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Critical Reviewsin Immunology, 30(1):129 (2010)

11040-8401/10/$35.00 2010 by Begell House, Inc.

Received: 8/17/09; Accepted: 11/11/09

Toll-Like Receptors and B-Cell Receptors Synergizeto Induce Immunoglobulin Class-Switch DNARecombination: Relevance to Microbial AntibodyResponses

Egest J. Pone, Hong Zan, Jinsong Zhang, Ahmed Al-Qahtani,Zhenming Xu, & Paolo Casali*

Institute or Immunology, School o Medicine and School o Biological Sciences, University oCaliornia, Irvine, Caliornia

*Address all correspondence to Dr. Paolo Casali, Institute or Immunology, 3028 Hewitt Hall, University o Caliornia, Irvine, CA 92697-4120;el.: 949- 824-4456; Fax: 949-824-2305; [email protected].

ABSTRACT: Dierentiation o nave B cells, including immunoglobulin class-switch DNA recombination, is criti-cal or the immune response and depends on the extensive integration o signals rom the B-cell receptor (BCR),tumor necrosis actor (NF) amily members, oll-like receptors (LRs), and cytokine receptors. LRs and BCRsynergize to induce class-switch DNA recombination in cell-dependent and cell-independent antibody responsesto microbial pathogens. BCR triggering together with simultaneous endosomal LR engagement leads to enhancedB-cell dierentiation and antibody responses. Te requirement o both BCR and LR engagement would ensureappropriate antigen-specic activation in an inection. Co-stimulation o LRs and BCR likely plays a signicant rolein anti-microbial antibody responses to contain pathogen loads until the cell-dependent antibody responses peak.Furthermore, the temporal sequence o dierent signals is also critical or optimal B cell responses, as exempliedby the activation o B cells by initial LR engagement, leading to the up-regulation o co-stimulatory CD80 andMHC-II receptors, which result in more ecient interactions with cells, thereby enhancing the germinal centerreaction and antibody anity maturation. Overall, BCR and LR stimulation and the integration with signals rom

the pathogen or immune cells and their products determine the ensuing B-cell antibody response.

Key woRdS: activation-induced (cytidine) deaminase, adaptive immunity, adjuvant, antibody, autophagy,B lymphocyte, B cell receptor, class-switch DNA recombination, CD40, CpG, cytokine, dendritic cell, germlinetranscription, immunoglobulin, innate immunity, LPS, microbe-associated molecular patterns, NF-B, patternrecognition receptors, somatic hypermutation, cell, oll-like receptor, vaccine.

ABBREVIATIONS

APC, antigen-presenting cell; CSR, class-switch DNA recombination; dC, dendritic cell; GC, germinal center;LPS, lipopolysaccharide; LRR, leucine-rich repeat; MAMP, microbe-associated molecular patterns; NK, naturalkiller; NLR, NOD-like receptor; PRR, pattern recognition receptors; SHM, somatic hypermutation; TCR, -cellreceptor; TLR, oll-like receptor; VLR, variable leucine-rich receptor

I. B-CELL DIFFERENTIATION VIA INNATEAND ADAPTIVE IMMUNE RECEPTORS

A. The Innate Immune Response

Te protection rom a nearly unlimited numbero pathogens encountered during the lietime o

an individual is achieved through the integrationo innate and adaptive immune responses, whichare intrinsically linked by crosstalk through cellsand molecules.1-6 Te immune system detectsand eliminates invading pathogenic microorgan-

isms, as well as inected and neoplastic sel-cells,by distinguishing them or associated molecules

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TABLE 1. Features of Innate and Adaptive Immunity

Feature Innate Immunity Adaptive Immunity

Specicity Low Very high

Receptor clonal distribution No Yes

Receptor diversity Encoded in germline Recombined gene segments

Response timeline No lag Lag time or induction

Eciency o response Moderately ecient Highly ecient

Memory No Yes

rom normal sel-cells and sel-molecules. Whilelower organisms have only an innate immunesystem, higher organisms, beginning with jawedvertebrates, in addition possess a more sophisti-cated system: the adaptive, or acquired, immunesystem.7-10 Innate immunity provides an early,though non-specic, initial response to patho-gens.7,11-14 In contrast, adaptive immunity requiresa lag period or activation, is specic, diverse andhighly ecient in targeting o pathogens8,9,15,16(able 1).

Te innate immune system is a universal andancient orm o host deense against inection.7,10,17In mammals, it is composed o diverse cell types,including macrophages, dendritic cells (DCs),neutrophils, basophils, mast cells, eosinophils,and natural killer (NK) cells; and molecules and

molecular systems, such as anti-microbial pep-tides, acute-phase proteins (collectins, colins,and pentraxins) and complement. 8,9,13 Innateimmune cells constantly sample the environ-ment or the presence o pathogens through theirgermline-encoded pattern recognition receptors(PRRs).3,7,9,13,18 PRRs primarily bind to non-sel molecules that have conserved eatures known asmicrobe-associated molecular patterns (MAMPs);an alternative term is pathogen-associated molecu-lar patterns (PAMPs). MAMPs are unique chemi-cal structures ound in the microorganism butgenerally absent in the higher hosts. For example,lipopolysaccharide (LPS) contains sugars and lip-ids composed o chemical bond arrangements notound in higher eukaryotes, thereby constituting aunique MAMP. Another MAMP is CpG (unm-ethylated, CG-containing DNA) rom bacteriaand viruses.19 LPS and CpG are recognized byan important subset o PRRs known as oll-likereceptors (LRs).3,20-24 Other PRR classes include

NOD-like receptors (NLRs), CARD helicases,C-type lectins, and scavenger receptors3,10,25 (able2). Some o these receptors may be unctional inB cells, however, their eects on B-cell antibodyresponses are not known; thereore, this review willocus on the unctions o LRs in B cells, whichhave been more extensively investigated. Atersensing pathogens via their PRRs, macrophagesand mast cells are activated to phagocytose thepathogen, display peptide ragments on MHCII(major histocompatibility II) receptors, and secretepro-inammatory cytokines and lipid mediators.Likewise, stimulation o PRRs on immature DCsleads to their maturation and activation, which inturn activates adaptive immunity.7,10,13,26

B. The Adaptive Immune Response

Evolutionarily, adaptive immunity emerged aterinnate immunity by the need to respond to spe-cic and mutating antigens through the use oantigen-specic receptors on the suraces o B and lymphocytes.1,4,15,27,28 Even though only jawedvertebrates possess a true adaptive immune system,recent evidence indicates that jawless vertebrates,such as lamprey and hagsh, contain lymphocyte-like immune cells expressing variable leucine-richreceptors (VLRs), which are triggered to rearrangeupon inection with a pathogen, and display aspecicity or antigen similar to antibodies o thejawed vertebrate adaptive immune system.29-36Te adaptive immune response enables highervertebrate animals to adapt their response to apathogen to be more specic and eective, andto elicit immunological memory, that is, to mounta aster and stronger response during subsequentencounters with the same pathogen.



