Phenotypic differences between mice deficient in XIAP and SAP, two factors targeted in X-linked lymphoproliferative syndrome (XLP) - PubMed (original) (raw)

Comparative Study

Phenotypic differences between mice deficient in XIAP and SAP, two factors targeted in X-linked lymphoproliferative syndrome (XLP)

Julie M Rumble et al. Cell Immunol. 2009.

Abstract

Mutations in the X-linked inhibitor of apoptosis (XIAP) have recently been identified in patients with the rare genetic disease, X-linked lymphoproliferative syndrome (XLP), which was previously thought to be solely attributable to mutations in a distinct gene, SAP. To further understand the roles of these two factors in the pathogenesis of XLP, we have compared mice deficient in Xiap with known phenotypes of Sap-null mice. We show here that in contrast to Sap-deficient mice, animals lacking Xiap have apparently normal NKT cell development and no apparent defect in humoral responses to T cell-dependent antigens. However, Xiap-deficient cells were more susceptible to death upon infection with the murine herpesvirus MHV-68 and gave rise to more infectious virus. These differences could be rescued by restoration of XIAP. These data provide insight into the differing roles of XIAP and SAP in the pathogenesis of XLP.

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Figures

Fig. 1

Fig. 1. No detectable interaction between XIAP and SAP

(A) XIAP and FLAG-tagged SAP were coexpressed with the cytoplasmic tail of SLAM-GST or GST alone in HEK293 cells. Glutathione-sepharose beads were added to lysates and bead-associated proteins were separated by SDS-PAGE and immunoblotted for FLAG, GST and XIAP. Additionally, the last panel shows immunoprecipitation using an anti-FLAG monoclonal antibody and IgA beads to assess binding of SLAM and XIAP to SAP. (B) HA-XIAP (both wildtype and a H467A point mutant) and FLAG-SAP were expressed in HEK293 cells in the presence of the tyrosine kinase Lck and either a GST-tagged cytoplasmic tail construct of the 2B4 receptor or GST alone. As in A, GST coprecipitations and immunoblots were performed assessing the ability of wildtype (WT) or D148A/W310A double mutant (MT) XIAP to bind SAP or 2B4. All samples include Lck.

Fig. 2

Fig. 2. Murine expression of XIAP and SAP

Thymocytes were harvested from XIAP (A) and SAP (B) WT and KO mice, lysed and immunoblotted for SAP, XIAP and β-actin. Asterisk (*) indicates a non-specific band.

Fig. 3

Fig. 3. NKT cells normal in XIAP KO mice

Splenocytes and thymocytes were isolated from three XIAP WT and three XIAP KO mice, and stained with anti-CD24, NK1.1+ and TCR-β+ (A and B), as well as PE-conjugated α-galactosylceramide-loaded CD1d tetramer, specific for NKT cells (C and D). NKT cells are defined as CD24low, NK1.1+, TCR-β+, and tetramer+. A and C show total results of at least 3 individual mice, error bars shown are standard error of the mean. B and D are representative FACS plots of the CD24low subpopulation.

Fig. 4

Fig. 4. Normal germinal center formation in XIAP KO mice

(A) XIAP WT and KO mice were injected with either SRBC or saline and spleens were harvested 6 days later. Frozen sections were stained with PNA-FITC and anti-B220 and viewed on an Olympus microscope. (B) Splenocytes were harvested from mice treated in A and stained with antibodies to B220, IgD and Fas. Subsets were also stained with PNA or antibodies to GL7 or CD38 to specifically identify germinal center B cells. Data are representative of at least three individual mice, and error bars shown are standard error of the mean.

Fig. 5

Fig. 5. _Xiap_-null cells are sensitive to virus-induced death

(A) The indicated MEF cell lines were infected with 0.1 pfu/cell MHV-68 and cultured for 72 hours, after which they were visualized with light microscope. (B) Cells were treated as in A, then floating and adherent cells were harvested and PI stained for viability by flow cytometry. Data represent at least three experiments, with error bars illustrating standard error, and significance (<0.001 indicated with an asterisk [*]) was calculated using a one-way ANOVA. (C) Supernatants from cells treated as in A were serially diluted and plated on 3T12 cells for quantitation by plaque assay. Three wells were counted from the 1:32,000 dilution of supernatant in each of two experiments.

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