Germinal-Center Organization and Cellular Dynamics
2007; Cell Press; Volume: 27; Issue: 2 Linguagem: Inglês
10.1016/j.immuni.2007.07.009
ISSN1097-4180
AutoresChristopher D.C. Allen, Takaharu Okada, Jason G. Cyster,
Tópico(s)T-cell and B-cell Immunology
ResumoGerminal centers (GCs) are important sites of antibody affinity maturation. In the classical model, the GC dark zone contains large centroblasts that are rapidly proliferating and undergoing somatic hypermutation of their antibody variable-region genes. Centroblasts give rise to smaller nonproliferating centrocytes in the light zone that compete for binding antigen on follicular dendritic cells. Recently, the approach of real-time imaging of GCs by two-photon microscopy of intact lymph nodes has provided new insights into GC dynamics that both support and challenge fundamental aspects of this model. Here we review recent and older findings on cell migration, proliferation, and interaction dynamics in the GC and discuss a model in which dark- and light-zone cells are morphologically similar, proliferation occurs in both zones, and GC B cells compete for T cell help as well as antigen. Germinal centers (GCs) are important sites of antibody affinity maturation. In the classical model, the GC dark zone contains large centroblasts that are rapidly proliferating and undergoing somatic hypermutation of their antibody variable-region genes. Centroblasts give rise to smaller nonproliferating centrocytes in the light zone that compete for binding antigen on follicular dendritic cells. Recently, the approach of real-time imaging of GCs by two-photon microscopy of intact lymph nodes has provided new insights into GC dynamics that both support and challenge fundamental aspects of this model. Here we review recent and older findings on cell migration, proliferation, and interaction dynamics in the GC and discuss a model in which dark- and light-zone cells are morphologically similar, proliferation occurs in both zones, and GC B cells compete for T cell help as well as antigen. The germinal center (GC) was first described in 1884 by Walther Flemming, who observed a site of large lymphocytes undergoing mitosis in the follicles of lymph nodes and other secondary lymphoid organs and proposed this site to be a major source of all lymphocytes in the body (reviewed in Nieuwenhuis and Opstelten, 1984Nieuwenhuis P. Opstelten D. Functional anatomy of germinal centers.Am. J. Anat. 1984; 170: 421-435Crossref PubMed Scopus (167) Google Scholar). Flemming's work prompted an intense study of the GC that continues today, although his original proposed function for the GC was eventually disproven. The GC is now known to be associated with T dependent antibody responses, and experimental evidence indicates that it is the main site in which high-affinity antibody-secreting plasma cells and memory B cells are generated. T dependent antibody responses are initiated when rare B and T cells specific for an incoming antigen cluster at the boundary between B cell follicles and T cell zones and engage in cognate interactions (Garside et al., 1998Garside P. Ingulli E. Merica R.R. Johnson J.G. Noelle R.J. Jenkins M.K. Visualization of specific B and T lymphocyte interactions in the lymph node.Science. 1998; 281: 96-99Crossref PubMed Scopus (604) Google Scholar, MacLennan et al., 1997MacLennan I.C. Gulbranson-Judge A. Toellner K.M. Casamayor-Palleja M. Chan E. Sze D.M. Luther S.A. Orbea H.A. The changing preference of T and B cells for partners as T-dependent antibody responses develop.Immunol. Rev. 1997; 156: 53-66Crossref PubMed Scopus (236) Google Scholar, Okada et al., 2005Okada T. Miller M.J. Parker I. Krummel M.F. Neighbors M. Hartley S.B. O'Garra A. Cahalan M.D. Cyster J.G. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells.PLoS Biol. 2005; 3: e150Crossref PubMed Scopus (410) Google Scholar). The activated B cells then can adopt one of two fates: (1) movement into extrafollicular areas and then proliferation and terminal differentiation into short-lived plasma cells that secrete antibody, or (2) movement into B cell follicles and then proliferation and the establishment of GCs (Jacob et al., 1991aJacob J. Kassir R. Kelsoe G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations.J. Exp. Med. 1991; 173: 1165-1175Crossref PubMed Scopus (584) Google Scholar, Liu et al., 1991Liu Y.J. Zhang J. Lane P.J. Chan E.Y. MacLennan I.C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.Eur. J. 