HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. DESIGN: Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. RESULTS: NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. CONCLUSION: Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Single-cell atlas of hepatic T cells reveals expansion of liver-resident naive-like CD4+ T cells in primary sclerosing cholangitis.

BACKGROUND & AIMS: Little is known about the composition of intrahepatic immune cells and their contribution to the pathogenesis of primary sclerosing cholangitis (PSC). Herein, we aimed to create an atlas of intrahepatic T cells and thereby perform an in-depth characterization of T cells in inflamed human liver. METHODS: Different single-cell RNA sequencing methods were combined with in silico analyses on intrahepatic and peripheral T cells from patients with PSC (n = 11) and healthy donors (HDs, n = 4). Multi-parameter flow cytometry and functional in vitro experiments were conducted on samples from patients with PSC (n = 24), controls with other liver diseases and HDs. RESULTS: We identified a population of intrahepatic naive-like CD4+ T cells, which was present in all liver diseases tested, but particularly expanded in PSC. This population had a transcriptome and T cell receptor repertoire similar to circulating naive T cells but expressed a set of genes associated with tissue residency. Their periductal location supported the concept of tissue-resident naive-like T cells in livers of patients with PSC. Trajectory inference suggested that these cells had the developmental propensity to acquire a T helper 17 (TH17) polarization state. Functional and chromatin accessibility experiments revealed that circulating naive T cells in patients with PSC were predisposed to polarize towards TH17 cells. CONCLUSION: We report the first atlas of intrahepatic T cells in PSC, which led to the identification of a previously unrecognized population of tissue-resident naive-like T cells in the inflamed human liver and to the finding that naive CD4+ T cells in PSC harbour the propensity to develop into TH17 cells. LAY SUMMARY: The composition of intrahepatic immune cells in primary sclerosing cholangitis (PSC) and their contribution to disease pathogenesis is widely unknown. We analysed intrahepatic T cells and identified a previously uncharacterized population of liver-resident CD4+ T cells which are expanded in the livers of patients with PSC compared to healthy liver tissue and other liver diseases. These cells are likely to contribute to the pathogenesis of PSC and could be targeted in novel therapeutic approaches.

Failure of thymic deletion and instability of autoreactive Tregs drive autoimmunity in immune-privileged liver.

The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II-restricted model peptide in hepatocytes by autoreactive CD4+ T cells. We found that the hepatic peptide was not expressed in the thymus, leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4+ T cells. In the liver, autoreactive CD4+ effector T cells accumulated within portal ectopic lymphoid structures and maturated toward pathogenic IFN-γ and TNF coproducing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs but not of nonspecific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation, and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared with that of nonspecific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.

IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Monocytes as Potential Mediators of Pathogen-Induced T-Helper 17 Differentiation in Patients With Primary Sclerosing Cholangitis (PSC).

BACKGROUND AND AIMS: T cells from patients with primary sclerosing cholangitis (PSC) show a prominent interleukin (IL)-17 response upon stimulation with bacteria or fungi, yet the reasons for this dominant T-helper 17 (Th17) response in PSC are not clear. Here, we analyzed the potential role of monocytes in microbial recognition and in skewing the T-cell response toward Th17. APPROACH AND RESULTS: Monocytes and T cells from blood and livers of PSC patients and controls were analyzed ex vivo and in vitro using transwell experiments with cholangiocytes. Cytokine production was measured using flow cytometry, enzyme-linked immunosorbent assay, RNA in situ hybridization, and quantitative real-time PCR. Genetic polymorphisms were obtained from ImmunoChip analysis. Following ex vivo stimulation with phorbol myristate acetate/ionomycin, PSC patients showed significantly increased numbers of IL-17A-producing peripheral blood CD4+ T cells compared to PBC patients and healthy controls, indicating increased Th17 differentiation in vivo. Upon stimulation with microbes, monocytes from PSC patients produced significantly more IL-1ß and IL-6, cytokines known to drive Th17 cell differentiation. Moreover, microbe-activated monocytes induced the secretion of Th17 and monocyte-recruiting chemokines chemokine (C-C motif) ligand (CCL)-20 and CCL-2 in human primary cholangiocytes. In livers of patients with PSC cirrhosis, CD14hiCD16int and CD14loCD16hi monocytes/macrophages were increased compared to alcoholic cirrhosis, and monocytes were found to be located around bile ducts. CONCLUSIONS: PSC patients show increased Th17 differentiation already in vivo. Microbe-stimulated monocytes drive Th17 differentiation in vitro and induce cholangiocytes to produce chemokines mediating recruitment of Th17 cells and more monocytes into portal tracts. Taken together, these results point to a pathogenic role of monocytes in patients with PSC.

The Transcription Factor Promyelocytic Leukemia Zinc Finger Protein Is Associated With Expression of Liver-Homing Receptors on Human Blood CD56bright Natural Killer Cells.

