Diminished Immune Cell Adhesion in Hypoimmune ICAM-1 Knockout Pluripotent Stem Cells

Abstract

Hypoimmune gene edited human pluripotent stem cells (hPSCs) are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive (e.g., T cell) immune responses, but have largely not addressed the innate immune cells (e.g., monocytes, neutrophils) that mediate inflammation and rejection processes occurring early after graft transplantation. We identified the adhesion molecule ICAM-1 as a novel hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In a series of studies, we found that ICAM-1 blocking or knock-out (KO) in hPSC-derived cardiovascular therapies imparted significantly diminished binding of multiple immune cell types. ICAM-1 KO resulted in diminished T cell proliferation responses in vitro and in longer in vivo retention/protection of KO grafts following immune cell encounter in NeoThy humanized mice. The ICAM-1 KO edit was also introduced into existing first-generation hypoimmune hPSCs and prevented immune cell binding, thereby enhancing the overall hypoimmune capacity of the cells. This novel hypoimmune editing strategy has the potential to improve the long-term efficacy and safety profiles of regenerative therapies for cardiovascular pathologies and a number of other diseases. HighlightsO_LIAntibody blocking of ICAM-1 on human pluripotent stem cell-derived cells inhibits immune cell adhesion C_LIO_LICRISPR/Cas9 knock-out of ICAM-1 ablates surface and secreted ICAM-1 protein and inhibits immune adhesion C_LIO_LIICAM-1 knock-out results in decreased T cell proliferative responses to human pluripotent stem cell-derived grafts in vitro, and resistance to immune-mediated graft loss in vivo C_LIO_LIAddition of ICAM-1 knock-out to first generation MHC knock-out human pluripotent stem cells confers protection against immune adhesion C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/597791v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@890b56org.highwire.dtl.DTLVardef@147449borg.highwire.dtl.DTLVardef@1e94f08org.highwire.dtl.DTLVardef@43c55c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO ICAM-1 Knock-out in Transendothelial Migration and at the Immune Synapse. Abbreviations: PSC-EC - pluripotent stem cell-derived endothelial cells; KO - knock-out; dSMAC - distal supramolecular activation complex; pSMAC - peripheral supramolecular activation complex; cSMAC - central supramolecular activation complex. C_FIG