviations from at least five independent experiments. Data were adapted from reference 2. for efficient isolation of NK cells from BALB/c mice with purities of up to 90%, whereas cells from C57BL/6 mice reached lower purities of up to 77%. This led us to modify the depletion IFN-γ (pg/ml) BALB/c C57BL/6 Figure 3 IFN-γ production by isolated NK cells. Cells were stimulated with IL-2 (50,000 Units/mL) over night. Cell culture supernatants were analyzed by an IFN-γ specific ELISA. One representative experiment of at least three independent experiments is shown. Experiments were performed in triplicates. Data were adapted from reference 2. IFN-γ (pg/ml) cocktail to achieve a more effective removal of residual T cells, B cells, and macrophages. Strikingly, the new depletion cocktail, i.e., NK Cell Isolation Kit II, resulted in high-purity NK CD49b (DX5) MicroBeads NK Cell Isolation Kit NK Cell Isolation Kit II Vol 15 1/2013 MACS & more 29

30 BALB/c C57BL/6 100 CT B16F10 % of specific tumor lysis % of specific tumor lysis CD49b (DX5) MicroBeads NK Cell Isolation Kit NK Cell Isolation Kit II Figure 4 Tumor lysis assay. For the crystal violet assay, 5,000 tumor cells were plated in 96-well plates and cocultured with isolated NK cells at an effector: target ratio of 10:1. One representative experiment of at least three independent experiments is shown. Experiments were performed in triplicates. Data were adapted from reference 2. cells from both C57BL/6 (approx. 90%) and BALB/c spleens (approx. 95%). Importantly, the subset distribution of CD27 + CD11b, CD27 + CD11b +, and CD27 CD11b + cells remained unaltered, regardless of whether the cells were isolated by positive selection or depletion (not shown). Functional analysis of isolated NK cells Next we tested whether the isolated cells retained their full functionality, i.e., the capacity to produce cytokines in response to stimulation with IL-2. Our results indicate that both BALB/c and C57BL/6 NK cells from all three isolation procedures secreted IFN-γ (fig. 3). NK cells prepared with the NK Cell Isolation Kit II showed slightly reduced IFN-γ production compared to the other approaches. This may be due to the higher NK cell purity in these preparations and thus a lower proportion of contaminating cells that might influence the IFN-γ production. Moreover, we analyzed the isolated NK cells for their capacity to lyse tumor cells. Our results from the crystal-violet assay show that BALB/c and C57BL/6 NK cells effectively lysed CT26 colon carcinoma and B16F10 melanoma cells, respectively (fig. 4). Conclusion Both CD49b (DX5) MicroBeads and the NK Cell Isolation Kit allow for the isolation of NK cells from BALB/c mouse spleens with high purity. The new NK Cell Isolation Kit II yields highly pure NK cells from BALB/c mice and represents the optimal solution for the isolation of C57BL/6 NK cells. Isolated NK cells are fully functional regardless of whether they were isolated by positive selection or depletion. References 1. Terme, M. et al. (2008) Nat. Immunol. 9: Meinhardt, K. et al. (2012) J. Immunol. Methods 378: MACS Product* automacs Pro Separator Starter Kit CD49b (DX5) MicroBeads, mouse Order no NK Cell Isolation Kit II, mouse * Products are for research use only. MACS Cytokines Consistent immune cell expansion and differentiation Superior quality Up to GMP grade Reliable Standardized high biological activity (IU/mg) Flexible Small, bulk, or customized fillings Time-saving Lot-specific activities available, no revalidation required macscytokines.com MACS GMP Cytokines, MACS GMP Media, and MACS GMP Antigens are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. The products are manufactured and tested under a certified ISO 9001 quality system and in compliance with relevant GMP guidelines. They are designed following the recommendations of USP <1043> on ancillary materials. 30 MACS & more Vol 15 1/2013

