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  1. 1. REVIEWS The who, how and where of antigen presentation to B cells Facundo D. Batista and Naomi E. Harwood Abstract | A functional immune system depends on the appropriate activation of lymphocytes following antigen encounter. In this Review, we summarize studies that have used high-resolution imaging approaches to visualize antigen presentation to B cells in secondary lymphoid organs. These studies illustrate that encounters of B cells with antigen in these organs can be facilitated by diffusion of the antigen or by the presentation of antigen by macrophages, dendritic cells and follicular dendritic cells. We describe cell-surface molecules that might be important in mediating antigen presentation to B cells and also highlight the key role of B cells themselves in antigen transport. Data obtained from the studies discussed here highlight the predominance, importance and variety of the cell-mediated processes that are involved in presenting antigen to B cells in vivo. Total internal reflection The immune system is highly dynamic and diverse, which and present antigen in association with MHC class II fluorescence microscopy enables it to effectively protect an individual from numer- molecules, thereby recruiting specific CD4 + T-cell A microscopy method that ous potentially pathogenic encounters. Early studies that help and stimulating B-cell proliferation and dif- allows for the identification combined techniques such as electron microscopy and ferentiation15,16 (BOX 2). Although the precise factors of fluorescence within immunofluorescence yielded valuable information on that determine the fate of activated B cells currently 100–200 nm of the interface between cells and their the static location and organization of the cells of the remain unclear, B cells can differentiate along two substrate (for example, lipid immune system, and inferred their dynamic properties distinct pathways. On the one hand, B cells can dif- bilayers or glass coverslips), (for a historical perspective and references to original ferentiate to form extrafollicular plasmablasts that thereby providing a high lateral articles see REFS 1–3). However, it is necessary to consider are essential for rapid antibody production and early and axial resolution at the cell–substrate interface and the development of immune responses in terms of both protective immune responses. On the other hand, the ability to observe intracellular and intercellular interactions in the appro- activated B cells can enter germinal centres, where they nanoscale movement of priate environment and in real time4–8. Indeed, the recent can differentiate into plasma cells, which can secrete signalling molecules during development of high-resolution imaging techniques, high-affinity antibody following affinity maturation, cellular activation. including confocal microscopy, total internal reflection or memory B cells, which confer long-lasting protection fluorescence microscopy and intravital multiphoton micro- from secondary challenge with antigen17,18. scopy, has provided insights into the spatio-temporal In this Review, we discuss recent insights into the dynamics of the molecular and cellular events that sites at which B cells encounter antigen in vivo, as well underlie immune responses to pathogenic infection. One as the mechanisms by which these interactions occur example of such an insight is the molecular description (for excellent reviews that include comprehensive Lymphocyte Interaction of the immunological synapse, a structure that is com- descriptions of antigen presentation to T cells in vivo, Laboratory, Cancer Research monly associated with lymphocyte activation following see REFS 2,19–21 ). It is now clear that B cells can UK London Research the recognition of a specific antigen9–12 (BOX 1). encounter and respond to antigen through many dif- Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn B-cell activation is initiated following engagement ferent mechanisms depending on the nature and size Fields, London WC2A 3PX, UK. of the B-cell receptor (BCR) by a specific antigen. of the antigen itself, as well as on the cellular context Correspondence to F.D.B. B cells can recognize and respond to both soluble and location in which antigen presentation occurs. e‑mail: facundo.batista@ and membrane-associated antigen, although recent This provides great versatility in terms of initiating doi:10.1038/nri2454 insights suggest that membrane-associated antigens responses that are appropriate to the particular antigen Published online are more important for B-cell activation in vivo 13,14. and are therefore the most effective for the protection 12 December 2008 Following antigenic stimulation, B cells can process of the host. nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 15 © 2009 Macmillan Publishers Limited. All rights reserved
  2. 2. REVIEWS Intravital multiphoton Sites of antigen encounter slOs are extremely well connected to the blood and microscopy As cells of the immune system can respond to a huge lymphatic systems, allowing them to continually sample A microscopy method that range of potential pathogenic challenges throughout the and concentrate antigens that are circulating through- combines the advanced whole body, these responses must be tightly coordinated out the body. The importance of slOs in organizing optical techniques of laser-scanning confocal to confer effective protection. Owing to this great anti- and coordinating immune responses is evident from microscopy with genic diversity, the event of antigen encounter with an the severe impairment in the activation of naive lym- long-wavelength multiphoton antigen-specific lymphocyte that can mount a rapid and phocytes in alymphoplastic (aly/aly) mice and asplenic fluorescence excitation to appropriate response would seem unlikely. To maximize (homebox 11-deficient) mice23. Accordingly, any mean- capture high-resolution, the probability of such an interaction, these events occur ingful investigation into the spatio-temporal dynamics three-dimensional images of living cells and/or tissues that in defined sites, such as the lymph nodes and spleen, and the mechanisms by which lymphocytes encounter have been labelled with which are collectively known as secondary lymphoid antigen in vivo must be considered within such highly fluorophores. It provides a organs (slOs). These sites possess a highly organized organized environments. greater tissue imaging depth microarchitecture that is necessary for the compartmen- (up to 350 µm depending on the tissue) and less talization of numerous cellular interactions and there- Lymph nodes. It is now widely understood that photobleaching and fore provide the optimal environment for the initiation beneath the protective outer collagenous capsule, a phototoxicity than of immune responses, including the determination of lymph node is divided into three discrete (but non- conventional