Analysis of T Cell Proliferating and Polarizing Potential of Murine Dendritic Cells in Allogeneic-mixed Leukocyte Reaction

Materials and Reagents

1. RPMI-1640 medium (HiMedia Laboratories, catalog number: AT028 )
   
2. Dulbecco’s Phosphate Buffered Saline (HiMedia Laboratories, catalog number: TS1006 )
   
3. Heat-inactivated fetal bovine serum (Biological industries, catalog number: 04-121-1A )
   
4. Antibiotic-antimycotic (penicillin-streptomycin) solution, 100x (HiMedia Laboratories, catalog number: A002A )
   
5. Round bottom, 96-well cell culture plates (Corning, catalog number: 3799 )
   
6. Dendritic cells (derived by culturing mouse bone marrow cells in the presence of recombinant GM-CSF) (PeproTech, catalog number: 315-03 )
   
7. Untouched CD4⁺ and CD8⁺ T cells from allogeneic mouse strain (isolated from spleen of Balb/c mice using CD4 T cell enrichment kit and CD8 T cell enrichment kit (BD, catalog number: 558131 and 558471 , respectively)
   
8. Fluorochrome-conjugated FITC anti-mouse CD3, PE anti-mouse CD4 and PE anti-mouse CD8 antibodies (BD Pharmingen, catalog number: 555274 , 553730 and 553032 , respectively)
   
9. Concanavalin A (Sigma-Aldrich, catalog number: C5275 )
   
10. ³H-thymidine (BARC)
    
11. Trypan Blue (Sigma-Aldrich, catalog number: T8154 )
    
12. RPMI-10 (see Recipes)
    

Equipment

1. Haemocytometer
   
2. Humidified CO₂ incubator
   
3. Laminar air flow bio-safety cabinet
   
4. Centrifuge
   
5. Gamma-irradiator
   
6. Microscope
   
7. Flow-cytometer
   

Procedure

1. Harvest mouse bone marrow-derived dendritic cells (BMDCs) from plates by gentle pipetting, give a wash with PBS and prepare the suspensions of 1.0 x 10⁵, 2.0 x 10⁵ and 4.0 x 10⁵ cells per ml in RPMI-10 medium.
   (BMDCs are derived by culturing mouse bone marrow cells in the presence of GM-CSF. Briefly, add 4 x 10⁶ bone-marrow cells per well of 6-well plate in RPMI-10 medium supplemented with 20 ng/ml GM-CSF. Remove culture medium along with non-adherent cells on day 3 and day 5, and fresh 4.0 ml GM-CSF-supplemented medium to each well. Harvest immature DCs on day 7 by gently pipetting. After giving a wash in RPMI-10 medium, cells can be used for subsequent experiments. The purity of DCs derived following this protocol is ~85%. These cells can be used directly in allo-MLR or can be further purified.)
   
2. To analyze the ability of DCs to induce T-cell proliferation, add 50 µl of DC suspensions (equivalent to 0.5 x 10⁴, 1.0 x 10⁴ and 2.0 x 10⁴ DCs) per well in a round bottom plate in triplicates.
   (It is necessary to plate the increasing number of DCs to achieve an increasing ratio of stimulator cells to responder cells. It is advised not to add DCs into outer wells of the plate because culture media tend to evaporate from these wells at higher rates. Instead, these wells can be filled with autoclaved distilled water).
   
3. To analyze the ability of DCs to induce T-cell polarization, similarly add 50 µl of 2.0 x 10⁵ cells/ml DC suspension (= 1.0 x 10⁴ cells) per well in a round bottom plate in triplicates.
   
4. Add the desired stimuli such as LPS or heat-killed mycobacteria to plated DCs and adjust final volume of total contents per well to 100 µl.
   [Dilute stock solution of LPS or mycobacterial suspension to required concentration in RPMI-10 medium. LPS could be used at a concentration of 0.1 to 1.0 µg/ml, whereas heat-killed bacteria (prepared by autoclaving) can be used preferably at a multiplicity of infection (MOI) of 5 to 10].
   
5. Keep plates in a humidified CO₂ incubator for 24 h.
   
6. Next day, isolate CD4⁺ and CD8⁺ T lymphocytes from the spleen of naïve allogeneic mice using a negative selection kit as suggested by manufacturer. Determine purity of lymphocytes using anti-mouse CD3/CD4 and CD3/CD8 antibodies by flow cytometry.
   Note: If DCs are derived from C57BL/6 mice, lymphocytes can be prepared from Balb/c mice.
   
7. Irradiate DCs with gamma-rays in a gamma-irradiation chamber (irradiation dose, 25 Gy). Irradiation will prevent the proliferation of DCs, which could otherwise give false results.
   
8. Adjust concentration of lymphocytes to 1.0 x 10⁶ cells/ml. Add 100 µl of cell suspension to irradiated DCs.
   
9. Set positive controls by stimulating CD4⁺ T cells and CD8⁺ T cells with Concanavalin A (final concentration, 5 µg/ml).
   
10. Keep plates at 37 °C in a humidified CO₂ incubator.
    
11. After 72 h, add 1.0 µCi ³H-thymidine per well of the plate set up with T cell proliferation assay. Keep plates back into the CO₂ incubator.
    
12. After 18 h, transfer plates to -20 °C. Plates can be thawed immediately or next day.
    
13. Harvest the cells onto a filter paper and wash them using an automated cell harvester.
    
14. Measure the ³H-thymidine levels on filter paper using a beta scintillation counter.
    
15. Collect culture supernatants from plate set up with T cell polarization assay, after 96 h. Store supernatants at -80 °C or immediately analyze for T_H1, T_H2, T_H17 signature cytokines by ELISA.
    

Recipes

1. RPMI-10
   RPMI-1640 base medium supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solution
   

References

1. Kumar, P., John, V., Marathe, S., Das, G. and Bhaskar, S. (2015). Mycobacterium indicus pranii induces dendritic cell activation, survival, and Th1/Th17 polarization potential in a TLR-dependent manner. J Leukoc Biol 97(3): 511-520.
   
2. Muul, L. M., Silvin, C., James, S. P. and Candotti, F. (2008). Measurement of proliferative responses of cultured lymphocytes. Curr Protoc Immunol Chapter 7: Unit 7 10 11-17 10 24.