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

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

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

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

Effects of cytokine stimulation on CD98 expression: Samples were compared using Wilcoxon matched-pairs signed rank tests and multiplicity was controlled for by FDR testing.

<p>Bars indicate the median, significance was defined as p≤0.05 (*). A. Representative histograms of CD98 expression on unstimulated and stimulated CD56^bright CXCR6⁺ (I, grey: unstimulated; purple: stimulated), CD56^dim CXCR6⁺ (II, grey: unstimulated; teal: stimulated), CD56^bright CXCR6^- (III, grey: unstimulated; purple: stimulated) and CD56^dim CXCR6^- (IV, grey: unstimulated; teal: stimulated) NK cells from blood (left), liver (middle) and spleen (right) samples. B. Expression (MdFI) of CD98 on unstimulated (grey) and CD56^brightCD16^- (purple) and CD56^dimCD16⁺ (teal) tissue-resident (TR), tissue-derived (TD) and peripheral blood (PB) NK cells from paired liver-blood (left diagram, n = 12) and spleen-blood (right diagram, n = 11) samples.</p

NK cell phenotype and baseline nutrient receptor expression: Samples were compared using Wilcoxon matched-pairs signed rank tests and multiplicity was controlled for by FDR testing.

<p>Bars indicate the median, significance was defined as p≤0.05 (*). A. viSNE representation of peripheral blood- (PBMC, top row), liver- (middle row) and spleen (bottom row) derived NK cells and their expression of CD56, CD16, CXCR6, CD57 and CD127. B. Expression (Median fluorescence intensity, MdFI) of Glut1 on CD56^brightCD16^- (purple) and CD56^dimCD16⁺ (teal) tissue-resident (TR), tissue-derived (TD) and peripheral blood (PB) NK cells from paired liver-blood (left diagram, n = 12) and spleen-blood (right diagram, n = 11) samples. C. Expression (MdFI) of CD98 on CD56^brightCD16^- (purple) and CD56^dimCD16⁺ (teal) tissue-resident (TR), tissue-derived (TD) and peripheral blood (PB) NK cells from paired liver-blood (left diagram, n = 12) and spleen-blood (right diagram, n = 11) samples. D. Expression (MdFI) of CD71 on CD56^brightCD16^- (purple) and CD56^dimCD16⁺ (teal) tissue-resident (TR), tissue-derived (TD) and peripheral blood (PB) NK cells from paired liver-blood (left diagram, n = 12) and spleen-blood (right diagram, n = 11) samples.</p

Relevant clinical data of the spleen cohort.

<p>Relevant clinical data of the spleen cohort.</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