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Innate B cell helpers reveal novel types of antibody responses

Carola G Vinuesa & Pheh-Ping Chang
Antibody responses are classified according to whether B cells receive help from T cellsthat is, whether they are thymusdependent (TD) responses or thymus-independent (TI) responses. The latter can be elicited by microbial ligands (TI type 1) or by extensive crosslinking of the B cell antigen receptor (BCR; TI type 2). The hallmark of a TD response is the induction of germinal centers in which follicular helper T cells (TFH cells) select B cells with somatically mutated high-affinity BCRs to become memory cells. Studies have shown that B cells can also receive innate TD help from natural killer T cells (NKT cells) and innate TI help from cells such as neutrophils but that the outcome of such help differs from conventional TD and TI responses. Here we update the classification of antibody responses to take into account these emerging types of B cell helpers. Antibodies are produced by antigen-activated B cells as they become plasmablasts and terminally differentiated plasma cells; the antibodies provide an important line of defense against infection, acting through various mechanisms to neutralize and/or eliminate microbes and infected cells. It is becoming clear that antibody responses are exquisitely tailored to the characteristics of the invading pathogen. B cells differ qualitatively and quantitatively in their differentiation program according to the nature of infection. The availability of T cell help for protein antigens, known as thymus-dependent (TD) antigens, has been known for many decades to define one of the key protective features of antibody responses: B cell memory. B cell memory occurs as a consequence of the T cells ability to initiate germinal center (GC) responses1. GCs have been regarded as dedicated microenvironments in which somatic hypermutation takes place to facilitate affinity maturation; those B cells that express B cell antigen receptors (BCRs) of the highest affinity can be selected to terminally differentiate into long-lived memory Bcells and memory plasma cells1,2. A growing number of nonclassical helper cell types have been shown to directly interact with B cells and provide the necessary signals for full activation and differentiation; in some cases this includes GC formation. It seems that different types of antigens activate specialized helper cells that can induce the antibody response type that will optimally clear the pathogen. Here we will critically review the prevalent understanding of antibody responses and summarize recently described responses mediated by nonclassical helper cells and pathways that generate B cell memory. Although the previous TD-TI paradigm shaped the understanding of how Bcell memory and antibody responses arise, the new findings suggest that revisions are needed that place more importance on the nature of
Department of Pathogens and Immunity, John Curtin School of Medical Research, Australian National University, Canberra, Australia. Correspondence should be addressed to C.G.V. (carola.vinuesa@anu.edu.au). Received 26 June 2012; accepted 4 December 2012; published online 18 January 2013; doi:10.1038/ni.2511

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the helper cells. This includes the incorporation of new categories of innate celldriven TD and TI antibody responses into the present classification (Fig. 1) Overview of TD and TI responses Studies of athymic (nude) mice or mice that had undergone removal of the thymus provided early insights into the cellular factors needed to elicit antibody responses to immunizing antigens. TD responses are absent in those mice because of the essential requirement for thymus-derived T cells for full activation and subsequent proliferation of B cells3 (Figs. 1 and 2 and Table 1). Such responses are typically elicited by protein antigens recognized specifically by the BCR (signal 1) in the presence of cognate help (signal 2) from T cells 4,5. After receiving a signal through the BCR, mature B cells migrate to the interface of the T cell zone (T zone) and the B cell follicle6 and present antigen-derived peptides on major histocompatibility complex (MHC) class II molecules for recognition by newly primed follicular helper T cells (TFH cells; Figs. 1 and 2a). The development of TFH cells depends on expression of the transcriptional repressor Bcl-6 that induces changes in the expression of chemokine receptors, including upregulation of CXCR5 and downregulation of CCR7, which position TFH cells at the T cellB cell interface7,8. Stable interactions between T cells and B cells facilitated by homotypic interactions between members of the SLAM family of proteins, which signal through the T cell adaptor SAP, promote the migration of T cells into follicles, where they become GC TFH cells9. TFH cells express the counterligand CD40L (CD154) and cytokines, such as interleukin 21 (IL-21), IL-4 or interferon-, that promote the proliferation of B cells, initiate the switching of immunoglobulin class and induce the differentiation of B cells into short-lived extrafollicular plasma cells, early memory B cells or GC B cells810 (Fig. 2a). Extrafollicular plasma cells typically localize at the boundaries between the T zone and the red pulp of the spleen or the medullary cords of lymph nodes6, peak in number at days 78 after immunization of mice with protein antigens in adjuvant and then undergo apoptosis10. This response is particularly important
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been further categorized according to whether they can be elicited in CBA/N mice, which have an X-linked immunological defect caused by deficiency in the kinase Btk19,20: antigens that do not elicit responses in CBA/N mice are designated TI type 2 (TI-2), whereas those that do elicit antibody responses in the absence of Btk are designated TI type 1 (TI-1). TI-1 antigens provide a second signal via Toll-like receptors (TLRs) specific for microbial products such as lipopeptides, lipopolysaccharide (LPS), microbial CpG DNA, viral RNA and certain viral coat proteins21. Although TLR ligands can polyclonally activate mouse B cells regardless of BCR specificity 22, in vitro and in vivo stimulation of splenocytes with conjugates of hapten-LPS and proteinCpG, respectively, have demonstrated that synergistic engagement of BCRs and TLRs is required for optimal B cell antibody responses 2325 (Fig. 3a). Resting human naive B cells, unlike their mouse counterparts, cannot be polyclonally stimulated by most ligands of TLR2 or TLR9, as they require prior stimulation of the BCR to acquire responsiveness21. Human naive B cells lack TLR4 expression21,26 and require priming by type I interferon to upregulate TLR7 (ref. 21). TI-1 responses arise faster than do TD antibody responses27, which is critical for preventing blood-borne infections from evolving into lifethreatening conditions. Previously TI-1 responses were not thought to generate memory B cells. That conclusion was drawn from the observation that secondary recall responses to the hapten dinitrophenol (DNP) can be raised in irradiated recipients after adoptive transfer of B cells from mice immunized with DNP coupled to the protein antigen KLH and subsequent challenge of the recipient mice with DNPKLH, but such responses cannot be raised in recipients given donor B cells from mice immunized with DNP-LPS28. Subsequent studies described below have nevertheless suggested that TI-1 antigens can also generate memory B cells. TI-2 antigens are typically multivalent antigens and, in contrast to TI-1 antigens, do not elicit responses in CBA/N mice19. Such antigens extensively crosslink BCRs and deliver a prolonged and persistent signal to the B cell, transmitted via Btk20 (Figs. 1 and 3b and Table 1). Typical TI-2 antigens are bacterial capsular polysaccharides, such as those found in Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis, but TI-2 responses can also develop in response to the highly repetitive motifs found in viral capsids29. TI-2 responses typically elicit potent extrafollicular antibody responses with no memory formation. Although GCs that formed in response to TI-2 antigens were observed in subsequent studies, they were reported to be nonproductive30,31 (Fig. 3b). Subsequent observations have suggested there may be a unique type of memory B cell response to TI-2 antigens, which we describe below. As with

