1. REVIEWS Molecular programming of B cell memory Michael McHeyzer-Williams, Shinji Okitsu, Nathaniel Wang and Louise McHeyzer-Williams Abstract | The development of high-affinity Bcell memory is regulated through three separable phases, each involving antigen recognition by specific Bcells and cognate Thelper cells. Initially, antigen-primed Bcells require cognate Tcell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, Bcells with variant Bcell receptors must access antigens and present them to germinal centre Thelper cells to enter long-lived memory Bcell compartments. Following antigen recall, memory Bcells require Tcell help to proliferate and differentiate into plasma cells. A recent surge of information resulting from dynamic Bcell imaging invivo and the elucidation of Tfollicular helper cell programmes has reshaped the conceptual landscape surrounding the generation of memory Bcells. In this Review, we integrate this new information about each phase of antigen-specific Bcell development to describe the newly unravelled molecular dynamics of memory Bcell programming.Tfollicular helper cells Most effective vaccines that are in use today gener- different classes of circulating antibodies engage separate(TFH cells). A distinct class of ate protective, antigen-specific Bcell memory. To be antigen-clearance mechanisms, providing multiple sero-Thelper cells specialized to effective, memory Bcells must target the right antigen, logical barriers to re-infection. Similarly, non-secretingregulate multiple stages of express the appropriate antibody class and bind to their memory Bcells can express affinity-matured BCRs ofantigen-specific Bcell antigen with sufficiently high affinity to provide the host different classes (either IgM or downstream antibodyimmunity through cognate cellcontact and the secretion of with long-term immune protection. These three cardinal isotypes following class switching). For example, IgG2a+cytokines. There are three attributes of antigen-specific Bcell memory emerge pro- memory Bcells express chemokine receptors that helpseparable TFH cell subsets gressively under the cognate guidance of Tfollicular helper them to traffic into inflamed tissues. IgA+ memorydefined in the current literature cells (TFH cells) following initial priming and secondary Bcells are found at mucosal surfaces in the gut and lungsthat correspond to the threephases of memory Bcell challenge with antigen invivo. Although we know a great after local infections. In the memory phase, these class-development. These are deal about circulating antibodies, little is understood specific memory Bcells proliferate robustly in responsepre-GC TFH cells, GC TFH cells about the development of high-affinity memory Bcells, to antigen re-exposure and promote the generation ofand memory TFH cells. which ultimately provide Bcell-mediated immune high-affinity plasma cells under the control of cognate protection invivo. memory Thelper cells. The exchange of information at Initial priming of naive B cells and subsequent each phase of antigen-specific engagement outlines the cognate contact with TFH cells initiates immunoglobulin molecular dynamics of memory Bcell programming. class switching and the differentiation of some Bcells In this Review, we evaluate recent findings on memoryDepartment of Immunology into plasma cells outside the germinal centre (GC); this Bcell programming and place them in their relevantand Microbial Sciences, is termed the pre-GC phase. This initial BcellTcell developmental context invivo. We consider the sequenceThe Scripps Research contact is also required to induce the GC reaction, which of molecular exchanges between antigen-presenting cellsInstitute, 10550 North TorreyPines Road, La Jolla, drives the maturation of memory Bcells. In the GC and Thelper cells at each stage, with emphasis on mouseCalifornia 92037, USA. phase, cycles of Bcell receptor (BCR) diversification models of adaptive immune responses; these sequencesCorrespondence to M.M.-W. and antigen-driven selection within the GC promote define major checkpoints in memory Bcell maturation.e-mail: the development and subsequent export of high-affinity The three main developmental phases of Bcell memorymcheyzer@scripps.edudoi:10.1038/nri3128 memory Bcells. Effective Bcell memory requires dif- are presented from the Bcell perspective. In each phase,Published online ferent functional classes of high-affinity plasma cells antigen recognition is followed by antigen presentation9 December 2011 and an array of non-secreting memory Bcells. The and cognate Tcell help. These two-step processes each24 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/immunol 2012 Macmillan Publishers Limited. All rights reserved

2. REVIEWS a b Secondary follicle formation c GC reaction d Antigen Secondary GC Primary SCS macrophage recall B cell follicle Follicular DC Antigen Memory TFH cell B cell BCRB cell zone Commitment to B cell B cell Naive Antigen memory B cell GC GC uptake B cell Anity Proliferation of maturation memory B cellsT cell zone Pre-GC Memory TFH cell Plasma cell TFH cell TCR Antibody Antibody secretion secretion Naive TH cell Antigen- CD4+ Memory primed DC T cell plasma cell CD4+ T cell Lymph Non-GC B cell Non-GC plasma cell proliferation node exit dierentiation dierentiation Figure 1 | TFH cell-regulated memory Bcell development. a | Local protein vaccination induces dendritic cell (DC) Nature Reviews Immunology maturation and migration to the Tcell zones of draining lymph nodes. DCs that express peptideMHC classII| complexes engage naive, antigen-specific CD4+ Tcells to induce their proliferation and differentiation into effector Thelper (TH) cells. In the Bcell zone, whole antigen is trapped by subcapsular sinus (SCS) macrophages and presented to naive follicular Bcells. Antigen-specific Bcells become activated, take up, process and present antigenic peptides and migrate towards the BcellTcell borders of the draining lymph node. Effector TH cells emerge in multiple forms; emigrant TH cells exit the lymph node to function at distal tissue sites and Tfollicular helper (TFH) cells relocate to BcellTcell borders and interfollicular regions. Cognate contact between pre-germinal centre (pre-GC) TFH cells and antigen-primed Bcells isCognate contact required for multiple programming events in the pathway to Bcell memory. b | Clonal expansion, antibody class switchingContact between a B cell and and non-GC plasma cell development proceeds in the extrafollicular regions of the lymph nodes. Secondary folliclea T follicular helper cell that formation and antibody class switching precede the initiation of the GC reaction, which forms the dominant