Isolation, phenotyping, and functional analysis of lymphocytes from human liver

Abstract

The isolation of mononuclear cells from human liver tissues was achieved by a simple method consisting of enzymatic and mechanical dissociation, density gradient centrifugation, and adherence to plastic. The method was optimized to obtain a nearly complete recovery of different lymphoid subpopulations. The mononuclear cells recovered from “normal” liver tissues consisted of 33 ± 9% (mean ± SD) small lymphocytes, 44 ± 6% large granular lymphocytes, 9 ± 2% monocytes/macrophages, 9 ± 3% granulocytes, and 5 ± 2% other cells as determined by microscopic analysis of May-Grünwald-Giemsa-stained cytocentrifuge smears. Phenotypic analysis of the liver-infiltrating lymphocytes (LIL) by two-color flow cytometry showed that CD3 −CD56 + NK cells represented 43 ± 6% (mean ± SD), CD3 + CD56 − T cells, 30 ± 9%, and CD19 + B cells 3 ± 1%. The predominant phenotype of the liver-infiltrating NK cells was CD3 −CD56 +CD16 − in contrast to that of circulating NK cells, which are largely CD3 −CD56 +CD16 +. The α β TCR + T cells were 42 ± 14% and γ δ T cells were infrequent (4 ± 4%). The proportions of the three major lymphoid populations (T, NK, and B cells) recovered from liver tissues of 10 patients with different liver diseases were altered. Tissue sections from the same specimens were stained by the immunoperoxidase method to compare the in situ cellular composition with that determined by flow cytometry. LIL recovered from normal (control) and virally infected (non-A, non-B hepatitis) liver tissues had high NK activity (up to 1000 LU 10 7 cells ) as measured against K-562 targets in 4-hr 51Cr-release assays. NK activity was significantly lower ( P < 0.05) in LIL recovered from other liver diseases. LIL had spontaneous cytotoxicity against NK-resistant Daudi targets which was significantly higher ( P < 0.05) in control and virally infected than in other liver tissues. Our results indicated that human LIL recovered from normal and diseased liver tissues contained a high proportion of functionally active NK cells and that NK and lymphokine-activated killer activities but not the percentages of CD56 + cells were decreased in end-stage primary biliary cirrhosis and primary sclerosing cholangitis. In contrast, the proportions of NK cells and NK activity remain high in non-A, non-B hepatitis.