REVIEWS

The immune system is highly dynamic and diverse, which enables it to effectively protect an individual from numer-ous potentially pathogenic encounters. Early studies that combined techniques such as electron microscopy and immunofluorescence yielded valuable information on the static location and organization of the cells of the immune system, and inferred their dynamic properties (for a historical perspective and references to original articles see REFS 1–3). However, it is necessary to consider the development of immune responses in terms of both intracellular and intercellular interactions in the appro-priate environment and in real time4–8. Indeed, the recent development of high-resolution imaging techniques, including confocal microscopy, total internal reflection fluorescence microscopy and intravital multiphoton micro-scopy, has provided insights into the spatio-temporal dynamics of the molecular and cellular events that underlie immune responses to pathogenic infection. One example of such an insight is the molecular description of the immunological synapse, a structure that is com-monly associated with lymphocyte activation following the recognition of a specific antigen9–12 (BOX 1).

B-cell activation is initiated following engagement of the B-cell receptor (BCR) by a specific antigen. B cells can recognize and respond to both soluble and membrane-associated antigen, although recent insights suggest that membrane-associated antigens are more important for B-cell activation in vivo13,14. Following antigenic stimulation, B cells can process

and present antigen in association with MHC class II molecules, thereby recruiting specific CD4+ T-cell help and stimulating B-cell proliferation and dif-ferentiation15,16 (BOX 2). Although the precise factors that determine the fate of activated B cells currently remain unclear, B cells can differentiate along two distinct pathways. On the one hand, B cells can dif-ferentiate to form extrafollicular plasmablasts that are essential for rapid antibody production and early protective immune responses. On the other hand, activated B cells can enter germinal centres, where they can differentiate into plasma cells, which can secrete high-affinity antibody following affinity maturation, or memory B cells, which confer long-lasting protection from secondary challenge with antigen17,18.

In this Review, we discuss recent insights into the sites at which B cells encounter antigen in vivo, as well as the mechanisms by which these interactions occur (for excellent reviews that include comprehensive descriptions of antigen presentation to T cells in vivo, see REFS 2,19–21). It is now clear that B cells can encounter and respond to antigen through many dif-ferent mechanisms depending on the nature and size of the antigen itself, as well as on the cellular context and location in which antigen presentation occurs. This provides great versatility in terms of initiating responses that are appropriate to the particular antigen and are therefore the most effective for the protection of the host.

Lymphocyte Interaction Laboratory, Cancer Research UK London Research Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK.Correspondence to F.D.B.e‑mail: [email protected]:10.1038/nri2454Published online 12 December 2008

Total internal reflection fluorescence microscopy A microscopy method that allows for the identification of fluorescence within 100–200 nm of the interface between cells and their substrate (for example, lipid bilayers or glass coverslips), thereby providing a high lateral and axial resolution at the cell–substrate interface and the ability to observe nanoscale movement of signalling molecules during cellular activation.

The who, how and where of antigen presentation to B cellsFacundo 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.

REVIEWS

nATuRE REvIEws | Immunology vOluME 9 | jAnuARy 2009 | 15

© 2009 Macmillan Publishers Limited. All rights reserved

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Intravital multiphoton microscopyA microscopy method that combines the advanced optical techniques of laser-scanning confocal microscopy with long-wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of living cells and/or tissues that have been labelled with fluorophores. It provides a greater tissue imaging depth (up to 350 µm depending on the tissue) and less photobleaching and phototoxicity than conventional imaging methods.

Germinal centre A highly specialized and dynamic microenvironment that gives rise to secondary B-cell follicles during an immune response. It is the main site of B-cell maturation, during which memory B cells and plasma cells that produce high-affinity antibody are generated.

Plasma cellA non-dividing, terminally differentiated, antibody- secreting cell of the B-cell lineage.

Memory B cellAn antigen-experienced B cell that expresses high-affinity antibodies and quickly differentiates into a plasma cell during antigen-recall responses.

Alymphoplastic (aly/aly) miceMice that are characterized by the absence of lymph nodes and Peyer’s patches. Alymphoplasia is caused by a spontaneous mutation in the gene that encodes nuclear-factor-κB-inducing kinase.

Sites of antigen encounterAs cells of the immune system can respond to a huge range of potential pathogenic challenges throughout the whole body, these responses must be tightly coordinated to confer effective protection. Owing to this great anti-genic diversity, the event of antigen encounter with an antigen-specific lymphocyte that can mount a rapid and appropriate response would seem unlikely. To maximize the probability of such an interaction, these events occur in defined sites, such as the lymph nodes and spleen, which are collectively known as secondary lymphoid organs (slOs). These sites possess a highly organized microarchitecture that is necessary for the compartmen-talization of numerous cellular interactions and there-fore provide the optimal environment for the initiation of immune responses, including the determination of cell fate22. Furthermore, within 24 hours lymphocytes that have been unsuccessful in their search for antigen recirculate throughout the body and between slOs to dramatically increase the probability of encountering cognate antigen3.

slOs are extremely well connected to the blood and lymphatic systems, allowing them to continually sample and concentrate antigens that are circulating through-out the body. The importance of slOs in organizing and coordinating immune responses is evident from the severe impairment in the activation of naive lym-phocytes in alymphoplastic (aly/aly) mice and asplenic (homebox 11-deficient) mice23. Accordingly, any mean-ingful investigation into the spatio-temporal dynamics and the mechanisms by which lymphocytes encounter antigen in vivo must be considered within such highly organized environments.

Lymph nodes. It is now widely understood that beneath the protective outer collagenous capsule, a lymph node is divided into three discrete (but non-rigid) regions that are defined by the expression of specific chemokines3,24 (FIG. 1a). Directly beneath the subcapsular sinus is a macrophage-rich sheet that sur-rounds the B-cell zone, which is also termed the cor-tex. B cells in this region are organized into aggregates,

Box 1 | The immunological synapse

Lymphocyte activation is initiated following the recognition of specific antigen by clonotypic immunoreceptors, such as the B‑cell receptor (BCR) and the T‑cell receptor (TCR). The recognition of specific antigen on a spatially constrained 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 a central supramolecular activation cluster (cSMAC) from the surrounding peripheral SMAC (pSMAC), which contains integrins, such as lymphocyte function‑associated antigen 1 (LFA1) (see the figure). The early molecular events that underlie the formation of the immunological synapse have been revealed by high‑resolution imaging techniques and are highly coordinated and tightly regulated. Following the recognition of specific antigen and before the formation of the immunological synapse, B cells rapidly spread over the antigen‑containing surface in a manner that is dependent on intracellular signalling and cytoskeletal rearrangements120. During B‑cell spreading, antigen–BCR microclusters are continually assembled at the periphery of the contact area between the cells14. As similar microclusters are crucial for sustained signalling in T cells121–123, we have proposed that these microclusters form functional signalling units that are common to both types of lymphocyte14.

A comprehensive genetic dissection of the requirements for B‑cell spreading identified an important role for the sequential recruitment of the kinases LYN and spleen tyrosine kinase (SYK) to antigen–BCR microclusters in the initiation of BCR‑induced signalling124. Furthermore, this study visualized the formation of microsignalosomes through the recruitment of 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

has been further supported through fluorescence resonance energy transfer (FRET)‑based assays125. B‑cell spreading facilitates the propagation of signalling through microclusters by allowing the BCR to engage more antigen, which promotes further localized signalling and cytoskeleton rearrangements120. The spreading response is followed by a prolonged contraction phase, which results in the accumulation of antigen–BCR microclusters in the cSMAC of the mature immunological synapse. The cSMAC functions as a site for internalization of antigen12,120, before it is processed and presented in association with MHC class II molecules; presentation of antigen–MHC molecules mediates the 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; FcR, Fc receptor.