imaging cell fate22. Furthermore, within 24 hours lymphocytes rigid) regions that are defined by the expression of methods. that have been unsuccessful in their search for antigen specific chemokines3,24 (FIG. 1a). Directly beneath the Germinal centre recirculate throughout the body and between slOs to subcapsular sinus is a macrophage-rich sheet that sur- A highly specialized and dramatically increase the probability of encountering rounds the B-cell zone, which is also termed the cor- dynamic microenvironment cognate antigen3. tex. B cells in this region are organized into aggregates, that gives rise to secondary B-cell follicles during an immune response. It is the main site of B-cell maturation, Box 1 | The immunological synapse during which memory B cells and plasma cells that produce Lymphocyte activation is initiated following the recognition of specific antigen by clonotypic immunoreceptors, such high-affinity antibody are as the B‑cell receptor (BCR) and the T‑cell receptor (TCR). The recognition of specific antigen on a spatially constrained generated. surface is associated with membrane reorganization that results in the formation of an immunological synapse9–12. The structure of the mature immunological synapse is characterized by the spatial segregation of immunoreceptors into Plasma cell a central supramolecular activation cluster (cSMAC) from the surrounding peripheral SMAC (pSMAC), which contains integrins, A non-dividing, terminally such as lymphocyte function‑associated antigen 1 (LFA1) (see the figure). The early molecular events that underlie the formation differentiated, antibody- of the immunological synapse have been revealed by high‑resolution imaging techniques and are highly coordinated and tightly secreting cell of the B-cell regulated. Following the recognition of specific antigen and before the formation of the immunological synapse, B cells rapidly lineage. spread over the antigen‑containing surface in a manner that is dependent on intracellular signalling and cytoskeletal Memory B cell rearrangements120. During B‑cell spreading, antigen–BCR microclusters are continually assembled at the periphery of the An antigen-experienced B cell contact area between the cells14. As similar microclusters are crucial for sustained signalling in T cells121–123, we have proposed that expresses high-affinity that these microclusters form functional signalling units that are common to both types of lymphocyte14. antibodies and quickly A comprehensive genetic dissection of the requirements for B‑cell spreading identified an important role for the sequential differentiates into a plasma recruitment of the kinases LYN and spleen tyrosine kinase (SYK) to antigen–BCR microclusters in the initiation of cell during antigen-recall BCR‑induced signalling124. Furthermore, this study visualized the formation of microsignalosomes through the recruitment of responses. signalling molecules and adaptors to the antigen–BCR microclusters and showed the importance of cooperation between VAV and phospholipase Cγ2 in mediating the spreading response. The role of LYN and SYK in the initiation of BCR signalling Alymphoplastic (aly/aly) has been further supported through mice B-cell contact area with APC Antigen–BCR microcluster: side view fluorescence resonance energy transfer Mice that are characterized (APC not shown) CR APC (FRET)‑based assays125. B‑cell spreading by the absence of lymph FcR nodes and Peyer’s patches. facilitates the propagation of signalling Alymphoplasia is caused by a Antigen through microclusters by allowing the BCR spontaneous mutation in the C3 Antibody to engage more antigen, which promotes gene that encodes nuclear- fragment further localized signalling and cytoskeleton factor-κB-inducing kinase. rearrangements120. The spreading response BCR Mature immunological synapse: is followed by a prolonged contraction top view Microsignalosome phase, which results in the accumulation B cell of antigen–BCR microclusters in the cSMAC of the mature immunological Immunological synapse: side view synapse. The cSMAC functions as a site for APC internalization of antigen12,120, before it is processed and presented in association pSMAC cSMAC with MHC class II molecules; presentation Integrin receptor of antigen–MHC molecules mediates the B-cell contact area recruitment of the CD4+ T‑cell help that is necessary for full B‑cell activation15,16. APC, antigen‑presenting cell; C3, complement component 3; CR, complement receptor; cSMAC pSMAC B cell FcR, Fc receptor. Nature Reviews | Immunology 16 | jAnuARy 2009 | vOluME 9 © 2009 Macmillan Publishers Limited. All rights reserved
  3. 3. REVIEWS T-cell-dependent antigen known as follicles, and are the largest population of The medulla, which is the innermost area of the A protein antigen that needs to IgMmedIgDhiCD21medCD23hi B cells in the body. Follicles lymph node, contains both B and T cells that are organ- be recognized by T helper cells are also rich in radiation-resistant follicular dendritic ized into medullary cords and is also rich in DCs and (in the context of MHC cells (FDCs) that express high levels of the adhesion macrophages. molecules) and requires cooperation between these molecules vascular cell-adhesion molecule 1 (vCAM1) lymph nodes are strategically positioned at branches antigen-specific T cells and and intercellular adhesion molecule 1 (ICAM1), as well throughout the lymphatic system to enable extensive B cells for a specific antibody as complement and Fc receptors25–28. FDCs are thought antigenic sampling of lymphatic fluid4. lymphatic fluid, response to be generated. to be of mesenchymal origin and thus form a popu- which contains various chemokines and antigens that lation of cells that is distinct from classic DCs 29. The have been collected from the body, enters the lymph Affinity maturation A process whereby the precise mechanism of FDC development is not yet node through the afferent lymph vessel and is subse- mutation of antibody fully understood, but is known to require the expres- quently filtered by the lymph node as it flows towards variable-region genes followed sion of various chemokine receptors and the presence the efferent lymph vessel. lymphatic fluid is prevented by selection for higher-affinity of B cells17,30. Following exposure to antigen, follicles from freely diffusing into the interior when it enters variants in the germinal centre leads to an increase in average may also contain specialized structures, known as the lymph node by the subcapsular sinus; instead, it antibody affinity for an antigen germinal centres, which consist of rapidly proliferat- travels to the medulla through structures known as as an immune response ing B cells within a network of FDCs. The formation trabecular sinuses. In addition, components of lym- progresses. The selection is of germinal centres is important during the develop- phatic fluid that have a molecular weight of below thought to be a competitive ment of humoral