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Critical Reviews™ in Immunology, 30(1):1–29 (2010)

Toll-Like Receptors and B-Cell Receptors Synergize


to Induce Immunoglobulin Class-Switch DNA
Recombination: Relevance to Microbial Antibody
Responses

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Egest J. Pone, Hong Zan, Jinsong Zhang, Ahmed Al-Qahtani,

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Zhenming Xu, & Paolo Casali*
Institute for Immunology, School of Medicine and School of Biological Sciences, University of

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California, Irvine, California

*Address all correspondence to Dr. Paolo Casali, Institute for Immunology, 3028 Hewitt Hall, University of California, Irvine, CA 92697-4120;

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Tel.: 949- 824-4456; Fax: 949-824-2305; pcasali@uci.edu.

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ABSTRACT: Differentiation of naïve B cells, including immunoglobulin class-switch DNA recombination, is criti-
cal for the immune response and depends on the extensive integration of signals from the B-cell receptor (BCR),
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tumor necrosis factor (TNF) family members, Toll-like receptors (TLRs), and cytokine receptors. TLRs and BCR
synergize to induce class-switch DNA recombination in T cell-dependent and T cell-independent antibody responses
to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced
B-cell differentiation and antibody responses. The requirement of both BCR and TLR engagement would ensure
appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role
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in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak.
Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified
by the activation of B cells by initial TLR engagement, leading to the up-regulation of co-stimulatory CD80 and
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MHC-II receptors, which result in more efficient interactions with T cells, thereby enhancing the germinal center
reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from
the pathogen or immune cells and their products determine the ensuing B-cell antibody response.
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Key words: activation-induced (cytidine) deaminase, adaptive immunity, adjuvant, antibody, autophagy,
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B lymphocyte, B cell receptor, class-switch DNA recombination, CD40, CpG, cytokine, dendritic cell, germline
transcription, immunoglobulin, innate immunity, LPS, microbe-associated molecular patterns, NF-κB, pattern
recognition receptors, somatic hypermutation, T cell, Toll-like receptor, vaccine.

I. B-CELL DIFFERENTIATION VIA INNATE an individual is achieved through the integration


AND ADAPTIVE IMMUNE RECEPTORS of innate and adaptive immune responses, which
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are intrinsically linked by crosstalk through cells


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A. The Innate Immune Response and molecules.1-6 The immune system detects
and eliminates invading pathogenic microorgan-
The protection from a nearly unlimited number isms, as well as infected and neoplastic self-cells,
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of pathogens encountered during the lifetime of by distinguishing them or associated molecules

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, natural
killer; NLR, NOD-like receptor; PRR, pattern recognition receptors; SHM, somatic hypermutation; TCR, T-cell
receptor; TLR, Toll-like receptor; VLR, variable leucine-rich receptor
Received: 8/17/09; Accepted: 11/11/09

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Table 1. Features of Innate and Adaptive Immunity

Feature Innate Immunity Adaptive Immunity


Specificity Low Very high
Receptor clonal distribution No Yes
Receptor diversity Encoded in germline Recombined gene segments
Response timeline No lag Lag time for induction
Efficiency of response Moderately efficient Highly efficient
Memory No Yes

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from “normal” self-cells and self-molecules. While NOD-like receptors (NLRs), CARD helicases,
lower organisms have only an innate immune C-type lectins, and scavenger receptors3,10,25 (Table

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system, higher organisms, beginning with jawed 2). Some of these receptors may be functional in
vertebrates, in addition possess a more sophisti- B cells, however, their effects on B-cell antibody

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cated system: the adaptive, or acquired, immune responses are not known; therefore, this review will
system.7-10 Innate immunity provides an early, focus on the functions of TLRs in B cells, which

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though non-specific, initial response to patho- have been more extensively investigated. After
gens.7,11-14 In contrast, adaptive immunity requires sensing pathogens via their PRRs, macrophages
a lag period for activation, is specific, diverse and TR and mast cells are activated to phagocytose the
highly efficient in targeting of pathogens8,9,15,16 pathogen, display peptide fragments on MHCII
(Table 1). (major histocompatibility II) receptors, and secrete
The innate immune system is a universal and pro-inflammatory cytokines and lipid mediators.
ancient form of host defense against infection.7,10,17 Likewise, stimulation of PRRs on immature DCs
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In mammals, it is composed of diverse cell types, leads to their maturation and activation, which in
including macrophages, dendritic cells (DCs), turn activates adaptive immunity.7,10,13,26
neutrophils, basophils, mast cells, eosinophils,
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and natural killer (NK) cells; and molecules and


molecular systems, such as anti-microbial pep- B. The Adaptive Immune Response
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tides, acute-phase proteins (collectins, ficolins,


