Transcriptional repressor Blimp-1 promotes CD8(+) T cell terminal differentiation and represses the acquisition of central memory T cell properties.

During acute infections, a small population of effector CD8(+) T cells evades terminal differentiation and survives as long-lived memory T cells. We demonstrate that the transcriptional repressor Blimp-1 enhanced the formation of terminally differentiated CD8(+) T cells during lymphocytic choriomeningitis virus (LCMV) infection, and Blimp-1 deficiency promoted the acquisition of memory cell properties by effector cells. Blimp-1 expression was preferentially increased in terminally differentiated effector and "effector memory" (Tem) CD8(+) T cells, and gradually decayed after infection as central memory (Tcm) cells developed. Blimp-1-deficient effector CD8(+) T cells showed some reduction in effector molecule expression, but primarily developed into memory precursor cells that survived better and more rapidly acquired several Tcm cell attributes, including CD62L and IL-2 expression and enhanced proliferative responses. These results reveal a critical role for Blimp-1 in controlling terminal differentiation and suppressing memory cell developmental potential in effector CD8(+) T cells during viral infection.

Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors.

Interleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo and furthermore revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4(-/-) T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4(-/-) mice showed impaired IL-21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.

Regulation of plasma-cell development.

Plasma cells are the terminally differentiated, non-dividing effector cells of the B-cell lineage. They are cellular factories devoted to the task of synthesizing and secreting thousands of molecules of clonospecific antibody each second. To respond to microbial pathogens with the necessary specificity and rapidity, B cells are exquisitely regulated with respect to both development in the bone marrow and activation in the periphery. This review focuses on the terminal differentiation of B cells into plasma cells, including the different subsets of B cells that become plasma cells, the mechanism of regulation of this transition, the transcription factors that control each developmental stage and the characteristics of long-lived plasma cells.

Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class switch.

Two transcription factors, Pax5 and Blimp-1, form a gene regulatory network (GRN) with a double-negative loop, which defines either B-cell (Pax5 high) or plasma cell (Blimp-1 high) status as a binary switch. However, it is unclear how this B-cell GRN registers class switch DNA recombination (CSR), an event that takes place before the terminal differentiation to plasma cells. In the absence of Bach2 encoding a transcription factor required for CSR, mouse splenic B cells more frequently and rapidly expressed Blimp-1 and differentiated to IgM plasma cells as compared with wild-type cells. Genetic loss of Blimp-1 in Bach2(-/-) B cells was sufficient to restore CSR. These data with mathematical modelling of the GRN indicate that Bach2 achieves a time delay in Blimp-1 induction, which inhibits plasma cell differentiation and promotes CSR (Delay-Driven Diversity model for CSR). Reduction in mature B-cell numbers in Bach2(-/-) mice was not rescued by Blimp-1 ablation, indicating that Bach2 regulates B-cell differentiation and function through Blimp-1-dependent and -independent GRNs.

Repression of BCL-6 is required for the formation of human memory B cells in vitro.

Memory B cells provide rapid protection to previously encountered antigens; however, how these cells develop from germinal center B cells is not well understood. A previously described in vitro culture system using human tonsillar germinal center B cells was used to study the transcriptional changes that occur during differentiation of human memory B cells. Kinetic studies monitoring the expression levels of several known late B cell transcription factors revealed that BCL-6 is not expressed in memory B cells generated in vitro, and gene expression profiling studies confirmed that BCL-6 is not expressed in these memory B cells. Furthermore, ectopic expression of BCL-6 in human B cell cultures resulted in formation of fewer memory B cells. In addition, the expression profile of in vitro memory B cells showed a unique pattern that includes expression of genes encoding multiple costimulatory molecules and cytokine receptors, antiapoptotic proteins, T cell chemokines, and transcription factors. These studies establish new molecular criteria for defining the memory B cell stage in human B cells.

Blimp1-mediated repression of negative regulators is required for osteoclast differentiation.

