In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Podsumowanie

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Streszczenie

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with γ-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

Wprowadzenie

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in controlling immune responses¹. While DCs are heterogeneous, they can be broadly classified into two populations, conventional DCs (cDCs) and plasmacytoid DCs (pDCs)^2,3. In addition to lymphoid organs, cDCs and pDCs are also found in tissues including lung, intestine, and skin². The morphology of cDCs and pDCs differs, with cDCs exhibiting dendrite-like projections and the shape of pDCs being more plasma cell-like. In addition, the common mouse DC marker, CD11c, is more highly expressed on cDCs than on pDCs. Moreover, cDCs can be further divided into CD11b⁺CD24^- cDCs and CD11b^-CD24⁺ cDCs, both of which express higher levels of MHC class II and costimulatory molecules than do pDCs². Mature pDCs, on the other hand, have been shown to selectively express Siglec-H and BST2⁴. Functionally, cDCs are better antigen presenting cells than are pDCs; however, pDCs can produce a vast amount of type I interferon upon virus infection or inflammatory stimulation⁵.

Both cDCs and pDCs are short-lived, and therefore, must be constantly replenished from progenitors within the bone marrow (BM)^6,7. Adoptive transfer of common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) into lethally-irradiated mice demonstrates that cDCs and pDCs can be generated from both lineages^8-10. However, there is a subset of pDCs that express RAG1/2 and possess rearranged D-J segments at the IgH locus^11,12. These cells also share molecular similarities with B lymphocytes, including expression of the B220 marker, nucleic acid-sensing receptors (TLR7/TLR9), and transcription factors (Spib and Bcl11a)¹³, features all believed to be signatures of the lymphoid lineage. Therefore, CLPs may be good choices for in vitro generation of pDCs because of lineage similarity.

While the frequency of both cDCs and pDCs in humans and mice is very low⁶, DCs, particularly cDCs, can be generated in vitro from BM or progenitors in the presence of cytokines, such as GM-CSF^11,14 or Flt3 ligand (FL) using feeder-free systems ^11,15,16. Unfortunately, however, it is not possible to produce large numbers of pDCs in vitro using FL^11,15,16. Previously we demonstrated that pDCs can be efficiently generated in vitro from CLPs using the AC-6 feeder system¹⁷. The advantage of using the AC-6 stromal cell line in the culture system is that it provides cell-cell contact and secretion of cytokines that support the generation of large numbers of pDCs from CLPs. Although production with this system is quite robust, careful replication of the procedures described below is required in order to ensure efficient generation of pDCs.

Protokół

C57BL/6 wild-type mice were purchased from the National Laboratory Animal Center (NLAC), Taiwan. All mice were bred and kept under specific pathogen-free conditions. Protocols and animal use procedures were reviewed and approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine (Permit Number: 20120075). In addition, researchers made every effort to reduce the potential for pain, suffering, or distress in the animals while performing experiments. All procedures described were carried out at RT while wearing gloves.

1. Preparation of AC-6 Feeder cells

Note: The AC-6.21 (AC-6 in short) stromal cell line¹⁸ (provided by I. Weissman, Stanford University) should be maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS). To serve as feeder cells, AC-6 cells are γ-irradiated with 3,000 rad (30Gy) one day before the co-culture to prevent their proliferation. Note that AC-6 cells tend to lose their differentiation-supporting ability if left overcrowded during maintenance, or if their passage number is over 20.

1. Wash AC-6 cells once with 1 ml Dulbecco's Phosphate-Buffered Saline (DPBS) and remove DPBS by aspiration. Treat AC-6 cells with 0.8 ml trypsin solution (0.05 % trypsin and 0.5 mM EDTA in DPBS) for 3-5 min at 37^oC, 5% CO₂ and then stop the reaction by diluting it with 10 volumes of culture medium to make single cell suspension.
2. Seed AC-6 cells at 5.9x10⁴ cells/well into 12-well plates and incubate at 37^oC, 5% CO₂ O/N.
   Note: The density of AC-6 cells is very important as a lower density will favor cDC generation. Seeding at a density of 5.9x10⁴ cells/well will allow AC-6 to reach confluency by the next day, which is optimal for derivation of pDCs from CLPs.
3. Irradiate AC-6 cells at 3,000 rad (30 Gy) using γ-irradiator.
   Note: The dose used to irradiate AC-6 is optimized to prevent the cells from proliferating and yet keep them viable long enough to provide the cytokines and cell-cell contact required for differentiation of DCs from CLPs.
4. Aspirate medium and replace with 1 ml complete RPMI (RPMI with 10 % heat-inactivated FBS, 50 µg/ml gentamycin and 50 µM β-mercaptoethanol).

