Generation of Human monocytafledte dendritiske celler fra fuldblod
Summary
Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.
Abstract
Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity.
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to naïve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.
Introduction
Dendritiske celler (DC'er) er de vigtigste specialiserede antigenpræsenterende celler i vores immunsystem. Umodne DCs (IDCS) bor i huden eller i slimhindevæv og er derfor blandt de første immunceller til at interagere med invaderende patogener. DC'er repræsenterer bro mellem det medfødte og det adaptive immunsystem 1, da de kan aktivere T- og B-celle-responser efter påvisning af patogener. Endvidere bidrager de til pro-inflammatoriske immunresponser på grund af udskillelse af store mængder af cytokiner, såsom IL-1β, IL-6 og IL-12. Udviklingslandene aktiverer også NK-celler og tiltrække andre immunceller til stedet for infektion med kemotaksi.
DC'er kan opdeles i umodne dendritiske celler (IDC'erne) og modne dendritiske celler (MDC'er) 2 baseret på deres morfologi og funktion. Efter anerkendelse af fremmede antigener af en af de mange mønstergenkendelse receptorer (f.eks toll-lignende receptorer, C-typelectiner, eller komplement receptorer) rigeligt udtrykt på celleoverfladen, IDC'erne undergå store forandringer og begynder at modnes. Under denne modningsproces, er receptorer for antigen capture nedreguleres, hvorimod molekyler er essentielle for antigenpræsentation er opreguleret 3. Modne DC'er opregulere major histocompatibility-komplekser I og II (MHC I og II), co-stimulerende molekyler såsom CD80 og CD86, som er essentielle for antigenpræsentation og aktivering af T-lymfocytter. Derudover er ekspressionen af kemokinreceptoren CCR7 på celleoverfladen induceres, som muliggør migration af DC'er fra perifert væv til lymfeknuderne. Migrationen lettes af "rullende" af DC'er langs en kemokinligand 19 (CCL19 / MIP-3b) og kemokinligand 21 (CCL21 / SLC) gradient til lymfeknuderne 4-6.
Efter migration, MDC'er præsentere den bearbejdede antigen til naive CD4 + og CD8 + T-celler, således initiating en adaptiv immunrespons mod de invaderende patogen 7. Denne interaktion med T-celler i lymfeknuder er også forbundet med spredning af virusset 8. Andre in vitro undersøgelser viste, at udviklingslandene effektivt fange og overføre HIV til T-celler, og at denne transmission resulterer i en kraftig infektion 9-12. Disse eksperimenter fremhæve, at in vivo HIV udnytter udviklingslandene som pendulfart fra periferien til lymfeknuderne. Under antigenpræsentation, DC'er udskiller centrale interleukiner der former differentieringen af effektor T-hjælperceller, og derfor er resultatet af hele immunrespons mod mikroben bestemt ved denne meget interaktion. Bortset fra type 1 (Th1) og type 2 (Th2) effektor T-celler, andre delmængder af CD4 + T-hjælper celler (f.eks, type 17 (Th17) og type 22 (Th22) T-celler) er blevet beskrevet, og deres induktion og funktion er blevet undersøgt grundigt. DC'er er endvidere involveret igenereringen af regulatoriske T-celler (tregs) 13,14. Disse celler er immunosuppressive og kan stoppe eller nedregulere induktion eller proliferation af effektor T-celler og er således af afgørende betydning for udvikling af immunitet og tolerance.
Humane konventionelle DCs (CDCS) omfatter flere delmængder af celler med en myeloid oprindelse (dvs. Langerhanske celler (LCS) og dermal og interstitielle DC'er) eller en lymfoid oprindelse (dvs. plasmacytoide DCs (pdCs)). For in vitro forsøg eller DC vaccinationsstrategier, er monocytafledte DCs rutinemæssigt anvendes som model for dermale udviklingslandene. Disse celler viser ligheder i fysiologi, morfologi, og funktion til konventionelle myeloide dendritiske celler. De genereres ved tilsætning af interleukin 4 (IL-4) og granulocyt-makrofag koloni-stimulerende faktor (GM-CSF) til monocytter isoleret fra raske donorer 12,15-18. Dendritiske celler kan også isoleres direkte fra dermale eller mukosale biopsier, eller kan endda be udviklet sig fra CD34 + hæmatopoietiske stamceller isoleret fra navlestrengen blodprøver opnået ex utero. Her viser vi, hvordan monocytter isoleres og stimuleres fra anti-koaguleret humant blod efter mononukleære perifere blodceller (PBMC) berigelse ved densitetsgradientcentrifugering. Efter inkubation i 5 dage, humane monocytter under særlige betingelser er differentieret i IDCS og er klar til forsøg procedurer i en ikke-kliniske omgivelser.
Protocol
Representative Results
Discussion
Denne protokol beskriver genereringen af monocytafledte dendritiske celler (MDDCs) gennem isolering af humane monocytter fra anti-koaguleret blod ved anvendelse af en magnetisk nanopartikel-baseret assay. I denne protokol, udføres de centrifugeringstrin opstrøms cellen isolation procedure, der fører til en berigelse af PBMC-fraktionen. Selvom celler tabes under centrifugering, for at overlejre indholdet af en fuldblod pack på densitetsgradient medium ville kræve 200-300 ml af densitetsgradient-medium og kan d…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank our technician Karolin Thurnes, Divison of Hygiene and Medical Microbiology, and Dr. Annelies Mühlbacher and Dr. Paul Hörtnagl, Central Institute for Blood Transfusion and Immunological Department, for their valuable help and support regarding this manuscript. We thank the Austrian Science Fund for supporting this work (P24598 to DW, P25389 to WP).
