CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Laura S Angelo,Lynn H Bimler,Silke Paust,Kevin Aviles-Padilla,Rana Nikzad

doi:10.3389/fimmu.2019.00469

Abstract

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet−/EOMES+) but differ from PB NK cells (T-bet+EOMES−). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6− counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6− and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

Highlights

NK cells are immune cells that kill infected or malignant cells and secrete cytokines and chemokines [1]
We found a much higher frequency of CD56bright NK cells in fetal liver (70%) than fetal spleen (46%), and in fetal NK cell preparations compared to adult PB NK cells (5%) (Figures 1A, S2–S6)
We used a combination of CXCR6, CD16, and killer immunoglobulin receptors (KIR) to identify CD56bright and dim subpopulations in fetal liver and spleen NK cells [32] (Figures S3, S4)

Summary

Introduction

NK cells are immune cells that kill infected or malignant cells and secrete cytokines and chemokines [1]. NK cells survey their environment through expression of activating or inhibitory cell surface receptors, as well as cytokine and chemokine receptors, and adhesion molecules [2, 3]. In human PB, 90% of all NK cells are considered mature NK cells that are highly cytotoxic, and identified phenotypically as T-bethigh, EOMESlow, CD56low, and CD16high. CD56high NK cells are generally considered less mature and less cytotoxic than CD56low NK cells and are robust cytokine producers, and generally do not express much CD16. Human adult liver contains T-bet and/or EOMES expressing NK cells, as well as a subset of EOMEShigh NK cells that co-express the G-protein coupled C-X-C Motif Chemokine Receptor 6 (CXCR6), and the activation or tissue residency marker CD69.

Methods

Results

Conclusion