Human liver-resident CD56^bright/CD16^neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways

Highlights

• Characterization of the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts (c-NK cells).

• Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56^bright lr-NK cells that localize within hepatic sinusoids.

• CD56^bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally for their migration to hepatic sinusoids expressing their putative ligands.

• The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56^bright NK cells that constitutively express CCR5 and CXCR6.

• This study releases the phenotypic and functional differences that distinguish lr-Nk cells from c-NK cells.

Abstract

Rationale

The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic.

Objectives

We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts.

Findings

Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56^bright lr-NK cells that localize within hepatic sinusoids. CD56^bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3^pos Kupffer cells, CXCL16^pos endothelial cells and CCL5^pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56^bright NK cells that constitutively express CCR5 and CXCR6. CD56^bright lr-NK cells co-exist with CD56^dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56^dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX₃CR1.

Conclusion

Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56^bright lr-NK cells will require modification of hepatic CCR5 and CXCR6.

Introduction

Natural killer (NK) cells are important effectors of the innate immune system that can lyse tumor-transformed or virus-infected cells in the absence of prior antigen sensitization. NK cells are also endowed with immune-regulatory functions at tissue sites of inflammation through the establishment of cellular interactions and the production of pro-inflammatory cytokines including IFN-γ, TNF-α, CCL3 (Mip1-α), CCL4 (Mip1-β)and CCL5 (RANTES) [1], [2]. Human peripheral blood NK (PB-NK) cells are divided into two functionally distinct subsets characterized by a different distribution of CD56 and CD16 surface markers. CD56^bright/CD16^neg-low (CD56^bright) NK cells account for 5–15% of all PB-NK cells and, while poorly cytotoxic, can produce large amounts of cytokines. CD56^dim/CD16^pos (CD56^dim) NK cells represent the majority of PB-NK cells (up to 95%) and serve primarily as cytotoxic effectors [3]. In order to spare autologous cells from cytotoxicity and ensure tolerance to self, NK cells receive inhibitory signals from a large family of inhibitory NK cell receptors (iNKRs), that include Killer cell immunoglobulin-like receptors (KIRs) and C-type lectins recognizing specific alleles of self MHC-class-I molecules (MHC-I). NK cell effector-functions are generally induced by the engagement of another family of activating NK cell receptors (aNKRs) that binds their ligands expressed on stressed, infected or tumor-transformed target cells that either lack or have a decreased expression of self-MHC-I [1], [4], [5], [6].

NK cells can account up to 50% of the total lymphocyte population in the human liver, which is in contrast to their lower frequency in blood (5–15%) or other peripheral tissues such as lymph nodes. Hence, great efforts have been placed over the recent years to understand the role of hepatic NK cells in the pathogenesis of liver disorders including fibrosis, viral infections, tumors and autoimmune diseases [7], [8], [9]. In this regard, a distinct subset of hepatic NK cells endowed with adaptive immune properties and exhibiting antigen-specific recall responses to viruses and haptens has been recently described in mice [10], [11]. This memory-like response is exerted by a unique subset of CD49a^pos/DX5^neg liver-resident NK (lr-NK) cells that are phenotypically and functionally distinct from peripheral blood CD49a^neg/DX5^pos conventional NK (c-NK) cells circulating throughout spleen and liver. Likewise, it has been reported that NK cells in human liver are different from their circulating counterparts [7], [12], [13], [14], [15], [16], thus suggesting that a unique lr-NK cell subset also exists in the human liver under homeostatic conditions. However, the overall frequency and the precise phenotype of human lr-NK cells are still being debated, while the distribution and the homing mechanisms regulating their retention in the liver are unknown. The present study characterizes CD56^bright lr-NK cells selectively located within hepatic sinusoids and accounting for half of the entire hepatic NK cell population. We demonstrate that CD56^bright lr-NK cells are phenotypically and transcriptionally distinct from PB-CD56^bright NK cells and constitutively express high levels of markers associated with tissue residency including CD69, CCR5 and CXCR6. This unique phenotype is functionally relevant as CD56^bright lr-NK cells migrate in response to CCL3, CCL5 and CXCL16, the CCR5 and CXCR6 ligands highly expressed within liver sinusoids on Kupffer cells, T and NK lymphocytes as well as endothelial cells.