Regulation of irreversible cell lineage commitment depends on a delicate balance between positive and negative regulators, which comprise a sophisticated network of transcription factors. Receptor activator of NF-kappaB ligand (RANKL) stimulates the differentiation of bone-resorbing osteoclasts through the induction of nuclear factor of activated T cells c1 (NFATc1), the essential transcription factor for osteoclastogenesis. Osteoclast-specific robust induction of NFATc1 is achieved through an autoamplification mechanism, in which NFATc1 is constantly activated by calcium signaling while the negative regulators of NFATc1 are suppressed. However, it has been unclear how such negative regulators are repressed during osteoclastogenesis. Here we show that B lymphocyte-induced maturation protein-1 (Blimp1; encoded by Prdm1), which is induced by RANKL through NFATc1 during osteoclastogenesis, functions as a transcriptional repressor of anti-osteoclastogenic genes such as Irf8 and Mafb. Overexpression of Blimp1 leads to an increase in osteoclast formation, and Prdm1-deficient osteoclast precursor cells do not undergo osteoclast differentiation efficiently. The importance of Blimp1 in bone homeostasis is underscored by the observation that mice with an osteoclast-specific deficiency in the Prdm1 gene exhibit a high bone mass phenotype caused by a decreased number of osteoclasts. Thus, NFATc1 choreographs the determination of cell fate in the osteoclast lineage by inducing the repression of negative regulators as well as through its effect on positive regulators.

B-1 B lymphocytes require Blimp-1 for immunoglobulin secretion.

B-1 B cells produce circulating natural antibodies that provide "innate-like" protection against bacterial and viral pathogens. They also provide adaptive responses to blood and air-borne pathogens. B lymphocyte-induced maturation protein 1 (Blimp-1) is a transcriptional repressor that is required for the formation of B-2-derived antibody-secreting plasma cells. In this study, we used mice lacking Blimp-1 in the B cell lineage to show that Blimp-1 is not necessary for the formation or self-renewal of B-1 B cells but that Blimp-1 is required for normal immunoglobulin (Ig) secretion by B-1 cells. B-1 cells lacking Blimp-1 do not repress Pax5 mRNA and do not induce X-box binding protein 1, and mu secreted mRNA normally, showing that B-1 and B-2 cells both use a common pathway for Ig secretion. Blimp-1-deficient B-1 B cells are also defective in providing early protection against influenza infection.

Blimp-1 is required for maintenance of long-lived plasma cells in the bone marrow.

Long-lived plasma cells, residing primarily in the bone marrow, continuously secrete antibody and provide an important component of humoral memory. However, when such cells secrete autoantibodies or become transformed, they can be pathogenic. We have shown recently that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is required for the formation of plasma cells. To determine what role Blimp-1 might play in maintenance of plasma cells, we generated mice in which the gene encoding Blimp-1 could be deleted in an inducible manner. Deletion of Blimp-1 either in vitro or in vivo leads to loss of previously formed B220(LO)CD138(HI) plasma cells. Using BrdU incorporation, we confirmed that Blimp-1 is required for the maintenance of nondividing, long-lived plasma cells in the bone marrow. Blimp-1 is also required for long-term maintenance of antigen-specific immunoglobulin in serum. Thus Blimp-1 is required not only for the formation but also for the maintenance of long-lived plasma cells. This finding provides the possibility of new drug design strategies for autoimmunity and multiple myeloma focused on blocking Blimp-1 expression or activity.

Activation-dependent induction of Blimp-1.

B lymphocyte induced maturation protein-1 (Blimp-1) mRNA is induced upon antigen-dependent activation of both T and B lymphocytes, in spite of the fact that it plays very different roles in the two lineages. B cells have at least four different mechanisms to repress Blimp-1 and repression is relieved before induction. Only one repressor, Bcl-6, is known in T cells. Activators must also be present to induce Blimp-1 in both T and B cells. Cytokines IL-21, IL-10, and IL-6, activating STAT3, are crucial in B cells along with toll-like receptor (TLR) signals, whereas IL-2 is crucial in T cells. AP-1, NF-kappaB, and IRF4 also activate Blimp-1.

Blimp-1/PRDM1 mediates transcriptional suppression of the NLR gene NLRP12/Monarch-1.