2. Isolate BM Cells from Mice

1. Sacrifice mice by CO₂ asphyxiation and cervical dislocation. Place the mice on a dissection tray and sterilize with 70% ethanol. Make an incision at the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.
2. Dissect mice in a semi-sterile hood. To release the femur and tibiae, clip the femur above and tibiae below the knee joint. Detach the muscles from the bones using scissors and place the bones in a 6 cm petri dish containing 5 ml complete RPMI.
3. Move to a sterile culture hood. Fill a 3 ml syringe connected to a 27G needle with complete RPMI. Cut off both ends of the bones using scissors, and insert the 27G needle to flush the marrow out of the bones by gently injecting complete RPMI once from each end.
4. Prepare a single cell suspension by gentle pipetting the cells up and down 3-5 times in the culture dish using the syringe without the needle.
5. Centrifuge the cells for 5 min at 500 x g and decant the supernatant.
6. Lyse red blood cells with 1 ml ACK buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) incubating for 1 min and stopping the reaction by diluting with 10 volumes of complete RPMI. Allow cells to stand for 5 min in order to pellet down dead cell clumps and tissue debris by gravity.
   Note: Do not stand more than 5 min as this will result in cell loss due to settling of viable cells.
7. Slowly decant cell-containing supernatant to a new tube leaving debris behind in the original tube.
8. Centrifuge for 5 min at 500 x g to pellet the cells, then remove and discard the supernatant.
9. Resuspend total BM cells (typically 4-6 x 10⁷ BM cells are obtained from one mouse) in 100 µl FACS buffer (1x PBS+ 2% FBS+ 1mM EDTA).

3. FACS Analysis and Sorting of CLPs

1. Add anti-CD16/32 (hybridoma supernant clone 2.4G2, 50 µl/reaction or 1-2 µg/reaction) into cells in FACS buffer (step 2.9) for 1-2 min in order to block Fc receptors.
   Note: Anti-CD16/32 is also called Fc block, which is added to prevent non-specific binding of antibodies to cells expressing Fc receptors including granulocytes, monocytes, and B lymphocytes.
2. Simultaneously stain cells with the following fluorescent dye-labeled antibodies (for 4x10⁷ cells) for 15 min on ice, avoiding ambient light. Antibodies: PE-conjugated lineage markers (0.2 µg each) including anti-CD3 (17A2), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), and anti-MHC-II (NIMR-4), 0.2 µg anti-c-Kit-PerCP-Cy5.5 (2B8), 1 µg anti-Sca-1-FITC (D7), 0.4 µg anti-M-CSFR-APC (AFS98), and 0.2 µg anti-IL-7Rα-PE-Cy7 (A7R34).
3. Wash cells with 3 ml FACS buffer, and centrifuge for 5 min at 500 x g.
4. Resuspend the cells in 300 µl FACS buffer and filter cells through a 40 µM cell strainer.
5. Wash the tube with an additional 100 µl FACS buffer to recover any remaining cells and filter into a sterile FACS sorting tube using the same cell strainer.
6. Perform flow cytometric analysis immediately after the staining using appropriate filters and voltages for signal detection. Use 488 nm laser to detect the FITC-, PerCP-Cy5.5-labeled antibodies, 561 nm laser to detect the PE- and PE-Cy7-labelded antibodies and 633 nm laser to detect the APC-labeled antibody.
7. Sort out CLPs according to the following markers lin^-c-kit^intSca-1^intM-CSFR^-IL-7Rα⁺ (as stained in step 3.2) using a cell sorter. Collect the sorted cells in a 15 ml tube containing 8 ml of complete RPMI as a cushion.
8. Record the absolute number of sorted cells as shown by the cell sorter at the end of the run. Typically, 5x10⁴ CLPs can be obtained from the BM of one mouse.

