Liver-resident memory CD8 T cells are present in the human liver but not in blood. (a) Frequencies of CD8 T cell differentiation/memory subsets (CD27⁺CD45RA⁺; CD27⁺CD45RA⁻; CD27⁻CD45RA⁻; and CD27⁻CD45RA⁺) in peripheral blood (n = 40 healthy controls) and intrahepatic samples (n = 11 healthy liver tissues; n = 19 healthy margins of resected tissue distant to colorectal carcinoma metastases [CRC margins], and n = 24 perfusates). (b) Percent CD69⁺CD103⁺ of healthy control peripheral (n = 40) and intrahepatic CD8 T cells (n = 10 healthy liver tissues, n = 20 CRC margins, and n = 27 perfusates). (c) Intrahepatic CD69⁺CD103⁺ CD8 T cells stratified by memory phenotype in healthy livers (n = 47). (d) Percent CD69⁺CD103⁺ within memory (CD45RA⁻) CD8 in healthy control PBMCs (n = 40) or livers (n = 54). (e) The dimensionality reduction method tSNE analysis was used to generate a two-dimensional map of T cells within perfusate samples from healthy livers with regard to their expression of key transcription factors and tissue-residency markers. tSNE was performed on the expression data for the markers CD69, CD103, Blimp-1, Eomes, and T-bet as measured by FACS on all CD3⁺CD8⁺CD45RA⁻ events concatenated from four perfusates. Manual gating was used to identify CD69⁺CD103⁺ (black), CD69⁺CD103⁻ (red), and CD69⁻CD103⁻ T cells (blue) as shown in the bottom left corner. These gated populations were then plotted on to the total tSNE map. (f and g) Representative immunofluorescence staining of frozen liver sections: hepatocytes surrounding a vessel stained with cytokeratin (green) and e-cadherin (red; f), and another vessel stained with CD8 (green) and CD103 (red; g). Arrows in g denote cells coexpressing CD8 and CD103 in situ. (h–l) Representative examples and summary data from circulating (white), intrahepatic CD45RA⁻CD69⁻CD103⁻ (gray), and intrahepatic CD45RA⁻CD69⁺CD103⁺ (black) memory CD8 T cells from healthy donors for CXCR6 (%; n = 24) and CXCR3 (MFI; n = 21; h), CD98 (MFI; n = 12) and CD14 (%; n = 20; i), perforin (MFI; n = 16) and granzyme B (MFI; n = 16; j), HLA-DR (%; n = 21; k), and IFNγ and IL-2 intracellular cytokine production (4 h anti-CD3 and anti-CD28; n = 15; l). Error bars indicate means ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; p-values were determined via MANOVA (a); Kruskal-Wallis test (ANOVA) with a Dunn’s post hoc test for pairwise multiple comparisons (b, c, h, i, j, k, and l); and Mann-Whitney t test (d).