Insights into the Absence of Lymphoma Despite Fulminant Epstein-Barr Virus Infection in Patients with XIAP Deficiency

Abstract

X-linked Lymphoproliferative Syndromes (XLP), which arise from mutations in the SH2D1A or XIAP genes, are characterized by the inability to control Epstein-Barr Virus (EBV) infection. While primary EBV infection triggers severe diseases in each, lymphomas occur at high rates with XLP-1 but not with XLP-2. Why XLP-2 patients are apparently protected from EBV-driven lymphomagenesis, in contrast to all other described congenital conditions that result in heightened susceptibility to EBV, remains a key open question. To gain insights, we cross-compared newly EBV infected versus immune stimulated B-cells from XLP-2 patients or upon XIAP CRISPR knockout, relative to healthy controls. XIAP perturbation impeded outgrowth of newly EBV-infected primary human B-cells, though had no impact on proliferation of B-cells stimulated by CD40 ligand and interleukin-21 or upon established EBV-immortalized lymphoblastoid cell lines (LCLs). B-cells from XLP-2 patients or in which XIAP was depleted by CRISPR editing exhibited a markedly lower EBV transformation efficiency than healthy control B-cells. Mechanistically, nascent EBV infection activated p53-mediated apoptosis signaling, whose effects on transforming B-cell death were counteracted by XIAP. In the absence of XIAP, EBV infection triggered high rates of apoptosis, not seen with CD40L/IL-21 stimulation. Moreover, inflammatory cytokines are present at high levels in XLP-2 patient serum with fulminant EBV infection, which heightened apoptosis induction in newly EBV-infected cells. These findings highlight the crucial role of XIAP in supporting early stages of EBV-driven B-cell immortalization and provide insights into the absence of EBV+ lymphoma in XLP-2 patients.

Figure 1.. XIAP inactivation impairs the outgrowth of newly EBV infected primary B-cells.

(A) Workflow for electroporation and EBV infection of primary human B-cells. B-cells purified from peripheral blood mononuclear cells (PBMC) were transduced with Cas9 ribonucleoprotein (RNP) complexes containing XIAP targeting or non-targeting control single guide RNA (sgRNA). 1 hour post electroporation, cells were infected with EBV or stimulated by CD40 ligand (CD40L) (50 ng/ml) and IL-21 (50 ng/ml). (B) Immunoblot analysis of whole cell lysates (WCL) from primary B-cells on day 3 post electroporation with Cas9 control (ctrl) or XIAP sgRNA containing RNPs. (C) XIAP editing does not alter EBV infection efficiency. FACS analysis of control versus XIAP edited B-cells at Day 2 post-infection by recombinant EBV that expresses a green fluorescence protein (GFP) marker. Mean + standard deviation (SD) GFP+ cell percentages from n=3 replicates are shown on the right. (D) Growth curve analysis of primary human B-cells transfected with the indicated sgRNA-containing Cas9 RNPs and treated with CD40L and IL-21 or infected with EBV. CD40L/IL-21 were replenished every 3 days. Mean ± SD fold change live cell numbers from n = 3 biological replicates, relative to Day 0 values, are shown. (E) Immunoblot analysis of WCL from primary B-cells with control or XIAP sgRNA-containing RNP on the indicated days post-EBV infection. (F) Growth curve analysis of primary B-cells treated with DMSO or the XIAP inhibitor embelin (5 μM) together with EBV infection (left) or CD40L/IL-21 treatment (right). Embelin, CD40L and IL-21 were replenished every 3 days. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. (G) Immunoblot and growth curve analysis of Cas9+ GM12878 LCLs expressing the indicated control or independent XIAP targeting sgRNAs. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. (H) Growth curves analysis of GM12878 LCLs treated with DMSO or embelin (5 μM), which were replenished every 3 days. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. (I) Growth curve analysis of primary human B-cells infected by EBV at Day 0 and then treated with embelin (5 μM) over the indicated times. Embelin was replenished every 3 days. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. Statistical significance was assessed by comparing each indicated groups with DMSO control groups. Statistical significance was assessed by two-tailed unpaired Student’s t test (C, D, F, H, I) or one-way ANOVA followed by Tukey’s multiple comparisons test (G). Blots are representative of n=3 replicates. **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

Figure 2.. XLP2 patient B-cells demonstrate impaired EBV but not CD40L/IL-21 driven outgrowth at early timepoints.