pathway forrecognizes the same antigen. the generation of memory Bcells. c | Polarization of the secondary follicle anatomically signifies the initiation of the GCThis contact requires antigen- cycle. The dark zone supports GC centroblast proliferation, class-switch recombination and Bcell receptor (BCR)receptor engagement by diversification through somatic hypermutation. Non-cycling GC centrocytes move to the light zone and continually scancell-associated antigens or follicular DC networks. Centrocytes that lose the ability to bind to the presented antigen undergo apoptosis, while thosepeptideMHC complexes and that express a variant BCR with a higher affinity can compete for binding to antigen-specific GC TFH cells. Cognate contactcan be modified by secondaryinteractions that can involve with GC TFH cells requires peptideMHC classII expression by the GC centrocytes. This contact can promote Bcell re-entryboth cell-associated and into the dark zone and the GC cycle or exit from the GC and entry into the affinity-matured memory Bcell compartmentssecreted molecules. Cognate of non-secreting memory Bcells and post-GC plasma cells. d | Following antigen re-challenge, memory Bcells presentcontact functions to initiate antigens to memory TFH cells to promote memory B cell clonal expansion with rapid memory plasma cell generation andbidirectional developmental the induction of a secondary GC reaction. Although related to the cellular and molecular activities of the primaryprogramming. response, as depicted, the memory-response dynamics remain poorly resolved. TCR, Tcell receptor.Immunoglobulin classswitchingA region-specific initiate and then consolidate a cellular reprogramming contact with SCS macrophages results in the movementrecombination process that event that ultimately propels naive Bcells into one of of antigen-specific Bcells to the BcellTcell bordersoccurs in antigen-activated the multiple compartments of class-specific high-affinity and induces antigen-specific Bcell responses to capturedBcells. It occurs between memory Bcells (FIG.1). antigens57. Hence, the SCS macrophages filtering theswitch-region DNA sequencesand results in a change in the lymphatic fluid not only protect from systemic infec-class of antibody that is Commitment to Bcell memory tion8, but also effectively initiate T helper cell-regulatedproduced from IgM to either Priming naive Bcells. Bcells can acquire soluble anti- antigen-specific Bcell immunity.IgG, IgA or IgE. This imparts gens that freely diffuse into lymphoid follicles 1 or Initial activation of naive Bcells through the BCR trig-flexibility to the humoralimmune response and allows that are transported through the lymphoid system of gers multiple gene expression programmes that enableit to exploit the different conduits2. Dynamic imaging has also captured early effective contact with cognate T helper cells. Dynamiccapacities of these antibody contact between naive Bcells and dendritic cells (DCs)3. contact with membrane-associated antigens determinesclasses to activate the However, populations of lymph node subcapsular sinus the amount of antigen that naive Bcells accumulate fol-appropriate downstream macrophages (SCS macrophages) appear to be the most lowing their first antigen exposure9. Effective cell con-effector mechanisms. effective at presenting cell-associated antigens to follicu- tact requires the expression of the signalling adaptorPlasma cells lar Bcells4. Bcells use complement receptors to take up DOCK8 (dedicator of cytokinesis protein8)10. MutationsTerminally differentiated, non-cognate antigens presented by SCS macrophages, in Dock8 disrupt the accumulation of integrin ligands inquiescent Bcells that develop and they then transport these antigens into follicular the immune synapse without altering BCR signallingfrom plasmablasts and arecharacterized by the capacity regions and transfer them to follicular DCs, which can events. Bcell-specific conditional ablation of calcineu-to secrete large amounts of serve as a source of antigens for priming naive Bcells4. rin regulatory subunit1 (Cnb1)11, myocyte enhancerantibodies. By contrast, priming with the cognate antigen on first factor2c (Mef2c)12,13, stromal interaction molecule1NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | JANUARY 2012 | 25 2012 Macmillan Publishers Limited. All rights reserved

3. REVIEWS (Stim1) or Stim2 (REF.14) has shown that calcium respon- crucial long-lasting interactions occur in the interfol- siveness is also necessary for cell cycle progression in licular zones of lymph nodes prior to GC formation33, these early stages. Hydrogen voltage-gated channel1 and persistent BCL6 expression in Bcells was requiredGerminal centre (HVCN1), which is internalized with the BCR, has also to maintain this effective cognate contact 31. Therefore,(GC). A lymphoid structurethat arises within lymph node been implicated in early Bcell programming events15. early TFH cell developmental programmes establish thefollicles after immunization Bcells exposed to a short-duration BCR signal only par- capacity for cognate contact, which is needed to promotewith, or exposure to, a tially activate nuclear factor-B (NF-B), but increase antigen-specific Bcell commitment to antibody classTcell-dependent antigen. their expression of CC-chemokine receptor7 (CCR7) and the subsequent maturation of BCR affinity (FIG.2).The GC is specialized for and MHC classII molecules and their responsiveness tofacilitating the development ofhigh-affinity, long-lived plasma CD40 to promote more effective cognate Tcell help16. Initial cognate contact appears to imprint antibodycells and memory Bcells. Severe defects in early Bcell proliferation have also impli- class. Antibody class switching in antigen-primed cated integrin binding by CD98 (REF.17) and the activa- Bcells is an irreversible genetic recombination event.GC reaction tion of extracellular signal-regulated kinase (ERK)18 in Briefly, sterile germline transcription through anti-(Germinal centre reaction).Acycle of activity preparing the antigen-primed Bcells to receive cognate body switch regions provides activation-induced cytidinecharacterized by three stages. Tcell help invivo. Hence, initial antigen recognition, deaminase (AID; also known as AICDA) with accessFirst, GC Bcells undergo clonal uptake, processing and presentation have a crucial impact to the single-stranded DNA template, enabling AID toexpansion and Bcell receptor on the early developmental fate of Bcells. deaminate cytosines34. This triggers the recruitment ofdiversification in the GC dark DNA damage machinery that removes the resulting ura-zone. The Bcells then scanfollicular dendritic cells