and pentraxins) and complement. 8,9,13 Innate Evolutionarily, adaptive immunity emerged after
innate immunity by the need to respond to spe-
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immune cells constantly sample the environ-


ment for the presence of pathogens through their cific and mutating antigens through the use of
germline-encoded pattern recognition receptors antigen-specific receptors on the surfaces of B and
(PRRs).3,7,9,13,18 PRRs primarily bind to “non-self ” T lymphocytes.1,4,15,27,28 Even though only jawed
molecules that have conserved features known as vertebrates possess a true adaptive immune system,
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microbe-associated molecular patterns (MAMPs); recent evidence indicates that jawless vertebrates,
an alternative term is pathogen-associated molecu- such as lamprey and hagfish, contain lymphocyte-
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lar patterns (PAMPs). MAMPs are unique chemi- like immune cells expressing variable leucine-rich
cal structures found in the microorganism but receptors (VLRs), which are triggered to rearrange
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generally absent in the higher hosts. For example, upon infection with a pathogen, and display a
lipopolysaccharide (LPS) contains sugars and lip- specificity for antigen similar to antibodies of the
ids composed of chemical bond arrangements not jawed vertebrate adaptive immune system.29-36
found in higher eukaryotes, thereby constituting a The adaptive immune response enables higher
unique MAMP. Another MAMP is CpG (unm- vertebrate animals to adapt their response to a
ethylated, CG-containing DNA) from bacteria pathogen to be more specific and effective, and
and viruses.19 LPS and CpG are recognized by to elicit immunological memory, that is, to mount
an important subset of PRRs known as Toll-like a faster and stronger response during subsequent
receptors (TLRs).3,20-24 Other PRR classes include encounters with the same pathogen.

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TLR and BCR Induction of CSR 3

Table 2. Pattern Recognition Receptors

PRR Ligand(s) Immune Cells

TLRs Several surface and intracellular MAMPs Neutrophils; monocytes/Mφ; DCs; B cells
NLRs MDP; anthrax toxin; RNA; flagellin Monocytes/Mφ; DCs
CARD helicases dsRNA Neutrophils; monocytes/Mφ; DCs; B cells
C-type lectins Glycosylated microbial ligands Monocytes/Mφ; DCs
Scavenger receptors Lipid-containing microbial ligands Monocytes/Mφ; DCs

Mφ: macrophages

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The mechanisms of generating antigen- Antibodies can directly inactivate pathogens,45-47
specific receptors in the adaptive system involve can opsonize pathogens to assist innate immune
extensive variability and rearrangement of receptor cells in eliminating pathogens, and, finally, can

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gene segments. During their development, B and activate complement.8,9,48 Conversely, B cells are
T lymphocytes rearrange IgH and Igκ/λ chains in directly and indirectly informed about the pres-

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B cells, and α and β chains in T cells, that code for ence and nature of pathogens by innate immune
BCRs and T-cell receptors (TCRs), respectively. elements such as TLRs,10,13,49 and are indirectly

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Lymphocytes display on the extracellular surface informed by complement factor C3d.50-52 Dysregu-
of their plasma membranes multiple (hundreds to lated and mis-targeted B cell antibody responses
thousands) identical copies of their BCR or TCR TR could result in autoimmunity, whereas impaired
with unique antigen specificity among the pool antibody responses during an actual infection
of total lymphocytes.37,38 The rearrangement and could result in an immune deficiency.
clonal distribution of antigen receptors is different In mammals, B cells begin their development
from the previously described PRRs, which do not in the bone marrow from lymphoid progeni-
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rearrange and usually have the same sequence in all tor cells, which in turn derive from pluripotent
cells that express them in an organism (Table 1). hematopoietic stem cells. B cells derive their name
from the bursa of Fabricius in chickens, which is
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When antigen receptors bind arrays of repetitive


antigens on a bacterium, virus, or parasite, they the equivalent organ of bone marrow in mammals.
cluster together, thereby bringing into proximity Antibody diversification during B-cell develop-
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membrane-associated and intracellular signaling ment occurs by sequential Ig gene recombination,


molecules, contributing to lymphocyte differentia- in which noncontiguous Ig variable (V), diversity,
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tion. Such an antigen receptor cross-linking process and joining ( J) gene segments are recombined into
only occurs in those B and T lymphocytes that functional V(D)J genes.37,38 The development of
express receptors with an above-threshold affinity mature B cells and the generation of a diversified
for antigen to ensure that only antigen-specific antigen receptor repertoire are crucial processes,
lymphocytes are amplified and differentiate into but they are beyond the scope of this review. Here
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effector and memory cells, which have the same we will focus on events that occur after B cells
specificity for the antigen that initiated and drove have completed their development in the bone
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the response.39,40 Antibodies (and BCRs) are fur- marrow and have matured in peripheral organs
ther diversified by class-switch DNA recombina- such as the spleen and lymph nodes, where they
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tion (CSR) and somatic hypermutation (SHM). are ready to respond to infection.
Upon encountering antigens, naïve mature B
cells (IgMlo IgDhi) exit the resting state, increase
C. B-Cell Differentiation metabolic activity, enter the cell cycle to initi-
ate proliferation, and undergo differentiation,
B lymphocytes play major roles in the immune eventually leading to fully differentiated effector
response by producing antibodies against antigens, cells that produce high-affinity antibodies against
by functioning as professional antigen-presenting pathogenic targets.53-56 Full differentiation of B
cells (APCs), and by producing cytokines.41-44 cells typically results in non-cycling short- or

