Helper-T-cell regulated B-cell differentiation. Phase I begins at the site of infection with acute inflammation that leads to the activation and

Helper-T-cell regulated B-cell differentiation. Phase I begins at the site of infection with acute inflammation that leads to the activation and emigration of DCs to the T- cell zones of the lymph nodes that drain the area of tissue. Antigen uptake, processing and presentation within the context of MHC II allows the activated DCs to contact and trigger naı ve Th cells expressing specific T-cell receptor (TCR) and initiating immune synapse I (Synapse I). B cells have the capacity to recognize soluble protein antigen; however, they are more efficiently activated by cell-bound antigen and may also initiate an immune synapse with an activated antigen-bearing DC. Following clonal expansion, antigen-activated Th cells migrate to the T B borders of the lymph node to initiate cognate contact with activated antigen-specific B cells. Phase II begins with immune synapse II formation (Synapse II) between these antigen-specific Th and B cells. Synapse II drives a major bifurcation in B-cell differentiation to either short-lived plasma-cell production, which progresses in the T-cell areas, or movement into the follicular areas and the formation of secondary lymphoid follicles. Phase III begins with the polarization of secondary follicles into light-zone and dark-zone regions of activity that typify the GC reaction. This dynamic cycle of activity involves clonal expansion, SHM of the BCR, antigenspecific selection for high affinity variants and then export of memory B cells. These memory B cells can either differentiate into long-lived plasma cells, or remain as non-secreting precursors for antigen recall. Synapse III interactions involve antigenspecific GC Th cells and GC B cells and are proposed to play a critical regulatory role in these late-stage B-cell developmental decisions. 3

Memory B-cell differentiation. At least two phenotypically-distinct types of nonsecreting memory B cells clonally expand in response to antigen recall, together with a rapid and massive production of antigen-specific plasma cells (B220-/CD138- antigen-binding). B220-CD138 antigen binding cells demonstrate greater proliferative capacity but lower differentiative potential than their B220CD138 counterparts over five days after adoptive transfer and antigen re-challenge. These experiments also indicate a parent-progeny relationship between the two memory B-cell subsets,as displayed above. Antigen was required for responsiveness of both memory B-cell subsets; however, the requirement for memory Th cells for each subset was not tested, as it was provided in all cases. Memory B-cell differentiation is generally regarded to be memory-th-cell regulated, suggesting that the formation of immune synapse IV is a critical checkpoint in Phase IV of TD immune responsiveness. 4

Relationship between immunological memory and protective immunity following smallpox vaccination. Two independent studies [20,24] have quantitated the duration of T-cell- and B- cell/antibody-mediated immunity over the course of several decades and came to remarkably similar conclusions: T-cell memory declines slowly over time, with a half-life of 8 15 years (representative thin line), whereas serum antibody responses (and B-cell memory; [20]) are maintained essentially for life with little or no observable decline (representative bold line). Immunological memory quantitated directly ex vivo does not necessarily demonstrate protective immunity; this can only be accomplished by natural exposure or experimental challenge experiments with the virulent pathogen of interest. In this regard, the protection afforded by smallpox vaccination was determined at the indicated intervals (bar graph inset) following immunization and shows that >90% of vaccinees are protected against lethal smallpox (normally 30% mortality in unvaccinated individuals) for at least 60 years post-vaccination [47,48]. Similar results showing long-term immunity were observed during imported smallpox outbreaks throughout Europe between 1950 and 1971 [49,50], decades after endemic smallpox had been eradicated [49]. 7

The formation of B-cell memory in response to antigen. a After activation by antigen, mature naive B cells (which are located in B-cell follicles in secondary lymphoid organs) migrate to the edge of the follicles, where they receive help from cognate T cells. If the B cells express the appropriate molecules, such as a combination of B-cell lymphoma 6 (BCL-6), inducible T-cell co-stimulator ligand (ICOSL), CD40 and B-lymphocyteinduced maturation protein 1 (BLIMP1), the interaction of the B cells and T cells leads to the formation of short-lived plasma cells and to the establishment of germinal centres in the follicles. In the germinal centre, proliferating antigen-specific B cells (known as centroblasts) are localized at one pole (the dark zone), whereas their non-proliferating immunoglobulin-expressing counterparts (known as centrocytes) localize at the other pole (the light zone). Centrocytes and centroblasts cycle within the germinal centre in a chemokine-driven process. Centrocytes can differentiate into memory B cells or plasma cells, or undergo apoptosis if they fail to receive an antigen-mediated survival signal. Although expression of BLIMP1 is crucial for the formation of plasma cells, the factors that control memory B-cell formation are less well defined. Memory B cells recirculate in the periphery, whereas germinal-centre-derived plasma cells accumulate preferentially in the bone marrow. b B1b cells can also generate memory B cells in response to T-cell-independent antigens. Exposure to antigen leads to the formation of plasma cells and to the clonal expansion and persistence of antigenspecific memory B1b cells with a phenotype that is indistinguishable from that of naive B1b cells. Conventional B cells have also been shown to be able to give rise to memory B cells in response to T-cell-independent antigens. 12

