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

## TL;DR

This study develops a method to measure how murine T cells stick to other cells using β2-integrins, showing that this process is influenced by ROS and calcium, and is relevant in heart disease.

## Contribution

A refined and specific assay for quantifying β2-integrin–mediated adhesion in murine T cells using whole blood.

## Key findings

- PMA and Mg2+/EGTA increase β2-integrin–mediated adhesiveness of T cells by 30-fold and 65-fold, respectively.
- ROS and Ca2+ are essential for chemokine-mediated β2-integrin activation in murine T cells.
- T cell adhesiveness is significantly elevated in mice after myocardial infarction.

## Abstract

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.

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.

Incubation of murine whole blood with PMA leads to 30‐fold and Mg2+/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.

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.

Integrin subunit β2 of murine T cells binds to the intercellular cell adhesion molecule 1 (ICAM‐1), which was fluorescently labeled to quantify β2‐integrin activation with fluorescence‐activated cytometry. Integrins remain inactive until they are activated. This activation process is called adhesion and can be induced through changes in receptor avidity or affinity. 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+. Specificity of the assay was proven by preincubation of a blocking antibody. Inhibitor experiments show that reactive oxygen species (ROS) and calcium (Ca2+) are crucial for chemokine (CCL‐17 and its receptor CCR7)‐mediated β2‐integrin activation.