Nature Reviews | Immunology

APC

Antigen

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Antibody

B cellMicrosignalosome

APC

B cell

B-cell contact area with APC (APC not shown)

Mature immunological synapse:top view

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Antigen–BCR microcluster: side view

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T-cell-dependent antigenA protein antigen that needs to be recognized by T helper cells (in the context of MHC molecules) and requires cooperation between these antigen-specific T cells and B cells for a specific antibody response to be generated.

Affinity maturation A process whereby the mutation of antibody variable-region genes followed by selection for higher-affinity variants in the germinal centre leads to an increase in average antibody affinity for an antigen as an immune response progresses. The selection is thought to be a competitive process in which B cells compete with free antibody to capture decreasing amounts of antigen.

µmt−/− miceA strain of mutant mice that carry a stop codon in the first membrane exon of the µ-chain constant region. They lack IgM+ B cells, and B-cell development is arrested before the differentiation stage at which IgD can be expressed.

known as follicles, and are the largest population of IgMmedIgDhiCD21medCD23hi B cells in the body. Follicles are also rich in radiation-resistant follicular dendritic cells (FDCs) that express high levels of the adhesion molecules vascular cell-adhesion molecule 1 (vCAM1) and intercellular adhesion molecule 1 (ICAM1), as well as complement and Fc receptors25–28. FDCs are thought to be of mesenchymal origin and thus form a popu-lation of cells that is distinct from classic DCs29. The precise mechanism of FDC development is not yet fully understood, but is known to require the expres-sion of various chemokine receptors and the presence of B cells17,30. Following exposure to antigen, follicles may also contain specialized structures, known as germinal centres, which consist of rapidly proliferat-ing B cells within a network of FDCs. The formation of germinal centres is important during the develop-ment of humoral immune responses to T-cell-dependent antigens, as they serve as sites for affinity maturation and the generation of long-lasting memory B cells.

The T-cell zone, or paracortex, which is adjacent to the follicles, contains high endothelial venules (HEvs) and numerous antigen-presenting cells. The HEvs are specialized capillaries that continually supply the lymph node with and drain it of lymphocytes from and to the periphery. Furthermore, HEvs in the paracortex can often mediate the recruitment of cells other than lymphocytes, such as antigen-presenting cells that have accumulated antigen in the periphery. The paracortex also contains a network of collagenous conduit fibres that allow the passage of low-molecular-mass components of lymphatic fluid, including chemokines, from the subcapsular sinus to HEvs (reviewed in REF. 31).

The medulla, which is the innermost area of the lymph node, contains both B and T cells that are organ-ized into medullary cords and is also rich in DCs and macrophages.

lymph nodes are strategically positioned at branches throughout the lymphatic system to enable extensive antigenic sampling of lymphatic fluid4. lymphatic fluid, which contains various chemokines and antigens that have been collected from the body, enters the lymph node through the afferent lymph vessel and is subse-quently filtered by the lymph node as it flows towards the efferent lymph vessel. lymphatic fluid is prevented from freely diffusing into the interior when it enters the lymph node by the subcapsular sinus; instead, it travels to the medulla through structures known as trabecular sinuses. In addition, components of lym-phatic fluid that have a molecular weight of below ~70 kDa (equivalent to a dynamic radius of ~5.5 nm) are allowed to move towards the HEv through the intri-cate meshwork of fibroblastic reticular cell (FRC)-lined collagenous conduits that are associated with DCs32,33 (FIG. 1b). Accordingly, early observations indicated that the paracortex did not contain unprocessed antigen, although some antigen could be detected in DCs34. The lymphatic fluid that exits the lymph node is enriched with lymphocytes35 and therefore provides a means for these cells to return to the circulation24.

Spleen. The organization of the lymphoid tissue of the spleen is similar to that of the lymph node, with B-cell follicles and T-cell zones that contain networks of retic-ular cells (also known as the periarteriolar lymphoid 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 µmt–/– 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 conjugates in the generation of sustained immunity has recently been highlighted through the investigation 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 probably low, this mechanism of antigen presentation to T cells by B cells contributes to the initiation of T‑cell responses in vivo.


Follicle

Follicle

T-cell zone

SCS

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7412&ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


T-cell-independent antigenAn antigen that directly activates B cells.

white pulp37 (FIG. 1a). The spleen also contains red pulp, which is richly supplied with blood and contains various antigen-presenting cells, lymphocytes and plasma cells38. The outer limit of the white pulp is separated from the red pulp by a region known as the marginal zone39. This area has extensive vasculature, and blood that enters the spleen through follicular arteriole branches in the marginal sinus reaches the marginal zone before its permeation through the red pulp40. The marginal zone also contains numerous macrophages and DCs, and a sizable population of non-recirculating B cells41.

These marginal-zone B cells form a population of cells that is distinct from follicular B cells, a large propor-tion of which are polyreactive clones. Marginal-zone B cells have an IgMhiIgDlowCD21hiCD23low phenotype and are thought to participate in the development of early immune responses to both T-cell-dependent and T-cell-independent antigens42. In contrast to lymph nodes, the spleen is not supplied with afferent lymphatic fluid (although it does contain efferent lymphatics); instead, it is specialized in mounting immune responses to blood-borne antigens43.


Subcapsular sinus

Afferent lymph

Efferent lymph

HEV

Follicle

Arteriolebranch

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Conduit networkConduit network

Conduit network

a

b c

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Collagenbundles

FRC Associated DC

Red pulp

White pulp

Central arteriole

T-cell zoneFollicle

Follicle

SCS

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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 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.

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Lymphocyte behaviour in ‘resting’ SLOsTo establish the sites and mechanisms of antigen encoun-ter with lymphocytes in vivo, it is important to understand the localization and dynamics of these cells in the resting lymphoid organs. Although information on cell dynam-ics in the past was inferred from studies using static approaches1–3, initial groundbreaking studies that directly visualized the spatio-temporal dynamics of lymphocytes in vivo were possible because of advances in the field of multiphoton microscopy (reviewed in REFS 5,19). This method allows for the investigation of cellular behaviour in real time and to a depth in living tissue that is prohib-ited by techniques such as confocal microscopy5. The ear-liest multiphoton microscopy investigations were carried out on explanted or isolated lymph nodes44–46, and many of the original observations have since been confirmed in lymph nodes in vivo following minimal surgical disrup-tion. However, high-resolution imaging of lymphocyte behaviour in splenic tissue is hampered by the technical challenges that are associated with the isolation of this vital and deeply embedded organ6.

The first surprising observation from these multi-photon microscopy studies was the highly motile nature of lymphocytes within the resting lymph node45–48. B and T cells that were highly polarized in shape were found to move within their restricted cellular zones (FIG. 1c) but along apparently random trajectories, with average velocities of around 6 µm (for B cells) and 12 µm (for T cells) per minute. Closer inspection of this movement fol-lowing the visualization of the conduit network provided evidence for an order to this process. using an elegant system involving chimeric mice that express green fluo-rescent protein, Bajénoff et al.49 could visualize the FDCs and FRCs in the lymph node before the introduction of a population of labelled lymphocytes. This study showed that the T cells in the paracortex are in close association with FRCs, which allows the T cells to be guided along a defined network. As B cells enter the lymph node through HEvs in the paracortex, it was also suggested that these cells could migrate along FRCs in this area before migrating in a chemokine-directed manner to the follicles, where a similar network composed of FDCs can be used to orchestrate their movement. such rapid move-ment across these networks suggests that lymphocytes in the lymph node are continually scanning their environ-ment and are ready to respond to antigen as soon as it is encountered. Interestingly, similar investigations have offered insight into the behaviour of CD11c+ DCs in the paracortex50. In contrast to lymphocytes, and as expected given their potential role in structural support of the con-duit network, these DCs were found to be sessile, show-ing movement at a velocity of less than 2 µm per minute. However, this restriction in motility did not prevent the rapid movement of their dendritic processes, indicating that these DCs retain the capacity to sample and respond to environmental signals.