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TLR and BCR Induction of CSR 3

he mechanisms o generating antigen-specic receptors in the adaptive system involveextensive variability and rearrangement o receptorgene segments. During their development, B and lymphocytes rearrange IgH and Ig/ chains inB cells, and and chains in cells, that code or

BCRs and -cell receptors (CRs), respectively.Lymphocytes display on the extracellular suraceo their plasma membranes multiple (hundreds tothousands) identical copies o their BCR or CRwith unique antigen specicity among the poolo total lymphocytes.37,38 Te rearrangement andclonal distribution o antigen receptors is dierentrom the previously described PRRs, which do notrearrange and usually have the same sequence in allcells that express them in an organism (able 1).When antigen receptors bind arrays o repetitiveantigens on a bacterium, virus, or parasite, they

cluster together, thereby bringing into proximitymembrane-associated and intracellular signalingmolecules, contributing to lymphocyte dierentia-tion. Such an antigen receptor cross-linking processonly occurs in those B and lymphocytes thatexpress receptors with an above-threshold anityor antigen to ensure that only antigen-speciclymphocytes are amplied and dierentiate intoeector and memory cells, which have the samespecicity or the antigen that initiated and drovethe response.39,40 Antibodies (and BCRs) are ur-ther diversied by class-switch DNA recombina-tion (CSR) and somatic hypermutation (SHM).

C. B-Cell Differentiation

B lymphocytes play major roles in the immuneresponse by producing antibodies against antigens,by unctioning as proessional antigen-presentingcells (APCs), and by producing cytokines.41-44

Antibodies can directly inactivate pathogens,45-47can opsonize pathogens to assist innate immunecells in eliminating pathogens, and, nally, canactivate complement.8,9,48 Conversely, B cells aredirectly and indirectly inormed about the pres-ence and nature o pathogens by innate immune

elements such as LRs,10,13,49

and are indirectlyinormed by complement actor C3d.50-52 Dysregu-lated and mis-targeted B cell antibody responsescould result in autoimmunity, whereas impairedantibody responses during an actual inectioncould result in an immune deciency.

In mammals, B cells begin their developmentin the bone marrow rom lymphoid progeni-tor cells, which in turn derive rom pluripotenthematopoietic stem cells. B cells derive their namerom the bursao Fabricius in chickens, which isthe equivalent organ o bone marrow in mammals.

Antibody diversication during B-cell develop-ment occurs by sequential Ig gene recombination,in which noncontiguous Ig variable (V), diversity,and joining ( J) gene segments are recombined intounctional V(D)J genes.37,38 Te development omature B cells and the generation o a diversiedantigen receptor repertoire are crucial processes,but they are beyond the scope o this review. Herewe will ocus on events that occur ater B cellshave completed their development in the bonemarrow and have matured in peripheral organssuch as the spleen and lymph nodes, where theyare ready to respond to inection.

Upon encountering antigens, nave mature Bcells (IgMlo IgDhi) exit the resting state, increasemetabolic activity, enter the cell cycle to initi-ate prolieration, and undergo dierentiation,eventually leading to ully dierentiated eectorcells that produce high-anity antibodies againstpathogenic targets.53-56 Full dierentiation o Bcells typically results in non-cycling short- or

TABLE 2. Pattern Recognition Receptors

PRR Ligand(s) Immune Cells

TLRs Several surace and intracellular MAMPs Neutrophils; monocytes/M; DCs; B cells

NLRs MDP; anthrax toxin; RNA; fagellin Monocytes/M; DCs

CARD helicases dsRNA Neutrophils; monocytes/M; DCs; B cells

C-type lectins Glycosylated microbial ligands Monocytes/M; DCs

Scavenger receptors Lipid-containing microbial ligands Monocytes/M; DCs

M: macrophages



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long-lived plasma cells (also reerred to as plas-macytes, to include cells at dierent stages odierentiation), which are specialized to producelarge amounts o antibodies, and memory B cells,which can be quickly dierentiated into plasmacells upon re-inection.

Prolonged and continuous engagement omultiple receptor types is required or B lympho-cytes to be activated, to prolierate, to dierentiate,and to maintain their survival.57,58 B cells beginto prolierate within 24 hours ater inductionrom stimuli, such as BCR cross-linking by anti-gens and engagement o other surace receptors,including CD40 and LRs (Fig. 1), and ater theinitial cell division, B cells divide every 6 to 10hours.55,59,60 DCs and cells require prolongedstimulation beore dierentiation,61,62 whereasinnate immune cells, such as macrophages and

neutrophils, respond aster upon sensing pathogensand are attracted to pathogens via chemotaxis byollowing concentration gradients o pathogeniccomponents,63,64 reecting the dierential roleso dierent types o immune cells in innate andadaptive immunity.

Te initial dierentiation o naive B cells insecondary lymphoid organs upon antigen recogni-tion can result in their quick dierentiation intoshort-lived plasmacytes, which secrete a burst omostly IgM antibodies, usually o low-anity, tolimit the spread o pathogens and blunt the inec-tion. Most activated B cells enter the germinalcenter (GC)reaction to undergo CSR to generateantibody isotypes with dierent biological eec-tor unctions, and SHM to generate antibodymutants as substrates or antigen-mediated posi-tive selection o higher-anity antibodies. Both

FIGURE 1. Four main signals or B-cell dierentiation and antibody responses. During an inection, nave matureB cells receive several types o stimuli that are then summed up beore determining the appropriate response.DCs and TH cells interact with B cells via surace receptors such as CD40 and by cytokine receptors such as ILreceptors. A pathogen would directly induce signals by cross-linking the BCR (as shown by the proximity o theO-saccharide o LPS) and by activating innate receptors such as the TLRs (as shown by the interaction o TLR4with the lipid A component o LPS). TLR ligands are sensed by the extracellular, or endosomal, sickle-shapeddomains, and signals are initially relayed to the nucleus via homotypic TIR-TIR interactions (orange spheres) withTIR adapters. These signals are integrated and initiate a response by inducing NF-B and AP-1, IFN-induciblegene transcription, and induction o AID activity, leading to CSR and SHM.



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CSR and SHM are initiated by the enzyme AID(activation-induced cytidine deaminase),65,66 whichis preerentially expressed in activated B cells,especially those in GCs.

Activated DCs in the secondary lymphoidorgans can process and present peptides to

ollicular helper cells (FH)67

in GCs68-71

ospleen, lymph nodes, Peyers patches, and isolatedlymphoid ollicles in the gut.72 Internalizationand processing o pathogenic proteins lead tothe display o antigen-derived peptides on theMHC II receptors o antigen-specic B cells,which then make cognate interactions with FHcells (that are specic or the same antigen) viaMHC II-antigenic peptide-CR interactions. Inaddition, B cells and cells urther contact eachother through the membrane protein receptorpairs CD28:CD80/CD86 and ICOS (inducible

co-stimulator): ICOS ligand (ICOSL), whichare located on the suraces o cells and B cells,respectively. Activation o FH cells by cognateB cells and DCs leads to up-regulation o themembrane protein receptor CD154 (CD40L)on cells. Te engagement o CD40, a membero the tumor necrosis actor (NF) amily otransmembrane receptors located on the suraceo B cells,73,74 by CD154 results in vigorous Bcell prolieration and dierentiation, as will bedetailed in section IIC. In addition to membraneprotein receptor interactions, several types o IL

(interleukin) molecules greatly inuence the natureo the dierentiation program or and B cells.

Te clonal expansion o a ew parental Blymphocytes that are activated and their concomi-tant dierentiation to plasmacytes, which secretethousands o antibodies per second,75-78 leadsto the secretion o millions o anity-maturedand class-switched antibody molecules targetingspecic antigens. Following antigen clearance,the expanded B-cell pool contracts, likely due toengagement o inhibitory FcRs (Fc receptors) on B

cells by excess antibodies,79-84

and by competitionor homing in the bone marrow stromal environ-ment,85,86 thereby ensuring that optimal titers ohigh-anity antibodies are produced.