Figure 2 A model for the generation of memory B cells and plasma cells in germinal centres during a primary immune response. Cells emigrate from the germinal centre throughout the immune response, as either plasma cells or memory B cells. Under the influence of B-cell receptor (BCR) stimulation, centrocytes with high affinity for antigen differentiate preferentially, but not exclusively, into plasma cells. These plasma cells migrate through the blood, and they accumulate in the bone marrow if they gain access to a survival niche. By contrast, memory B cells that emigrate from the germinal centre constitute a random sampling of centrocytes with various affinities. The size of the memory B-cell pool is finite, so the survival of memory B cells is competitive. Prolonged survival in the germinal centre, which correlates with increased affinity for antigen, improves the competitiveness also known as the fitness of the memory B-cell emigrants and, consequently, their representation in the ultimate memory B-cell population. Increasing intensity of colour corresponds to increasing affinity and fitness. 13

Figure 2 Model of homeostasis in the memory pool. a For each new memory cell that is generated, one must be deleted. This should be on the basis of their ability to access or respond to survival factors, such as antigen or cytokines, both of which replenish the memory pool and can balance homeostatic deletion. b A simple model of how this might work is suggested: the expression of receptors for the survival factor (for example, IL-15 receptor; IL-15R) decays over time after antigen stimulation. So, the longer the time from encounter with antigen, the more likely the cells will be lost from the system. 14

Possible regulation of plasma cell homeostasis by survival niches. Probably recruited by the chemokine receptors indicated in bold, plasma cells formed in secondary lymphoid tissues such as spleen and Peyer s patches migrate into lamina propria, bone marrow or inflamed tissue. Here, their survival depends on the availability of factors provided in a limited number of survival niches. The relatively high numbers of plasma cells competing for survival niches that are present in mucosa-associated tissues result in tough competition conditions and in consequence in the observed short average lifetimes of plasma cells in the lamina propria. The short half-life of plasma cells outside survival niches is probably due to the lack of specific survival signals; however, active elimination can not be excluded. 15

Figure 6 Long-lived plasma cells in the bone marrow. Postgerminal-centre plasma cells, which express somatically mutated, classswitched immunoglobulin, lose expression of CXC-chemokine receptor 5 (CXCR5), facilitating their exit from the germinal centre. These cells then increase their expression of CXCR4, which helps them to home to the bone marrow, where stromal cells produce high amounts of CXCchemokine ligand 12 (CXCL12). Endothelial-cell selectin (E-selectin) and vascular cell-adhesion molecule 1 (VCAM1) expressed at the surface of bone-marrow stromal cells are important for the retention of plasma cells in the bone marrow, through association with polysaccharides and integrins expressed at the surface of the plasma cells. Plasma cells induce the stromal cells to produce interleukin-6 (IL-6). B-cell-activating factor (BAFF), probably produced by macrophages or dendritic cells, activates the receptor B-cell maturation antigen (BCMA) and, together with IL-6, provides crucial survival signals to the plasma cells. BLIMP1, B-lymphocyte-induced maturation protein 1; IL-6R, IL-6 receptor; SDC1, syndecan; XBP1, X-box-binding protein 1. 17

Figure 1. Distinct sets of transcriptional regulators control commitment to the plasma cell differentiation pathway during the humoral immune response. Naïve B cells are activated by antigen (Ag) in the presence of CD4 T-cell help. The activated B cells then continue down one of two divergent pathways: plasma cell (PC) differentiation, or initiation of a GC reaction. MBCs and LLPCs are generated in the GC. When MBCs are re-exposed to antigen they divide rapidly and differentiate into either PCs or more MBCs. The commitment of B cells to the PC differentiation pathway is regulated by the transcription factors Blimp-1, XBP-1 and IRF-4. These factors repress the gene-expression program that defines B-cell identity (Pax5, MITF etc.) and activate a program that drives terminal differentiation and antibody secretion. 19

Figure 3 Transcriptional repression inforces mutually exclusive B-cell and plasma-cell gene-expression programmes. Several transcription factors BCL-6 (B-cell lymphoma 6), MTA3 (metastasisassociated 1 family, member 3), MITF (microphthalmia-associated transcription factor) and PAX5 (paired box protein 5) repress plasmacytic development by repressing BLIMP1 (B-lymphocyte-induced maturation protein 1), XBP1 (X-box-binding protein 1) and IRF4 (interferon-regulatory factor 4). In plasma cells, BLIMP1 represses B-cell gene-expression programmes. This mutual repression prevents the unelicited formation of plasma cells in the germinal centre and prevents the reversion of plasma cells to a B-cell stage. BCR, B-cell receptor; TLR, Toll-like receptor. BCL-6, MTA3, PAX5 and MITF also regulate the expression of genes that are required for B-cell and germinal-centre functions, which are outlined in the pink box. BLIMP1, XBP1 and IRF4 induce the expression of genes that are required for plasma cells, which are outlined in the blue box. 20