immune responses to T-cell-dependent ~70 kDa (equivalent to a dynamic radius of ~5.5 nm) process in which B cells compete with free antibody antigens, as they serve as sites for affinity maturation and the are allowed to move towards the HEv through the intri- to capture decreasing amounts generation of long-lasting memory B cells. cate meshwork of fibroblastic reticular cell (FRC)-lined of antigen. The T-cell zone, or paracortex, which is adjacent to collagenous conduits that are associated with DCs32,33 the follicles, contains high endothelial venules (HEvs) (FIG. 1b). Accordingly, early observations indicated that µ mt−/− mice A strain of mutant mice that and numerous antigen-presenting cells. The HEvs are the paracortex did not contain unprocessed antigen, carry a stop codon in the first specialized capillaries that continually supply the lymph although some antigen could be detected in DCs34. The membrane exon of the µ-chain node with and drain it of lymphocytes from and to the lymphatic fluid that exits the lymph node is enriched constant region. They lack periphery. Furthermore, HEvs in the paracortex can often with lymphocytes35 and therefore provides a means for IgM+ B cells, and B-cell mediate the recruitment of cells other than lymphocytes, these cells to return to the circulation24. development is arrested before the differentiation stage at such as antigen-presenting cells that have accumulated which IgD can be expressed. antigen in the periphery. The paracortex also contains Spleen. The organization of the lymphoid tissue of the a network of collagenous conduit fibres that allow the spleen is similar to that of the lymph node, with B-cell passage of low-molecular-mass components of lymphatic follicles and T-cell zones that contain networks of retic- fluid, including chemokines, from the subcapsular sinus ular cells (also known as the periarteriolar lymphoid to HEvs (reviewed in REF. 31). sheath) and a similar conduit system36 in the splenic Box 2 | B cells mediate antigen presentation to antigen-specific T cells B cells can have a role in initiating T‑cell responses by functioning as antigen‑presenting cells (APCs). When compared with dendritic cells, B cells are relatively poor APCs, and therefore the importance of B cells in antigen presentation to T cells has been controversial. However, their presentation potential can be enhanced following specific recognition of antigen126. Furthermore, it has been recently shown that reconstitution of chimeric µ mt–/– mice with B cells that lack MHC class II molecules results in impairment of T‑cell responses following antigen stimulation127, which suggests that presentation of antigen by B cells does contribute to T‑cell activation. Tracking and intravital microscopy of antigen‑specific lymphocytes in lymph nodes (see the figure) showed that around 24 hours following antigen encounter, B cells (green) migrated towards the B‑cell–T‑cell boundary at the edge of the follicles70 in a CC‑chemokine receptor‑7‑dependent manner48. When they reached this boundary, B cells were found to engage antigen‑specific T cells (red) for up to 1 hour. A similar migration pattern to the B‑cell–T‑cell boundary was observed in response to stimulation with particulate antigen or immune complexes following their accumulation at the subcapsular sinus (SCS)64,67. B‑cell–T‑cell conjugates are suprisingly motile and migrate through the lymph node at the same rate as unconjugated B cells. The importance of these long‑lived B‑cell–T‑cell Follicle conjugates in the generation of sustained immunity has recently been highlighted through the investigation SCS of mice that are deficient in SLAM‑associated protein (SAP)128. Although the CD4+ T cells from SAP‑deficient mice exhibit typical dendritic‑cell‑mediated activation, they cannot interact with cognate B cells, resulting in severely impaired formation and function of germinal centres. Thus, it seems that although the number of naive antigen‑specific B cells in vivo is T-cell probably low, this mechanism of antigen presentation zone to T cells by B cells contributes to the initiation of T‑cell responses in vivo. Follicle Nature Reviews | Immunology nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 17 © 2009 Macmillan Publishers Limited. All rights reserved
  4. 4. REVIEWS a Lymph node Spleen Afferent lymph Red pulp Trabecular sinus Paracortex White Subcapsular pulp sinus Follicle HEV Marginal zone Medulla Marginal Efferent sinus lymph PALS Blood Central Conduit network arteriole Conduit network Follicle Arteriole branch b Conduit network c SCS Follicle T-cell zone Collagen bundles FRC Associated DC Follicle Figure 1 | The organization of secondary lymphoid organs. a | A schematic representation of secondary lymphoid organs (SLOs). The lymph node is organized into three discrete (but non-rigid) regions: the medulla, the paracortex (also known as the T-cell zone) and the follicles (also known as the B-cell zone). Antigen-laden lymphatic fluid flows from the afferent lymph vessel Nature Reviews | Immunology into the subcapsular sinus and through the trabecular sinuses to the medulla, where it exits through the efferent lymph vessel. Lymphatic fluid also flows through the conduit network in the paracortex, allowing passage of low-molecular-mass components, including chemokines and antigen, from the subcapsular sinus to high endothelial venules (HEVs). HEVs in the paracortex also allow for the entry and exit of lymphocytes to and from the lymph node. The white pulp of the spleen consists of the paracortex (also known as the periarteriolar lymphoid sheath; PALS), B-cell follicles, a central arteriole and the marginal zone. Blood arrives at the spleen at the marginal sinus through branches of the central arteriole and from there it flows to the red pulp through the marginal zone or to the white pulp through the conduit network. b | The conduit network in the lymph node is composed of a collagen core and is lined with fibroblastic reticular cells (FRCs). Dendritic cells (DCs) are often associated with FRCs and can extend short protrusions into the conduits, possibly to sample the lymphatic fluid that is transported through this network. c | A snapshot of a video generated using multiphoton microscopy on the popliteal lymph node. The distribution of seminaphtho-rhodafluor (SNARF)-labelled B cells (red) within the follicles (delineated by a dashed line) beneath the subcapsular sinus (SCS; solid line) and of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T cells (green) in the paracortex is shown in the absence of antigen. Scale bar represents 100 µm. white pulp37 (FIG. 1a). The spleen also contains red pulp, These marginal-zone B cells form a population of cells which is richly supplied with blood and contains various that is distinct from follicular B cells, a large propor- antigen-presenting cells, lymphocytes and plasma cells38. tion of which are polyreactive clones. Marginal-zone The outer limit of the white pulp is separated from the B cells have an IgMhiIgDlowCD21hiCD23low phenotype red pulp by a region known as the marginal zone 39. and are thought to participate in the development of This area has extensive vasculature, and blood that early immune responses to both T-cell-dependent and enters the spleen through follicular arteriole branches T-cell-independent antigens42. In contrast to lymph nodes, in the marginal sinus reaches the marginal zone before the spleen is not supplied with afferent lymphatic fluid T-cell-independent antigen its permeation through the red pulp40. The marginal (although it does contain efferent lymphatics); instead, An antigen that directly zone also contains numerous macrophages and DCs, it is specialized in mounting immune responses to activates B cells. and a sizable population of non-recirculating B cells41. blood-borne antigens43. 18 | jAnuARy 2009 | vOluME 9 © 2009 Macmillan Publishers Limited. All rights reserved