D. CSR in Humoral Immune Responses

Te prominence o antibodies in immunity isillustrated by the act that all current clinical vac-

cines unction at least in part by eliciting protectiveantibodies,87,88 which are usually class-switchedand o high anity, similar to those generatedduring natural inections. Nave B cells displayIgM and IgD on their surace, and CSR substitutesthe original IgM isotype with IgG, IgA, and IgE

by replacing the constant region o heavy chain(CH) o antibodies65,89-97 (Fig. 2).

Te rst antibodies to be produced are othe IgM class and are likely encoded in thegermline, as the B cells that produce them havenot yet completed the GC reaction or undergoneSHM. Te intrinsic afnity o the initial IgMor antigen is relatively low, however, the overallavidity o IgM or antigen is relatively high, asits pentameric structure containing a total o 10identical Fab arms results in a gain in local entropywhen antigen binding has been initiated in any

one o the 10 arms, enabling these early IgMs toeciently coat bacterial and viral antigens duringthe early stages o inection.98,99 Te large size othe initially produced pentameric IgM restricts itsdistribution mainly to the blood and prevents itrom crossing eciently into extravascular spacesand binding pathogens systemically.

In contrast to IgM, all other antibody isotypesdiuse eciently to extravascular sites, and there-ore their generation is critical during an inec-tion. Class-switched antibodies possess diversebiological eector unctions, including direct

pathogen or toxin/virulence actor neutralization,opsonization, complement activation, sensitizationo mast and NK cells, extravascular diusion,improved transport properties, and longer hal-lives.9,92,96,97,100,101 A deciency in CSR, whetherdue to B-cell intrinsic or extrinsic causes, wouldlead to an impaired antibody response during aninection, as occurs in humans with the hyper-IgMsyndrome.97,100-102 Tese individuals are highlysusceptible to inection and require injections oIgG ractions rom pooled sera o many healthy

donors to provide passive antibody-mediatedprotection against potential pathogens.B cells express both the exquisite receptor or

adaptive immunity, i.e., BCR, and receptors orinnate immunity, such as LRs. In addition tobeing stimulated by -dependent signals, B cellscan be directly stimulated by engagement o theirLRs by the respective ligands. Innate and adap-tive immune signals can thereore be integratedin the same B cell.17,54



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II. THE ROLES OF BCR, CD40, TLRs,AND CYTOKINE RECEPTORS IN CSR

Signaling pathways rom BCR, CD40, LRs, andcytokine receptors play dominant roles in B-cell

antibody responses. While -cell help, throughboth surace receptor (e.g., CD40) engagementand cytokine secretion, is critical or optimalB-cell dierentiation, including CSR and SHM,recent evidence indicates that -independentsignals rom BCR and LRs also signicantlycontribute to the antibody responses againstmicrobial pathogens. Mutations that interere withBCR signaling, such as those in genes encoding

several tyrosine kinases, PI3K (phosphoinositide3 kinase), and PLC2 (phospholipase C2) andtheir several signaling adapters, while they resultin varying deciencies in B-cell development,compromise antigen recognition and subsequent

signaling, resulting in severe impairment o anti-body production and CSR.16,103-105 Humans106-110and mice111-114 carrying mutations in LR genesor in genes encoding IR-domain adapters (suchas MyD88115-118 and RIF119) or LR-regulatorymolecules (such as CD14,120,121 which is a co-receptor or LR4, and Unc93b1,113,122,123 whichis involved in correct endosomal LR tracking)display varying impairments o innate and adap-

FIGURE 2. Overview o CSR and SHM. a, Human B cells diversiy the variable portion o their antigen receptorsduring development in the bone marrow through V(D)J recombination, which is mediated by recombinationactivating gene 1 (RAG1) and RAG2 recombinases. b, During an inection, nave mature B cells in the spleenand lymph nodes undergo rearrangement o the constant portion o the IgH (i.e, CSR) to endow it with newbiological eector unctions, as well point mutations in the variable regions (i.e, SHM) to urther increase its

anity, both o which depend on AID. Each CH region is indicated in blue, beginning with C on the let. Theintronic IgH enhancer (iE, in light green), which is essential or optimal IgH gene expression, is located upstreamo C. The dierent orange-colored thin segments just upstream o each CH region are the IH promoters ollowedby the S regions. IH promoters are activated in response to particular cytokines, and serve to drive germline tran-scription through S and CH regions, possibly opening up local chromatin structure or AID activity, or deliveringAID to the S regions by RNA pol II or other trans-acting actors.



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tive immune responses, as reviewed recently.124-127However, the extent to which the deect directlyimpairs B-cell responses remains unclear.20,49,128,129While one signal alone can directly activate B cells,the response is limited in terms o the antibodyisotypes produced, and combining multiple sig-

nals together broadens the scope o the antibodyresponse by leading to increased B-cell dieren-tiation. It is likely that B cells are optimally di-erentiated by more than one signal,17,54 with BCRand LR signaling contributing to the generationo optimal antibody responses.

A. BCR Signals

One o the primary B-cell activating signals isprovided by BCR cross-linking, where several

BCR units composed o the membrane Ab, Ig,Ig, CD19, CD21, CD81, and associated adapters(Fig. 1) are brought together into close proxim-ity to initiate downstream signaling.104,130,131BCR cross-linking leads to phosphorylationo IAM (immunotyrosine-based activationmoti) Ig and Ig receptors, which in turnactivate several enzymes, in particular PI3K andPLC2. Tese two enzymes generate secondmessenger molecules, such as PtdIns (phospho-rylated phosphatidylinositols) by PI3K, and IP3(inositol triphosphate) and DAG (diacylglycerol)

by PLC2, leading to the release o ree intra-cellular calcium ions (Ca2+). Subsequent NF-B(nuclear actor bound to B) activation results inan increase in the levels o transcription actorsrequired or entering the cell cycle and dier-entiation pathways. However, in the absence oother stimulating signals, B-cell prolieration isollowed by activation-induced cell death,56,132-135a process probably involved in the elimination oB cells activated by sel-antigens in the absenceo an inection. Tus, BCR signaling per se is

insucient or ull B-cell dierentiation, includ-ing CSR, but it synergizes with strong signalsrom dierent receptor amilies, such as CD40or LRs, to induce CSR.

B. Cytokine Receptor Signals

Cytokines, such as IL-4, IFN-, and GF-,inuence B-cell dierentiation by combining with

other signals to direct the B-cell response, such asCSR to specic isotypes.67,136-138 Cytokine receptor

signaling in B cells acts in part by inducing IgHgermline transcription (IH-CH) o heavy chain(CH) regions participating in CSR.

91,92,96,136,138Each CH gene cluster consists o three distinct

and sequential components: the IH promotersor germline transcript initiation, the switch (S)regions where DNA breaks or CSR occur, and

nally, the CH genes themselves (Fig. 2B).IL-4 induces the activation o the transcription

actors NF-B and SA-6, whose DNA-bindingsites include those located in I1 and I promoters.Ater NF-B and SA-6 bind to these promot-

ers, germline I1-C1 and I-C transcription orIgG1 and IgE, respectively, is initiated. Likewise,

I2b and I promoters contain binding sites orthe transcription actors Smud and Runx, whichare induced upon stimulation with GF- to

mediate CSR to these two classes.91,92,96,139 Because

cytokines are mainly, although not exclusively,secreted by H1 and H2 cells, and can be clas-sied into H1 (IFN- and GF-) and H2

(IL-4) classes,138 the antibody isotypes directedby cytokines can also be classied, in the mouse,

into H1 (IgG2a, IgG2b, IgG3) and H2 (IgG1,IgE) isotypes, with IgM and IgA not alling exactly

into either category.Germline IH-CH transcription plays a criticalrole in CSR, likely by either opening up DNA

and/or by routing RNA polymerase II to deliverAID to S regions. S regions contain an unusual

abundance o the 5-WRCY-3 hotspot,140,141and particularly the 5-AGC-3 iteration. Tese

sequences could play important roles in recruitingCSR actors, including AID, and could also serveas the preerred substrate or AID.142 In S regions,AID deaminates cytosine (dC) to uracil (dU).96,142

dU can be excised by apurinic/apyrimidinicendonuclease (APE). Excision o nearby dUs onopposite DNA strands results in dsDNA breaks,

which are then thought to be re-ligated mostly bycomponents o the nonhomologous end-joiningmachinery.96,143,144 Te entire process replaces oneCH gene with a downstream CH gene; that is, itreplaces the C gene with the C1, in the case

o CSR to IgG195,96,141,145 (Fig. 2B).