for Early TFH cell programmes. TFH cells have emerged as a cils and of mismatch repair factors that then generateantigens, and finally make new class of T helper cells specialized to regulate Bcell double-strand breaks (DSBs). Non-homologous end joiningcontact with cognate GC TFH immune responses19,20. The central attribute of TFH cells (NHEJ) completes the class-switch recombination (CSR)cells in the GC light zone. is the capacity to secure contact with cognate antigen- event. AID expression is largely restricted to antigen-Positive selection continuesthe GC cycle with re-entry primed Bcells. Expression of CXC-chemokine recep- activated Bcells, although there is some evidence for lowinto the dark zone or promotes tor5 (CXCR5) and the loss of CCR7 expression positions levels of AID in the bone marrow. Recent evidence indi-exit from the GC into the antigen-primed TFH cells in follicular Bcell regions of the cates that, following antigen stimulation, AID expressionmemory Bcell compartment. lymph node. Recent studies showed that the transcription is regulated in Bcells by paired box protein5 (PAX5), factor Bcell lymphoma6 (BCL6) is expressed by antigen- Ebox proteins35, homeoboxC4 (HOXC4)36 and fork-Class-specific memoryBcells specific TFH cells and that Blymphocyte-induced matura- head boxO1 (FOXO1)37. The adaptor protein 1433 isNon-secreting memory Bcells tion protein1 (BLIMP1; also known as PRDM1), which recruited with AID to switch regions38, and polymerase-that express either IgM or has an opposing function, is expressed by other Tcells has been implicated in the repair process associated withdownstream non-IgM antibody in the lymph node21. BCL6 is required for the develop- CSR39. Peripheral Bcells undergoing CSR in the absenceclasses following Thelpercell-regulated class-switch ment of the TFH cell programme2224, and its expression of the XRCC4 (X-ray repair cross-complementing pro-recombination. is reinforced in TFH cells following contact with pre-GC tein4) component of the DSB repair machinery are also Bcells25,26. Interleukin21 (IL21) also has a major role in highly susceptible to translocation events and oncogenicSubcapsular sinus TFH cell function, as a substantial loss of Bcell immunity transformation40. Hence, antibody class switching is amacrophages occurs in its absence. More recently, the transcription destabilizing and potentially dangerous cellular event(SCS macrophages).ACD11b+CD169+ factors MAF and BATF were shown to act with BCL6 that is likely to be resolved early during the generationmacrophage subset that to program TFH cell development 27,28. Hence, the distinct of antigen-specific memory Bcells.populates the subcapsular transcriptional programming of unique cellular functions Cytokines and innate stimuli alone can drive naivesinus region of lymph nodes. directs early TFH cell development, and this is central to IgM+ Bcells to switch antibody class. However, typicalThese cells function to trapparticulate antigens from the subsequent memory Bcell generation. vaccination responses require cognate Tcell help to gen-lymph and present antigens The T FH cell programme is one developmental erate antigen-specific non-IgM antibodies. Early staticto follicular Bcells. option adopted by naive T helper cells following initial imaging studies demonstrated coordinated antibody antigen-specific priming by DCs21. Expression of induc- class switching in both the non-GC and GC pathways,Follicular DCs ible Tcell co-stimulator ligand (ICOSL) on DCs appears suggesting that the earliest events of class switching are(Follicular dendritic cells).Specialized non- to be necessary to induce the TFH cell programme over controlled at the pre-GC phase, following initial con-haematopoietic stromal cells more typical effector Thelper cell options29. BCL6 tact with TFH cells41. Early hybridoma studies furtherthat reside in the lymphoid and BLIMP1 expression is mutually exclusive across supported this notion with evidence that antibodyfollicles and germinal centres. these two Thelper cell populations; this distinction is class switching can occur without somatic hypermuta-These cells possess longdendrites and carry intact already evident by the second cell division invivo and is tion. Reporter mouse models have revealed that Tcellsantigens on their surface. associated with differential expression of IL2 receptor produce cytokines at sites of antigen-specific contactThey are crucial for the subunit- (IL2R)29. Depending on the type of anti- with cognate Bcells, and Tcell-derived cytokines canoptimal selection of Bcells gen, even Bcells can be the priming cells for the TFH be visualized in the follicular regions following initialthat produce antigen-binding cell programme, as in the case of priming with particu- Tcell contact with Bcells4244. Different cytokines haveantibodies. late virus-like particles30. Dynamic imaging has placed been reported to drive commitment to different anti-Activation-induced cytidine initial contact between TFH cells and antigen-primed body classes. For example, IL4 promotes IgG1 and IgEdeaminase Bcells within the follicular regions of lymphoid tis- class switching 45; interferon (IFN) induces IgG2a45;(AID). An enzyme that is sue31. The expression of the adaptor molecule SAP and transforming growth factor- (TGF) directs com-required for two crucialevents in the germinal centre: (SLAM-associated protein) can regulate BcellTFH mitment to IgA46. In this manner, pre-GC BcellTFH cellsomatic hypermutation and cell contact duration and affect antigen-specific Bcell contact, involving the delivery of different cytokines,class-switch recombination. fate32. More recently, dynamic imaging has shown that can imprint antibody class among the progeny of the26 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/immunol 2012 Macmillan Publishers Limited. All rights reserved

