Differentiation of human and mouse THGM cells in vitro

Differentiation of mouse T_HGM cells

1. Isolate naïve (CD62L^hiCD44^-CD25^-CD4⁺) splenic T cells of wild-type (WT) or 2D2 mice by FACS.
2. Prepare antigen presenting cells (APCs) by removing CD3⁺ T cells in WT splenocytes by magnetic beads separation. 
3. Plate naïve T cells and APCs (1:4 ratio) at a density of 1x10⁶ cell/mL in IMDM supplemented with 10% heat-inactivated FBS, Penicillin-Streptomycin-Glutamine, and 1% 2-mercaptoethanol. Activate for 72 h WT T cells with anti-CD3 and anti-CD28 Abs (1 μg/mL each), or 2D2 T cells with MOG_35-55 peptide (25 μg/mL) in T_HGM-polarizing condition: IL-1β (10 ng/mL), and anti-IFN-γ, -IL-12, -IL-4 Abs (10 μg/mL each). 
4. Reactivate for 48 h WT or 2D2 T cells with anti-CD3 and anti-CD28 Abs (1 μg/mL each) at a density of 1x10⁶ cell/mL in the same differentiation condition as in the first stimulation.

Expected results 

• 40-50% of T cells express GM-CSF after the first simulation, and 60-80% after the second stimulation.
• 5-10% of cells express IFN-γ and/or IL-17A (1).
• GM-CSF concentration in culturing media is 1-3 ng/ml after the first stimulation, and 8-15 ng/ml after the second stimulation.

Differentiation of human T_HGM cells

1. Coat a 96-well flat-bottom cell culture plate with anti-CD3 Ab (3 mg/mL) in PBS and incubate it at 37°C overnight.
2. Isolate PBMCs using Ficoll-Paque Plus density gradient centrifugation. 
3. Isolate naïve (CD25^-CCR7⁺CD45RA⁺CD4⁺) T cells from PBMCs by FACS.
4. Remove anti-CD3 Ab from the plate and plate naïve T cells at a density of 1x10⁶ cell/mL in IMDM, containing: 5% heat-inactivated human AB serum, Penicillin-Streptomycin-Glutamine, and 1% 2-mercaptoethanol. Activate naïve T cells for five days with soluble anti-CD28 Ab (1 μg/mL) in T_HGM -polarizing condition: IL-6 (50 ng/mL), and anti-IFN-γ and anti-IL-4 Abs (10 μg/mL each). 
5. Reactivate T cells for three days with soluble anti-CD3 and anti-CD28 Abs (2 μg/mL each) at a density of 1x10⁶ cell/mL in the same polarizing condition as in the first stimulation. 

Expected results 

• 40-50% of cells polarized using this protocol express GM-CSF after the first and second stimulation.
• Approximately 5% of cells express IFN-γ and/or IL-4 (1).

Reagents: 

Mouse

1. CD62L, CD44, CD25, and CD4 flow cytometry Abs, Biolegend
2. CD3 isolation kit, Miltenyi Biotec
3. IL-1β, R&D Systems
4. Anti-CD3, anti-CD28, anti-IFN-γ, anti-IL-12, and anti-IL-4 Abs, BioXcell

Human

1. CD25, CCR7, CD45RA, CD4 flow cytometry Abs, Biolegend and BD Biosciences 
2. IL-6, R&D Systems
3. Anti-CD3, anti-CD28, eBioscience 
4. Anti-IFN-γ and IL-4 Abs, BioXcell

Reference

1.           J. Rasouli, G. Casella, S. Yoshimura, W. Zhang, D. Xiao, J. Garifallou, M. V. Gonzalez, A. Wiedeman, A. Kus, E. R. Mari, P. Fortina, H. Hakonarson, S. A. Long, G. X. Zhang, B. Ciric, A. Rostami, A distinct GM-CSF(+) T helper cell subset requires T-bet to adopt a TH1 phenotype and promote neuroinflammation. Sci Immunol 5,  (2020).