CD21(-/low) B cells in human blood are memory cells.

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co-receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21(-/low) ) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21(-/low) B cell subset in peripheral blood from healthy donors. Here, we show that CD21(-/low) cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21(-/low) subset can be divided into CD38(-) 24(+) and CD38(-) 24(low) cells, where most of the CD38(-) 24(+) are CD27(+) immunoglobulin (Ig)M(+) IgD(+) and the CD38(-) 24(low) are switched CD27(-) . Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21(-/low) cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll-like receptor (TLR)-7/8 and interleukin (IL)-2 induces proliferation and differentiation of the CD21(-/low) B cells comparable to CD21(+) CD27(+) memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38(-) 24(low) subset, implying that some memory B cells require not only TLR but also BCR triggering. We conclude that the CD21(-/low) cells in healthy donors are memory B cells.

CD21 -/low B cells: A Snapshot of a Unique B Cell Subset in Health and Disease.

B cells represent one of the cellular components of the immune system that protects the individual from invading pathogens. In response to the invader, these cells differentiate into plasma cells and produce large amounts of antibodies that bind to and eliminate the pathogen. A hallmark of autoimmune diseases is the production of autoantibodies i.e. antibodies that recognize self. Those that are considered pathogenic can damage tissues and organs, either by direct binding or when deposited as immune complexes. For decades, B cells have been considered to play a major role in autoimmune diseases by antibody production. However, as pathogenic autoantibodies appear to derive mainly from T cell dependent responses, T cells have been the focus for many years. The successful treatment of patients with autoimmune diseases with either B cell depletion therapy (rituximab) or inhibition of B cell survival (belimumab), suggested that not only the autoantibodies but also other B cell features are important. This has caused a surge of interest in B cells and their biology resulting in the identification of various subsets e.g. regulatory B cells, several memory B cell subsets etc. Also, in other conditions such as chronic viral infections and primary immunodeficiency, several B cell subsets with unique characteristics have been identified. In this review, we will discuss one of these subsets, a subset that is expanded in conditions characterized by chronic immune stimulation. This B cell subset lacks, or expresses low, surface levels of the complement receptor 2 (CD21) and has therefore been termed CD21(-/low) B cells.

Memory B cells in mouse models.

One of the principles behind vaccination, as shown by Edward Jenner in 1796, and host protection is immunological memory, and one of the cells central to this is the antigen-experienced memory B cell that responds rapidly upon re-exposure to the initiating antigen. Classically, memory B cells have been defined as progenies of germinal centre (GC) B cells expressing isotype-switched and substantially mutated B cell receptors (BCRs), that is, membrane-bound antibodies. However, it has become apparent over the last decade that this is not the only pathway to B cell memory. Here, we will discuss memory B cells in mice, as defined by (1) cell surface markers; (2) multiple layers; (3) formation in a T cell-dependent and either GC-dependent or GC-independent manner; (4) formation in a T cell-independent fashion. Lastly, we will touch upon memory B cells in; (5) mouse models of autoimmune diseases.

The pre-B cell receptor; selecting for or against autoreactivity.

Antibodies represent a crucial component of humoral immunity as protection against invading pathogens, to which they bind and thereby trigger mechanisms that lead to the disposal of the pathogen. Antibodies are assembled from Ig heavy chains (HCs) and light chains (LCs) and are found in both a secreted and a membrane-bound form, termed B cell receptors (BCRs), where the latter allows the 'right' B cell to respond upon recognition of its cognate antigen. The antibody repertoire is almost unlimited because of a process in which germ line V(D)J gene segments, encoding the variable (antigen-binding) region of the antibody HCs and LCs, are recombined. As this process is random, it is apparent that it results in a vast variety of antibodies, those that recognize foreign but also those that recognize self- (auto-) antigens. Control mechanisms are, therefore, in place to ensure that as few autoreactive B cells as possible are allowed to proceed in development. This counter-selection takes place through various mechanisms and at several stages as the cells develop from pre-B cells to antibody-secreting plasma cells. At the first major checkpoint, at the pre-BI to pre-BII cell transition, antibody HCs assemble with the invariant surrogate LC (SLC) forming a pre-BCR. Herein, we will discuss the role of the pre-BCR in the selection at this stage, how a dysfunctional pre-BCR affects selection and its effects on later stages, and whether the pre-BCR selects for or against autoreactivity.