4. REVIEWS a IFN IL-4 IL-21 IL-10 IL-17 IL-12 IL-5 TH1-like TFH cell TH2-like TFH cell IL-21 producing IL-10 producing TH17-like TFH cell TFH cell TFH cell b Pre-GC TFH cell Antigen-primed B cell c IgG1 IgG2a IgA CXCR5 BCR Bcl6 Bcl6 OX40 OX40L Bcl6 Cytokine Cytokine receptor ICOS ICOSL GC pathway Bcl6 Peptide TCR Non-GC pathway MHC class II CD40L CD40 Blimp1 IL-21 IL-21R Blimp1 Blimp1 SLAM SLAM Cognate IgA contact Plasma cell IgG2a IgG1 Figure 2 | Pre-GC phase: commitment to memory. a | Multiple subsets of antigen-specific pre-germinal centre (pre-GC) Tfollicular helper (TFH) cells are produced to regulate Bcell immunity. So far, the organization of theseReviewsremains Nature subsets | Immunology speculative; there is evidence for distinct TFH cell populations that secrete different cytokines and regulate commitment to separate antibody classes, as well as for other types of TFH cells that regulate non-GC plasma cell differentiation. Expression of Bcell lymphoma6 (BCL6) and CXC-chemokine receptor5 (CXCR5) is thought to be a common feature of all TFH cell subsets. b | Antigen-primed Bcells must process and present peptideMHC classII complexes to receive cognate help from pre-GC TFH cells. Upregulation of the molecules involved in TFH cell contact is a poorly resolved component of early antigen-driven Bcell maturation. Cognate contact between antigen-specific Tcell receptors (TCRs) and peptideMHC classII complexes focuses the intercellular exchange of molecular information between pre-GC Bcells and TFH cells. The modifying interactions that occur at first contact are known to involve co-stimulatory molecule interactions (for example, CD40LCD40 and inducible Tcell co-stimulator (ICOS)ICOSL), accessory molecule interactions (for example, SLAM family interactions and OX40OX40L) and interactions between cytokines and their receptors (for example, interleukin4 (IL4)IL4R, interferon- (IFN)IFNR and IL21IL21R). The distribution of these functional attributes in pre-GC TFH cell compartments is not yet well resolved invivo. c | The non-GC pathway to plasma cell development permits antibody class-switch recombination without somatic hypermutation, and the outcome depends largely on the cytokine stimulus provided by pre-GC TFH cells. Blymphocyte-induced maturation protein1 (BLIMP1) expression is required for plasma cell commitment across all antibody classes. The GC pathway to memory Bcell development begins with extensive Bcell proliferation in secondary follicles that polarize into dark and light zones to initiate the GC reaction. The GC pathway is associated with BCL6 upregulation and AID expression to support both class-switch recombination and somatic hypermutation. These GC features enable the generation of all antibody classes and require a long duration of productive contact with pre-GC TFH cells. TH, T helper.Non-homologous endjoining(NHEJ). A mechanism for antigen-responsive Bcells. This commitment to anti- Finally, Ikaros regulates antibody class decisions by dif-repairing double-strand DNA body class appears to define an early and distinct devel- ferentially controlling the transcriptional accessibility ofbreaks that does not requirehomologous sequences for opmental fate for antigen-primed Bcells with functional constant region genes50. How the initial commitmentligation. NHEJ is used to consequences that remain poorly understood. to antibody class is maintained and propagated duringcomplete recombination during In antigen-responsive Bcells, the molecular machin- clonal expansion, BCR diversification and affinity-basedantibody class switching. ery that regulates CSR is deployed in an antibody class- selection within the GC reaction remains an importantSomatic hypermutation specific manner. The global CSR machinery is targeted by but unresolved issue. However, it remains plausible thatA process in which point transcription factors downstream of the cytokine recep- functional reprogramming accompanies CSR and createsmutations are generated in the tors that control specific antibody classes. For example, separable lineages of class-specific memory Bcells invivo.immunoglobulin IFN activates signal transducer and activator of tran-variable-region gene segments scription1 (STAT1) downstream of the IFN receptor Cognate contact initiates GC formation. Initial pre-of cycling centroblasts. Somemutations might generate a to induce Tbet and promote IgG2a class switching 47,48. GC contact between Bcells and antigen-specific TFHbinding site with increased Similarly, TGF signals through the TGF receptor to cells promotes a major division in the developing Bcellaffinity for the specific antigen, activate SMAD and RUNX transcription factors that response. Some antigen-primed Bcells proceed towardsbut others can lead to loss of promote IgA class switching 49. Furthermore, the tran- plasma cell differentiation at this early stage. This earlyantigen recognition by theBcell receptor or the scriptional regulator BATF, which is required for TFH cell Bcell fate occurs via an extrafollicular Bcell pathway,generation of a self-reactive development, is also required in Bcells to generate germ and thus these Bcells do not enter a GC reaction51.Bcell receptor. line switch transcripts and to promote AID expression28. There is also recent evidence for an early memory BcellNATURE REVIEWS | IMMUNOLOGY VOLUME 12 | JANUARY 2012 | 27 2012 Macmillan Publishers Limited. All rights reserved

5. REVIEWS pathway that does not involve the GC reaction25,52. CSR then promote antigen-specific clonal expansion and proceeds in these non-GC pathways, supporting the BCR diversification followed by positive selection of notion of an early, pre-GC commitment to antibody high-affinity BCR variants51. Within the GC, Bcells scan class. The GC pathway to memory Bcell development antigens presented by follicular DCs and, following suc- is the other developmental fate imprinted at this early cessful antigen binding, make contact with cognate GC stage of the Bcell response, and this is the major focus TFH cells62. GCs must also delete ineffective BCR variants of thisReview. and guard against self-reactivity, a feature of the GC that Effective, antigen-specific contact between pre-GC remains poorly understood. Under the control of cognate Bcells and TFH cells is required for the non-GC plasma GC TFH cells, Bcells that express high-affinity BCR vari- cell pathway, although the short-duration BcellTFH cell ants are exported from the GC to build multiple facets of interactions that occur in the absence of SAP appear antigen-specific Bcell memory. In this manner, the GC to be sufficient 32. ERK signalling in Bcells is needed cycle of activity regulates clonal composition and ulti- to induce the transcriptional repressor BLIMP1 and mately the long-term immune function of class-specific plasma cell differentiation18. Regulation of the unfolded- high-affinity memory Bcells (FIG.3). protein response by Xbox-binding protein1 (XBP1) is not needed for plasma cell development but is necessary Clonal expansion and BCR diversification. Early mod- for antibody secretion53,54. EpsteinBarr virus-induced els report intense Bcell clonal expansion in follicular Gprotein-coupled receptor2 (EBI2) also appears to be regions that locally exclude naive Bcells to form sec- essential for Bcell movement to extrafollicular sites and ondary follicles63. Polarization of secondary follicles the non-GC plasma cell response55,56. into light zones that are rich in follicular DCs and GC In addition, EBI2 