Eomeshi NK Cells in Human Liver Are Long-Lived and Do Not Recirculate but Can Be Replenished from the Circulation

Abstract

Human liver contains an Eomes^hi population of NK cells that is not present in the blood. In this study, we show that these cells are characterized by a molecular signature that mediates their retention in the liver. By examining liver transplants where donors and recipients are HLA mismatched, we distinguish between donor liver-derived and recipient-derived leukocytes to show that Eomes^lo NK cells circulate freely whereas Eomes^hi NK cells are unable to leave the liver. Furthermore, Eomes^hi NK cells are retained in the liver for up to 13 y. Therefore, Eomes^hi NK cells are long-lived liver-resident cells. We go on to show that Eomes^hi NK cells can be recruited from the circulation during adult life and that circulating Eomes^lo NK cells are able to upregulate Eomes and molecules mediating liver retention under cytokine conditions similar to those in the liver. This suggests that circulating NK cells are a precursor of their liver-resident counterparts.

FIGURE 1.

Eomes^hi NK cells are present in human liver and have a distinct phenotype. (A) PBLs were isolated from healthy volunteers and NK cells identified by gating on live cells, scatter, CD3⁻, and CD56⁺. NK cells were examined for their expression of Eomes and T-bet. (B) Leukocytes were isolated from the perfusion fluid of healthy human livers destined for transplantation and stained as in (A). (C) Summary data showing Eomes^hi NK cells as a percentage of total NK cells across 11 samples. Significance was determined using a Mann–Whitney U test. (D) RNASeq data from Eomes^lo and Eomes^hi NK cells sorted from the perfusion fluid from n = 5 healthy livers. Differentially expressed genes were identified using paired t tests. The top 10 most differentially expressed genes and the top most significantly enriched canonical pathways are shown. (E) Flow cytometry for key proteins expected to differ between Eomes^lo (blue dashed line) and Eomes^hi (red solid line) liver NK cells. Histograms are representative of n = 4 independent samples. (F) Overlap between top upregulated genes in Eomes^lo and Eomes^hi liver NK cells, compared with tonsil NK cells and ILC1 (23).

FIGURE 2.

Eomes^lo NK cells exit the liver but Eomes^hi NK cells do not. (A and B) Example of staining from a single transplant in which an HLA-A3–negative liver (A) was transplanted into an HLA-A3–positive recipient. Twenty-four hours after transplant, PBLs were isolated from the recipient’s blood and circulating liver-derived cells were distinguished from recipient-derived cells on the basis of HLA-A3 staining (B). NK cells were examined for their expression of Eomes and T-bet. (C) Summary data from n = 7 transplants mismatched at either HLA-A2 or HLA-A3. Significance was determined using a paired t test. (D and E) Example staining from a single transplant in which an HLA-A2–positive liver (D) was transplanted into an HLA-A2–negative recipient. Three hours posttransplant, a second biopsy was taken from the liver and liver-derived cells were distinguished from recipient-derived cells on the basis of HLA-A2 staining (E). (F) Summary data from n = 3 transplants, all mismatched at HLA-A2. The gates used to produce the summary data (C and F) are highlighted.

FIGURE 3.

Eomes^hi NK cells are long-lived in the liver, but they can be recruited from the circulation during adult life. (A) An HLA-A2–positive liver was transplanted into an HLA-A2–negative recipient and removed 8 d later. Liver leukocytes were isolated from a biopsy and liver-derived cells were distinguished from recipient-derived cells on the basis of HLA-A2 staining. Liver- and recipient-derived NK cells present in the liver were examined for their expression of Eomes and T-bet. (B) An HLA-A2–positive liver was transplanted into an HLA-A2–negative recipient and removed 3 y later. (C) An HLA-A2–positive liver was transplanted into an HLA-A2–negative recipient and removed 6 y later. (D) An HLA-A3–positive liver was transplanted into an HLA-A3–negative recipient and removed 6 y later. (E) An HLA-A2–positive liver was transplanted into an HLA-A2–negative recipient and removed 13 y later.

FIGURE 4.

Eomes^lo NK cells can become Eomes^hi. (A and B) NK cells were sorted from perfusion fluid [(A), Eomes^lo; (B), Eomes^hi)] and cultured for 7 d in the indicated conditions. At the end of the culture period, the cells were examined for their expression of Eomes and T-bet. (C) Sorted blood NK cells were cultured as above. (D–F) Summary data showing the percentage of Eomes^hi NK cells in n = 4 independent experiments, starting with Eomes^lo liver NK cells (D), Eomes^hi liver NK cells (E), or Eomes^lo peripheral blood NK cells (F). Groups that are significantly different (p < 0.05 by one-way ANOVA) are indicated by different letters.

FIGURE 5.

Eomes upregulation is associated with increased expression of mediators of tissue retention. (A) Sorted blood NK cells were cultured for 7 d in IL-15 and TGF-β to produce in vitro–derived Eomes^hi NK cells. Eomes^lo NK cell expression of the indicated proteins before culture (gray dashed line) and Eomes^hi NK cell expression after culture (black solid line) is shown. (B) NKL cells were transduced with Eomes or vector control and gated on transduced (dsRed⁺) cells. Expression of the indicated proteins in vector-transduced cells (gray dashed line) and Eomes-transduced cells (black solid line) is shown. The histograms show a single experiment, representative of n = 4.