4. Coculture CLPs and AC-6

1. Centrifuge the sorted cells from step 3.7 for 10 min at 500 x g, remove the supernatant and resuspend the cell pellet with enough complete RPMI to obtain a cell density around 5x10⁴ cells/ml. Seed 500 cells/well into the 12-well plate containing feeder cells prepared in step 1.4.
2. Add 100 ng/ml FL¹⁹ (hu-Flt3L-Ig generated in-house using an expression system provided by M. Manz, University Hospital Zürich, Switzerland) and incubate at 37^oC, 5% CO₂ with periodic visual monitoring of DC development under a microscope.
   Note: Commercially available recombinant human FL or mouse FL can be used as a substitute for hu-Flt3L-Ig to support DC development.
3. Collect the cells in the supernatant at day 12 and wash the wells once with 0.5 ml complete RPMI medium combining the resulting supernatants. Add 0.5 ml fresh medium and scrap the adherent cells with a cell scraper.
4. Combine the cell-containing medium from both parts and centrifuge for 5 min at 500 x g.
5. Decant supernatant and resuspend cells in 50 µl FACS buffer. Add 50 µl anti-CD16/32 hybridoma supernatant and incubate for 1-2 min to block Fc receptors.
6. Enumerate and stain all the cells with the following antibodies (0.05 µg each) anti-CD11c-APC (N418), anti-CD11b-FITC, and anti-B220-PE.
7. Wash and centrifuge the cells as described in step 3.3.
8. Resuspend the cells in 100 µl FACS buffer, gate on CD11c⁺ and analyze for cDCs (CD11c⁺CD11b⁺B220^-) and pDCs (CD11c⁺CD11b^-B220⁺)¹⁷.

Wyniki

A total of 4-6x10⁷ BM cells are typically isolated from femurs and tibiae of one 6-8 wk-old, wild-type C57BL/6 mouse. To sort out CLPs, total BM cells are stained with PE-conjugated antibodies against lineage markers (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119, and MHC-II), anti-c-Kit-PerCP/Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC and anti-IL-7Rα-PE/Cy7, analyzed and sorted with a cell sorter. The sorting strategy for CLP is shown in Figure 1. Typically, 5x10⁴ CLPs...

Dyskusje

Here we describe an in vitro culture system for the robust generation of DCs, and pDCs in particular, from a small number of CLPs. The uniqueness of this culture system is due to the use of AC-6 cells, a stromal cell line, as feeders. This approach has been shown to provide not only the cytokines, such as IL-7, SCF, M-CSF and FL²⁰, but also the cell-cell contact²¹ necessary to support DC development. AC-6 cells have been used previously to facilitate the study of in vitro DC develo...

Materiały

Name	Company	Catalog Number	Comments
Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5	Biolegend	108408	linage marker
Anti-mouse NK1.1 (PE), clone PK136	Biolegend	108708	linage marker
Anti-mouse  CD11b (PE), cloneM1/70	Biolegend	101208	linage marker
Anti-mouse CD19 (PE), clone eBio1D3	Biolegend	115508	linage marker
Anti-mouse B220 (PE), clone RA3-6B2	Biolegend	103208	linage marker/FACS
Anti-mouse CD3 (PE), clone 17A2	Biolegend	100308	linage marker
Anti-mouse CD8a (PE), clone 53-6.7	Biolegend	100707	linage marker
Anti-mouse MHC-II (PE), clone NIMR-4	Biolegend	107608	linage marker
Anti-mouse Ter119 (PE), clone TER-119	Biolegend	116208	linage marker
Anti-mouse Thy1.1 (PE), clone HIS51	eBioscience	12-0900-83	linage marker
Anti-mouse M-CSFR (APC), clone AFS98	Biolegend	135510	FACS
Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8	Biolegend	105824	FACS
Anti-mouse Sca-1 (FITC), clone D7	Biolegend	108106	FACS
Anti-mouse IL-7Ra (PE/Cy7), clone A7R34	Biolegend	135014	FACS
Anti-mouse CD11c (PerCP/Cy5.5), clone N418	Biolegend	117328	FACS
Anti-mouse CD11b (FITC), clone M1/70	Biolegend	101206	FACS
FACSAria III	BD Biosciences		Cell sorter
FACS sorting tube	BD Biosciences	352054
FlowJo	FlowJo LLC		Flow analysis sofrware