Quantitative analysis of immune synapse formation using imaging flow cytometry. (130.18)

Abstract

Abstract Antigen-specific immune responses are initiated via direct cellular contact between an antigen presenting cell (APC) and an effector cell. Early events in the interaction between these two cells involve reorganization of the actin cytoskeleton and recruitment of adhesive and signaling molecules to the immunological synapse (IS), which ultimately results in effector cell activation. Because manual image acquisition and quantitative image analysis are time consuming processes, microscopic analyses of protein recruitment to the IS have remained either qualitative or statistically limited, despite the rich information content inherent in high resolution digital images. Here we describe an objective, statistically robust microscopy-based method for quantifying molecular recruitment to the IS using multispectral imaging flow cytometry, which enables quantitative image analysis of large populations of automatically collected images. The analysis technique uses morphology-based features to identify conjugates, followed by measurement of fluorescence specifically at the conjugate junction. Two models are described, including: 1) actin recruitment to the IS following T cell activation by artificial APC coated with anti-CD3; 2) antigen-specific recruitment of ADAP to the IS within TCR transgenic T cells. These data outline an objective and statistically robust method to rapidly quantify molecular recruitment to the immune synapse using automated high speed microscopy.