Generation of Aβ-specific T cell lines and in vivo Transfer

Materials and Reagents

1. C57BL/6 mice (adult, >6 weeks old; typically 4 per experiment)
   
2. Aβ_1-42 peptide (Life Technologies, Invitrogen^TM, catalog number: 03112 )
   
3. CpG (CpG-oligodeoxynucleotide 1668; 5′-tccatgacgttccgatgct-3′; Sigma-Genosys)
   
4. PBS (Sigma-Aldrich, catalog number: D8537 )
   
5. IL-1 (Immunotools, catalog number: 12340013 )
   
6. IL-2 (Immunotools, catalog number: 12340024 )
   
7. IL-4(Immunotools, catalog number: 12340043 )
   
8. IL-12 (Miltenyi, catalog number: 130-096-707 )
   
9. IL-23 (Miltenyi, catalog number: 130-096-676 )
   
10. Anti–IFNγ (BD, catalog number: 554430 )
    
11. Dexamethasone (Sigma-Aldrich, catalog number: D4902 )
    
12. High-performance liquid chromatography (HPLC)-grade water (sterile ddH₂O)
    
13. RPMI medium (Sigma-Aldrich, catalog number: R0883 )
    
14. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333 )
    
15. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
    
16. FBS (Sigma-Aldrich, catalog number: F9665 )
    
17. Complete RPMI medium (see Recipes)

Equipment

1. Shaker capable of 200 rpm at 37 °C
   
2. 24-well cell culture plates (Greiner Bio-one, catalog number: 662160 )
   
3. 1 ml tuberculin syringes (BD, catalog number: 300013 )
   
4. 70 μm nylon mesh filter (Corning, catalog number: 352350 )
   
5. Tissue culture facilities including class II laminar flow hood
   
6. Centrifuge

Procedure

1. Dissolve Aβ_1-42 peptide in HPLC-grade water to provide a 12 mg/ml stock solution, which is diluted to 2 mg/ml using sterile PBS and allowed to aggregate for 48 h at 37 °C and with agitation of 200 rpm. Aβ_1-42 is used immediately or stored at -20 °C.
   
2. Immunize C57BL/6 mice by subcutaneous injection into the rear footpad with Aβ_1-42 (75 μg/mouse) and CpG (25 μg/mouse) in a total volume of 50 μl i.e. 25 μl per foot.
   
3. Administer a sec (booster) immunization with the same doses of antigen and adjuvant after 21 days.
   
4. After a further 7 days, sacrifice the mice and remove the popliteal lymph nodes (the draining lymph node; see Figure 1) and spleens.
   
   
   Figure 1. A schematic of popliteal lymph node
   
   
5. Dissociate lymph node and spleen tissue through a sterile 70 μm nylon mesh filter, wash with complete RPMI and centrifuged at 280 x g for 5 min and perform a cell count.
   
6. Stimulate the cells at 2 x 10⁶ cells per ml with Aβ_1-42 (25 μg/ml) in the presence of cytokines and antibodies depending on the type of cell line required. To generate Th1 cells, the cells from the lymph nodes and spleens are stimulated with Aβ_1-42 (25 μg/ml) and IL-12 (10 ng/ml). Th2 cells are amplified using dexamethasone (1 x 10^-8 M), IL-4 (10 ng/ml), and anti-IFNγ (5 mg/ml), and Th17 cells are generated with IL-1 (10 ng/ml), IL-23 (10 ng/ml) and anti-IFNγ (5 mg/ml).
   
7. After 4 days, add IL-2 (5 ng/ml) to the Th1 and Th2 cell cultures, or medium only to the Th17 cells and incubation continued for a further 7 days.
   
8. Wash cells with complete RPMI, centrifuge at 280 x g for 5 min and count.
   
9. Confirm that cells are polarized to Th1, Th2 or Th17 either by performing intracellular cytokine staining for IFN-γ, IL-5 or IL-17 and FACS analysis or by assessing the quantity of these cyokines in supernatants by ELISA.
   
10. Inject cells i.p. into recipient mice (typically 1.5 x 10⁷ cells/mouse) in 100 μl serum-free medium or PBS.
    

Recipes

1. Complete RPMI medium
   RPMI medium supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% FBS

Acknowledgments

This work was funded by a PI grant to Kingston Mills from Science Foundation Ireland.

References

1. Browne, T. C., McQuillan, K., McManus, R. M., O'Reilly, J. A., Mills, K. H. and Lynch, M. A. (2013). IFN-gamma Production by amyloid beta-specific Th1 cells promotes microglial activation and increases plaque burden in a mouse model of Alzheimer's disease. J Immunol 190(5): 2241-2251.
   
2. McQuillan, K., Lynch, M. A. and Mills, K. H. (2010). Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its regulation by Th2 cells. Brain Behav Immun 24(4): 598-607.