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.
Estrogen-induced Syrian hamster tumors in the kidney represent a useful model to gain insight into the role of estrogens in oncogenic processes. We provided evidence that early tumor foci in the kidney arise from interstitial ectopic uterine-like germinal stem cells, and that early tumor foci and well-established tumors are highly aneuploid (92-94%). The molecular mechanisms whereby estrogens mediate this process are unclear. Here, we report that estrogen treatment induced significant increases in Aurora A protein expression (8.7-fold), activity (2.6-fold), mRNA (6.0-fold), and Aurora B protein expression (4.6-fold) in tumors, compared with agematched cholesterol-treated kidneys. Immunohistochemistry revealed that this increase in Aurora A and B protein expression was essentially confined to cells within early and large tumor foci at 3.5 and 6 months of estrogen treatment, respectively. Upon estrogen withdrawal or coadministration of tamoxifen for 10 days, a 78% to 79% and 81% to 64% reduction in Aurora A and B expression, respectively, were observed in primary tumors compared with tumors continuously exposed to estrogens. These data indicate that overexpressed Aurora A and B in these tumors are under estrogen control via estrogen receptor A. Aurora A coenriched with the centrosome fraction isolated from tumors in the kidney. Centrosome amplification (number and area/cell) was detected in early tumor foci and large tumors but not in adjacent uninvolved or age-matched control kidneys. Taken together, these data indicate that persistent overexpression of Aurora A and B is under estrogen control, and is coincident with centrosome amplification, chromosomal instability, and aneuploidy, and represent an important mechanism driving tumorigenesis.
Key Points Natural killer cell granzyme B, A, and K delivery and subsequent caspase activation is rapid after conjugation with tumor target cells. Natural killer cells also induce caspase activation through death receptor ligation that can be monitored in real time.
K.R., M.J., and P.M. share senior authorship.Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/-transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.
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