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Several decades ago, it was observed that after immunization with a foreign antigen, the average affinity of serum antibody for the foreign antigen increases over time (Eisen and Siskind, 1964Eisen H.N. Siskind G.W. Variations in affinities of antibodies during the immune response.Biochemistry. 1964; 3: 996-1008Crossref PubMed Scopus (675) Google Scholar). This process was later termed affinity maturation and shown to be due to somatic mutations in the antibody variable region (reviewed in Tarlinton and Smith, 2000Tarlinton D.M. Smith K.G. Dissecting affinity maturation: A model explaining selection of antibody-forming cells and memory B cells in the germinal centre.Immunol. Today. 2000; 21: 436-441Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Subsequent work showed that most short-lived plasma cells in primary extrafollicular foci lacked somatic mutations, whereas a high frequency of somatic mutations was evident in GC B cells, suggesting that a hypermutation mechanism is activated in GC B cells (Berek et al., 1991Berek C. Berger A. Apel M. Maturation of the immune response in germinal centers.Cell. 1991; 67: 1121-1129Abstract Full Text PDF PubMed Scopus (730) Google Scholar, Jacob et al., 1991bJacob J. Kelsoe G. Rajewsky K. Weiss U. Intraclonal generation of antibody mutants in germinal centres.Nature. 1991; 354: 389-392Crossref PubMed Scopus (870) Google Scholar). These somatic mutations were clustered in the regions of antibodies that form the interface with the antigens. A high ratio of amino acid replacement to silent mutations was observed, indicating that selection had taken place. In addition, many of the GC B cells picked out from individual GCs appeared to be clonally related, and indeed some GCs appeared to be dominated by cells derived from a single clone. These observations suggest that affinity maturation occurs in GCs, through the processes of clonal proliferation, somatic hypermutation, and selection. GCs do not appear to be absolutely required for affinity maturation, however, because mice deficient in GCs exhibit measurable affinity maturation with certain types of immunization (Futterer et al., 1998Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues.Immunity. 1998; 9: 59-70Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, Koni and Flavell, 1999Koni P.A. Flavell R.A. Lymph node germinal centers form in the absence of follicular dendritic cell networks.J. Exp. 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Dissecting affinity maturation: A model explaining selection of antibody-forming cells and memory B cells in the germinal centre.Immunol. Today. 2000; 21: 436-441Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Many of the long-lived plasma cells are found in the bone marrow, where they secrete antibody for several weeks or longer. Notably, the antibody variable genes in most long-lived plasma cells and memory B cells exhibit a high degree of somatic mutations that show evidence of selection, suggesting that these cells were derived from GCs (Blink et al., 2005Blink E.J. Light A. Kallies A. Nutt S.L. Hodgkin P.D. Tarlinton D.M. Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization.J. Exp. Med. 2005; 201: 545-554Crossref PubMed Scopus (201) Google Scholar, McHeyzer-Williams et al., 2006McHeyzer-Williams L.J. Malherbe L.P. McHeyzer-Williams M.G. Checkpoints in memory B-cell evolution.Immunol. 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Despite these variables, however, some common principles of GC formation have emerged. It is thought that the GC is seeded by activated B cells specific for foreign antigen within a few days after the initiation of an immune response. These activated B cells first move to the center of B cell follicles in secondary lymphoid organs and proliferate within the follicular dendritic cell (FDC) network, as was established by careful kinetic studies in the rat (Liu et al., 1991Liu Y.J. Zhang J. Lane P.J. Chan E.Y. MacLennan I.C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.Eur. J. Immunol. 1991; 21: 2951-2962Crossref PubMed Scopus (592) Google Scholar) and recently confirmed in the mouse (Wang and Carter, 2005Wang Y. Carter R.H. CD19 regulates B cell maturation, proliferation, and positive selection in the FDC zone of murine splenic germinal centers.Immunity. 2005; 22: 749-761Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). FDCs are radiation-resistant stromal cells that form a network of processes in B cell follicles and have the ability to capture large amounts of antigen in the form of immune complexes in highly ordered units termed iccosomes (Cyster et al., 2000Cyster J.G. Ansel K.M. Reif K. Ekland E.H. Hyman P.L. Tang H.L. Luther S.A. Ngo V.N. Follicular stromal cells and lymphocyte homing to follicles.Immunol. Rev. 2000; 176: 181-193Crossref PubMed Scopus (315) Google Scholar, Kosco-Vilbois, 2003Kosco-Vilbois M.H. Are follicular dendritic cells really good for nothing?.Nat. Rev. Immunol. 