Cell Culture

scientificprotocols authored about 3 years ago

Authors: Nicolas Manel 

Introduction

Human CD4+ Th-17 cells produce inflammatory cytokines and have been implicated in the development of several inflammatory pathologies. The transcription factor RORgammaT is though to establish Th-17 cell differentiation. Expression of IL-17A is a hallmark of Th-17 cells.

We have used RORgammaT overexpression as an approach to identify factors that induce Th-17 cell differentiation. This led us to the observation that TGF-beta is essential for induction of RORgammaT. However, unknown factors present in serum inhibit Th-17 cell differentiation. Thus, it is important to cultivate cells in serum-free conditions in order to generate Th-17 cells from naive CD4+ T cells. In serum-free conditions, we found that a combination of TGF-beta, IL-1beta and either IL-6, IL-21 or IL-23 is able to induce Th-17 cell differentiation.

Here is described a procedure to generate human Th-17 cells from naive CD4+ T cells isolated from adult or cord blood.

Reagents

  1. Miltenyi human CD4 microbeads
  2. Fluorescently labelled ant-CD3, anti-CD4, anti-CD45RA, anti-CD25, anti-HLA-DR, anti-IL-17
  3. Recombinant human IL-1beta, IL-23, IL-2, TGF-beta
  4. Serum-free media (e.e Lonza XVIVO-20)
  5. Anti-CD3/CD28 activation beads
  6. Phorbol 12-myristate 13-acetate (PMA)
  7. Ionomycin
  8. BD GolgiStop
  9. BD Perm/Fix and BD Perm/Wash Intracellular staining buffers

Equipment

  1. Magnetic cell separator
  2. Cell sorter
  3. Flow cytometer

Procedure

DAY 1

  • 1. Isolate mononculear cells from adult or cord blood on a FicollPAQUE gradient
  • 2. Use Miltenyi human CD4+ beads and procedure to isolate CD4+ T cells
  • 3. Isolate naive CD4+ T cells by cell storing. Human naive CD4+ T cells are typically defined as CD3+CD4+CD45RA+CD25-HLA-DR- but more complex sorting schemes can been utilized (1).
    • TIP: Coat tubes with serum-free media for harvesting cells during sort.
  • 4. Count cells and resuspend in fresh serum-free media at a concentration of 250,000 to 500,000 cells per mL
  • 5. Add 10 U/ml of IL-2, 10 ng/mL of IL-1beta, 10 ng/ml of IL-23, 1 µg/ml of anti-IL-4, 1 µg/mL of anti-IFNgamma and anti-CD3/CD28 activation beads at a ratio of 1 bead per cell
  • 6. In U-bottom 96-well plates, aliquote 200 µL per well
  • 7. Add an increasing concentration of TGF-beta to a serie of four wells : 0, 0.1, 1 and 10 ng/mL
    • TIP: TGF-beta is a highly hydrophobic protein and its activity can be variable. Thus, it is important to titrate TGF-beta systematically in each experiment.

DAY 3

  • TIP: At day 3, cells spontaneously gather to the center of each well and should be visible macroscopically.
    • 8. Spin plates, remove media and replace with fresh media containing all cytokines and antibodies

DAY 5

  • 9. Split each well in half
  • 10. Spin plates, remove media and replace with fresh media containing all cytokines and antibodies

DAY 6

  • 11. Activate cells for 5 hours in the presence of 50 ng/ml of PMA, 500 ng/ml of ionomycin and 1x of BD GolgiStop
  • 12. Fix cells in BD Perm/Fix and proceed for intracellular staining of IL-17A and IFNgamma in BD Perm/Wash buffer
  • 13. Analyse cells by flow cytometry
    • TIP: In the presence of TGF-beta, 1% to 15% of IL-17A+ cells are typically observed

References

  1. De Rosa, S.C., Herzenberg, L.A., Herzenberg, L.A. & Roederer, M. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat Med 7, 245-248 (2001).

Associated Publications

The differentiation of human TH-17 cells requires transforming growth factor- and induction of the nuclear receptor RORt, Nicolas Manel, Derya Unutmaz, and Dan R Littman, Nature Immunology 9 (6) 641 - 649 04/05/2008 doi:10.1038/ni.1610

Author information

Nicolas Manel, New York University

Correspondence to: Nicolas Manel ([email protected])

Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.122. Originally published online 3 June 2008.

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