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4 Pone et al.

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FIGURE 1. Four main signals for B-cell differentiation and antibody responses. During an infection, naïve mature
B cells receive several types of stimuli that are then summed up before determining the appropriate response.
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DCs and TH cells interact with B cells via surface receptors such as CD40 and by cytokine receptors such as IL
receptors. A pathogen would directly induce signals by cross-linking the BCR (as shown by the proximity of the
O-saccharide of LPS) and by activating innate receptors such as the TLRs (as shown by the interaction of TLR4
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with the lipid A component of LPS). TLR ligands are sensed by the extracellular, or endosomal, sickle-shaped
domains, and signals are initially relayed to the nucleus via homotypic TIR-TIR interactions (orange spheres) with
TIR adapters. These signals are integrated and initiate a response by inducing NF-κB and AP-1, IFN-inducible
gene transcription, and induction of AID activity, leading to CSR and SHM.
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long-lived plasma cells (also referred to as plas- neutrophils, respond faster upon sensing pathogens
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macytes, to include cells at different stages of and are attracted to pathogens via chemotaxis by
differentiation), which are specialized to produce following concentration gradients of pathogenic
large amounts of antibodies, and memory B cells, components,63,64 reflecting the differential roles
which can be quickly differentiated into plasma of different types of immune cells in innate and
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cells upon re-infection. adaptive immunity.


Prolonged and continuous engagement of The initial differentiation of naive B cells in
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multiple receptor types is required for B lympho- secondary lymphoid organs upon antigen recogni-
cytes to be activated, to proliferate, to differentiate, tion can result in their quick differentiation into
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and to maintain their survival.57,58 B cells begin short-lived plasmacytes, which secrete a burst of
to proliferate within 24 hours after induction mostly IgM antibodies, usually of low-affinity, to
from stimuli, such as BCR cross-linking by anti- limit the spread of pathogens and blunt the infec-
gens and engagement of other surface receptors, tion. Most activated B cells enter the germinal
including CD40 and TLRs (Fig. 1), and after the center (GC) reaction to undergo CSR to generate
initial cell division, B cells divide every 6 to 10 antibody isotypes with different biological effec-
hours.55,59,60 DCs and T cells require prolonged tor functions, and SHM to generate antibody
stimulation before differentiation,61,62 whereas mutants as substrates for antigen-mediated posi-
innate immune cells, such as macrophages and tive selection of higher-affinity antibodies. Both

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TLR and BCR Induction of CSR 5

CSR and SHM are initiated by the enzyme AID cines function at least in part by eliciting protective
(activation-induced cytidine deaminase),65,66 which antibodies,87,88 which are usually class-switched
is preferentially expressed in activated B cells, and of high affinity, similar to those generated
especially those in GCs. during natural infections. Naïve B cells display
Activated DCs in the secondary lymphoid IgM and IgD on their surface, and CSR substitutes
organs can process and present peptides to T the original IgM isotype with IgG, IgA, and IgE
follicular helper cells (TFH)67 in GCs68-71 of by replacing the constant region of heavy chain
spleen, lymph nodes, Peyer’s patches, and isolated (CH) of antibodies65,89-97 (Fig. 2).
lymphoid follicles in the gut.72 Internalization The first antibodies to be produced are of
and processing of pathogenic proteins lead to the IgM class and are likely encoded in the

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the display of antigen-derived peptides on the germline, as the B cells that produce them have
MHC II receptors of antigen-specific B cells, not yet completed the GC reaction or undergone

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which then make cognate interactions with TFH SHM. The intrinsic affinity of the initial IgM
cells (that are specific for the same antigen) via for antigen is relatively low, however, the overall

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MHC II-antigenic peptide-TCR interactions. In avidity of IgM for antigen is relatively high, as
addition, B cells and T cells further contact each its pentameric structure containing a total of 10
other through the membrane protein receptor identical Fab arms results in a gain in local entropy

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pairs CD28:CD80/CD86 and ICOS (inducible when antigen binding has been initiated in any
co-stimulator): ICOS ligand (ICOSL), which one of the 10 arms, enabling these early IgMs to