Materials
| APC Mouse Anti-Human CD19 Clone HIB19 | BD Biosciences | 555415 | |
| APC Mouse Anti-Human CD83 Clone HB15e | BD Biosciences | 551073 | |
| BD Imag Anti-Human CD14 Magnetic Particles | BD Biosciences | 557769 | |
| BD Imagnet | BD Biosciences | 552311 | |
| BSA (Albumin Fraction V) | Carl Roth | EG-Nr 2923225 | |
| Costar 6 Well Clear TC-Treated Multiple Well Plates | Costar | 3506 | |
| Density gradient media: Ficoll-Paque Premium | GE Healthcare Bio-Sciences | 17-5442-03 | |
| Dulbecco’s Phosphate Buffered Saline (D-PBS) | Sigma-Aldrich | D8537 | |
| Falcon 10mL Serological Pipet | Corning | 357551 | |
| Falcon 25mL Serological Pipet | Corning | 357525 | |
| Falcon 50mL High Clarity PP Centrifuge Tube | Corning | 352070 | |
| Falcon Round-Bottom Tubes | Corning | 352054 | |
| FITC Mouse Anti-Human CD3 Clone HIT3a | BD Biosciences | 555339 | |
| Ghost Dye Violet 510 (Cell Viability Reagent) | Tonbo biosciences | 13-0870 | |
| GM-CSF | MACS Miltenyi Biotec | 130-093-862 | |
| Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America) | Gibco | 10500-064 | |
| Hettich Rotanta 460R | Hettich | — | |
| IL-4 CC | PromoKine | C-61401 | |
| Isolation buffer: BD IMag Buffer (10X) | BD Biosciences | 552362 | |
| L-Glutamine solution | Sigma-Aldrich | G7513 | |
| Microcentrifuge tubes, 1,5 ml, SuperSpin | VWR | 211-0015 | |
| PE Mouse Anti-Human CD14 Clone M5E2 | BD Biosciences | 555398 | |
| PE Mouse Anti-Human CD209 Clone DCN46 | BD Biosciences | 551265 | |
| RPMI-1640 medium | Sigma-Aldrich | R0883 | |
| UltraPure 0.5M EDTA, pH 8.0 | Invitrogen | 15575020 |
References
- Banchereau, J., Steinman, R. M. Dendritic cells and the control of immunity. Nature. 392, 245-252 (1998).
- Steinman, R. M., Hemmi, H. Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol. 311, 17-58 (2006).
- Steinman, R. M., Inaba, K., Turley, S., Pierre, P., Mellman, I. Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies. Hum Immunol. 60, 562-567 (1999).
- Schwarz, J., Sixt, M. Quantitative Analysis of Dendritic Cell Haptotaxis. Methods Enzymol. 570, 567-581 (2016).
- Weber, M., Sixt, M. Live cell imaging of chemotactic dendritic cell migration in explanted mouse ear preparations. Methods Mol Biol. 1013, 215-226 (2013).
- Sixt, M., Lammermann, T. In vitro analysis of chemotactic leukocyte migration in 3D environments. Methods Mol Biol. 769, 149-165 (2011).
- Randolph, G. J., Angeli, V., Swartz, M. A. Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol. 5, 617-628 (2005).
- Wilflingseder, D., Banki, Z., Dierich, M. P., Stoiber, H. Mechanisms promoting dendritic cell-mediated transmission of HIV. Mol Immunol. 42, 229-237 (2005).
- Pope, M., Gezelter, S., Gallo, N., Hoffman, L., Steinman, R. M. Low levels of HIV-1 infection in cutaneous dendritic cells promote extensive viral replication upon binding to memory CD4+ T cells. J Exp Med. 182, 2045-2056 (1995).
- McDonald, D., et al. Recruitment of HIV and its receptors to dendritic cell-T cell junctions. Science. 300, 1295-1297 (2003).
- Pruenster, M., et al. C-type lectin-independent interaction of complement opsonized HIV with monocyte-derived dendritic cells. Eur J Immunol. 35, 2691-2698 (2005).
- Wilflingseder, D., et al. IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells. J Immunol. 178, 7840-7848 (2007).
- Kapsenberg, M. L. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol. 3, 984-993 (2003).
- Mills, K. H. TLR-dependent T cell activation in autoimmunity. Nat Rev Immunol. 11, 807-822 (2011).
- Banki, Z., et al. Complement as an endogenous adjuvant for dendritic cell-mediated induction of retrovirus-specific CTLs. PLoS Pathog. 6, e1000891 (2010).
- Posch, W., et al. Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes. J Allergy Clin Immunol. 130, 1368-1374 (2012).
- Posch, W., et al. Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells. PLoS Pathog. 11, e1005005 (2015).
- Wilflingseder, D., et al. Immediate T-Helper 17 Polarization Upon Triggering CD11b/c on HIV-Exposed Dendritic Cells. J Infect Dis. 212, 44-56 (2015).
- Grassi, F., et al. Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules. J Leukoc Biol. 64, 484-493 (1998).
