Selective Induction of DNA Repair Pathways in Human B Cells Activated by CD4+ T Cells

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID), GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID -/- mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells

Transcriptional and post-transcriptional mechanisms of BAFF-receptor dysregulation in human B lineage malignancies

Together, circulating BAFF and dominant receptor BAFF-R homeostatically regulate the humoral immune system. Consistently aberrant BAFF-R expression in leukemic cells reveals an intimate connection of these cells' malignant physiology to the BAFF/BAFF-R axis and also provides an additional survival mechanism to the expressing cells. In this study, we used primary cells and cell lines to interrogate the mechanisms underlying aberrant BAFF-R expression in precursor B acute lymphoblastic leukemia (precursor B-ALL) and mature B chronic lymphocytic leukemia (CLL). Here we demonstrate the aberrant expression of BAFF-R in precursor B-ALL cell lines and reveal that these cells acquire BAFF-R expression through premature transcriptional activation of the BAFF-R promoter in coordination with regulatory transcription factor c-Rel. Investigations using primary CLL cells provide a crucial counterpoint through their paucity of BAFF-R relative to their benign mature B cell counterparts, which we establish as functionally significant in its depletion of the CLL cells' BAFF-binding capacity. Furthermore, BAFF-R downregulation in CLL patients is revealed here to be restricted to the malignant compartment and mediated post-transcriptionally in order to compensate for the consistently unchanged levels of transcription factor c-Rel and BAFF-R mRNA. Finally, we present evidence that CLL cells retain endogenous mechanisms of BAFF-R regulatory control despite active receptor dysregulation

DNA repair proteins are selectively overexpressed in GC B cells.

<p><b>A.</b> IHC staining of tonsillar tissue sections with antibodies against DNA mismatch repair proteins MSH2, MSH6, MLH1, and AID. <b>B.</b> Western detection of various DNA repair proteins in total tonsillar B cells (presort), naïve (N), GC, and memory (M) B cells. The data are representative of three independent experiments.</p

<i>Ex vivo</i> induction of SHR and AID.

<p><b>A.</b> Purified peripheral blood B cells were stimulated with CpG, CD40 ligand, and/or anti-Ig, in the presence of IL2 and IL10 for 3 days, or coculture with anti-CD3 and anti-CD28 activated CD4 T cells for 3 days. <b>B.</b> B cells were activated by anti-CD3 and anti-CD28 activated CD4 T cells through direct coculture, transwell coculture, or activated with CpG for 3 days. Expression of select representative SHR genes were analyzed in activated B cells and activated T cells as a control (B cells and T cells were repurified when T/B direct coculture was the mode of activation) by real-time RT-PCR.</p

Real-time RT-PCR quantification of mRNA of SHR repair genes in tonsillar naïve (N), GC centroblast (CB), GC centrocyte (CC) and memory (M) B cells.

<p>Three tonsil pools each prepared from 6 individual tonsils were used. Genes induced transcriptionally in GC B cells are marked with the red circles while the ones not induced are highlighted with the blue circles. Statistical significance: *<i>p</i><0.05, **<i>p</i><0.01.</p