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are located on the surfaces of T cells and B cells, efficiently coat bacterial and viral antigens during
respectively. Activation of TFH cells by cognate the early stages of infection.98,99 The large size of
B cells and DCs leads to up-regulation of the the initially produced pentameric IgM restricts its
membrane protein receptor CD154 (CD40L)
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on T cells. The engagement of CD40, a member from crossing efficiently into extravascular spaces
of the tumor necrosis factor (TNF) family of and binding pathogens systemically.
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transmembrane receptors located on the surface In contrast to IgM, all other antibody isotypes
of B cells,73,74 by CD154 results in vigorous B diffuse efficiently to extravascular sites, and there-
cell proliferation and differentiation, as will be fore their generation is critical during an infec-
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detailed in section IIC. In addition to membrane tion. Class-switched antibodies possess diverse
protein receptor interactions, several types of IL biological effector functions, including direct
(interleukin) molecules greatly influence the nature pathogen or toxin/virulence factor neutralization,
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of the differentiation program for T and B cells. opsonization, complement activation, sensitization
The clonal expansion of a few parental B of mast and NK cells, extravascular diffusion,
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lymphocytes that are activated and their concomi- improved transport properties, and longer half-
tant differentiation to plasmacytes, which secrete lives.9,92,96,97,100,101 A deficiency in CSR, whether
thousands of antibodies per second,75-78 leads due to B-cell intrinsic or extrinsic causes, would
to the secretion of millions of affinity-matured lead to an impaired antibody response during an
and class-switched antibody molecules targeting infection, as occurs in humans with the hyper-IgM
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specific antigens. Following antigen clearance, syndrome.97,100-102 These individuals are highly
the expanded B-cell pool contracts, likely due to susceptible to infection and require injections of
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engagement of inhibitory FcRs (Fc receptors) on B IgG fractions from pooled sera of many healthy
cells by excess antibodies,79-84 and by competition donors to provide passive antibody-mediated
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for homing in the bone marrow stromal environ- protection against potential pathogens.
ment,85,86 thereby ensuring that optimal titers of B cells express both the exquisite receptor for
high-affinity antibodies are produced. adaptive immunity, i.e., BCR, and receptors for
innate immunity, such as TLRs. In addition to
being stimulated by T-dependent signals, B cells
D. CSR in Humoral Immune Responses can be directly stimulated by engagement of their
TLRs by the respective ligands. Innate and adap-
The prominence of antibodies in immunity is tive immune signals can therefore be integrated
illustrated by the fact that all current clinical vac- in the same B cell.17,54

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6 Pone et al.

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FIGURE 2. Overview of CSR and SHM. a, Human B cells diversify the variable portion of their antigen receptors
during development in the bone marrow through V(D)J recombination, which is mediated by recombination
activating gene 1 (RAG1) and RAG2 recombinases. b, During an infection, naïve mature B cells in the spleen
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and lymph nodes undergo rearrangement of the constant portion of the IgH (i.e, CSR) to endow it with new
biological effector functions, as well point mutations in the variable regions (i.e, SHM) to further increase its
affinity, both of which depend on AID. Each CH region is indicated in blue, beginning with Cµ on the left. The
intronic IgH enhancer (iEµ, in light green), which is essential for optimal IgH gene expression, is located upstream
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of Cµ. The different orange-colored thin segments just upstream of each CH region are the IH promoters followed
by the S regions. IH promoters are activated in response to particular cytokines, and serve to drive germline tran-
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scription through S and CH regions, possibly opening up local chromatin structure for AID activity, or delivering
AID to the S regions by RNA pol II or other trans-acting factors.

II. THE ROLES OF BCR, CD40, TLRs, several tyrosine kinases, PI3K (phosphoinositide
AND CYTOKINE RECEPTORS IN CSR 3 kinase), and PLCγ2 (phospholipase Cγ2) and
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their several signaling adapters, while they result


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Signaling pathways from BCR, CD40, TLRs, and in varying deficiencies in B-cell development,
cytokine receptors play dominant roles in B-cell compromise antigen recognition and subsequent
antibody responses. While T-cell help, through
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signaling, resulting in severe impairment of anti-


both surface receptor (e.g., CD40) engagement body production and CSR.16,103-105 Humans106-110
and cytokine secretion, is critical for optimal and mice111-114 carrying mutations in TLR genes
B-cell differentiation, including CSR and SHM, or in genes encoding TIR-domain adapters (such
recent evidence indicates that T-independent as MyD88115-118 and TRIF119) or TLR-regulatory
signals from BCR and TLRs also significantly molecules (such as CD14,120,121 which is a co-
contribute to the antibody responses against receptor for TLR4, and Unc93b1,113,122,123 which
microbial pathogens. Mutations that interfere with is involved in correct endosomal TLR trafficking)
BCR signaling, such as those in genes encoding display varying impairments of innate and adap-

