4th Palermo Conference on INNOVATIVE THERAPIES FOR LYMPHOID MALIGNANCIES
Published online 13 August 2009
Haematologica, Vol 95, Issue 2, 214-223 doi:10.3324/haematol.2009.011650
Copyright © 2010 by Ferrata Storti Foundation
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Paroxysmal Nocturnal Hemoglobinuria

Phenotypic and functional characterization of a mouse model of targeted Pig-a deletion in hematopoietic cells

Valeria Visconte1, Nalini Raghavachari2, Delong Liu3, Keyvan Keyvanfar1, Marie J. Desierto1, Jichun Chen1, Neal S. Young1

1 Hematology Branch, National Heart, Lung, and Blood Institute
2 Genomics Core Facility, Pulmonary and Vascular Medicine Branch, National Heart, Lung, and Blood Institute
3 Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD, USA

Correspondence: Valeria Visconte, Phd, Hematology Branch, NHLBI, NIH, Building 10, CRC, 3E-5232, Bethesda MD, 20892-1202 USA., E-mail: viscontev{at}mail.nih.gov

Background: Somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) causes glycosyl phosphatidylinositol anchor deficiency in human patients with paroxysmal nocturnal hemoglobinuria.

Design and Methods: We produced an animal model of paroxysmal nocturnal hemoglobinuria by conditional Pig-a gene inactivation (Pig-a–/–) in hematopoietic cells; mice carrying two lox sites flanking exon 6 of the Pig-a gene were bred with mice carrying the transgene Cre-recombinase under the human c-fes promoter. We characterized the phenotypic and functional properties of glycosyl phosphatidylinositol-deficient and glycosyl phosphatidylinositol-normal hematopoietic cells from these Pig-a–/– mice using gene expression microarray, flow cytometry, bone marrow transplantation, spectratyping, and immunoblotting.

Results: In comparison to glycosyl phosphatidylinositol-normal bone marrow cells, glycosyl phosphatidylinositol-deficient bone marrow cells from the same Pig-a–/– animals showed up-regulation of the expression of immune function genes and contained a significantly higher proportion of CD8 T cells. Both characteristics were maintained when glycosyl phosphatidylinositol-deficient cells were transplanted into lethally-irradiated recipients. Glycosyl phosphatidylinositol-deficient T cells were inactive, showed pronounced Vβ5.1/5.2 skewing, had fewer {gamma}-interferon-producing cells after lectin stimulation, and contained fewer CD4+CD25+FoxP3+ regulatory T cells. However, the levels of T-cell receptor signaling proteins from glycosyl phosphatidylinositol-deficient cells were normal relative to glycosyl phosphatidylinositol-normal cells from wild type animals, and cells were capable of inducing target cell apoptosis in vitro.

Conclusions: Deletion of the Pig-a gene in hematopoietic cells does not cause frank marrow failure but leads to the appearance of clonally-restricted, inactive yet functionally competent CD8 T cells.

Key words: Pig-a deletion, paroxysmal nocturnal hemoglobinuria, glycosyl phosphatidylinositol, T-cell mediated immunity.