Source of signal 2

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TD-1 TFH

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TI-3 Innate cell (NBH)

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Figure 1 Updated classification of antibody responses. Subtypes of TD and TI antibody responses, highlighting (in orange) the following two newly identified antibody responses driven by innate cells: TD-2 responses driven by NKTFH cells, and TI-3 responses driven by cells such as NBH cells.

in the early control of infection11. B cells that adopt the Bcl-6hi GC fate downregulate the chemokine receptor EBI2, which promotes migration toward the center of the follicle to initiate GC reactions12. GC B cells undergo somatic hypermutation, which introduces random point mutations mainly in the immunoglobulin variable (V)-region gene segments that can change the affinity of the BCR1,13. Those cells with BCRs of higher affinity are thought to take up more antigen from follicular dendritic cells and present more peptideMHC class II antigens on their surface, thereby outcompeting cells of lower affinity for selecting survival and differentiation signals by limiting GC TFH cells. This TFH celllimited selection of high-affinity B cells has been demonstrated through an elegant in vivo imaging experiment in mice with delivery of ovalbumin protein to hapten NPspecific GC B cells expressing the dendritic cellspecific scavenging receptor DEC-205 via chimeric antibody to DEC-205 fused to ovalbumin, resulting in presentation of ovalbumin peptides to T cells by B cells independently of BCR signaling13. In those experiments, DEC-205-sufficient (wildtype) GC B cells, but not DEC-205-deficient GC B cells present in the same mice, were selected to reenter the dark zone, clonally expand and differentiate into plasmablasts. These results established that the availability of Tcell help is the limiting factor for all such B cell fate decisions. The selection of GC B cells to become long-lived memory B cells and memory plasma cells (Fig. 2a) is likewise regulated by TFH cells and involves signaling via CD40L14, PD-1 (ref. 15) and cytokines such as IL-4, IL-10 and IL-2 (refs. 1618). A wave of memory B cells can also form before entry into the GC and independently of the signals required for the formation of GCs. Athymic mice can mount antibody responses to nonprotein TI antigens (Figs. 1 and 3 and Table 1). Traditionally TI responses have
Figure 2 TD antibody responses. (a) TD-1 responses are elicited by cognate interactions of TFH cells and B cells after recognition of peptide MHC class II (MHCII) complexes on B cells. (b) TD-2 TD responses are elicited by cognate interactions of NKTFH cells and B cells after recognition of CD1d-glycolipid complex on B cells. Although both responses generate GCs, only TD-1 responses give rise to long-lived memory B cells and plasma cells. B, B lymphocyte; EF-PC, extrafollicular plasma B cell; GC B, germinal center B cell; mem PC, memory plasma cell; -GalCer, -galactosylceramide.

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Table 1 Characteristics and outcomes of antibody responses
Antibody response TD-1 TD-2 TI-1 Antigen Protein Glycoprotein Responding B cell Follicular Source of help GC Somatic hypermutation Yes, leads to afnity maturation Memory B cell response Yes, pre-GC and post-GC memory Minimal B-1 cellderived memory, GC independent B-1b cellderived memory, GC independent ? Long-lived plasma cells Yes No Not reported Extrafollicular response Yes, delay due to T cell priming Yes, early and robust Yes, early and robust

TFH cells, CD40L, IL-21, Yes, productive SLAM family members

CD1d+ (MZ?) NK cellTFH cell, IL-21, SLAM family members TLR signaling in B cells

Yes, early, small, Limited, minimal nonproductive afnity maturation Possibly Occurs extrafollicularly in MRLlpr mice Limited, no afnity maturation Yes, not known if it leads to afnity maturation

LPS, CpG, MZ, B-1 lipopeptides, RNA Bacterial capsular polysaccharides, viral capsids Blood-borne or gut bacteria MZ, B-1

TI-2

Extensive BCR crosslinking, Btk

Can occur, nonproductive

Some bone Yes, early marrow plasma cells ? Yes, early

TI-3

MZ

Myeloid cells (NBH cells, No basophils, mast cells, eosinophils) APRIL, BAFF, CD40L, IL-21

MZ, marginal zone.

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TI-1 responses, human responses to encapsulated bacteria are very different from the responses in mice. In humans, responding Bcells are IgM+IgD+CD27+ memory B cells of uncertain ontogeny, as these cells seem to be GC independent yet carry somatic mutations32. In summary, in the TD-TI paradigm, T cells provide help for the formation of GCs that are essential for affinity maturation and memory formation, which are not major features of TI antibody responses elicited by nonprotein antigens because of the lack of T cell help. Division of labor among B cell subsets Mature peripheral B cells include three distinct subsets: follicular Bcells, marginal zone B cells and B-1 cells33. Most mature B cells are recirculating cells that home mainly to B cell follicles in secondary lymphoid organs and are known as follicular B cells. Follicles are located adjacent to T zones where the follicular B cells participate mainly in TD responses to protein antigens and respond less well to TLR agonists than do marginal zone B cells or B-1 cells34. Follicular B cells recirculate through the bone marrow, where they are positioned in organized aggregates around bone marrow sinusoids and can respond in a TI manner to blood-borne pathogens35. Marginal zone B cells reside between the marginal sinus and the red pulp and are thus located at the first line of defense against bloodborne particulate pathogens36,37. They are heterogeneous, composed of a mixture of naive B cells and memory B cells, and mediate rapid humoral responses to TI-1 and TI-2 antigens, rapidly differentiating into short-lived extrafollicular plasma cells27,38,39 (Fig. 3). Memory B cells located in the marginal zones can also rapidly participate in recall TD responses after immunization with hapten-protein conjugates37. In vivo experiments using adoptive transfer of marginal zone B cells that are polyclonal or specific for hen egg lysozyme (HEL), followed by immunization with a conjugate of NP and chicken -globulin in alum or a conjugate of HEL and sheep red blood cells, respectively,
Figure 3 TI antibody responses. (a) In TI-1 responses, B cells receive second signals through their TLRs. (b) In TI-2 antibody responses, extensive crosslinking of the BCR provides essential signals through Btk. (c) TI-3 antibody responses are elicited when B cells receive help from bone marrowderived myeloid cells (neutrophils (N BH cells), monocytes, mast cells and basophils). Ag, antigen; PS, polysaccharide; B1-b mem, B-1b memory B cell; B1-b mem PC, B-1b memory plasma cell; MZ, marginal zone; AID, activation-induced cytidine deaminase; SHM, somatic hypermutation; FcR, receptor for IgE.