  5. 5. REVIEWS Lymphocyte behaviour in ‘resting’ SLOs diffusion of antigen into the lymphoid tissue; how- To establish the sites and mechanisms of antigen encoun- ever, these encounters are usually mediated through ter with lymphocytes in vivo, it is important to understand macrophages, DCs and FDCs (FIG. 2). the localization and dynamics of these cells in the resting lymphoid organs. Although information on cell dynam- Encountering of soluble antigen. Antigen can be rap- ics in the past was inferred from studies using static idly supplied to the lymph node through the afferent approaches1–3, initial groundbreaking studies that directly lymph vessels. Indeed, antigen arrival at the subcapsu- visualized the spatio-temporal dynamics of lymphocytes lar sinus can be detected within minutes of subcutane- in vivo were possible because of advances in the field of ous administration51. As most B cells are mainly located multiphoton microscopy (reviewed in REFS 5,19). This in the follicles, lymph-borne antigens must gain access method allows for the investigation of cellular behaviour to follicular B cells in a manner that is independent in real time and to a depth in living tissue that is prohib- of the conduit system, which is present predominantly in ited by techniques such as confocal microscopy 5. The ear- the paracortex. liest multiphoton microscopy investigations were carried Interestingly, several electron microscopy stud- out on explanted or isolated lymph nodes44–46, and many ies have identified pores of ~0.1–1 µm diameter in the of the original observations have since been confirmed in regions of the subcapsular sinus that are adjacent to lymph nodes in vivo following minimal surgical disrup- the lymph-node parenchyma52–54. These pores might tion. However, high-resolution imaging of lymphocyte allow small soluble antigen, such as low-molecular-mass behaviour in splenic tissue is hampered by the technical toxins, that enter the lymph node through the afferent challenges that are associated with the isolation of this lymph vessel to directly pass into the follicle and gain vital and deeply embedded organ6. access to B cells55 (FIG. 2a). Indeed, using immunohisto- The first surprising observation from these multi- chemistry of frozen lymph-node sections, it was shown photon microscopy studies was the highly motile nature that this rapid diffusion of antigen was independent of of lymphocytes within the resting lymph node45–48. B and DCs and did not require migration of B cells from their T cells that were highly polarized in shape were found follicular location. These observations are consistent to move within their restricted cellular zones (FIG. 1c) with a previous study, which showed that immuniza- but along apparently random trajectories, with average tion of mice with soluble antigen initially resulted in a velocities of around 6 µm (for B cells) and 12 µm (for T substantial decrease in the motility of antigen-specific cells) per minute. Closer inspection of this movement fol- B cells in the follicle48. Furthermore, in the 24 hours lowing the visualization of the conduit network provided following immunization these B cells were shown to evidence for an order to this process. using an elegant present antigen in association with MHC class II mol- system involving chimeric mice that express green fluo- ecules on their surface (BOX 2) and to migrate towards rescent protein, Bajénoff et al.49 could visualize the FDCs the boundary between the follicle and the paracortex. and FRCs in the lymph node before the introduction of a Therefore, small low-molecular-mass antigens may gain population of labelled lymphocytes. This study showed access to cells in the follicles without requiring cell- that the T cells in the paracortex are in close association mediated presentation, through direct diffusion from with FRCs, which allows the T cells to be guided along pores in the subcapsular sinus. However the existence a defined network. As B cells enter the lymph node of these pores remains controversial, as they may simply through HEvs in the paracortex, it was also suggested be sites generated by cells that have recently migrated that these cells could migrate along FRCs in this area through the sinus wall. Furthermore, there is a discrep- before migrating in a chemokine-directed manner to the ancy between the dimensions of the observed pores follicles, where a similar network composed of FDCs can and the radius of the antigens that have been excluded be used to orchestrate their movement. such rapid move- from accessing the follicle by diffusion. In view of this, it ment across these networks suggests that lymphocytes in seems reasonable to postulate the existence of an alter- the lymph node are continually scanning their environ- native means of mediating the movement of smaller ment and are ready to respond to antigen as soon as it antigens into the follicles, perhaps in a manner that is is encountered. Interestingly, similar investigations have reminiscent of diffusion from the conduit system, as offered insight into the behaviour of CD11c+ DCs in the observed in the paracortex. paracortex 50. In contrast to lymphocytes, and as expected given their potential role in structural support of the con- Macrophage-mediated presentation. Although diffu- duit network, these DCs were found to be sessile, show- sion of antigen from the subcapsular sinus provides one ing movement at a velocity of less than 2 µm per minute. mechanism for exposing follicular B cells to small soluble However, this restriction in motility did not prevent the antigens, the access of larger particulate antigens to B cells rapid movement of their dendritic processes, indicating in the follicle is limited. It was therefore suggested that that these DCs retain the capacity to sample and respond specialized cells, possibly located in the subcapsular sinus, to environmental signals. might transport large antigens to the B-cell follicles53,56–58. Early microscopy studies revealed a population of macro- Presentation of antigen to B cells phages that reside directly beneath the subcapsular B cells can promptly detect and mount responses sinus52,53 and can extend their processes through it to gain to antigen after immunization. In the case of small soluble access to afferent lymphatic fluid53,56,58. As these macro- antigens, responses can be mounted following a simple phages have been shown to bind a soluble recombinant nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 19 © 2009 Macmillan Publishers Limited. All rights reserved