2003; 3: 764-769Crossref PubMed Scopus (124) Google Scholar, Szakal and Tew, 1991Szakal A.K. Tew J.G. Significance of iccosomes in the germinal centre reaction.Res. Immunol. 1991; 142: 261-263Crossref PubMed Scopus (8) Google Scholar). As the GC matures, two main compartments, which were first described in 1930 through the careful study of cross-sections of cat lymph nodes, become evident (Röhlich, 1930Röhlich K. Beitrag zur Cytologie der Keimzentren der Lymphknoten.Z. Mikrosk. Anat. Forsch. 1930; 20: 287-297Google Scholar). These compartments were termed dark and light zones on the basis of their histological appearance, in which much of the light zone is occupied by FDC processes, whereas few processes extend into the dark zone, where lymphocytes are closely packed (Figure 1). The FDCs of the light zone acquire features that distinguish them from FDCs in the primary follicles, including the upregulation of the integrin ligand VCAM-1 and the antibody Fc receptor FcγRIIB (Balogh et al., 2002Balogh P. Aydar Y. Tew J.G. Szakal A.K. Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1.Anat. 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CD19 regulates B cell maturation, proliferation, and positive selection in the FDC zone of murine splenic germinal centers.Immunity. 2005; 22: 749-761Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The light zone appears to be strategically positioned in secondary lymphoid organs toward the source of foreign antigens (Millikin, 1966Millikin P.D. Anatomy of germinal centers in human lymphoid tissue.Arch. Pathol. 1966; 82: 499-505PubMed Google Scholar). In the spleen, the light zone pole of the GC is proximal to the marginal sinus where blood-borne antigens enter the tissue. In lymph nodes, the light zone is positioned close to the subcapsular sinus, which receives afferent lymphatic drainage from the skin, mucosa, or viscera. In the Peyer's patches, tonsils, and appendix, the light zone is oriented toward the mucosal surface. This orientation of the GC is concordant with evidence that antigen, often in the form of immune complexes, can be transported rapidly to GC light zones (Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar, Szakal et al., 1989Szakal A.K. Kosco M.H. Tew J.G. Microanatomy of lymphoid tissue during humoral immune responses: Structure function relationships.Annu. Rev. Immunol. 1989; 7: 91-109Crossref PubMed Scopus (225) Google Scholar). Follicular B cells might have a role in this transport process (Phan et al., 2007Phan T.G. Grigorova I. Okada T. Cyster J.G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells.Nat. Immunol. 2007; (in press. Published online July 29, 2007)https://doi.org/10.1038/ni1494Crossref Scopus (447) Google Scholar). The chemokine receptor, CXCR4, is needed for GC B cell positioning in the dark zone, and its ligand, SDF-1 (CXCL12), is more abundant in this zone than it is in the light zone (Allen et al., 2004Allen C.D. Ansel K.M. Low C. Lesley R. Tamamura H. Fujii N. Cyster J.G. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5.Nat. Immunol. 2004; 5: 943-952Crossref PubMed Scopus (492) Google Scholar). SDF-1 appears to be produced locally by stromal cells (Allen et al., 2004Allen C.D. Ansel K.M. Low C. Lesley R. Tamamura H. Fujii N. Cyster J.G. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5.Nat. Immunol. 2004; 5: 943-952Crossref PubMed Scopus (492) Google Scholar) (Figure 1). Stromal cells have been described in the dark zone, although these apparently do not form a dense network (Opstelten et al., 1982Opstelten D. Stikker R. Deenen G.J. Nieuwenhuis P. Germinal centers and the B-cell system. VII. Complement receptors, antigen receptors, immunoglobulin and alkaline phosphatase in germinal centers of the rabbit appendix and popliteal lymph nodes.Cell Tissue Res. 1982; 224: 505-516Crossref PubMed Scopus (6) Google Scholar, Rademakers, 1992Rademakers L.H. Dark and light zones of germinal centres of the human tonsil: An ultrastructural study with emphasis on heterogeneity of follicular dendritic cells.Cell Tissue Res. 1992; 269: 359-368Crossref PubMed Scopus (35) Google Scholar). CXCL13 is more abundant in the light zone, where it accumulates on the processes of FDCs, and the CXCL13-CXCR5 chemokine and receptor pair is needed for GC B cells to accumulate normally in the light zone (Allen et al., 2004Allen C.D. Ansel K.M. Low C. Lesley R. Tamamura H. Fujii N. Cyster J.G. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5.Nat. Immunol. 2004; 5: 943-952Crossref PubMed Scopus (492) Google Scholar, Cyster et al., 2000Cyster J.G. Ansel K.M. Reif K. Ekland E.H. Hyman P.L. Tang H.L. Luther S.A. Ngo V.N. Follicular stromal cells and lymphocyte homing to follicles.Immunol. Rev. 2000; 176: 181-193Crossref PubMed Scopus (315) Google Scholar) (Figure 1). Immunohistochemical staining shows that CXCR4 is more abundant in the dark zone than it is in the light zone, and combined flow-cytometric and BrdU-incorporation analysis (discussed further below) reveals a profile of BrdU labeling in CXCR4hi and CXCR4lo GC B cells that corresponds to the kinetics of labeling in dark and light zones, respectively (Allen et al., 2004Allen C.D. Ansel K.M. Low C. Lesley R. Tamamura H. Fujii N. Cyster J.G. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5.Nat. Immunol. 2004; 5: 943-952Crossref PubMed Scopus (492) Google Scholar, Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar). In contrast with the differences in CXCR4 expression in dark and light zones, the GC B cell population as a whole has rather uniform CXCR5 expression (Allen et al., 2004Allen C.D. Ansel K.M. Low C. Lesley R. Tamamura H. Fujii N. Cyster J.G. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5.Nat. Immunol. 2004; 5: 943-952Crossref PubMed Scopus (492) Google Scholar). Although these data were largely collected in the mouse, the staining of human tonsil also suggests that CXCR4 is higher on dark zone cells (Forster et al., 1998Forster R. Kremmer E. Schubel A. Breitfeld D. Kleinschmidt A. Nerl C. Bernhardt G. Lipp M. Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: Rapid internalization and recycling upon activation.J. Immunol. 1998; 160: 1522-1531PubMed Google Scholar). Taken together, these findings suggest that CXCR4 protein expression is tightly controlled on GC B cells and is a major factor controlling cell positioning in the dark zone versus the light zone. Bringing together a century of observations from fixed tissue analysis and in vitro studies, MacLennan, 1994MacLennan I.C. Germinal centers.Annu. Rev. Immunol. 1994; 12: 117-139Crossref PubMed Scopus (1643) Google Scholar presented a model 13 years ago for the mechanism of GC organization and function. GC B cells in dark and light zones were historically defined as centroblasts and centrocytes, respectively. Centroblasts were named on the basis of the observation of large, mitotically active cells in the dark zone that lacked surface immunoglobulin (Ig). These cells were proposed to undergo a rapid process of proliferation and somatic hypermutation of their antibody variable-region genes. Centroblasts were then suggested to exit the cell cycle, re-express surface Ig, and become smaller centrocytes that traveled to the light zone. This process was proposed to result in the generation of centrocytes expressing surface antibodies with a wide range of affinities for a given antigen. Centrocytes were then proposed to compete for binding to antigen in the form of immune complexes displayed on the surface of FDCs in the light zone. The presence of macrophages that have phagocytosed large numbers of GC B cells that were recently in cell cycle (referred to as “tingible body macrophages” because of the intense staining characteristics of the engulfed cells) suggests that most B cells die during this selection process. Selected centrocytes could then present antigen to helper T cells in the light zone, and this could enhance survival or promote differentiation into antibody-secreting plasma cells or memory B cells. This now-classical model provided an important framework for further experimentation, and several features of the model have been well validated. However, some studies, including recent real-time-imaging experiments, have led to findings that are not fully consistent with the original version of the model. In the sections below we discuss these findings, and in the summary paragraph (and Figure 1, Figure 2) we present a revised version of the model that attempts to incorporate this new information. A striking feature that emerged in the real-time imaging studies was that GC B cells were highly motile and exhibited a dendritic morphology as they moved (Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar, Hauser et al., 2007Hauser A.E. Junt T. Mempel T.R. Sneddon M.W. Kleinstein S.H. Henrickson S.E. von Andrian U.H. Shlomchik M.J. Haberman A.M. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.Immunity. 