The transcription factor promyelocytic leukemia zinc finger protein (PLZF) is involved in the development of natural killer (NK) cells and innate lymphoid cells, including liver-resident NK cells in mice. In human NK cells, the role of PLZF in liver residency is still unknown. Expression of PLZF in matched human peripheral blood- and liver-derived NK cells and the association of PLZF expression with surface molecules and transcription factors relevant for tissue residency were investigated using multiparameter flow cytometry and assessing single-cell messenger RNA (mRNA) levels. Intrahepatic cluster of differentiation (CD)56bright NK cells expressed significantly higher levels of PLZF than peripheral blood CD56bright NK cells, which were predominantly PLZFlo. Expression of PLZF was highest within C-X-C motif chemokine receptor 6 (CXCR6)+CD69+ liver-resident NK cells among intrahepatic CD56bright NK cell populations. Association of PLZF with liver-residency markers was also reflected at mRNA levels. A small PLZFhiCD56bright NK cell population was identified in peripheral blood that also expressed the liver-residency markers CXCR6 and CD69 and shared functional characteristics with liver-resident NK cells. Conclusion: PLZF is implicated as part of a transcriptional network that promotes liver residency of human NK cells. Expression of liver-homing markers on peripheral blood PLZFhiCD56bright NK cells identifies an intermediate population potentially contributing to the maintenance of liver-resident NK cells.

CD49a Expression Identifies a Subset of Intrahepatic Macrophages in Humans.

Macrophages play central roles in inflammatory reactions and initiation of immune responses during infections. More than 80% of total tissue macrophages are described to be located in the liver as liver-resident macrophages, also named Kupffer cells (KCs). While studies in mice have established a central role of liver-resident KCs in regulating liver inflammation, their phenotype and function are not well-characterized in humans. Comparing paired human liver and peripheral blood samples, we observed significant differences in the distribution of macrophage (Mφ) subsets, with lower frequencies of CD14hiCD16lo and higher frequencies of CD14int-hiCD16int Mφ in human livers. Intrahepatic Mφ consisted of diverse subsets with differential expression of CD49a, a liver-residency marker previously described for human and mice NK cells, and VSIG4 and/or MARCO, two recently described human tissue Mφ markers. Furthermore, intrahepatic CD49a+ Mφ expressed significantly higher levels of maturation and activation markers, exhibited higher baseline levels of TNF-α, IL-12, and IL-10 production, but responded less to additional in vitro TLR stimulation. In contrast, intrahepatic CD49a- Mφ were highly responsive to stimulation with TLR ligands, similar to what was observed for CD49a- monocytes (MOs) in peripheral blood. Taken together, these studies identified populations of CD49a+, VSIG4+, and/or MARCO+ Mφ in human livers, and demonstrated that intrahepatic CD49a+ Mφ differed in phenotype and function from intrahepatic CD49a- Mφ as well as from peripheral blood-derived monocytes.

Interferon-γ-dependent immune responses contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an idiopathic, chronic cholestatic liver disorder characterized by biliary inflammation and fibrosis. Increased numbers of intrahepatic interferon-γ- (IFNγ) producing lymphocytes have been documented in patients with PSC, yet their functional role remains to be determined. METHODS: Liver tissue samples were collected from patients with PSC. The contribution of lymphocytes to liver pathology was assessed in Mdr2-/- x Rag1-/- mice, which lack T and B cells, and following depletion of CD90.2+ or natural killer (NK)p46+ cells in Mdr2-/- mice. Liver pathology was also determined in Mdr2-/- x Ifng-/- mice and following anti-IFNγ antibody treatment of Mdr2-/- mice. Immune cell composition was analysed by multi-colour flow cytometry. Liver injury and fibrosis were determined by standard assays. RESULTS: Patients with PSC showed increased IFNγ serum levels and elevated numbers of hepatic CD56bright NK cells. In Mdr2-/- mice, hepatic CD8+ T cells and NK cells were the primary source of IFNγ. Depletion of CD90.2+ cells reduced hepatic Ifng expression, NK cell cytotoxicity and liver injury similar to Mdr2-/- x Rag1-/- mice. Depletion of NK cells resulted in reduced CD8+ T cell cytotoxicity and liver fibrosis. The complete absence of IFNγ in Mdr2-/-x Ifng-/- mice reduced NK cell and CD8+ T cell frequencies expressing the cytotoxic effector molecules granzyme B and TRAIL and prevented liver fibrosis. The antifibrotic effect of IFNγ was also observed upon antibody-dependent neutralisation in Mdr2-/- mice. CONCLUSION: IFNγ changed the phenotype of hepatic CD8+ T cells and NK cells towards increased cytotoxicity and its absence attenuated liver fibrosis in chronic sclerosing cholangitis. Therefore, unravelling the immunopathogenesis of PSC with a particular focus on IFNγ might help to develop novel treatment options. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by biliary inflammation and fibrosis, whose current medical treatment is hardly effective. We observed an increased interferon (IFN)-γ response in patients with PSC and in a mouse model of sclerosing cholangitis. IFNγ changed the phenotype of hepatic CD8+ T lymphocytes and NK cells towards increased cytotoxicity, and its absence decreased liver cell death, reduced frequencies of inflammatory macrophages in the liver and attenuated liver fibrosis. Therefore, IFNγ-dependent immune responses may disclose checkpoints for future therapeutic intervention strategies in sclerosing cholangitis.