Presentation of antigen to B cellsB cells can promptly detect and mount responses to antigen after immunization. In the case of small soluble antigens, responses can be mounted following a simple

diffusion of antigen into the lymphoid tissue; how-ever, these encounters are usually mediated through macrophages, DCs and FDCs (FIG. 2).

Encountering of soluble antigen. Antigen can be rap-idly supplied to the lymph node through the afferent lymph vessels. Indeed, antigen arrival at the subcapsu-lar sinus can be detected within minutes of subcutane-ous administration51. As most B cells are mainly located in the follicles, lymph-borne antigens must gain access to follicular B cells in a manner that is independent of the conduit system, which is present predominantly in the paracortex.

Interestingly, several electron microscopy stud-ies have identified pores of ~0.1–1 µm diameter in the regions of the subcapsular sinus that are adjacent to the lymph-node parenchyma52–54. These pores might allow small soluble antigen, such as low-molecular-mass toxins, that enter the lymph node through the afferent lymph vessel to directly pass into the follicle and gain access to B cells55 (FIG. 2a). Indeed, using immunohisto-chemistry of frozen lymph-node sections, it was shown that this rapid diffusion of antigen was independent of DCs and did not require migration of B cells from their follicular location. These observations are consistent with a previous study, which showed that immuniza-tion of mice with soluble antigen initially resulted in a substantial decrease in the motility of antigen-specific B cells in the follicle48. Furthermore, in the 24 hours following immunization these B cells were shown to present antigen in association with MHC class II mol-ecules on their surface (BOX 2) and to migrate towards the boundary between the follicle and the paracortex. Therefore, small low-molecular-mass antigens may gain access to cells in the follicles without requiring cell-mediated presentation, through direct diffusion from pores in the subcapsular sinus. However the existence of these pores remains controversial, as they may simply be sites generated by cells that have recently migrated through the sinus wall. Furthermore, there is a discrep-ancy between the dimensions of the observed pores and the radius of the antigens that have been excluded from accessing the follicle by diffusion. In view of this, it seems reasonable to postulate the existence of an alter-native means of mediating the movement of smaller antigens into the follicles, perhaps in a manner that is reminiscent of diffusion from the conduit system, as observed in the paracortex.

Macrophage-mediated presentation. Although diffu-sion of antigen from the subcapsular sinus provides one mechanism for exposing follicular B cells to small soluble antigens, the access of larger particulate antigens to B cells in the follicle is limited. It was therefore suggested that specialized cells, possibly located in the subcapsular sinus, might transport large antigens to the B-cell follicles53,56–58. Early microscopy studies revealed a population of macro-phages that reside directly beneath the subcapsular sinus52,53 and can extend their processes through it to gain access to afferent lymphatic fluid53,56,58. As these macro-phages have been shown to bind a soluble recombinant

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Metallophilic macrophageA macrophage that is located at the border of the white pulp and the marginal zone of the spleen. These macrophages express high levels of CD169 but lack expression of the mannose receptor.

protein that contains domains of the mannose receptor, it has been suggested that they may capture and concen-trate antigen in a similar way to metallophilic macrophages that reside in the splenic marginal zone59–61.

The macrophages in the subcapsular sinus are a dis-tinct population to those in the medulla and have lim-ited phagocytic activity, which enables them to present

intact antigen on their cell surface56,58. Indeed, these macrophages have been shown to capture and retain antigen for up to 72 hours following the initial antigen exposure62. However, whether this involves the simple retention of antigen on the macrophage surface or anti-gen internalization into non-degradative intracellular compartments before it is recycled to the cell surface


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DC-SIGN

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FcR

AntibodyImmune complex

C3 fragmentAntigen

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Antigen non-specific B cells

MAC1

CR

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 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.

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Clodronate-loaded liposomeA liposome that contains the drug dichloromethylene diphosphonate. These liposomes are ingested by macrophages, resulting in cell death.

is not clear. In addition, macrophages are known to express a wide range of cell-surface receptors that could participate in the presentation of unprocessed antigen, including complement receptors, pattern-recognition receptors and/or carbohydrate-binding scavenger receptors63 (TABLE 1). Indeed, macrophage receptor 1 (MAC1; also known as αMβ2 integrin and CD11b–CD18 dimer), which is a receptor for complement component 3 (C3) that is expressed by macrophages, has been suggested to contribute to the retention of antigen on the cell surface64. Alternatively, the inhibitory low-affinity receptor for IgG (FcγRIIB) might mediate the internalization and recycling of IgG-containing immune complexes to the macrophage cell surface, as has been shown in DCs65. Finally, the C-type lectin DC-specific ICAM3-grabbing non-integrin (DC-sIGn; also known as CD209) could participate in the retention of glycosylated antigens, which is con-sistent with the observation that mice deficient in the mouse homologue of DC-sIGn, sIGnR1, fail to mount humoral immune responses following infection with Streptococcus pneumoniae66.

A crucial and newly described role for subcapsular-sinus macrophages in the presentation of antigen to follicular B cells has been recently established by three independent studies64,67,68. These studies identified that, soon after antigen administration, subcapsular-sinus macrophages are responsible for the rapid accumula-tion of various larger antigens, such as those found in immune complexes (with antibody and/or complement fragments), particulates, bacteria and viruses (FIG. 2a,b). These macrophages might favour the retention of anti-gen on their surface through their expression of sul-phated glycoproteins, such as CD169, and the lack of expression of the mannose receptor, which is usually associated with phagocytosis of opsonized antigen68. Depletion of macrophages, including those in the sub-capsular sinus and the medulla, through treatment with clodronate-loaded liposomes rendered mice unable to capture and retain vesicular stomatitis virus at the sub-capsular sinus, resulting in systemic dissemination of

this virus68. Furthermore, as the retention of antigen was not impaired following the depletion of C3, complement-independent mechanisms might also contribute to the maintenance of antigen on the macrophage surface68.

The accumulated antigen is subsequently presented in its intact form by macrophages for recognition by neighbouring follicular B cells. Accordingly, following administration of antigen, it was shown that antigen-specific B cells in the follicle exhibited reduced migration over time and made stable contacts with macrophages in the subcapsular sinus64,67,68. As ICAM1 and vCAM1 are expressed in the subcapsular sinus68, it is expected that they may facilitate B-cell adhesion in this region and thereby lower the threshold of antigen that is required for B-cell activation13,69. Together, these studies provide the first clear demonstration of a role for macrophages in the initiation of follicular B-cell responses. The macro-phage–B-cell interactions at the subcapsular sinus allow antigen-specific B cells to acquire and internalize antigen through their BCR before their migration to the B-cell–T-cell boundary67, where they may receive specific T-cell help48,70 (BOX 2). Interestingly, it has recently been noted that the response of B cells and CD4+ T cells to large viral particles is deterministically linked to their antigen spe-cificities71, suggesting that antigen uptake by the B cell may not involve internalization of whole viral pathogens at the subcapsular sinus.