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TLR and BCR Induction of CSR 7

tive immune responses, as reviewed recently.124-127 other signals to direct the B-cell response, such as
However, the extent to which the defect directly CSR to specific isotypes.67,136-138 Cytokine receptor
impairs B-cell responses remains unclear.20,49,128,129 signaling in B cells acts in part by inducing IgH
While one signal alone can directly activate B cells, germline transcription (IH-CH) of heavy chain
the response is limited in terms of the antibody (CH) regions participating in CSR.91,92,96,136,138
isotypes produced, and combining multiple sig-
Each CH gene cluster consists of three distinct
nals together broadens the scope of the antibody
and sequential components: the IH promoters
response by leading to increased B-cell differen-
tiation. It is likely that B cells are optimally dif- for germline transcript initiation, the switch (S)
ferentiated by more than one signal,17,54 with BCR regions where DNA breaks for CSR occur, and

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and TLR signaling contributing to the generation finally, the CH genes themselves (Fig. 2B).
of optimal antibody responses. IL-4 induces the activation of the transcription

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factors NF-κB and STAT-6, whose DNA-binding
sites include those located in Iγ1 and Iε promoters.

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A. BCR Signals After NF-κB and STAT-6 bind to these promot-
ers, germline Iγ1-Cγ1 and Iε-Cε transcription for
One of the primary B-cell activating signals is

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IgG1 and IgE, respectively, is initiated. Likewise,
provided by BCR cross-linking, where several Iγ2b and Iα promoters contain binding sites for
BCR units composed of the membrane Ab, Igα,

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the transcription factors Smud and Runx, which
Igβ, CD19, CD21, CD81, and associated adapters
are induced upon stimulation with TGF-β to
(Fig. 1) are brought together into close proxim-
ity to initiate downstream signaling.104,130,131
TR mediate CSR to these two classes.91,92,96,139 Because
BCR cross-linking leads to phosphorylation cytokines are mainly, although not exclusively,
of ITAM (immunotyrosine-based activation secreted by TH1 and TH2 cells, and can be clas-
motif ) Igα and Igβ receptors, which in turn sified into TH1 (IFN-γ and TGF-β) and TH2
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activate several enzymes, in particular PI3K and (IL-4) classes,138 the antibody isotypes directed
PLCγ2. These two enzymes generate second by cytokines can also be classified, in the mouse,
messenger molecules, such as PtdIns (phospho- into TH1 (IgG2a, IgG2b, IgG3) and TH2 (IgG1,
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rylated phosphatidylinositols) by PI3K, and IP3 IgE) isotypes, with IgM and IgA not falling exactly
(inositol triphosphate) and DAG (diacylglycerol) into either category.
by PLCγ2, leading to the release of free intra-
Germline IH-CH transcription plays a critical
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cellular calcium ions (Ca2+). Subsequent NF-κB


role in CSR, likely by either opening up DNA
(nuclear factor bound to κB) activation results in
and/or by routing RNA polymerase II to deliver
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an increase in the levels of transcription factors


required for entering the cell cycle and differ- AID to S regions. S regions contain an unusual
entiation pathways. However, in the absence of abundance of the “5-WRCY-3” hotspot,140,141
other stimulating signals, B-cell proliferation is and particularly the “5-AGCT-3” iteration. These
followed by activation-induced cell death,56,132-135 sequences could play important roles in recruiting
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a process probably involved in the elimination of CSR factors, including AID, and could also serve
B cells activated by self-antigens in the absence as the preferred substrate for AID.142 In S regions,
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of an infection. Thus, BCR signaling per se is AID deaminates cytosine (dC) to uracil (dU).96,142
insufficient for full B-cell differentiation, includ- dU can be excised by apurinic/apyrimidinic
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ing CSR, but it synergizes with strong signals endonuclease (APE). Excision of nearby dUs on
from different receptor families, such as CD40
opposite DNA strands results in dsDNA breaks,
or TLRs, to induce CSR.
which are then thought to be re-ligated mostly by
components of the nonhomologous end-joining
B. Cytokine Receptor Signals machinery.96,143,144 The entire process replaces one
CH gene with a downstream CH gene; that is, it
Cytokines, such as IL-4, IFN-γ, and TGF-β, replaces the Cµ gene with the Cγ1, in the case
influence B-cell differentiation by combining with of CSR to IgG195,96,141,145 (Fig. 2B).

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8 Pone et al.