have indicated that marginal zone B cells can also differentiate to become GC B cells in response to TD antigens, albeit with a delay relative to the development of follicular Bcells40,41. Marginal zone B cells can shuttle between the marginal zone and follicles to deliver blood-borne antigen to follicular dendritic cells42. In addition, the high expression of the antigen-presenting molecule CD1d on marginal zone B cells allows them to act as antigen-presenting cells for the activation

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TI-3: Myeloid cell dependent Mucosal microbe BAFF APRIL SHM MZ TLR Monocyte Microbial TLR-ligand Mast cell FcR Antigen IgE Basophil Whole bacteria AID IL-21 TACI B CD40LCD40 B1 IL-6 EF-PC IgE Marina Corral EF-PC IgA IgE EF-PC IgM IgA IgM IgG IgA EF-PC

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of natural killer T cells (NKT cells)43, which is probably important in responses dependent on NKTFH cells described below. B-1 cells are the main B cell subpopulation in the pleural and peritoneal cavities44 and are present in small numbers in the spleen. Unlike the conventional B-2 cells, which are produced throughout life in the bone marrow, B-1 cells are thought to arise early in ontogeny from dedicated progenitors found in the greatest abundance in fetal bone marrow45. They can be further subcategorized as B-1a (CD5+) cells and B-1b (CD5) cells. B-1 cells are the main producers of the natural immunoglobulin M (IgM) important for early protection against bacterial infection. They are highly sensitive to stimulation with LPS and also participate in TI-2 responses to bacterial capsular polysaccharides, differentiating into extrafollicular plasma cells (Fig. 3b,c and Table 1). B-1b cells give rise to TI memory B cells, as has been shown in experiments involving rechallenge with Borrelia hermsii46. B-1b cells can also generate long-lived memory plasma cells in a thymus-independent manner after immunization with the model TI-2 antigen NP-Ficoll and the pneumococcal capsular polysaccharide PS3 (ref. 47; Fig. 3b). B-1 cells have a prominent role in immune defense against mucosal pathogens and produce IgA in a thymus-independent fashion, as shown by studies in which chimeras in which the T cell antigen receptor -chain and -chain are knocked out are reconstituted with B-1 cells and host-derived bone marrow cells4850. The resultant mice express a BCR repertoire enriched for polyspecific receptors that often bind to both self antigens and microbial antigens51. Innate celldriven TD antibody responses: TD-2 CD4+ T cells, and specifically TFH cells, have long been known to be specialized helpers of B cells. Accumulating evidence suggests that thymus-derived and bone marrowderived innate-like cells have essential roles as helpers of B cells in response to nonprotein antigens. Glycolipid antigens have been shown to elicit B cell responses independently of conventional help from TFH cells but still dependent on cognate interaction with thymus-derived invariant NKT cells (iNKT cells)5255 (Figs. 1 and 2b and Table 1). These iNKT cells express an invariant T cell antigen receptor -chain that is V14-J18 in mice and V24-J18 in humans56. Unlike conventional CD4+ T cells or CD8+ T cells that recognize peptide presented by MHC class II or MHC class I, respectively, iNKT cells recognize synthetic, self and microbial glycolipids and self phospholipid antigens presented by CD1d57. The maturation of iNKT cells is thymus dependent, although it can be completed outside the thymus58. After administration of iNKT cell ligands such as -galactosylceramide into C57BL/6 mice, the activation of iNKT cells is initiated by -galactosylceramidepresenting dendritic cells through signaling via the costimulatory molecule CD40 and IL-12 (ref. 59). Such interactions elicit a CXCR5hiPD-1hiBTLAhi subset of CD4+ iNKT cells in both humans and mice called NKTFH cells. These NKTFH cells co-opt the TFH-differentiation pathway by expressing Bcl-6 and form cognate interactions with CD1d-expressing B cells in a SAP-dependent manner53,54. During an in vivo antibody response to NP-galactosylceramide, which occurs independently of conventional CD4+ T cells, initial help provided to B cells by cognate iNKT cells requires costimulatory signaling provided by CD40CD40L interactions and interactions between the coreceptor CD28 and its ligands (B7-1 and B7-2)55. The outcome is a rapid extrafollicular plasmablast response that leads to the rapid production of IgM and IgG (Fig. 2b). Notably, iNKT cells reacting to glycolipid antigens also induce rapid formation of GCs53,60 (Fig. 2b). Both the extrafollicular and GC responses are dependent on IL-21 derived from iNKT cells53,60. However, the antibody responses elicited by NKTFH cells induce affinity maturation that is more limited than the
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conventional TD responses to protein antigens and do not generate long-lived memory B cells or memory plasma cells53,60,61 (Fig. 2b). That finding has prompted the designation of NKTFH celldriven responses as TD type 2 (TD-2)60. The iNKT cells can also provide help to CD4+ T cells that in turn provide help to B cells, either through licensing of dendritic cells or the production of cytokines such as IL-4 and interferon-62. Innate celldriven TI antibody responses: TI-3 Bone marrowderived innate cells (typically myeloid cells) have been shown to drive and provide essential accessory signals during certain TI antimicrobial responses (Fig. 3c). Because of their unique characteristics, including the requirement for costimulatory signals different from those associated with TI-1 and TI-2 responses, we propose the designation TI-3 for such responses. An elegant study has identified a subset of neutrophils, called B cell helper neutrophils (NBH cells), found to colonize the human splenic perimarginal zone areas and provide help to marginal zone B cells in the context of innate TI responses (Figs. 1 and 3c and Table 1). Conventional neutrophils acquire Bcell helper functions to become NBH cells after exposure to IL-10, which is induced by the stimulation of splenic sinusoidal endothelial cells and perifollicular macrophages with LPS. Splenic NBH cells (but not circulating NBH cells) contain microbial genetic material and interact with marginal zone B cells via neutrophil extracellular traplike structures63. NBH cells suppress T cell proliferation and provide both contact-dependent and contact-independent help to marginal zone B cells, inducing their differentiation into antibody-producing cells. This help is dependent on production of B cellactivating factor (BAFF), the proliferation-inducing ligand APRIL, IL-21 and possibly CD40L63 (Fig. 3c). BAFF and APRIL promote the survival and differentiation of B cells and immunoglobulin-secreting plasma cells and trigger IgM production and class switching from IgM to IgG or IgA independently of CD40L64,65. Rather unexpectedly, NBH cells have been found to induce expression of activation-induced cytidine deaminase in marginal zone Bcells. As a consequence, marginal zone B cells cultured together with NBH cells undergo isotype switching and accumulate mutations in heavy-chain variable-region genes suggestive of somatic hypermutation63 (Fig. 3c). Whether those mutations change the affinity of the responding B cells is still unknown. Because localization of NBH cells to splenic perimarginal zone areas becomes more prominent after the colonization of neonatal mucosa by bacteria and occurs in a noninflammatory manner, it has been suggested that interactions between NBH cells and marginal zone B cells provide an additional innate layer of postnatal antimicrobial humoral immunity63. That hypothesis is supported by the demonstration of fewer marginal zone B cells, lower mutation frequencies among marginal zone B cells and fewer antibodies reactive to TI antigens in neutropenic patients due to mutations in SBDSP1 (Shwachman-BodianDiamond syndrome pseudogene 1) or in genes encoding elastase, WASp (Wiskott-Aldrich syndrome protein), the chemokine receptor CXCR4, the respiratory-burst protein p91-PHOX and the 2 integrin chain CD18. This work provides beautiful and powerful evidence of the importance of neutrophils in innate TI marginal zone B cell responses. Macrophage-derived signals have long been known as being important for B cell activation in vitro in response to both TI-1 and TI-2 antigens66. There is also in vivo evidence that monocyte-derived cells and/or dendritic cells provide important accessory signals for the activation and/or survival of B cells in response to TI antigens6770. After administration of heat-killed S. pneumoniae to mice, monocyte-derived dendritic cells interact with marginal zone B cells in the bridging channels of the spleen to promote their survival, population expansion and