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C. TNFR Superfamily Signals

Te NF receptor superamily consists o memberssuch as CD40, BAFFR (B-cell activating actoro the NF amily receptor), BCMA (B cellmaturation antigen), and ACI (transmembrane

activator and calcium signaling modulating andcyclophilin ligand [CAML] interactor).146 Eachmember is a homotrimeric type II membrane pro-tein consisting o an extracellular ligand-bindingdomain, a membrane-spanning domain, and anintracellular signaling domain. Tere is a dier-ence in the ligands that these members bind, withCD40 engaging CD154 on the surace o cells,and BAFFR, BCMA, and ACI binding one orboth o the soluble proteins BAFF and APRIL(a prolieration-inducing ligand).

BAFF and APRIL are produced mainly by

innate immune cells as well as by epithelial cells.BAFFR binds BAFF, BCMA binds APRIL, andACI binds both BAFF and APRIL.147,148 BAFFand APRIL unction to promote the maturationo 1 immature transitional B cells to matureB cells to maintain appropriate levels o matureB cells in a negative eedback ashion, to helpwith the establishment o plasma cells to theirniches in the bone marrow,147-150 and, nally, toinduce or enhance B cell dierentiation, includ-ing CSR.151-155 One report suggested the directinduction o CSR by engagement o ACI or

BAFFR,152 however, in our experiments, BAFFdid not induce CSR, although BAFF enhancedLPS- or CD40-driven CSR in mouse B cellsstimulated in vitro. Tus, BAFF may enhanceCSR when B cells are activated by an additionalstimulus, as evidence suggests in the case o B-cellactivation by viral components151,153,155 or epithelialcell-secreted BAFF and APRIL,156-159 as will bedescribed in section D1 below. Finally, BAFFcan be produced by IgD-armed basophils uponbinding bacteria, thus establishing a link between

initial IgD production and sustained, local B-celldierentiation, including CSR.160

In B cells, the main stimulatory surace recep-tor belonging to the NF receptor superamilythat induces robust B-cell dierentiation is CD40.CD40 is ound on the suraces o other APCs,and is generally required or their activation.Tis receptor has been ound to be essential inmediating a broad variety o immune responses,including -dependent CSR, SHM, the GC

reaction, and plasma and memory B-cell orma-tion.74,161-163 CD40 signals are transmitted byinteraction o CD40 cytoplasmic tail with RAF(NF receptor-associated actor) adapter proteins,including RAF2, RAF3, RAF5, RAF6, andRAP,164,165 which ultimately results in the acti-

vation o transcription actors such as NF-B andAP-1 (activator protein 1).163 Te most importantRAFs that transduce CD40 signals are RAF2and RAF6. Tese subsequently interact withGCK (germinal center kinase), which then activatesthe JNK (c-Jun N-terminal kinase) and MAPK(mitogen-activated protein kinase) pathways.165RAFs mediate NF-B induction by activatingthe IKK (inhibitor o NF-B kinase) complex tophosphorylate the IB (inhibitor oB) proteins,thereby releasing NF-B or nuclear translocationand binding to its target DNA166 (Fig. 1). NF-B

then orchestrates a transcriptional program, whichincludes the induction o molecules necessaryor B-cell dierentiation, including induction oAID or initiation o CSR and SHM.164 As the-dependent B-cell response in vivois MHC-IIrestricted, in vitrostudies o B-cell dierentiationby CD40 should perhaps also involve simultaneousMHC-II engagement.167,168

D. TLR Signals

1. TLRs in Immune Cells

Te involvement o LRs in mammalian immu-nity was rst discovered based on their homologyto the Drosophila oll receptor,where oll playsa dual role in the dorso-ventral segmentationduring development and in the production oantiungal peptides during an inection.54,169 Mostmammalian species contain 10 to 15 dierentLRs, each o which senses one or a limitednumber o MAMPs (able 3), and, in a ew

cases, host-derived molecules.21,170-172

LRs areexpressed in innate immune cells, B cells, and insome non-immune cell types, including epithelialcells. In particular, epithelial cells sense bacteriavia LRs, and then secrete BAFF or APRIL,which activates B cells residing in the peripheryand promotes their CSR, thereby containing thebacteria.156-159 Non-immune unctions o LRs,as in development, cancer, or tissue injury,173 areoutside the scope o this review.



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LRs consist o an extracellular or endosomalsensing domain that contains leucine-rich repeats(LRRs), a transmembrane region, and a cytoplas-

mic IR (oll/interleukin I/resistance protein)domain.23,174,175 LRRs old into a sickle-shaped-coil domain and coner specicity in sensing aparticular class o ligands, mainly through theirconcave, leucine-rich surace.22 Te intracellularand variable IR domain coners signaling speci-cities to dierent LRs, enabling each o themto interact with a limited number o IR-domainsignaling adaptor proteins via homotypic IR-IRinteractions.21,22,24,174,176,177Comparative sequenceanalysis o the human LRs have revealed thatthe members o the LR amily can be divided

into ve subamilies, as represented by LR2,LR3, LR4, LR5, and LR9. Te LR2 am-ily consists o LR1, LR2, LR6, and LR10;the LR9 subamily consists o LR7, LR8,and LR9; the other three subamilies, LR3,LR4, and LR5, contain only the one indicatedmember.178

Upon engagement by their ligands, LRsare thought to signal as homodimers (or pos-

sibly homomultimers), with the exception oLR1/LR2 and LR2/LR6, which signalas heterodimers. Signaling pathways emanat-

ing rom dierent LRs are complex and otenredundant (Fig. 1). It is generally accepted thatthere are two main transmission pathways in thepropagation o LR signals, mediated by twoIR domain-containing adaptors: the MyD88(myeloid dierentiation actor 88) pathway and theRIF (IR domain-containing adaptor inducingintereron-) pathway, respectively. Te other threeknown IR domain-containing adapters are MAL(MyD88-adaptor-like, also known as IRAP, oll/interleukin 1 receptor domain containing adaptorprotein), RAM (RIF-related adaptor molecule),

and the recently discovered SARM (sterile andarmadillo motis). LR4 signals through boththe MyD88 pathway, as bridged by IRAP, andthe RIF pathway, as bridged by RAM; LR9signals directly and only through the MyD88pathway. With the exception o SARM, the otherour adaptors utilize IRAKs (IL-1 receptor associ-ated kinases) to transmit signals to RAFs, whichultimately leads to the nuclear translocation and