guides recently activated Bcells to TFH cells and dark zones that contain many proliferat- interfollicular lymph node regions and then to outer fol- ing Bcells provides an anatomical definition of an active licular areas as a prelude to GC formation. Futhermore, GC microenvironment 62. Although Tcell-independent there appears to be an early, pre-GC proliferative phase GClike structures can emerge when there are increased at the perimeter of follicles that also precedes GC for- numbers of Bcell precursors, these structures are short- mation and BCR diversification57. Interestingly, recent lived and do not support the diversification or positive dynamic imaging studies indicate that TFH cells migrate selection of BCRs. Hence, the capacity for affinity matu- to the follicle interior, even before the accumulation of ration and memory Bcell development can be consid- GC Bcells33. ered as an integral functional component of a dynamic It has been unclear how differential BCR affinity can GC reaction invivo (FIG.4). affect the early fate of antigen-primed Bcells. Bcells The discovery of AID provided crucial insight into of very low affinity are capable of forming GCs58 but the molecular machinery that drives somatic hyper fail to do so in the presence of high-affinity competi- mutation and BCR diversification64. Similarly to its func- tion59. By contrast, there is evidence that the highest tion in CSR, AID is required for cytosine deamination affinity Bcells preferentially enter the non-GC plasma to generate uracils that recruit the somatic hypermuta- cell pathway, leaving lower affinity Bcells to mature tion machinery. The initial changes target sequence- in the GC cycle60. This issue has been addressed more specific hotspots within the rearranged variable regions recently using intravital imaging to examine the early, of antibody genes. Following uracil excision by uracil pre-GC selection events61. In this model, access to anti- DNA glycosylase (UNG), the DNA is processed by error- gens was not affected by BCR affinity, but the capacity prone DNA replication to introduce point mutations in of Bcells to present antigens to pre-GC TFH cells was the actively transcribed immunoglobulin locus. Error- associated with BCR affinity. Increased Tcell help pro- prone processing using mismatch repair and base exci- moted greater access to both the plasma cell pathway sion repair factors is selectively offset with high-fidelity and the GC reaction. Thus, BCR affinity thresholds processing to protect genome stability 65. The range of regulate Bcell fate at the earliest pre-GC junctures of sequences that are targeted by AID (as determined by the antigenspecific BcellTFH cell interactions. enzymes active site) can be altered to modify the somatic Effective priming by antigens initiates the pre-GC hypermutation of variable-region gene segments66 and phase of memory Bcell programming. Naive antigen- the rate of antibody diversification67. AID stability in the specific Bcells must take up, process and present anti- cytoplasm of Ramos Bcell lines can be regulated by heat gens to receive cognate help by antigen-specific TFH cells. shock protein90 (HSP90); specific inhibition of HSP90 These early TFH cell programmes drive commitment to leads to destabilized AID68, and this provides a means to antibody class, non-GC plasma cell differentiation and modify the rate of antibody diversification. The details GC formation to influence crucial facets of adaptive of the mutating complex that contains AID, its action Bcell immunity and long-term Bcellmemory. and its regulation in the GC reaction are active areas ofUnfolded-protein response research that have been reviewed in detail elsewhere64.A response that increases the Affinity maturation BCR diversification is dependent on DNA rep-ability of the endoplasmic The GC cycle. GCs are dynamic microanatomical struc- lication and is largely restricted to GC Bcells in thereticulum to fold and tures that arise in the follicular regions of secondary pathway to memory. Earlier studies using dynamictranslocate proteins, decreasesthe synthesis of proteins, and lymphoid tissues to support the generation of high- imaging indicated that GC Bcell proliferation occurscan cause cell cycle arrest and affinity Bcell memory. As discussed, entry into the GC in both the light zone and the dark zone of the GCapoptosis. reaction is regulated by antigen-specific TFH cells20. GCs reaction6971. There was also evidence for significant28 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/immunol 2012 Macmillan Publishers Limited. All rights reserved

6. REVIEWS zonal movement and cellular exchange between these ICOSICOSL interactions are important throughout this areas69,72. More recently, labelling of Bcells based on pathway, at the early DC contact 29 and pre-GC contact 75 their GC zonal location (using a photoactivatable stages and probably during the GC reaction itself. IL21 green fluorescent protein (GFP) tag) provided more and its receptor appear to be of continued importance conclusive evidence for these activities invivo73. These elegant studies indicated that proliferation was largely GC reaction restricted to the dark zone and that this was followed by a net movement to the light zone. Importantly, movement back into the dark zone and re-initiation Follicular DC of proliferation was controlled by antigen presentation to GC TFH cells73. These studies provide experimental evidence that the reiterative cycles of BCR diversifi- scanning Cog DC na cation and positive selection are central events during lar te icu affinity maturation that drive clonal evolution in the co ll Fo nta antigenspecific memory Bcell compartment. ct Light B cell zone TFH cell Antigen scanning on follicular DCs. Affinity maturation Apoptotic refers to the rising affinity of antigen-specific antibodies B cell Dark that can be measured over time following infection or zone n Clas vaccination. Cell death is a prevalent outcome of the GC nsio cycle51, and myeloid cell leukaemia sequence1 (MCL1) s sw pa has emerged as a major anti-apoptotic factor controlling Ex h it c B cell GC Bcell formation and survival16,74. Positive selection zone of variant GC Bcells must be a major driving force in the D iv ersi c a tio n GC and is based on the increased capacity of the mutated BCR to bind to its antigen. Direct imaging studies pro- Figure 3 | The antigen-specific GC reaction. The germinal vided the first dynamic view of GC Bcell and follicular centre (GC) cycle is initiated throughReviews | Immunology Nature the pre-GC contact DC interactions6971. All groups reported a continuous of Bcells with cognate Tfollicular helper (TFH) cells, as