Christian Vettermann

CTCF-binding elements mediate control of V(D)J recombination

Immunoglobulin heavy chain (IgH) variable region exons are assembled from VH, D and JH gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the VH and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of DH-proximal VH gene segments and promoting rearrangement of distal VH segments. IGCR1 maintains ordered and lineage-specific VH(D)JH recombination by suppressing VH joining to D segments not joined to JH segments, and VH to DJH joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal VH-to-DJH recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all VH genes to be brought in proximity with DH-JH segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the VH region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a DH-JH-Eμ-CH domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the DH region and parts of the VH locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

Interaction of Murine Precursor B Cell Receptor with Stroma Cells Is Controlled by the Unique Tail of λ5 and Stroma Cell-Associated Heparan Sulfate

Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane μH chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igαβ. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and λ5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igαβ in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of λ5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of λ5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.

Allelic exclusion of immunoglobulin genes: Models and mechanisms

Summary:  The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B‐cell development, the relevance of monospecificity to B‐cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Igκ and Igλ light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.

Lipid Rafts Associate with Intracellular B Cell Receptors and Exhibit a B Cell Stage-Specific Protein Composition

Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits α-1 and β-2, vimentin, γ-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

Immunoglobulin μ Heavy Chains Do Not Mediate Tyrosine Phosphorylation of Igα from the ER-cis-Golgi

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early μ heavy chain (μHC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a μHC, a surrogate L chain, and the signal dimer Igαβ. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a μHC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Igα in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing μHC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing μHC assembled with the Igαβ dimer; however, in contrast to a pairing μHC, the nonpairing μHC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Igα after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of μHC, Igαβ, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.

The pre-B cell receptor: turning autoreactivity into self-defense

The first step in establishing the antibody repertoire in humans and mice is the rearrangement of immunoglobulin heavy chain (HC) genes in early B lineage cells. These cells then assemble microHCs with surrogate light chains (SLC) into a pre-B cell receptor (pre-BCR). We propose that the pre-BCR has evolved from an ancient autoreactive BCR, since the SLC is an autoreactive entity that binds to the pre-BCR itself and to other self-antigens. Abrogation of autoreactivity in the SLC diminishes pre-BCR signaling and impairs the clonal expansion of pre-B cells producing functional microHCs. Since SLC expression is restricted to pre-B cells, the autoreactivity encoded by the pre-BCR can be utilized to pre-select the antibody repertoire, while simultaneously avoiding the formation of autoreactive B lymphocytes.

A colloidal silver staining–destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.

The Proximal J Kappa Germline-Transcript Promoter Facilitates Receptor Editing through Control of Ordered Recombination

V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.

APOBEC3 enzymes restrict marginal zone B cells

In general, a long‐lasting immune response to viruses is achieved when they are infectious and replication competent. In the mouse, the neutralizing antibody response to Friend murine leukemia virus is contributed by an allelic form of the enzyme Apobec3 (abbreviated A3). This is counterintuitive because A3 directly controls viremia before the onset of adaptive antiviral immune responses. It suggests that A3 also affects the antibody response directly. Here, we studied the relative size of cell populations of the adaptive immune system as a function of A3 activity. We created a transgenic mouse that expresses all seven human A3 enzymes and compared it to WT and mouse A3‐deficient mice. A3 enzymes decreased the number of marginal zone B cells, but not the number of follicular B or T cells. When mouse A3 was knocked out, the retroelement hitchhiker‐1 and sialyl transferases encoded by genes close to it were overexpressed three and two orders of magnitude, respectively. We suggest that A3 shifts the balance, from the fast antibody response mediated by marginal zone B cells with little affinity maturation, to a more sustained germinal center B‐cell response, which drives affinity maturation and, thereby, a better neutralizing response.