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

Unsupervised analysis of intrahepatic CD49a+ and CD49a- NK cells.

<p><b>(A)</b> Gated CD49a+ and <b>(B)</b> CD49a- NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed.</p

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

<div><p>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 <i>in vitro</i> 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.</p></div

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

Immune phenotyping of combined peripheral and intrahepatic NK cells.

<p>Gated NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. <b>(A)</b> peripheral and <b>(B)</b> intrahepatic NK cells are shown. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed. <b>(C)</b> Proportion of NK cells derived from the liver (ihNK) and the peripheral blood (pNK) on the liver transplantation cohort expressing CD49a (pNK median (IQR): 0.9 (0.3–3.9); ihNK median (IQR): 34.4 (27.6–40.5); p<0.0001), CD34 (pNK median (IQR): 2.2 (1–4.7); ihNK median (IQR): 12 (6.8–20.9); p<0.0001), CXCR4 (pNK median (IQR): 9.8 (4.9–22.2); ihNK median (IQR): 3.4 (1.3–7.7); p = 0.0024), CD57 (pNK median (IQR): 19 (22–38.5); ihNK median (IQR): 13.7 (9.4–23.3); p<0.0001) and DNAM-1 (pNK median (IQR): 79.6 (51.5–85.6); ihNK median (IQR): 26.5 (8.5–32.1); p<0.0001) (n = 19). <b>(D)</b> Proportion of NK cells from the tumor-free liver resections expressing CD49a, CD34, CD57, DNAM-1, CXCR3 and CXCR4 within the IHLs NK cells and pNK cells (n = 5). <b>(E)</b> Frequency of CD49a+ NK cell population within the IHLs NK cells in tumor-free liver resection cohort (HLR) and the liver retransplant cohort (cirrhotic livers, CL). Data is depicted as scatter plot, with each dot corresponding to a participant. Bars indicate median and IQR. Wilcoxon signed rank tests with adjustment of p-values by false discovery rate.</p

Unsupervised analysis of intrahepatic CD49a+ and CD49a- NK cells.

<p><b>(A)</b> Gated CD49a+ and <b>(B)</b> CD49a- NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. Color coding indicates the expression intensity of the surface marker, pink being higher expressed and green being lower expressed.</p

Demographics and clinical characteristics from explanted liver tissue samples.

<p>Demographics and clinical characteristics from explanted liver tissue samples.</p

Immunophenotyping of intrahepatic CD49a+ and CD49a- NK cells.

<p>CD49a expression on bulk, CD56^dim and CD56^bright NK cells in the liver transplantation cohort <b>(A)</b> and in the tumor-free liver resection cohort <b>(B)</b>. Proportion of cells expressing specific markers in ihNK cells once gated on CD49a+ and CD49a- NK cells in the liver transplantation cohort <b>(C)</b> with CD25+ (CD49a+ NK cell median (IQR): 14.7 (7.1–22.7); CD49a- NK cell median (IQR): 2.5 (1.6–3.8); p<0.0001), CD34+ (CD49a+NK cell median (IQR): 17.4 (10–24.1); CD49a- NK cell median (IQR): 6.8 (4.2–16.3); p = 0.0107) and CXCR3+ (CD49a+ NK cell median (IQR): 14.8 (8.1–19.4); CD49a- NK cell median (IQR): 4.5 (2.2–10); p = 0.0002). <b>(D)</b> Proportion of cells expressing specific markers in ihNK cells once gated on CD49a+ and CD49a- in tumor-free liver resections. All data is depicted as scatter plot, with each dot corresponding to a participant. Bars indicate median and IQR. Wilcoxon signed rank tests with adjustment of p-values by false discovery rate.</p

Relevance of CD49a+CD25+ ihNK cells.

<p><b>(A)</b> Boolean gating of CD49a, CD25 and CD34 markers on ihNK cells. <b>(B)</b> Pie chart representing the frequency of each of the 7 possible combinations of the 3 markers, CD49a, CD25 and CD34. The median percentage of each population is represented. <b>(C)</b> Alanine Aminotransferase (ALT) serum levels correlation with the proportion of intrahepatic CD49a+CD25+ NK cells in the liver transplantation cohort. Data in <b>(A)</b> is depicted as scatter plot showing all individuals, the bar represents the median and the deviation is depicted as interquartile range.</p

Functional assessment of liver-resident CD49a+ CD25 ihNK cells.

<p><b>(A)</b> Representative histograms 7 days post-cell tracing with CFSE on 5 livers, LTXHH24, LTXHH26, LTXHH29, LTXHH30 and LTXHH32 (gated on live cells). Intrahepatic NK cells were sorted using a BD FACSARIA FUSION according to their expression levels of CD49a and CD25. Recovered cells were CFSE-cell stained and cultured for 7 days with low amounts of IL-2 (red: CD49a+CD25+, blue: CD49a+CD25-, orange: CD49a-CD25+, green: CD49a-CD25-). <b>(B)</b> Summary of the cell proliferation data from the four sorted populations. Percentage of CFSE+ cells was determined by setting the gate on the lower part of the CD49a-CD25- population (in green) as shown by the dashed lines. Data in <b>(B)</b> is depicted as scatter plot showing all individuals, the bar represents the median and the deviation is depicted as interquartile range.</p