  6. 6. REVIEWS a Afferent lymph vessel Antigen C3 fragment Antibody Subcapsular sinus Immune MAC1 complex DC-SIGN FcR SCS macrophage BCR Small soluble antigen CR Antigen-specific Follicular B cell dendritic cell Primary follicle b c SCS Antigen-specific Paracortex B cell HEV Recently migrated DC FRC Resident DC Antigen-specific B cells Antigen non-specific B cells T cell Antigen Figure 2 | B-cell encounters with specific antigen in the lymph node. a | B cells in follicles have been found to encounter small soluble antigens from the lymphatic fluid as they diffuse from the subcapsular sinus (SCS) to the follicles55. Large antigens, immune complexes and viruses can be presented to follicular B cells at the macrophage- rich SCS64,67,68. In addition, follicular B cells may recognize antigen that is presented on the surface of follicular dendritic Nature Reviews | Immunology cells (FDCs). b | Snapshot of a video that was generated using multiphoton intravital microscopy on the popliteal lymph node. The solid line identifies the position of the SCS. A magnified view of antigen–B-cell conjugates is shown, in which conjugates appear yellow as a result of the merge of the red antigen-specific B cells and green antigen (P. Barral and F. B., unpublished observations). c | Schematic view of the paracortex to illustrate where antigen-specific B cells encounter antigen at this site. B cells entering the lymph node can encounter unprocessed antigen on the surface of resident or recently migrated DCs, in close proximity to the high-endothelial venules (HEVs)73,77–79. The conduit system, which is lined with FRCs and DCs, transports low-molecular-mass components of the lymphatic fluid through the lymph node; B cells and T cells have been shown to migrate in association with the FRC network49. BCR, B-cell receptor; C3, complement component 3; CR, complement receptor; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; FcR, Fc receptor; ICAM3, intercellular adhesion molecule 3; MAC1, macrophage receptor 1. Metallophilic macrophage protein that contains domains of the mannose receptor, intact antigen on their cell surface 56,58. Indeed, these A macrophage that is located it has been suggested that they may capture and concen- macrophages have been shown to capture and retain at the border of the white pulp trate antigen in a similar way to metallophilic macrophages antigen for up to 72 hours following the initial antigen and the marginal zone of the that reside in the splenic marginal zone59–61. exposure62. However, whether this involves the simple spleen. These macrophages express high levels of CD169 The macrophages in the subcapsular sinus are a dis- retention of antigen on the macrophage surface or anti- but lack expression of the tinct population to those in the medulla and have lim- gen internalization into non-degradative intracellular mannose receptor. ited phagocytic activity, which enables them to present compartments before it is recycled to the cell surface 20 | jAnuARy 2009 | vOluME 9 © 2009 Macmillan Publishers Limited. All rights reserved
  7. 7. REVIEWS Table 1 | Cell-surface molecules that are implicated in presenting antigen to B cells Presenting cell Receptor Antigen presented Presentation strategy Macrophage MAC1 Complement-coated antigen Remains on the cell surface FcγRIIB IgG-coated antigen Internalized in neutral endosomes and recycled? DC-SIGN Carbohydrate-containing antigen Internalized in neutral endosomes and recycled? DC FcγRIIB IgG-coated antigen Internalized in neutral endosomes and recycled? DC-SIGN Carbohydrate-containing antigen Internalized in neutral endosomes and recycled? FDC CR1 and CR2 Complement-coated antigen Remains on the cell surface FcγRIIB IgG-coated antigen Internalized in neutral endosomes and recycled? Marginal-zone CR1 and CR2 Complement-coated antigen Remains on the cell surface B cell Follicular B cell CR1 and CR2 Complement-coated antigen Remains on the cell surface FcγRIIB IgG-coated antigen Internalized in neutral endosomes and recycled? CR, complement receptor; DC, dendritic cell; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; FcγRIIB, low-affinity Fc receptor for IgG; FDC, follicular DC; ICAM3, intercellular adhesion molecule 3; MAC1, macrophage receptor 1. is not clear. In addition, macrophages are known to this virus68. Furthermore, as the retention of antigen was express a wide range of cell-surface receptors that not impaired following the depletion of C3, complement- could participate in the presentation of unprocessed independent mechanisms might also contribute to the antigen, including complement receptors, pattern- maintenance of antigen on the macrophage surface68. recognition receptors and/or carbohydrate-binding The accumulated antigen is subsequently presented scavenger receptors63 (TABLE 1). Indeed, macrophage in its intact form by macrophages for recognition by receptor 1 (MAC1; also known as αMβ2 integrin neighbouring follicular B cells. Accordingly, following and CD11b–CD18 dimer), which is a receptor for administration of antigen, it was shown that antigen- complement component 3 (C3) that is expressed by specific B cells in the follicle exhibited reduced migration macrophages, has been suggested to contribute to the over time and made stable contacts with macrophages retention of antigen on the cell surface64. Alternatively, in the subcapsular sinus64,67,68. As ICAM1 and vCAM1 the inhibitory low-affinity receptor for IgG (FcγRIIB) are expressed in the subcapsular sinus68, it is expected might mediate the internalization and recycling of IgG- that they may facilitate B-cell adhesion in this region and containing immune complexes to the macrophage cell thereby lower the threshold of antigen that is required surface, as has been shown in DCs65. Finally, the C-type for B-cell activation13,69. Together, these studies provide lectin DC-specific ICAM3-grabbing non-integrin the first clear demonstration of a role for macrophages (DC-sIGn; also known as CD209) could participate in the initiation of follicular B-cell responses. The macro- in the retention of glycosylated antigens, which is con- phage–B-cell interactions at the subcapsular sinus allow sistent with the observation that mice deficient in the antigen-specific B cells to acquire and internalize antigen mouse homologue of DC-sIGn, sIGnR1, fail to mount through their BCR before their migration to the B-cell– humoral immune responses