Microfluidic‐based, live‐cell analysis allows assessment of NK‐cell migration in response to crosstalk with dendritic cells

Abstract

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.Keywords: Bone marrow-derived dendritic cells r Chemo-repulsion r Chemotaxis r Microfluidic device r Natural killer cell r NK-DC crosstalk Additional supporting information may be found in the online version of this article at the publisher's web-site Introduction NK cells are motile bone marrow (BM)-derived lymphocytes that play a key role in innate immunity against viral, microbial infections, and transformed cells [1][2][3]. They are capable of killing transformed or infected cells [1,2,4,5], and/or producing cytokines/chemokines that can profoundly influence the quality and magnitude of the adaptive immune responses [6][7][8][9]. They Correspondence: Dr. Sam K. P. Kung e-mail: Sam.Kung2@med.umanitoba.ca acquire specific chemokine surface receptors during development and maturation [2,[10][11][12][13][14][15]. Chemokine receptors such as CCR7, CCR5, and CXCR3 are involved in the preferential migrations and localization of NK cells into the LNs [8,[16][17][18][19], whereas NK cells reside in blood, liver, and spleen exhibit higher CXCR1 and CX 3 CR1 expression [8,9,[20][21][22][23][24][25]. Nonchemokine family proteins such as chemerin and SIP 5 are also involved in the regulation of NK-cell trafficking [26,27]. Collectively, they highlight the complexity of the environmental regulation of NK-cell migrations in physiological and pathological conditions. NK-cell activation and functions are regulated by cytokine/ chemokine and/or DC in the microenvironments [18,[28][29][30] [36,37]. In this report, we demonstrated an application of such microfluidic device in examining how soluble factors produced by DC regulated NK-cell migrations in vitro. Results A microfluidic platform to perform live-cell imaging of NK-cell migrationsWe used the established Y-shaped microfluidic device to examine chemotactic or chemo-repulsive movements of NK cells ...

Advances in the Studies of NK Cell Migrations Using Microfluidic Devices

“…We identified that CXCR3 is a key receptor on NK cells that mediated the migration. More interestingly, a surprising function of GM-CSF in repulsing murine NK cell migratory response was revealed in this microfluidic assay [34]. This study highlighted also key feature and difference of the two migration assays (trans-well and microfluidic assays): cell migration parameters (speed and chemotactic index) were analyzed in the microfluidic assay, at the single-cell level on a 2D plane under a stable chemical gradient; in contrast, chemotaxis was measured as a net downward movement of a population of the migratory cells in the trans-well assay.…”

“…Our study demonstrated the effective use of a simple microfluidic device in experimental studies of directed NK cell migration in controlled gradient environments and for quantitative cell migration data analysis at the single cell level [34]. The study revealed interesting insights regarding migratory interactions between NK cells and dendritc cells.…”

“…Through close collaboration between the Kung lab and the Lin lab at the University of Manitoba, we recently established the first application of this Y-shaped device in the imaging and analyses of the abilities of the immature and mature DC to regulate murine IL-2 activated NK cell migrations in vitro [34]. We generated immature and lipopolysaccharide (LPS)-matured bone marrow derived DC (BMDC) using the established Granulocyte macrophage colony stimulating factor (GM-CSF) culture condition.…”

“…conditioned medium from cell cultures) [34]. In a similar way, we have studied chemotactic migration of human neutrophils as well as stem cells to diseased tissue samples such as sputum samples from patients with chronic pulmonary disease or tissue extract from injured rat heart respectively [46,47].…”

Bidirectional Interactions of NK Cells and Dendritic Cells in Immunotherapy: Current and Future Perspective

“…One might derive exosomes from a specific DC preparation to activate NK cells in vitro to augment the ability of these NK cells in the activation of DC in vivo to promote the NK-DC crosstalk in the induction of specific immunological outcomes in future development of immunotherapy. Third, as migrating immune cells at tumor sites are critical in mounting effective antitumor activity, understanding the regulation of NK-cell or DC-cell migrations in NK-DC crosstalk can be critical in future designs of antitumor immunotherapy [59]. Abilities to upregulate specific chemokine receptors for NK-cell trafficking into the tumors will promote infiltration of NK cells at the tumor microenvironment.…”