The TNF-Family Cytokine TL1A/Death Receptor 3 System Reduces Metabolic Activity in Chronic Lymphocytic Leukemia B Cells

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily, expressed on dendritic cells, macrophages, lymphocytes, and endothelial cells. It is the only described ligand for death receptor 3 (DR3), a death-domain containing receptor of the TNF-receptor superfamily, mainly expressed on lymphocytes, natural killer (NK) cells, and NK-T cells. TL1A–DR3 interaction results in co-stimulatory signaling for activated T cells, leading to amplification of inflammation and immune responses, correlated with greater pathogenicity in diverse autoimmune diseases. In contrast, in activated B cells the TL1A/DR3 system exerts inhibitory effect on cell proliferation, suggesting that TL1A may also have modulatory and homeostatic functions on B-cell expansion. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults in Western countries. It is well documented that several elements within the tumor microenvironment, including antigens, cytokines, adhesion molecules, and surface receptors, play a fundamental role in supporting the growth of CLL. In contrast, little is known on regulatory mechanisms of CLL growth. In this study, we have investigated the possible regulatory role of the TL1A/DR3 system in B cells from CLL patients.

CLL patients from the Hematology Unit at the University Hospital of Verona (Italy) were included in this study (n=37). Disease characteristics and demographic variables were collected on all patients. Purified B cells were obtained by negative selection. DR3 expression was measured on peripheral blood B cells by flow cytometry and western blot at baseline and following B cell receptor (BCR) stimulation with anti-human IgM. DR3 expression was confirmed on lymph-node CLL specimens by immunofluorescence. Metabolic activity of CLL B cells was analyzed by MTT assay. Apoptosis was analyzed by Annexin V assay. TL1A serum levels in CLL patients were measured by ELISA. Baseline analysis in vitro showed that DR3 was expressed at low levels in CLL B cells. Cell activation through stimulation of the BCR induced a significant increase of DR3 expression in a fraction of CLL B cells (p<0.001). Higher levels of DR3 expression were associated with early-stage (Rai 0) disease (p=0.019). The relevance of these findings was confirmed by immuofluorescence analysis of B-CLL lymph-node specimens showing that DR3 was expressed at high levels in some CLL B cells in vivo. Treatment of purified CLL B cells with exogenous recombinant TL1A in vitro, in the presence of BCR stimulation, induced a decreased metabolic activity in 3/8 B-CLL cell samples (37.5%). No change in CLL metabolic activity was observed following treatment with TL1A alone, in the absence of BCR stimulation. Treatment of B-CLL samples with TL1A, either in the presence or absence of BCR stimulation, induced no changes in cell viability, thus ruling out that decreased metabolic activity was due to reduced survival. A soluble form of TL1A was detected in the sera of CLL patients 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).


In summary, this study shows that B-CLL metabolic activity can be reduced through the activity of the TL1A/DR3 system, in the presence of the BCR stimulation. Furthermore, TL1A and DR3 levels are higher in patients with early-stage disease. Taken together, these findings suggest that the TL1A/DR3 system in vivo, in the presence of antigen stimulation, may modulate leukemic cell metabolism in early-stage CLL, thus influencing the clinical course of disease.