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In vitro differentiation of Th cells from naïve CD4⁺ T cells is achieved by stimulating their T cell receptor (TCR) in the presence of appropriate cytokines. Here we describe a comprehensive protocol for in vitro differentiation of Th0, Th1, Th2, Th17 and iTreg T cells from mouse CD4⁺ T cells.

Reagents:

• Antibodies: anti-CD28, anti-CD3, anti-IL-4 and anti-IFN-γ antibodies
• Cytokines: IL-2, IL-4, IL-6, IL-12, TGF-β
• RPMI1640
• Sterile PBS

Procedures:

Day 1: Coat wells with anti-CD3 and CD28 antibody

1. Prepare anti-CD3 and anti-CD28 antibodies at 1μg/mL in sterile PBS.

2. Add 250~500 μL total volume per well to 24-well plates.

3. Cover and wrap in parafilm to prevent evaporation and contamination, then incubated at 4 °C.
Note: Concentration of anti-CD3 at 2 μg/ml and anti-CD28 at 1 μg/ml is optimal for Th17 generation.

Day 2: Differentiation of Th subtypes

4. Aspirate anti-CD3 and anti-CD28 antibodies solution from the wells of the coated plates, wash each well with 1 mL of sterile PBS.

5. Prepare differentiating culture medium:

• Th0: 5 ng/mL IL-2 in RPMI1640.
• Th1: 5 ng/mL IL-2, 15 ng/mL IL-12 and 1μg/mL anti-IL-4 antibodies in RPMI1640.
• Th2: 5 ng/mL IL-2, 10 ng/mL IL-4, 0.5 μg/mL and 1μg/mL anti-IFN-γ antibodies in RPMI1640.
• Th17: 20 ng/mL IL-6, 3 ng/mL TGF-β, 1μg/mL anti-IL-4 and 1μg/mL anti-IFN-γ antibodies in RPMI1640.
• iTreg: 5 ng/mL IL-2, 15 ng/mL TGF-β, 1μg/mL anti-IL-4 and 1μg/mL anti-IFN-γ antibodies in RPMI1640.

6. Dissolve 1 × 10⁶ naïve CD4⁺ T cells with 500 μL of differentiating culture medium.

Learn more about naïve CD4⁺ T cells isolation

7. Plate cells in each well of anti-CD3 and anti-CD28 coated 24-well plate.

8. Incubate cells in 5 % CO₂ incubator at 37 °C for 4~5 days.

Day 7: Analysis

9. Generally, differentiated Th cells are analyzed via cytokines detection and/or transcription factors identification. IFN-γ, IL-4, IL-17, and Foxp3 are typically used markers for Th1, Th2, Th17, and iTreg differentiations, respectively. The following approaches are alternative for Th subsets analysis:

• Cytokine analysis by flow cytometry
• Protein quantification by ELISA
• Gene expression analysis by real time PCR

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