VPB0899 - Mechanisms of HLA-KO iPSC-platelets immune escape from NK cells

Background: In some cases, patients can become refractory to platelet transfusion due to qllo-immune responses. Therefore, to make iPS-derived platelets suitable for the widest majority, we chose to edit out the expression of HLA class-I on the surface of induced pluripotent stem cell-derived platelets (iPSC-platelets). While we observed that their HLA-KO precursor cells elicited an NK cell response, the HLA-KO platelets did not.

Aims: We propose to identify factors which influence the difference in sensitivity to NK cell cytotoxicity between HLA-KO iPSC-platelets and their precursors, HLA-KO iPSC-derived immortalized megakaryocytes progenirot cell line (imMKCLs).

Methods: We used both discovery and hypothesis-driven methodologies. In the discovery approach, we used improved mass spectrometry analysis to compare the proteome of both cell types, then a LIMMA-based statistical analysis extracts differentially represented pathways. In vitro assays subsequently validated relevant candidates. We also used live-imaging to characterize contact and killing events, as well as visualise the formation of immunological synapses between NK and target cells.

Results: Proteins involved in repression of immune processes were up-regulated in HLA-KO platelets compared to HLA-KO imMKCLs. This includes integrin pathways and known NK cells inhibitors. Moreover, platelet precursor cells formed immunological synapses with NK cells, ending in either tolerance or killing.

Conclusion(s): For iPSC-derived therapies to move toward clinical trials, considerations about their HLA-compatibility, as well as their deep functional characterization are fundamental. HLA-KO iPSC-platelets display a stronger immuno-regulatory proteome than their precursor cells and this work has identified crucial pathways governing NK cell toxicity and tolerance to both platelets and their precursor cells. These findings represent important insights for future clinical trials of universal, HLA knockout platelets.