this scanning activity of GC Bcells over follicular DC net- promotes the extensive proliferation of antigen-primed works that were laden with immune complexes. These Bcells. The GC cycle is thought to begin when an IgD GC Bcell movements were more reminiscent of stromal secondary follicle polarizes to form two microanatomically scanning activity than of cognate immune synapse paus- distinct regions: the Tcell zone-proximal dark zone ing by T helper cells on antigen-presenting cells (APCs). (which contains proliferating centroblasts) and the Tcell These images show the stage at which variant GC Bcells zone-distal light zone (which contains centrocytes, are most likely to contact antigens to test the binding antigen-laden follicular dendritic cell (DC) networks and antigen-specific GC TFH cells). The clonal expansion of properties of their mutatedBCRs. antigen-specific GC Bcells in the dark zone is accompanied by Bcell receptor (BCR) diversification through somatic Cognate contact with GC TFH cells. After scanning fol- hypermutation, which introduces point mutations into the licular DCs, only a few GC Bcells were shown to make variable-region segments of antibody genes. Antibody stable, immune synapse-like contacts with GC TFH cells, class-switch recombination can also proceed under as determined by two-photon imaging 69. These early these circumstances. Both somatic hypermutation and images gave rise to the notion that competition for GC class-switch recombination are associated with TFH cells may be the limiting factor in GC Bcell selection transcriptionally active gene loci, require DNA replication of variant high-affinity BCRs62. More recently, antigen and repair machinery and occur during the cell cycle. presentation by GC Bcells without BCR engagement was Hence, these activities have been associated with the dark-zone phase of the GC cycle. Exit from the cell cycle shown to dominate the selection mechanism in GCs73. coincides with the relocation of non-cycling GC Bcells to GC Bcells that were capable of presenting higher lev- the light zone. Continual scanning of follicular DCs that are els of antigen exited the GC reaction rapidly and pro- coated with immune complexes is observed in the light duced more post-GC plasma cells than GC Bcells that zone and has been associated with the potential for GC were less efficient at antigen presentation. These studies Bcells to test their variant BCRs for antigen-binding ability. implicated similar mechanisms to those of the pre-GC Loss of antigen binding can lead to death by apoptosis and selection event 61 and argued strongly that antigen pres- the clearance of dead cells by tingible body macrophages entation to GC TFH cells is the rate-limiting event during in the light zone. Positive signals through the BCR during affinity maturation in the GCcycle. the scanning of follicular DCs program GC Bcells to It remains technically difficult to manipulate cellular compete for contact with cognate GC TFH cells. Productive contact with GC TFH cells can induce re-entry into the GC and molecular activities in the GC cycle without inter- cycle; this involves movement back into the dark zone, the fering with the developmental programmes that initiate induction of the cell cycle and BCR re-diversification. the GC reaction in the first place. Many of the molecules Alternatively, affinity-matured GC Bcells can exit the GC, associated with pre-GC TFH cell function may also func- either as non-secreting memory Bcell precursors for the tion within the GC. BCL6 expression itself is reinforced memory response, or as secreting long-lived memory in TFH cells following contact with pre-GC Bcells31. plasma cells that contribute to serological memory.NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | JANUARY 2012 | 29 2012 Macmillan Publishers Limited. All rights reserved

7. REVIEWSc Follicular DC scanning d Cognate control Follicular DC IFN IL-4 FcR CR2 CD40L IL-17 IL-12 IL-5 TH1-like TH2-like TH17-like C3 TFH cell TFH cell TFH cell IgG Adhesion BCR molecule GC TFH cell B cell CD40 IL-21R Chemokine CXCR5 Peptide MHC class II Chemokine PD-1 receptor BCR SLAM GC reaction Bcl6 Adhesion Adhesion Co-stimulatory Co-stimulatory B cell Light Bcl6 Peptide Bcl6 zone TCR MHC class II Accessory Accessory Cytokine Cytokine receptor Dark zone SLAM SLAMa Commitment to antibody class e Dark zone IgG1 re-entry f GC exit B cell b Cell cycle, CSR and SHM Memory plasma cell Bcl6 Blimp1 Memory B cell Cell cycle proteins IgG2a IgG1 AID Expansion Blimp1 IgG1 Bcl6 Transcription Error-prone factor polymerase DNA repair IgA IgG2a SHM CSR IgG2a AID AID Blimp1 Transcription Bcl6 factor Transcription factor IgA IgA Figure 4 | Memory B cell evolution. a | Cues from pre-germinal centre (pre-GC) cognate Tfollicular helper (TFH) cells instruct Nature Reviews | Immunology antigen-primed Bcells to initiate the GC reaction. It is likely that the commitment to antibody class is pre-programmed at this initial juncture and that all classes of Bcells can seed the primary GC response. b | Molecular control of the cell cycle is an integral component of dark-zone Bcell dynamics and involves the expression of Bcell lymphoma6 (BCL6), although the ways in which BCL6 contributes to this regulation remain poorly resolved. The expression and activity of activation-induced cytidine deaminase (AID) and uracil DNA glycosylase (UNG) are required to initiate somatic hypermutation (SHM), which is targeted to single-stranded DNA. Following uracil excision, the DNA is processed by error-prone DNA polymerases to introduce point mutations into the variable regions of the rearranged antibody genes. Class-switch recombination (CSR) can also occur during this dark-zone phase using AID to target DNA cleavage to antibody switch regions; the DNA double strand breaks that are generated trigger the DNA damage machinery, which completes the CSR event. The associations between cell cycle control, SHM and CSR are not clearly resolved invivo. c | To scan folicullar dendritic cells (DCs) for antigens, GC Bcells continuously move along follicular DC processes that are laden with mature immune complexes. These interactions are more similar to stromal cell-associated trafficking behaviour than to stable immune synapse-like interactions. The affinity of the Bcell receptor (BCR) for antigens may influence antigen uptake and peptideMHC classII presentation at this juncture of development. Programmes of gene expression for molecules that are able to modify cognate contact may also be differentially induced as a result of BCR signal strength during follicular DC scanning. d | B cells then make contact for a longer duration with cognate GC TFH