Identification and Isolation of Type II NKT Cell Subsets in Human Blood and Liver

Abstract

BackgroundSteatotic livers are more prone to rejection, but are often transplanted owing to the shortage of available organs. Type II NKT (T2NKT) cells are liver-resident lymphocytes that react to lipids presented by CD1d. The role of T2NKT cells in rejection of fatty liver transplants is unclear, partly because of a lack of T2NKT cell markers and their very low frequency in blood. Here, we quantify human T2NKT cells in blood and liver tissue by flow cytometry and provide a strategy for their enrichment and expansion.MethodsHuman T2NKT cells were identified as CD3+ CD56+ CD161+ TCR-γᵹ- TCRVα7.2- and TCRVα24- cells. T2NKT cells were enriched from blood by sequential positive selection using CD56 and CD3 microbeads. These were subsequently FACS-sorted to purity then expanded in vitro for 3 weeks using anti-CD3/CD28 beads and TGF-β1.ResultsThe frequency of human T2NKT cells in blood was very low (0.8 ± 0.4% of CD3+ T cells) but they were a more abundant population in liver (6.3 ± 0.9%). Enriched T2NKT cells expressed the transcription factor PLZF. A novel subset of FoxP3+ T2NKT cells was discovered in blood and liver tissue. T2NKT cells were expanded in culture by 15- to 28-fold over 3 weeks, during which time they maintained expression of all identifying markers, including PLZF and FoxP3.ConclusionsOur work defines new strategies for identifying and isolating T2NKT cells from human blood and liver tissue. We showed that this rare population can be expanded in vitro in order to obtain experimentally amenable cell numbers. Further, we identified a novel T2NKT cell subset that stably expresses FoxP3, which might play a role in regulating innate-like lymphocyte responses in steatotic liver transplants.

Innate immunity and early liver inflammation

“…NKT cells are a rare subset of T lymphocytes comprising less than 1% of human peripheral blood T lymphocytes but enriched in the liver ( 8 , 40 ). NKT cells are known to express NK cell markers like CD56, CD16 and CD161, and produce granzyme ( 40 , 41 ). Their restricted TCR repertoire recognizes antigenic lipids presented by the MHC class I-like molecule CD1d ( 42 , 43 ).…”

“…It is difficult to study T2NKT cells because there is a lack of tools to identify and characterize them. Recently, we proposed a novel strategy to isolate and characterize T2NKT cells in humans but the low number of cells in blood is still a limitation ( 40 ). The role of iNKT cells and T2NKT cells in liver diseases have been mainly studied using transgenic mice models of CD1d-knockouts or TCRVα14-knockouts, which lack iNKT cells.…”

“…These studies suggest that, in general, iNKT cells play a pro-inflammatory phenotype whereas T2NKT cells suppress inflammation through direct and indirect inhibition of inflammatory cells, including iNKT cells ( 46 – 49 ). We described a novel subpopulation of T2NKT cells that expresses regulatory T cell markers such as FoxP3 and CD25 ( 40 ). FoxP3 + T2NKT cells were found both in the periphery and in the liver and may explain some of the regulatory functions reported previously.…”

“…Another study described the crosstalk of T2NKT cells with plasmacytoid dendritic cells and recruitment of anergic iNKT cells to the mouse liver via IL-12 and MIP-2 ( 126 ). As mentioned above, our group recently identified a novel population of human FoxP3 + T2NKT cells that might exert immunoregulatory functions in this scenario ( 40 ). Alcoholic-related cirrhosis and severe alcoholic hepatitis patients had a dramatic depletion and hyperactivated circulating MAIT cells ( 127 , 128 ).…”

Natural Killer T (NKT) Cells in Autoimmune Hepatitis: Current Evidence from Basic and Clinical Research.

“…Conversely, type II NKT cells lack any reliable positive markers. Therefore, their identification is challenging and could be based on the exclusion of the TCR-Vα24 + type I NKT cells (and, possibly, TCR-Vα7.2 + mucosal-associated invariant T cells as well) in the CD3 + CD56 + CD161 + TCRγδ − T cell population, for instance [20]. Type I NKT cells are relatively abundant in mice, representing between 1% and 3% of T cells in most tissues.…”