In vitro differentiation of human Th-17 CD4+ T cells

doi:10.1038/nprot.2008.122

Method Article

In vitro differentiation of human Th-17 CD4+ T cells

https://doi.org/10.1038/nprot.2008.122

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted

You are reading this latest protocol version

Biological techniques

human Th-17

IL-17

IL-17A

RORgammaT

RORgT

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.

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

Magnetic cell separator
Cell sorter
Flow cytometer

DAY 1

Isolate mononculear cells from adult or cord blood on a FicollPAQUE gradient
Use Miltenyi human CD4+ beads and procedure to isolate CD4+ T cells
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.
Count cells and resuspend in fresh serum-free media at a concentration of 250,000 to 500,000 cells per mL
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
In U-bottom 96-well plates, aliquote 200 µL per well
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.

Spin plates, remove media and replace with fresh media containing all cytokines and antibodies

DAY 5

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

DAY 6

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

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).

Download PDF

Version 1

posted

You are reading this latest protocol version

In vitro differentiation of human Th-17 CD4+ T cells

Status:

Version 1

Introduction

Reagents

Equipment

Procedure

References

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

In vitro differentiation of human Th-17 CD4+ T cells

Status:

Version 1

Introduction

Reagents

Equipment

Procedure

References

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

Manage Cookie Preferences