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A chemokine-driven positive feedback loop organizes lymphoid follicles.

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyers patches. In addition to mediating chemoattraction, BLC induces B cells to upregulate membrane lymphotoxin α1β2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin α1β2 expression being induced by a mechanism independent of BLC.

A B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1

Secondary lymphoid organs (spleen, lymph nodes and Peyers patches) are divided into compartments, such as B-cell zones (follicles) and T-cell zones, which provide specialized environments for specific steps of the immune response. Migration of lymphocyte subsets into these compartments is essential for normal immune function, yet the molecular cues guiding this cellular traffic are poorly defined. Chemokines constitute a family of chemotactic cytokines that have been shown to direct the migration of leukocytes during inflammation, and which may be involved in the constitutive homing of lymphocytes into follicles and T-cell zones. Here we describe a novel chemokine, B-lymphocyte chemoattractant (BLC), that is strongly expressed in the follicles of Peyers patches, the spleen and lymph nodes. BLC strongly attracts B lymphocytes while promoting migration of only small numbers of T cells and macrophages, and therefore is the first chemokine to be identified that is selective towards B cells. An orphan chemokine receptor, Burkitts lymphoma receptor 1 (BLR-1), has been found to be required for B-cell migration into lymphoid follicles. We show that BLC stimulates calcium influx into, and chemotaxis of, cells transfected with BLR-1. Our results indicate that BLC functions as a BLR-1 ligand and may guide B lymphocytes to follicles in secondary lymphoid organs.

Chemokine Requirements for B Cell Entry to Lymph Nodes and Peyer's Patches

B cell entry to lymph nodes and Peyers patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4−/− B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyers patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyers patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyers patches.

Membrane-Bound TNF Supports Secondary Lymphoid Organ Structure but Is Subservient to Secreted TNF in Driving Autoimmune Inflammation

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.

Initiation of Cellular Organization in Lymph Nodes Is Regulated by Non-B Cell-Derived Signals and Is Not Dependent on CXC Chemokine Ligand 13

The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-α1β2 (LTα1β2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTα1β2 expression. At this time, CD45+CD4+CD3− cells are the predominant LTα1β2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTα1β2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTα1β2, a role for CD45+CD4+CD3− cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.

Chemokines and B-cell Homing to Follicles

B cells that bind autoantigen in the periphery may be excluded from lymphoid follicles and rapidly eliminated (Cyster, 1997). To understand the basis for follicular exclusion we considered whether Gi coupled chemokine receptors might play a role by testing the effect of treatment with pertussis toxin (PTX), an inhibitor of Gi signaling, on B cell migration into splenic follicles. Strikingly, PTX treated B cells were unable to migrate into follicles or the white pulp cords of the spleen, whereas cells treated with buffer alone or with the oligomer B subunit of PTX could migrate into follicles normally (Cyster and Goodnow 1995). These observations led us to consider which chemokine receptors and chemokines might have a role in B cell positioning within lymphoid organs. We focused on two orphan receptors, BLR1 and EBI1, because these had been shown to be constitutively expressed by B cells in humans (Birkenbach et al. 1993; Dobner et al. 1992). To track expression of the mouse receptors, the amino-terminal ectodomains were expressed as GST fusion proteins and used to immunize rabbits. An antiserum against BLR1 was isolated and affinity purified using the same BLR1 fragment expressed as a fusion protein with mannose-binding protein. Flow cytometric analysis of mouse lymphoid tissues showed BLR1 expression on all mature B cells (Schmidt et al. 1998) with slightly higher surface expression on B cells with a CD21hiIgDlo marginal zone phenotype (Fig. 1). BLR1 expression was also observed on B220+CD5+ peritoneal B-l cells (Fig. 1). In B cell development, there was little or no BLR1 detectable on B220+IgM- pro/pre-B cells, whereas B220+IgM+ immature B cells showed weak expression (Fig. 1; note that as BLR1 is detected with a polyclonal antiserum it is necessary to be cautious in interpreting the significance of weak signals such as seen on many of the cells in bone marrow gate G4). BLR1 expression became strongly upregulated on immature B cells at about the same time as surface IgD and CD21 (Fig. 1). The low expression by immature B cells is consistent with findings that immature B cells are inefficient at entering follicles (Cyster 1997) and suggests that BLR1 upregulation may be an important part of the immature to mature B cell transition.

Traffic Patterns of B Cells and Plasma Cells

B cells at various stages of development and activation undergo specific migration events that are important for their function. A majority of the immature B cells emerging from the bone marrow home to spleen and after a short period of further maturation become competent for migration into B cell follicles. Most B cells join the conventional or B2 subset and recirculate between lymphoid follicles of secondary lymphoid organs. Smaller numbers of cells take a different path, differentiating to B1 cells which preferentially lodge within the body cavities. Following encounter with antigen, conventional B cells redistribute from follicles to T cell zones where they interact with helper T cells. Antigen-stimulated B cells that differentiate to IgM- or IgG-secreting plasma cells leave the T and B cell areas and migrate to splenic red pulp, lymph node medullary cords or bone marrow. Each of these migration events contributes to regulate B cell fate and understanding the factors controlling B cell migration has been a major goal of our work. Here we describe our recent findings showing the roles played by chemokines (Cyster, 1999; Zlotnik and Yoshie, 2000) in controlling the positioning of B cells and plasma cells.

