Transcription factors of the alternative NF-κB pathway are required for germinal center B-cell development.

The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.

Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B cells.

Signaling through the canonical nuclear factor-κB (NF-κB) pathway is critical for the generation and maintenance of mature B cells and for antigen-dependent B-cell activation. c-REL (rel) and RELA (rela) are the downstream transcriptional activators of the canonical NF-κB pathway. Studies of B cells derived from constitutional rel knockout mice and chimeric mice repopulated with rela-/- fetal liver cells provided evidence that the subunits can have distinct roles during B-cell development. However, the B cell-intrinsic functions of c-REL and RELA during B-cell generation and antigen-dependent B-cell activation have not been determined in vivo. To clarify this issue, we crossed mice with conditional rel and rela alleles individually or in combination to mice that express Cre-recombinase in B cells. We here report that, whereas single deletion of rel or rela did not impair mature B-cell generation and maintenance, their simultaneous deletion led to a dramatic reduction of follicular and marginal zone B cells. Upon T cell-dependent immunization, B cell-specific deletion of the c-REL subunit alone abrogated the formation of germinal centers (GCs), whereas rela deletion did not affect GC formation. T-independent responses were strongly impaired in mice with B cell-specific deletion of rel, and only modestly in mice with RELA-deficient B cells. Our findings identify differential requirements for the canonical NF-κB subunits c-REL and RELA at distinct stages of mature B-cell development. The subunits are jointly required for the generation of mature B cells. During antigen-dependent B-cell activation, c-REL is the critical subunit required for the initiation of the GC reaction and for optimal T-independent antibody responses, with RELA being largely dispensable at this stage.

Functional dissection of the chromosome 13q14 tumor-suppressor locus using transgenic mouse lines.

Deletion of chromosomal region 13q14 represents the most common genetic aberration in B-cell chronic lymphocytic leukemia (CLL). 13q14 deletions are commonly large and heterogeneous in size and affect multiple genes. We recently found that targeted deletion in mice of the 0.11 megabase (mb)-long minimal deleted region (MDR) encompassing the DLEU2/miR-15a/16-1 cluster recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5(+) monoclonal B-cell lymphocytosis, and CD5(-) non-Hodgkin lymphomas. In the present study, we demonstrate that additional deletion of the 0.69-mb large genomic region telomeric to the MDR called the common deleted region (CDR) changed the spectrum of lymphoproliferations developing in CDR- versus MDR-deleted mice in that the number of CLL among B-cell lymphoproliferations was significantly elevated in the former. In addition, CDR-deleted mice seemed to succumb to their disease faster than MDR-deleted mice. Comparing HCDR3 regions of CD5(+) lymphoproliferations derived from this and published CLL mouse models, 44% (29 of 66) of junctions could be assigned to 8 sets of highly similar HCDR3 regions, demonstrating that CLL developing in mice frequently expresses almost identical, stereotypic Ag receptors. These results suggest that the size of 13q14 deletions influences the phenotype of the developing lymphoproliferations and potentially the severity of disease, suggesting a tumor-suppressor function for genetic elements in addition to DLEU2/miR-15a/16-1.

The arthritis-associated HLA-B*27:05 allele forms more cell surface B27 dimer and free heavy chain ligands for KIR3DL2 than HLA-B*27:09.