DC-mediated presentation. B cells enter the lymph node through the HEvs, so the paracortex would seem the ideal site for the immediate presentation of antigen to these cells. Furthermore, this region contains both resident DCs in close association with FRCs and recently migrated DCs that have collected antigen from peripheral tis-sues. DCs are widely considered to be the most efficient professional antigen-presenting cells and therefore are particularly important for the presentation of peptides in a complex with MHC molecules to naive T cells72–74. However, B cells recognize antigen in its unprocessed native state, and consequently presentation to B cells would necessitate a mechanism whereby DC-accumulated

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


Marginal-zone B cell

CR1 and CR2 Complement-coated antigen Remains on the cell surface

Follicular B cell CR1 and CR2 Complement-coated antigen Remains on the cell surface


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.

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Endosome A vacuolar compartment where large molecules are transported after being engulfed by endocytosis. The endosome can then mature and fuse with lysosomes, which contain degrading enzymes. Endosomal and phagosomal pathways are interconnected.

B-1 cellAn IgMhiIgDlowMAC1+

B220lowCD23– cell that is dominant in the peritoneal and pleural cavities. B-1-precursor cells develop in the fetal liver and omentum, and in adult mice the size of the B-1-cell population is kept constant owing to the self-renewing capacity of these cells. B-1 cells recognize self components, as well as common bacterial antigens, and they secrete antibodies that tend to have low affinity and broad specificity.

antigen is either stably displayed on the cell surface or is resistant to intracellular degradation65,75 (TABLE 1). Indeed, it has been shown that FcγRIIB can mediate the intern-alization of antigen-containing immune complexes into non-degradative intracellular compartments65, and it has also been suggested that DC-sIGn might allow the accumulation of intact antigen in neutral endosomes in DCs. Concomitant with this hypothesis, sIGnR1 has been implicated in the internalization of HIv-1 into a non-lysosomal compartment of DCs and the subsequent delivery of intact HIv-1 virions to slOs76.

A population of DCs in the paracortex that can present intact antigen to B cells has been identified77–79. A detailed characterization of this DC population by intravital multiphoton microscopy showed that these cells are mainly located around HEvs so that migrat-ing B cells can survey their antigenic contents79 (FIG. 2c). Following the administration of antigen-loaded DCs to mice, recently migrated antigen-specific B cells show decreased motility and an increase in their resi-dency time on DCs. Interestingly, these DCs retained the capacity to stimulate B cells following treatment with pronase (a mixture of proteinases) to remove cell- surface-exposed antigen, suggesting that DCs can inter-nalize and then recycle intact antigen to their cell surface. The DC-mediated presentation of antigen to B cells in the paracortex is the ideal environment in which to receive the necessary T-cell help for their maximal acti-vation79. In support of this, DCs, B cells and T cells have been shown to colocalize in this region of the lymph node50. This DC-mediated activation of B cells may give rise to extrafollicular plasma cells that mount early antibody responses to antigen. Alternatively, following activation by CD4+ T cells, B cells might migrate to the follicle and mediate germinal-centre formation. It will also prove valuable to characterize the in vivo dynamics of a population of mannose-receptor-binding DCs that accumulate soluble antigen in the follicles and present it to follicular B cells80.

Technical constraints have limited the application of intravital multiphoton microscopy in the study of lympho cyte dynamics in the spleen. However, as the con-duit system in the white pulp36 and the core organization of B- and T-cell zones in the spleen are similar to those in the lymph node, the mechanisms of antigen presen-tation to B cells might be similar in both tissues. The splenic marginal-zone B-cell population has been shown to rapidly produce antibody in response to blood-borne antigen, and therefore these cells have an important role in the initiation of early immune responses81. However, the participation of marginal-zone B cells at later stages of the immune response has been less well character-ized. static in vitro imaging approaches have been used to study DC-mediated antigen presentation to splenic marginal-zone B cells82 and have shown that this activa-tion leads to the rapid generation of plasma cells that produce IgM independently of T-cell help, allowing for the rapid and enhanced formation of antigen-containing immune complexes. Intriguingly, a population of B cells with similar characteristics to marginal-zone B cells has been detected in human lymph nodes83.

FDC-mediated presentation. Classically, FDCs have been considered to be the cells that are mainly respon-sible for antigen presentation to B cells in slOs. Early autoradiography analysis combined with high-resolution electron microscopy showed that extracellular antigen in a plasma-membrane-associated form is retained in the follicles for prolonged periods after immunization51,84. subsequently, it was established that FDCs in the fol-licles are responsible for mediating the retention of anti-gen in the form of immune complexes26,85,86. In addition to their role in antigen retention and presentation, it is known that FDCs function as potent accessory cells dur-ing B-cell activation, possibly through mechanisms that involve the secretion of chemokines and/or the expres-sion of FcγRIIB87.

The distinctive phenotype of FDCs supports the retention of immune complexes in the follicles through two different mechanisms (TABLE 1). The first mechanism depends on the complement system85,88,89. FDCs express high levels of complement receptor 1 (CR1) and CR2 (also known as CD35 and CD21, respectively), which bind to various fragments of C3. Chimeric mice that lack expression of CR1 and CR2 in the radioresistant stromal- cell compartment (including FDCs) cannot deposit antigen on the surface of FDCs90,91, and FDCs from mice that are unable to produce CR2 ligands cannot present antigen to B cells92.

The second mechanism of antigen deposition involves the retention of immune complexes that contain IgG fol-lowing their binding to Fc receptors, such as FcγRIIB, that are expressed on the surface of FDCs in germinal centres28,93. Consistent with this, FcγRIIB-deficient mice show severely reduced trapping of immune complexes in the lymph node and spleen, and FcγRIIB-deficient mice that were reconstituted with wild-type, antigen-specific lymphocytes exhibit severely impaired recall responses to immune complexes28.

Deposition of antigen on the surface of FDCs through either or both of these mechanisms would therefore be expected to increase the initiation of immune responses. Indeed, preformed immune complexes58,94 and antigen coated with C3d fragments95 are both associated with the stimulation of B-cell activation, potentially as a result of increased deposition of antigen on the surface of FDCs96. Furthermore, following their binding to antigen, natural IgM antibodies that are produced mainly by peritoneal B-1 cells97 induce the formation of immune complexes and accelerate the deposition of antigen on the surface of FDCs27. Mice with a targeted deletion in the carboxy- terminal tail of IgM fail to express serum IgM and show delayed humoral immune responses, suggesting a role for FDCs in the initiation of immune responses98,99.

Despite the ability of FDCs to present unprocessed antigen, the importance of this pathway for the activation of naive B cells during the initiation of primary immune responses, as well as its underlying mechanism, have yet to be firmly established. Interestingly, it has been postu-lated that FDCs may be particularly important for the presentation of microbial antigens, in response to which pre-existing natural IgM antibodies could facilitate the rapid formation of immune complexes, thereby resulting

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Somatic hypermutation (SHM). A unique mutation mechanism that is targeted to the variable regions of rearranged immunoglobulin gene segments. Combined with selection for B cells that produce high-affinity antibody, SHM leads to affinity maturation of B cells in germinal centres.

Cobra venom factorThe complement-activating glycoprotein component of cobra venom, which is functionally analogous to the mammalian complement factor C3b.

in microbial-antigen presentation by Fc receptors on the surface of FDCs100. However, it is known that FDCs in primary follicles do not express high levels of FcγRIIB28 and therefore this receptor cannot have an important role in antigen retention and presentation during the initiation of immune responses.

FDCs can also act as antigen ‘depots’ in the slOs such that they can present antigen even after the initial ‘wave’ of antigen has passed26–28,101. This is important for the development of an effective and long-lasting immune response, as the initial encounter of antigen with naive B cells in the slOs is likely to be of low affinity. under these conditions, activated B cells then enter germinal centres, where they undergo affinity maturation17. This process allows the selection of B-cell clones with higher affinity for antigen during a T-cell-dependent antibody response and starts a few days after the initiation of the response.