2007; 26: 655-667Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Schwickert et al., 2007Schwickert T.A. Lindquist R.L. Shakhar G. Livshits G. Skokos D. Kosco-Vilbois M.H. Dustin M.L. Nussenzweig M.C. In vivo imaging of germinal centres reveals a dynamic open structure.Nature. 2007; 446: 83-87Crossref PubMed Scopus (347) Google Scholar). The overall motility rate was partially dependent on the chemokine CXCL13 (Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar). Unexpectedly, the morphology and velocities of cells in light and dark zones were similar. Moreover, real-time imaging showed that cell division and cell death occur in both compartments and not just in one zone (Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar). The latter findings will be discussed further in later sections, but an important implication of these combined observations is that dark- and light-zone cells are more similar than the terms centroblast and centrocyte might suggest, a theme that we will continue to develop throughout this review. An important feature of the classical model is that GC B cells migrate from the dark zone to the light zone (MacLennan, 1994MacLennan I.C. Germinal centers.Annu. Rev. Immunol. 1994; 12: 117-139Crossref PubMed Scopus (1643) Google Scholar). Evidence for this movement was originally obtained at the cell population level in studies in which cells were labeled in the S phase of the cell cycle with thymidine analogs, such as 3H-thymidine or bromodeoxyuridine (BrdU), and then their distribution was measured at various time points (Hanna, 1964Hanna Jr., M.G. An autoradiographic study of the germinal center in spleen white pulp during early intervals of the immune response.Lab. Invest. 1964; 13: 95-104PubMed Google Scholar, Koburg, 1966Koburg, E. (1966). Cell Production and Cell Migration in the Tonsil. Paper presented at: Germinal Centers in Immune Responses (University of Bern, Switzerland, Springer-Verlag New York Inc.).Google Scholar, Liu et al., 1991Liu Y.J. Zhang J. Lane P.J. Chan E.Y. MacLennan I.C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.Eur. J. Immunol. 1991; 21: 2951-2962Crossref PubMed Scopus (592) Google Scholar). The enrichment of labeled cells in the dark zone after several hours was followed by the appearance of labeled cells in the light zone, suggesting that cells had moved from the dark zone to the light zone. Indirect experimental results and theoretical modeling also suggested that some centrocytes might return to the dark zone to complete an additional round of mutation and selection (Kelsoe, 1996Kelsoe G. Life and death in germinal centers (redux).Immunity. 1996; 4: 107-111Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, Kepler and Perelson, 1993Kepler T.B. Perelson A.S. Cyclic re-entry of germinal center B cells and the efficiency of affinity maturation.Immunol. Today. 1993; 14: 412-415Abstract Full Text PDF PubMed Scopus (206) Google Scholar). The migration of cells between dark and light zones has now been directly observed by real-time imaging (Allen et al., 2007Allen C.D. Okada T. Tang H.L. Cyster J.G. Imaging of germinal center selection events during affinity maturation.Science. 2007; 315: 528-531Crossref PubMed Scopus (542) Google Scholar, Hauser et al., 2007Hauser A.E. Junt T. Mempel T.R. Sneddon M.W. Kleinstein S.H. Henrickson S.E. von Andrian U.H. Shlomchik M.J. Haberman A.M. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.Immunity. 2007; 26: 655-667Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Schwickert et al., 2007Schwickert T.A. Lindquist R.L. Shakhar G. Livshits G. Skokos D. Kosco-Vilbois M.H. Dustin M.L. Nussenzweig M.C. In vivo imaging of germinal centres reveals a dynamic open structure.Nature. 2007; 446: 83-87Crossref PubMed Scopus (347) Google Scholar). In these studies, the light-zone FDC network was visualized by the injection of fluorescently labeled antibodies or through the deposition of fluorescent immune complexes. The studies differed in the criteria used to define cells that crossed between dark and light zones. Two groups focused on GCs that were of a size and orientation that allowed the dark and light zones to be seen in the xy plane and then drew a line dividing the two zones and reported the frequency of cells that crossed this line (Hauser et al., 2007Hauser A.E. Junt T. Mempel T.R. Sneddon M.W. Kleinstein S.H. Henrickson S.E. von Andrian U.H. Shlomchik M.J. Haberman A.M. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.Immunity. 2007; 26: 655
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