Liver-Derived TGF-β Maintains the EomeshiTbetlo Phenotype of Liver Resident Natural Killer Cells

The adult human liver hosts a complex repertoire of liver resident and transient natural killer (NK) cell populations with diverse phenotypes and functions. Liver resident NK cells are CD56bright NK cells defined by a unique expression profile of transcription factors and cell surface markers (EomeshiTbetloTIGIT+CD69+CXCR6+CD49e−). Despite extensive characterization of the phenotype of liver resident NK cells, it remains unclear how factors within the liver microenvironment induce and maintain this unique phenotype. In this study, we have explored the factors regulating the phenotype of liver resident NK cells. Isolation of healthy liver resident NK cells from donor liver perfusate and in vitro culture results in the gradual loss of the characteristic Tbetlo phenotype, with the cells increasing Tbet expression significantly at day 7. This phenotypic loss could be halted through the dose-dependent addition of liver conditioned media (LCM), generated from the ex vivo culture of liver biopsies from healthy organ donors. TGF-β, but not IL-10, replicated the Tbet suppressive effects of LCM in both liver resident and peripheral blood NK cells. Furthermore, blocking TGF-β receptor signaling using the inhibitor SB431542, reversed the effect of LCM treatment on liver resident NK cells, causing the loss of tissue resident Eomeshi Tbetlo phenotype. Our findings identify liver-derived TGF-β as an important component of the liver microenvironment, which acts to regulate and maintain the phenotype of liver resident NK cells.


INTRODUCTION
Large populations of tissue resident natural killer (NK) cells have been identified in a diverse range of tissues including liver, lung, lymph node, bone marrow, and uterus (1). However, the tissuespecific factors regulating resident NK cell phenotype and function remain largely unknown. The adult human liver hosts a diverse repertoire of liver resident and transient NK cell populations with differing phenotypes and functions, whose roles in liver homeostasis and disease remain poorly defined.
Liver resident NK cells are enriched in the CD56 bright NK cell subpopulation and are defined by their unique expression of the transcription factors Eomes and Tbet, and a range of cell surface markers including TIGIT, CD69, CXCR6, and the absence of CD49e (Eomes hi Tbet lo Hobit + TIGIT + CD69 + CXCR6 + CD49e − ) (2)(3)(4)(5)(6). This liver resident phenotype is absent in peripheral blood NK cell populations. Expression of the chemokine receptor CXCR6 is thought to be particularly important for the retention of resident populations within the liver due to constitutive expression of the ligand (CXCL16) in the liver sinusoid.
Despite extensive characterization of the phenotype of liver NK cells, it remains unclear how factors within the liver microenvironment contribute to the induction and maintenance of this unique phenotype. Our group has previously described the wide range of cytokines and growth factors present in healthy adult liver which can maintain and propagate NK cells (7)(8)(9)(10). However, this work did not explore specific liver resident NK cell phenotypes. Previous research identified that liver resident NK cells produce significantly less IFN-γ compared with corresponding peripheral blood NK cell populations, and liver conditioned media (LCM) was sufficient to suppress IFN-γ in peripheral blood CD56 bright cells (2,3), suggesting that liver-derived soluble factors are capable of regulating NK cell populations.
In this study we demonstrate that LCM can maintain the liver resident NK cell phenotype ex vivo, as well as suppress Tbet and Eomes expression in peripheral NK cells (which mirrors the Tbet phenotype observed in liver resident NK cells). Furthermore, we show TGF-β, produced in the liver microenvironment, suppresses Tbet and Eomes expression in liver resident NK cells and peripheral blood NK cells. Understanding how the liver microenvironment induces and maintains liver specific immune cell populations may identify novel therapeutic targets capable of regulating local immunity and tissue repair.

Collection of Liver Perfusate During Orthotopic Liver Transplantation (OLT)
Samples were collected from donor livers (n = 10) during orthotopic liver transplantation at St. Vincent's University Hospital. During retrieval, the donor aorta and superior mesenteric vein were flushed with University of Wisconsin (UW) solution (Bristol-Myers Squibb, Uxbridge, UK) at the time of exsanguination. The liver was flushed again with UW solution after excision of the organ until all blood was removed and the perfusate appeared clear, at which time the liver was placed in a container with UW solution and packed on ice for transportation. Donor livers were transplanted within 12 h. At implantation, after completion of the upper inferior cava anastomosis, livers were flushed with normal saline through the portal vein to wash out the UW before reperfusion. This wash-out fluid was collected from the inferior vena cava; the UW transportation solution was also collected. A matched donor blood sample was taken at the time of organ retrieval. Peripheral blood was obtained from anonymised blood donors from the Irish Blood transfusion Board (IBTS