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IgM production in a manner dependent on the transmembrane activator TACI6870 (Fig. 3c). In in vivo experiments in mice, monocytederived dendritic cells have also been found to recognize commensal bacteria through the TLR and regulate the production of IgA by plasma B cells in mucosa-associated lymphoid tissues via APRIL and BAFF6971 (Fig. 3c). Human macrophages produce BAFF in vitro, which enhances the proliferation of tonsil B cells stimulated via the BCR72. Mast cells have been found in the parafollicular zone extending into the medullary cords of lymph nodes showing reactive follicular hyperplasia and in close contact with IgA-expressing plasma cells in inflamed human tissues73. Coculture assays of mouse bone marrow mast cells and splenic B cells have shown that through direct contact with B cells, mast cellderived IL-6 can enhance the proliferation of naive, CD40- and IgM-activated B cells. Furthermore, IgE-sensitized mast cells stimulated with DNP have been shown to drive the differentiation of splenic B cells into IgA-producing plasma cells in vitro; this involves CD40-CD40L signaling and IL-6 derived from mast cells73 (Fig. 3c). That observation has led to the speculation that mast cells may be specialized in helping B-1 cells. Human lung mast cells pre activated with the ligand for the stem-cell factor receptor c-Kit or with a combination of the phorbol ester PMA and ionomycin have also been shown to be able to replace T cells in vitro in the provision of help to purified tonsil B cells to produce IgE 74 (Fig. 3c). Parallel findings have been obtained with preactivated basophils from human blood74 (Fig. 3c). It is still possible that the responding cells in those studies were memory B cells, given the report that mouse IgE+ cells can derive from post-GC B cells activated in a TD manner75. The studies described above have collectively demonstrated the existence of an important type of TI responses, TI-3 responses, that require bone marrowderived innate cells as helpers. Accessory signals modulate antibody responses Emerging evidence indicates an increasing number of cells and signals that, although not essential for B cell activation, have important accessory roles in both TD and TI B cell responses. Accumulating evidence suggests that innate-type cells can influence practically every stage of TD B cell differentiation. IL-4-producing eosinophils have been shown to prime mouse splenic B cells in the context of TD antibody responses for rapid IgM production in response to alum-precipitated NP-ovalbumin76,77. Mast cells, basophils, eosinophils and monocytes can also provide indirect help to B cells through the provision of help from CD4+ T cells by serving as antigen-presenting cells or via secretion of T cellactivating cytokines7881. Mucosal dendritic cells have been shown to promote IgA switching82. Studies of cocultures of sorted human bone marrow eosinophils and plasma cells and in vivo studies of mice that lack mature eosinophils (dblGATA-1 mice) have demonstrated that eosinophils help maintain bone marrow plasma cells through their ability to produce IL-6 and APRIL83. Such a role in the maintenance of plasma cells is also common to APRIL- and IL-6producing bone marrow megakaryocytes, as has been demonstrated in mice with either fewer megakaryocytes (Mpl/ mice) or transiently greater numbers of megakaryocytes (thrombopoietin-treated mice)18. Basophils can bind intact antigen84 and present processed antigen to CD4+ T cells79,85; they are one of the main source of IL-4 and IL-6 in mouse spleen and bone marrow. Basophils enhance the memory antibody response in mice during immunization with the model antigen allophycocyanin (a fluorescent protein)86. There have also been suggestions that in addition to enabling TI responses, TLR signals can enhance the activation of B cells in response to TD antigens. Invitro experiments have shown that the addition of the TLR9 agonist CpG potently increases the proliferation of human naive B cells in the
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resence of BCR- and T cellderived signals and allows their survival p and differentiation into antibody-secreting plasma cells87. It is still not clear whether such signals are as important in vivo. Mouse studies have provided conflicting results88,89, and humans with defective TLR signaling seem to mount normal primary responses to vaccines against tetanus and diphtheria toxin90,91. T cells and TLR signals can also serve important accessory roles during TI antibody responses. Although there is clear evidence that the activation, differentiation and isotype switching of Bcells in the presence of BCR signaling and ligation of the TLR (TI-1 responses) can occur independently of T cells in vivo, work has shown that T cellderived CD40L and IL-21 can substantially enhance the extrafollicular TI plasma cell response92. This has been demonstrated in the autoreactive response of AM14 mice with transgenic expression of rheumatoid factor, in which rheumatoid factorproducing B cells are activated by chromatincontaining immune complexes. Accessory roles for T cells and TLR ligands have also been described in the TI-2 response to S. pneumoniae that involves antibody to polysaccharide. Although T cells and TLR signals are not essential for the generation of IgM responses in mice, both signals have been shown to be required for the more protective IgG responses93,94. The key TLR signals are delivered in part, but not exclusively, by bacterial lipopeptides that activate TLR2 (ref. 94), although it is still not clear that these signals are delivered directly to B cells. Humans with defective TLR signaling due to deficiency in the kinase IRAK4 also have selective defects in response to the polysaccharide vaccine against pneumococcus in the presence of intact TD responses90,91. B cell responses can also be negatively influenced by regulatory T cells. Both suppressive CD8+ T cells restricted to the nonclassical MHC class Ib molecule Qa-1 (ref. 15) and CD4 + follicular regulatory T cells (TFR cells) that express the transcription factor Foxp3 (ref. 9597) have been found to repress GC responses. Both types of regulatory T cells are CD44+ICOSL+CXCR5+PD1hi cells and can be found in GCs. Qa-1-restricted CD8+ regulatory T cells repress TFH cells that express Qa-1 in complex with peptides, and disruption of this repression leads to lupus-like disease in mice15. In vivo experiments with chimeric mice, adoptive-transfer systems and C57BL/6 mice lacking molecules important for the formation of T FH cells have shown that CD4+ TFR cells derive from CD4+ Foxp3+ thymic precursors and, as has been described for TFH cells, differentiate to become follicular-localizing cells in a manner dependent on signaling through CD28 and the inducible costimulator ICOS, upregulation of Bcl-6, and SAP-mediated cognate interaction with B cells95,97. TFR cells have the characteristic activated regulatory T cell phenotype, with high expression of IL-10 and the immunomodulatory receptors CTLA-4 (CD152) and GITR97. Experiments involving depleting mice of TFR cells or the adoptive transfer of Cxcr5/ regulatory T cells that cannot give rise to TFR cells in response to immunization have demonstrated that the absence of TFR cells lead to a greater abundance of TFH cells and GC B cells9597, with a prominent overrepresentation of non antigen-specific B cells97. It is thus likely that CD4+ TFR cells suppress TD responses by acting on both TFH cells and GC B cells. What distinguishes TD responses from TI responses? Better understanding of the heterogeneous outcomes of TD and TI antibody responses, together with descriptions of unique types of TD and TI responses considered unconventional because of their requirement for help from innate cell types, is gradually changing the way that antibody responses are conceptualized. The initial postulation that TD responses, by virtue of their ability to induce GCs, are best suited to generate B cell memory, whereas TI antigens cannot elicit a long-lived or recall antibody response has been challenged by several
