EXPRESSION AND FUNCTIONAL ACTIVITY OF THE TNFR-SUPERFAMILY MEMBER DEATH RECEPTOR 3 IN CHRONIC LYMPHOCYTIC LEUKEMIA B CELLS ACTIVATED BY THE BCR

Background: Growing evidences suggest a dynamic balance of B-cell chron- ic lymphocytic leukemia (B-CLL) cells between the blood and lymphoid tissues, which represent permissive niches for cell proliferation and survival. Within lymphoid tissue sites, molecules of the tumor necrosis factor (TNF) superfam- ily have been shown to play a supportive role and contribute to the pathogen- esis of B-CLL. Thereby, investigating expression and functions of novel TNFR- superfamily members in B-CLL would allow us to gain deeper insights into molecular crosstalk within leukemic microenvironments. Death receptor (DR) 3 is a TNFR-superfamily member expressed in lymphocyte-enriched tissues that binds to the TNF-like ligand 1A (TL1A), a TNF superfamily member. The TL1A/DR3 axis is implicated in regulatory mechanisms of adaptive immune response under physiological and pathological settings. Further evidence for the implication of TL1A in regulatory mechanisms arises from our recent data show- ing an inhibitory function of TL1A on B-cell proliferation. In leukemia cells, DR3 expression and functions have not been explored so far. CLL patients and to explore the possible role of TL1A/DR3 axis in the disease. Methods: B cells were purified from PBMC of 37 B-CLL patients by negative selection with magnetic beads. DR3 surface expression was measured by flow cytometry at baseline and following stimulation with F(ab’)2 anti-human IgM con- jugated to latex microspheres. DR3 expression was confirmed by western blot and immunofluorescence analysis on B-CLL lymph nodes. DR3 function was studied by MTT assay and Annexin V assay. TL1A serum levels were measured by ELISA. Results: Under basal conditions CLL B cells in vitro expressed low levels of DR3. Stimulation of the B cell receptor (BCR) with anti-IgM antibodies induced a sta- tistically significant increase of DR3 expression in CLL B cells (p<0.001). Induced DR3 expression showed great variability amongst B-CLL cells (variance (σ2)=6.38). Flow cytometry data were confirmed by Western blot analysis. The relevance of these findings was further confirmed by immuofluorescence analy- sis of B-CLL lymph-node specimens showing that in vivo high levels of DR3 were expressed by a number of B-CLL cells. To assess whether the anti-IgM-induced DR3 molecule was functionally active in CLL B cells, we examined the ability of DR3 to modulate their metabolic activity. Stimulation of DR3 with TL1A in the presence of BCR engagement showed that TL1A induced an equal or greater than 25% decrease of metabolic activity in 37.5% of B-CLL cell samples. In these samples the modulation is not due to reduced survival, as assessed by Annexin V assay. No change in metabolic activity was observed following TL1A treatment, in the absence of anti-IgM. Higher levels of DR3 expression were significantly associated with early-stage (Rai 0) disease (p=0.019) and higher serum levels of TL1A were significantly associated with early-stage (Rai 0) disease (p=0.023) and absence of CD38 expressions (p=0.035). CLL cells activated by the BCR stimulation express DR3 and TL1A reduces meta- bolic activity in some B-CLL cells. Herein, our data show a novel regulatory role for TL1A that, in the presence of antigen stimulation, may modulate leukemic cell metabolism through DR3 in early-stage B-CLL and suggest that homeostatic functions of TL1A may influence the clinical course of B-CLL disease.