High-Level Expression of the T Cell Chemokines CCL3 and CCL4 by Chronic Lymphocytic Leukemia B Cells in Nurselike Cell Co-Cultures and in Response to BCR Stimulation.
Authors: Burger Jan A. , Niedermeier Matthias , Bürkle Andrea , Hartmann Elena , Wierda William G. , Keating Michael J. , Rosenwald Andreas 
Published: October 2019

Abstract
Despite their apparent longevity in vivo, isolated Chronic Lymphocytic Leukemia (CLL) B cells generally undergo spontaneous apoptosis in vitro when cultured under conditions that support the growth of human B cell lines. This suggests that interactions between CLL cells and a distinct tissue microenvironment in the marrow and the lymphatic tissues, where CLL cells are in close contact with accessory cells (mesenchymal stromal cells, and CD68+ “nurselike cells”/NLC), are critical for the progression of the disease. NLC can be detected in secondary lymphoid tissues from CLL patients and appear to be an integral part of the CLL microenvironment, comparable to lymphoma-associated macrophages in follicular lymphoma. The molecules involved in CLL-NLC cross talk are only partially understood. Therefore, we examined the gene expression profile of purified CLL B cells using Affymetrix U133 Plus 2.0 Arrays to define distinct expression profiles induced in purified CLL B cells by co-culture with NLC. When compared to freshly isolated blood CLL B cells, we found that CLL B cells co-cultured for 14 days with NLC displayed high level expression of two T cell chemoattractants, macrophage inflammatory protein-1a and b (MIP-1 a, MIP-1b, also called CCL3 and CCL4). CCL3 and CCL4 expression levels correlated with ZAP-70 expression by the CLL cells, suggesting that B cell receptor signaling is involved in inducing the expression of these chemokines. Supernatants from CLL-NLC co-cultures harvested 7 and 14 days after initiation of the cultures revealed high CCL3 and CCL4 protein levels (up to >30 ng/ml) by ELISA, predominantly in the same CLL cases that displayed high CCL3, CCL4 and ZAP-70 expression by expression profiling. Moreover, serum samples from CLL patients were tested for CCL3 and CCL4 protein expression by ELISA. These studies demonstrated higher CCL3 and CCL4 serum levels in CLL patients when compared to healthy volunteers. CCL3 and CCL4 serum levels were found to be higher in CLL patients that were CD38+, displayed non-mutated IgVH genes, and b2 microglobulin levels that are > 4 mg/L. In vitro, B cell receptor (BCR) triggering of CLL cells, using anti-IgM antibodies, induced a rapid and robust induction of CCL3 and CCL4 protein production by CLL B cells. In contrast, CD40 triggering did not induce expression of these chemokines. These studies reveal a novel mechanism of cross-talk between CLL B cells and their microenvironment, namely the induction of two T cell chemokines, CCL3 and CCL4, by CLL-NLC interaction. Thus, we provide the first evidence that neoplastic B cells are an important source of T cell chemokines, that can induce recruitment of T cells of helper/effector phenotype to sites of cognate T cell-CLL interactions. Besides inducing the outgrowth of NLC, this is another mechanism how CLL cells actively create a microenvironment that favors their growth and survival.
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