Abstract
The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca2+ levels. Ca2+ is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ∼6 µm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca2+ levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.
Highlights
The capability of T lymphocytes to migrate is a crucial part of the adaptive immune response
Activated T cells migrated with a mean velocity of 5. 060.1 mm/min (n = 198 from 12 independent donors; mean 6 SEM), with a minimum velocity of 1.5 mm/min, maximum velocity of 12.7 mm/min and a median velocity of 5.1 mm/min (Fig. 2A–B), which was comparable to earlier in-vitro studies [34]
KCa3.1 and transient receptor potential melastatin 7 (TRPM7) localize in the uropod and control the cell migration velocity, while Kv1.3, CRAC and TRPM4 accumulate in the cell front and do not facilitate cell motility
Summary
The capability of T lymphocytes to migrate is a crucial part of the adaptive immune response. T cells migrate continuously in and out of the bloodstream to lymphoid organs and to tissues in search of specific antigens to carry out immune surveillance [1,2]. The leading-edge contains chemokine receptors, like C-X-C chemokine receptor type 4 (CXCR-4), T cell receptor (TCR) and filamentous actin (F-actin) [3,4]. These allow sensing of chemoattractants, adhesion and Factin polymerization to drive forward movement [4]. Other proteins, including the adhesion proteins intercellular adhesion molecule-1 (ICAM-1) and CD44, together with ERM proteins (ezrin, radixin and moesin) and the mitochondria, instead polarize to the uropod, which undergoes cycles of attachment, release and retraction, completing the crawling process that allows cell motility [3,4]
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