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subsequent discoveries. First, there is accumulating evidence of the existence of a pre-GC or GC-independent pathway for the generation of memory B cells that is still T cell dependent. Adoptively transferred HEL-specific B cells can develop into early memory B cells before GC formation. Those cells were predominantly IgM and had entered the cell cycle in response to immunization with HEL and cognate T cell help98. Parallel studies of Bcl-6-deficient mice, IL-21-deficient mice and mice treated with mAb to ICOS have also demonstrated the existence of nonGC-derived memory B cells to TD antigens that can form in the absence of the formation of TFH cells or TFH cellderived ICOS and IL-21 signals and do not undergo somatic hypermutation17,99,100. Second, we have recently become aware of the existence of TD responses that fail to induce memory. Until very recently, the term thymus dependent was synonymous with T cell dependent, and TD responses were defined as those elicited by protein antigens. The description above of antibody responses to purified glycolipid antigens elicited by thymus-derived iNKT cells that are therefore thymus dependent calls for broadening of the definition of TD responses to include those elicited by lipids presented by CD1d. Furthermore, those TD-2 responses fundamentally differ from the classic TD responses. Despite the demonstration of GC formation during the course of NKTFH celldependent responses to glycolipids, memory B cells and memory plasma cells, the traditional hallmarks of TD responses, do not develop53,60 (Fig. 2b). Third, several studies have now demonstrated that TI antigens can generate a second memory compartment that consists of either resting cells long-lived memory cells or long-lived memory bone marrow plasma cells, both of which were once thought to develop as a consequence of T cell help and require GC formation. Immunization with B. hermsii induces long-lived TI memory B cells that originate from B-1b cells (Fig. 3a); functional memory has been demonstrated after the adoptive transfer of B-1b cells from T celldeficient mice convalescing from infection with whole B. hermsii into mice deficient in Rag1, followed by rechallenge of the recipient mice with whole B. hermsii46. Unmutated IgM efficiently combats the relapsing fever caused by these bacteria46. It was noted over a decade ago that NP-specific plasma blasts can persist in the spleen for up to 3 months after immunization with TI-2 antigens such as haptenated Ficoll29. That observation was later explained by the demonstration that resting B-1b memory clones can sustain continued growth of NP-specific extrafollicular plasmablasts and the accumulation of NP-specific B cells in follicles101,102 (Fig. 3b). Plasma cells generated in response to NP-Ficoll can also home to the bone marrow103. Furthermore, plasma cells generated in response to the pneumococcal capsular polysaccharide PS3 have been shown to last for at least 6 months in the absence of replenishment by naive or memory B cells in patients treated with rituximab (a monoclonal antibody to the B cellspecific surface antigen CD20)104, which provides further support for the proposal that memory plasma cells develop during TI-2 responses. Fourth, TI antigens were initially thought to give rise only to extrafollicular responses, but two independent studies have demonstrated that the prototypic TI-2 antigen NP-Ficoll induces GCs in nude mice30,31 (Fig.3b). In those studies, the size and frequency of the GCs was proportional to the frequency and possibly the affinity of responding B cells: GCs were rare in wild-type mice31 but were much more abundant in quasi-monoclonal mice, in which a high proportion of B cells bind NP with high affinity because of sequence encoding a rearranged variable-diversity-joining segment knocked in to the immunoglobulin heavy-chain locus (the heavy chain, when paired with any -light chain, is specific for the hapten NP)30. For the formation of such GCs, CD40 signals not derived from T cells are
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required and are thought to be provided by the complement component C4 held by immune complexes on follicular dendritic cells105. Unlike TD GCs, B cells in TI-2 GCs do not survive beyond the first wave of centrocyte generation and undergo limited affinity maturation30,106 (Fig. 3b), which supports the proposal that T cellderived help is required for the maintenance of GC reactions. Finally, as mentioned above, marginal zone B cells can also acquire somatic mutations during TI responses that are helped by neutrophils63 (Fig. 3c). Concluding remarks The fact that some antigens can induce antibody responses without thymus-derived T cells, whereas others cannot, and that TD responses are fundamentally different from TI responses, is still a central tenet in immunology. The TD-TI paradigm was based on the observation of differences between two mouse strainsnude mice and CBA/N micein their immune responses to protein, LPS and polysaccharide antigens, and for many years immunologists have tried to fit data into this paradigm. The classification of B cell responses as TD and TI proved useful in understanding the different pathways by which B cells are activated. However, the discovery of additional and unconventional B cell helpers that drive types of immune responses that do not fit into the classical functional differences once ascribed to TD responses versus TI responses, the realization that most pathogens share properties of TD and TI antigens, and fundamental differences between mice and humans in TI immune responses are gradually removing the emphasis from the TD-TI paradigm and placing more weight on the characteristics of the B cell helpers. Among such helpers, innate cells are receiving greater recognition for the varied and important roles they seem to have in protective antibody responses and in the regulation of memory plasma cell niches in the bone marrow.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.
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1. 2. 3. 4. 5. 6. 7. 8. 9. MacLennan, I.C. Germinal centers. Annu. Rev. Immunol. 12, 117139 (1994). Tarlinton, D.M. Evolution in miniature: selection, survival and distribution of antigen reactive cells in the germinal centre. Immunol. Cell Biol. 86, 133138 (2008). Gershon, R.K. & Kondo, K. Cell interactions in the induction of tolerance: the role of thymic lymphocytes. Immunology 18, 723737 (1970). Bretscher, P. & Cohn, M. A theory of self-nonself discrimination. Science 169, 10421049 (1970). Noelle, R.J. & Snow, E.C. Cognate interactions between helper T cells and B cells. Immunol. Today 11, 361368 (1990). Cyster, J.G. B cell follicles and antigen encounters of the third kind. Nat. Immunol. 11, 989996 (2010). Crotty, S. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29, 621663 (2011). Vinuesa, C.G., Tangye, S.G., Moser, B. & Mackay, C.R. Follicular B helper T cells in antibody responses and autoimmunity. Nat. Rev. Immunol. 5, 853865 (2005). Cannons, J.L. et al. Optimal germinal center responses require a multistage Tcell: B cell adhesion process involving integrins, SLAM-associated protein, and CD84. Immunity 32, 253265 (2010). Smith, K.G., Hewitson, T.D., Nossal, G.J. & Tarlinton, D.M. The phenotype and fate of the antibody-forming cells of the splenic foci. Eur. J. Immunol. 26, 444448 (1996). MacLennan, I.C. et al. Extrafollicular antibody responses. Immunol. Rev. 194, 818 (2003). Vinuesa, C.G. & Cyster, J.G. How T cells earn the follicular rite of passage. Immunity 35, 671680 (2011). Victora, G.D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable uorescent reporter. Cell 143, 592605 (2010). Liu, Y.J. et al. Mechanism of antigen-driven selection in germinal centres. Nature 342, 929931 (1989). Good-Jacobson, K.L. et al. PD-1 regulates germinal center B cell survival and the formation and afnity of long-lived plasma cells. Nat. Immunol. 11, 535542 (2010).