NLR (nucleotide-binding domain, leucine-rich repeat) proteins are intracellular regulators of host defense and immunity. One NLR gene, NLRP12 (NLR family, pyrin domain containing 12)/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in human myeloid cells. This is supported by genetic analysis linking the loss of a functional NLRP12 protein to hereditary periodic fever. NLRP12 transcription is diminished by specific TLR stimulation and myeloid cell maturation, consistent with its role as a negative regulator of inflammation. The NLRP12 promoter contains a novel Blimp-1 (B lymphocyte-induced maturation protein-1)/PRDM1 (PR domain-containing 1, with ZNF domain) binding site, and Blimp-1 reduces NLRP12 promoter activity, expression, and histone 3 acetylation. Blimp-1 associates with the endogenous NLRP12 promoter in a TLR-inducible manner and mediates the down-regulation of NLRP12 expression by TLR agonists. As expected, the expression of NLRP12 and Blimp-1 is inversely correlated. Analysis of Blimp-1(-/-) murine myeloid cells provides physiologic evidence that Blimp-1 reduces NLRP12 gene expression during cell differentiation. This demonstrates a novel role for Blimp-1 in the regulation of an NLR gene.

Blimp-1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression.

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.

Transcription factors controlling the beginning and end of B-cell differentiation.

Powerful techniques--including gene targeting, genomic and proteomic analyses--are allowing a more complete understanding of the transcription factors that drive B-cell development. Regulatory cascades involving multiple steps have been identified at many decision points. In addition, feedback loops and transcriptional inhibition of alternative fates have been found to be important both for commitment to the B lymphocyte lineage and for terminal B-cell differentiation.

Plasma cell differentiation and multiple myeloma.

Microarray analyses and gene targeting have recently enhanced the understanding of factors involved in normal plasma cells and multiple myeloma. Plasma cells develop from marginal zone or germinal center B cells following stimulation by antigen, microbial products, TNF family signals and cytokines. Transcription factors, B-lymphocyte-induced maturation protein 1 (Blimp-1) and X-box binding protein 1 (XBP-1) are required for plasma cell development. They regulate sets of genes that induce immunoglobulin secretion, halt proliferation and block alternative B-cell fates. In multiple myeloma, transforming events lead to proliferation and survival, but programs for plasma cell differentiation and the inhibition of B-cell genes appear to be largely intact.

Blimp-1-dependent repression of Pax-5 is required for differentiation of B cells to immunoglobulin M-secreting plasma cells.

B-cell lineage-specific activator protein (BSAP), encoded by the Pax-5 gene, is critical for B-cell lineage commitment and B-cell development but is not expressed in terminally differentiated B cells. We demonstrate a direct connection between BSAP and B-lymphocyte-induced maturation protein 1 (Blimp-1), a transcriptional repressor that is sufficient to drive plasmacytic differentiation. Blimp-1 binds a site on the Pax-5 promoter in vitro and in vivo and represses the Pax-5 promoter in a binding-site-dependent manner. By ectopically expressing Blimp-1 or a competitive inhibitor of Blimp-1, we show that Blimp-1 is both necessary and sufficient to repress Pax-5 during plasmacytic differentiation of primary splenic B cells. Blimp-1-dependent repression of Pax-5 is sufficient to regulate BSAP targets CD19 and J chain and is necessary but not sufficient to induce XBP-1. We further show that repression of Pax-5 is required for Blimp-1 to drive differentiation of splenocytes to immunoglobulin M-secreting cells. Thus, repression of Pax-5 plays a critical role in the Blimp-1-dependent program of plasmacytic differentiation.

Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development.

Although V(D)J recombination is thought to be regulated by changes in the accessibility of chromatin to the recombinase machinery, the mechanisms responsible for establishing "open" chromatin are poorly understood. We performed a detailed study of the acetylation status of histones associated with 11 V(H) gene segments, their flanking regions, and various intergenic elements during B-cell development and ontogeny, when V(D)J recombination is highly regulated. Histone H4 shows higher and more-regulated acetylation than does histone H3 in the V(H) locus. In adult pro-B cells, V(H) gene segments are acetylated prior to V(D)J rearrangement, with higher acetylation associated with J(H)-distal V(H) gene segments. While large regions of the V(H) locus have similar patterns of histone acetylation, acetylation is narrowly confined to the gene segments, their flanking promoters, and recombinase signal sequence elements. Thus, histone acetylation in the V(H) locus is both locally and globally regulated. Increased histone acetylation accompanies preferential recombination of J(H)-proximal V(H) gene segments in early B-cell ontogeny, and decreased histone acetylation accompanies inhibition of V-DJ recombination in a transgenic model of immunoglobulin heavy-chain allelic exclusion. Thus, changes in histone acetylation appear to be important for both promotion and inhibition of V-DJ rearrangement during B-cell ontogeny and development.

