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

Abstract

Background: Steatotic 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.

Methods: Human 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.

Results: The 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.

Conclusions: Our 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.

Keywords: FoxP3; NKT; expansion; isolation; liver; steatotic.

Figure 1

Flow cytometry gating strategy for quantification of human type II NKT cells. (A) Human T2NKT cells were defined as CD3⁺ CD56⁺ CD161⁺ TCRγδ^- TCRVα7.2^- TCRVα24^- lymphocytes. Gating of human PBMC resulted in a very low frequency of circulating T2NKT cells (0.3% of total CD3⁺ T cells). (B) Gating of human intrahepatic lymphocytes (IHL) revealed a higher frequency of T2NKT cells in non-steatotic liver (6.5% of total CD3⁺ T cells). Intrahepatic T2NKT cells could be subdivided into CD4⁺ and CD8⁺ cells. (C) Frequency of human T2NKT cells with respect to CD3⁺ T cells from 3 PBMC and 3 liver samples (steatosis 0%, n = 2; steatosis 15%, n = 1).

Figure 2

Two-step magnetic-activated cell sorting (MACS) strategy for enrichment of human T2NKT cells. (A) Two-step MACS strategy consisted first in a CD56 positive selection from human PBMC with CD56 REAlease microbeads. These beads were then detached from the cell surface after separation. In the second step, NKT cells were enriched using CD3 microbeads. The outcome of this procedure was a CD3⁺ CD56⁺ cell isolate enriched in NKT cells. (B) Summary table indicating the frequency of cells relative to preMACS values and absolute (Abs) cell numbers after each MACS step. (C) Gating of human leucocytes enriched for CD56⁺ CD3⁺ cells using the two-step MACS protocol. The sample was enriched for iNKT cells (2% of total CD3⁺ T cells) and T2NKT cells (5.3% of total CD3⁺ T cells). (D) Expression of the transcription regulator promyelocytic leukemia zinc finger (PLZF) in subsets of human lymphocytes. Differential expression of PLZF was observed between conventional T cells (blue line) and NK cells (black line). There was similar PLZF expression in T2NKT cells (red line) and NK cells, whereas iNKT cells (green line) showed higher expression. (E) A CD4⁺ FoxP3⁺ CD25⁺ CD127^low subpopulation of CD3⁺ CD56⁺ CD161⁺ TCRγδ^- TCRVα7.2^- TCRVα24^- T2NKT cells after the two-step enrichment process was identified in all 3 donors (one donor is shown as an example).

Figure 3

In vitro expansion of FoxP3⁺ T2NKT cells. (A) Gating strategy to flow-sort T2NKT cell after double-step MACS enrichment. From left to right, cells were gated for lymphocytes based on FSC/SSC and singlets. Biotin FITC excluded cells labeled with TCRVα7.2, TCRVα24 or TCRγᵹ antibody. Cells were then gated for CD3⁺ CD4⁺ CD56⁺ CD161⁺. (B) Schematic map depicting each step of the expansion protocol. (C) Cell counting after 3 weeks expansion using Flow Cytometry Counting Beads. Counting beads were positive for PE-Cy7, cells were separated from MACSiBeads based on FSC. (D) Quality control analysis of T2NKT cell expansion after 3 weeks. Lymphocytes were separated from MACSiBeads and dead cells by FCS/SSC. MAIT cells, γᵹ T cells and iNKT cells were excluded by staining with Biotin FITC. Within the CD3⁺ T cell population, PLZF was highly expressed (80.7% of the total lymphocyte population). These cells were mainly CD56⁺ CD161⁺ (75.7% of the total lymphocyte population) and a subset expressed regulatory-like phenotype FoxP3⁺ CD25⁺ CD127^low (31% of the total lymphocyte population). (E) Summary table indicating absolute number of cells before expansion (day 0) and after expansion (day 21). Percentage of FoxP3⁺ cells after expansion is included.