npg

2013 Nature America, Inc. All rights reserved.

10.

11. 12. 13. 14. 15.

VOLUME 14 NUMBER 2 FebruArY 2013 NATURE IMMUNOLOGY

REVIEW
16. Bryant, V.L. et al. Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells. J. Immunol. 179, 81808190 (2007). 17. Linterman, M.A. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353363 (2010). 18. Winter, O. et al. Megakaryocytes constitute a functional component of a plasma cell niche in the bone marrow. Blood 116, 18671875 (2010). 19. Mosier, D.E., Mond, J.J. & Goldings, E.A. The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect. J. Immunol. 119, 18741878 (1977). 20. Mond, J.J., Lees, A. & Snapper, C.M. T cell-independent antigens type 2. Annu. Rev. Immunol. 13, 655692 (1995). 21. Bekeredjian-Ding, I. & Jego, G. Toll-like receptorssentries in the B-cell response. Immunology 128, 311323 (2009). 22. Peng, S.L. Signaling in B cells via Toll-like receptors. Curr. Opin. Immunol. 17, 230236 (2005). 23. Alugupalli, K.R., Akira, S., Lien, E. & Leong, J.M. MyD88- and Brutons tyrosine kinase-mediated signals are essential for T cell-independent pathogen-specic IgM responses. J. Immunol. 178, 37403749 (2007). 24. Eckl-Dorna, J. & Batista, F.D. BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specic plasma cell formation. Blood 113, 39693977 (2009). 25. Minguet, S. et al. Enhanced B-cell activation mediated by TLR4 and BCR crosstalk. Eur. J. Immunol. 38, 24752487 (2008). 26. Lanzavecchia, A. & Sallusto, F. Toll-like receptors and innate immunity in B-cell activation and antibody responses. Curr. Opin. Immunol. 19, 268274 (2007). 27. Martin, F., Oliver, A.M. & Kearney, J.F. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14, 617629 (2001). 28. Braley-Mullen, H. Antigen requirements for induction of B-memory cells: studies with dinitrophenyl coupled to T-dependent and T-independent carriers. J. Exp. Med. 147, 18241831 (1978). 29. Garcia de Vinuesa, C., OLeary, P., Sze, D.M., Toellner, K.M. & MacLennan, I.C. T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is conned to extrafollicular foci. Eur. J. Immunol. 29, 13141323 (1999). 30. Garcia de Vinuesa, C. et al. Germinal centers without T cells. J. Exp. Med. 191, 485494 (2000). 31. Lentz, V.M. & Manser, T. Cutting edge: germinal centers can be induced in the absence of T cells. J. Immunol. 167, 1520 (2001). 32. Weller, S. et al. Human blood IgM memory B cells are circulating splenic marginal zone B cells harboring a prediversied immunoglobulin repertoire. Blood 104, 36473654 (2004). 33. Allman, D. & Pillai, S. Peripheral B cell subsets. Curr. Opin. Immunol. 20, 149157 (2008). 34. Genestier, L. et al. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses. J. Immunol. 178, 77797786 (2007). 35. Cariappa, A. et al. Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes. Immunity 23, 397407 (2005). 36. Martin, F. & Kearney, J.F. Marginal-zone B cells. Nat. Rev. Immunol. 2, 323335 (2002). 37. Liu, Y.J., Oldeld, S. & MacLennan, I.C. Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. Eur. J. Immunol. 18, 355362 (1988). 38. Zandvoort, A. & Timens, W. The dual function of the splenic marginal zone: essential for initiation of anti-TI-2 responses but also vital in the general rst-line defense against blood-borne antigens. Clin. Exp. Immunol. 130, 411 (2002). 39. Guinamard, R., Okigaki, M., Schlessinger, J. & Ravetch, J.V. Absence of marginal zone B cells in Pyk-2-decient mice denes their role in the humoral response. Nat. Immunol. 1, 3136 (2000). 40. Song, H. & Cerny, J. Functional heterogeneity of marginal zone B cells revealed by their ability to generate both early antibody-forming cells and germinal centers with hypermutation and memory in response to a T-dependent antigen. J. Exp. Med. 198, 19231935 (2003). 41. Phan, T.G., Gardam, S., Basten, A. & Brink, R. Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen. J. Immunol. 174, 45674578 (2005). 42. Cinamon, G., Zachariah, M.A., Lam, O.M., Foss, F.W. Jr. & Cyster, J.G. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat. Immunol. 9, 5462 (2008). 43. Zietara, N., Lyszkiewicz, M., Krueger, A. & Weiss, S. ICOS-dependent stimulation of NKT cells by marginal zone B cells. Eur. J. Immunol. 41, 31253134 (2011). 44. Hayakawa, K., Hardy, R.R. & Herzenberg, L.A. Progenitors for Ly-1 B cells are distinct from progenitors for other B cells. J. Exp. Med. 161, 15541568 (1985). 45. Montecino-Rodriguez, E., Leathers, H. & Dorshkind, K. Identication of a B-1 B cell-specied progenitor. Nat. Immunol. 7, 293301 (2006). 46. Alugupalli, K.R. et al. B1b lymphocytes confer T cell-independent long-lasting immunity. Immunity 21, 379390 (2004). 47. Defrance, T., Taillardet, M. & Genestier, L. T cell-independent B cell memory. Curr. Opin. Immunol. 23, 330336 (2011). 48. Baumgarth, N., Tung, J.W. & Herzenberg, L.A. Inherent specicities in natural antibodies: a key to immune defense against pathogen invasion. Springer Semin. Immunopathol. 