The classic model that described the mechanism of antigen presentation to B cells in germinal centres was based on several biochemical and histological observa-tions. In this model, the germinal centre was divided into two functionally distinct zones17. The dark zone was thought to contain centroblasts and to be the site of rapid B-cell proliferation, whereas the FDC-rich light zone functioned as the site for the selection of B-cell clones that express BCRs of the highest affinity follow-ing somatic hypermutation. Recently, three independent studies examining the spatio-temporal dynamics of B cells have questioned the original model and the abso-lute ‘division of labour’ between the two zones of the germinal centre102–104. Interestingly, each of these studies showed that 6 days after antigen administration B cells moved rapidly within the germinal centre, which indi-cated they did not make prolonged contacts with antigen presented on the surface of FDCs. It was therefore sug-gested that the mode of antigen recognition by B cells occurring during the later stages of an immune response may operate differently to that observed during early antigen encounters. Indeed, these studies showed that germinal-centre B cells adopted an unusual morphology with many extended processes102–104. These processes may be responsible for mediating antigen internaliza-tion as the B cells migrate along the surface of the FDCs. under antigen-limiting conditions, B cells expressing BCRs with higher affinity can accumulate more antigen and consequently compete more effectively to recruit specific T-cell help for their development, with only the highest affinity clones being selected for survival in the germinal centre102,105. However, it is important to note that CD4+ T cells reside exclusively within the light zone, so the complete absence of functional segregation in the germinal centre is unlikely.

Antigen presentation by FDCs through binding of immune complexes to FcRs or complement receptors may be important for the development of high-affinity antibody and long-lasting memory responses106. surprisingly, how-ever, the process of affinity maturation and the survival of memory B cells, although impaired, can occur in the absence of detectable antigen deposition on FDCs107,108. This observation led to the hypothesis that in addition

to antigen retention, FDCs might have another role during affinity maturation109. However, these studies did not exclude the possibility that competition among B cells for antigen could have a role in the selection of high-affinity B-cell clones in the germinal centre. In view of this, it would prove extremely informative to characterize the spatio-temporal dynamics of cells that retain antigen within immune complexes, which would establish the precise role of deposits of antigen on FDCs during the process of affinity maturation.

How does antigen gain access to FDCs? As a consequence of the role of FDCs in mediating antigen presentation to B cells, much effort has been invested in addressing the apparent anomaly of how antigen in the form of immune complexes can gain rapid access to FDCs, which are con-fined to the B-cell follicles. As the subcapsular sinus and the conduit system restrict the diffusion of large antigens from the lymphatic fluid, a cell-mediated mechanism must operate to transport antigen to FDCs.

In the spleen, marginal-zone B cells have been impli-cated in this process59,110 (FIG. 3a). The location of B cells in the marginal zone makes them ideally positioned to sample and mount rapid responses to blood-borne antigen. Indeed, displacement of these B cells by treat-ment with endotoxins, such as lipopolysaccharide, was accompanied by impaired accumulation of immune complexes on the surface of FDCs in the follicles59,111,112. Moreover, it has been shown that the accumulation of IgM-containing immune complexes on FDCs was abro-gated in the absence of functional marginal-zone B cells. This suggests that marginal-zone B cells facilitate the adjuvant activity of IgM113.

Marginal-zone B cells express complement receptors (TABLE 1), and these may be important for their function in transporting antigen to FDCs. Indeed, early investi-gations using cobra venom factor and antibodies against C3 showed that the transport of immune complexes from the marginal zone to the FDCs depends on com-ponents of the complement system85,89,114. In the absence of CR2 expression by marginal-zone B cells, immune complexes were associated predominantly with macro-phages in the marginal zone, as the B cells failed to transport antigen to the FDCs115. The mechanism by which marginal-zone B cells transport antigen from the marginal zone to the follicles was revealed by a recent study involving the treatment of chimeric mice with the sphingosine 1-phosphate receptor 1 (s1PR1) antagonist FTy720 (REF. 116). Marginal-zone B cells were found to continually shuttle to and from the follicles in a process that depended on the expression of CXC-chemokine receptor 5 (CXCR5) and s1PR1, respectively. As mar-ginal-zone B cells can bind immune complexes through CR1 and CR2, this shuttling process could be the way in which antigen is effectively delivered from the blood to FDCs. Interestingly, it has been postulated that the actual transfer of immune complexes from marginal-zone B cells to FDCs occurs through proteolysis of CR2 on the surface of the marginal-zone B cells117. However, it is not known whether marginal-zone B cells can pass free antigen directly to follicular B cells.

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similarly, it has been suggested that follicular B cells themselves function as antigen transporters in the lymph nodes and spleen89,118 (FIG. 3b). This activity does not depend on antigen-specific recognition and therefore is mediated by receptors other than the BCR (TABLE 1). It has been proposed that antigen can be acquired by other follicular B cells in the absence of antigen-specific B cells64,67, and this may depend on the expression of complement receptors on the B-cell surface64. As FDCs express higher levels of complement receptors than follicular B cells, it would be expected that they could compete effectively with B cells for antigen binding27. It remains to be determined whether other receptors of the innate immune system have a role in the transport of antigen by B cells within slOs. CD23+ mature B cells have in fact been shown to mediate the transport of IgE-containing immune complexes into the follicle in a BCR-independent manner119. Although such a situation is unlikely to occur in physiological conditions, it does indicate that alternative receptors might be involved in antigen transport.

Conclusions and perspectivesOverall, it is clear that B cells can respond rapidly to antigenic stimulation through several mechanisms, and they also have a unique strategic role in the transport of antigen to FDCs in the germinal centre. This wide variety in potential antigen-presentation mechanisms is promoted by functional specialization within slOs and provides invaluable flexibility in mounting appropriate immune responses to antigens. As such, the mechanism that is used to present a specific antigen can be effec-tively tailored both to the antigen itself and to the way the antigen is delivered to the B cell. The predominance of cell-based strategies for the presentation of antigen to B cells allows for extensive regulation and coordination of the resulting immune responses.

In spite of significant recent progress in character-izing the sites and dynamics of antigen presentation to B cells in vivo, some issues remain to be addressed. These include a definitive molecular description of the mechanisms of antigen presentation to B cells, includ-ing the retention and internalization strategies that are


Immune complex

Antigen non-specific BCR

CR

Marginal-zone B cell

Marginal zone

a Splenic marginal-zone B cells transport antigen to FDCs b Follicular B cells in the lymph node transport antigen to FDCs

Primary follicle Primary follicle

SCS macrophage

Follicular dendritic cell

Follicular B cell ?

?

Subcapsular sinusS1Phi

CXCL13low

S1Plow

CXCL13hi

C3fragment

FcR

DC-SIGN

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 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.

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used by the antigen-presenting cells. In addition, it would prove informative to track the fate of B cells fol-lowing antigenic stimulation, in terms of the particular differentiation pathway that is initiated. such progress may be possible by improving the resolution that is achievable by imaging techniques, in combination with the generation of animal models that are deficient in or have altered expression of the different components that may be involved. Furthermore, advances in the field of high-resolution intravital imaging may allow for the visualization of in vivo antigen encounters at greater tissue depth and for more extended periods of time. These developments may also offer insight into potential

roles of the interactions between follicular B cells in the immune response and into the importance of FDCs in the presentation of antigen to naive B cells. Finally, it is important to establish that the in vivo observations that have been collected so far in transgenic animal-model systems reflect those that occur during the stimulation of the natural repertoire of B cells during infection or following vaccination.

