BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo

Abstract

A chronic antigenic stimulation is believed to sustain the leukemogenic development of chronic lymphocytic leukemia (CLL) and most of lymphoproliferative malignancies developed from mature B cells. Reproducing a proliferative stimulation ex vivo is critical to decipher the mechanisms of leukemogenesis in these malignancies. However, functional studies of CLL cells remains limited since current ex vivo B cell receptor (BCR) stimulation protocols are not sufficient to induce the proliferation of these cells, pointing out the need of mandatory BCR co-factors in this process. Here, we investigated benefits of several BCR co-stimulatory molecules (IL-2, IL-4, IL-15, IL-21 and CD40 ligand) in multiple culture conditions. Our results demonstrated that BCR engagement (anti-IgM ligation) concomitant to CD40 ligand, IL-4 and IL-21 stimulation allowed CLL cells proliferation ex vivo. In addition, we established a proliferative advantage for ZAP70 positive CLL cells, associated to an increased phosphorylation of ZAP70/SYK and STAT6. Moreover, the use of a tri-dimensional matrix of methylcellulose and the addition of TLR9 agonists further increased this proliferative response. This ex vivo model of BCR stimulation with T-derived cytokines is a relevant and efficient model for functional studies of CLL as well as lymphoproliferative malignancies.

Proliferative Signals in Chronic Lymphocytic Leukemia; What Are We Missing?

“…CD40 stimulation in combination with IL-21 or IL-4 is sufficient to induce CLL proliferation in vitro whereas for BCR stimulation this is not the case ( 6 , 7 ). However, CLL cells treated with ibrutinib in vivo did not proliferate upon CD40/IL-21 stimulation in vitro , an effect which can be recapitulated by in vitro inhibition of either BTK, PI3K or SYK, indicating the requirement of BCR kinases in CD40-mediated proliferation ( 107 ).…”

“…Aside from BCR, CD40, and TLR stimulation, various other in vitro stimulations and culture conditions have been applied in the context of CLL proliferation, including factors like BAFF and APRIL, as well as co-culture with stromal cells, follicular dendritic cells or nurse-like cells ( 1 ). These and other candidates are certainly of interest, yet the interaction with feeder cells combined with their secretion of cytokines makes the identification of essential factors difficult ( 6 ). In addition, our previous efforts have not managed to show a direct role for BAFF or APRIL in human CLL proliferation ( 25 , 26 ).…”

“…Accordingly, CLL cells within proliferation centers of the LN showed high expression of cyclin D2 and downregulation of p27 ( 111 ). Therefore, CLL proliferation may depend on costimulatory signals such as those delivered through CD40 or TLRs, possibly in combination with cytokines ( 6 , 7 , 16 ). The interleukins are a family of cytokines that serve as key regulatory elements within the immune system and a number of specific interleukins have been identified as being associated with the proliferation of CLL cells in vitro ( 43 ).…”

“…However, CLL cells treated with ibrutinib in vivo did not proliferate upon CD40/IL-21 stimulation in vitro , an effect which can be recapitulated by in vitro inhibition of either BTK, PI3K or SYK, indicating the requirement of BCR kinases in CD40-mediated proliferation ( 107 ). CLL proliferation is significantly increased further when BCR stimulation is added to the combined stimulation of CD40 and cytokines whereas for healthy B cells this is not the case, showing that these separate signaling nodes may potentially cooperate to drive CLL proliferation in vivo ( 6 ).…”

“…Strong evidence exists for a crucial role of BCR signaling to drive CLL disease progression, especially the success of inhibitors targeting BCR-associated kinases ( 5 ). However, in vitro BCR triggering only leads to low-level activation without induction of proliferation, suggesting that additional factors that play a role in vivo are missing ( 6 ). Several other receptors are known to mediate interactions between CLL cells and the microenvironment, such as CD40 or Toll-like receptor (TLRs), in combination with cytokine receptors which have been shown to induce proliferation upon in vitro stimulation ( 7 , 8 ).…”