CCL21-expression and accumulation of CCR7+ NK cells in livers of patients with primary sclerosing cholangitis.

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7-ligand CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7+ NK cells in the inflamed liver tissue.

Human liver-derived CXCR6+ NK cells are predominantly educated through NKG2A and show reduced cytokine production.

NK cells have been implicated to affect the outcome of numerous liver diseases. In particular, members of the killer-cell Ig-like receptor (KIR) family, predominantly expressed by NK cells, have been associated with the outcome of hepatitis C virus infection and clearance of hepatocellular carcinoma. Inhibitory KIRs tune NK cell function through interaction with HLA class I, a process termed education. Nevertheless, the impact of the hepatic environment on NK cell education is incompletely understood. Therefore, we investigated the composition and function of hepatic KIR-expressing NK cells. Matched PBMC and hepatic lymphocytes were isolated from 20 individuals undergoing liver surgery and subsequently phenotypically analyzed for expression of KIRs and markers for tissue residency using flow cytometry. NK cell function was determined by co-culturing NK cells with the target cell line 721.221 and subsequent assessment of CD107a, IFN-γ, and TNF-α expression. Liver-resident CXCR6+ /CD56Bright NK cells lacked KIRs and were predominantly educated through NKG2A, while CXCR6- /CD16+ NK cells expressed KIRs and resembled peripheral blood NK cells. Hepatic NK cells showed lower response rates compared to peripheral blood NK cells; in particular, CXCR6+ NK cells were hyporesponsive to stimulation with target cells. The high proportion of educated NK cells in both subsets indicates the importance of self-inhibitory receptors for the balance between maintenance of self-tolerance and functional readiness. However, the reduced functionality of hepatic NK cells may reflect the impact of the tolerogenic hepatic environment on NK cells irrespective of NK cell education.

Interactions Between KIR3DS1 and HLA-F Activate Natural Killer Cells to Control HCV Replication in Cell Culture.

Killer-cell immunoglobulin-like receptors (KIRs) are transmembrane glycoproteins expressed by natural killer (NK) cells. Binding of KIR3DS1 to its recently discovered ligand, HLA-F, activates NK cells and has been associated with resolution of hepatitis C virus (HCV) infection. We investigated the mechanisms by which KIR3DS1 contributes to the antiviral immune response. Using cell culture systems, mice with humanized livers, and primary liver tissue from HCV-infected individuals, we found that the KIR3DS1 ligand HLA-F is up-regulated on HCV-infected cells, and that interactions between KIR3DS1 and HLA-F contribute to NK cell-mediated control of HCV. Strategies to promote interaction between KIR3DS1 and HLA-F might be developed for treatment of infectious diseases and cancer.

Tissue-resident NK cells differ in their expression profile of the nutrient transporters Glut1, CD98 and CD71.

Metabolism is a critical basis for immune cell functionality. It was recently shown that NK cell subsets from peripheral blood modulate their expression of nutrient receptors following cytokine stimulation, demonstrating that NK cells can adjust to changes in metabolic requirements. As nutrient availability in blood and tissues can significantly differ, we examined NK cells isolated from paired blood-liver and blood-spleen samples and compared expression of the nutrient transporters Glut1, CD98 and CD71. CD56bright tissue-resident (CXCR6+) NK cells derived from livers and spleens expressed lower levels of Glut1 but higher levels of the amino acid transporter CD98 following stimulation than CD56bright NK cells from peripheral blood. In line with that, CD56dim NK cells, which constitute the main NK cell population in the peripheral blood, expressed higher levels of Glut1 and lower levels of CD98 and CD71 compared to liver CD56bright NK cells. Our results show that NK cells from peripheral blood differ from liver- and spleen-resident NK cells in the expression profile of nutrient transporters, consistent with a cell-adaptation to the different nutritional environment in these compartments.

Mechanistic peculiarities of activation-induced mobilization of cytotoxic effector proteins in human T cells.

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might 'sense' which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.

Proliferative capacity exhibited by human liver-resident CD49a+CD25+ NK cells.

The recruitment and retention of Natural Killer (NK) cells in the liver are thought to play an important role during hepatotropic infections and liver cirrhosis. The aims of this study were to determine differences between liver-derived and peripheral blood-derived NK cells in the context of liver inflammation and cirrhosis. We conducted a prospective dual-center cross-sectional study in patients undergoing liver transplantation or tumor-free liver resections, in which both liver tissue and peripheral blood samples were obtained from each consenting study participants. Intrahepatic lymphocytes and PBMCs were stained, fixed and analyzed by flow cytometry. Our results showed that, within cirrhotic liver samples, intrahepatic NK cells were particularly enriched for CD49a+ NK cells when compared to tumor-free liver resection samples. CD49a+ liver-derived NK cells included populations of cells expressing CD25, CD34 and CXCR3. Moreover, CD49a+CD25+ liver-derived NK cells exhibited high proliferative capacity in vitro in response to low doses of IL-2. Our study identified a specific subset of CD49a+CD25+ NK cells in cirrhotic livers bearing functional features of proliferation.