A pTalpha-negative subpopulation of CD25+ TN thymocytes revealed by a transgenic marker.

We have recently generated 5'lambda5-huTAC mice, which express the human CD25 (huTAC) gene under the control of the 5'-flanking region of the mouse lambda5-gene. The huTAC-transgene was expressed in pre-B cells but neither in mature B cells nor in T cells of these mice. In this report we demonstrate that the transgene is also transiently expressed by adult CD25+ CD3-CD4-CD8- (triple negative, TN) thymocytes and in fetal thymocytes. The huTAC+, in contrast to the huTAC- subpopulation of the CD44+CD25+ TN cells, was unexpectedly found not to express the pTalpha-gene. Still the huTAC+CD44+CD25+ TN cells reconstituted the development of both alphabeta and gammadelta lineage cells equally efficiently as the pTalpha-expressing huTAC- fraction, demonstrating that this pTalpha-negative subpopulation contained precursors for both T-cell lineages. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments demonstrated that also in normal mice only a fraction of CD44+CD25+ and CD44-CD25+ TN cells expressed this gene. Taken together, these data indicate that huTAC transgene expression revealed a truly pTalpha-negative fraction of the CD44+CD25+ TN cells. The observation that not all precursors in the CD25+ TN population express the pTalpha-gene has important implications for the understanding of early T-cell development and T-cell lineage commitment.

PEBP2 and c-myb sites crucial for lambda5 core enhancer activity in pre-B cells.

The lambda5 gene is expressed exclusively in precursor (pre-) B cells where its gene product, as part of the pre-B cell receptor, is crucial for the proliferation of these cells. Several DNA regions regulate the activity and expression pattern of the lambda5 gene. Amongst these is an enhancer, B(lambda5), located 5' of the gene. Here we analyze the lambda5 enhancer core, b(lambda5), which in earlier experiments was demonstrated to retain 50% of the enhancer activity, and show that this activity is restricted to pre-B cells. We identify a DNA element within b(lambda5), PEBP2(lambda5), which is essential for enhancer activity: mutation within this site dramatically reduces core enhancer activity in pre-B cells. The PEBP2(lambda5) site binds bacterially produced polyoma enhancer binding proteins (PEBP) (Runx/AML/CBFA). Furthermore, PEBP2 proteins present in nuclear extracts from murine pre-B cells bind to the PEBP2(lambda5) element. PEBP2 proteins in mature B cells also bind to the PEBP2(lambda5 )element, implying that if PEBP2 proteins are responsible for the stage-specific expression, they have to be non-activating or inhibiting in mature B cells. We also demonstrate that a described partner of PEBP2, c-myb, binds to a sequence termed myb(lambda5) located just upstream of the PEBP2(lambda5) site in the core enhancer. The myb(lambda5) element is also crucial for enhancer activity, since mutating the myb site reduces core enhancer activity to the same extent as mutating the PEBP2 site. Earlier reports have shown that c-myb is expressed at high levels in pre-B cell lines whereas its expression is down-regulated in more mature B cell lines. Thus, c-myb may be involved in determining the stage-specific expression of the lambda5 gene.

Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice.

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.

Lymphoid-restricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands.

