Overview
This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.
Protocol
1. Evaluate the Function of TolDCs In Vitro and In Vivo
- T-cell Syngeneic Proliferation Assay
- Sterilize all the surgical instruments via autoclaving and perform the experiment in a class II biological safety cabinet with appropriate safety procedures.
- Prepare MACS buffer using 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 500 ml PBS. Sterilize the buffer by filtering through a 0.2 µm filter.
NOTE: Keep the buffer on the ice during the following experiment. - To obtain CD4+ T cells, euthanize OT-II T-cell receptor (TCR) transgenic mice 8-10 weeks of age by using a CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps.
- Put the spleen with 2 ml PBS in a 6 cm culture dish and use the back of the push-stick of a 3 ml syringe to mince the spleen by passing a 40 μm cell strainer.
- Collect the cell suspension and centrifuge at 300 x g for 5 mins.
- Resuspend the cell pellet in 400 μl of MACS buffer.
- Add 100 μl of CD4+ T Cell Biotin-Antibody Cocktail at 4°C for 5 mins.
NOTE: The antibody cocktail binds on other cell types except CD4+ T cells, such as CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, Anti-MHC class II, Ter-119, and TCRγ/δ. - Add 300 μl of MACS buffer and 200 μl of Anti-Biotin beads (Table of Materials) at 4°C for 10 mins.
- Place the column composed of ferromagnetic spheres (Table of Materials) and Pre-Separation Filter together in the magnetic field and rinse it with 3 ml of MACS buffer.
- Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.
- Resuspend the cell pellet in 3 ml of MACS buffer and apply it to the column. Collect flow-through containing CD4+ T cells.
- Wash the column with another 3 ml of MACS buffer and also collect the flow-through.
- To obtain splenic pan DCs, euthanize C57BL/6 mice 8-10 weeks of age by using a CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps. Put the spleen in a 6cm culture dish containing 2 ml of collagenase D solution (2 mg/ml collagenase D dissolved in HBSS containing calcium, and magnesium).
- Inject 1 ml of collagenase D solution into the spleen two times with a 1 ml syringe and a 25G needle. Cut the spleen into small pieces with small scissors.
- Shake and incubate at room temperature for 25 mins.
- Add 500 μl of 0.5 M EDTA at room temperature for 5 mins.
NOTE: The steps from 1.1.13-1.1.14 are critical for increasing the yield of DCs. - Now use the back of the push-stick of the 3 ml syringe to mince the spleen slurry by passing a 40 μm cell strainer and collect the cell suspension by centrifuging at 300 x g for 5 mins.
- Resuspend the cell pellet in 350 μl of MACS buffer, 50 μl of FcR Blocking Reagent, and 100 μl of Pan Dendritic Cell Biotin-Antibody Cocktail at 4°C for 10 mins.
NOTE: The antibody cocktail against antigens that are not expressed by DCs. - Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.
- Resuspend the cell pellet in 800 μl of MACS buffer and add 200 μl of Anti-Biotin beads at 4°C for 10 mins.
- Repeat the steps from 1.1.9-1.1.11 to collect DCs.
- Wash the column with another 3 ml of MACS buffer two times and also collect the flow-through.
- Treat the 2 x 105 DCs/ml in the presence or absence of 100-400 nM CDDO-DFPA at 37°C for 1 hr. Separately, label the CD4+ T cells (1 x 107/ml) with 1 μM CFSE at 37°C for 15 mins, wash with PBS, and readjust the volume to get the final concentration at 2 x 106 T cells/ml.
- Next, in a 96-well plate, co-culture 100 μl of the treated dendritic cells with the same volume of CFSE-labeled CD4+ T cells, both collected from the above steps to get a 1:10 ratio. Now add 100 ng/mL ovalbumin (OVA) peptide 323-329 per well and measure the CFSE intensity of the T cells by flow cytometry after 2-3 days of incubation.
NOTE: The cell numbers and ratio of DCs and T cells have been optimized from our previous work.
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Materials
Name | Company | Catalog Number | Comments |
CDDO-DFPA (RTA-408) | Reata Pharmaceuticals | in house synthesis | Cell culture |
Mouse GM-CSF | Peprotech Inc | 315-03 | BMDC differentiation |
Pan dendritic cell isolation kit | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
Pan Dendritic Cell Biotin-Antibody Cocktail | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
CD4+ T cell isolation kit | Miltenyi Biotec Inc | 130-104-454 | T cell proliferation assay |
CFSE | BioLegend | 423801 | T cell proliferation assay |
FcR Blocking Reagent | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
Anti-Biotin MicroBeads | Miltenyi Biotec Inc. | 130-104-454 | T cell proliferation assay |
1 ml syringe | BD Biosciences | 309626 | T cell proliferation assay |
BSA | Sigma Aldrich Inc | A2058 | T cell proliferation assay |
EDTA | ThermoFisher Scientific | 15575020 | T cell proliferation assay |
LS Column | Miltenyi Biotec Inc. | 130-042-401 | T cell proliferation assay |
Pre-Separation Filter | Miltenyi Biotec Inc | 130-095-823 | T cell proliferation assay |
Collagenase D | Sigma Aldrich Inc. | 11088858001 | T cell proliferation assay |
HBSS | ThermoFisher Scientific | 14025076 | T cell proliferation assay |
Ovalbumin (OVA) peptide 323–329 | Sigma Aldrich Inc. | O1641 | T cell proliferation assay |