Germinal centers

Abstract

This article introduces a series of reviews covering Germinal Centers appearing in Volume 247 of Immunological Reviews. Germinal centers (GCs) are clusters of highly proliferative B cells that form within otherwise quiescent B-cell follicles in response to certain types of antigenic stimulation. GCs form after a delay of several days following immunization, and the responses typically do not reach peak size until 1.5–2 weeks postimmunization. In mice, immunized with protein antigen in adjuvant, the GC response can persist detectably for more that 8 weeks, long after typical pathogens would be cleared. It has been appreciated for some time that this delayed and persistent response is more concerned with development of optimal long-lived immunity rather than immediate pathogen clearance (1). Clonal selection typically elicits very low-affinity B cells to initiate immune responses and even to seed the GC (2-4). Within the GC, a process of intense selection of B cells yields a population with ever-increasing affinity for antigen as well as increased diversity. Somatic hypermutation of V (variable) regions, which adds as many as one mutation per cell per division (5, 6), provides a substrate for selection of higher affinity and more diverse populations of cells. From these cells differentiate two long-lived populations: memory B and plasma cells. Hence, the GC is largely responsible for generating both components of long-lived humoral immunity and optimizing affinity and diversity of these cellular compartments. The processes that determine these outcomes are among the most longstanding mysteries of the immune system, both because of the unique nature of events such as somatic hypermutation as well as the critical importance of them for phenomena as important as vaccination, immune memory, autoimmunity, and cancer. For these reasons, GCs have been studied consistently over the last 30 or more years, since it was originally recognized that GCs contain the precursors of memory B cells (7). However, progress has been episodic. Pioneering studies proved that GCs were a prominent site of somatic hypermutation (8, 9), and inhibition of CD40L (10-12) demonstrated that GCs required ongoing T-cell signals and also strongly suggested that long-lived plasma cells were derived from the GC. A key observation from the last decade demonstrated the requirement of the transcription factor Bcl-6 for GC development (13, 14). Connected with this finding has been the appreciation that the cell of origin for many types of B-cell malignancies is the GC (15, 16). Nonetheless, many important questions have remained: how selection for high affinity works in the GC, how cells migrate in the GC, what the functions of zonal differences in the GC are, the nature of T–B interactions and of the T cells in the GC, and how GC cells differentiate into either memory B or plasma cells. The last 7 or so years have been a period of increased activity in GC-related research. This has been fueled by several factors including technical advances such as microarray-based transcriptional analysis, ChiP and ChiP-seq to assess transcriptional networks, bioinformatics, tissue-specific gene deletion, and the ability to image cells in vivo. This volume has assembled reviews from many of the leaders in these diverse approaches to GC biology. Recently, all have contributed in exciting ways to the field and several have contributed consistently over the past 2 decades. Their topic areas cover the most fundamental and current topics in GC biology. They can be grouped loosely together based on their perspectives on the study of the GC reaction. In this context, the GC can be seen as an incubator of long-term immunity, a model system for understanding the roles and controls of cell movements during the immune response, a system for study of molecules that control developmental decisions and survival, and a source of B-cell lymphoproliferative disorders. The accompanying figure (Fig. 1) diagrammatically represents the reviews in the context of the B-cell immune response and its malignant transformation. The stages or processes on which each review focuses are indicated on the cartoon, with some reviews appearing in multiple places. This ‘visual abstract’ will allow the reader interested in a particular aspect to direct oneself to the relevant reviews. If this is not enough, read on. ‘Visual abstract’ of the reviews in this volume of Immunological Reviews in the context of the B-cell immune response and its malignant transformation. The stages or processes on which each review focuses are indicated on the cartoon, with some reviews appearing in multiple places. Several of the reviews in this volume have a perspective on how the GC promotes long-term immunity. The reviews from both the Brink (17) and Shlomchik (18) groups deal with the critical issue of how higher affinity cells are selected in the GC and what promotes differential cell survival or proliferation. Chan and Brink (17) provide a comprehensive history and context for the questions, going on to review their laboratory’s many contributions to our understanding of this issue using their SWHEL Ig V knockin mouse. They provide hypothetical models for both positive and negative selection in the GC. Weisel and Shlomchik (18) cover similar ground, with emphasis on the origins of memory and plasma cells as well as on how affinity-based selection occurs in the GC. This article includes a critical review of the literature, defining the persistent areas of disagreement and as yet inconclusive data. Drawing from these studies, as well as their own, Weisel and Shlomchik also propose a novel model to explain how the GC generates both memory B and plasma cells. Klein (19) and Tarlinton (20) take a more molecular view, focusing on IRF-4 in the former