TH1/TH2 Cytokine Ratios by Flow Cytometry with IVIG

The ratio of TH1:TH2 cytokines in the cytoplasm of CD3+CD4+ lymphocytes can be determined by flow cytometry. Lymphocytes (mononuclear cells) are stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of GolgiPlug (brefeldin A), a Golgi transport inhibitor, and IVIg (a preparation of immunoglobulin derived from human plasma) for 18 hours. The cells are then harvested, permeabilized, and stained with phycoerythrin-conjugated antibodies against TNF-α, IFN-γ, or IL-10. Four-color flow cytometry is used to measure the percentages of intracellular cytokines in CD3+CD4+ lymphocytes.

Stimulation down-regulates CD4 expression on the cell surface, preventing direct identification. Therefore, CD3+CD4+ cells are identified by negative gating, using ECD-anti-CD3 and FITC-anti-CD8. Lymphocytes that are CD3+CD8− represent the CD3+CD4+ population. The percentages of cytokine-positive CD3+CD8− cells are determined for each of the three cytokines, and ratios are calculated for TNF-α:IL-10 and IFN-γ:IL-10.

Results of this test are compared with those from the TH1:TH2 Intracellular Cytokine Ratio assay performed without IVIG to assess the ability of IVIG to inhibit CD3+CD4+ lymphocyte activation.

Send at room temperature. Do not refrigerate. Deliver to the laboratory within 24 hours.

• Cold specimens (refrigerated or shipped on ice)
• Specimens with extensive clotting or hemolysis
• Specimens older than 48 hours

If a specimen is older than 48 hours, lymphocytes will be isolated and viability assessed. If viability is >80%, the assay will be performed; if <80%, the specimen will be rejected.

The TH1:TH2 Intracellular Cytokine Ratio with IVIG may only be performed in conjunction with the TH1:TH2 Intracellular Cytokine Ratio test.

Flow Cytometry

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• Sacks GP, Clover LM, Bainbridge DR, Redman CW, Sargent IL. Flow cytometric measurement of intracellular Th1 and Th2 cytokine production by human villous and extravillous cytotrophoblast. Placenta. 22:550–559; 2001.
• Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 188:117–128; 1995.
• Schuerwegh AJ, Stevens WJ. Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1β, interleukin-6, and tumor necrosis factor-α in monocytes. Cytometry. 46:172–176; 2001.

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