following infection with T-cell boundary 67, where they may receive specific T-cell Streptococcus pneumoniae66. help48,70 (BOX 2). Interestingly, it has recently been noted A crucial and newly described role for subcapsular- that the response of B cells and CD4+ T cells to large viral sinus macrophages in the presentation of antigen to particles is deterministically linked to their antigen spe- follicular B cells has been recently established by three cificities71, suggesting that antigen uptake by the B cell independent studies64,67,68. These studies identified that, may not involve internalization of whole viral pathogens soon after antigen administration, subcapsular-sinus at the subcapsular sinus. macrophages are responsible for the rapid accumula- tion of various larger antigens, such as those found in DC-mediated presentation. B cells enter the lymph node immune complexes (with antibody and/or complement through the HEvs, so the paracortex would seem the ideal fragments), particulates, bacteria and viruses (FIG. 2a,b). site for the immediate presentation of antigen to these These macrophages might favour the retention of anti- cells. Furthermore, this region contains both resident DCs gen on their surface through their expression of sul- in close association with FRCs and recently migrated phated glycoproteins, such as CD169, and the lack of DCs that have collected antigen from peripheral tis- expression of the mannose receptor, which is usually sues. DCs are widely considered to be the most efficient Clodronate-loaded liposome associated with phagocytosis of opsonized antigen68. professional antigen-presenting cells and therefore are A liposome that contains the Depletion of macrophages, including those in the sub- particularly important for the presentation of peptides drug dichloromethylene capsular sinus and the medulla, through treatment with in a complex with MHC molecules to naive T cells72–74. diphosphonate. These clodronate-loaded liposomes rendered mice unable to However, B cells recognize antigen in its unprocessed liposomes are ingested by macrophages, resulting in capture and retain vesicular stomatitis virus at the sub- native state, and consequently presentation to B cells cell death. capsular sinus, resulting in systemic dissemination of would necessitate a mechanism whereby DC-accumulated nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 21 © 2009 Macmillan Publishers Limited. All rights reserved
  8. 8. REVIEWS antigen is either stably displayed on the cell surface or is FDC-mediated presentation. Classically, FDCs have resistant to intracellular degradation65,75 (TABLE 1). Indeed, been considered to be the cells that are mainly respon- it has been shown that FcγRIIB can mediate the intern- sible for antigen presentation to B cells in slOs. Early alization of antigen-containing immune complexes into autoradiography analysis combined with high-resolution non-degradative intracellular compartments65, and it electron microscopy showed that extracellular antigen in has also been suggested that DC-sIGn might allow the a plasma-membrane-associated form is retained in the accumulation of intact antigen in neutral endosomes in follicles for prolonged periods after immunization51,84. DCs. Concomitant with this hypothesis, sIGnR1 has subsequently, it was established that FDCs in the fol- been implicated in the internalization of HIv-1 into a licles are responsible for mediating the retention of anti- non-lysosomal compartment of DCs and the subsequent gen in the form of immune complexes26,85,86. In addition delivery of intact HIv-1 virions to slOs76. to their role in antigen retention and presentation, it is A population of DCs in the paracortex that can known that FDCs function as potent accessory cells dur- present intact antigen to B cells has been identified77–79. ing B-cell activation, possibly through mechanisms that A detailed characterization of this DC population by involve the secretion of chemokines and/or the expres- intravital multiphoton microscopy showed that these sion of FcγRIIB87. cells are mainly located around HEvs so that migrat- The distinctive phenotype of FDCs supports the ing B cells can survey their antigenic contents79 (FIG. 2c). retention of immune complexes in the follicles through Following the administration of antigen-loaded DCs two different mechanisms (TABLE 1). The first mechanism to mice, recently migrated antigen-specific B cells depends on the complement system85,88,89. FDCs express show decreased motility and an increase in their resi- high levels of complement receptor 1 (CR1) and CR2 dency time on DCs. Interestingly, these DCs retained (also known as CD35 and CD21, respectively), which the capacity to stimulate B cells following treatment bind to various fragments of C3. Chimeric mice that lack with pronase (a mixture of proteinases) to remove cell- expression of CR1 and CR2 in the radioresistant stromal- surface-exposed antigen, suggesting that DCs can inter- cell compartment (including FDCs) cannot deposit nalize and then recycle intact antigen to their cell surface. antigen on the surface of FDCs90,91, and FDCs from mice The DC-mediated presentation of antigen to B cells that are unable to produce CR2 ligands cannot present in the paracortex is the ideal environment in which to antigen to B cells92. receive the necessary T-cell help for their maximal acti- The second mechanism of antigen deposition involves vation79. In support of this, DCs, B cells and T cells have the retention of immune complexes that contain IgG fol- been shown to colocalize in this region of the lymph lowing their binding to Fc receptors, such as FcγRIIB, node50. This DC-mediated activation of B cells may that are expressed on the surface of FDCs in germinal give rise to extrafollicular plasma cells that mount early centres28,93. Consistent with this, FcγRIIB-deficient mice antibody responses to antigen. Alternatively, following show severely reduced trapping of immune complexes in activation by CD4+ T cells, B cells might migrate to the the lymph node and spleen, and FcγRIIB-deficient mice follicle and mediate germinal-centre formation. It will that were reconstituted with wild-type, antigen-specific also prove valuable to characterize the in vivo dynamics lymphocytes exhibit severely impaired recall responses of a population of mannose-receptor-binding DCs that to immune complexes28. accumulate soluble antigen in the follicles and present it Deposition of antigen on the surface of FDCs through Endosome to follicular B cells80. either or both of these mechanisms would therefore be A vacuolar compartment Technical constraints have limited the application expected to increase the initiation of immune responses. where large molecules are transported after being of intravital multiphoton microscopy in the study of Indeed, preformed immune complexes58,94 and antigen engulfed by endocytosis. The lymphocyte dynamics in the spleen. However, as the con- coated