C. TNFR Superfamily Signals reaction, and plasma and memory B-cell forma-
tion.74,161-163 CD40 signals are transmitted by
The TNF receptor superfamily consists of members interaction of CD40 cytoplasmic tail with TRAF
such as CD40, BAFFR (B-cell activating factor (TNF receptor-associated factor) adapter proteins,
of the TNF family receptor), BCMA (B cell including TRAF2, TRAF3, TRAF5, TRAF6, and
maturation antigen), and TACI (transmembrane TTRAP,164,165 which ultimately results in the acti-
activator and calcium signaling modulating and vation of transcription factors such as NF-κB and
cyclophilin ligand [CAML] interactor).146 Each AP-1 (activator protein 1).163 The most important
member is a homotrimeric type II membrane pro- TRAFs that transduce CD40 signals are TRAF2
tein consisting of an extracellular ligand-binding and TRAF6. These subsequently interact with

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domain, a membrane-spanning domain, and an GCK (germinal center kinase), which then activates
intracellular signaling domain. There is a differ- the JNK (c-Jun N-terminal kinase) and MAPK

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ence in the ligands that these members bind, with (mitogen-activated protein kinase) pathways.165
CD40 engaging CD154 on the surface of T cells, TRAFs mediate NF-κB induction by activating

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and BAFFR, BCMA, and TACI binding one or the IKK (inhibitor of NF-κB kinase) complex to
both of the soluble proteins BAFF and APRIL phosphorylate the IκB (inhibitor of κB) proteins,
(a proliferation-inducing ligand). thereby releasing NF-κB for nuclear translocation

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BAFF and APRIL are produced mainly by and binding to its target DNA166 (Fig. 1). NF-κB
innate immune cells as well as by epithelial cells. then orchestrates a transcriptional program, which

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BAFFR binds BAFF, BCMA binds APRIL, and includes the induction of molecules necessary
TACI binds both BAFF and APRIL.147,148 BAFF for B-cell differentiation, including induction of
and APRIL function to promote the maturation AID for initiation of CSR and SHM.164 As the
of T1 immature transitional B cells to mature
TR T-dependent B-cell response in vivo is MHC-II
B cells to maintain appropriate levels of mature restricted, in vitro studies of B-cell differentiation
B cells in a negative feedback fashion, to help by CD40 should perhaps also involve simultaneous
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with the establishment of plasma cells to their MHC-II engagement.167,168
niches in the bone marrow,147-150 and, finally, to
induce or enhance B cell differentiation, includ-
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ing CSR.151-155 One report suggested the direct D. TLR Signals


induction of CSR by engagement of TACI or
BAFFR,152 however, in our experiments, BAFF 1. TLRs in Immune Cells
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did not induce CSR, although BAFF enhanced


LPS- or CD40-driven CSR in mouse B cells The involvement of TLRs in mammalian immu-
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stimulated in vitro. Thus, BAFF may enhance nity was first discovered based on their homology
CSR when B cells are activated by an additional to the Drosophila Toll receptor, where Toll plays
stimulus, as evidence suggests in the case of B-cell a dual role in the dorso-ventral segmentation
activation by viral components151,153,155 or epithelial during development and in the production of
cell-secreted BAFF and APRIL,156-159 as will be antifungal peptides during an infection.54,169 Most
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described in section D1 below. Finally, BAFF mammalian species contain 10 to 15 different


can be produced by IgD-armed basophils upon TLRs, each of which senses one or a limited
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binding bacteria, thus establishing a link between number of MAMPs (Table 3), and, in a few
initial IgD production and sustained, local B-cell cases, host-derived molecules.21,170-172 TLRs are
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differentiation, including CSR.160 expressed in innate immune cells, B cells, and in


In B cells, the main stimulatory surface recep- some non-immune cell types, including epithelial
tor belonging to the TNF receptor superfamily cells. In particular, epithelial cells sense bacteria
that induces robust B-cell differentiation is CD40. via TLRs, and then secrete BAFF or APRIL,
CD40 is found on the surfaces of other APCs, which activates B cells residing in the periphery
and is generally required for their activation. and promotes their CSR, thereby containing the
This receptor has been found to be essential in bacteria.156-159 Non-immune functions of TLRs,
mediating a broad variety of immune responses, as in development, cancer, or tissue injury,173 are
including T-dependent CSR, SHM, the GC outside the scope of this review.

