A protocol for the role of TNF-α on regulatory T cell function

doi:10.1038/protex.2015.088

Method Article

A protocol for the role of TNF-α on regulatory T cell function

https://doi.org/10.1038/protex.2015.088

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In vitro Treg suppression assays are performed to determine the functional effect of TNF-α on Treg cells. In addition to the co-culture condition to measure the effect of TNF-α on human Treg by mixing them with Teff cells, parallel experiments were conducted where TNF-α was washed away prior to co-culture (pre-treatment condition).

Biological techniques

TNF-α

Treg cells

Teff cells

-Recombinant human TNF-α (R&D Systems, Cat: 210-TA-020), rhIL-2 (Roche, Cat: 11011456001), CFSE (Invitrogen, Cat: C34554), anti-CD3/CD28 beads (Life Technologies, Cat: 111.31D), Anti-Human CD4 PerCP-Cyanine5.5 (eBioscience, Cat: 45-0048), Anti-Human CD25 APC (eBioscience, Cat: 17-0259), Anti-Human CD127 PE (eBioscience, Cat: 12-1278), Human CD4⁺ T Cell Isolation Kit (Miltenyi Biotec, Cat: 130-096-533), Lymphoprep (Axis-shield, Cat: 1114546)

-Culture medium: RPMI-1640 (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco).

-MACS Buffer: 1×PBS supplemented with 0.5% BSA and 2 mM EDTA.

-FACS Buffer: 1×PBS supplemented with 2% FBS.

FACSAria II (BD Biosciences) for sorting

FACSCanto II (BD Biosciences) for analysis

FACS tubes round bottom (BD Biosciences)

96-well U-bottom plates (Corning Costar)

LS columns (Miltenyi Biotec)

CO₂ incubator (Thermo)

Isolation of Treg and Teff cells

Obtain fresh blood specimens from healthy donors and isolate PBMC by Lymphoprep density gradient medium.
Isolate total CD4⁺ T cells using the CD4⁺ T cell Isolation Kit as the following steps in brief. (1) Resuspend total PBMC in 40 µl of MACS buffer per 10⁷ cells, and incubate with 10 µl of Biotin-Antibody Cocktail per 10⁷ cells for 10 min at 4°C.

(2) Subsequently, add 30 µl of MACS buffer per 10⁷ cells, and incubate cells with 20 µl of MicroBead Cocktail per 10⁷ cells for 15 min at 4°C.

(3) Wash cells with MACS buffer and resuspend cells in 1 ml of MACS buffer.

(4) Prepare LS column by rinsing with 3 ml of MACS buffer.

(5) Apply cell suspension onto the column and collect unlabeled cells that passed through.

Wash CD4⁺ T cells (negative fraction) with MACS buffer.
Resuspend CD4⁺ T cells with 100 µl of FACS buffer, and add 10 µl anti-CD4, anti-CD25, anti-CD127, respectively, mix and incubate for 15 min at 4°C.
Wash cells twice with 3 ml of FACS buffer, follow by FACS sorting for CD4⁺CD25^hiCD127^low/- as Treg cells and CD4⁺CD25^- T cells as Teff cells. The purity of Treg cells and Teff cells ranged 95%-98%.

Assay setup

Treat Treg cells with TNF-α (50 ng/ml) or leave them in medium alone (control) in 96-well U-bottom plates (0.5-1.0×10⁵ cells per well) in the presence of IL-2 (100 U/ml) for 24 hs.
Wash the resulting Treg cells twice with culture medium, and count the cells.
Label Teff cells with CFSE at 2 µM, 37°C for 8 min, then wash for three times with culture medium.
Inhibition assay in different conditions as follows. (1) TNF-α pre-treatment assay: culture CFSE labeled Teff cells (1×10⁴ cells per well) alone or with Treg cells (1×10⁴ cells per well) that were either untreated or pretreated with TNF-α (50 ng/ml) in 96-well U-bottom plates. Maintain the culture in the presence of 2×10³ anti-CD3/CD28 beads for 4 days.

(2) TNF-α co-culture assay: culture CFSE labeled Teff cells (1×10⁴ cells per well) alone or with Treg cells (1×10⁴ cells per well) which were not pre-exposed to TNF-α, in the presence or absence of TNF-α (50 ng/ml) in 96-well U-bottom plates. Maintain the culture in the presence of 2×10³ anti-CD3/CD28 beads for 4 days.

Measure proliferation of Teff cells by CFSE dilution in flow cytometry analyses. Express the results as percent inhibition: (1 − (experimental CFSE dilution/control CFSE dilution)) × 100%.

The authors declare no competing financial interests.

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A protocol for the role of TNF-α on regulatory T cell function

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