Role of T Cell-Derived IL-21 in the Proliferation of Chronic Lymphocytic Leukemia Cells,

Lymph nodes (LN) from chronic lymphocytic leukaemia (CLL) patients contain characteristic proliferation centres, which are interspersed with CD154+ CD4+ T cells. We have previously shown that in vitro stimulation of peripheral blood (PB) CLL cells with CD154-expressing fibroblasts (3T40L) results in an apoptotic profile similar to the one found in LN (Smit et al, Blood 2007 Feb 15;109(4):1660; Kater et al, Br J Haematol 2004;127(4):404). However this stimulus fails to induce proliferation of CLL cells. In fact, the signals involved in CLL proliferation in vivo remain largely unknown. It has recently been described that IL-21 which is produced by activated T cells, has an essential role in activation and proliferation of normal B cells. The aim of this work was to analyze the contribution of IL-21 to the proliferation of CLL cells, both in vitro and in vivo.

We stimulated CLL cells with IL-21 in the presence or absence of 3T40L cells and assessed proliferation 5 days later. We observed an increase in the proliferation of CLL cells after the combined stimulation with CD40L and IL-21. In this setting, CLL cells divided once or twice. However, when fresh 3T40L cells and IL-21 were provided every 3–4 days, CLL cells could proliferate passed the fifth division. To analyze the contribution of IL-21 to proliferation in an in vitro setting better resembling the in vivo situation, we then studied the interaction between CLL cells and autologous activated T cells. CLL cells were positively selected from PB and cultured with autologous T cells, activated with CD3/CD28 antibodies (Tact), in the presence/absence of blocking antibodies against CD40L or IL-21 receptor (IL-21R). Proliferation was assessed 2 days later. Co-culture with Tact led to a CD40L- and IL-21-dependent increase in Ki67⁺CLL cells.

Next, we assessed the gene expression profile of CLL cells stimulated with CD40L and/or IL-21 by microarray analysis. CLL cells were stimulated overnight with medium, 3T40L cells, IL-21 or the combination, and then RNA was obtained and analyzed with Affimetrix U133 2.0 microarrays. In CD40L-stimulated cells more than 30 genes were up-regulated by IL-21 (fold induction>4; p<0.005), among which there were components of the JAK-STAT pathway like STAT3, and molecules related to cell proliferation like BCL3 and GS02. This information will allow us to generate an IL-21 signalling signature related to CLL cell proliferation that we will use to interrogate gene expression changes in CLL cells from LN samples.

Finally, we wished to ascertain whether IL-21 is being produced in vivo in CLL. For this, we performed IHC stainings on paraffin LN samples from untreated patients. We were able to observe IL-21 production by large cells, scattered among small lymphocytes, which are currently being characterized.

Our results indicate that IL-21 might play a role in the proliferation of CLL cells in vivo. This is not only important for understanding the biology of CLL but might also open new venues to treatment.

No relevant conflicts of interest to declare.