Ex vivo purification and characterization of human invariant Natural Killer T cells

Abstract

Natural Killer T (NKT) cells have gained widespread attention among immunologists because of their distinct ability to regulate anti-tumor responses and to influence the outcome of infections or autoimmunity. Type I (also called invariant) NKT cells (iNKT) are best characterized mainly because of the availability of lipid antigen-loaded CD1d-tetramer detection reagents. Human iNKT cells present important phenotypic differences relative to their murine counterpart, restricting the extrapolation of findings from experimental murine models to human health and disease states. Particularly, the ontogeny and early life phenotype of iNKT cells largely differ between human and mice, indicating divergent functional properties between species. The high therapeutic potential offered by manipulation of iNKT cells in disease warrants a better understanding of human iNKT cell biology. Here, we discuss characteristics of human iNKT cells and present an efficient and rapid method for their ex vivo purification and characterization.

Figure 1. Gating strategy for flow cytometry identification of iNKT cells among blood mononuclear cells

Lower panels: PBS57/CD1d-tetramer staining on (A) CD3^positive cells or (B) CD19^positive cells. Note the significant background PBS57/CD1d-tetramer staining on (CD19-expressing) B cells warranting use of an anti-CD19 antibody as a dump channel.

Figure 2. Multi-step protocol for purification of human iNKT cells

The low proportion of iNKTs present in human mononuclear cells (A) can be enriched using an initial magnetic bead column purification step followed by (B) Fluorescent Activated Cell Sorting (FACS).

Figure 3. Characterization of purified human iNKT cells

(A) Positive staining of CD1d tetramer-purified iNKT-cell with the 6B11 antibody. (B) iNKT cell proliferation when stimulated with plate-bound anti-CD3 (OKT3) and soluble anti-CD28 antibodies (as described in Ladd et al., 2010). (C) Cytokine profile of purified iNKT cells reconstituted (1 iNKT:1000 mononuclear cells) with CD1d-tetramer-depleted (FACS) autologous cord blood (CB) or adult peripheral blood (APB) mononuclear cells following stimulation with α-galactosylceramide (100 ng/mL; 96 hours). Cytokine levels are presented (mean, error bars: standard deviation of duplicate ELISA analyses) after subtraction of unstimulated cells.