Identification of E-box factor TFE3 as a functional partner for the E2F3 transcription factor.

Various studies have demonstrated a role for E2F proteins in the control of transcription of genes involved in DNA replication, cell cycle progression, and cell fate determination. Although it is clear that the functions of the E2F proteins overlap, there is also evidence for specific roles for individual E2F proteins in the control of apoptosis and cell proliferation. Investigating protein interactions that might provide a mechanistic basis for the specificity of E2F function, we identified the E-box binding factor TFE3 as an E2F3-specific partner. We also show that this interaction is dependent on the marked box domain of E2F3. We provide evidence for a role for TFE3 in the synergistic activation of the p68 subunit gene of DNA polymerase alpha together with E2F3, again dependent on the E2F3 marked box domain. Chromatin immunoprecipitation assays showed that TFE3 and E2F3 were bound to the p68 promoter in vivo and that the interaction of either E2F3 or TFE3 with the promoter was facilitated by the presence of both proteins. In contrast, neither E2F1 nor E2F2 interacted with the p68 promoter under these conditions. We propose that the physical interaction of TFE3 and E2F3 facilitates transcriptional activation of the p68 gene and provides strong evidence for the specificity of E2F function.

Regulatory events in early and late B-cell differentiation.

We are studying transcriptional control of critical developmental decision points in B lymphocytes. Commitment to the B-lymphocyte lineage is dependent on the transcriptional regulator Pax5 and committed B lymphocytes represent the first developmental stage when V(H)-to-DJ recombination occurs in the immunoglobulin (Ig) heavy chain locus. We summarize our recent studies showing that methylation of histone H3 lysine 9, a heterochromatic chromatin modification, is present in the Ig V(H) region in hematopoietic progenitors and in non-B lineage hematopoietic cells. Pax5 is both necessary and sufficient to remove this heterochromatic mark in B cells. Using genetically altered mice, we have shown that terminal differentiation of B cells to memory and Ig-secreting plasma cells depends on the transcriptional repressor Blimp-1. Recent studies demonstrating a requirement for Blimp-1 in the formation of pre-plasma memory B cells, Ig-secreting plasma cells as well as preliminary data suggesting a requirement for Blimp-1 in the maintenance of long-lived plasma cells are summarized. We also summarize our recent studies on the regulation of Blimp-1, showing direct repression by Bcl-6 and providing evidence for activation by NF-kappaB following toll-like receptor signaling.

The dynamic expression pattern of B lymphocyte induced maturation protein-1 (Blimp-1) during mouse embryonic development.

We have analyzed the spatial and temporal patterns of B lymphocyte-induced maturation protein-1 (Blimp-1) expression during mouse embryonic development. Blimp-1 expression is induced early in the anterior definitive endoderm, mesoderm of head process, and prechordal plate. In ectoderm-derived tissues at later stages, Blimp-1 expression is found in the primitive photoreceptors of neural retina, in differentiated epithelial cells of epidermis, tongue, oral and nasal cavities, and in the precursors of internal root sheaths of hair follicles. In mesoderm-derived tissues, Blimp-1 expression is observed in splanchnopleure, a subset of somatopleure-derived cells in limb buds, and myotomes of somites. Blimp-1 is also expressed in mesenchyme of developing hand plates, digits, branchial arches, nasal processes, and external genitalia. Blimp-1 is present in mesenchyme-derived chondroblasts, supporting cells of taste buds, and papilla of teeth, hair follicles and taste buds. In endoderm-derived tissues, Blimp-1 expression in the foregut region is restricted to a subset of epithelial cells at the headfold stage while expression in the endodermal epithelium of midgut and hindgut persists from the headfold stage to birth. Finally, Blimp-1 is expressed in the migrating primordial germ cells.