Isolation of Hepatic Mononuclear Cells From Liver Perfusate and Peripheral Blood
Hepatic mononuclear cells (HMNCs) were isolated from donor liver perfusates, as previously described (11), by filtration through 70 µm filters (BD Biosciences, Erembodegem, Belgium) followed by centrifugation at 1,200 rpm for 10 min. The supernatant was aspirated and the cells resuspended in RPMI 1640 medium, supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Gibco, Wicklow, Ireland). HMNCs were separated from this suspension by density gradient centrifugation using Ficoll-Paque TM PLUS (GE Healthcare, Uppsala, Sweden) and residual red blood cells were removed by adding red cell lysis solution (Sigma, Wicklow, Ireland). Matched peripheral blood mononuclear cells (PBMCs) were also isolated by density centrifugation.

Preparation of Liver Conditioned Media
Wedge biopsies, taken at the time of transplantation, were used to generate tissue conditioned media. Tissue samples were measured and weighed. Tissue was then cut into sections measuring approximately 0.5 cm × 0.5 cm × 0.5 cm. These were placed in a 24-well-culture plate and 500 µl of X-VIVO (Lonza Biologics, Slough, UK) media was added to each well and incubated for 72 h at 37 • C. Following incubation, the supernatant was centrifuged to remove cell debris and stored at −20 • C until use.

Isolation of NK Cells From Liver Perfusate and Peripheral Blood
CD3 − CD56 + NK cells were isolated from fresh liver perfusates and healthy donor peripheral blood by negative selection using NK cell isolation kits (130-092-657; Miltenyi Biotech, Teterow, Germany, and 480054; BioLegend, CA, USA), as per the manufacturers protocol. Briefly, mononuclear cells were labeled with a biotin conjugated antibody cocktail against lineage specific targets. Anti-biotin microbeads were then added, and the NK cells were separated using a magnetic cell sorting (MACS) LS column. Cells were then cultured in RPMI 1640 supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. In addition, NK cells were sorted into two populations CD56 bright CD16 +/− and CD56 dim CD16 ++ using a FACS Aria cell sorter (BD Biosciences).

Statistical Analysis
Statistical analysis was carried out using Prism GraphPad Version 5.0. For comparison of two unmatched groups, Mann Whitney U-test was used. Comparison of more than two unmatched groups was performed using Kruskal-Wallis test, with Dunn's multiple comparison test. For paired comparisons, Friedman test with Dunns multiple comparison tests was used for more than two groups, Wilcoxon signed rank test was used for comparison of two matched groups. Within an experiment, * p < 0.05, * * p < 0.01, and * * * p < 0.001, respectively.  Figure 1C). Liver CD56 bright cells also express CXCR6, which is almost undetectable on peripheral blood counterparts (LP: 52.1 ± 10.1%, PB: 3.1 ± 1.9%, p = 0.0223, Figure 1D). In contrast, CD56 dim NK cells from liver perfusate resemble their peripheral blood counterparts with no significant differences in Eomes, Tbet, or CXCR6 (Figures 1E-G).

Liver
In order to determine whether the phenotype of hepatic NK cells is permanently altered by residing within the liver microenvironment, NK cells were isolated from liver perfusate and cultured in RPMI supplemented with human AB serum, to replicate the peripheral blood microenvironment. It was necessary to supplement this culture with IL-15 (2 ng/ml) in order to maintain NK cell viability and expression of CD56 (12,13). NK cells were cultured for 7 days with the culture media replaced every 2-3 days.
In CD56 dim hepatic NK cells, Tbet remained stable during culture with the MFI ranging between MFI 2007 at day 0 and MFI 1550 at day 7 (p = 0.69, Figure 2E). Eomes expression increased between day 0 (MFI 1463 ± 516) and day 7 (MFI 3364 ± 1807, Figure 2F) but this did not reach statistical significance. CXCR6 expression in CD56 dim hepatic NK cells increased marginally, however this increase was not statistically significant and even at day 7 only represents a small minority of the total CD56 dim population (Day 0 2.5 ± 1.3% vs. day 7 6.6 ± 2.4%; p = 0.3, Figure 2G).