with C3d fragments95 are both associated with the endosome can then mature duit system in the white pulp36 and the core organization stimulation of B-cell activation, potentially as a result of and fuse with lysosomes, which of B- and T-cell zones in the spleen are similar to those increased deposition of antigen on the surface of FDCs96. contain degrading enzymes. in the lymph node, the mechanisms of antigen presen- Furthermore, following their binding to antigen, natural Endosomal and phagosomal pathways are interconnected. tation to B cells might be similar in both tissues. The IgM antibodies that are produced mainly by peritoneal splenic marginal-zone B-cell population has been shown B-1 cells97 induce the formation of immune complexes B-1 cell to rapidly produce antibody in response to blood-borne and accelerate the deposition of antigen on the surface An IgMhiIgDlowMAC1+ antigen, and therefore these cells have an important role of FDCs27. Mice with a targeted deletion in the carboxy- B220lowCD23– cell that is dominant in the peritoneal and in the initiation of early immune responses81. However, terminal tail of IgM fail to express serum IgM and show pleural cavities. B-1-precursor the participation of marginal-zone B cells at later stages delayed humoral immune responses, suggesting a role cells develop in the fetal liver of the immune response has been less well character- for FDCs in the initiation of immune responses98,99. and omentum, and in adult ized. static in vitro imaging approaches have been used Despite the ability of FDCs to present unprocessed mice the size of the B-1-cell to study DC-mediated antigen presentation to splenic antigen, the importance of this pathway for the activation population is kept constant owing to the self-renewing marginal-zone B cells82 and have shown that this activa- of naive B cells during the initiation of primary immune capacity of these cells. B-1 tion leads to the rapid generation of plasma cells that responses, as well as its underlying mechanism, have yet cells recognize self produce IgM independently of T-cell help, allowing for to be firmly established. Interestingly, it has been postu- components, as well as the rapid and enhanced formation of antigen-containing lated that FDCs may be particularly important for the common bacterial antigens, and they secrete antibodies immune complexes. Intriguingly, a population of B cells presentation of microbial antigens, in response to which that tend to have low affinity with similar characteristics to marginal-zone B cells has pre-existing natural IgM antibodies could facilitate the and broad specificity. been detected in human lymph nodes83. rapid formation of immune complexes, thereby resulting 22 | jAnuARy 2009 | vOluME 9 © 2009 Macmillan Publishers Limited. All rights reserved
  9. 9. REVIEWS in microbial-antigen presentation by Fc receptors on the to antigen retention, FDCs might have another role surface of FDCs100. However, it is known that FDCs in during affinity maturation109. However, these studies primary follicles do not express high levels of FcγRIIB28 did not exclude the possibility that competition among and therefore this receptor cannot have an important B cells for antigen could have a role in the selection of role in antigen retention and presentation during the high-affinity B-cell clones in the germinal centre. In initiation of immune responses. view of this, it would prove extremely informative to FDCs can also act as antigen ‘depots’ in the slOs characterize the spatio-temporal dynamics of cells that such that they can present antigen even after the initial retain antigen within immune complexes, which would ‘wave’ of antigen has passed26–28,101. This is important establish the precise role of deposits of antigen on FDCs for the development of an effective and long-lasting during the process of affinity maturation. immune response, as the initial encounter of antigen with naive B cells in the slOs is likely to be of low How does antigen gain access to FDCs? As a consequence affinity. under these conditions, activated B cells then of the role of FDCs in mediating antigen presentation to enter germinal centres, where they undergo affinity B cells, much effort has been invested in addressing the maturation17. This process allows the selection of B-cell apparent anomaly of how antigen in the form of immune clones with higher affinity for antigen during a T-cell- complexes can gain rapid access to FDCs, which are con- dependent antibody response and starts a few days after fined to the B-cell follicles. As the subcapsular sinus and the initiation of the response. the conduit system restrict the diffusion of large antigens The classic model that described the mechanism of from the lymphatic fluid, a cell-mediated mechanism antigen presentation to B cells in germinal centres was must operate to transport antigen to FDCs. based on several biochemical and histological observa- In the spleen, marginal-zone B cells have been impli- tions. In this model, the germinal centre was divided cated in this process59,110 (FIG. 3a). The location of B cells into two functionally distinct zones17. The dark zone in the marginal zone makes them ideally positioned was thought to contain centroblasts and to be the site to sample and mount rapid responses to blood-borne of rapid B-cell proliferation, whereas the FDC-rich light antigen. Indeed, displacement of these B cells by treat- zone functioned as the site for the selection of B-cell ment with endotoxins, such as lipopolysaccharide, was clones that express BCRs of the highest affinity follow- accompanied by impaired accumulation of immune ing somatic hypermutation. Recently, three independent complexes on the surface of FDCs in the follicles59,111,112. studies examining the spatio-temporal dynamics of Moreover, it has been shown that the accumulation of B cells have questioned the original model and the abso- IgM-containing immune complexes on FDCs was abro- lute ‘division of labour’ between the two zones of the gated in the absence of functional marginal-zone B cells. germinal centre102–104. Interestingly, each of these studies This suggests that marginal-zone B cells facilitate the showed that 6 days after antigen administration B cells adjuvant activity of IgM113. moved rapidly within the germinal centre, which indi- Marginal-zone B cells express complement receptors cated they did not make prolonged contacts with antigen (TABLE 1), and these may be important for their function presented on the surface of FDCs. It was therefore sug- in transporting antigen to FDCs. Indeed, early investi- gested that the mode of antigen recognition by B cells gations using cobra venom factor and antibodies against occurring during the later stages of an immune response C3 showed that the transport of immune complexes may operate differently to that observed during early from the marginal zone to the FDCs depends on com- antigen encounters. Indeed, these