Ligand-complex–based quantification of β2-integrin–mediated affinity and avidity of murine T cells

Abstract

Background: Integrins facilitate binding to the extracellular matrix and other cells. Their subunit β2 is exclusively expressed by leukocytes, binds to the intercellular cell adhesion molecule 1 (ICAM-1), and is pivotal for their recruitment to sites of inflammation such as the atherosclerotic plaque.

Methods: To investigate β2-integrin–mediated adhesiveness, a well-established assay for human whole blood was adapted for the analysis of murine T cell subsets. Changes in avidity and affinity were assessed by incubation of murine complexes ICAM-1 in murine whole blood and consecutive stimulation with PMA and Mg²⁺/EGTA. Underlying signaling pathways in β2-integrin–mediated adhesiveness upon chemokine stimulation with CCL-19 were identified by incubation with reducing substances, and a Ca²⁺ chelator and ROS and Ca²⁺ measurements were carried out.

Results: Incubation of murine whole blood with PMA leads to 30-fold and Mg^2+/EGTA to 65-fold increase in β2-integrin–mediated adhesiveness of T cells. Specificity of the assay was proven by preincubation of a blocking antibody, leading to a 60% reduction in adhesion capacity. ROS species and Ca²⁺ are crucial for chemokine-mediated β2-integrin activation. In vivo relevance was proven by induction of T cell adhesiveness in whole blood of mice upon myocardial infarction.

Conclusions: Our assay allows specific quantification of β2-integrin–mediated affinity and avidity of T cells in whole blood samples. In congruence to human adhesion, these mechanisms are ROS and Ca²⁺ dependent and significantly elevated after myocardial infarction. Our refined and robust assay may be of particular use in phenotyping involved mechanisms in T cell activation in atherosclerotic cardiovascular disease.

Keywords

affinity / avidity / ICAM-1 / T cells / T helper cells / β2-integrin

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2026 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.