26, 347362 (2005). 49. Fagarasan, S. & Honjo, T. Intestinal IgA synthesis: regulation of front-line body defences. Nat. Rev. Immunol. 3, 6372 (2003). 50. Macpherson, A.J. et al. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 288, 22222226 (2000). 51. Hardy, R.R. B-1 B cells: development, selection, natural autoantibody and leukemia. Curr. Opin. Immunol. 18, 547555 (2006). 52. Barral, P. et al. B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo. Proc. Natl. Acad. Sci. USA 105, 83458350 (2008). 53. Chang, P.P. et al. Identication of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat. Immunol. 13, 3543 (2012). 54. Detre, C. et al. SAP expression in invariant NKT cells is required for cognate help to support B-cell responses. Blood 120, 122129 (2012). 55. Leadbetter, E.A. et al. NK T cells provide lipid antigen-specic cognate help for B cells. Proc. Natl. Acad. Sci. USA 105, 83398344 (2008). 56. Bendelac, A., Savage, P.B. & Teyton, L. The biology of NKT cells. Annu. Rev. Immunol. 25, 297336 (2007). 57. Venkataswamy, M.M. & Porcelli, S.A. Lipid and glycolipid antigens of CD1drestricted natural killer T cells. Semin. Immunol. 22, 6878 (2010). 58. Godfrey, D.I. & Berzins, S.P. Control points in NKT-cell development. Nat. Rev. Immunol. 7, 505518 (2007). 59. Kitamura, H. et al. The natural killer T (NKT) cell ligand -galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells. J. Exp. Med. 189, 11211128 (1999). 60. King, I.L. et al. Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner. Nat. Immunol. 13, 4450 (2012). 61. Tonti, E. et al. Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4+ T cell help. J. Immunol. 188, 32173222 (2012). 62. Fujii, S., Shimizu, K., Hemmi, H. & Steinman, R.M. Innate V14+ natural killer T cells mature dendritic cells, leading to strong adaptive immunity. Immunol. Rev. 220, 183198 (2007). 63. Puga, I. et al. B cell-helper neutrophils stimulate the diversication and production of immunoglobulin in the marginal zone of the spleen. Nat. Immunol. 13, 170180 (2012). 64. Litinskiy, M.B. et al. DCs induce CD40-independent immunoglobulin class switching through BLyS and APRIL. Nat. Immunol. 3, 822829 (2002). 65. Mackay, F. & Schneider, P. Cracking the BAFF code. Nat. Rev. Immunol. 9, 491502 (2009). 66. Mosier, D.E. & Subbarao, B. Thymus-independent antigens: complexity of B-lymphocyte activation revealed. Immunol. Today 3, 217222 (1982). 67. Garcia De Vinuesa, C. et al. Dendritic cells associated with plasmablast survival. Eur. J. Immunol. 29, 37123721 (1999). 68. Balazs, M., Martin, F., Zhou, T. & Kearney, J. Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses. Immunity 17, 341352 (2002). 69. Randolph, G.J., Jakubzick, C. & Qu, C. Antigen presentation by monocytes and monocyte-derived cells. Curr. Opin. Immunol. 20, 5260 (2008). 70. Leon, B. & Ardavin, C. Monocyte-derived dendritic cells in innate and adaptive immunity. Immunol. Cell Biol. 86, 320324 (2008). 71. Tezuka, H. et al. Regulation of IgA production by naturally occurring TNF/iNOSproducing dendritic cells. Nature 448, 929933 (2007). 72. Craxton, A., Magaletti, D., Ryan, E.J. & Clark, E.A. Macrophage- and dendritic celldependent regulation of human B-cell proliferation requires the TNF family ligand BAFF. Blood 101, 44644471 (2003). 73. Merluzzi, S. et al. Mast cells enhance proliferation of B lymphocytes and drive their differentiation toward IgA-secreting plasma cells. Blood 115, 28102817 (2010). 74. Gauchat, J.F. et al. Induction of human IgE synthesis in B cells by mast cells and basophils. Nature 365, 340343 (1993). 75. Erazo, A. et al. Unique maturation program of the IgE response in vivo. Immunity 26, 191203 (2007). 76. Shinkai, K., Mohrs, M. & Locksley, R.M. Helper T cells regulate type-2 innate immunity in vivo. Nature 420, 825829 (2002). 77. Wang, H.B. & Weller, P.F. Pivotal advance: eosinophils mediate early alum adjuvant-elicited B cell priming and IgM production. J. Leukoc. Biol. 83, 817821 (2008). 78. Henz, B.M., Maurer, M., Lippert, U., Worm, M. & Babina, M. Mast cells as initiators of immunity and host defense. Exp. Dermatol. 10, 110 (2001). 79. Sokol, C.L. et al. Basophils function as antigen-presenting cells for an allergeninduced T helper type 2 response. Nat. Immunol. 10, 713720 (2009). 80. Spencer, L.A. & Weller, P.F. Eosinophils and Th2 immunity: contemporary insights. Immunol. Cell Biol. 88, 250256 (2010). 81. Tacke, F. et al. Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery. J. Exp. Med. 203, 583597 (2006). 82. Chorny, A., Puga, I. & Cerutti, A. Innate signaling networks in mucosal IgA class switching. Adv. Immunol. 107, 3169 (2010). 83. Chu, V.T. et al. Eosinophils are required for the maintenance of plasma cells in the bone marrow. Nat. Immunol. 12, 151159 (2011).

