Table 1.
Demographics and clinical characteristics from explanted liver tissue samples.
Table 2.
Demographics and clinical characteristics from liver resection samples.
Fig 1.
Immune phenotyping of combined peripheral and intrahepatic NK cells.
Gated NK cells from 19 donors were concatenated and represented in t-SNE maps for the expression of chemokine receptors, activation and residency markers. (A) peripheral and (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. (C) 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). (D) 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). (E) 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.
Fig 2.
Unsupervised analysis of intrahepatic CD49a+ and CD49a- NK cells.
(A) Gated CD49a+ and (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.
Fig 3.
Immunophenotyping of intrahepatic CD49a+ and CD49a- NK cells.
CD49a expression on bulk, CD56dim and CD56bright NK cells in the liver transplantation cohort (A) and in the tumor-free liver resection cohort (B). Proportion of cells expressing specific markers in ihNK cells once gated on CD49a+ and CD49a- NK cells in the liver transplantation cohort (C) 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). (D) 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.
Fig 4.
Relevance of CD49a+CD25+ ihNK cells.
(A) Boolean gating of CD49a, CD25 and CD34 markers on ihNK cells. (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. (C) Alanine Aminotransferase (ALT) serum levels correlation with the proportion of intrahepatic CD49a+CD25+ NK cells in the liver transplantation cohort. Data in (A) is depicted as scatter plot showing all individuals, the bar represents the median and the deviation is depicted as interquartile range.
Fig 5.
Functional assessment of liver-resident CD49a+ CD25 ihNK cells.
(A) 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) 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) is depicted as scatter plot showing all individuals, the bar represents the median and the deviation is depicted as interquartile range.