Background. In the microenvironments where B-CLL develops, complex molecular networks propagate signals that confer growth advantage to CLL. Within this context, interplaying between TNF superfamily members and their cognate receptors has been shown to play a relevant role in controlling B-CLL growth and survival. TNF-like protein 1A (TL1A) is a recently discovered member of the TNF superfamily expressed on various cell types, including macrophages, dendritic cells, and endothelial cells. The TL1A cognate receptor, death receptor 3 (DR3), is a member of the TNF receptor superfamily expressed on various cell types, including activated T cells and macrophages. TL1A-DR3 interactions have been shown to modulate immune and inflammatory functions. So far, the expression of DR3 on B-lineage cells as well as possible roles of TL1A-DR3 interplaying in CLL biology has not been explored. Aims. The objective of the study was to investigate DR3 expression and function(s) in B-CLL. Methods. B cells were purified from PBMC of 23 B-CLL patients and 13 healthy donors by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and various time points following stimulation with F(ab’)2 anti-human IgM conjugated to latex microspheres. DR3 mRNA was detected by quantitative RT-PCR. Phosphorylation states of NF-kappaB, Erk1/2, and TBK1 were measured by using phospho-specific antibodies and flow cytometry in basal condition and following DR stimulation of cells with agonistic antibody at different time points (15, 30, 60, 180 minutes). Results. Although both healthy and CLL B cells did not express DR3 in basal conditions, stimulation of B cell receptor (BCR) induced a statistically significant increase of DR surface expression in healthy as well as malignant B cells (p<0.001 for both cells). Time course analysis showed that DR3 expression peaked at 24 hour after stimulation. DR3 expression was confirmed also at the mRNA level. Time course analysis showed that DR3 mRNA in B-CLL cells peaked at 2.5 hour following anti-IgM stimulation (4-fold change with respect to basal conditions). Anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells if compared with healthy B cells (p<0.05). Interestingly, when B-CLL patients were stratified by IGVH mutational status, anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells harboring unmutated IGVH genes compared with cells with mutated IGVH genes. To assess whether the anti-IgM-induced DR3 molecule was functionally active in B-CLL cells, we examined the ability of DR3 to trigger phosphorylation events in biologically relevant signaling nodes (i.e. Erk1/2, NF-kappaB, and TBK1) by stimulating cells with anti-DR3 agonistic antibodies. Phospho-specific flow cytometry analysis showed that DR3 engagement induced phosphorylation of Erk1/2 but not NF-kappaB or TBK1. Conclusions. We described for the first time the expression of functional DR3 molecules in B-lineage cells activated by BCR stimulation in healthy and pathological cells (i.e. B-CLL cells). The findings that anti-IgM-induced DR3 expression was higher in B-CLL cells if compared with healthy B cells and that, among B-CLL patients, this expression was higher in cells with unmutated IGVH genes, suggest that DR3 stimulation may have a role in B-CLL pathogenesis.

B-cell chronic lymphocytic leukemia cells espress functional death receptor 3 following stimulation of the B cell receptor

CAVALLINI, Chiara;LOVATO, Ornella;NICHELE, Ilaria;MALPELI, Giorgio;BERTOLASO, Anna;AMBROSI, GIORGINA;ZAMO', Alberto;SCARPA, Aldo;PIZZOLO, Giovanni;SCUPOLI, Maria
2011

Abstract

Background. In the microenvironments where B-CLL develops, complex molecular networks propagate signals that confer growth advantage to CLL. Within this context, interplaying between TNF superfamily members and their cognate receptors has been shown to play a relevant role in controlling B-CLL growth and survival. TNF-like protein 1A (TL1A) is a recently discovered member of the TNF superfamily expressed on various cell types, including macrophages, dendritic cells, and endothelial cells. The TL1A cognate receptor, death receptor 3 (DR3), is a member of the TNF receptor superfamily expressed on various cell types, including activated T cells and macrophages. TL1A-DR3 interactions have been shown to modulate immune and inflammatory functions. So far, the expression of DR3 on B-lineage cells as well as possible roles of TL1A-DR3 interplaying in CLL biology has not been explored. Aims. The objective of the study was to investigate DR3 expression and function(s) in B-CLL. Methods. B cells were purified from PBMC of 23 B-CLL patients and 13 healthy donors by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and various time points following stimulation with F(ab’)2 anti-human IgM conjugated to latex microspheres. DR3 mRNA was detected by quantitative RT-PCR. Phosphorylation states of NF-kappaB, Erk1/2, and TBK1 were measured by using phospho-specific antibodies and flow cytometry in basal condition and following DR stimulation of cells with agonistic antibody at different time points (15, 30, 60, 180 minutes). Results. Although both healthy and CLL B cells did not express DR3 in basal conditions, stimulation of B cell receptor (BCR) induced a statistically significant increase of DR surface expression in healthy as well as malignant B cells (p<0.001 for both cells). Time course analysis showed that DR3 expression peaked at 24 hour after stimulation. DR3 expression was confirmed also at the mRNA level. Time course analysis showed that DR3 mRNA in B-CLL cells peaked at 2.5 hour following anti-IgM stimulation (4-fold change with respect to basal conditions). Anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells if compared with healthy B cells (p<0.05). Interestingly, when B-CLL patients were stratified by IGVH mutational status, anti-IgM-induced DR3 expression levels were significantly higher in B-CLL cells harboring unmutated IGVH genes compared with cells with mutated IGVH genes. To assess whether the anti-IgM-induced DR3 molecule was functionally active in B-CLL cells, we examined the ability of DR3 to trigger phosphorylation events in biologically relevant signaling nodes (i.e. Erk1/2, NF-kappaB, and TBK1) by stimulating cells with anti-DR3 agonistic antibodies. Phospho-specific flow cytometry analysis showed that DR3 engagement induced phosphorylation of Erk1/2 but not NF-kappaB or TBK1. Conclusions. We described for the first time the expression of functional DR3 molecules in B-lineage cells activated by BCR stimulation in healthy and pathological cells (i.e. B-CLL cells). The findings that anti-IgM-induced DR3 expression was higher in B-CLL cells if compared with healthy B cells and that, among B-CLL patients, this expression was higher in cells with unmutated IGVH genes, suggest that DR3 stimulation may have a role in B-CLL pathogenesis.
B-cell chronic lymphocytic leukemia; death receptor 3; DR3
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/359192
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