TABLE 3. TLRs and Their Ligands

TLR L igand(s) Microorganism TIR Adapters Immune Cells

TLR1 Triacyl lipopeptides,e.g., Pam3CSK4

Bacteria, mycobacteria MyD88; TIRAP Neutrophils; monocytes/M;mDC; B cells

TLR2 Diacyl lipopeptides,e.g., Pam2CSK4;lipotechoic acid;zymosan

Bacteria, mycobacteria,yeasts

MyD88; TIRAP Neutrophils; monocytes/M;mDCs; B cells

TLR3 dsRNA Viruses TRIF mDCs; M; B cells

TLR4 LPS Gram-negative acteria MyD88; TIRAP;TRAM; TRIF

Neutrophils; monocytes/M;mDCs; B cells

TLR5 Flagellin Bacteria MyD88 Monocytes/M; mDCs; B cells

TLR6 Diacyl lipopeptides Mycoplasma MyD88; TIRAP Neutrophils; monocytes/M;mDCs; B cells

TLR7 ssRNA Bacteria and viruses MyD88 Neutrophils; monocytes/M;pDCs; B cells

TLR8 ssRNA Bacteria and viruses MyD88 Neutrophils; monocytes/M;

mast cells; B cellsTLR9 Unmethylated CpG-

containing DNABacteria and viruses MyD88 Neutrophils; monocytes/M;

pDCs; B cells

TLR11 Prolin Toxoplasma MyD88 Monocytes/M; B cells

M: macrophages; mDCs: myeloid DCs; pDCs: plasmacytoid DCs.



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target gene promoter binding o transcription ac-tors such as NF-B and AP-1, thereby initiatinggene transcription o IRF-3 and SA-1, and theinduction o AID expression to mediate CSR andSHM in activated B cells.176,177 SARM has beensuggested to negatively modulate signaling,174 pos-

sibly to restore the pre-activation non-signalingstate or to dampen the response.In monocytes, in which LRs are highly

expressed, LR engagement induces their dier-entiation into macrophages or DCs, and urtherLR stimulation can modulate immune unctionso these cells, including enhancement o phago-cytosis o pathogens.179,180 In DCs, LRs appearto be expressed in a cell subset-dependent manner:myeloid DCs in peripheral tissues express mostLRs except LR9, while plasmacytoid DCs inblood express mostly LR1, LR6, LR7, and

LR9.26,181,182 Binding o the appropriate ligandinduces DC maturation, including enhancedantigen presentation and cytokine secretion, andup-regulation o the stimulatory receptor CD86,all o which greatly inuence the adaptive immuneresponse.181 Te presence and potential unctiono LRs in lymphocytes, which generallysense antigen presented by DCs and B cells, iscurrently unclear, though there is some evidenceindicating that LR2 engagement may removethe reg-suppressive unctions during ongoinginection.183,184

2. TLR Signaling in B-Cell AntibodyResponses

B cells express high levels o most LRs andrespond to stimulation by LR ligands associatedwith pathogens, likely those present in patho-gens phagocytosed by B cells, as well as released,soluble LR ligands. Te LR signal transduc-tion pathways in cells such as macrophages and

DCs21,22,185

described in the previous section arelikely relevant to B cells as well (Fig. 1). LRengagement by MAMPs such as LPS or CpGinduces potent B-cell dierentiation,19 similar inmagnitude to that induced by CD40, and muchstronger than that induced by BCR cross-linkingalone. Even though direct B-cell dierentiationand antibody production in response to LRligands such as LPS or agellin was reportedsince the 1960s,17,54,186-189 only recently has the

signicance and scope o these data received dueattention.10,17,20,49,54,177 While LRs are clearlyunctional in B cells, relatively little is knownabout how LRs inuence B-cell dierentiation,including CSR and SHM.176,177,190

Te role o LRs in B-cell antibody responses

has been somewhat controversial, at least inpart due to dierent outcomes rom dierentexperimental settings. In one study, LR signal-ing in B cells was ound to be indispensable oroptimal B-cell antibody responses against nativeproteins using LPS or agellin as LR ligandsin immunizations employing wild-type, LR4-,or MyD88-decient B cells adoptively trans-erred into B-cell-decient mice.49 However, inother studies, antibody responses to haptenatedproteins mixed with dierent adjuvants, many o

which contain LR agonists, was not signicantlyimpaired in mice doubly decient in MyD88 andRIF.128,175,191 One explanation or these data isthat antibody responses to haptenated proteinsmay be dierent rom those against natural andunhaptenated proteins, perhaps due to the alterna-tion o the protein structures by the haptenationprocess.175,191 Alternatively, it has been proposedthat the repetitive arrays o haptens cross-linkBCR with high eciency such that LR signal-ing may not be essential or the B-cell responses,whereas soluble protein antigens that cannoteciently cross-link the BCR may require LRstimulation to elicit a robust response.192 Teact that B cells highly express most LRs andare eciently activated by engagement o theirligands points to a role or LRs in optimalB-cell dierentiation, as will be detailed in thesubsequent sections.

LR stimulation o B cells in combinationwith cytokines has been used to drive CSR tospecic isotypes. In act, well beore the discoveryo CD154 as one o the main H cell receptors,

LPS was used to activate mouse B cells,187 andwas subsequently ound to do so by engagingLR4.111,193 LPS stimulation o B cells also led tothe historical discovery o NF-B.194-196 BecauseLPS stimulation o B cells induces NF-B andAID, and results in CSR to IgG3, the specicityo CSR could be partially inuenced by LRligands such as LPS, but is greatly inuenced bycytokines, as was mentioned earlier.



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III. TLR AND BCR SIGNALINGSYNERGIZE TO INDUCE CSR

A. Surface TLRs in the Induction of CSR

Te surace LR1, LR2, LR4, LR5, and

LR6 bind MAMPs that are expressed on thesurace o microorganisms, while the endosomalLR3, LR7, LR8, and LR9 bind nucleic acidMAMPs located inside microorganisms. LR4,which is expressed on the plasma membrane,binds LPS present on the surace o bacteria andis internalized together with LPS,197,198 whichcould be a general eature o LR recognition anddownstream tracking and signaling events.199-202LR agonists are potent drivers o B-cell dier-entiation, including NF-B activation and AIDexpression, which coupled with cytokine-directed

germline IH-CH transcription, induces CSR tospecic isotypes. Experimentally, LPS stimulationo mouse B cells induces CSR to IgG3; LPS incombination with IL-4 induces CSR to IgG1 andIgE; LPS in combination with IFN- inducesCSR to IgG2a; nally, LPS in combination withGF- induces CSR to IgG2b and IgA.91,92,96Likewise, we have recently ound that stimulationo LR1/2 by Pam3CSK4 results in CSR to theisotypes specied by cytokines (Pone and Casali,unpublished data). hereore, LR signalingresults in context-dependent modulation o both

the magnitude and the ne structure o the B-cellantibody responses.

LR ligands may stimulate not only theircognate LRs, but they may also happen tobind and cross-link the BCR in a small ractiono B cells, thereby activating an entirely dierentstimulation pathway.203-206 In particular, LRligands are thought to behave in two dierentways: a I-I (-independent type I) and a I-II(-independent type II) way.8,9,54,189,204,207 Atsaturating doses, I-I antigens, including LPS,

will bind all B cells via their LR4, and thereorevirtually all B cells will be (polyclonally) activated(mitogenic stimulation). At much lower LPSconcentrations, those ewer B cells whose BCRalso happens to bind the O-saccharide componento LPS with high anity are thought to receiveenough signals rom both LR4 (engaged bythe lipid A component o LPS) and BCR. I-IIligands are highly repetitive antigens, such as thepolysaccharides dextran and coll, which cross-link

the BCR with greater eciency than antigens withlower valency do. Even though LR4 engagementby LPS does not require BCR cross-linking toinduce B cell dierentiation, BCR cross-linkingcan urther enhance LR4-induced B-cell di-erentiation198,208 and CSR; the same holds or

LR1/2-induced CSR. Furthermore, whilein vitro stimulation o B cells with high (mitogenic)doses o LPS, in the presence o cytokines caninduce CSR to specic isotypes, the contribu-tion o BCR cross-linking by LPS under thesein vitro conditions may skew the response to aI-II response (LR + BCR), which engagesthe BCR and thus results in the amplication oantigen-specic B cells, rather than a genuine I-I(LR alone) response. Polymerized agellin is aneven better example in that it possesses both I-Ieatures (e.g., it cross-links LR5 in the whole

B- cell population) and I-II eatures (e.g., it maycross-link the BCR, with variable eciency, in araction o the B-cell population).

