in vivo 20: 239-242 (2006)

Isolation of Human Myeloid Dendritic Cells from Tumor Tissue and Peripheral Blood

CARSTEN BROCKS*, HENDRIK GRAEFE*, HENNING FRENZEL, RALPH PRIES and BARBARA WOLLENBERG

Department of Otorhinolaryngology, University of Schleswig-Holstein Campus Lübeck, 23538 Lübeck, Germany

Abstract. Background: Human myeloid dendritic cells (MDC) antigen-1, BDCA-1) and were determined to be negative for have been identified in human solid tumor tissue of head and neck lineage markers (CD3, CD16, CD19, CD20, CD56) (2). squamous cell carcinoma (HNSCC), their cellular functions being DCs, as well as other types of immune cells, have been strongly affected in this environment. The characterization of MDC shown to infiltrate human solid tumor tissues, such as head and differentiation and functions requires the establishment of highly neck squamous cell carcinoma (HNSCC) (9-11). HNSCC is one efficient isolation procedures. Materials and Methods: Human of the most frequent cancers in the world and, over the last 40 MDC were isolated from peripheral blood and solid HNSCC using years, standard treatment has only marginally improved the density gradient centrifugation or preparation of single cell 5-year survival rate of patients with this disease. It is supposed suspensions, respectively. The cells were isolated by “magnetic bead that tumor production of various immune suppressive separation” using magnetically-labelled antibodies and were mediators contributes to the massively impaired immune analyzed by flow cytometry. Results: The isolation of human MDC functions in patients with head and neck cancer (12, 13). from peripheral blood or solid HNSCC tissue resulted in highly Since monocyte-derived DCs are known to show enriched cell populations and revealed cell purities of about 90%. significant differences compared to in vivo matured MDCs Conclusion: The scale of molecular investigations on human (14, 15), it is essential to establish effective as well as gentle myeloid dendritic cells is restricted by the limited proportion of this MDC isolation procedures to investigate the influence of cell type. Thus, highly efficient and gentle MDC isolation HNSCC on distinct cellular functions of human MDCs. procedures are essential to characterize in vivo matured MDC. In this work, human MDCs were directly isolated from peripheral blood and HNSCC tissue. Our data Dendritic cells (DCs) are the most potent of the antigen- demonstrated the isolation yields and cell purities which presenting cells and, therefore, essential for the initiation a finally define the scale of possible molecular investigations. primary immune response. DCs are CD34+ bone marrow- derived leukocytes and can be subdivided into subtypes, such Materials and Methods as plasmacytoid and myeloid DCs (PDC, MDC), as well as the Langerhans cells of the skin (1-3). A complicated MDC isolation from peripheral blood. MDCs were isolated from trafficking system leads them from the bone marrow, through human peripheral blood provided by the blood bank of the the bloodstream to distinct peripheral tissues where they University Hospital, Lübeck, Germany. The blood donors were fulfill their antigen-capturing function. Finally they migrate healthy 18-65 years old who were tested negative for allergies. to lymphoid organs in order to present processed antigens to Additional exclusion criteria were manifest infections during the previous 4 weeks, fever and medication of any kind. PBMCs lymphocytes and, thus, to stimulate adequate immune (peripheral blood mononuclear cells) were obtained from buffy responses (2, 4-8). MDC show a monocytoid morphology and coats by Ficoll-Hypaque density gradient centrifugation, as express CD11c and CD1c (human blood dendritic cell described previously (16). The MDCs were isolated by magnetic bead separation using magnetically-labelled anti-BDCA-1 antibodies (Miltenyi, Bergisch Gladbach, Germany). The isolated cells were analyzed by flow cytometry using FSC and SSC properties *Both authors contributed equally to this work. and identified as a population of lineage-negative, CD11c-positive and HLA-DR-positive cells, as described previously (17). Correspondence to: Barbara Wollenberg, UK-SH, Klinik für HNO, To isolate more MDCs, leukaphereses can be used for Ratzeburger Allee 160, D - 23538 Lübeck, Germany. Tel: ++49 automated cell isolation (autoMACSì Separator, Miltenyi). 451 500 2241, e-mail: [email protected] Preparation of single-cell suspension. The HNSCC specimens were Key Words: Myeloid dendritic cells, HNSCC, peripheral blood. washed several times and carefully minced into small pieces in sterile

0258-851X/2006 $2.00+.40 239 in vivo 20: 239-242 (2006)

Figure 1. Isolation of myeloid dendritic cells (MDCs) from peripheral blood and tumor tissue. Human MDCs were isolated from peripheral blood and solid head and neck squamous cell carcinoma using density gradient centrifugation or preparation of single cell suspensions, respectively. The MDCs were isolated by magnetic bead separation using magnetically-labelled anti-BDCA-1 antibodies and were analyzed by flow cytometry.

