Cutting Edge: Human CD49e- NK Cells Are Tissue Resident in the Liver

Abstract

Most knowledge on NK cells is based on studies of what are now known as conventional NK cells in the mouse spleen or human peripheral blood. However, recent studies in mice indicate the presence of tissue-resident NK cells in certain organs, such as the liver, that display different markers and transcription factor dependencies as compared with conventional NK cells. In this study, we provide evidence from cytometry by time-of-flight analysis and humanized mice indicating that human CD49e^- NK cells are tissue resident in the liver. Thus, these studies indicate that tissue-resident NK cells are evolutionarily conserved in humans and mice, providing a foundation to explore their role in human disease.

Figure 1

Multidimensional analysis of venous blood (PBMC) or liver post-excision flush (L-PxF) isolated NK cells identifies a unique liver-specific subset lacking expression of CD49e. (A) viSNE plots of NK cells from 3 donors were generated by clustering with the markers CD11b, CD16, CD45, CD56, and CD57. Gates were manually drawn in the contour plots based on the distribution of cells from the PBMC fraction. Numbers indicate percentage of cells in respective gates. (B) Average percentage of indicated marker expression derived from the gates in (A). Bars indicate SD, * = p<0.05, ** = p<0.01, Student t test after Bonferroni correction. (C) viSNE plots showing CD49e expression for the 3 donors in (A). Color intensity indicates CD49e expression intensity. (D) viSNE plots with CD49e expression from donor 1 that includes CD49e in the list of clustering markers. (E) Summary of cell percentages in liver-specific gates from PBMC and L-PxF when CD49e was excluded (-) or included (+) from clustering. * = p<0.05, Student paired t test.

Figure 2

Differential cell surface marker expression on CD49e⁺ and CD49e⁻ NK cells. (A) Expression of CD49e and CD56 on isolated single cell viable CD3⁻ CD19⁻ lymphocytes from healthy donor PBMC and L-PxF samples. Summary of percentage of NK cells from the 2 subsets of CD49e⁺ and CD49e⁻ cells from venous blood and L-PxF is shown on the right. **** = p<0.0001, Student t test. Bars indicate mean ± SD. (B) Representative scatter plots (upper panel) and summary graph (lower panel) of indicated cell surface markers of NK cells from PBMC and L-PxF. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = not significant, Student t test. Bars indicate mean ± SD

Figure 3

Differential expression of transcription factors but not functional profile of CD49e⁺ and CD49e⁻ NK cells. (A) Expression and MFI summary of T-bet and Eomes of PBMC and L-PxF derived NK cells. ** = p<0.01, *** = p<0.001, ns = not significant, Student t test. (B) Representative intracellular cytokine staining for IFNγ and TNFα production by NK cells from PBMC and L-PxF after 6 hours stimulation with PMA/ionomycin. Panels also show CD107a expression after stimulation. Summary graphs of intracellular cytokine production are shown at the bottom. ns = not significant. Bars indicate mean ± SD.

Figure 4

Human CD49e⁻ NK cells are preferentially found in the liver of humanized NSG mice. Representative contour plots and summary graphs of humanized-NSG mice NK cells isolated from venous blood (PBMC) and liver homogenate (n= 3). Single cell viable hCD45⁺, CD3⁻, CD19⁻, lymphocytes were gated on before determining the expression of CD56 and CD49e. **** = p<0.0001, Student t test. Bars indicate SD.