31 Measuring prolyl aminopeptidase activity in extracts prepared from magnetically purified malaria parasites Fabio L. da Silva, Donald L. Gardiner, and Katharine R. Trenholme Department of Biology, Queensland Institute of Medical Research, Brisbane, Australia Introduction Despite extensive eradication campaigns malaria remains a major cause of morbidity and mortality worldwide with an estimated 1 3 million deaths each year¹. The prevention and treatment of this disease is becoming increasingly difficult due to the spread of drugresistant parasites and, therefore, there is an urgent need to discover novel targets for new drug development. The aminopeptidases are a group of enzymes identified within the genome of the most lethal malaria parasite, Plasmodium falciparum, and which may have the potential to become new targets for antimalarial drug development. These enzymes catalyze the cleavage of the N-terminal amino acids from proteins and peptides and one such enzyme, the prolyl aminopeptidase (PfS33PAP) is currently being investigated in our laboratory. As a first step in characterizing the PfS33PAP, it was necessary to identify the enzyme activity in extracts of malaria parasites. However, for biochemical studies it is often crucial to obtain pure preparations of parasite-infected red blood cells (RBCs) that are relatively free of uninfected cells. P. falciparum passes through a series of morphologically distinguishable stages as they mature within RBCs, developing from ring stages into trophozoites and then into schizontstage parasites. Each parasite contains up to 16 daughter merozoites, which are released into the blood stream to invade new RBCs. During its 48-hour development cycle the parasite digests 65 70% of the host cell hemoglobin producing insoluble hemozoin as a byproduct. The magnetic properties of hemozoin allow the isolation of parasitized erythrocytes. Techniques for the in vitro cultivation of P. falciparum are well established and relatively high parasitemia can be achieved, though this is dependent on the particular parasite line and skill of operator. In this report we present a protocol for the rapid magnetic purification of P. falciparum infected RBCs from in vitro cultures using the automacs Pro Separator. Extracts prepared from purified parasites exhibited prolyl aminopeptidase activity. Materials and methods Preparation of parasite cultures P. falciparum parasites were cultured and synchronized in vitro using established methods²,³. An in vitro culture of trophozoitestage parasites at 3 5% parasitemia was used for further processing. Ten ml of the parasite culture (equivalent to 500 µl packed cell volume) were centrifuged at 400 g for 5 minutes, and the supernatant was discarded. Cells were washed twice with pre-warmed (37 C) RPMI. Subsequently, the 500-µL cell sediment was resuspended in 50 ml RPMI (equivalent to a 1% hematocrit), and incubated in a water bath at 37 C for 30 minutes. Purification of P. falciparum infected erythrocytes using the automacs Pro Separator Following the 30-minute incubation in RPMI, the cells were gently resuspended. The sample was split into five 10-mL aliquots in 15-mL tubes and kept at 37 C prior to separation. The first sample was placed in position A1 of a Chill 10 Rack at room temperature. Empty 15-mL tubes for the collection of the negative and positive fractions were place in position B1 and C1, respectively. The rack was placed onto the MACS MiniSampler of the automacs Pro Separator. Cell enrichment was performed using program PMalaria. The remaining samples were consecutively processed in the same way. Prior to separation each sample was gently mixed to resuspend cells. Assessment of yield All positive fractions were combined and centrifuged at 400 g for 5 minutes. The supernatant was discarded. Approximately 3 μl of the cell pellet was smeared onto a microscope slide, fixed with methanol and stained with Giemsa solution. Parasitemia was determined microscopically. Prolyl aminopeptidase activity assay Purified parasites were hypotonically lysed with distilled water to obtain active malarial extract. Enzymatic activity of the prolyl aminopeptidase, PfS33PAP, in malaria Vol 15 1/2013 MACS & more 31

32 Submitting your abstract is easy I want to be a Pro! Abstract Awards Miltenyi Biotec is happy to announce a new grant program aimed at accelerating innovative research. Submit an abstract describing how you would use an automacs Pro Separator to advance your research and you could win a substantial credit towards its purchase. Become a Pro today! Entering is as easy as Visit the official I want to be a Pro! Abstract Awards website 2. Tell us in 500 words or less how you would use the automacs Pro Separator to advance your research 3. Submit your abstract Submissions must be received by midnight, July 15, Winners will be announced via on July 30, miltenyibiotec.com 32 MACS & more Vol 15 1/2013