The two tyrosine kinase receptors, c-kit and flt3, and their respective ligands KL and FL, have been demonstrated to play key and nonredundant roles in regulating the earliest events in hematopoiesis. However, their precise roles and potential interactions in promoting early lymphoid commitment and development remain unclear. Here we show that most if not all murine Lin(-/lo)Sca1(+)c-kit(+) bone marrow (BM) cells generating B220(+)CD19(+) proB-cells in response to FL and interleukin-7 (IL-7) also have a myeloid potential. In contrast to FL + IL-7, KL + IL-7 could not promote proB-cell formation from Lin(-/lo)Sca1(+)c-kit(+) cells. However, KL potently enhanced FL + IL-7-stimulated proB-cell formation, in part through enhanced recruitment of FL + IL-7-unresponsive Lin(-/lo)Sca1(+)c-kit(+) progenitors, and in part by enhancing the growth of proB-cells. The enhanced recruitment (4-fold) in response to KL occurred exclusively from the Lin(-/lo)Sca1(+)c-kit(+)flt3(-) long-term repopulating stem cell population, whereas KL had no effect on FL + IL-7-stimulated recruitment of Lin(-/lo)Sca1(+)c-kit(+)flt3(+) short-term repopulating cells. The progeny of FL + IL-7-stimulated Lin(-/lo)Sca1(+)c-kit(+) cells lacked in vitro and in vivo myeloid potential, but efficiently reconstituted both B and T lymphopoiesis. In agreement with this FL, but not KL, efficiently induced expression of B220 and IL-7 receptor-alpha on Lin(-/lo)Sca1(+)c-kit(+)flt3(+) cells. Thus, whereas KL appears crucial for recruitment of FL + IL-7-unresponsive candidate (c-kit(+)flt3(-)) murine stem cells, FL is essential and sufficient for development toward lymphoid restricted progenitors from a population of (c-kit(+)flt3(+)) multipotent short-term reconstituting progenitors.

Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice.

The surrogate light chain (SL) is composed of two polypeptides, Vpre-B and lambda5. In large pre-BII cells the SL chain associates with Ig mu heavy chain (muH) to form the pre-B cell receptor (pre-BCR). In mice there are two Vpre-B genes which are 98% identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with lambda5. However, it is not known whether both gene products serve the same function in vivo. Here we have established mice that lack the Vpre-B1 gene (VpreB1(-/-)), but still express the Vpre-B2 gene, both as RNA and protein. In Vpre-B1(-/-) mice, the bone marrow cellularity and the percentage of B220+ cells is normal. However, among the B220+ cells, the percentage of pre-BI cells is increased, and the percentage of pre-BII and immature B cells is slightly decreased, suggesting that the lack of Vpre-B1 causes a partial block at the transition from pre-BI to pre-BII cells, i.e. into the pre-BCR stage. The number of cells that produce a functional pre-BCR is thus lower, but the cells that reach this stage are normal as they can be expanded by proliferation and then differentiate into more mature cells. The spleens of Vpre-B1 homozygous mutant mice show normal numbers of B and T lymphocytes. Moreover, the Ig loci are allelicly excluded and the homozygous mutant mice respond with normal levels of antigen-specific antibodies to T-dependent antigens. These results demonstrate that VpreB2 alone is capable of supporting B lymphocyte development in the bone marrow and can give rise to immuno-competent cells in the periphery.

Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse.

During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathke's pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Identification of a tissue- and differentiation stage-specific enhancer of the VpreB1 gene.

The VpreB and lambda 5 genes encode proteins that associate non-covalently to form the so-called surrogate light (SL) chain. The SL chain complexes with the immunoglobulin heavy chain to form the pre-B cell receptor, which plays a critical role in B cell development. Expression of the murine SL genes is regulated at the level of transcription initiation. Here, we show that a VpreB1 enhancer is located within the 356 bp immediately upstream of the coding sequence. Interestingly, this region exhibits 96% identity to the upstream region of VpreB2. Deletion mapping located the enhancer to between positions -214 and -47 (+1 is the 5'-most transcription initiation site). The enhancer is tissue and differentiation stage specific, and is composed of several DNA elements that are important for its activity. We also show that a transcription factor, early B cell factor, binds to two such elements, and that at least one of these sites is involved in determining enhancer activity.