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TLR and BCR Induction of CSR 9

Table 3. TLRs and Their Ligands

TLR Ligand(s) Microorganism TIR Adapters Immune Cells


TLR1 Triacyl lipopeptides, Bacteria, mycobacteria MyD88; TIRAP Neutrophils; monocytes/Mφ;
e.g., Pam3CSK4 mDC; B cells

TLR2 Diacyl lipopeptides, Bacteria, mycobacteria, MyD88; TIRAP Neutrophils; monocytes/Mφ;


e.g., Pam2CSK4; yeasts mDCs; B cells
lipotechoic acid;
zymosan
TLR3 dsRNA Viruses TRIF mDCs; Mφ; B cells

N
TLR4 LPS Gram-negative acteria MyD88; TIRAP; Neutrophils; monocytes/Mφ;
TRAM; TRIF mDCs; B cells

O
TLR5 Flagellin Bacteria MyD88 Monocytes/Mφ; mDCs; B cells
TLR6 Diacyl lipopeptides Mycoplasma MyD88; TIRAP Neutrophils; monocytes/Mφ;

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

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TLR8 ssRNA Bacteria and viruses MyD88 Neutrophils; monocytes/Mφ;
mast cells; B cells

IB
TLR9 Unmethylated CpG- Bacteria and viruses MyD88 Neutrophils; monocytes/Mφ;
containing DNA pDCs; B cells
TLR11 Profilin Toxoplasma MyD88 Monocytes/Mφ; B cells

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


TR
IS

TLRs consist of an extracellular or endosomal sibly homomultimers), with the exception of


sensing domain that contains leucine-rich repeats TLR1/TLR2 and TLR2/TLR6, which signal
D

(LRRs), a transmembrane region, and a cytoplas- as heterodimers. Signaling pathways emanat-


mic TIR (Toll/interleukin I/resistance protein) ing from different TLRs are complex and often
domain.23,174,175 LRRs fold into a sickle-shaped redundant (Fig. 1). It is generally accepted that
R

β-coil domain and confer specificity in sensing a there are two main transmission pathways in the
particular class of ligands, mainly through their propagation of TLR signals, mediated by two
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concave, leucine-rich surface.22 The intracellular TIR domain-containing adaptors: the MyD88
and variable TIR domain confers signaling speci- (myeloid differentiation factor 88) pathway and the
ficities to different TLRs, enabling each of them TRIF (TIR domain-containing adaptor inducing
to interact with a limited number of TIR-domain interferon-β) pathway, respectively. The other three
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signaling adaptor proteins via homotypic TIR-TIR known TIR domain-containing adapters are MAL
interactions.21,22,24,174,176,177 Comparative sequence (MyD88-adaptor-like, also known as TIRAP, Toll/
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analysis of the human TLRs have revealed that interleukin 1 receptor domain containing adaptor
the members of the TLR family can be divided protein), TRAM (TRIF-related adaptor molecule),
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into five subfamilies, as represented by TLR2, and the recently discovered SARM (sterile and
TLR3, TLR4, TLR5, and TLR9. The TLR2 fam- armadillo motifs). TLR4 signals through both
ily consists of TLR1, TLR2, TLR6, and TLR10; the MyD88 pathway, as bridged by TIRAP, and
the TLR9 subfamily consists of TLR7, TLR8, the TRIF pathway, as bridged by TRAM; TLR9
and TLR9; the other three subfamilies, TLR3, signals directly and only through the MyD88
TLR4, and TLR5, contain only the one indicated pathway. With the exception of SARM, the other
member.178 four adaptors utilize IRAKs (IL-1 receptor associ-
Upon engagement by their ligands, TLRs ated kinases) to transmit signals to TRAFs, which
are thought to signal as homodimers (or pos- ultimately leads to the nuclear translocation and

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10 Pone et al.

target gene promoter binding of transcription fac- significance and scope of these data received due
tors such as NF-κB and AP-1, thereby initiating attention.10,17,20,49,54,177 While TLRs are clearly
gene transcription of IRF-3 and STAT-1, and the functional in B cells, relatively little is known
induction of AID expression to mediate CSR and about how TLRs influence B-cell differentiation,
SHM in activated B cells.176,177 SARM has been including CSR and SHM.176,177,190
suggested to negatively modulate signaling,174 pos- The role of TLRs in B-cell antibody responses
sibly to restore the pre-activation non-signaling has been somewhat controversial, at least in
state or to dampen the response. part due to different outcomes from different
In monocytes, in which TLRs are highly experimental settings. In one study, TLR signal-
expressed, TLR engagement induces their differ-

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ing in B cells was found to be indispensable for
entiation into macrophages or DCs, and further optimal B-cell antibody responses against native
TLR stimulation can modulate immune functions

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proteins using LPS or flagellin as TLR ligands
of these cells, including enhancement of phago-
in immunizations employing wild-type, TLR4-,
cytosis of pathogens.179,180 In DCs, TLRs appear
or MyD88-deficient B cells adoptively trans-

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to be expressed in a cell subset-dependent manner:
myeloid DCs in peripheral tissues express most ferred into B-cell-deficient mice.49 However, in
TLRs except TLR9, while plasmacytoid DCs in other studies, antibody responses to haptenated

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blood express mostly TLR1, TLR6, TLR7, and proteins mixed with different adjuvants, many of
TLR9.26,181,182 Binding of the appropriate ligand which contain TLR agonists, was not significantly