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125

REVIEW
84. Mack, M. et al. Identication of antigen-capturing cells as basophils. J. Immunol. 174, 735741 (2005). 85. Kim, S., Shen, T. & Min, B. Basophils can directly present or cross-present antigen to CD8 lymphocytes and alter CD8 T cell differentiation into IL-10-producing phenotypes. J. Immunol. 183, 30333039 (2009). 86. Denzel, A. et al. Basophils enhance immunological memory responses. Nat. Immunol. 9, 733742 (2008). 87. Ruprecht, C.R. & Lanzavecchia, A. Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur. J. Immunol. 36, 810816 (2006). 88. Gavin, A.L. et al. Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science 314, 19361938 (2006). 89. Pasare, C. & Medzhitov, R. Control of B-cell responses by Toll-like receptors. Nature 438, 364368 (2005). 90. Day, N. et al. Interleukin receptor-associated kinase (IRAK-4) deciency associated with bacterial infections and failure to sustain antibody responses. J. Pediatr. 144, 524526 (2004). 91. Ku, C.L. et al. IRAK4 and NEMO mutations in otherwise healthy children with recurrent invasive pneumococcal disease. J. Med. Genet. 44, 1623 (2007). 92. Sweet, R.A. et al. Facultative role for T cells in extrafollicular Toll-like receptordependent autoreactive B-cell responses in vivo. Proc. Natl. Acad. Sci. USA 108, 79327937 (2011). 93. Snapper, C.M. et al. Distinct types of T-cell help for the induction of a humoral immune response to Streptococcus pneumoniae. Trends Immunol. 22, 308311 (2001). 94. Khan, A.Q., Chen, Q., Wu, Z.Q., Paton, J.C. & Snapper, C.M. Both innate immunity and type 1 humoral immunity to Streptococcus pneumoniae are mediated by MyD88 but differ in their relative levels of dependence on toll-like receptor 2. Infect. Immun. 73, 298307 (2005). 95. Chu, V.T. & Berek, C. Immunization induces activation of bone marrow eosinophils required for plasma cell survival. Eur. J. Immunol. 42, 130137 (2012). 96. Wollenberg, I. et al. Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells. J. Immunol. 187, 45534560 (2011). 97. Linterman, M.A. et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 17, 975982 (2011). 98. Chan, T.D. et al. Antigen afnity controls rapid T-dependent antibody production by driving the expansion rather than the differentiation or extrafollicular migration of early plasmablasts. J. Immunol. 183, 31393149 (2009). 99. Inamine, A. et al. Two waves of memory B-cell generation in the primary immune response. Int. Immunol. 17, 581589 (2005). 100. Toyama, H. et al. Memory B cells without somatic hypermutation are generated from Bcl6-decient B cells. Immunity 17, 329339 (2002). 101. Hsu, M.C., Toellner, K.M., Vinuesa, C.G. & Maclennan, I.C. B cell clones that sustain long-term plasmablast growth in T-independent extrafollicular antibody responses. Proc. Natl. Acad. Sci. USA 103, 59055910 (2006). 102. Obukhanych, T.V. & Nussenzweig, M.C. T-independent type II immune responses generate memory B cells. J. Exp. Med. 203, 305310 (2006). 103. Nie, Y. et al. The role of CXCR4 in maintaining peripheral B cell compartments and humoral immunity. J. Exp. Med. 200, 11451156 (2004). 104. Taillardet, M. et al. The thymus-independent immunity conferred by a pneumococcal polysaccharide is mediated by long-lived plasma cells. Blood 114, 44324440 (2009). 105. Gaspal, F.M. et al. The generation of thymus-independent germinal centers depends on CD40 but not on CD154, the T cell-derived CD40-ligand. Eur. J. Immunol. 36, 16651673 (2006). 106. Toellner, K.M. et al. Low-level hypermutation in T cell-independent germinal centers compared with high mutation rates associated with T cell-dependent germinal centers. J. Exp. Med. 195, 383389 (2002).

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