Abstract 3898: Octamer Binding Protein-2 (Oct-2): A non-oncogene addiction in germinal center derived lymphomas and a promising therapeutic target

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnTo identify genes required for the proliferation and survival of Diffuse Large B Cell Lymphomas (DLBCL) we conducted an “Achilles Heel” RNA interference screen in cell lines model of ABC (Activated B Cell-like) and GCB (Germinal Center B-cell like) DLBCL subtypes. One of the most toxic shRNAs in this screen targeted Oct2, encoding a POU domain transcriptional activator, primarily lymphoid restricted. It was identified due its ability to bind the DNA octamer motif (ATGCAAAT) within immunoglobulin (Ig) genes promoters. The B cell specific coactivator OCA-B interacts with Oct-2 enhancing its transactivation potential. Although Oct2 and OCA-B are dipensable for Ig transcription, they are essential for germinal center (GC) development. To understand the apoptotic cell death of DLBCL cells following shRNA Oct2 induction we investigated the genetic pathways controlled by Oct2. We profiled gene expression changes in DLBCL cells upon Oct2 knocked down and merged this data with genome-wide assessment of Oct2 and OCA-B binding sites, coupling chromatin immunoprecipitation (ChiP) with high-throughput sequencing (ChIPSeq). More than 60% of the Oct2 target genes also showed OCA-B biding. The Oct2/OCA-B overlapping set of targets was enriched for genes selectively expressed in GC B cells. We found that Oct2/OCA-B lie upstream many transcription factors known to play critical roles in GC development such as BCL6, MTA3, PU.1, IRF8 and SpiB. Oct2 regulates these genes in DLBCL cells and in centroblasts. Among Oct-2 downstream effectors, BCL6 could rescue DLBCL cells from the Oct2 shRNA lethal effect. The Oct2/OCA-B binding of BCL6 promoter was confirmed by single locus ChIP in primary GC B-cells and derived lymphomas. Gel shifts experiments showed that Oct2 binds a non canonical octamer site at the BCL6 transcription start site. Our findings uncovered a novel aspect of the Oct2 biology believed to regulate the activity of octamer containing promoters. Interestingly, Oct2 controls the expression of GC specific genes that do not harbor a canonical octamer motif. By array CGH, amplification of Oct2 and OCA-B was found in less than 1% of non Hodgkin lymphoma (NHL) patient samples. Nonetheless, lymphoma cells become dependent on the Oct2 controlled network to survive, being an example of non oncogene addiction. This turns Oct2 into an attractive therapeutic target for NHL treatment. Oct2 and OCA-B lie upstream of BCL6, widely considered a master regulator of the GC response, suggesting that Oct2 directed therapy should kill the same DLBCLs as BCL6 Peptide Inhibitor. Furthermore, all GC and Post-GC B cells tested require Oct2 for survival, indicating that Oct2 directed therapy might have a broder activity spectrum than the BCL6 directed therapy. The Oct2/OCA-B binding interface would be amenable to attack with potential manageable toxicity since this interaction is exclusively required in GC B cells.nnNote: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3898.

Casein kinase 1α governs antigen receptor-induced NF-κB and human lymphoma cell survival

The transcription factor NF-κB is required for lymphocyte activation and proliferation as well as the survival of certain lymphoma types. Antigen receptor stimulation assembles an NF-κB activating platform containing the scaffold protein CARMA1 (also called CARD11), the adaptor BCL10 and the paracaspase MALT1 (the CBM complex), linked to the inhibitor of NF-κB kinase complex, but signal transduction is not fully understood. We conducted parallel screens involving a mass spectrometry analysis of CARMA1 binding partners and an RNA interference screen for growth inhibition of the CBM-dependent ‘activated B-cell-like’ (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Here we report that both screens identified casein kinase 1α (CK1α) as a bifunctional regulator of NF-κB. CK1α dynamically associates with the CBM complex on T-cell-receptor (TCR) engagement to participate in cytokine production and lymphocyte proliferation. However, CK1α kinase activity has a contrasting role by subsequently promoting the phosphorylation and inactivation of CARMA1. CK1α has thus a dual ‘gating’ function which first promotes and then terminates receptor-induced NF-κB. ABC DLBCL cells required CK1α for constitutive NF-κB activity, indicating that CK1α functions as a conditionally essential malignancy gene—a member of a new class of potential cancer therapeutic targets.