Differentiation of Chronic Lymphocytic Leukemia B Cells into Immunoglobulin Secreting Cells Decreases LEF-1 Expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation

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

Stimulation of Cell Division by Argon and Nd:YAG Loser Trabeculoplasty in Cynomolgus Monkeys

Although laser treatment of the trabecular meshwork is the most common form of surgery for glaucoma, the tissue response to this therapy is still incompletely understood. We applied argon or Nd:YAG laser to the trabecular meshwork of six monkeys. Cell division was identified by injecting tritiated thymidine into the anterior chamber 24 hr after laser application. Autoradiography of tissue sections revealed significantly more labelled cells in eyes treated with laser than in the untreated controls. In addition, cells in neighboring tissues such as iris, ciliary body and sclera showed labelling in association with laser application. Furthermore, comparison of argon-induced lesions with those caused by pulsed Nd.YAG suggests that there are quantitative and qualitative differences in the response of trabecular meshwork and surrounding tissues to these two forms of laser energy. Invest Ophthalmol Vis Sci 31:115-124,199

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

Stage-Specific Non-Coding RNA Expression Patterns during In Vitro Human B Cell Differentiation into Antibody Secreting Plasma Cells

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies

<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