The Eomes hi Tbet lo Phenotype of Liver Resident NK Cells Can be Maintained ex vivo by Supplementing With Liver Conditioned Media
Hepatic CD56 bright NK cells have a unique pattern of Eomes and Tbet expression, which is lost upon long term culture. In order to determine the effect of the liver microenvironment on these transcription factors, we assessed Eomes and Tbet expression after ex vivo culture supplemented with LCM (n = 5). LCM significantly reduced the expression of Tbet in liver resident NK cells, in a dose dependent manner, to a level similar to that seen at day 0. At day 7, the MFI of Tbet in untreated CD56 bright NK cells was 724.3 ± 170.4 MFI, rising from 323.7 ± 34.3 MFI at day 0. This was reduced to 391.0 ± 29.8 MFI with 5% LCM and further reduced to 261.7 ± 11.9 with 10% LCM (p = 0.017, Figures 3A,B). Similarly, the increase in Eomes expression upon ex vivo culture of CD56 bright NK cells was significantly reduced by treatment with LCM in a dose dependent manner. By day 7, Eomes MFI in untreated NK cells had risen to 3,149 ± 389.9 from 1,343 ± 204.4 at day 0. Treatment with 5% LCM reduced the MFI to 1,774 ± 630.6, with a further reduction to 983.3 ± 243.1 MFI with 10% LCM (p = 0.0278, Figure 3C). At day 7, CXCR6 was expressed on 45.5 ± 6.4% of CD56 bright NK cells. This level increased to 58.0 ± 15.5% and 65.3 ± 10.8% with 5 and 10% LCM, respectively (p = 0.21, Figure 3D).

TGF-β, Which Is Present in the Liver Microenvironment, Is Capable of Suppressing Tbet ex vivo in Liver Resident and Blood NK Cells
We next attempted to identify the factor present in LCM responsible for maintaining this liver resident phenotype. IL-10 and TFG-β are two immunosuppressive cytokines which can suppress IFN-γ production in NK cells and T cells (14)(15)(16). Our group has previously reported the presence of several NK related cytokines, including the essential IL-15, activatory IL-12 and IL-18, and regulatory IL-10 and TGF-β in healthy liver tissue (9). Secreted TGF-β and IL-10 were detected in all LCM samples at concentrations of 270.3 ± 63.1 pg/ml and 124.3 ± 22.3 pg/ml, respectively ( Figure 5A). Next, we assessed the effect of IL-10 and TGF-β on the phenotype on hepatic NK cells in culture.

Inhibiting TGF-β Signaling Partially Reverses the Effect of LCM Treatment on Liver Resident and Blood NK Cells
We next inhibited TGF-β receptor signaling using the inhibitor SB431542, which inhibits the TGF-β type I receptor activin receptor-like kinase (ALK)5 and related genes ALK4 and ALK7. Liver CD56 bright NK cells were pre-treated with SB431542 for 2 h before being treated with LCM. As before, treatment with LCM significantly reduced the expression of Tbet compared to untreated cells (Figures 6A,B). Pre-treatment with SB431542 prevented the reduction in Tbet expression (LCM: MFI 356 ± 49.6, LCM+SB431542: MFI 552.8 ± 69.6, p = 0.0239, Figure 6B). The addition of SB431542 also significantly increased the expression of Eomes compared to LCM treated cells ( Figure 6C).
In order to confirm that this effect was specific to TGF-β receptor signaling we next treated magnetic bead-purified blood NK cells with LCM and 5 µg/ml of a TGF-β1 blocking antibody (clone 19D8, BioLegend) or isotype control (clone MOPC-21, BioLegend). Blocking TGF-β1 prevented the inhibition of Tbet in CD56 bright NK cells (Figures 6D,E). Blocking TGF-β1 also prevented the inhibition of Eomes by LCM although this did not reach statistical significance (Figure 6F).

