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

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.

Keywords: Cherokees Chemokine receptors; Homing of hepatic NK cells; Liver immunology.

Figure 1. Distribution of NK cell subsets in peripheral blood, liver tissues and liver perfusates

(A,B) Flow cytometric contour plots (A) and histogram (B) graphs from a representative example showing the phenotypic distribution of CD56 and CD16 (A) and the mean fluorescence intensity (MFI) of CD56 (B) on CD56^pos/CD16^neg (black line) and CD56^pos/CD16^pos (dashed gray line) NK cell subsets freshly purified from peripheral blood (left), healthy liver tissue (middle) and healthy liver perfusates (right). (C) Summary graphs of statistical dot plots with p values and means showing the relative frequencies of CD56^pos/CD16^neg NK cell subset within total NK cell populations purified from peripheral blood and healthy liver tissue of matching donors. Data are presented as percentages and are representative of 20 donors. (D) Summary graphs of statistical histogram bars with p values and SD showing the MFI of CD56 on CD56^pos/CD16^neg NK cells freshly purified from peripheral blood (left), healthy liver tissues (middle) and healthy liver perfusates (right). Data are representative of 3 donors. (E) Summary graphs of statistical histogram bars with p values and SD showing the relative frequencies of CD56^pos/CD16^neg NK cell subset within total NK cell populations purified from healthy liver tissues (left) and healthy liver perfusates (right). Data are presented as percentages and are representative of 3 donors. * = p < 0,05 – ** = p < 0,01 – *** = p < 0,001

Figure 2. Transcriptional and phenotypic profiles of CD56^bright hepatic NK cells

(A) Heat map showing the unsupervised clustering of 441 genes differently expressed in the Nanostring dataset on CD56^bright NK cell subset freshly purified from perfusates (Perf.) of healthy liver compared to that of CD56^bright NK cell from peripheral blood (PB) and to CD56^dim Perf- and PB-NK cells. (B) Flow cytometric contour plots from a representative example showing the percentages of L-selectin, CCR7, α₅ integrin, α_x integrin, DNAM-1 and CD161 on CD56^bright NK cells from peripheral blood (PB-NK cells in the upper line) and healthy liver (H-NK cells in the lower line) of the same donor. (C) Summary graphs of statistical histogram bars with p values and SD showing the surface expression of L-selectin, CCR7, α₅ integrin, α_x integrin, DNAM-1 and CD161 on CD56^bright NK cells from peripheral blood (black bars) and healthy livers (white bars) of matching donors. Data are presented as percentages and are representative of 5 human donors. ** = p < 0,01 – *** = p < 0,001

Figure 3. Repertoire of chemokine receptors and CD69 expression on hepatic NK cell subsets compared to their circulating counterparts

(A) Flow cytometric contour plots graphs showing the surface expression of the chemokine receptors CXCR6, CCR5 and CD69 on CD56^bright (first column) and CD56^dim (second column) purified from liver (upper line) and peripheral blood (lower line) of matching donors. (B) Summary graphs of statistical histogram bars with p values and SD showing the surface expression of CCR3, CCR4, CCR5, CCR7, CXCR2, CXCR3, CXCR4, CXCR6, CX₃CR1 and CD69 on CD56^bright and CD56^dim NK cells from healthy livers (white bars) and peripheral blood (black bars) of matching donors. Data are presented as percentages and are representative of 5 donors. * = p < 0,05 – ** = p < 0,01 – *** = p< 0,001

Figure 4. Functional relevance and migration patterns of hepatic NK cell subsets

(A) Flow cytometric contour plots graphs from the same representative donor showing the percentages of IFN-γ^pos/CD56^bright (left column) and IFN-γ^pos/CD56^dim (right column) NK cells purified from liver (upper line) and peripheral blood (lower line) (B) Summary graphs of statistical histogram bars with p values and SD from matching donors showing the percentages of IFN-γ^pos/CD56^bright and IFN-γ^pos/CD56^dim NK cells purified from healthy livers (white bars) and peripheral blood (black bars). Data are presented as percentages and are representative of 6 human donors. (C) Summary graphs of statistical histogram bars with p values and SD showing the percentages of CD56^bright (black bars) and CD56^dim (white bars) H-NK cells from matching donors migrating in response to CCL3, CCL4, CCL5, CXCL16 and fractalkine. Data are representative of 5 human donors. * = p < 0,05 – ** = p < 0,01 – *** = p < 0,001

Figure 5. Distribution of CCR5 ligands in healthy human liver

(A) Representative immunohistochemistry image (magnification of 20X) showing-CCL3^pos (brown indicated with arrows) cells in healthy liver. The hepatic sinusoids are indicated with S. (B) Representative fluorescence microscopic image showing the expression of CCL3 (purple on the left) and CD68 (green in the middle) alone or co-localized on the same cells (right). DAPI in blue labels cell nuclei. The hepatic sinusoids are indicated with S (C) Summary graphs of statistical histogram bars with p values and SD showing the mean number for field of CCL3^pos, CCL4^pos and CCL5^pos cell in the in parenchyma of healthy liver determined by immunohistochemistry experiments. Cells were counted at 20X magnification and a least 10 fields per specimen were analyzed. Data are representative of 5 donors. (D) Representative immunohistochemistry image showing the CCL5^pos (brown indicated with arrows) cells in parenchyma of healthy liver. The hepatic sinusoids are indicated with S. (e) Summary graphs of statistical histogram bars with p values and SD showing the percentage of CCL5^pos/CD3^pos T cells, CCL5^pos/CD3^neg/CD56^pos NK cells and CCL5^pos/CD19^pos B cells freshly purified from healthy liver and analyzed by flow cytometry. Data are representative of 5 donors. ** = p < 0,01 – *** = p < 0,001

Figure 6. CD56^bright NK cells are preferentially located in hepatic sinusoids

(A) Summary graphs of statistical histogram bars with p values and SD showing the percentage of expression of NKp46 on CD56^bright and CD56^dim H-NK cells from healthy liver and analyzed by flow cytometry. Data are representative of 5 donors. (B) Representative immunohistochemistry image showing NKp46^pos H-NK cells (brown indicated with arrows) cells in sections of parenchyma (left) and portal spaces (right) of healthy liver. The hepatic sinusoids are indicated with S, bile ducts with BD, hepatic arteries with HA and hepatic veins with HV. (C) Summary graphs of statistical dot plots with means and P value showing the mean number for field of NKp46^pos H-NK cells in the in parenchyma of healthy liver determined in immunohistochemistry experiments. Cells were counted at 40X magnification and a least 10 fields per specimen were analyzed. Data are representative of 3 donors. (D) Representative fluorescence microscopic image showing the expression of CCR5 (green on the left) and NKp46 (green in the middle) alone or co-localized on the same cells (right) within hepatic sinusoids labeled with Lyve-1 (red). DAPI in blue labels cell nuclei.