Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells

Protocol

1. Evaluate the Function of TolDCs In Vitro and In Vivo

1. T-cell Syngeneic Proliferation Assay
   1. Sterilize all the surgical instruments via autoclaving and perform the experiment in a class II biological safety cabinet with appropriate safety procedures.
   2. 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.
   3. 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.
   4. 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.
   5. Collect the cell suspension and centrifuge at 300 x g for 5 mins.
   6. Resuspend the cell pellet in 400 μl of MACS buffer.
   7. 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.
   8. Add 300 μl of MACS buffer and 200 μl of Anti-Biotin beads (Table of Materials) at 4°C for 10 mins.
   9. 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.
   10. Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.
   11. Resuspend the cell pellet in 3 ml of MACS buffer and apply it to the column. Collect flow-through containing CD4+ T cells.
   12. Wash the column with another 3 ml of MACS buffer and also collect the flow-through.
   13. 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).
   14. 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.
   15. Shake and incubate at room temperature for 25 mins.
   16. 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.
   17. 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.
   18. 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.
   19. Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.
   20. Resuspend the cell pellet in 800 μl of MACS buffer and add 200 μl of Anti-Biotin beads at 4°C for 10 mins.
   21. Repeat the steps from 1.1.9-1.1.11 to collect DCs.
   22. Wash the column with another 3 ml of MACS buffer two times and also collect the flow-through.
   23. 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.
   24. 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