cells in the light zone, and this can be visualized directly invivo. As in earlier, pre-GC events, these contacts must focus around Tcell receptor (TCR)peptideMHC classII interactions and can be modified by a multitude of intercellular exchanges of molecular information. There is still little detailed analysis of these interactions invivo. We depict the classes of molecules that can be associated with this crucial programming event, but the organization of these interactions and their precise developmental imprint are not yet clear. e | Antigen presentation by Bcells can influence re-entry into the dark zone and the re-initiation of BCR diversification (which involves cell proliferation, SHM and CSR). f | GC cognate contact can also initiate Bcell exit from the GC into the distinct non-secreting memory Bcell and post-GC long-lived memory plasma cell compartments. BLIMP1, Blymphocyte-induced maturation protein1; CR2, complement receptor 2; CXCR5, CXC-chemokine receptor5; IFN, interferon; IL, interleukin; PD1, programmed cell death protein1; TH, Thelper.30 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/immunol 2012 Macmillan Publishers Limited. All rights reserved

8. REVIEWS at the pre-GC stage and during the GC reaction 25,26. Genetic labelling of AID-expressing cells with yellow Sphingosine-1phosphate receptor2 (S1P2) has an impor- fluorescent protein (YFP) has allowed memory Bcells tant role in confining GC Bcells to the GC niche invivo76. to be monitored over long periods81. Surprisingly, it was In addition, elevated expression levels of programmed shown that primary-response GC reactions could per- cell death protein1 (PD1) correlate with GC localization sist for extended periods of time (over 8months after of the TFH cell compartment 77, and the absence of PD1 priming) following immunization with certain types of ligand2 on Bcells affects plasma cell generation and antigen. In these studies, class-switched memory Bcells affinity maturation78. Most interestingly, the associa- rapidly promoted plasma cell generation, whereas their tion between cytokine production and class-specific GC IgM+ counterparts promoted secondary GC reactions. Bcells appears to continue in the GC long after the origi- Depending on the form of antigen delivery and the nal CSR event43. This surprising functional pairing of GC combination of innate stimuli provided with the anti- TFH cells and class-switched GC Bcells for example, gen, Bcell responses could be skewed towards memory IL4+ TFH cells with IgG1+ GC Bcells and IFN+ TFH cells formation with extended GC reactions, which can last with IgG2a+ GC Bcells hints at the extended level of over 1.5years82. Hence, it is possible that persistent GCs heterogeneity that exits in the GC cycle of memory Bcell can continuously produce non-secreting memory Bcells development. Hence, it is likely that each separable class- well after the initial primingevent. specific GC Bcell compartment requires cognate contact High-affinity antibody-producing plasma cells that with separate class-specific GC TFHcells. emerge from the GC reaction can also be considered an integral part of antigen-specific Bcell memory. High- Clonal evolution in the GC. Evidence connecting BCR affinity GC Bcells preferentially assort into the plasma signal strength in the GC Bcell compartment and affinity cell compartment and produce high-affinity circulat- maturation has been lacking. BCR signalling and anti- ing antibodies83. In the lymph nodes, affinity-matured gen presentation are required to initiate the GC reaction plasma cells dwell in paracortical areas to mature84 and and thus are difficult to manipulate specifically in the then migrate towards the medullary regions before GC. Early GCs still develop in the absence of DOCK8, export 85. CD93 is expressed at this early stage and is despite the defects in early immune synapse formation10. required for plasma cell survival in the bone marrow 86. However, without DOCK8 these GCs do not persist and Clearly, the circulating antibodies that are produced by GC Bcells do not undergo affinity maturation. Calcium post-GC plasma cells contribute to ongoing serological influx as a consequence of BCR signalling also appears immune protection87. to be dispensable for affinity maturation under various We have recently demonstrated that post-GC Tcell-dependent priming conditions invivo. Although antibody-secreting Bcells not only express BCRs, but Bcells deficient for the calcium sensors STIM1 and also present antigens and can modulate cognate Thelper STIM2 or for CNB1 exhibit profound defects in prolif- cell responses88. These surprising studies further dem- eration invitro14,11, these signalling molecules are dispen- onstrate that plasma cells negatively regulate the expres- sable for the maturation of antibody responses invivo. sion of BCL6 and IL21 in antigen-specific TFH cells88. Downstream of BCR signals, the transcription factor Thus, plasma cells are not only the producers of anti- MEF2C is necessary for early Bcell proliferation and bodies; they can also engage in antigen-specific immune GC formation12,13, but the pre-GC versus GC functions regulation. Signals through the BCR or MHC classII of MEF2C remain unresolved. Bcell-specific deletion of molecules on post-GC plasma cells may serve to regulate nuclear factor of activated Tcells, cytoplasmic1 (Nfatc1) the ongoing production of high-affinity antibodies in also compromises Bcell responses invivo79, but the level the serum. The long-term antigen-presenting or regula- of the defect remains unclear. Nevertheless, as BCR signal tory function of post-GC plasma cells has not yet been strength must drive affinity maturation at some level, it elucidated. remains important to resolve the Bcell-intrinsic mecha- nisms that help to shape the affinity of the memory Bcell Antigen persistence. Tonic signalling through the compartment. BCR and the downstream activation of phospho inositide3kinase (PI3K), together with signalling by Bcell memory and antigen recall Bcell-activating factor (BAFF) through the BAFF recep- Bcell memory. The population of non-immunoglobulin- tor, are required for the survival of naive Bcells in the secreting cells that is produced in the GC reaction dur- periphery 89,90. Similarly, inducible deletion of phospho ing a primary response largely comprises class-specific lipaseC2 (Plcg2) after the generation of antigen- affinity-matured memory Bcells. There are reports of specific memory Bcells substantially depleted the early memory Bcell development that does not occur memory Bcell compartment and suggested a BCR in GCs25,52, although how well these germline BCR- signalling requirement for memory 91. Nevertheless, expressing memory Bcells compete with post-GC mem- earlier genetic studies indicated that cognate BCR ory Bcells in the antigen recall response remains to be specificity was not required to provide the tonic sur- evaluated. Affinity-matured IgM+ memory Bcells can vival signal after the generation of memory Bcells89. emerge from the GC reaction and persist for long periods Thus, persistent antigen does not appear to be invivo80. These non-switched memory cells appear to required for the survival of antigen-specific memory be more active in secondary responses in the absence of Bcells, although memory Bcell function has not been circulating antibodies. addressed in thismodel.NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | JANUARY 2012 | 31 2012 Macmillan Publishers Limited. All rights reserved