(A) Schematic diagram highlighting the XIAP mutation shared by XLP2 Patients #1 and #2. (B) Growth curve analysis of primary B-cells from XLP2 patients or from controls that were infected with EBV. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. The annotations represent the results of statistical comparisons between XLP2 samples and Control #1. (C) Growth curve analysis of primary B-cells from XLP2 patients or controls treated with CD40L and IL-21, which was replenished every 3 days. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. The annotations represent the results of statistical comparisons between XLP2 samples and Control #1. (D) Growth curves of lymphoblastoid cells established from B cells from either two XLP2 patients or three controls. Mean ± SD fold change live cell numbers from n=3 replicates, relative to Day 0 values, are shown. The annotations represent the results of statistical comparisons between XLP2 samples and Control #1. (E) EBV B-cell transformation assay workflow. CD19+ B-cells purified from PBMCs were plated and infected with serial dilutions of the Akata EBV strain, using a range of 0 – 100 EBV transforming units/well. Wells with B-cell outgrowth were scored 4 weeks later. Outgrowth wells on one of the three replicate plates were displayed. (F) EBV transformation assays of primary human B-cells from XLP2 patient or healthy controls, as in (E). Shown are the mean ± SD percentages of wells with B-cell outgrowth from n=3 replicates. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons test (B-D, F). *p<0.05, **p<0.01, ***p<0.001, ****, p<0.0001, ns, non-significant.

Figure 3.. EBV but not CD40L/IL-21 triggers apoptosis within the first week of XLP2 B-cell infection.

(A) FACS analysis of control versus XIAP edited primary human B-cells at Day 4 post-EBV infection or CD40L/IL-21 stimulation. Shown are representative FACS plots from n=3 replicates of cells stained with CFSE prior to EBV infection or CD40L/IL-21 treatment. 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 as in (A) of EBV infection versus CD40L/IL-21 stimulation. (C) Mean + SD % 7AAD+ cells from n=3 replicates of control or XIAP edited primary B-cells on Day 4 post-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 post-EBV infection or CD40L/IL-21 treatment. (E) Mean + SD caspase 3/7 activity from n=3 replicates of control or XLP2 primary B-cells on day 4 post-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 vehicle or the pan-caspase inhibitor zVAD-fmk (20 μM) on day 4 post-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 post-EBV infection or CD40L/IL21 treatment. (H) Growth curve analysis of control versus XIAP edited primary B-cells infected with EBV on Day 0 and cultured with DMSO vehicle or zVAD-Fmk (20 μM). Mean ± SD fold change live cell numbers, relative to uninfected values, are shown. DMSO or zVAD-Fmk were replenished every 3 days. Statistical significance was assessed by two-tailed unpaired Student’s t test (B-D, H) or one (E) or two-way (F and G) ANOVA followed by Tukey’s multiple comparisons test. In A-G, CD40L and IL21 were replenished on Day 3. **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

Figure 4.. EBV but not CD40L/IL-21 activates p53- and BAX-dependent apoptosis in newly infected XIAP deficient B-cells.

(A) Heatmap of RNAseq analysis of primary B-cells from XLP2 patient or controls on Day 7-post EBV infection or CD40L/IL-21 treatment. Z-scores of normalized reads of the indicated mRNAs from n=3 replicates are shown. Columns show data from two XLP2 patients or three controls. (B) Multiplexed tandem mass tag proteomic analysis of primary B-cells from XLP2 patients or healthy controls on Day 7 post-EBV infection or CD40L/IL-21 treatment. Unstimulated cells were harvested on Day 0. Z-scores of relative protein abundances of the indicated mRNAs from n=3 replicates are shown. Columns show data from two XLP2 patients or three controls. (C) Mean + standard deviation (SD) TP53 and BAX mRNA expression from n=3 replicates of RNAseq analysis of primary human B-cells on the indicated days post EBV infection . (D) Schematic diagram illustrating p53 that p53 target genes PUMA and NOXA can each upregulate BAX, which in turn induces the intrinsic apoptosis pathway. Red stars denote upregulation at Day 7 post-EBV infection relative to CD40L/IL-21 levels. (E) Mean + SD caspase 3/7 activity from n=3 replicates of primary B-cells expressing control or XIAP targeting sgRNAs and treated with BAI1 (5 μM) on day 4 post EBV infection. BAI1 was added from Day 0 onwards, replenished every 3 days. (F) Growth curve analysis of control versus XIAP edited primary B-cells and cultured with DMSO, zVAD-Fmk (20 μM) or BAI1 (5 μM) from Day 0 onwards. Mean ± SD fold change live cell numbers, relative to uninfected values, are shown. DMSO, BAI and zVAD-Fmk were replenished every 3 days. (G) Mean + SD caspase 3/7 activity from n=3 replicates of control versus XIAP edited primary B-cells cultured with DMSO or pifithrin-α at 5 or 20 μM on Day 4 post-infection. Statistical significance was assessed by two-tailed unpaired Student’s t test (F) or two-way ANOVA followed by Tukey’s multiple comparisons test (E and G). *p<0.05, ***p<0.001, ****p<0.0001.

