Generation of iNKT cells with hematopoiesis-promoting properties from human iPSCs

Abstract Human invariant natural killer T (iNKT) cells are a conserved population of innate-like T cells that are activated by glycolipid antigens presented by non-classical CD1d molecules. They follow a distinct developmental pathway marked by PLZF expression, which imparts them with innate-like functional properties. In addition to their well-known role in anti-tumor function, iNKT cells are also involved in regulating and maintaining hematopoiesis in the bone marrow. Here, we present the reprogramming of human CD4+Vα24+ iNKT cells into induced pluripotent stem cells (iPSCs) and describe a chemically defined, feeder-free 3D spheroid method for generating CD34+ hematopoietic progenitors where iPSC suspension is apportioned into separate droplets hanging from the surface and then cultured on Collagen IV in the presence of morphogens, growth factors and cytokines. Floating hematopoietic progenitors collected on day 9 of differentiation were cultured on OP9-DLL4 cultures for 3-4 weeks to generate lymphocytes most of which (~87%) were TCRVa24+TCRVb11+ double-positive. The i-iNKT cells showed specific binding to CD1d tetramers loaded with the lipid antigen a-galactosylceramide and had a similar transcription factor profile to that of somatic CD4+ iNKT cells for expression of T-bet, E4BP4, and GATA-3, which control production of IFN-g, IL-13, IL-4, respectively. In response to CD3 stimulation, the i-iNKT cells consistently produced GM-CSF at levels similar to those of somatic iNKT cells, and also clearly produced IL-3, IFN-g, and IL-13. However, in contrast to somatic iNKT cells, the i-iNKT cells showed no detectable secretion of IL-4. i-iNKT cells further demonstrated pro-hematopoietic activity for MACS-enriched CD34+ cells from cord blood, where exposure to trans-wells containing anti-CD3-activated i-iNKT cells resulted in significantly increased total numbers of differentiated cells in the lower wells. Interestingly, exposure to activated i-iNKT but not somatic iNKT cells also led to a significantly increased number of cells that retained CD34 and were negative for lineage markers. These findings suggest the feasibility of using iPSCs as off-the-shelf i-iNKT cell sources to enhance the hematopoietic activity of bone marrow after hematopoietic stem cell (HSC) transplantation.