case and Bcl-2 family member function in the latter, both emphasizing how these promote development and survival of long-lived progeny. The Cyster laboratory has long been a leader in defining how cell movement is controlled during the immune response as a whole, and in recent years, they have focused considerable attention on the GC itself as well as cellular interactions leading up to it (21-25). This review (26) brings us right up to date with a focus on coordinated expression of receptors and ligands that control B and T-cell movement throughout the process. Particularly emphasized are this groups recent and emerging data on two ‘new’ players in this area, EBI2 (27, 28) and S1PR2. An integrated model is proposed to explain GC B and T-cell choreography based on the expression of these receptors and their ligands, along with more well-known receptors CXCR4, CXCR5, and CCR7 and their ligands. In addition, Green and Cyster (26) discuss how S1PR2 may also signal for cell survival and thereby influence positionally dependent selection events. Takaharu Okada, working with Cyster, began his study in the field by providing pivotal data on early movements that precede the GC reaction and then went on to define cell movements within the GC in live tissue (21, 23). Herein (29), along with two colleagues from his independent laboratory at the RIKEN in Japan, he reviews and describes his most recent study on the early events in both B and T cells, in this case using a novel Bcl6 protein-reporter mouse that they constructed (30). These data, along with data from other laboratories using more traditional staining and in vivo microscopy methods (31, 32), have led to new insights into how the GC forms and the order and nature of the cellular interactions that promote GC development. It has long been appreciated that a significant minority population in the GC is comprised of CD4+ T cells. However, only recently has the identity and the functions of these cells been unraveled. It is thus fitting, in the context of key cellular interactions in the GC, that three of the reviews – all from groups that have made important contributions in the last few years – center on these so-called T follicular helper (Tfh) cells. Each review nonetheless takes a different perspective. Crotty (33) provides a conceptual framework for the complex molecular interactions that underlie Tfh (or for that matter any immune cell type’s) function, specification (as in cell type identity), and induction (as in cellular interactions and external signals). In a sense, the review by Linterman and Vinuesa (34) – two individuals (along with colleague Liston) that together have made a series of pioneering discoveries about the origins, functions, and diversity of Tfh-like cells – hits a similar note. They cover recent data from their own and other laboratories that demonstrate functional diversity within the Tfh-like compartment. Among cells T cells that express Bcl6, which is required for Tfh differentiation, not all are classical Tfh in phenotype or function. Strikingly, as shown recently by their laboratory and two others (35-37), some Bcl6+ cells in the GC express forkhead box protein 3 (FoxP3) and serve to regulate the size, duration, and quality of the GC B-cell compartment. In addition, invariant natural killer T cells can also express Bcl6 and provide B helper function. Moreover, as shown by both the Vinuesa and Craft group (38, 39), some CD4+ T-helper cells expressing Bcl6 also express CXCR4 and hence are located in extrafollicular sites, where they help the local plasmablast response. In their review, Craft and colleagues (40) cover both Tfh and their extrafollicular counterpart, which he has termed Tefh (38). However, their perspective differs importantly from the other reviews that discuss these related T cell lineages. They focus on the role of these cell types in autoimmunity, with emphasis on human T cells and circulating counterparts found in the blood. This review (40), thus, nicely rounds out a group of papers that examine novel aspects of Tfh biology. Notably, for the reader learning about this topic, the review by Okada and colleagues (29) also touches on Tfh development, with a focus on Bcl6. Bcl6 has been the poster child for a ‘master-regulator’ of GC B-cell development. Certainly, GC B cells do not develop without it (13, 14). As noted, Bcl6 remarkably plays important and complex roles in a variety of T-cell types that interact with B cells. In this volume, Dalla-Favera (41), whose laboratory has contributed greatly over many years to our understanding of Bcl6 at every level, updates us on expression and function of this molecule in health and disease, emphasizing human biology. Similarly, the review by Okada and colleagues (29) joins this group by reviewing data on Bcl6 expression in vivo during the B-cell immune response, as visualized by their new reporter mouse. Recent study has identified a variety of other transcription factors as well as cell signaling pathways that interact with Bcl6 and each other to determine GC B-cell fate. Several of the reviews in this volume tackle some of these lesser known, but fascinating molecules, including those by Klein (IRF-4; 19) and Maeda (LRF/PLZF/Zbtb7a; 42). Both of these molecules play key, but distinct roles in GC development. Although IRF-4 is important in early B-cell development [and hence required Klein to develop a conditional allele to study its role in GCs (43)], GCs form relatively normally in IRF-4-deficient mice, but such GC B cells do not make long-lived PCs. The review by Klein (19) represents a definitive assessment of the role of IRF-4 throughout B-cell development. It covers and tries to reconcile sometimes conflicting data and provides models of integrated signaling networks, with particular emphasis on PC development from GC