B. Endosomal TLRs in the Inductionof CSR

Te endosomal location o LR3, LR7, LR8,and LR9 may be optimal in allowing their sens-ing o internalized nucleic acid MAMPs romviruses and bacteria, while likely avoiding sensing

host nucleic acids.202,209 While the dynamics odistribution and maturation o endosomal LRsis still a matter o active research,210 several reportsindicate that each endosomal LR may not belocated in exclusive endosomes, but rather theymay be located to common endosomes hostingseveral dierent LRs, where each LR sensesits respective ligand as it is gradually exposed ina disintegrating microorganism.211,212

Stimulation o the endosomal LR9 byunmethylated CpG DNA has been thought to

suppress class switching to IgG1 and IgE iso-types but promote class switching to IgG2a inB cells activated by LPS or CD154 and IL-4,likely by regulating germline transcription.213However, there has been a great variability inthe data o dierent research groups, particu-larly in the magnitude and the identity o theLR9-enhanced or -suppressed isotypes,213-218and the controversial involvement o -bet (box expressed in cells),213,219-221 a transcription



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actor important or H1 cell polarization. CpGstimulation was reported to suppress CSR toIgG1 and IgE induced by LPS or CD40 andthe cytokine IL-4, and at the same time promoteCSR to the H1 isotype IgG2a under these H2conditions,213 suggesting that CpG stimulation

could somehow divert the CSR machinery toother target S regions, which is a role usuallyplayed by cytokines.

CpG can also modulate B-cell dierentiationto plasmablasts/plasmacytes,86,222 thereby resultingin complex changes in the titer o isotype-switchedantibodies in culture supernatants, and thereorecomplicating the monitoring o CSR. Further-more, IFN- and GF-, two cytokines criticalor CSR to IgG2a and IgG2b, respectively, areknown to decrease B-cell prolieration and survival,whereas CpG is a potent prolieration and survival

stimulus, which adds additional complexity to theoverall antibody response.

C. BCR Signaling Synergizes With TLRSignaling

LR-induced CSR depends on the correct cellulartracking and localization o the engaged LRs,as well as whether other signals, particularly thoserom the BCR, are also activated.6,199-202,223,224LRligands such as LPS or agellin can elicit antibody

responses directed against them, complicating thedelineation o the respective roles o BCR andLRs in anti-microbial antibody responses. B cellsphagocytose antigen in a FcR- or BCR-dependentway,225-228 eciently process internalized antigenicproteins, as well as sense various MAMPs presentin the phagocytosed pathogen. BCR and LR9signaling synergize in the induction o NF-B inB cells,199,223,229 and BCR signaling recruits LR9-containing endosomes to autophagosomes,199,223possibly as a way o eciently coordinating the

sensing o nucleic acid LR ligands present whena B cell internalizes pathogens.Signaling cross-talk between IAM receptors

and LRs could modulate responses in immunecells.6,224 For example, DCs responded to ssRNAby producing IFN- only i the viruses had beenpreviously opsonized with antibodies, whichcross-link Fc receptors on the DC membrane230;cross-linking o activating FcRs in DCs andother cells can serve the same unction as the

cross-linking o BCR on B cells, as both areIAM-bearing receptors. In another study, VSV(vesicular stomatitis virus) induced IFN- inDCs upon its internalization to autophagosomesand simultaneous engagement o LR7 by viralRNA.231

In our studies, we have ound that CpG stimu-lation alone results in robust B-cell prolieration,but not in signicant CSR, as indicated by the lowrequency o B cells expressing surace IgG, IgE,or IgA measured by ow cytometry. However, inthe presence o BCR cross-linking (and appro-priate cytokines), CpG stimulation could lead torobust CSR to all respective isotypes, as well asto plasma cell ormation and antibody secretion.Furthermore, contrary to previous reports,213,217,232our data indicate that LR9 stimulation in theabsence o BCR cross-linking suppresses LPS- (or

CD154-) and cytokine-induced CSR not onlyto H1 but also to H2 isotypes, and suppressesplasma cell ormation. However, in the presenceo BCR cross-linking, LPS and CpG, as wellas LPS and Pam3CKS4, synergized in inducingCSR in a dose-dependent way (Pone and Casali,unpublished observations). Similar synergisticand antagonistic eects have been reported orcytokine production by B cells43 and PBMCs233stimulated with specic pairwise combinations oLR ligands.

CpG and other surace and endosomal LR

ligands, such as Pam3CSK4 and R848, might notdisplay a H1 vs H2 bias in inducing B cells toundergo CSR to specic Ig isotypes (like cytokinesdo), but like LPS, these may act as inducers oNF-B and AID, with the latter initiating CSRto isotypes specied by cytokine-induced germlinetranscription. Te mechanism by which CpGantagonizes LPS- and CD40-induced CSR inthe absence o BCR cross-linking could be due tosequestration o signaling adapters or due to signalparalysis. Another possibility could be that in the

absence o strong BCR cross-linking, CpG mayenter the cell via BCR-mediated uptake, settingo weak BCR signaling (low IAM signaling),which has been proposed to negatively regulateLR and cytokine signaling in macrophages.6,224Conversely, during dual stimulation with BCRcross-linking and CpG (mimicking the internal-ization o microorganisms by BCRs that havehigh-anity or their external epitopes), the strongIAM signaling synergizes with LR9 signaling,



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and results in dierentiation, as hallmarked byCSR (Fig. 3).

Considering CpG as a general inducer o B

cells to undergo CSR to isotypes directed by thedierent cytokines, without itsel displaying anintrinsic H1 vs H2 isotype bias, does not con-tradict the reported H1 vs H2 polarizing eecto CpG in other innate immune cell types, and itsuse as a predominantly H1 vaccine adjuvant.

234-238It is likely that cells such as DCs and macrophagessense the pathogen type present in an inection byengagement o various PRRs (including LRs)

by corresponding pathogenic MAMPs, and then

produce H1 or H2 cytokines that initiate the

induction o H

1 or H

2 cells, which then ampliy

the polarization and induce B-cell dierentiation

and CSR to specic H1 or H2 isotypes.13,26,239-241

Whether B cells, ater directly sensing the type o

pathogen via engagement o their LRs by the

corresponding pathogenic MAMPs, can undergo

CSR to the most appropriate isotypes, as is the

case or LPS-induced CSR to IgG3, remains to

be determined.

FIGURE 3. The interplay o surace TLR4 and endosomal TLR9 in CSR. a, LPS induces CSR to isotypes, asdirected by cytokines. b, CpG by itsel does not induce signicant CSR; however, in the presence o BCR cross-linking (indicated by a virus bound by BCR; experimentally it can be mimicked by reagents that cross-link theBCR polyclonally), it behaves similar to LPS in inducing CSR to the isotypes specied by cytokines. c, CpG sup-presses LPS-induced CSR; however, CpG synergizes with LPS in a dose-dependent way in the presence o BCRcross-linking (d).