Bidirectional effect of interleukin-10 on early murine B-cell development: stimulation of flt3-ligand plus interleukin-7-dependent generation of CD19(-) ProB cells from uncommitted bone marrow progenitor cells and growth inhibition of CD19(+) ProB cells.

B-cell commitment and early development from multipotent hematopoietic progenitor cells has until recently been considered to be dependent on direct interaction with stromal cells. We recently showed that the flt3 ligand (FL) has a unique ability to interact with interleukin-7 (IL-7) to directly and selectively promote B-cell development from murine bone marrow progenitor cells with a combined myeloid and lymphoid potential. Here we report that whereas IL-10 alone has no ability to stimulate growth of primitive (Lin-Sca-1(+)c-kit+) bone marrow progenitor cells, it potently enhances FL + IL-7-induced proliferation (sevenfold). This enhanced proliferation results from recruitment of progenitors unresponsive to FL + IL-7 alone, as well as from increased growth of individual clones, resulting in a 7,000-fold cellular expansion over 12 days. Single cell cultures and delayed addition studies suggested that the stimulatory effect of IL-10 was directly mediated on the progenitor cells. The cells generated in response to FL + IL-7 + IL-10 appeared to be almost exclusively proB cells, as shown by their expression of B220, CD24, CD43, and lack of expression of c mu, myeloid, erythroid, and T-cell surface antigens. Although IL-10 also enhanced kit ligand (KL) + IL-7-induced proliferation of Lin-Sca-1(+)c-kit+ progenitor cells, the resulting cells were predominantly myeloid progeny. Accordingly, FL + IL-7 + IL-10 was 100-fold more efficient in stimulating production of proB cells than KL + IL-7 + IL-10. In contrast to its ability to stimulate the earliest phase of proB cell formation and proliferation, IL-10 inhibited growth of proB cells generated in response to FL + IL-7. Analysis of CD19 expression on cells generated in FL + IL-7 + IL-10 showed that almost all cells generated under these conditions lacked expression of CD19, in contrast to cells generated in the absence of IL-10, which were predominantly CD19(+). Replating of sorted CD19(+) and CD19(-) proB cells in FL + IL-7 or FL + IL-7 + IL-10 showed that IL-10 efficiently blocked growth of CD19(+), but not CD19(-) cells. Both CD19(-) and CD19(+) cells expressed lambda5 and VpreB , shown to be specific for B-cell progenitors. In addition, sorted CD19(-) cells generated CD19(+) cells in response to FL + IL-7. Thus, IL-10 has a dual regulatory effect on early B-cell development from primitive murine bone marrow progenitor cells in that it enhances FL + IL-7-induced proB-cell formation and growth before acquisition of CD19 expression, whereas growth of CD19(+) proB cells is inhibited.

A transgenic marker for mouse B lymphoid precursors.

Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5' of the precursor (pre)-B cell-specific lambda5 gene. All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19-lambda5+ precursor of the B220+CD19+lambda5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5' region of the lambda5 gene into the mouse genome confers tissue and differentiation stage-specific expression of a transgene.

Early B cell factor binds to a site critical for lambda5 core enhancer activity.

The pre-B cell-specific expression of the lambda5 gene is regulated at the level of transcription. The 5' region of the lambda5 gene has been shown to contain an enhancer that activates heterologous promoters. Here, we show that this enhancer, B(lambda5), also acts as a lineage- and tissue-restricted enhancer on its own promoter. We define the enhancer core, b(lambda5), that carries around 50% of the total enhancer activity. We also demonstrate that the transcription factor early B cell factor (EBF) binds to a DNA motif in the lambda5 core enhancer which is crucial for enhancer activity, suggesting that lambda5 is a second target gene of EBF.