33 parasites was determined by addition of extract aliquots to the fluorogenic substrate H-Pro- NHMec. Reactions were carried out in 96-well microtiter plates (100 µl total volume, 60 min incubation at 37 C) using a spectrofluorimeter with excitation at 370 nm and emission at 460 nm. Initial rates were obtained over a range of substrate concentrations in 50 mm Tris-HCl, ph 7.5. To ensure the prolyl aminopeptidase activity was not due to malarial or human metalloproteases, aliquots of malarial extract were pre-incubated (10 min, 37 C) with 2 mm of the metal chelator o-phenanthroline in 50 mm Tris-HCl and then assayed for activity against H-Pro-NHMec, H-Ala-NHMec, H-Leu-NHMec, and H-Glu-NHMec. To distinguish the PfS33PAP activity from a second putative P. falciparum prolyl aminopeptidase, extracts from a PfS33PAP knockout parasite line were tested for H-Pro- NHMec activity and compared to 3D7 wild type parasites. Assays were performed in the presence of 2 mm phenathroline to inhibit metalloprotease activity. Results and discussion Enrichment of P. falciparum infected RBCs Cultures of trophozoite stage P. falciparum infected RBCs at between 3 and 5% parasitemia were enriched to give a parasitemia of 80 85% following one round of enrichment using the automacs Pro Separator (fig. 1). Relative activity (%) H-Ala-NHMec H-Ala-NHMec + o-phenanthroline H-Leu-NHMec H-Leu-NHMec + o-phenanthroline H-Pro-NHMec H-Pro-NHMec + o-phenanthroline Figure 2 H-Pro-NHMec hydrolysis in P. falciparum extracts. Hydrolysis was measured in the presence or absence of the metal chelator o-phenanthroline (2 mm) as described in the materials and methods section. Data reflect the mean relative hydrolysis activity (n=2) of 3D7 extract with 200 µm H-Pro-NHMec, 10 µm H-Ala-NHMec, and 5 µm H-Leu-NHMec as substrates. P. falciparum extracts contain prolyl aminopeptidase activity Soluble extracts of hypotonically lysed wild type parasites exhibited prolyl aminopeptidase activity towards H-Pro-NHMec, which was only partially inhibited by the metal chelator o-phenanthroline (fig. 2). In contrast, aminopeptidase activities towards the substrates H-Ala-NHMec, H-Leu-NHMec were completely inhibited by the chelator. This result indicates that at least part of the prolyl aminopeptidase activity was not due to malarial or human metalloproteases. To distinguish between PfS33PAP activity and that of a second putative malarial prolyl aminopeptidase, we compared the enzymatic activity in extracts of wild type and a PfS33PAP knockout strain and found that o-phenanthroline-insensitive prolyl aminopeptidase activity was reduced in the knockout (fig. 3). The remaining activity in the knockout might be due to a second as yet uncharacterized prolyl aminopeptidase. 100 H-Pro-NHMec + o-phenanthroline Relative activity (%) Wild type PfS33PAP knockout Figure 1 P. falciparum infected erythrocytes isolated using the automacs Pro Separator. The image shows a light-microscopic view of a Giemsastained thin smear of enriched infected cells. Figure 3 H-Pro-NHMec hydrolysis in extracts from 3D7 wild type and PfS33PAP knockout. Prolyl aminopeptidase activity was measured using extracts prepared from wild type and the PfS33PAP knockout strain. Tests were performed in the presence of 2 mm o-phenanthroline. Vol 15 1/2013 MACS & more 33

