T-lymphocyte functionality assessed by analysis of cytokine receptor expression, intracellular cytokine expression, and femtomolar detection of cytokine secretion by quantitative flow cytometry

Abstract

Assessing the functionality of T lymphocytes is important in determining progression rate in human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS), transplant rejection, and autoimmune disease. Activation of T-cells in response to antigen results in expression of cytokines and cytokine receptors, proliferation, and development of effector function. Multiplexed flow cytometric analyses were developed to measure cytokine receptor expression, internal cytokine expression, and cytokine secretion by activated T-cells in vitro. Receptor expression was determined by the binding of phycoerythrin-labeled cytokines. Internal cytokine was determined by intracellular labeling with anti-cytokine antibodies. Cytokine secretion was determined by a flow cytometry-based immunofluorescence assay. The assays could be multiplexed, measuring up to six cytokines simultaneously and measuring cellular receptor expression simultaneously with cytokine secretion. The immunoassays were sensitive in the femtomolar range, allowing determination of normal serum levels of cytokines (<100 fg/ml). Using granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion as a marker for activation, it was determined that at peak secretion (68 h post activation) an average of 1,150 molecules of GM-CSF were produced per cell per hour. Active infection with several viruses reduced the ability of T-cells to be activated. Activated T-cells (1 x 10(6)) normally produced 4-8 pg/ml/h GM-CSF after 20 h of activation, impaired T-function resulted in a decrease to the 0.2-2.0 pg/ml/h range.