Taken together, a more comprehensive description of the mechanisms by which B cells sense and respond to antigen will have implications for the future tailoring of vaccination strategies and also in the rational design of treatments for infectious and autoimmune diseases.

1. Bajénoff, M. & Germain, R. Seeing is believing: a focus on the contribution of microscopic imaging to our understanding of immune system function. Eur. J. Immunol. 37, S18–S33 (2007).

2. Halin, C., Rodrigo Mora, J., Sumen, C. & von Andrian, U. In vivo imaging of lymphocyte trafficking. Annu. Rev. Cell Dev. Biol. 21, 581–603 (2005).

3. von Andrian, U. & Mempel, T. Homing and cellular traffic in lymph nodes. Nature Rev. Immunol. 3, 867–878 (2003).References 1–3 are three excellent reviews detailing key historical insights into the function of the immune system and have served as the foundation for subsequent high-resolution imaging investigations.

4. Catron, D., Itano, A., Pape, K., Mueller, D. & Jenkins, M. Visualizing the first 50 hr of the primary immune response to a soluble antigen. Immunity 21, 341–347 (2004).

5. Germain, R., Miller, M., Dustin, M. & Nussenzweig, M. Dynamic imaging of the immune system: progress, pitfalls and promise. Nature Rev. Immunol. 6, 497–507 (2006).

6. Mempel, T., Scimone, M., Mora, J. & von Andrian, U. In vivo imaging of leukocyte trafficking in blood vessels and tissues. Curr. Opin. Immunol. 16, 406–417 (2004).

7. Cahalan, M. & Parker, I. Imaging the choreography of lymphocyte trafficking and the immune response. Curr. Opin. Immunol. 18, 476–482 (2006).

8. Bousso, P. & Robey, E. Dynamic behavior of T cells and thymocytes in lymphoid organs as revealed by two-photon microscopy. Immunity 21, 349–355 (2004).

9. Grakoui, A. et al. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221–227 (1999).

10. Krummel, M., Sjaastad, M., Wülfing, C. & Davis, M. Differential clustering of CD4 and CD3z during T cell recognition. Science 289, 1349–1352 (2000).

11. Monks, C., Freiberg, B., Kupfer, H., Sciaky, N. & Kupfer, A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395, 82–86 (1998).

12. Batista, F., Iber, D. & Neuberger, M. B cells acquire antigen from target cells after synapse formation. Nature 411, 489–494 (2001).This paper describes the formation of the immunological synapse in response to membrane-bound antigen and the necessity of internalization of antigen through the central supramolecular activation cluster for maximal B-cell activation.

13. Carrasco, Y. R. & Batista, F. D. B cell recognition of membrane-bound antigen: an exquisite way of sensing ligands. Curr. Opin. Immunol. 18, 286–291 (2006).

14. Depoil, D. et al. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand. Nature Immunol. 9, 63–72 (2008).This paper visualizes the formation of antigen–BCR microclusters and, by identifying an essential role for CD19 in B-cell activation by membrane-bound antigen, it indicates the importance of the recognition of this type of antigen in vivo during the development of immune responses.

15. Lanzavecchia, A. Antigen-specific interaction between T and B cells. Nature 314, 537–539 (1985).

16. Rock, K., Benacerraf, B. & Abbas, A. Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors. J. Exp. Med. 160, 1102–1113 (1984).

17. MacLennan, I. Germinal centers. Annu. Rev. Immunol. 12, 117–139 (1994).

18. Rajewsky, K. Clonal selection and learning in the antibody system. Nature 381, 751–758 (1996).

19. Cahalan, M. & Parker, I. Choreography of Cell. Motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26, 585–626 (2008).

20. Celli, S., Garcia, Z., Beuneu, H. & Bousso, P. Decoding the dynamics of T cell–dendritic cell interactions in vivo. Immunol. Rev. 221, 182–187 (2008).

21. Germain, R. et al. Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging. Immunol. Rev. 221, 163–181 (2008).

22. Junt, T., Scandella, E. & Ludewig, B. Form follows function: lymphoid tissue microarchitecture in antimicrobial immune defence. Nature Rev. Immunol. 8, 764–775 (2008).

23. Karrer, U. et al. On the key role of secondary lymphoid organs in antiviral immune responses studied in alymphoplastic (aly/aly) and spleenless (Hox11–/–) mutant mice. J. Exp. Med. 185, 2157–2170 (1997).

24. Cyster, J. Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu. Rev. Immunol. 23, 127–159 (2005).

25. Klaus, G., Humphrey, J., Kunkl, A. & Dongworth, D. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53, 3–28 (1980).

26. Mandel, T., Phipps, R., Abbot, A. & Tew, J. The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53, 29–59 (1980).References 25 and 26 are two excellent reviews that summarize much of the early work that characterized the role of FDCs in the presentation and retention of antigen and described how FDCs influence immune responses.

27. Carroll, M. The role of complement and complement receptors in induction and regulation of immunity. Annu. Rev. Immunol. 16, 545–568 (1998).

28. Qin, D. et al. Fcγ receptor IIB on follicular dendritic cells regulates the B cell recall response. J. Immunol. 164, 6268–6275 (2000).

29. Cyster, J. et al. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193 (2000).

30. Fu, Y. & Chaplin, D. Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17, 399–433 (1999).

31. Gretz, J., Anderson, A. & Shaw, S. Cords, channels, corridors and conduits: critical architectural elements facilitating cell interactions in the lymph node cortex. Immunol. Rev. 156, 11–24 (1997).

32. Gretz, J., Norbury, C., Anderson, A., Proudfoot, A. & Shaw, S. Lymph-borne chemokines and other low molecular weight molecules reach high endothelial

venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J. Exp. Med. 192, 1425–1440 (2000).This paper examines the distribution of various fluorescently labelled soluble antigens in the draining lymph node following subcutaneous administration, showing that low-molecular-mass molecules, but not larger antigens, can gain access to the interior of the lymph node by direct diffusion from the conduit network.

33. Sixt, M. et al. The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node. Immunity 22, 19–29 (2005).

34. Miller, J. & Nossal, G. Antigens in immunity. VI. The phagocytic reticulum of lymph node of follicles. J. Exp. Med. 120, 1075–1086 (1964).

35. Gowans, J. The effect of the continuous re-infusion of lymph and lymphocytes on the output of lymphocytes from the thoracic duct of unanaesthetized rats. Brit J. Exp. Pathol. 38, 67–78 (1957).

36. Nolte, M. et al. A conduit system distributes chemokines and small blood-borne molecules through the splenic white pulp. J. Exp. Med. 198, 505–512 (2003).

37. Cesta, M. Normal structure, function, and histology of the spleen. Toxicol. Path. 34, 455–465 (2006).

38. Saito, H. et al. Reticular meshwork of the spleen in rats studied by electron microscopy. Am. J. Anat. 181, 235–252 (1988).

39. Kraal, G. Cells in the marginal zone of the spleen. Int. Rev. Cytol. 132, 31–74 (1992).

40. Schmidt, E., MacDonald, I. & Groom, A. Comparative aspects of splenic microcirculatory pathways in mammals: the region bordering the white pulp. Scanning Microsc. 7, 613–628 (1993).

41. Kumararatne, D., Bazin, H. & MacLennan, I. Marginal zones: the major B cell compartment of rat spleens. Eur. J. Immunol. 11, 858–864 (1981).