DISCUSSION
In this study, we have shown that the liver microenvironment is essential for maintaining the unique Eomes hi Tbet lo phenotype of liver resident NK cells. NK cells isolated from liver perfusate lose their unique Eomes hi Tbet lo phenotype when cultured, but this can be reversed by the addition of LCM. The liver is rich in immunoregulatory cytokines, including TGF-β and IL-10, produced by Kupffer cells and immature dendritic cells (9,(17)(18)(19). Liver resident NK cells cultured with TGF-β, but not IL-10, maintain their Eomes hi Tbet lo phenotype, indicating that TGF-β is essential for maintaining liver resident populations. Blocking TGF-β signaling through pre-treatment with SB431542 reverses this effect and shifts the phenotype of liver resident NK cells to a peripheral blood-like state. Furthermore, Tbet expression in peripheral blood NK cells can be modulated by treatment with both LCM and TGF-β.
Culture with LCM can maintain CXCR6 expression, but it does not appear to induce expression of CXCR6 on peripheral blood NK cells. Cytokine stimulation (IL-12, IL-15) of peripheral blood NK cells can induce CXCR6 or CD49a expression, however the level of CXCR6 expression was highly variable and less than half of the stimulated NK cells showed expression, suggesting additional stimuli are required for sustained CXCR6 expression (20). NK cells may acquire CXCR6 expression in the periphery, through cytokine activation, and migrate to the liver. Here CXCL16, RANTES, and CCL3, present in the liver sinusoid, may retain activated NK cells (4,21) and possibly sustain CXCR6 expression. Following recruitment to the liver sinusoid, TGF-β (produced by resident macrophages and dendritic cells) may alter transcription factor expression and repress the production of pro-inflammatory cytokines (IFN-γ).
Significant work has been performed investigating the effect of TGF-β on NK cells. TGF-β has been shown to have potent immunosuppressive effects and alters NK cell development (15,22). Furthermore, TGF-β has been shown to suppress glycolysis and inhibit the pro-inflammatory effector functions of CD56 bright NK cells, such as IFN-γ production (23). The mechanism by which TGF-β inhibits Tbet and IFN-γ has previously been elucidated in T cells, where TGF-β signaling induces the expression of SHP-1 which in turn inhibits STAT1/4 and Tbet expression (24,25). Recently, TGF-β has been shown to be involved in the conversion of NK cells to ILC1-like cells, via a JNK-dependent, Smad4-independent pathway resulting in reduced cytotoxicity in murine tumor models (26,27). Why this process does not appear to occur in liver resident NK cells warrants further investigation. Intriguingly, Smad4 appears to suppress the JNK-dependent non-canonical TGFβ signaling associated with differentiation into ILC1-like cells within tissues, indicating that canonical TGF-β signals may be required to maintain NK cell phenotype. It is important to note there is a marked differences in the phenotype and function of liver resident NK cells between humans and mice (28). Furthermore, single cell RNA-seq analysis of human and murine NK cells have highlighted similarities but also significant differences in peripheral blood and splenic samples (29). Therefore, TGF-β may have divergent functions between species and care must be taken when comparing murine and human data.
In this study, we identified TGF-β as a factor required for the induction and maintenance of the tissue resident phenotype of liver resident NK cells in humans. Liver resident NK cells are unique in that they can provide immunosurveillance without producing large quantities of IFN-γ with its potential to drive tissue damaging pathology. In this context, liver resident NK cells can remove dysplastic or virally infected cells without perturbing the tolerogenic milieu of the liver, which so often leads to chronic inflammatory conditions. Further evidence for the importance of TGF-β in the establishment and maintenance of tissue resident NK cell populations in humans comes from analysis of uterine NK cells. Uterine NK cells share many phenotypic characteristics with liver resident NK cells (CD56 bright Eomes hi CD69 + ) (30). The local uterine microenvironment is rich in TGF-β and treatment of blood NK cells with either endometrial stromal cell supernatants or TGF-β alone can induce phenotypic changes resembling decidual NK cells (31).
Our results suggest that disruption of TGF-β levels in the liver microenvironment in disease could lead to a loss of local immune regulation and the promotion of tissue inflammation and damage. While the roles of liver resident NK cells in maintaining homeostasis or mediating liver disease have yet to be established in humans, a reduction in TGF-β levels during liver disease could drive resident NK cell populations to a more conventional pro-inflammatory phenotype and contribute to chronic inflammation. In this situation, restoring homeostatic cytokine levels would be essential to regulate local immunity and tissue repair in the liver. Interestingly, the use of a strong pro-inflammatory signal, such as PMA, appears to overcome this TGF-β mediated repression and restore IFN-γ production (2,5). This appears similar to the mechanism by which TGF-β imprinted peripheral blood NK cells become hyper-producers of IFN-γ in vitro (32). While in healthy liver these resident NK cells have suppressed pro-inflammatory function, it appears this can be overcome with sufficient stimulation.
The liver is a naturally tolerogenic environment, which maintains unique anti-inflammatory status even in the presence of immune activating dietary antigens and bacterial components. It is therefore no surprise to find that liver resident NK cells are profoundly changed by residing in this microenvironment. TGF-β is one of the chief mediators of the tolerogenic hepatic environment and we have shown here that this regulation extends to the phenotype and function of NK cells. Through the suppression of Tbet, liver resident NK cells have reduced pro-inflammatory potential but maintain their ability to perform immunosurveillance in an organ prone to infection and malignancy.

ETHICS STATEMENT
All protocols were approved by St. Vincent's University Hospital Ethics Committee and the Trinity College Dublin School of Medicine Research Ethics Committee, in accordance with the ethical guidelines of the 1975 Declaration of Helsinki. Patients were consented pre-operatively by DH, EH, or JG. All patients were supplied with a information packet informing them of the project aims, their rights to withdraw at any time and the length of time samples and data would be retained. All patients were over the age of 18 and had no conditions which would preclude them from giving informed consent.

AUTHOR CONTRIBUTIONS
CO, MR, and CH contributed to the conception and design of the study. DH, EH, and JG acquired data and managed clinical samples. CH, GJ, and DA designed and carried out experimental procedures. Statistical analysis was performed by CH, GJ, and MR. CO, MR, CH, DH, EH, and JG were involved in interpretation of data and manuscript preparation. Manuscript drafting was performed by CH, MR, and CO. All authors reviewed and approved the final manuscript.