SUMMARY The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. However, 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 new transgenic photoactivatable green fluorescent protein (PA-GFP) 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, and that there is a net vector of B cell movement from the DZ to the LZ. The decision to return from the LZ to the DZ and undergo clonal expansion is controlled by T cells, which discern between LZ B cells based on the amount of antigen captured, providing a mechanism for GC selection.

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 is unknown. We show that DC development progresses from the macrophage and DC precursor (MDP), to common DC precursors (CDPs) 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 HEVs and later disperse and integrate into the DC network. Further cDC development involves cell division, controlled in part by regulatory T cells (Treg) and fms-related tyrosine kinase-3 (Flt3).Dendritic cells (DCs) are immune cells specialized to capture, process and present antigens to T lymphocytes to induce immunity or tolerance (1). Where commitment to DC development takes place, at what stage the monocyte lineage diverges from DCs, and the precise nature of the migrating DC precursor that moves from the bone marrow to the peripheral lymphoid organs is not known. These questions have been difficult to resolve in part because DC subsets are functionally and phenotypically diverse (2). For example, classical spleen DCs (cDCs) include two major functionally distinct subsets distinguished by the expression of a variety of C-type lectins and CD8 (2-4). Spleen and other tissues also contain plasmacytoid DCs (pDCs) that primarily initiate immune responses to nucleic acids (5,6).Lymphoid tissue cDCs, pDCs, and monocytes share a common progenitor called the macrophage and DC precursor (MDP) identified by its surface phenotype (Lin − cKit hi CD115 + CX 3 CR1 + Flt3 + ) (7,8), whereas a distinct progenitor called the common DC precursor (CDP) (Lin − cKit lo CD115 + Flt3 + ) is restricted to producing cDCs and pDCs (9,10). Although monocytes can develop many of the phenotypic features of DCs under inflammatory conditions (11-13), the cDC, pDC and monocyte lineages separate by the time they reach tissues and neither monocytes nor pDCs develop into cDCs under steady state conditions (8,14). Unlike monocytes and pDCs, cDCs in lymphoid tissues are thought to emerge from the bone marrow as immature cells that must further differentiate and divide in lymphoid organs (15,16 We searched for MDPs and CDPs in the blood and spleen by flow cytometry but could only detect them in the bone marrow ( Fig. 1A and Fig. S1). Although pre-cDCs can be identified in the spleen by combining density centrifugation and flow cytometry (18), we speculated that these cells could be identified directly by expression of Flt3 and the chemokine receptor, CX 3 CR1, which are expressed on other DC progenitors and also on mature cDCs (7,10,19). Indeed, we found a small but distinct population of lineage negative CD11c + MHC class II − SIRP-α int Flt3 + cells (pre-cDCs) in the bone marrow (0.2%), blood (0.03%), spleen (0.05%) and lymph nodes (LN) (0.03%) (Fig. 1B).To determine ...

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.

Entry into the germinal center requires antigen-bearing B cells to compete for cognate T cell help at the T–B border.

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.

Ikaros is an essential regulator of lymphopoiesis. Here, we studied the B-cell-specific function of Ikaros by conditional Ikzf1 inactivation in pro-B cells. B-cell development was arrested at an aberrant 'pro-B' cell stage characterized by increased cell adhesion and loss of pre-B cell receptor signaling. Ikaros was found to activate genes coding for pre-BCR signal transducers and to repress genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of Aiolos expression could 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, Ikaros binding and target gene expression was dynamically regulated at distinct stages of early B-lymphopoiesis.

T cells survey antigen-presenting dendritic cells (DCs) by migrating through DC networks, arresting and maintaining contact with DCs for several hours after encountering high-potency complexes of peptide and major histocompatibility complex (pMHC), leading to T cell activation. The effects of low-potency pMHC complexes on T cells in vivo, however, are unknown, as is the mechanism controlling T cell arrest. Here we evaluated T cell responses in vivo to high-, medium- and low-potency pMHC complexes and found that regardless of potency, pMHC complexes induced upregulation of CD69, anergy and retention of T cells in lymph nodes. However, only high-potency pMHC complexes expressed by DCs induced calcium-dependent T cell deceleration and calcineurin-dependent anergy. The pMHC complexes of lower potency instead induced T cell anergy by a biochemically distinct process that did not affect T cell dynamics.

SUMMARY 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.