34 Conclusion The automacs Pro Separator allows the quick and efficient isolation of P. falciparum infected RBCs to high purities. Use of the automacs Pro Separator minimizes manual handling steps during separation, enabling operator-independent separation results. Enzyme activity can be measured in extracts prepared from P. falciparum malaria parasites purified with the automacs Pro Separator. PfS33 PAP enzyme activity was detected in extracts prepared from wild type P. falciparum parasites, and was reduced in extracts prepared from a transgenic PfS33 PAP knockout parasite line. References 1. World Malaria Report Available at malaria2008.pdf 2. Trager, W. and Jensen, J.B. (1976) Science 193: Lambros, C. and Vanderberg, J.P. (1979) J. Parasitol. 65: MACS Product* automacs Pro Separator Starter Kit * Products are for research use only. Order no Easy access. Anytime. MACS PICK UP POINT A smarter way to manage inventory MACS PICK UP POINT is a fully automated, on-site inventory management program that centers around intelligent refrigerator or freezer units. MACS PICK UP POINT gives you access to Miltenyi Biotec research products at any time, all with a swipe of a card. Save time and costs Get 24/7 product availability Customize it to fit your needs Focus on what really matters your research Currently available in Germany and the U.S. miltenyibiotec.com/macspickup 34 MACS & more Vol 15 1/2013

35 MACS&more Vol. 15 1/2013 Germany/Austria/ Switzerland Miltenyi Biotec GmbH Friedrich-Ebert-Straße Bergisch Gladbach Germany Phone Fax USA/Canada Miltenyi Biotec Inc Lindbergh Street Auburn, CA 95602, USA Phone 800 FOR MACS Phone Fax Australia Miltenyi Biotec Australia Pty. Ltd. Unit 16A, 2 Eden Park Drive Macquarie Park NSW 2113 Australia Phone Fax macs@miltenyibiotec.com.au Benelux Miltenyi Biotec B.V. Schipholweg 68 H 2316 XE Leiden The Netherlands macs@miltenyibiotec.nl Customer service The Netherlands Phone Fax Customer service Belgium Phone Fax Customer service Luxembourg Phone Fax China Miltenyi Biotec GmbH Shanghai Office Rm No. 319 Xianxia Rd. Shanghai , P.R. China Phone Fax macs@miltenyibiotec.com.cn France Miltenyi Biotec SAS 10 rue Mercoeur Paris, France Phone Fax macs@miltenyibiotec.fr Italy Miltenyi Biotec S.r.l. Via Persicetana, 2/D Calderara di Reno (BO) Italy Phone Fax macs@miltenyibiotec.it Japan Miltenyi Biotec K.K. Nittsu-Eitai Building 5F Fuyuki, Koto-ku, Tokyo , Japan Phone Fax macs@miltenyibiotec.jp Nordics and Baltics Miltenyi Biotec Norden AB Scheelevägen Lund Sweden macs@miltenyibiotec.se Customer service Sweden Phone Fax Customer service Denmark Phone Fax Customer service Norway, Finland, Iceland, and Baltic countries Phone Fax Singapore Miltenyi Biotec Asia Pacific Pte Ltd. 100 Beach Road #28-06 to Shaw Tower Singapore Phone Fax macs@miltenyibiotec.com.sg South Korea Miltenyi Biotec Korea Petra building 659 Yeoksam-dong Gangnam-gu Seoul, South Korea macs@miltenyibiotec.kr Spain Miltenyi Biotec S.L. C/Luis Buñuel 2 Ciudad de la Imagen Pozuelo de Alarcón (Madrid) Spain Phone Fax macs@miltenyibiotec.es United Kingdom Miltenyi Biotec Ltd. Almac House, Church Lane Bisley, Surrey GU24 9DR, UK Phone Fax macs@miltenyibiotec.co.uk Miltenyi Biotec provides products and services worldwide. Visit /local to find your nearest Miltenyi Biotec contact. Vol 15 1/2013 MACS & more 35

36 Cell separation renewed Next-generation cell isolation kits Reliability now comes with improved performance Miltenyi Biotec introduces the next generation of cell isolation kits. All the reliability you expect. All the flexibility you need. Now with improved performance higher purity, greater recovery, and faster protocols. Read the news article on page 5 or visit for more information. Better performance Exceptional purity and unmatched recovery Reliable technology MACS Technology the solid foundation for your research Faster protocol Isolate cells in as little as 25 minutes miltenyibiotec.com