Role of T Cell-Derived IL-21 in the Proliferation of Chronic Lymphocytic Leukemia Cells,
Authors: Pascutti Maria F. , Tromp Jacqueline M. , Jak Margot , Derks Ingrid A.M. , van Lier Rene A.W. , Pals Steven T. , Van Oers Marinus H.J. , Kater Arnon P , Eldering Eric 
Published: October 2019

Abstract
Abstract 3871

Introduction:
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.

Results:
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.

Conclusion:
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.

Disclosures:
No relevant conflicts of interest to declare.