Blimp-1, a transcriptional repressor, drives the terminal differentiation of B cells to plasma cells. Using DNA microarrays, we found that introduction of Blimp-1 into B cells blocked expression of a remarkably large set of genes, while a much smaller number was induced. Blimp-1 initiated this cascade of gene expression changes by directly repressing genes encoding several transcription factors, including Spi-B and Id3, that regulate signaling by the B cell receptor. Blimp-1 also inhibited immunoglobulin class switching by blocking expression of AID, Ku70, Ku86, DNA-PKcs, and STAT6. These findings suggest that Blimp-1 promotes plasmacytic differentiation by extinguishing gene expression important for B cell receptor signaling, germinal center B cell function, and proliferation while allowing expression of important plasma cell genes such as XBP-1.

(14). BSAP is also important for proliferation and isotype switching in germinal center B cells (4,11,16,20,30). BSAP is expressed throughout B-cell development until the terminally differentiated plasma cell stage (1, 4). BSAP can either activate or repress transcription (33). Targets of BSAP activation include VpreB, 5, CD19, and blk (B lymphoid kinase) (8,15,17,22,35). BSAP represses J chain, the immunoglobulin heavy-chain 3ЈC␣ enhancer and XBP-1 (12,25,28).B-lymphocyte-induced maturation protein 1 (Blimp-1, encoded by the prdm1 gene) is a critical regulator of plasma cell differentiation, induced during cytokine-dependent differentiation of a B-cell lymphoma line (BCL-1) (29) and after lipopolysaccharide (LPS) treatment of primary murine splenocytes (2). Blimp-1 is expressed in all plasma cells and in a subset of germinal center B cells with a partial plasma cell phenotype but not in memory B cells (3). Ectopic expression of Blimp-1 in BCL-1 cells and in primary splenic B cells is sufficient to cause terminal differentiation and immunoglobulin M (IgM) secretion (2,19,26,29).Blimp-1 is a transcriptional repressor. Its DNA-binding activity is conferred by five zinc-finger motifs (7), whereas association with histone deacetylases (34) and hGroucho (24) is required for transcriptional repression. One important target of Blimp-1 repression is c-myc (10). Although repression of c-myc is necessary for terminal differentiation of BCL-1 cells, it is not sufficient, suggesting the existence of additional Blimp-1 targets (9). Indeed, MHC2A, encoding CIITA, a coactivator for major histocompatibility class II (MHC-II) transcription, was recently identified as a Blimp-1 target, providing a mechanism for extinction of MHC-II expression during plasma cell differentiation (19). We demonstrate here that Blimp-1 represses Pax-5 and show that Blimp-1-dependent repression of Pax-5 is required for plasma cell differentiation. MATERIALS AND METHODSCell culture. BCL-1 (CW13.20-3B3, ATCC CRL 1669), P3X (P3X63Ag8), 18-81 Raji, and primary splenocytes were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products, Inc.), 20 g of gentamicin (Gemini)/ml, and 50 M ␤-mercaptoethanol. To induce differentiation of BCL-1, cells (5 ϫ 10 5 cells/ml) were stimulated with interleukin-2 (IL-2) and IL-5, as described previously (29), for various times. 3T3 and Phoenix cells (G. Nolan, Stanford University) were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and 20 g of gentamicin/ ml. WI-L2 transfectants were cultured in the phenol red-free RPMI medium supplemented with 10% charcoal-dextran-treated FBS (HyClone) and penicillin-streptomycin (Gibco-BRL) and cultured in the presence of the selection antibiotic, hygromycin B (500 g/ml; Gibco-BRL). 4-Hydroxytomaxifen was dissolved in 70% ethanol (1 M) and CdSO 4 (5 M) from Sigma.Plasmids. To generate a Blimp-1 binding site mutated reporter, a wild-type luciferase reporter dependent on the Pax-5 promoter (BSAP-Luc) (18) was used as ...

