The aim of the present study was to validate a rapid 96 wells microplate flow cytometry assay (Cytometric Bead Array, BD Biosciences) to detect a set of cytokines (TH1/Th2) released following T lymphocytes activation. To validate this test, we stimulated purified CD4+ T cells with various systems (PMA, anti-CD3 and anti-CD3/anti-CD28) and we verified the activity of anti-inflammatory compounds (Cyclosporin A and Tacrolimus).

Biological model:
Primocultures of purified normal human T lymphocytes (CD4+ T cells) isolated from blood freshly recovered from a healthy donor.

Treatements and stimulations:

• Unstimulated control
• PMA
• Anti-CD3 stimulation.
• Anti-CD3 and anti-CD28 co-stimulation
• Anti-CD3 and anti-CD28 co-stimulation + Cyclosporin A
• Anti-CD3 and anti-CD28 co-stimulation + Tacrolimus

For CD3 stimulation, micro-plates were coated with an anti-CD3 IgG1 antibody before cell seeding. Anti-CD28 was added directly to the culture medium. Cells were incubated for 24 hours in RPMI medium at 37°C, 5% CO2 and RH > 95-99 %. All treatments were performed in triplicate (n=3).

Cytokine analysis:
Multiple cytokines (IL-2, IL-4, IL-5, IL-10, IFN-gamma, TNF-alpha) were measured by Cytometric Bead Array (Th1/Th2 Cytokine Kit) using the BD FACSArray™ bioanalyzer in agreement with supplier's specifications.

The results are shown in Figures 1 and 2

Stimulation by PMA induced a significant release of IL-2, IL-4, IL-5, IL-10, IFN-gamma and TNF-alpha.

Stimulation by anti-CD3 alone (mimics the basic antigen stimulation) significantly induced the secretion of IFN-gamma and TNF-alpha. The concentrations of IL-2, IL-4, IL-5 and IL-10 were too low to be detected in this assay.

The double stimulation anti-CD3/anti-CD28 (co-stimulation) was more potent and induced a significant release of IL-2, IL-4, IL-5, IL-10, IFN-gamma and TNF-alpha.These results are comparable to those obtained by conventional ELISA assays. Please note that the stimulation levels observed are strongly donor dependent.

Figure 1: Effects of various stimulating factors on the release of IL-2, IL-4, IL5, IL-10, IFNgamma and TNFalpha in culture medium of CD4+ T lymphocytes (Contact time, 24h).

The anti-inflammatory references “Cyclosporin A” and “Tacrolimus” at 0.1 µM completely blocked the cytokine release (without specific selectivity regarding cytokines).

Figure 2: effects of Cyclosporin A and Tacrolimus on the release of IL-2, IL-4, IL5, IL-10, IFNgamma and TNFalpha in culture medium of activated CD4+ T lymphocytes (Contact time, 24h).

Conclusion

Cytometric Bead Array is well adapted for the rapid assay of stimulating conditions or compounds (96 wells microplate format) for multiple cytokine release. Moreover, this technology allows multiplex detection in small sample volume (<50µl) and is especially suitable to precious samples (i.e. from clinical studies).

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