Background

The secondary LN play a fundamental role in immune responses at different locations in the body as they are tactically localized to drain antigens via the lymphatic system. This implies in situ, the recruitment of immune cells, antigen encountering, and the mounting of immune responses (Tan and Watanabe, 2010; Tavares et al., 2014). B cell recirculation through the LN begins with its capture and L-selectin-dependent rolling, along the high endothelial venules (HEV); subsequently, B cells firmly arrest through G-protein coupled receptors (GPCR)-mediated recognition of optimal concentrations of chemokines presented on the luminal surface of the HEV. B cell arrest critically depends on the acquisition of the high-affinity conformation of the integrins LFA-1, VLA-4 (Park et al., 2016). After arrest, B cells crawl to search for a proper site for extravasation into the parenchyma of the LN, where they can recognize antigens and mount an immune response (Pereira et al., 2010; Mesin et al., 2016). The release of B cells from the LN also requires chemokine gradient sensing and interaction with the endothelium of the lymphatic sinuses; however, their exit through the efferent lymph is still poorly understood (Park et al., 2016).

The migration of B cells relies on actin cytoskeletal remodeling and is tightly controlled by signaling molecules such as kinases, GTPases, and motor proteins. Morphological changes resulting from this process are indispensable for rolling, adhesion, migration, motility, filipodia formation, and transmigration (De Pascalis and Etienne-Manneville, 2017). To study these events in vivo, IVM of the LN is a powerful technique that allows in real-time, the visualization of the earliest steps of B-cell extravasation, as well as their behavior and morphological changes during migration (Miller et al., 2003). Although, variations of this technique have been used before to visualize lymphocyte trafficking to Peyer patches or spleen, the inguinal LN has the advantage of being easily accessible for IVM without much surgical effort. Less surgical manipulation allows for better preservation of vascular integrity and blood flow, and better visibility during video recording for the quantification of migratory parameters (von Andrian, 1996; Lafouresse et al., 2015).

In previous work, we focused on the evaluation of the long-tailed class-I myosin, Myo1e, as a regulator of migration of activated B cells in the inguinal LN. We found that the absence of Myo1e causes defective B cell recruitment to the LN (Giron-Perez et al., 2020). In particular, the number of rolling B cells and their rolling velocity were increased in the different HEVs of types I and II, whereas slow-rolling and adhesion were reduced in the HEVs of types III and IV. Moreover, the absence of Myo1e diminished B cell spreading and transmigration (Giron-Perez et al., 2020).

Here we describe a protocol to analyze B cell migration into the inguinal LN by IVM. We provide a detailed description from the purification of B cells to data analysis. This protocol may be adapted to explore the migration of other cell types and the effect of different treatments in this context. Our method can be easily established and multiple adaptations are feasible including application of blocking antibodies or pharmaceuticals to interfere with B cell migration in vivo.