LFA-1 signals to promote actin polymerization and upstream migration in T cells

Abstract

T cell entry into inflamed tissue requires firm adhesion, cell spreading, and migration along and through the endothelial wall. These events require the T cell integrins LFA-1 and VLA-4 and their endothelial ligands ICAM-1 and VCAM-1, respectively. T cells migrate against the direction of shear flow on ICAM-1 and with the direction of shear flow on VCAM-1, suggesting that these two ligands trigger distinct cellular responses. However, the contribution of specific signaling events downstream of LFA-1 and VLA-4 has not been explored. Using primary mouse T cells, we found that engagement of LFA-1, but not VLA-4, induces cell shape changes associated with rapid 2D migration. Moreover, LFA-1 ligation results in activation of the phosphoinositide 3-kinase (PI3K) and ERK pathways, and phosphorylation of multiple kinases and adaptor proteins, whereas VLA-4 ligation triggers only a subset of these signaling events. Importantly, T cells lacking Crk adaptor proteins, key LFA-1 signaling intermediates, or the ubiquitin ligase cCbl (also known as CBL), failed to migrate against the direction of shear flow on ICAM-1. These studies identify novel signaling differences downstream of LFA-1 and VLA-4 that drive T cell migratory behavior.This article has an associated First Person interview with the first author of the paper.

Natural Killer Cell Integrins and Their Functions in Tissue Residency

“…As in the liver ( 216 ) expression of CXCR6 is high, and expression of CCR9 is low on α1 hi , αE hi , αM lo , αX lo NK cells from the tonsil ( 88 , 216 ). Interestingly, NEDD9 (CasL), a protein associated with inside-out integrin signaling via its interaction with FAK, is also very highly expressed in these cells, suggesting that adhesion regulators may also mediate integrin functions associated with residency ( 88 , 140 , 217 ). In addition to its well-described role in liver residency, CXCR6 is associated with specificity for epithelial tissues, including the airways of the lung ( 61 , 218 , 219 ).…”

“…In migrating T cells, integrins are differentially localized along the front-rear axis based upon their conformation, with high affinity β1 integrins found primarily in the uropod, whereas LFA-1 is found in high-affinity conformation in the lamellipodia and mid-body focal zone, and low affinity conformation in the uropod ( 138 , 139 ). T cell integrin spatial localization is linked to differential usage, which in the case of VLA-4 and LFA-1 includes mediating up- and down-stream mechanotaxis under shear flow, respectively ( 139 , 140 ). Engagement of LFA-1 or VLA-4 to ICAM-1 or VCAM-1 leads to distinct signaling pathways that help shape these specified responses ( 140 ).…”

“…T cell integrin spatial localization is linked to differential usage, which in the case of VLA-4 and LFA-1 includes mediating up- and down-stream mechanotaxis under shear flow, respectively ( 139 , 140 ). Engagement of LFA-1 or VLA-4 to ICAM-1 or VCAM-1 leads to distinct signaling pathways that help shape these specified responses ( 140 ). Thus, at least in T cells migrating under flow, differential cellular localization of integrin species direct different functions in the same context, with the passive uropod acting as a windvane in LFA-1 mediated upstream migration, and the lamellipodia passively focusing downstream migration that is mediated by VLA-4 ( 139 , 141 ).…”

Automated cell tracking using StarDist and TrackMate

“…To highlight that our pipeline can also be used to analyze brightfield images, we generated a StarDist model (Training dataset after 5x augmentation: 209 paired images, 31200 labelled objects) to track T cells migrating on ICAM-1 or VCAM-1 (Figure 2C-E; Extended data: Video 2 16 ). Importantly, automated analysis of these data could reproduce the results obtained via manual tracking 19 . Finally, we used our pipeline to automatically track non-adherent cancer cells flowing in a microfluidic chamber (Figure 2F and G; Extended data: Video 3 16 ).…”

“…were coated with 2 µg/mL ICAM-1 or VCAM-1 overnight at 4°C 19 . Activated primary mouse CD4+ T cells were washed and resuspended in L-15 media containing 2 mg/mL D-glucose.…”