Antibody specificity and diversity is generated in B cells during germinal center maturation through clonal expansion while they undergo class-switch recombination and somatic hypermutation. Here we demonstrate that the transcriptional repressor Bcl-6 mediates this phenotype by directly repressing ATR in centroblasts and lymphoma cells. ATR is critical in replication and DNA damage-sensing checkpoints. Bcl-6 allowed B cells to evade ATR-mediated checkpoints and attenuated the response of the B cells to exogenous DNA damage. Repression of ATR was necessary and sufficient for those Bcl-6 activities. CD40 signaling 'rescued' B cells from those effects by disrupting the Bcl-6 transcription-repression complex on the promoter of the gene encoding ATR. Our data demonstrate a transcriptional regulatory loop whereby Bcl-6 mediates the centroblast phenotype through transient silencing of ATR.

A diverse antibody repertoire is essential for an effective adaptive immune response to novel molecular surfaces. Although past studies have observed common patterns of V-segment use, as well as variation in V-segment use between individuals, the relative contributions to variance from genetics, disease, age, and environment have remained unclear. Using high-throughput sequence analysis of monozygotic twins, we show that variation in naive V H and D H segment use is strongly determined by an individual's germ-line genetic background. The inherited segment-use profiles are resilient to differential environmental exposure, disease processes, and chronic lymphocyte depletion therapy. Signatures of the inherited profiles were observed in class switched germ-line use of each individual. However, despite heritable segment use, the rearranged complementarity-determining region-H3 repertoires remained highly specific to the individual. As it has been previously demonstrated that certain V-segments exhibit biased representation in autoimmunity, lymphoma, and viral infection, we anticipate our findings may provide a unique mechanism for stratifying individual risk profiles in specific diseases.heritable variation | next generation sequencing | V-gene S pecific biases in the antibody repertoire have been found in many diseases, from viral infections to cancers to autoimmune disorders (1-15). Although it is possible that heritable variation in the composition of the antibody repertoire could alter inherent risk to specific diseases, the diversity of the antibody repertoire has hindered direct characterization of heritable influences.Early twin studies provided some evidence of genetic variation affecting reactive titers from the antibody repertoire. Multiple studies observed both total Ig and antigen-specific titers to be more correlated in monozygotic twins than dizygotic twins or unrelated individuals (16)(17)(18). In some cases of monozygotic twins discordant for autoimmune diseases, the healthy twin often shared high autoantibody reactive titers with their affected twin (16,19,20).Early sequencing studies were able to identify some systematic biases in the antibody repertoire with limited sampling depth. The first sequencing studies to characterize V(D)J diversification mechanisms identified the gene segment recombination process, but also implied a repertoire too diverse to exhaustively interrogate by traditional sequencing technologies (21). Complete characterization of V-segment loci established ∼50 V H , 40 V κ , and 30 V λ segments in an individual, with a number of allelic variants for the majority of segments (22)(23)(24). Evaluation of use across individuals revealed biased V-gene representation that preceded selection (25)(26)(27). Quantitative PCR of V-gene families showed family use largely stable over time, with fluctuations in use correlated to antigen-specific responses (28). In the T-cell receptor (TCR) repertoire, TCRB-V use was more highly correlated in healthy monozygotic twins than unrelated individua...

In multiple sclerosis (MS) pathogenic B cells likely act on both sides of the blood-brain barrier (BBB). However, it is unclear whether antigen-experienced B cells are shared between the CNS and the peripheral blood (PB) compartments. We applied deep repertoire sequencing of IgG heavy chain variable region genes (IgG-VH) in paired cerebrospinal fluid and PB samples from patients with MS and other neurological diseases to identify related B cells that are common to both compartments. For the first time to our knowledge, we found that a restricted pool of clonally related B cells participated in robust bidirectional exchange across the BBB. Some clusters of related IgG-VH appeared to have undergone active diversification primarily in the CNS, while others have undergone active diversification in the periphery or in both compartments in parallel. B cells are strong candidates for autoimmune effector cells in MS, and these findings suggest that CNS-directed autoimmunity may be triggered and supported on both sides of the BBB. These data also provide a powerful approach to identify and monitor B cells in the PB that correspond to clonally amplified populations in the CNS in MS and other inflammatory states.

