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

Abstract

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.

Figure 1

B cell lines derived from early B cell cancers aberrantly express BAFF-R. (A) Flow cytometric analysis of BAFF-R staining (empty histogram) and isotype control staining (filled histogram) in precursor B-ALL-derived cell lines Reh and NALM-6 compared to the Burkitt's lymphoma-derived mature B cell lines Loukes and RAMOS. Ratio of geometric mean fluorescence intensity of specific staining to isotype control staining (ΔMFI) is inset. (B) Quantitative RT-PCR analysis of c-Rel mRNA levels in the precursor B-ALL and Burkitt's lymphoma lines. (C) Immunoblot for c-Rel and nuclear loading control Histone H1. Approximately 50 µg of nuclear extracts were loaded and blotted for the labeled proteins.

Figure 2

BAFF-R-expressing early B cell lines show significant BAFF-R promoter reporter activity. The genomic region spanning the 0.5 kb upstream of TNFRSF13C, the gene encoding BAFF-R, ending 6 bp 5′ of the start codon, was cloned and inserted into firefly luciferase reporter vector pGL3-Basic. This reporter was co-transfected into the target cell lines by electroporation with a fixed amount of control Renilla luciferase reporter vector pRL-TK. Specific promoter activity is reported as relative light units, the ratio of firefly: Renilla luciferase activity normalized to the relative activity of the empty pGL3-Basic vector. **p < 0.01.

Figure 3

CLL cells lose significant BAFF-binding capacity coincident with downregulation of BAFF-R surface expression. (A) Normal B cells and CLL cells were stained for BAFF-R and an isotype-matched control antibody. The ratio of the geometric mean fluorescence intensity in specific to control antibody staining is reported here (ΔMFI). CLL samples were divided into mutated and unmutated groups reflecting the amount of somatic hypermutation undergone at the immunoglobulin locus as determined by sequencing. (B) The same CLL samples tested for BAFF-R expression in (A) and independent normal B cell controls were tested for BAFF binding by incubation with an anti-BAFF antibody labeled with biotin and stained secondarily with streptavidin bound to fluorophore. ΔMFI was determined as above relative to an isotype-matched control primary antibody. BAFF-binding was determined both immediately after isolation (“Endogenous BAFF”) and after 20 minute incubation with an excess of recombinant BAFF (“Exogenous BAFF”). (C) BAFF-binding in the normal and CLL B cells represented as matched pairs before and after the addition of recombinant BAFF. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

The normal CD5⁻ B cell compartment in CLL patients retains greater BAFF-R expression than the malignant clone. (A) Representative histogram of normal peripheral blood B cells (far right histogram) and malignant CLL cells (center and left histograms) isolated with Ficoll-Hypaque separation and co-stained with antibodies to CD19, CD5 and either BAFF-R (black outlined histograms) or an isotype-matched control antibody (filled histogram without outline). (B) Matched CD5⁺ (CLL) and CD5⁻ (normal peripheral blood B) cell BAFF-R expression relative to the isotype-matched control antibody, ΔMFI. *p < 0.05.

Figure 5

The regulation of BAFF-R expression in CLL is primarily post-transcriptional. (A) Relative expression of BAFF-R and NFκB family member c-Rel in CLL and normal peripheral blood B cells. Expression levels were determined by qRT-PCR and normalized to 18S rRNA. (B) The relationship between c-Rel and BAFF-R mRNA levels in CLL and normal B cells. (C) Immunoblot for BAFF-R and β-actin in five CLL and four normal peripheral blood B cell samples. Densitometric analysis was performed and quantified as a ratio of adjusted intensity volume of BAFF-R to β-actin and normalized to a scale of 0 to 10. Relative values are noted above the BAFF-R blot.

Figure 6

CLL cells employ native BAFF-R regulatory pathways in response to CpG stimulation. Isolated CLL cells were stimulated with CpG and IL-2 in vitro for two days (empty histogram) or not stimulated (dark gray histogram) and stained for BAFF-R expression or an isotype-matched control antibody (light gray histogram).