studies showed that ponents of the complement system85,89,114. In the absence germinal-centre B cells adopted an unusual morphology of CR2 expression by marginal-zone B cells, immune with many extended processes102–104. These processes complexes were associated predominantly with macro- may be responsible for mediating antigen internaliza- phages in the marginal zone, as the B cells failed to tion as the B cells migrate along the surface of the FDCs. transport antigen to the FDCs115. The mechanism by under antigen-limiting conditions, B cells expressing which marginal-zone B cells transport antigen from the Somatic hypermutation BCRs with higher affinity can accumulate more antigen marginal zone to the follicles was revealed by a recent (SHM). A unique mutation and consequently compete more effectively to recruit study involving the treatment of chimeric mice with the mechanism that is targeted specific T-cell help for their development, with only sphingosine 1-phosphate receptor 1 (s1PR1) antagonist to the variable regions of the highest affinity clones being selected for survival FTy720 (REF. 116). Marginal-zone B cells were found to rearranged immunoglobulin gene segments. Combined with in the germinal centre102,105. However, it is important to continually shuttle to and from the follicles in a process selection for B cells that note that CD4+ T cells reside exclusively within the light that depended on the expression of CXC-chemokine produce high-affinity antibody, zone, so the complete absence of functional segregation receptor 5 (CXCR5) and s1PR1, respectively. As mar- SHM leads to affinity in the germinal centre is unlikely. ginal-zone B cells can bind immune complexes through maturation of B cells in Antigen presentation by FDCs through binding of CR1 and CR2, this shuttling process could be the way germinal centres. immune complexes to FcRs or complement receptors may in which antigen is effectively delivered from the blood Cobra venom factor be important for the development of high-affinity antibody to FDCs. Interestingly, it has been postulated that the The complement-activating and long-lasting memory responses106. surprisingly, how- actual transfer of immune complexes from marginal- glycoprotein component of ever, the process of affinity maturation and the survival zone B cells to FDCs occurs through proteolysis of CR2 cobra venom, which is functionally analogous to the of memory B cells, although impaired, can occur in the on the surface of the marginal-zone B cells117. However, mammalian complement absence of detectable antigen deposition on FDCs107,108. it is not known whether marginal-zone B cells can pass factor C3b. This observation led to the hypothesis that in addition free antigen directly to follicular B cells. nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 23 © 2009 Macmillan Publishers Limited. All rights reserved
  10. 10. REVIEWS similarly, it has been suggested that follicular B cells Conclusions and perspectives themselves function as antigen transporters in the lymph Overall, it is clear that B cells can respond rapidly to nodes and spleen89,118 (FIG. 3b). This activity does not antigenic stimulation through several mechanisms, and depend on antigen-specific recognition and therefore they also have a unique strategic role in the transport is mediated by receptors other than the BCR (TABLE 1). of antigen to FDCs in the germinal centre. This wide It has been proposed that antigen can be acquired by variety in potential antigen-presentation mechanisms is other follicular B cells in the absence of antigen-specific promoted by functional specialization within slOs and B cells64,67, and this may depend on the expression of provides invaluable flexibility in mounting appropriate complement receptors on the B-cell surface64. As FDCs immune responses to antigens. As such, the mechanism express higher levels of complement receptors than that is used to present a specific antigen can be effec- follicular B cells, it would be expected that they could tively tailored both to the antigen itself and to the way compete effectively with B cells for antigen binding 27. the antigen is delivered to the B cell. The predominance It remains to be determined whether other receptors of of cell-based strategies for the presentation of antigen to the innate immune system have a role in the transport B cells allows for extensive regulation and coordination of antigen by B cells within slOs. CD23+ mature B cells of the resulting immune responses. have in fact been shown to mediate the transport of In spite of significant recent progress in character- IgE-containing immune complexes into the follicle in a izing the sites and dynamics of antigen presentation BCR-independent manner 119. Although such a situation to B cells in vivo, some issues remain to be addressed. is unlikely to occur in physiological conditions, it does These include a definitive molecular description of the indicate that alternative receptors might be involved in mechanisms of antigen presentation to B cells, includ- antigen transport. ing the retention and internalization strategies that are a Splenic marginal-zone B cells transport antigen to FDCs b Follicular B cells in the lymph node transport antigen to FDCs S1Phi Immune Marginal zone Subcapsular sinus CXCL13low complex SCS macrophage C3 fragment CR Antigen FcR non-specific DC-SIGN BCR Marginal-zone B cell Follicular B cell ? ? Follicular dendritic cell S1Plow CXCL13hi Primary follicle Primary follicle Figure 3 | B cells mediate antigen transport to follicular dendritic cells using complement receptors. a | Marginal-zone B cells in the spleen can bind to immune complexes that contain antigen and are coated in complement fragments, using complement receptors (CR) in a manner that is independent of B-cell receptor (BCR) specificity. These Nature Reviews | Immunology marginal-zone B cells can shuttle to the follicular region, which is rich in CXC-chemokine ligand 13 (CXCL13), in a CXC- chemokine-receptor-5-dependent manner116. Follicular dendritic cells (FDCs) in the follicle, which express high levels of CR, can then compete for binding to antigen that is presented by marginal-zone B cells. The marginal-zone B cells then migrate back to the marginal zone, where there are high levels of sphingosine 1-phosphate (S1P), and this depends on their expression of the receptors for S1P1 and S1P3. b | Follicular B cells in the lymph node can bind to particulate antigen on the surface of subcapsular sinus (SCS) macrophages. This binding does not depend on the specificity of the BCR and is mediated by CRs. It has been suggested that these follicular B cells can then mediate the transport of antigen to FDCs, which express higher levels of CRs64,67. C3, complement component 3; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; FcR, Fc receptor; ICAM3, intercellular adhesion molecule 3. 24 | jAnuARy 2009 | vOluME 9 © 2009 Macmillan Publishers Limited. All rights reserved