Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

Ikaros prevents autoimmunity by controlling anergy and Toll-like receptor signaling in B cells.

The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318, which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88-nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling.

Essential role for the transcription factor Bhlhe41 in regulating the development, self-renewal and BCR repertoire of B-1a cells.

Innate-like B-1a cells provide a first line of defense against pathogens, yet little is known about their transcriptional control. Here we identified an essential role for the transcription factor Bhlhe41, with a lesser contribution by Bhlhe40, in controlling B-1a cell differentiation. Bhlhe41-/-Bhlhe40-/- B-1a cells were present at much lower abundance than were their wild-type counterparts. Mutant B-1a cells exhibited an abnormal cell-surface phenotype and altered B cell receptor (BCR) repertoire exemplified by loss of the phosphatidylcholine-specific VH12Vκ4 BCR. Expression of a pre-rearranged VH12Vκ4 BCR failed to 'rescue' the mutant phenotype and revealed enhanced proliferation accompanied by increased cell death. Bhlhe41 directly repressed the expression of cell-cycle regulators and inhibitors of BCR signaling while enabling pro-survival cytokine signaling. Thus, Bhlhe41 controls the development, BCR repertoire and self-renewal of B-1a cells.

Enhancers of the PAIR4 regulatory module promote distal VH gene recombination at the Igh locus.

While extended loop extrusion across the entire Igh locus controls VH -DJH recombination, local regulatory sequences, such as the PAIR elements, may also activate VH gene recombination in pro-B-cells. Here, we show that PAIR-associated VH 8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5' region, which reduced distal VH gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal VH gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate VH gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal VH gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.

Stage-specific control of early B cell development by the transcription factor Ikaros.

The transcription factor Ikaros is an essential regulator of lymphopoiesis. Here we studied its B cell-specific function by conditional inactivation of the gene encoding Ikaros (Ikzf1) in pro-B cells. B cell development was arrested at an aberrant 'pro-B cell' stage characterized by increased cell adhesion and loss of signaling via the pre-B cell signaling complex (pre-BCR). Ikaros activated genes encoding signal transducers of the pre-BCR and repressed genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, binding of Ikaros and expression of its target genes were dynamically regulated at distinct stages of early B lymphopoiesis.

Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

Flexible long-range loops in the VH gene region of the Igh locus facilitate the generation of a diverse antibody repertoire.

The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH-rearranged gene segment in the 3' proximal Igh domain. By using high-resolution mapping of long-range interactions, we demonstrate that local interaction domains established the three-dimensional structure of the extended Igh locus in lymphoid progenitors. In pro-B cells, these local domains engaged in long-range interactions across the Igh locus, which depend on the regulators Pax5, YY1, and CTCF. The large VH gene cluster underwent flexible long-range interactions with the more rigidly structured proximal domain, which probably ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire. These long-range interactions appear to be an intrinsic feature of the VH gene cluster, because they are still generated upon mutation of the Eµ enhancer, IGCR1 insulator, or 3' regulatory region in the proximal Igh domain.

Development and migration of plasma cells in the mouse lymph node.

In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. Pre-PCs exhibited a unique migration pattern characterized by long, linear paths that were randomly oriented. Although chemotaxis via Galphai coupled-receptors has been implicated in PC migration, treatment with Pertussis toxin (Ptx), which ablates these signals, did not prevent movement of pre-PCs while it arrested other lymphocytes. In vitro, pre-PCs displayed processive amoeboid locomotion on surfaces coated with integrin ligand, whereas fully differentiated PCs moved slowly or were arrested. Both PC arrest and differentiation occurred in the medullary cords. Ptx treatment before PC differentiation blocked their accumulation in the medullary cords but pre-PCs still differentiated in other lymph node regions. Taken together, we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ.

MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.

In vivo imaging of germinal centres reveals a dynamic open structure.

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.

Thymocyte-dendritic cell interactions near sources of CCR7 ligands in the thymic cortex.

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals.

Control of antigen receptor diversity through spatial regulation of V(D)J recombination.

Lymphocytes recognize a vast variety of pathogens by expressing a diverse repertoire of antigen receptor genes that are assembled by V(D)J recombination in immature B cells (Igh, Igk) and T cells (Tcrb, Tcra/d). V(D)J recombination takes place in the 3' proximal domain containing the D, J, and C gene segments, whereas 31 (Tcrb) to 200 (Igh) V genes are spread over a large region of 0.67 (Tcrb) to 3 (Igk) Mb pairs. All antigen receptor loci undergo reversible contraction at the developmental stage, where they engage in V-(D)J recombination. This long-range looping promotes the participation of all V genes in V-(D)J recombination by juxtaposing distant V genes next to (D)J segments in the proximal recombination center. The B-cell-specific Pax5, ubiquitous YY1, and architectural CTCF/cohesin proteins promote Igh locus contraction in pro-B cells by binding to multiple sites in the VH gene cluster. These regulators also control the pro-B-cell-specific activity of the distally located PAIR elements, which are likely involved in the regulation of VH-DJH recombination by mediating locus contraction. Notably, the large VH gene cluster of the Igh locus undergoes flexible long-range looping that ensures similar participation of all VH genes in VH-DJH recombination to generate a diverse antibody repertoire.

A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center.

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide-major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T-B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells.

Germinal center reutilization by newly activated B cells.

Germinal centers (GCs) are specialized structures in which B lymphocytes undergo clonal expansion, class switch recombination, somatic hypermutation, and affinity maturation. Although these structures were previously thought to contain a limited number of isolated B cell clones, recent in vivo imaging studies revealed that they are in fact dynamic and appear to be open to their environment. We demonstrate that B cells can colonize heterologous GCs. Invasion of primary GCs after subsequent immunization is most efficient when T cell help is shared by the two immune responses; however, it also occurs when the immune responses are entirely unrelated. We conclude that GCs are dynamic anatomical structures that can be reutilized by newly activated B cells during immune responses.

In vivo analysis of dendritic cell development and homeostasis.

Dendritic cells (DCs) in lymphoid tissue arise from precursors that also produce monocytes and plasmacytoid DCs (pDCs). Where DC and monocyte lineage commitment occurs and the nature of the DC precursor that migrates from the bone marrow to peripheral lymphoid organs are unknown. We show that DC development progresses from the macrophage and DC precursor to common DC precursors that give rise to pDCs and classical spleen DCs (cDCs), but not monocytes, and finally to committed precursors of cDCs (pre-cDCs). Pre-cDCs enter lymph nodes through and migrate along high endothelial venules and later disperse and integrate into the DC network. Further cDC development involves cell division, which is controlled in part by regulatory T cells and fms-like tyrosine kinase receptor-3.

Regulation of class switch recombination and somatic mutation by AID phosphorylation.

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.