42. Martin, F. & Kearney, J. Marginal-zone B cells. Nature Rev. Immunol. 2, 323–335 (2002).

43. Bohnsack, J. & Brown, E. The role of the spleen in resistance to infection. Annu. Rev. Med. 37, 49–59 (1986).

44. Bousso, P., Bhakta, N., Lewis, R. & Robey, E. Dynamics of thymocyte–stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002).

45. Miller, M., Wei, S., Parker, I. & Cahalan, M. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).

46. Stoll, S., Delon, J., Brotz, T. & Germain, R. Dynamic imaging of T cell–dendritic cell interactions in lymph nodes. Science 296, 1873–1876 (2002).References 44–46 are three key papers published back-to-back that describe the first use of two-photon microscopy for the investigation of lymphocyte behaviour, visualizing the unexpected and rapid migration of lymphocytes in the absence of antigen.

47. Bousso, P. & Robey, E. Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes. Nature Immunol. 4, 579–585 (2003).

R E V I E W S



48. Okada, T. et al. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biol. 3, e150 (2005).This paper uses multiphoton microscopy to observe the dynamics of B cells and their interaction with antigen-specific T cells within the lymph node following antigen exposure.

49. Bajénoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).In this paper, the authors develop an elegant chimeric system to visualize the highly organized migration of B and T cells within lymph nodes across networks of FDCs and FRCs, respectively.

50. Lindquist, R. et al. Visualizing dendritic cell networks in vivo. Nature Immunol. 5, 1243–1250 (2004).

51. Nossal, G., Abbot, A., Mitchell, J. & Lummus, Z. Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles. J. Exp. Med. 127, 277–290 (1968).

52. Clark, S. The reticulum of lymph nodes in mice studied with the electron microscope. Am. J. Anat. 110, 217–257 (1962).

53. Farr, A., Cho, Y. & De Bruyn, P. The structure of the sinus wall of the lymph node relative to its endocytic properties and transmural cell passage. Am. J. Anat. 157, 265–284 (1980).

54. van Ewijk, W., Brekelmans, P., Jacobs, R. & Wisse, E. Lymphoid microenvironments in the thymus and lymph node. Scanning Microsc. 2, 2129–2140 (1988).

55. Pape, K., Catron, D., Itano, A. & Jenkins, M. The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity 26, 491–502 (2007).This study tracks the diffusion of a low-molecular- mass fluorescent antigen from the subcapsular sinus and shows the rapid acquisition of this antigen by follicular B cells within minutes of administration.

56. Szakal, A., Holmes, K. & Tew, J. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131, 1714–1727 (1983).

57. Szakal, A., Kosco, M. & Tew, J. Microanatomy of lymphoid tissue during humoral immune responses: structure function relationships. Annu. Rev. Immunol. 7, 91–109 (1989).

58. Fossum, S. The architecture of rat lymph nodes. IV. Distribution of ferritin and colloidal carbon in the draining lymph nodes after foot-pad injection. Scand. J. Immunol. 12, 433–441 (1980).

59. Gray, D., Kumararatne, D., Lortan, J., Khan, M. & MacLennan, I. Relation of intra-splenic migration of marginal zone B cells to antigen localization on follicular dendritic cells. Immunology 52, 659–669 (1984).

60. Humphrey, J. & Grennan, D. Different macrophage populations distinguished by means of fluorescent polysaccharides. Recognition and properties of marginal-zone macrophages. Eur. J. Immunol. 11, 221–228 (1981).

61. Martínez-Pomares, L. et al. Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers. J. Exp. Med. 184, 1927–1937 (1996).

62. Unanue, E., Cerottini, J. & Bedford, M. Persistence of antigen on the surface of macrophages. Nature 222, 1193–1195 (1969).

63. Taylor, P. et al. Macrophage receptors and immune recognition. Annu. Rev. Immunol. 23, 901–944 (2005).

64. Phan, T., Grigorova, I., Okada, T. & Cyster, J. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nature Immunol. 8, 992–1000 (2007).

65. Bergtold, A., Desai, D. D., Gavhane, A. & Clynes, R. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23, 503–514 (2005).

66. Koppel, E. et al. Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection. Eur. J. Immunol. 35, 2962–2969 (2005).

67. Carrasco, Y. & Batista, F. B cells acquire particulate antigen in a macrophage-rich area at the boundary

between the follicle and the subcapsular sinus of the lymph node. Immunity 27, 160–171 (2007).

68. Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).References 64, 67 and 68 reveal a role for macrophages within the subcapsular sinus in the presentation of larger antigens, such as particulates, immune complexes and viruses, to follicular B cells, using multiphoton microscopy to visualize the distribution of B cells in vivo over time following antigen administration.

69. Carrasco, Y., Fleire, S., Cameron, T., Dustin, M. & Batista, F. LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20, 589–599 (2004).

70. Garside, P. et al. Visualization of specific B and T lymphocyte interactions in the lymph node. Science 281, 96–99 (1998).

71. Sette, A. et al. Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities. Immunity 28, 847–858 (2008).

72. Delamarre, L., Pack, M., Chang, H., Mellman, I. & Trombetta, E. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science 307, 1630–1634 (2005).

73. Itano, A. et al. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47–57 (2003).

74. Dudziak, D. et al. Differential antigen processing by dendritic cell subsets in vivo. Science 315, 107–111 (2007).

75. Huang, N., Han, S., Hwang, I. & Kehrl, J. B cells productively engage soluble antigen-pulsed dendritic cells: visualization of live-cell dynamics of B cell-dendritic cell interactions. J. Immunol. 175, 7125–7134 (2005).

76. Kwon, D., Gregorio, G., Bitton, N., Hendrickson, W. & Littman, D. DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity 16, 135–144 (2002).

77. Wykes, M., Pombo, A., Jenkins, C. & MacPherson, G. Dendritic cells interact directly with naive B lymphocytes to transfer antigen and initiate class switching in a primary T-dependent response. J. Immunol. 161, 1313–1319 (1998).

78. Colino, J., Shen, Y. & Snapper, C. Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms. J. Exp. Med. 195, 1–13 (2002).

79. Qi, H., Egen, J. G., Huang, A. Y. & Germain, R. N. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science 312, 1672–1676 (2006).This paper uses two-photon microscopy to visualize the presentation of intact antigen by DCs to B cells as they enter the lymph node.

80. Berney, C. et al. A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein. J. Exp. Med. 190, 851–860 (1999).

81. Lopes-Carvalho, T., Foote, J. & Kearney, J. Marginal zone B cells in lymphocyte activation and regulation. Curr. Opin. Immunol. 17, 244–250 (2005).

82. Balázs, 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, 341–352 (2002).

83. Stein, H. et al. Immunohistologic analysis of the organization of normal lymphoid tissue and non-Hodgkin’s lymphomas. J. Histochem. Cytochem. 28, 746–760 (1980).

84. Mitchell, J. & Abbot, A. Ultrastructure of the antigen-retaining reticulum of lymph node follicles as shown by high-resolution autoradiography. Nature 208, 500–502 (1965).

85. Chen, L., Frank, A., Adams, J. & Steinman, R. Distribution of horseradish peroxidase (HRP)–anti-HRP immune complexes in mouse spleen with special reference to follicular dendritic cells. J. Cell Biol. 79, 184–199 (1978).

86. Tew, J., Phipps, R. & Mandel, T. The maintenance and regulation of the humoral immune response: persisting antigen and the role of follicular antigen-binding dendritic cells as accessory cells. Immunol. Rev. 53, 175–201 (1980).

87. Tew, J., Wu, J., Fakher, M., Szakal, A. & Qin, D. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22, 361–367 (2001).