Figure 5 Gene expression in immunoglobulin-secreting plasma cells is regulated by BLIMP1 and XBP1. Induction of B-lymphocyteinduced maturation protein 1 (BLIMP1) expression in developing plasma cells leads to decreased expression of paired box protein 5 (PAX5) and increased expression of immunoglobulin. These two events are required for the expression of mrna that encodes X-box-binding protein 1 (XBP1) and for the activation of the endoribonuclease inositol-requiring 1 (IRE1 ), which processes Xbp1 mrna to yield mrna that encodes a different carboxyl terminus and a more active and stable protein. XBP1 then induces the expression of many genes that are involved in the secretory pathway, which generates a physiological unfolded-protein response. The unfolded-protein response allows the continuous production and secretion of high levels of antibody by plasma cells. ER, endoplasmic reticulum; IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; J chain, joining chain; M, transmembrane form of the IgH; S, secretory form of the IgH; PERK, RNA-dependent protein kinase (PKR)-like ER kinase. 21

Proposed model of how BCR affinity controls long-lived humoral immune responses. During an oligoclonal B-cell response, antigen-specific B cells ranging in affinity are activated and undergo proliferation. (a) From this oligoclonal response, B cells with initially low affinity towards antigen will form germinal centers, undergo somatic hypermutation, and differentiate to long-lived memory B (Bmem) cells, plasma cell precursors (PCpre), and bone marrow plasma cells (PCs), each of which has improved antibody affinity. (b) Oligoclonal responding B cells that have moderate affinity for antigen will also form germinal centers yet show a skewing in terminal differentiation whereby they can become long-lived PCpre and PCs, but do not commit to Bmem. (c) When the innate BCR affinity on a naı ve B cell exceeds a certain threshold for antigen, its differentiation fate is to become a short-lived PC. Shuttling intrinsically high-affinity B cells towards an extrafollicular pathway provides rapid local antibody production with the best possible protection early during an immune response while preventing the development of B-cell memory. 22

The transcriptional development of plasma cells is mediated by the total signal strength on the B cell. We propose that the sum signal strength induces high IRF-4 expression in a subset of light-zone centrocytes exiting the germinal center (GC). These signals are composed of BCR signal strength and controlled by BCR affinity to antigen as well as complement receptor and CD40 engagement. Low IRF-4 expression induces AID and thus may play a role in GC B cells, although this link has not yet been made in vivo. (a) Within GC B cells, AID expression is induced by Pax5, which is also responsible for enhancing Bcl-6 expression. To maintain GC B-cell character, Pax5 suppresses XBP-1 whereas Bcl-6 represses Blimp-1, prohibiting differentiation into PCs. (b) High expression of IRF-4 in light-zone centrocytes would lead to the induction of Blimp-1, which represses Pax5 and Bcl-6 and allows for the expression of XBP-1 and terminal differentiation into a plasma cell (PC) secreting immunoglobulin (c). The second major cellular fate of GC B cells is the generation of Bmem (d). IRF-4 does not appear to be required for this fate and the transcriptional pathway driving and sustaining Bmem remains unknown. The Bmem pool supplies the body with a burst of PCs upon secondary antigen encounter and continually replenishes the PC pool through polyclonal stimulation such as TLR ligation and bystander T-cell help. During the transition from a Bmem to a PC, IRF-4 again induces the PC transcriptional pathway through Blimp-1 23

The classical pathway of complement activation. Antigen-antibody complexes that activate the classical pathway may be soluble, fixed on the surface of cells (as shown), or deposited on extracellular matrices. The classical pathway is initiated by the binding of C1 to antigen-complexed antibody molecules, which leads to the production of C3 and C5 convertases attached to the surfaces where the antibody was deposited. The C5 convertase cleaves C5 to begin the late steps of complement activation

Late steps of complement activation and formation of the MAC. A schematic view of the cell surface events leading to formation of the MAC is shown. Cell-associated C5 convertase cleaves C5 and generates C5b, which becomes bound to the convertase. C6 and C7 bind sequentially, and the C5b,6,7 complex becomes directly inserted into the lipid bilayer of the plasma membrane, followed by stable insertion of C8. Up to 15 C9 molecules may then polymerize around the complex to form the MAC, which creates pores in the membrane and induces cell lysis. C5a released on proteolysis of C5 stimulates inflammation.

Functions of complement. The major functions of the complement system in host defense are shown. Cell-bound C3b is an opsonin that promotes phagocytosis of coated cells (A); the proteolytic products C5a, C3a, and (to a lesser extent) C4a stimulate leukocyte recruitment and inflammation (B); and the MAC lyses cells (C).