In vitro Human Th17 development
Lab/Group: De Waal Malefyt Lab
Related Journal & Article Information
Journal: Nature Immunology
Article Title: Development, cytokine profile and function of human interleukin 17–producing helper T cells
Introduction
This protocol describes the isolation of naive human T cells and culture conditions that result in the development of IL-17 producing helper Th17 cells. The addition of IL-23 or IL-1 is essential for this process as is T cell receptor activation.
Materials
Reagents
-Recombinant hIL-1β (50 ng/ml), rhIL-4 (10 ng/ml), rhIL-12 (5 ng/ml), rhIL-6 (30 ng/ml), rhTGFβ (10 ng/ml), and rhIL-2 (100 U/ml) were purchased from R&D Systems. Recombinant hIL-23 was generated in house.
-Medium : Yssel’s medium with 1% human serum AB was from Gemini Bio-Products
-Human CD4+ T cell isolation kit II (#130-091-155), human CD45RO microbeads (#130-046-001), and human T cell activation/expansion kit (#130-091-441) were from Miltenyi Biotec.
Equipment
AutoMACS, Milteniy Biotec
Procedure
1. Human PBMC were isolated from buffy coats of healthy donors by Ficoll hypaque density centrifugation.
2. Total CD4+ T cells were isolated using the CD4+ T cell Isolation Kit II from Miltenyi Biotec:
- Total PBMC were resuspended in 40µl of MACS buffer (PBS 0.5% BSA 2mM EDTA) per 107 cells, and incubated with 10 µl of Biotin-Antibody cocktail per 107 cells for 10 min at 4°C.
-. Subsequently, 30 µl of MACS buffer per 107 cells was added, and cells were incubated with 20 µl of Anti-Biotin Microbeads per 107 cells for 15 min at 4°C.
- Cells were washed with MACS buffer and resuspended up to 108 cells in 500µl of MACS buffer.
- CD4+ T cells were sorted by two rounds of magnetic depletion on the AutoMACS (program Deplete S).
3. CD4+ T cells (negative fraction) were washed with MACS buffer and resuspended in 80µl of MACS buffer per 107 cells. Then, 20µl of CD45RO microbeads per 107 cells was added for 15 min at 4°C. Naïve CD45RO- T cells were isolated by two rounds of depletion on the AutoMACS (as in step 2.).
4. Naïve CD4+ CD45RO- T cells were cultured at 1 × 105 cells/well in 96-well flat-bottom plates or 5 × 105 cells/well in 24-well plates (Falcon) for 5-6 days in Yssel’s medium containing 1% human AB serum (Gemini Bio Products) with the appropriate cytokines and beads coated with anti-CD3, anti-CD28, and anti-CD2 antibodies (T Cell Activation/Expansion Kit, Miltenyi Biotec) (1 bead/10 cells).
5. After 5-6 days, cells were collected, counted, washed and cultured at 1 × 105 cells/well in 96-well flat-bottom plates or 5 × 105 cells/well in 24-well plates for an additional period of 5-6 days in the presence of the indicated cytokines and IL-2.
6. After 10-12 days of culture, cells were counted and stimulated at 5 × 105 cells/ml with anti-CD3, anti-CD28, and anti-CD2 antibodies in the presence of IL-2 for 24 h to analyze gene expression or 48 h to assess cytokine level in cell-free supernatants.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
Human
Th17 cells
T cell differentation
T cell development
cytokine
Interleukin-23