88. Klaus, G. & Humphrey, J. The generation of memory cells. I. The role of C3 in the generation of B memory cells. Immunology 33, 31–40 (1977).

89. Papamichail, M. et al. Complement dependence of localisation of aggregated IgG in germinal centres. Scand. J. Immunol. 4, 343–347 (1975).

90. Fang, Y., Xu, C., Fu, Y., Holers, V. & Molina, H. Expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J. Immunol. 160, 5273–5279 (1998).

91. Barrington, R., Pozdnyakova, O., Zafari, M., Benjamin, C. & Carroll, M. B lymphocyte memory: role of stromal cell complement and FcγRIIB receptors. J. Exp. Med. 196, 1189–1199 (2002).

92. Qin, D. et al. Evidence for an important interaction between a complement-derived CD21 ligand on follicular dendritic cells and CD21 on B cells in the initiation of IgG responses. J. Immunol. 161, 4549–4554 (1998).

93. Yoshida, K., van den Berg, T. & Dijkstra, C. Two functionally different follicular dendritic cells in secondary lymphoid follicles of mouse spleen, as revealed by CR1/2 and FcRγII-mediated immune-complex trapping. Immunology 80, 34–39 (1993).

94. Nossal, G., Ada, G., Austin, C. & Pye, J. Antigens in immunity. 8. Localization of 125-I-labelled antigens in the secondary response. Immunology 9, 349–357 (1965).

95. Dempsey, P., Allison, M., Akkaraju, S., Goodnow, C. & Fearon, D. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271, 348–350 (1996).

96. Heyman, B. Regulation of antibody responses via antibodies, complement, and Fc receptors. Annu. Rev. Immunol. 18, 709–737 (2000).

97. Herzenberg, L. et al. The Ly-1 B cell lineage. Immunol. Rev. 93, 81–102 (1986).

98. Boes, M., Prodeus, A., Schmidt, T., Carroll, M. & Chen, J. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188, 2381–2386 (1998).

99. Ehrenstein, M., O’Keefe, T., Davies, S. & Neuberger, M. Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response. Proc. Natl Acad. Sci. USA 95, 10089–10093 (1998).

100. MacLennan, I. Holding antigen where B cells can find it. Nature Immunol. 8, 909–910 (2007).

101. Kosco-Vilbois, M. Are follicular dendritic cells really good for nothing? Nature Rev. Immunol. 3, 764–769 (2003).

102. Allen, C., Okada, T., Tang, H. & Cyster, J. Imaging of germinal center selection events during affinity maturation. Science 315, 528–531 (2007).

103. Hauser, A. et al. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns. Immunity 26, 655–667 (2007).

104. Schwickert, T. et al. In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446, 83–87 (2007).Studies 102–104, published concurrently, provide a description of the cellular events in the germinal centre, calling into question the classic light-zone and dark-zone model of germinal-centre function.

105. Batista, F. & Neuberger, M. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8, 751–759 (1998).

106. Allen, C. & Cyster, J. Follicular dendritic cell networks of primary follicles and germinal centers: phenotype and function. Semin. Immunol. 20, 14–25 (2008).

107. Anderson, S., Hannum, L. & Shlomchik, M. Memory B cell survival and function in the absence of secreted antibody and immune complexes on follicular dendritic cells. J. Immunol. 176, 4515–4519 (2006).

108. Hannum, L., Haberman, A., Anderson, S. & Shlomchik, M. Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells. J. Exp. Med. 192, 931–942 (2000).

109. Haberman, A. & Shlomchik, M. Reassessing the function of immune-complex retention by follicular dendritic cells. Nature Rev. Immunol. 3, 757–764 (2003).

R E V I E W S



110. Brown, J., De Jesus, D., Holborow, E. & Harris, G. Lymphocyte-mediated transport of aggregated human γ-globulin into germinal centre areas of normal mouse spleen. Nature 228, 367–369 (1970).

111. Groeneveld, P., Erich, T. & Kraal, G. In vivo effects of LPS on B lymphocyte subpopulations. Migration of marginal zone-lymphocytes and IgD-blast formation in the mouse spleen. Immunobiology 170, 402–411 (1985).

112. Groeneveld, P., Erich, T. & Kraal, G. The differential effects of bacterial lipopolysaccharide (LPS) on splenic non-lymphoid cells demonstrated by monoclonal antibodies. Immunology 58, 285–290 (1986).

113. Ferguson, A., Youd, M. & Corley, R. Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells. Int. Immunol. 16, 1411–1422 (2004).

114. Van den Berg, T., Döpp, E., Daha, M., Kraal, G. & Dijkstra, C. Selective inhibition of immune complex trapping by follicular dendritic cells with monoclonal antibodies against rat C3. Eur. J. Immunol. 22, 957–962 (1992).

115. Youd, M., Ferguson, A. & Corley, R. Synergistic roles of IgM and complement in antigen trapping and follicular localization. Eur. J. Immunol. 32, 2328–2337 (2002).

116. Cinamon, G., Zachariah, M., Lam, O., Foss, F. & Cyster, J. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nature Immunol. 9, 54–62 (2007).In this paper, the authors demonstrate the continual shuttling of splenic marginal-zone B cells into the follicle and suggest that this is a mechanism for the capture and delivery of antigen to follicular B cells.

117. Whipple, E., Shanahan, R., Ditto, A., Taylor, R. & Lindorfer, M. Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice. J. Immunol. 173, 2297–2306 (2004).

118. Heinen, E. et al. Transfer of immune complexes from lymphocytes to follicular dendritic cells. Eur. J. Immunol. 16, 167–172 (1986).

119. Hjelm, F., Karlsson, M. & Heyman, B. A novel B cell-mediated transport of IgE-immune complexes to the follicle of the spleen. J. Immunol. 180, 6604–6610 (2008).

120. Fleire, S. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–741 (2006).

121. Bunnell, S. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002).

122. Campi, G., Varma, R. & Dustin, M. Actin and agonist MHC–peptide complex-dependent T cell receptor microclusters as scaffolds for signaling. J. Exp. Med. 202, 1031–1036 (2005).

123. Yokosuka, T. et al. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nature Immunol. 6, 1253–1262 (2005).

124. Weber, M. et al. Phospholipase C-γ2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J. Exp. Med. 205, 853–868 (2008).

125. Sohn, H. W., Tolar, P. & Pierce, S. K. Membrane heterogeneities in the formation of B cell receptor–Lyn kinase microclusters and the immune synapse. J. Cell Biol. 182, 367–379 (2008).

126. Sallusto, F. & Lanzavecchia, A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor α. J. Exp. Med. 179, 1109–1118 (1994).

127. Crawford, A., Macleod, M., Schumacher, T., Corlett, L. & Gray, D. Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells. J. Immunol. 176, 3498–3506 (2006).

128. Qi, H., Cannons, J. L., Klauschen, F., Schwartzberg, P. L. & Germain, R. N. SAP-controlled T–B cell interactions underlie germinal centre formation. Nature 455, 764–769 (2008).

AcknowledgementsWe thank members of the Lymphocyte Interaction Laboratory for critical reading of the manuscript, particularly P. Barral for providing multiphoton microscopy images.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneC3 | CR1 | CR2 | CXCR5 | DC-SIGN | ICAM1 | MAC1 | S1PR1 | VCAM1

FURTHER INFORMATIONFacundo Batista’s homepage: http://science.cancerresearchuk.org/research/loc/london/lifch/batistaf

All lInks ARe AcTIve In The onlIne Pdf

R E V I E W S



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene










http://science.cancerresearchuk.org/research/loc/london/lifch/batistaf

http://science.cancerresearchuk.org/research/loc/london/lifch/batistaf

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