Expression Profile and Regulation of BAFF and APRIL Receptors in Chronic Lymphocytic Leukemia,

Abstract 3880

The two TNF family proteins, B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL], and their three receptors, transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R] are critical regulators of normal B-cell development and survival. In CLL, both proteins can rescue CLL cells from apoptosis as shown in in vitro studies. We and others have previously shown that patients with CLL and other B-cell chronic lymphoproliferative disorders show abnormal BAFF and APRIL serum levels. Additionally, a few reports indicate that CLL cells can express BAFF and APRIL receptors. Nevertheless, there is no a meaningful and comparative analysis of BAFF-R, TACI and BCMA levels in CLL. We therefore quantitatively assessed BAFF-R, TACI and BCMA on B-cells from patients with CLL and healthy controls. The expression of BAFF-R, TACI and BCMA was analyzed by flow cytometry in purified peripheral blood B cells from 42 patients with CLL and 13 healthy controls. BAFF receptor was the most highly expressed receptor in both CLL and normal B cells (MFI ratios, 161.5 and 179.1, respectively). TACI was heterogeneously expressed and almost undetectable in 15 CLL and 5 normal B cells. BCMA was expressed on all CLL and normal B cells. Furthermore, the expression of TACI and BCMA was significantly higher on CLL cells than on normal B cells (p=0.034 and p<0.001, respectively). A correlation was also observed between the expression of BAFF and APRIL receptors and prognostic factors (i.e, IGHV mutational status and cytogenetics). Higher BCMA expression on leukemic cells were detected in patients with unmutated IGHV gene and high-risk genetics (17p-, 11q-, trisomy 12 or 3 or more cytogenetics abnormalities) (p=0.045 and p=0.068). Additionally, patients with higher levels of BCMA on the CLL cells also had a significantly higher risk of disease progression, with a median progression-free interval of 57 vs. 206 months (p=0.021). We further examined whether activation signals can modulate BAFF-R, TACI and BCMA expression and thereby the sensitivity of CLL cells to respond to BAFF and APRIL. In 10 CLL patients (5 mutated, 5 unmutated) and six healthy controls, receptors expression was measured at 48 hours after stimulation with F(ab')² antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling of CD69 and Annexin V/TO-PRO-3, were evaluated at 48 and 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). After 48h culture, an increase of all three receptors was observed in normal B-cells in response to either BCR stimulation or CD40 ligation. In CLL cells, CD40 ligation induced a significant up-regulation of TACI expression (p=0.007) and a significant reduction of BCMA expression (p=0.007). In contrast, BCR stimulation induced almost no variation in CLL receptors expression. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). Of note, no differences were observed in the regulation of BAFF and APRIL receptors nor in the activation or viability on CLL cells according to the IGHV mutational status. The addition of exogenous soluble BAFF or APRIL resulted in an increase in the viability of normal B-cells at 72 hours independently of cells stimulation through BCR or CD40 ligation. The viability of CLL cells was significantly increased upon CD40 stimulation whereas in non-stimulated or BCR-stimulated CLL cells the addition of BAFF and APRIL had a modest effect on their viability. Altogether, these results indicate that BAFF and APRIL receptors are differentially expressed on CLL and normal B-cells, with TACI and BCMA being highly expressed on leukemic cells. In addition, higher BCMA expression was significantly associated with poor prognostic variables (i.e, unmutated IGHV genes, poor cytogenetics) and higher risk of disease progression. Interestingly, activatory signals including BCR and CD40 ligation influenced BAFF and APRIL receptor expression and responsiveness to either BAFF or APRIL. CD40 ligation on CLL cells induced TACI up regulation by positively impacting on the survival promoting effect of BAFF and APRIL. Collectively, these findings link positive regulatory signals of BCR and CD40 ligation with the pro-survival effect of BAFF and APRIL and their receptors on leukemic CLL cells.