9. REVIEWS More recently, there has been evidence of persis- confining the antigen-specific memory TFH cell com- tent peptideMHC classII complexes in the context of partment to lymph nodes that drained the site of initial antiviral responses invivo92, leading to local activation priming 20. Although it has been known for some time of naive Thelper cells even after the clearance of the that follicular DC networks are capable of trapping virus. We recently demonstrated a similar persistence whole antigens as immune complexes for extended peri- of peptideMHC classII complexes for longer than ods of time62, the nature of the long-lived local APCs 100days following vaccination with a protein antigen remains unresolved. in a non-depot adjuvant 93. The depots of peptideMHC classII complexes were restricted to the lymph nodes Recalling Bcell memory that drained the initial vaccination site, and persistent Antigen recall responses by memory Bcells promote antigen presentation induced naive Thelper cell pro- accelerated clonal expansion and rapid differentiation liferation93. We proposed that peptideMHC classII to high-affinity plasma cells. IgG1 BCRs show enhanced complexes on immunocompetent APCs had a role in signal initiation and microclustering at the single-cell level compared with IgM BCRs owing to membrane- proximal regions in the cytoplasmic tails of IgG1 BCRs Memory response Secondary GC (REF.94). The cytoplasmic tails of these BCRs in class- switched memory Bcells can contribute substantially to Follicular a Antigen DC the increased burst of clonal expansion that is associ- uptake ated with re-triggering by antigen95. There is evidence f Memory Memory for distinct changes in BCR signalling pathways9699. B cell e The increased affinity of the BCR on memory cells must B cell zone also contribute to memory Bcell sensitivity to low-dose c soluble antigens that do not induce a primary immune b response. In addition to these intrinsic attributes, cir- TCR MHC class II d culating high-affinity antibodies contribute to the differential management of antigens invivo. Rapid pres- T cell zone Memory entation of immune complexes to the memory Bcells TFH cell is enhanced. Furthermore, memory Bcells require Memory regulation by antigen-specific Thelper cells to initiate plasma cell secondary immune responses100. These issues have not 1 5 been well studied but remain central to the capacity of Days after recall memory Bcell populations to expand and self-replenish Figure 5 | Memory response to antigen recall. and to boost the levels of high-affinity plasma cells and a|Memory Bcell responses can emerge in the absence circulating antibodies that provide long-term immune Nature Reviews | Immunology of innate inflammatory stimuli. In this case, the main protection (FIG.5). antigen-presenting cells are the affinity-matured memory There has been recent evidence that memory Bcells Bcells themselves. b | The memory Bcell response to Tcell-dependent antigens still requires Thelper (TH) can re-initiate a GC reaction following antigen recall. cell-mediated regulation following antigen recall. When the The type of antigen appears to have an impact on the priming and recall antigens are identical, memory TH cells persistence of the primary-response GC, with particu- are the rapid responders and are thought to emerge late antigens more likely to promote GC longevity 81. preferentially over their low-frequency naive counterparts. Moreover, innate immune stimuli differentially affect Regarding the regulation of memory Bcell responses, persistent GC structures, with combinations of Toll-like antigen-specific memory Tfollicular helper (TFH) cells are receptor4 (TLR4) and TLR7 signals more effective than the most likely candidates for rapid cognate regulation. single stimuli82. Whether the secondary GC is a con- c|Cognate contact at this developmental juncture occurs tinuation and re-expansion of a primary GC remains across sets of memory Bcells and memory TFH cells, but the unclear. More importantly, it remains to be determined organization and kinetics of this process remain poorly resolved invivo. There is rapid and vigorous local clonal whether these secondary or persistent GClike struc- expansion during the first 23days after antigen tures support the re-diversification of affinity-matured exposure in both the memory Bcell and memory TFH cell BCRs and the selection of clonotypes with even higher compartments. d|Proliferation of affinity-matured memory affinities. These issues are central to the future manage- plasma cells occurs very quickly, and evidence suggests that ment of primeboost vaccination protocols and have most memory plasma cells have already undergone affinity substantial practical impact in thisfield. maturation. e | There is evidence for memory Bcell subsets that have a germinal centre (GC) phenotype and create Cognate contact with memory TFH cells. As discussed GClike structures following antigen recall. Whether these above, we have provided evidence for the local persis- structures are residual from the primary-response GC or tence of an antigen-specific memory TFH cell compart- re-emerge with GC activities following recall has not been resolved. f | Increased numbers of memory Bcells and ment. CXCR5+ TFH cells bind to peptideMHC classII memory-response plasma cells persist after antigen recall. complexes with higher affinity, express lower levels Itremains unclear whether these cells are the product of of ICOS and have lost the capacity to express mRNAs memory GC reactions or of the extrafollicular, non-GC encoding a range of cytokines, as compared with effec- memory response. DC, dendritic cell; TCR, Tcell receptor. tor Thelper cells21. These putative memory TFHcells32 | JANUARY 2012 | VOLUME 12 www.nature.com/reviews/immunol 2012 Macmillan Publishers Limited. All rights reserved

10.