CXCR4 coordinates adhesion, migration, and development of human NK cells
Shira E. Eisman, Francesca E. Grossberg, Batya S. Koenigsberg, David H. McDermott, Frédérique van den Haak, Luis A. Pedroza, Everardo Hegewisch-Solloa, Philip M. Murphy, Emily M. Mace
Shira E. Eisman, Francesca E. Grossberg, Batya S. Koenigsberg, David H. McDermott, Frédérique van den Haak, Luis A. Pedroza, Everardo Hegewisch-Solloa, Philip M. Murphy, Emily M. Mace
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Research Article Cell biology Immunology

CXCR4 coordinates adhesion, migration, and development of human NK cells

Abstract

Natural killer (NK) cells undergo stepwise differentiation from multipotent progenitors within secondary lymphoid tissues. Despite the central importance of the tissue microenvironment in their development, little is known about cell-cell interactions that regulate human NK cell trafficking and maturation. Here, we identify the chemokine receptor CXCR4 and its ligand CXCL12 as regulators of stromal–NK cell interactions required for NK cell maturation. We demonstrate that CXCR4 is expressed throughout human NK cell development in peripheral blood and tonsil, and CXCL12 is enriched in stromal niches containing developing NK cells. Pharmacologic blockade or genetic disruption of CXCR4 resulted in diminished adhesion to integrin ligands, and high-resolution imaging demonstrated crosstalk between CXCR4 and integrins, providing a mechanistic basis for chemokine-dependent modulation of adhesion. Further, CXCR4 blockade resulted in altered contact-dependent motility on stromal cells and integrin ligands, with decreased stable stromal engagement and increased cell speed. Consistent with a requirement for these interactions, treatment with the CXCR4 antagonist plerixafor (AMD3100) impaired NK cell generation from CD34+ precursors. Analysis of NK cells from patients with WHIM syndrome with CXCR4 gain-of-function mutations treated with plerixafor revealed similar defects in migration and adhesion, supporting the in vivo relevance of CXCR4-dependent regulation of NK cell adhesion and motility.

Authors

Shira E. Eisman, Francesca E. Grossberg, Batya S. Koenigsberg, David H. McDermott, Frédérique van den Haak, Luis A. Pedroza, Everardo Hegewisch-Solloa, Philip M. Murphy, Emily M. Mace

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Figure 5

CXCR4-CXCL12 interactions are required for effective NK cell differentiation from CD34+ cells.

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CXCR4-CXCL12 interactions are required for effective NK cell differentia...
(A) Schematic of NK cell in vitro differentiation protocol. Human CD34+ cells were isolated from cord blood using density centrifugation and fluorescence-activated cell sorting (FACS). Sorted CD34+ cells were plated on EL08.1D2 stromal cells with cytokines as indicated. Cultures were treated with 10 μM AMD3100 or vehicle at weekly media exchanges. (B) Fold increase in the absolute number of live CD45+ cells from AMD3100-treated (orange) and vehicle-treated (green) cultures. n = 4–10 donors. (C) Frequency of CD34+ cells gated on CD45+ cells at days 7 and 14. n = 7–10 donors. (D) Absolute number (×103) of NK cells at day 14. n = 5 donors. (E) Frequency of Lin–CD117+ NK/ILC progenitors gated on CD45+ live cells by week. n = 7–9 donors. For B–E, each dot represents an individual donor, and lines connect paired samples. Statistical comparisons between vehicle and AMD3100 conditions at each time point were performed using Wilcoxon’s matched-pairs signed-rank tests. (F) Representative flow plots from days 14 and 28 showing frequencies of CD56+ Lin– NK cells. Previously gated on CD45+ live cells. (G) Frequencies of CD94+ conventional NK cells at days 14, 21, and 28. n = 7–9 donors. (H) Absolute number (×105) of CD94+ conventional NK cells at day 28. n = 9 donors. (I) Frequencies of granzyme B+ (GzmB) and EOMES+ NK cells at day 28. n = 4–7 donors.