Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometrybased assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgMmodulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.Association between B-cell receptor responsiveness and disease progression in B-cell chronic lymphocytic leukemia: results from single cell network profiling studies

B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced HO is thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous HO in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous HO in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous HO in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL.

TNF-like ligand 1A (TL1A) and its unique receptor death receptor 3 (DR3) acts as broad T-cell costimulator involved in regulatory mechanisms of adaptive immune response under physiological and pathological settings. Moreover, we have recently shown that TL1A negatively regulates B-cell proliferation. Despite increasing interest on the TL1A/DR3-axis functions, very little is known on its expression and role in leukemia. In this study, we investigated the expression and function of TL1A/DR3 axis in chronic lymphocytic leukemia (CLL). DR3 was differentially expressed in activated CLL cells and predominantly detected in patients with early clinical stage disease. Soluble TL1A has been revealed in the sera of CLL patients where higher TL1A levels were associated with early stage disease. T cells, monocytes and leukemic B cells have been identified as major sources of TL1A in CLL. The relevance of these findings has been sustained by functional data showing that exogenous TL1A reduces CLL proliferation induced by stimulation of the B cell receptor. Overall, these data document the expression of the TL1A/DR3 axis in early-stage CLL. They also identify a novel function for TL1A as a negative regulator of leukemic cell proliferation that may influence the CLL physiopathology and clinical outcome at an early-stage disease.

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