Figure 5.. Embelin XIAP inhibition perturbs EBV-mediated primary B-cell outgrowth and sensitizes newly-infected cells to IFNγ-triggered apoptosis.

(A) FACS analysis of CD4+ or CD8+ T cell, CD56⁺ NK cell and CD19+ B-cell subsets from PBMCs of a control donor, infected with EBV and treated with DMSO or embelin (5 μM) on Day 7 post-EBV infection. DMSO and embelin were added starting from Day 0 and replenished every 3 days. (B) Mean + SD percentages of CD4+ or CD8+ T cell, CD56⁺ NK cell and CD19+ B-cells from (A) are shown. (C) Mean + SD % 7AAD+ cells from n=3 replicates of DMSO or embelin treated primary B-cells on Day 4 post-EBV infection or CD40L/IL21 treatment. DMSO and embelin were added starting from Day 0 and replenished every 3 days. (D) FACS analysis of CD19+ B-cells CellTrace Violet (CTV) dye dilution, whose abundance is reduced by half with each mitosis, from PBMCs treated with DMSO (control) or embelin (5 μM) on Day 7 post-EBV infection. The whole PBMC cell cultures were stained with CTV and infected with EBV and treated with DMSO or embelin (5 μM) as in (A). Cells were stained with anti-CD19 antibodies on day 7 and CTV on CD19+ B cell was analyzed. DMSO and embelin were added starting from Day 0 and replenished every 3 days. (E) Mean + SD percentages of 7AAD+ cells of primary human B-cells from controls, cultured alone or co-cultured with autologous PBMC in the presence of DMSO versus embelin (5 μM) for 4 days. B-cells were stained with CFSE prior to PBMC co-culture, which served as a cell trace marker to allow their FACS gating within mixed PBMC cultures. (F) Mean + SD caspase 3/7 activity from n=3 replicates of EBV-infected DMSO or embelin treated cells that were co-cultured with PBS vehicle, IFNg (50 ng/mL), TNFa (50 ng/mL), IL-6 (50 ng/mL) or IL-18 (50 ng/mL) on Day 4 post-infection. (G) Growth curve analysis of control primary human B-cells infected by EBV and treated with DMSO or embelin (5 μM) together with IFNγ, TNFα, IL-6 or IL-18 as indicated. Shown are mean ± SD fold change live cell numbers from n=3 replicates. Statistical significance was assessed by comparing each cytokine treated group with PBS control group. Statistical significance was assessed by two-tailed unpaired Student’s t test (B, C, G) or two-way ANOVA followed by Tukey’s multiple comparisons test (E and F). In all experiments, DMSO, Embelin and cytokines were refreshed every 3 days. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ns, not significant.

Figure 6.. Schematic model of key anti-apoptotic XIAP role in newly EBV-infected B-cells.

EBV drives rapid proliferation of newly infected B-cells, which triggers DNA damage, upregulation of p53 and of downstream NOXA, PUMA and BAX. XIAP blocks caspase activity and apoptosis in most settings, including with XLP1, enabling newly EBV-infected B-cells to undergo transformation and in XLP1 to cause high rates of lymphomas. Lymphomas are not observed in XLP2 patients, where the absence of XIAP enables caspase 3/7 activation and apoptosis induction over the first week of EBV infection, which is exacerbated by the inflammatory cytokine milieu, in particular IFNγ, restraining lymphomagenesis.