precursors. LRF is a member of the large BTB-POZ family of transcriptional repressors, whose genes are named in the ‘Zbtb’ gene series. Notably, Bcl6 is another family member. Although the latter is well known for its roles in GC development, Maeda and colleagues (44) recently discovered that LRF plays a very critical role in GC development. In this volume, Sung-Uk and Maeda (42) review the role of LRF throughout B-cell development, emphasizing GCs. They further put this in the context of a broader look at the roles of BTB-POZ family members in lymphoid development, which represents a fascinating perspective. Linked to differentiation and selection is the control of survival. This is a major issue in the GC and in fact throughout lymphocyte responses. Intense proliferation and selection required to optimize clonal selection-based systems that begin with off-the-shelf receptors for pathogen epitopes cannot work unless most of the cells do not survive. How is selective survival controlled? It has long been known that Bcl2 (B-cell lymphoma-2) family members play important roles in controlling cell survival in every tissue, but the first clues to functions of these proteins came from study of B cells. This family and its molecular inhibitors are complex, and it is only now being appreciated how and when individual proteins of this group function during B-cell development and immune responses. Tarlinton and colleagues (45-47) have recently made important contributions to this field, using novel genetic and inhibitor approaches. The role of Bcl2 family members in B-cell responses is the focus of their review in this volume (20). A second molecular angle on the GC reaction comes from consideration of signaling molecules and cytokines that help to initiate, modify, and coordinate the response. These are numerous, of course, and many are well known. For example, CD40L and ICOS are two T-cell–expressed molecules critical for normal GC formation and maintenance (10-12, 48, 49). In this volume, three molecules that influence the GC reaction and that are the subject of recent and ongoing studies are central topics for review. Hai Qi (50), who while working with Ron Germain beautifully elucidated the role that SAP and CD150 family signaling plays in the development of the GC (51), reviews how these molecular signals coordinate the GC reaction. This discussion culminates in a synthesis of how bi-directional signals between T and B cells and T and dendritic cells both initiate and sustain the GC reaction. DeFranco and colleagues (52) have explored the role of Toll-like receptor (TLR) signaling in GC development. This has been an area of considerable controversy and conflicting results (53-56). Recently, DeFranco’s laboratory (52) has revisited this issue using precise tools – a floxed MyD88 allele along with appropriate Cre mice – and a comprehensive analysis of the immune response along with different stimulatory conditions. This has led to new insights into this topic. Although conceptual and experimental gaps remain, the review by DeFranco (57) provides a well-integrated review of the existing data and a synthesis of how TLRs influence both GC and extrafollicular reactions in health and disease, emphasizing how crosstalk between the nature of B cell receptor and TLR signaling could influence outcome. It is thought that a number of B-cell malignancies are likely to have a GC B-cell origin, including follicular lymphoma and at least some types of diffuse large cell lymphoma, as well as Hodgkin’s lymphoma (58). Perhaps not surprisingly, genes important for GC development can also serve as oncogenes, usually as a result of translocation events, in these malignancies. Indeed, activation-induced cytidine deaminase (AID), the protein responsible for somatic hypermutation and class switch DNA rearrangement and which is highly expressed in GC B cells, is involved in both rearrangements and point mutations that activate key oncogenes and is required for oncogenesis in murine models (59-62). Bcl2 was identified first in a B-cell lymphoma (63). In their review, the Tarlinton group (20) not only covers the broader family’s roles in normal development and responses but also in various B-cell malignancies. Bcl6 translocations are commonly found in diffuse and to some extent follicular B-cell lymphomas. The role of Bcl6 in oncogenesis is the second major topic of the review by Basso and Dalla-Favera (41). In a striking parallel, the other BTB-POZ family member reviewed in this volume (lymphoma/leukemia-related factor [LRF]), is not only required for normal GC function but also is involved in B cell and myeloid malignancy. Like Bcl6, overexpression of this transcriptional repressor is linked to lymphomagenesis. These topics and their detailed mechanisms are described by Sung-Uk and Maeda (42). Finally, IRF-4 has been in recent years increasingly implicated in various B- and plasma cell malignancies (58, 64). These roles are complex and varied, as in some cases IRF-4 expression can promote and in other suppress malignant transformation and cell growth. The review by Klein (19) carefully discusses and helps to reconcile data from multiple sources in multiple malignancies. Overall, this volume encompasses reviews that attack the mystery of the GC reaction and its allied processes from multiple directions. Taken together, they update us on this rapidly moving and centrally important area of immunology that impinges on topics as diverse as development, vaccines and memory, autoimmunity, and malignant transformation. Individually and together these reviews do a wonderful job of integrating these seemingly disparate yet clearly related topics at both the molecular and cellular levels. Supported by NIH R01-AI043603 to M.J.S. and a DFG Research Fellowship to F.W. The authors declare no conflicts of interest.