Insights into absence of lymphoma despite fulminant Epstein-Barr virus infection in patients with XIAP deficiency
Yizhe Sun, Janet Chou, Kevin D. Dong, Steven P. Gygi, Benjamin E. Gewurz
Yizhe Sun, Janet Chou, Kevin D. Dong, Steven P. Gygi, Benjamin E. Gewurz
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Research Article Infectious disease Virology

Insights into absence of lymphoma despite fulminant Epstein-Barr virus infection in patients with XIAP deficiency

Abstract

X-linked Lymphoproliferative Syndromes (XLP), arising from mutations in SH2D1A or XIAP genes, are characterized by fulminant Epstein-Barr virus (EBV) infection. Lymphomas occur frequently in XLP-1 and in other congenital conditions with heightened EBV susceptibility, but not in XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis remains a key open question. To gain insights, newly EBV-infected versus receptor-stimulated primary B cells from XLP-2 patients or with XIAP CRISPR editing were compared with healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected B cells, but not of CD40 ligand and interleukin-21–stimulated B cells. XLP-2–deficient B cells showed significantly lower EBV transformation efficiency than cells from healthy controls. Interestingly, EBV-immortalized lymphoblastoid cell proliferation was not impaired by XIAP knockout, implicating a XIAP role in early EBV B cell transformation. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, which was counteracted by XIAP in control cells. With XIAP deficiency, EBV markedly elevated apoptosis rates over the first 2 weeks of infection. IFN-γ, whose levels are increased with severe XLP2 EBV infection, markedly increased newly EBV-infected B cell apoptosis. These findings underscored XIAP’s crucial role in support of the earliest stages of EBV-mediated B cell immortalization and provide insights into the curious absence of EBV+ lymphoma in patients with XLP-2.

Authors

Yizhe Sun, Janet Chou, Kevin D. Dong, Steven P. Gygi, Benjamin E. Gewurz

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Figure 3

EBV, but not CD40L/IL-21, triggers apoptosis within the first week of XLP-2 B cell infection.

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EBV, but not CD40L/IL-21, triggers apoptosis within the first week of XL...
(A) FACS analysis of control versus XIAP-edited primary B cells at Day 4 after EBV infection or CD40L/IL-21 stimulation. Shown are representative FACS plots from n = 3 replicates of cells stained with CFSE on Day 0. Live cells were gated by absence of 7-AAD vital dye uptake. (B) Mean + SD percentages of cells with the indicated number of mitoses from n = 3 replicates of EBV infection versus CD40L/IL-21 stimulation, as in A. (C) Mean + SD % 7AAD+ cells from n = 3 replicates of control or XIAP-edited primary B cells on Day 4 after EBV infection or CD40L/IL21 treatment. (D) Mean + SD caspase 3/7 activity from n = 3 replicates of control or XIAP-edited primary B cells on day 4 after EBV infection or CD40L/IL-21 treatment. (E) Mean + SD caspase 3/7 activity from n = 3 replicates of control or XLP-2 primary B cells on day 4 after EBV infection or CD40L/IL-21 treatment. (F) Mean + SD caspase 3/7 activity from n = 3 replicates of control or XIAP edited primary B cells incubated with DMSO or the pan-caspase inhibitor zVAD-fmk (20 μM) on day 4 after EBV infection or CD40L/IL-21 treatment. (G) Mean + SD % 7AAD+ cells from n = 3 replicates of control or XIAP-edited primary B cells on Day 4 after EBV infection or CD40L/IL21 treatment. (H) Growth curve analysis of control versus XIAP-edited B cells infected with EBV on Day 0 and cultured with DMSO or zVAD-Fmk (20 μM). Mean ± SD fold-change live cell numbers, relative to uninfected values, are shown. Statistical significance was assessed by 2-tailed unpaired Student’s t test (H) or 1- (E) or 2-way (B–D, F, and G) ANOVA followed by Tukey’s multiple comparisons test. CD40L (50 ng/mL), IL-21 (50 ng/mL), DMSO and zVAD-Fmk were replenished every 3 days (A–H). **P < 0.01, ***P < 0.001, ****P < 0.0001.