OBJECTIVES: HLA-B*27:05 is associated with AS whereas HLA-B*27:09 is not associated. We hypothesized that different interactions with KIR immune receptors could contribute to the difference in disease association between HLA-B*27:05 and HLAB*27:09. Thus, the objective of this study was to compare the formation of ß2m-free heavy chain (FHC) including B27 dimers (B272) by HLA-B*27:05 and HLA-B*27:09 and their binding to KIR immunoreceptors. METHODS: We studied the formation of HLA-B*27:05 and HLA-B*27:09 heterotrimers and FHC forms including dimers in vitro and in transfected cells. We investigated HLA-B*27:05 and HLA-B*27:09 binding to KIR3DL1, KIR3DL2 and LILRB2 by FACS staining with class I tetramers and by quantifying interactions with KIR3DL2CD3ε-reporter cells and KIR3DL2-expressing NK cells. We also measured KIR expression on peripheral blood NK and CD4 T cells from 18 HLA-B*27:05 AS patients, 8 HLA-B27 negative and 12 HLA-B*27:05+ and HLA-B*27:09+ healthy controls by FACS staining. RESULTS: HLA-B*27:09 formed less B272 and FHC than HLA-B*27:05. HLA-B*27:05-expressing cells stimulated KIR3DL2CD3ε-reporter T cells more effectively. Cells expressing HLA-B*27:05 promoted KIR3DL2+ NK cell survival more strongly than HLA-B*27:09. HLA-B*27:05 and HLA-B*27:09 dimer tetramers stained KIR3DL1, KIR3DL2 and LILRB2 equivalently. Increased proportions of NK and CD4 T cells expressed KIR3DL2 in HLA-B*27:05+ AS patients compared with HLA-B*27:05+, HLA-B*27:09+ and HLA-B27- healthy controls. CONCLUSION: Differences in the formation of FHC ligands for KIR3DL2 by HLA-B*27:05 and HLA-B*27:09 could contribute to the differential association of these alleles with AS.

Soluble ephrin a1 is necessary for the growth of HeLa and SK-BR3 cells.

BACKGROUND: Ephrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. In malignancy, the precise role of EFNA1 and its preferred receptor, EPHA2, is controversial. Several studies have found that EFNA1 may suppress EPHA2-mediated oncogenesis, or enhance it, depending on cell type and context. However, little is known about the conditions that influence whether EFNA1 promotes or suppresses tumorigenicity. EFNA1 exists in a soluble form as well as a glycophosphatidylinositol (GPI) membrane attached form. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2. RESULTS: Using a RNAi strategy to reduce the expression of endogenous EFNA1 and EPHA2, we found that both EFNA1 and EPHA2 are required for growth of HeLa and SK-BR3 cells. The growth defects could be rescued by conditioned media from cells overexpressing soluble EFNA1. Interestingly, we found that overexpression of the membrane attached form of EFNA1 suppresses growth of HeLa cells in 3D but not 2D. Knockdown of endogenous EFNA1, or overexpression of full-length EFNA1, resulted in relocalization of EPHA2 from the cell surface to sites of cell-cell contact. Overexpression of soluble EFNA1 however resulted in more EPHA2 distributed on the cell surface, away from cell-cell contacts, and promoted the growth of HeLa cells. CONCLUSIONS: We conclude that soluble EFNA1 is necessary for the transformation of HeLa and SK-BR3 cells and participates in the relocalization of EPHA2 away from sites of cell-cell contact during transformation.

Alpha-sarcin catalytic activity is not required for cytotoxicity.

BACKGROUND: Alpha-sarcin is a protein toxin produced by Aspergillus giganteus. It belongs to a family of cytotoxic ribonucleases that inactivate the ribosome and inhibit protein synthesis. alpha-Sarcin cleaves a single phosphodiester bond within the RNA backbone of the large ribosomal subunit, which makes the ribosome unrecognizable to elongation factors and, in turn, blocks protein synthesis. Although it is widely held that the protein synthesis inhibition caused by the toxin leads to cell death, it has not been directly shown that catalytically inactive mutants of alpha-sarcin are non-toxic when expressed directly within the cytoplasm of cells. This is important since recent studies have cast doubt on whether protein synthesis inhibition is sufficient to initiate apoptosis. RESULTS: In this report, we assay alpha-sarcin cytotoxicity and ability to inhibit protein synthesis by direct cytoplasmic expression. We show that mutations in alpha-sarcin, which impair alpha-sarcin's ability to inhibit protein synthesis, do not affect its cytotoxicity. The mutants are unable to activate JNK, confirming that the sarcin-ricin loop remains intact and that the alpha-sarcin mutants are catalytically inactive. In addition, both mutant and wildtype variants of alpha-sarcin localize to the nucleus and cytoplasm, where they co-localize with ribosomal marker RPS6. CONCLUSION: We conclude that although protein synthesis inhibition likely contributes to cell death, it is not required. Thus, our results suggest that alpha-sarcin can promote cell death through a previously unappreciated mechanism that is independent of rRNA cleavage and JNK activation.

Germinal center B cell maintenance and differentiation are controlled by distinct NF-κB transcription factor subunits.

Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.

IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity.

The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity.