Many key features of helper T cell–dependent antibody responses, including affinity maturation and the generation of long-lived plasma cells and memory B cells, develop in organized structures called germinal centers that are created within lymphoid follicles during T-dependent immune responses. The differentiation and selection of B cells that occur in these sites constitute the germinal center reaction. Germinal centers begin to develop in secondary lymphoid organs approximately 4 to 7 days after the initiation of a T-dependent B-cell response. Long before the functional significance of this anatomical structure was understood, it was named the germinal center by morphologists because the presence of many mitotic figures in the region suggested that new cells were generated (“germinated”) there. Germinal centers consist of two distinct regions (Fig. 1): a dark zone that is densely packed with rapidly proliferating B cells and appears dark in histologic sections stained with certain dyes, and a light zone that stains weakly with the same dyes, where high affinity B cells are selected to survive and differentiate further but many cells die. The cellular composition of these zones is different. Tfh cells are present only in the light zone, which also contains FDCs. FDCs, briefly mentioned earlier in this chapter, are stromal cells that express complement receptors (CR1, CR2, and CR3) and Fc receptors and these cells are found only in lymphoid follicles. The Fc receptor and complement receptors on FDCs are involved in displaying antigens for the selection of germinal center B cells, as described below. FDCs do not express MHC-II molecules and are not derived from progenitors in the bone marrow. Thus, in spite of their name, they are distinct from the MHC-I and -II–expressing DCs that present peptides to T lymphocytes. The long cytoplasmic processes of FDCs form a meshwork around which germinal centers are formed. B cells in the dark zone and light zone are sometimes called centroblasts and centrocytes, respectively; however, dark zone and light zone B cells have very similar patterns of gene expression, differing in the expression of just a few genes, so they represent transiently altered states of germinal center B cells and not different cell types. These zones are not fixed, since B cells move between the dark zone and the light zone. The rim of naive B cells in the follicle surrounding the germinal center makes up the mantle zone.
Fig1. Germinal centers in secondary lymphoid organs. (A) The germinal center is within the follicle and includes a dark zone (DZ) and an adjacent light zone (LZ). (B) Germinal center B cells express B-cell lymphoma 6 (BCL-6) (as do T follicular helper cells), inducible costimulator (ICOS)–expressing helper T cells reside mainly in the LZ and in the extrafollicular area, and activation-induced cytidine deaminase (AID) is expressed at its highest levels in germinal center B cells in the DZ (A, Courtesy Dr. James Gulizia, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts. B, Courtesy Dr. Naoki Kaneko, Ragon Institute of Mass General, MIT and Harvard, Cambridge Massachusetts.)
The germinal center reaction requires the entry into the follicle of Tfh cells, as a result of high levels of CXCR5 expression, and these Tfh cells help induce the architecture of the germinal center. When Tfh cells enter the follicle from the T-B border, a few previously activated extrafollicular B cells that have re expressed CXCR5 also return into the follicle. Activated B cells that have been triggered by Tfh cells through CD40L-CD40 interactions downregulate the oxysterol chemoattractant receptor EBI2, and move to the center of the follicle. Each germinal center contains B cells derived from as little as a few to up to about a hundred antigen-specific clones.
The germinal center reaction consists of a number of sequential steps (Fig. 2).
1. B-cell proliferation and somatic hypermutation. B cells that have been triggered by Tfh cells (via CD40L-CD40 interactions) repeatedly proliferate, forming the dark zone of the germinal center. Proliferating cells express the CXCR4 chemokine receptor, which helps retain them in the dark zone because the stromal cells in the dark zone secrete higher levels of CXCL12, the ligand for CXCR4, compared to the light zone. The doubling time of these proliferating germinal center B cells is estimated to be 6 to 12 hours, so that within 5 days, a single lymphocyte may give rise to as many as 5000 progeny. Proliferating dark zone B cells mutate their Ig V genes at a very high rate as part of a process called somatic hypermutation. This process is initiated by the enzyme AID, which is expressed at high levels in dark zone B cells; its mechanism is described later. AID is also required for class switching. Although most class switching occurs extrafollicularly, it can continue when B cells are in the light zone of the germinal center.
After multiple divisions, dark zone B cells stop dividing and shut off expression of the CXCR4 chemokine receptor. The loss of CXCR4 expression results in the migration of these heavily mutated nondividing B cells to the adjacent light zone that contains Tfh cells and FDCs. These nondividing dark zone B cells still express CXCR5 and are drawn towards the higher concentration of CXCL13 in the light zone.
2. Capture of antigen by light zone B cells. FDCs display anti gens in immune complexes bound to complement receptors or to Fc receptors. FDCs are the major source of the chemokine CXCL13, which, as described earlier, draws B cells into the germinal center. The activated B cells enter the light zone and B cells with BCRs with a higher affinity for the antigen in the immune complexes most efficiently bind, capture and ingest the antigen.
3. Selection of high-affinity B cells. In the light zone, B cells that have undergone somatic hypermutation, have acquired high affinity BCRs, and have successfully captured the antigen from FDCs then present the relevant MHC-II–peptide com plex on the cell surface. This surface expression of the same antigen that was initially presented by DCs to helper T cells allows Tfh cells in the light zone to recognize and positively select high-affinity B cells. This process of cognate interactions of selected B cells with Tfh cells results in the delivery of CD40 and cytokine signals to the B cells. The B cells also re express the CXCR4 chemokine receptor and thus return to the dark zone. Germinal center B cells can undergo repeated rounds of mutation and selection, migrating back and forth from light to dark zone if they are positively selected. This process contributes to the affinity maturation of the antibody response. B cells that fail selection undergo apoptotic death in the light zone.
4. Differentiation into memory cells. After just a few rounds of selection, and relatively early in the germinal center response, B cells of moderate affinity start to exit the germinal center as memory B cells that acquire the ability to recirculate and migrate from one secondary lymphoid organ to another. Memory B cells can respond rapidly to antigen and T-cell help on subsequent exposures. Memory B-cell formation in the germinal center peaks about a week before the peak of long-lived plasma cell formation, but both processes occur over many days. The relatively early exit from the germinal center after only a few rounds of selection ensures that many memory B cells are not too narrowly focused only on the original antigen that induced them but they retain “immunological breadth,” the potential to respond to variant or mutant versions of the original antigen, as may often be encountered by the host.
5. Differentiation into long-lived plasma cells. After many rounds of selection, relatively late in the germinal center response, B cells of high affinity develop into plasma cells that exit the germinal center and migrate to the bone mar row. In most infectious contexts and with some, but not all, vaccines, these bone marrow plasma cells are long-lived and may secrete high-affinity antibodies for decades. However, strong germinal center responses do not always result in long-lived plasma cells. Plasma cell durability is discussed later in this chapter. Fully differentiated plasma cells lack sur face BCR expression and are unable to recognize or respond to antigen.
Fig2. Sequential events in the germinal center reaction. Activated B cells migrate into the follicle and proliferate, forming the dark zone of the germinal center while recently generated T follicular helper (Tfh) cells migrate into the light zone. Activated dark zone B cells undergo somatic hypermutation of immunoglobulin (Ig) V genes and migrate into the light zone, where they encounter follicular dendritic cells (FDCs) displaying antigen. B cells with higher affinity B-cell antigen receptors capture the antigen from FDCs. B cells with the highest affinity Ig receptors can most efficiently present major histocompatibility complex class II–peptide complexes to Tfh cells and are selected to survive; they cycle back to the dark zone where they undergo more somatic hypermutation. Selected cells can go back and forth multiple times and are thus repeatedly mutated and selected. After a few rounds of selection, some B cells differentiate into memory B cells, leave the germinal center, and enter the recirculating lymphocyte pool. After many rounds of mutation and selection, other B cells finally differentiate into antibody-secreting cells that leave the germinal center and migrate to and reside in the bone marrow as long-lived plasma cells.
In germinal center B cells, signals delivered through CD40 and the IL-21 receptor induce the expression of BCL-6, which functions as a transcriptional repressor to maintain the germinal center reaction, particularly the massive proliferation of germinal center B cells. BCL-6 is also the transcription fac tor required for activated CD4+ T cells to assume a Tfh fate, as described above. In germinal center B cells, BCL-6 represses the expression of cyclin-dependent kinase inhibitors and thus cooperates with transcriptional activators, such as c-MYB, to orchestrate rapid cell cycle entry of the B cells. BCL-6 also represses p53, a transcription factor that mediates cell cycle arrest and apoptotic cell death after DNA damage. As a result, dark zone B cells can tolerate the DNA breaks that accompany class switching and somatic hypermutation and do not undergo apoptosis. BCL-6 antagonizes another transcriptional repressor called BLIMP1 (B lymphocyte–induced maturation protein 1), which is required for plasma cell development (see later), and thus prevents cells in the germinal center from prematurely differentiating into plasma cells during the massive proliferation that is characteristic of the germinal center reaction.
How the decision is made for selected light zone B cells to not return to the dark zone but to instead exit the germinal center and differentiate into memory B cells or plasma cells, especially at different stages of the germinal center reaction, is not known. What is known is that once this decision is made, light zone B cells that have committed to a differentiated fate re-express EBI2 and thus leave the germinal center. FDCs that reside in the center of the light zone express very high levels of the complement receptor CR2, and these cells contribute to the extended persistence of antigens in the germinal center, for many months, and possibly years, sequestered within immune complexes.
Germinal center formation is defective in humans (and in genetically engineered mice) with genetic defects in T-cell development or activation or with mutations of either CD40 or its ligand, discussed earlier.
Now that we have described the basic characteristics of the extrafollicular response and the germinal center reaction, we will discuss some of the individual cellular and molecular events in these processes.