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While the role o LRs in autoimmunity isbeyond the scope o this review, the reader is reerredto several recent reviews on the subject.172,242-249Briey, it appears that under conditions in whichLRs sense host ligands that resemble MAMPs,such as phospholipids that resemble LPS and could

engage LR4, or sel RNA or DNA that couldengage LR3, LR7/8, and LR9, autoreactive Bcells are activated to produce autoantibodies.250-253Tis could be particularly important in the genera-tion o anti-DNA class-switched autoantibodies,which are the main cause o systemic lupus ery-thematosus.

IV. TLR- AND BCR-INDUCED B-CELLANTIBODY RESPONSES AGAINSTMICROBIAL PATHOGENS

A. TLR Signaling in T-Independent B-CellDifferentiation

1. TLR Signaling During the Initial Phaseof B-Cell Differentiation

LRs were likely one o the rst immune recep-tors that recognized conserved molecular pat-terns o pathogens.4 A recent report describedthe rearrangement o lamprey antigen receptors,where an APOBEC-related enzyme was ound

to deaminate the gene segments encoding thesereceptors, leading to their rearrangement and thusdiversication to a limited number o congura-tions.32 Another report on lamprey immunityrevealed the presence o B- and -like cells, whichsense pathogens via membrane-expressed VLRs,with the B-like cells progressing through blastormation and secreting soluble antigen-specicVLRs.36 Tus, it is possible that LRs, VLRs, orrelated primordial PRRs were the original antigenreceptors, and during evolution were replaced by

the newer and more and versatile BCRs, whichare based on the Ig domain old. LRs continuedto provide inormation regarding the presenceand type o pathogen to innate immune cellsand B cells.

LR-mediated dierentiation o B cells maybe a critical component o -independent, butearly and eective B-cell clonal amplication anddierentiation leading to the production o classswitched, potentially hypermutated, protective

antibodies during early inection when pathogenloads are relatively low and cell help is notyet ully available. LPS stimulation o mouseB cells in vitro results in IgM and IgG3 antibodyproduction. Te rst antibodies to be producedin vivo against an antigen encountered or the

rst time are IgM, which can contain the patho-gen in the rst hours to days o inection. Studiesin mice selectively decient in secreted IgM,254suggest that the initial -independent productiono IgM serves not only to contain pathogens, butby orming complexes with pathogens, serves asan autocrine actor or continued local B-cellprolieration via IgM-mediated multimeriza-tion o soluble antigen that can now ecientlycross-link the BCR. In the absence o timely-cell help, other cell types, such as olliculardendritic cells, may provide a level o stimulation

sucient or B-cell activation and early IgM,255and likely IgG, secretion.

2. TLR Signaling During B-CellDifferentiation in the Absenceof T-Cell Help

Even though optimal CSR requires -cell help,some CSR does occur without -cell help ol-lowing challenge with viral, bacterial, and ungalpathogens (able 4). While the titers o class-

switched antibodies in the absence o -cell helpare usually much lower than the optimal titersproduced in the presence o normal -cell help,in several cases the titers are enough to conersome protection against inection. Class-switchedantibodies can be generated against invadingpathogens without -cell help because the enzymeAID can be induced in part by the conservedhomeodomain transcription actor HoxC4145,256,257in a -independent ashion.17,54,176,177,189,192,258-260

CSR that occurs in the absence o H cells261

is particularly applicable to antiviral antibodyresponses.258,262 Immunization o -cell-decientmice with polyoma virus resulted in the produc-tion o virus-specic class switched IgG, althoughin lower titers compared with wild-type mice,whereas immunization with either a viral antigenor VLPs (virus-like particles) did not result in IgGproduction, possibly due to the presence o dualLR and BCR engagement in the case o wholevirus and lack o LR engagement in the case o



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antigen alone or VLP immunization.263 In other

studies, natural IgA antibodies were produced inresponse to LR ligands o commensal gut bac-teria,264 natural IgE antibodies were generated toactivate innate immune mast cells without -cellhelp,265 and IgG2a antibodies were produced out-side GC ollicles three days ater immunization.266Considering the low-level CSR that occurs in theabsence o -cell help, it is possible that this canhave unctional signicance in helping to containthe low pathogen loads during the early stages oinection when -cell help has not yet peaked198,267

(Fig. 4).

B. TLR Signaling May Play Roles Duringthe T-Dependent Phase of B-cellDifferentiation

Initial IgM and IgG production by B cells is likelydue to their activation by various LRs and theBCR, since antigen-specic helper cell numbers

do not peak until several days ater their initial

activation. IgM production, in addition to provid-ing some early and direct protection by targetingpathogenic epitopes, also serves in the activationo complement, which then results in microbecoating with C3d, which binds the CD21/CD35receptors on the B-cell surace and enhances B-cellactivation.267 Te antibody-pathogen complexesare drained in secondary lymphoid organs, wherethey are destroyed by proessional phagocytes suchas macrophages, and in the process stimulate the-dependent GC reaction.99

Direct LR stimulation o B cells duringthe GC reaction may help to sustain and shapethe processes o antibody anity maturation byacting as a reporter o pathogen load and inec-tion conditions.10,20,49,175,191 For example, initialLR-mediated activation o B cells leads to theup-regulation o co-stimulatory CD80/CD86 andMHC-II receptors, thereby priming B cells ormore abundant and ecient interactions with cells and DCs.268 -cell help alone may not be

TABLE 4. T Cell-Independent Anti-Microbial Class Switched Antibody Responses

Pathogen/Antigen Mouse Model Ig Produced Reference

Polyoma virus TCR/ x /B cell transer in SCIDCR2/ B cell transer inSCID

IgM, IgGIgM, IgGIgM, IgG

316317

318

VSV TCR/; TCR/ IgM, IgG2a 319

Rotavirus TCR/ x /TCR/

IgM, IgG, IgAIgA

320321

LCMV, VSV, Pichinde virus CD154/ IgM, IgG2a 322

Infuenza virus CD4/CD40/; MHCII/

IgM, IgG1, IgG2b, IgG3IgM, IgG

323324

Ehrlichia muris CD4/; CD8/; MHCII/ IgM, IgG1, IgG2c, IgG2b, IgG3 325

Ehrlichia chafeensis CD4/ IgG1, IgG2a, IgG2b, IgG3 326

Borrelia burgdorferi TCR/; TCR/ x / IgM, IgG3 327

Bacillus anthracis TCR/ IgG 328

Yersinia enterocolitica nude IgG 329Citrobacter rodetium CD4/ IgM, IgG1, IgG2c, IgG2b, IgG3, IgA 330

Porphyromonas gingivalis TCR/ IgG, IgA 331

Melanin (C. neoformans) nude IgM, IgG1, IgG2b, IgG3 332

Cryptosporidium parvum nude IgM, IgA 333

Protoscolex (E. granulosus) CD4/ IgG1, IgG2a, IgG2b, IgG3 334

Echinococcus multilocularis nude; TCR/;MHCI/; MHCII/

IgM, IgG1, IgG2a, IgG2b, IgG3 335



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sucient or optimal antibody responses, withdirect LR stimulation o B cells contributingto optimal antibody titers and CSR,49 thoughthis issue remains unclear.128,129,175,191 Duringthe GC reaction, LR signals in B cells arelikely integrated with signals rom the BCR,

cells, and DCs, eventually giving rise to plasmaand memory B cells, whose Ig genes encodeantigen-specic, high-anity, and class-switchedantibodies.199,223,269

Immunization with CpG and hepatitis antigenresulted in higher antibody anities than immu-

FIGURE 4. Model on the role o endosomal TLRs in B-cell antibody responses. a, B cells in the secondary lymphoidorgans can be directly activated by viruses that cross-link the BCR. This then leads to their phagocytosis, double-membrane autophagosome ormation, and tracking o the autophagosomes to TLR-containing endosomes. Iany present viral TLR ligands are sensed by TLR3, TLR7/8, or TLR9, an appropriate response is then mountedby the B cells, which includes their dierentiation (and in some cells, T-independent CSR), the up-regulation oreceptors that interact with TH cells and DCs, thus enhancing the GC reaction (b). The result o T-independent (a)and T-dependent (b) B cell dierentiation is the ormation o antigen-specic plasma cells, and memory B cells,which can be quickly dierentiated to plasma cells upon subsequent re-inection (c).



