The T-Cell/CLL/Macrophage Triad Shapes a Supportive Tumor Microenvironment in CLL
Authors: van Attekum Martijn H.A. , Terpstra Sanne , Reinen Emilie , Von Lindern Marieke , Slinger Erik , Eric Eldering , Kater Arnon P. 
Published: October 2019

Abstract
Survival of CLL cells critically depends on heterotypic communication with benign bystanders cells in micro-environmental niches such as lymph node (LN) tissue. Here, mesenchymal stromal cells and macrophages, in concert with CD40L expressing T cells, are thought to participate in the dialog with the neoplastic B cells, but the mechanisms of this intricate interplay remain largely unknown. Moreover, whether CLL cells actively participate in shaping their prosurvival niche is poorly understood. We aimed to study 1) whether CD40 stimulation initiates active recruitment of monocytes by CLL cells, 2) whether CLL cells are able to differentiate these monocytes towards a supporting phenotype and 3) by which mechanism macrophages induce CLL survival.
We first studied the chemokinome of CLL cells after T cell stimulation using both microarray and Luminex techniques. Co-culture of autologous activated T cells with CLL cells resulted in induction of mRNA expression of CCL2,3,4,5,22 and IL10, which are known chemo-attractants for monocytes. These effects could be mimicked by CD40 activation of CLL cells. Protein screens of supernatants of CD40 activated CLL cells by Luminex assays confirmed increased protein expression of these chemo-attractants. Indeed, transwell assays showed enhanced migration of primary monocytes towards supernatants of CD40L stimulated CLL cells. Inhibitor experiments furthermore showed that the migratory effects of these chemokines was largely governed via the CCR2 and CCR3 receptors.
We next examined and compared polarization patterns of monocytes after differentiation with serum derived from CLL patients (N=25) or pooled healthy donor serum and found that CLL serum was able to differentiate macrophages towards a tumor supporting M2 phenotype. This finding was confirmed ex vivo by IHC, as M2 marker CD206 co-localizes with CD68 cells in CLL LNs, while the majority of macrophages in non-CLL derived LNs are CD80+ (M1 type).
Lastly, we examined how these macrophages exert their pro-survival effect on CLL. From a variety of Bcl-2 family proteins investigated, only Mcl-1 protein expression levels increased after interaction with macrophages. The relevance of Mcl-1 upregulation was verified by MCL-1 siRNA interference studies. The mechanism of induction of Mcl-1 was independent on NF-κB signaling, Mcl-1 mRNA transcription levels or protein stability, but rather unexpectedly appeared as a result of recruitment of polysomes to Mcl-1 mRNA, resulting in an increase in translation. This increase was accompanied by an increased phosphorylation of the rate-limiting translation initiation factor 4E-BP1 and ribosomal protein S6. The increase in Mcl-1 translation could be attributed to macrophage-induced Akt signaling.
In conclusions, these studies shed light on reciprocal cellular interactions in the CLL LN that shape pro-tumor differentiation of supporting cells, that in turn cause survival by changing the apoptotic balance. These interactions can be targeted at different levels, creating new treatment venues for this still incurable disease.

Disclosures
No relevant conflicts of interest to declare.