Abstract
Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.
Highlights
Hemophagocytic lymphohistiocytosis (HLH) represents a spectrum of inherited and acquired conditions characterized by severe immune dysregulation.[1]
We show that gene transfer using self-inactivating lentiviral vectors (LVs) results in expression of perforin in T and NK cells and leads to a significant correction of the cytotoxic defects both in vitro and in vivo in murine models of HLH
The two functional perforin-expressing vectors (PGK.perforin gene (PRF) and PRF.PRF) were tested for expression of GFP and perforin in human cell lines, and high levels of expression were observed in all cell lines using the PGK promoter–driven vector, while expression from the vector with PRFprom was restricted to T (Jurkat) and NK (YT) cell lines (Supplementary Figure S1)
Summary
Hemophagocytic lymphohistiocytosis (HLH) represents a spectrum of inherited and acquired conditions characterized by severe immune dysregulation.[1] The inherited form is termed familial hemophagocytic lymphohistiocytosis (FHL) and arises from genetic defects that lead to abnormalities of T and NK cell cytotoxicity that in turn result in hypercytokinemia, macrophage activation, hemophagocytosis and a clinical syndrome of fever, coagulopathy, hepatosplenomegaly, and often central nervous system involvement.[2] Five different genetic loci have been identified (FHL 1–5), and of these, perforin deficiency (FHL-2) is the most common and best characterized.[3]. Perforin is contained within these granules and upon release forms pores in target cell membranes, allowing the passage of granzymes which initiate apoptotic pathways, eventually leading to target cell death.[4] Mutations in the perforin gene (PRF) lead to failure of pore formation and target cell cytotoxicity in effector cells, resulting in proliferation of highly activated cytotoxic T and NK cells, which in turn lead to hypercytokinemia and the subsequent clinical sequelae of HLH. Correction of immune dysregulation in this model by transplantation with wildtype (WT) bone marrow has been demonstrated, and in these studies, the prevention of HLH development is critically dependent upon the engraftment of functional CD8+ T cells.[6]
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