DETECTION OF INTRACYTOPLASMIC CYTOKINE USING FLOW-CYTOMETRY AND DIRECTLY CONJUGATED ANTI-CYTOKINE ANTIBODIES

Recently, there have been several reports demonstrating improvements i n the flow cytometric detection of intracellular cytokines. These adva nces, although significant, have not yielded techniques that have easi ly been translated into broad use. To address this issue, we have coup led a fixation and permeabilization method with the use of directly la belled monoclonal anti-cytokine antibodies, providing both improved si gnal and simpler staining. The kinetics of in situ cytokine production in both CD4 and CD8 cells are shown for IL-2, IL-4, IL-5 and IFN-gamm a. Based on these data, 6 h was chosen for optimal detection of this c ombination of cytokines. We show the specificity of this technique by blocking cytokine staining using a molar excess of recombinant cytokin e. Additionally, unlabelled anti-cytokine antibodies are demonstrated to block specific staining of labelled antibody, providing an objectiv e means to place statistical markers. Using such controls, we routinel y detected as few as 0.1% false positive cells, allowing the flow cyto metric detection of IL-5, which is below the threshold of detection of published methods. To further prove the specificity of staining, we s tained using two anti-IL-5 mAbs known to recognize different epitopes and demonstrate that the same cells stain with both antibodies. Withou t permeabilization we could detect a fraction of cells with low intens ity staining for cytokine. This staining was further examined using di fferential two color staining for intracellular and extracellular cyto kine, clearly demonstrating no cells staining exclusively for extracel lular cytokine, confirming a lack of passive transfer of cytokine to n earby cells. We show that cytokine flow cytometry is useful in examini ng the increased IL-5 production characteristic of eosinophilic states and that IL-5 production is limited to the CD27 negative subpopulatio n. These data illustrate the unique capability of cytokine flow cytome try to correlate cytokine expression with cell surface phenotype witho ut cell separation. In summary, using directly conjugated anti-cytokin e antibodies, cytokine flow cytometry becomes a specific and versatile technique for the assessment of complex cytokine production phenotype s in fresh ex vivo T cell subpopulations.