In multiple sclerosis (MS), an exchange of lymphocytes, in particular B cells, between the central nervous system (CNS) and periphery is believed to be required for the maintenance of active disease. Therapeutic monoclonal antibodies that prevent lymphocytes from crossing the blood-brain barrier (BBB) or induce near-complete peripheral B cell depletion rapidly mitigate MS disease activity. Using next-generation sequencing technology, we recently found that clonally related B cells exist in the cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients, establishing the existence of an immune axis across the BBB. However, it remains unclear which subpopulations of the highly diverse peripheral B cell compartment share antigen-specificity with intrathecal B cell repertoires, and whether their antigen stimulation occurs on both sides of the BBB. To address these questions, we combined flow cytometry sorting of PB B cell subsets with deep immune repertoire sequencing of CSF and PB B cells. Immunoglobulin (IgM and IgG) heavy chain variable (VH) region repertoires of five PB B cell subsets from MS patients (n=8) were compared with their CSF Ig-VH transcriptomes. In 6 of 8 patients, we identified peripheral CD27+IgD−memory B cells, CD27hiCD38hi plasma cells/plasmablasts, or CD27−IgD− B cells providing an immune connection to the CNS compartment. Pinpointing Ig class-switched B cells as key component of the immune axis thought to contribute to ongoing MS disease activity strengthens the rationale of current therapeutic strategies and may lead to more targeted approaches.

The transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) is expressed in some differentiated cells and is required for terminal differentiation of B cells. To facilitate identification of Blimp-1 target genes, we have determined the optimal DNA recognition sequence for Blimp-1. The consensus is very similar to a subset of sites recognized by IFN regulatory factors (IRFs) that contain the sequence GAAAG. By binding competition and determination of equilibrium dissociation constants, we show that Blimp-1, IRF-1, and IRF-2 have similar binding affinities for functionally important regulatory sites containing this sequence. However, Blimp-1 does not bind to all IRF sites, and specifically does not recognize IRF-4/PU.1 or IRF-8 sites lacking the GAAAG sequence. Chromatin immunoprecipitation studies showed that Blimp-1, IRF-1, and IRF-2 all bind the IFN-β promoter in vivo, as predicted by the in vitro binding parameters, and in cotransfections Blimp-1 inhibits IRF-1-dependent activation of the IFN-β promoter. Thus, our data suggest that Blimp-1 competes in vivo with a subset of IRF proteins and help predict the sites and IRF family members that may be affected.

CD47 is a widely expressed cell surface protein that functions as an immune checkpoint in cancer. When expressed by tumor cells, CD47 can bind SIRPα on myeloid cells, leading to suppression of tumor cell phagocytosis and other innate immune functions. CD47-SIRPα signaling has also been implicated in the suppression of adaptive antitumor responses, but the relevant cellular functions have yet to be elucidated. Therapeutic blockade of the CD47 pathway may stimulate antitumor immunity and improve cancer therapy. To this end, a novel CD47-blocking molecule, ALX148, was generated by fusing a modified SIRPα D1 domain to an inactive human IgG1 Fc. ALX148 binds CD47 from multiple species with high affinity, inhibits wild type SIRPα binding, and enhances phagocytosis of tumor cells by macrophages. ALX148 has no effect on normal human blood cells in vitro or on blood cell parameters in rodent and non-human primate studies. Across several murine tumor xenograft models, ALX148 enhanced the antitumor activity of different targeted antitumor antibodies. Additionally, ALX148 enhanced the antitumor activity of multiple immunotherapeutic antibodies in syngeneic tumor models. These studies revealed that CD47 blockade with ALX148 induces multiple responses that bridge innate and adaptive immunity. ALX148 stimulates antitumor properties of innate immune cells by promoting dendritic cell activation, macrophage phagocytosis, and a shift of tumor-associated macrophages toward an inflammatory phenotype. ALX148 also stimulated the antitumor properties of adaptive immune cells, causing increased T cell effector function, pro-inflammatory cytokine production, and a reduction in the number of suppressive cells within the tumor microenvironment. Taken together, these results show that ALX148 binds and blocks CD47 with high affinity, induces a broad antitumor immune response, and has a favorable safety profile.