serum-free RPMI medium (RPMI 1640 supplemented with 100 after density gradient centrifugation (for a model see Figure 1). units/ml penicillin, 1 mM glutamine and 100 units/ml streptomycin). Apart from MDCs, CD1c (BDCA-1) is also expressed on a The tumor tissue was digested with collagenase type VIII (1.5 mg/ml; Sigma) and DNase type I (1.0 Ìg/ml) for 120 min at 37ÆC with gentle subpopulation of CD19+ small resting B lymphocytes. agitation. The resulting cell suspensions were washed in phosphate- Therefore, CD19 MicroBeads were used for the depletion of B buffered saline (PBS), resuspended in PBS containing trypsin/EDTA cells before enriching BDCA-1+ MDCs. Light microscopy was and filtered through a 40-Ìm nylon cell strainer (Falcon; Becton used to calculate cell numbers as well as to analyze the viability Dickinson Labware) into cold RPMI medium containing 10% fetal of the isolated cells by Trypan-blue staining of dead cells. calf serum (FCS). Single cell suspensions were used for isolation of Microscopic analysis illustrated the characteristic shape and MDCs as described above. morphology, as well as vitality, of the huge majority of cells Flow cytometry. Surface antigen staining was performed as described (Figure 2). The isolated cells were analyzed by flow cytometry previously (4). The cells were stained with fluorescein-5- using FSC and SSC properties. The expression of characteristic isothiocyanate (FITC-), phycoerythrin- (PE), peridinin-chlorophyll- surface antigens was investigated and MDCs were identified by protein- (PerCP) conjugated antibodies by incubation on ice for their lack of lineage (lin) markers as well as their expression of 15 min, followed by washing with PBS. Fluorescence-labelled HLA-DR and CD11c, as described before (Figure 3). Magnetic monoclonal antibodies against CD11c, Lin-markers and HLA-DR bead separation of MDCs resulted in average cell counts of were purchased from BD Biosciences (Becton Dickinson, Heidelberg, about 3x108 cells per 500 ml of peripheral blood. Flow Germany). TO-PRO-3 iodide (2 nM; Molecular Probes, Leiden, The Netherlands) was used to determine dead cells. The samples were cytometric analysis revealed cell purities between 80 and 90%. analyzed on a FACSCanto (Becton Dickinson). Data acquisition and analysis were performed using the FACS DIVA software. Isolation of MDCs from solid HNSCC. To analyze the frequency and function of tumor-infiltrating MDCs, single Results cell suspensions of solid HNSCC were prepared. Therefore, tissue specimens were split into small pieces, digested and Isolation of MDCs from peripheral blood. MDCs were isolated filtered (for a model see Figure 1). from the peripheral blood of healthy donors by magnetic bead The frequencies of HNSCC-infiltrating MDCs were separation using magnetically labelled anti-BDCA-1 antibodies determined by flow cytometry of these single cell suspensions.

240 Brocks et al: Isolation of Myeloid Dendritic Cells

Figure 2. Light microscopic analysis illustrates the characteristic shape and morphology of the isolated myeloid dendritic cells (1, 2). Dead cells were determined by Trypan-blue staining as indicated by an arrow in panel 3.

Figure 3. Flow cytometric analysis of myeloid dendritic cells (MDCs). The isolation of MDCs using magnetic bead separation resulted in purities of about 90%. The MDCs were identified by flow cytometry as a population of lineage-negative as well as HLA-DR- and CD11c-positive cells.

Figure 4. Flow cytometric analysis of single cell suspensions of head and neck squamous cell carcinoma tissue revealed a myeloid dendritic cell (MDC) proportion of 0.2% of total cells. MDCs were analyzed with respect to SSC and FSC properties and identified as lineage (lin)-negative as well as CD11c- and HLA-DR-positive cells. TO-PRO-3 iodide was used to determine dead cells.

Our data revealed average MDC frequencies of about 0.2% blood. Single cell suspensions were used for magnetic bead of total cells (Figure 4), which was significantly lower separation as described for the isolation of MDC from compared to a frequency of about 0.5% in human peripheral peripheral blood.

241 in vivo 20: 239-242 (2006)