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nization with antigen alone.270 When LR ligandsin a RSV (respiratory syncytial virus) vaccine wereinactive, the resulting antibodies were low-anitydue to impaired SHM and were not protective.271Tese reports suggest that LRs play a role inSHM, since their engagement contributes to the

generation o high-anity antibodies, though themolecular and cellular mechanisms responsiblehave not yet been elucidated. Te immune system,including B cells, need to continually recognizeand bind pathogens in order to ultimately clearthem. In particular, nucleic acid LR ligands maybe important signals or SHM, because they wouldsignal the presence o replicating, and potentiallyhypermutating, microorganisms.272

C. TLR and BCR Signaling in VaccineResearch

1. Vaccine Adjuvants

Well beore the discovery o the LRs, vac-cinologists empirically used adjuvants such asFreunds complete adjuvant, Freunds incompleteadjuvant, and Ribbis adjuvant, which, in addi-tion to containing oils or orming depots upon

injection, also contain mycobacterial components(which include various LR ligands) in order toelicit robust immune responses to soluble proteinantigens.7,10,175,273,274 Since the discovery o thecrucial adjuvanting properties o LR and otherPRR ligands, there has justiably been an explo-

sion in the application o dened adjuvants oruse in vaccine development.274-297

All current clinical vaccines elicit protectiveantibodies,87,88 requently also eliciting the mobili-zation o other immune cells, including NK cells,cytotoxic cells, H cells, DCs, macrophages,and so orth.274 As mentioned previously, severalstudies have reported that CpG or antigen-CpG usion immunization in humans and miceresult in higher antibody titers and/or antibodyanities or antigen and/or longer-lasting protec-

tion,19,234,235,269,270,298-303 possibly due to enhancedCSR and SHM and expansion o high-anitymemory B cells. Te reader may reer to a recentreview or an extensive list o examples whereprior CpG administration has resulted in someprotection beore challenge with pathogens.304 Acomprehensive list o current clinical and experi-mental vaccines that target -independent bacte-rial antigens has also been recently reviewed.305

FIGURE 5. The design eatures o modular vaccines. a, Virus-mimicking biodegradable particles with avorablepharmacokinetics, which by themselves are not immunogenic, are used as scaolds or the incorporation o pro-tein antigens rom one or several pathogens. Initial screens determine the most immunogenically suitable proteinantigens (brown triangles) to use. In addition, combinations o one or more natural or synthetic TLR ligands areincluded, usually mimicking their occurrence in the actual pathogen(s). b, Particles mimicking bacteria are coatedwith bacterial protein antigens as well as TLR ligands characteristic o each bacterium. These composite vaccines(a, b) activate both innate immune cells as well as B lymphocytes, which will dierentiate to plasma cells thatsecrete antigen-specic antibodies, as well as to memory B cells that can be activated upon potential uturere-inection. c, Legend indicates the protein antigens and the various TLR ligands used in modular vaccines.



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2. New Generation Modular Vaccines

Based on the promise o dened LR agonistsor use in vaccines, a new generation o vaccines iscurrently being developed. Tese vaccines employparticles made o biodegradable materials, lipid

vesicles, or sel-assembling protein subunits, that inaddition to containing protein antigen, are inusedwith internal or external LR ligands192,306-312 (Fig.5). Tis approach allows or modular, systematic,and controlled testing o the best combinations oprotein antigens rom the pathogens and naturalor synthetic LR agonists, taking into accountthe dual LR and BCR induction o B-cell di-erentiation that was described in the previoussection. Generally, a rigid array o a minimumo 15 to 20 repeating antigens induces optimalBCR signaling.192

While modular vaccine design is still underdevelopment, as a general approach, the particleand the LR ligand combinations used shouldmimic the pathogen as closely as possible; orexample, it may not be benecial to use agellinto develop modular viral vaccines because it doesnot naturally occur in viruses. Te LR ligandscan be LPS, Pam3CSK4, agellin, and so orth,which should be incorporated on the outside othe vaccine particle, and CpG, ssRNA, dsRNA,or agonists such as imiquimod or R848, whichshould be best incorporated inside the particle.

Future studies may, however, report benecial,synergistic combinations o LR ligands that arenot normally ound in the same pathogen, whichmay apply especially in the cases o co-inectionby two or more pathogens.

aking this approach one step urther, recentstudies have explored the easibility o develop-ing broad-spectrum vaccines, where immunityis provided against more than one pathogenicstrain, or against several related or even unrelatedpathogens.313,314 In this case, antigens rom several

dierent pathogens are incorporated in the sameparticle or separately in mixtures o dierent par-ticles, with LR ligands typical o each pathogenalso incorporated in the particles. In principle,this approach should allow or mobilization oB cells that produce antibodies specic or eachpathogen. Issues remaining to be resolved relateto the saety and ecacy o CpG and other LRagonists in eliciting broad-spectrum immunity toemerging and re-emerging pathogens.313,315

V. CONCLUSIONS

Te synergy o BCR and LR signaling criti-cally regulates B-cell antibody responses, and inparticular CSR. BCR cross-linking acilitates theencounter o endosomes containing LR9, and

likely other LRs, to autophagosomes as a wayo surveilling the presence o nucleic acid LRligands during an inection. BCR stimulationprovides inormation on the anity o antigenor antibody, and the spacing and topology oantigenic arrays on the pathogen, whereas LRstimulation may instruct B cells on the natureo the pathogen, e.g., the presence o a Gram+veor Gram-ve bacterium or o an RNA or DNAvirus. It would be particularly useul or B cellsto sense multiple LR signals rom a pathogen

in a combinatorial ashion, thereby leading to amore appropriate and specic response.Te dual presence o BCR and LRs on B cells

allows them to respond to a greater variety andcombination o signals than previously thought,including traditionally innate signals, therebyallowing or ne-tuned responses that best dealwith the pathogenic threat. Te interplay andsynergy between BCR, LRs, and -dependentsignals is also proving to be both challenging andrewarding. Te ultimate task remains to predictB-cell behavior during an inection in vivo basedon studies o responses to dened combinationso stimuli under dierent conditions in vitro andin vivo. Knowledge o undamental B-cell signalsand typical responses would be benecial or thedesign o therapies or immune deciency orautoimmunity, and or the development o eec-tive vaccines.

ACKNOWLEDGMENTS

Owing to space limitations, we could cite only araction o the papers relevant to the topics dis-cussed in this review article. We apologize to theother authors o publications that were not cited.We would like to thank members o the Casalilaboratory or invaluable discussions and criticalreading o this manuscript. Tis work was sup-ported by NIH grants AI 045011, AI 0790705,and AI 060573 to P.C.



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