Germinal centers develop in the B cell follicles of secondary lymphoid tissues during T cell-dependent (TD) antibody responses. The B cells that give rise to germinal centers initially have to be activated outside follicles, in the T cell-rich zones in association with interdigitating cells and T cell help. After immunization with a single dose of protein-based antigen, the germinal centers formed are oligoclonal; on average three B blasts colonize each follicle. These blasts undergo massive clonal expansion and activate a site-directed hypermutation mechanism that acts on their immunoglobulin-variable (Ig-v)-region genes. Mature germinal centers are divided into dark and light zones. The proliferating blasts, centroblasts, occupy the dark zone and give rise to centrocytes that are not in cell cycle and fill the light zone. The light zone contains a rich network of follicular dendritic cells (FDC) that have the capacity to take up antigen and hold this on their surface for periods of more than a year. The antigen is held as an immune complex in a native unprocessed form; but the antigen may be taken up from FDC by B cells, which can process this and present it to T cells. Centrocytes appear to be selected by their ability to interact with antigen held on FDC. There is a high death rate among centrocytes in vivo, and when these cells are isolated in vitro, they undergo apoptosis within hours on culture. The onset of apoptosis can be delayed by cross-linking centrocytes' surface Ig, and long-term survival is achieved by signalling through their surface CD40. After activation through CD40 the centrocytes increase their surface Ig and acquire characteristics of memory and processing of antigen held on FDC and its presentation to T cells that can be induced to express CD40 ligand at the point of cognate interaction. Other signals that induce a proportion of germinal center cells to become plasma cells have also been described. Germinal centers persist for about 3 weeks following immunization, but after this, memory B blasts continue to proliferate in follicles throughout the months of T cell-dependent antibody responses. These cells are probably the source of plasma cells and memory cells required to maintain long-term antibody production and memory after the first 3 weeks of T cell-dependent antibody responses.

Although T cell help for B cells was described several decades ago, it was the identification of CXCR5 expression by B follicular helper T (Tfh) cells and the subsequent discovery of their dependence on BCL6 that led to the recognition of Tfh cells as an independent helper subset and accelerated the pace of discovery. More than 20 transcription factors, together with RNA-binding proteins and microRNAs, control the expression of chemotactic receptors and molecules important for the function and homeostasis of Tfh cells. Tfh cells prime B cells to initiate extrafollicular and germinal center antibody responses and are crucial for affinity maturation and maintenance of humoral memory. In addition to the roles that Tfh cells have in antimicrobial defense, in cancer, and as HIV reservoirs, regulation of these cells is critical to prevent autoimmunity. The realization that follicular T cells are heterogeneous, comprising helper and regulatory subsets, has raised questions regarding a possible division of labor in germinal center B cell selection and elimination.

microRNA-155 (miR-155) is expressed by cells of the immune system after activation and has been shown to be required for antibody production after vaccination with attenuated Salmonella. Here we show the intrinsic requirement for miR-155 in B cell responses to thymus-dependent and -independent antigens. B cells lacking miR-155 generated reduced extrafollicular and germinal center responses and failed to produce high-affinity IgG1 antibodies. Gene-expression profiling of activated B cells indicated that miR-155 regulates an array of genes with diverse function, many of which are predicted targets of miR-155. The transcription factor Pu.1 is validated as a direct target of miR155-mediated inhibition. When Pu.1 is overexpressed in wild-type B cells, fewer IgG1 cells are produced, indicating that loss of Pu.1 regulation is a contributing factor to the miR-155-deficient phenotype. Our results implicate post-transcriptional regulation of gene expression for establishing the terminal differentiation program of B cells.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)