Discussion 6 D'Amico G, Bianchi G, Bernasconi S, Bersani L, Piemonti L, Sozzani S, Mantovani A and Allavena P: Adhesion, HNSCC is known to be infiltrated by various kinds of immune transendothelial migration, and reverse transmigration of in vitro cultured dendritic cells. Blood 92: 207-214, 1998. cells, but effective immune responses are greatly impaired by 7 Sallusto F, Schaerli P, Loetscher P, Schaniel C, Lenig D, Mackay the HNSCC microenvironment (9-11). Thus, the DC function CR, Qin S and Lanzavecchia A: Rapid and coordinated switch caused by abnormal differentiation of these cells represents in chemokine receptor expression during dendritic cell an important immune escape mechanism. Previously, it was maturation. Eur J Immunol 28: 2760-2769, 1998. suggested that large numbers of immature MDCs participate 8 Dieu MC, Vanbervliet B, Vicari A, Bridon JM, Oldham E, Ait- in decreased Ag-specific T cell responses in cancer patients. Yahia S, Briere F, Zlotnik A, Lebecque S and Caux C: Selective It is supposed that an increased infiltration of HNSCC by DCs recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med correlates with a worse prognosis (9, 18). Correspondingly, the 188: 373-386, 1998. T-lymphocyte and monocyte functions of peripheral blood 9 Hartmann E, Wollenberg B, Rothenfusser S, Wagner M, mononuclear cells from patients with HNSCC have been Wellisch D, Mack B, Giese T, Gires O, Endres S and Hartmann shown to be predictive factors for outcome (11) . G: Identification and functional analysis of tumor-infiltrating Since patient survival in HNSCC has not changed plasmacytoid dendritic cells in head and neck cancer. Cancer Res significantly in many years, despite progress in surgical, 63: 6478-6487, 2003. radiotherapy and chemotherapy techniques, the development 10 Veltri RW, Rodman SM, Maxim PE, Baseler MW and Sprinkle PM: Immune complexes, serum proteins, cell-mediated of immune-modulating therapies against HNSCC represents immunity, and immune regulation in patients with squamous cell a rapidly progressing alternative cancer treatment (19). carcinoma of the head and neck. Cancer 57: 2295-2308, 1986. Therefore, the use of effective DC isolation techniques is 11 Heimdal JH, Aarstad HJ and Olofsson J: Peripheral blood essential for the investigation of the influence of the HNSCC T-lymphocyte and monocyte function and survival in patients microenvironment on DC function and differentiation, as well with head and neck carcinoma. Laryngoscope 110: 402-407, 2000. as for the modulation of DCs for novel immunotherapeutic 12 Chin D, Boyle GM, Theile DR, Parsons PG and Coman WB: strategies. Molecular introduction to head and neck cancer (HNSCC) carcinogenesis. Br J Plast Surg 57: 595-602, 2004. 13 Whiteside TL: Immunobiology of head and neck cancer. Cancer Acknowledgements Metastasis Rev 24: 95-105, 2005. 14 Hartmann G and Krieg AM: Mechanism and function of a newly Special thanks to Nicole Bohnert for her skillful technical support in identified CpG DNA motif in human primary B cells. J Immunol some parts of this work and all the members of the Department of 164: 944-953, 2000. Otorhinolaryngology for helpful discussions and a comfortable 15 Jakob T, Walker PS, Krieg AM, Udey MC and Vogel JC: atmosphere. This work was supported by the Mildred Scheel-Stiftung Activation of cutaneous dendritic cells by CpG-containing (Deutsche Krebshilfe), the Monika-Kutzner-Stiftung and the Rudolf- oligodeoxynucleotides: a role for dendritic cells in the Bartling-Stiftung, Germany. augmentation of Th1 responses by immunostimulatory DNA. J Immunol 161: 3042-3049, 1998. References 16 Hartmann G, Weiner GJ and Krieg AM: CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA 96: 9305-9310, 1999. 1 Cella M, Sallusto F and Lanzavecchia A: Origin, maturation and 17 Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah antigen presenting function of dendritic cells. Curr Opin K, Ho S, Antonenko S and Liu YJ: The nature of the principal Immunol 9: 10-16, 1997. type 1 interferon-producing cells in human blood. Science 284: 2 Hart DN: Dendritic cells: unique leukocyte populations which 183-187, 1999. control the primary immune response. Blood 90: 3245-3285, 1997. 18 Almand B, Clark JI, Nikitina E, van Beynen J, English NR, Knight 3 Steinman RM: The dendritic cell system and its role in SC, Carbone DP and Gabrilovich DI: Increased production of immunogenicity. Annu Rev Immunol 9: 271-296, 1991. immature myeloid cells in cancer patients: a mechanism of 4 Sozzani S, Luini W, Borsatti A, Polentarutti N, Zhou D, immunosuppression in cancer. J Immunol 166: 678-689, 2001. Piemonti L, D'Amico G, Power CA, Wells TN, Gobbi M, 19 Dunn G, Oliver KM, Loke D, Stafford ND and Greenman J: Allavena P and Mantovani A: Receptor expression and Dendritic cells and HNSCC: a potential treatment option? responsiveness of human dendritic cells to a defined set of CC (Review). Oncol Rep 13: 3-10, 2005. and CXC chemokines. J Immunol 159: 1993-2000, 1997. 5 Sozzani S, Sallusto F, Luini W, Zhou D, Piemonti L, Allavena P, Van Damme J, Valitutti S, Lanzavecchia A and Mantovani A: Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol 155: 3292- Received January 23, 2006 3295, 1995. Accepted February 15, 2006

242