The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co-expressed during B cell development.

The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16-17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre-B cell receptor complexes.

The c-myc protein represses the lambda 5 and TdT initiators.

The lambda 5 promoter initiates transcription at multiple sites and confers expression in all cell types. Two lambda 5 promoter-derived oligonucleotides (Inr lambda 5:1 and Inr lambda 5:2), each with a transcription start site, could promote transcription in transient transfection assays. In contrast, a third oligonucleotide (+90 lambda 5), without a transcription initiation site, was inactive. The Inr lambda 5:1 and Inr lambda 5:2 oligonucleotides formed a major DNA-protein complex B' in gel retardation analyses; no protein-DNA complexes were observed with the inactive +90 lambda 5 oligonucleotide. The B' complexes of Inr lambda 5:1 and Inr lambda 5:2 each contained c-myc and myn (murine homologue of Max) proteins. The c-myc and myn proteins were also found to bind the TdT initiator (InrTdT). Using mutated oligonucleotides, we found that the c-myc/myn proteins bound to the transcription initiation site of both Inr lambda 5:1 and InrTdT, however, these mutated oligonucleotides were inactive in transfection assays. This suggested that, in this system, transcription depended both on a transcription initiation site and appropriate flanking sequences. The significance of c-myc binding to the respective initiator was analysed by overexpressing c-myc in co-transfection assays. Under these conditions the transcriptional activity of both the lambda 5 and the TdT initiator was repressed.

The structure of an alternate form of complement C3 that displays costimulatory growth factor activity for B lymphocytes.

In this study, the structure of a novel 1.9-kb transcript coding for complement component 3 (C3) is described. This alternate C3 is identical to the 3' end of the C3 message beginning at position 3300 of the C3 cDNA. Its transcription appears to be driven by an alternate promoter located within intron 8 of the C3 gene. This alternate C3 message contains an open reading frame that may encode a 536-amino acid-long protein identical to the 3' part of the C3 alpha chain. The resulting protein contains the complement receptor CR2 binding site. The suggested 5' end of coding region of the alternate C3 includes information for a potential hydrophobic leader peptide that would allow secretion of the protein. In vitro assays with macrophage-depleted mouse splenic B cells indicate that an activity is secreted from cell lines transfected with the alternate C3 cDNA. Together with Sepharose-bound immunoglobulin M-specific monoclonal antibodies and interleukin 2, it costimulates the proliferation of B cells. Implications for possible in vivo functions are discussed.

Pre-B cell-specific lambda 5 gene expression due to suppression in non pre-B cells.

The lambda 5 gene is expressed specifically in pre-B cells; during B lymphocyte differentiation the expression of the lambda 5 gene is turned on in pre-B cells and turned off again at the mature B cell stage. No other cell type has yet been found to express the lambda 5 gene. The pre-B cell-specific expression of the lambda 5 gene is regulated at the level of transcription. We asked whether the region 5' of the lambda 5 gene (5' lambda 5) could explain the stage- and tissue-specific expression. The lambda 5 promoter lacks a TATA box and transcription is initiated at multiple sites. In the presence of a heterologous enhancer, 5' lambda 5 confers pre-B cell-specific expression on the reporter gene chloramphenicol acetyl-transferase. Deletion analysis of 5' lambda 5 defines two separate regions, referred to as A lambda 5 and B lambda 5. Region B lambda 5 suppresses the expression in non pre-B cells since deletion of B lambda 5 allows A lambda 5 to promote transcription of the reporter gene in in all cell types tested. In addition, B lambda 5 acts as an enhancer on the heterologous kappa light chain promoter in pre-B cells but not in B cells. Thus region A lambda 5 functions as a basal promoter in all cell types tested. Region B lambda 5, in concert with a heterologous enhancer, acts as a suppressing region in non-pre-B cells and therefore confers pre-B cell specificity on the expression of the lambda 5 gene.