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induces DC maturation, including enhanced impaired in mice doubly deficient in MyD88 and
antigen presentation and cytokine secretion, and TRIF.128,175,191 One explanation for these data is
up-regulation of the stimulatory receptor CD86, that antibody responses to haptenated proteins
all of which greatly influence the adaptive immune
TR may be different from those against natural and
response.181 The presence and potential function unhaptenated proteins, perhaps due to the alterna-
of TLRs in T lymphocytes, which generally tion of the protein structures by the haptenation
IS
sense antigen presented by DCs and B cells, is process.175,191 Alternatively, it has been proposed
currently unclear, though there is some evidence that the repetitive arrays of haptens cross-link
indicating that TLR2 engagement may remove BCR with high efficiency such that TLR signal-
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the Treg-suppressive functions during ongoing ing may not be essential for the B-cell responses,
infection.183,184 whereas soluble protein antigens that cannot
efficiently cross-link the BCR may require TLR
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stimulation to elicit a robust response.192 The


2. TLR Signaling in B-Cell Antibody fact that B cells highly express most TLRs and
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Responses are efficiently activated by engagement of their


ligands points to a role for TLRs in optimal
B cells express high levels of most TLRs and B-cell differentiation, as will be detailed in the
respond to stimulation by TLR ligands associated
subsequent sections.
with pathogens, likely those present in patho-
TLR stimulation of B cells in combination
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gens phagocytosed by B cells, as well as released,


with cytokines has been used to drive CSR to
soluble TLR ligands. The TLR signal transduc-
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tion pathways in cells such as macrophages and specific isotypes. In fact, well before the discovery
DCs21,22,185 described in the previous section are of CD154 as one of the main TH cell receptors,
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likely relevant to B cells as well (Fig. 1). TLR LPS was used to activate mouse B cells,187 and
engagement by MAMPs such as LPS or CpG was subsequently found to do so by engaging
induces potent B-cell differentiation,19 similar in TLR4.111,193 LPS stimulation of B cells also led to
magnitude to that induced by CD40, and much the historical discovery of NF-κB.194-196 Because
stronger than that induced by BCR cross-linking LPS stimulation of B cells induces NF-κB and
alone. Even though direct B-cell differentiation AID, and results in CSR to IgG3, the specificity
and antibody production in response to TLR of CSR could be partially influenced by TLR
ligands such as LPS or flagellin was reported ligands such as LPS, but is greatly influenced by
since the 1960s,17,54,186-189 only recently has the cytokines, as was mentioned earlier.

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TLR and BCR Induction of CSR 11

III. TLR AND BCR SIGNALING the BCR with greater efficiency than antigens with
SYNERGIZE TO INDUCE CSR lower valency do. Even though TLR4 engagement
by LPS does not require BCR cross-linking to
A. Surface TLRs in the Induction of CSR induce B cell differentiation, BCR cross-linking
can further enhance TLR4-induced B-cell dif-
The surface TLR1, TLR2, TLR4, TLR5, and ferentiation198,208 and CSR; the same holds for
TLR6 bind MAMPs that are expressed on the TLR1/2-induced CSR. Furthermore, while
surface of microorganisms, while the endosomal in vitro stimulation of B cells with high (mitogenic)
TLR3, TLR7, TLR8, and TLR9 bind nucleic acid doses of LPS, in the presence of cytokines can
MAMPs located inside microorganisms. TLR4, induce CSR to specific isotypes, the contribu-

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which is expressed on the plasma membrane, tion of BCR cross-linking by LPS under these
binds LPS present on the surface of bacteria and in vitro conditions may skew the response to a

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is internalized together with LPS,197,198 which TI-II response (TLR + BCR), which engages
could be a general feature of TLR recognition and the BCR and thus results in the amplification of

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downstream trafficking and signaling events.199-202 antigen-specific B cells, rather than a genuine TI-I
TLR agonists are potent drivers of B-cell differ- (TLR alone) response. Polymerized flagellin is an
entiation, including NF-κB activation and AID even better example in that it possesses both TI-I

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expression, which coupled with cytokine-directed features (e.g., it cross-links TLR5 in the whole
germline IH-CH transcription, induces CSR to B- cell population) and TI-II features (e.g., it may

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specific isotypes. Experimentally, LPS stimulation cross-link the BCR, with variable efficiency, in a
of mouse B cells induces CSR to IgG3; LPS in fraction of the B-cell population).
combination with IL-4 induces CSR to IgG1 and
IgE; LPS in combination with IFN-γ induces
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CSR to IgG2a; finally, LPS in combination with B. Endosomal TLRs in the Induction
TGF-β induces CSR to IgG2b and IgA.91,92,96 of CSR
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Likewise, we have recently found that stimulation
of TLR1/2 by Pam3CSK4 results in CSR to the The endosomal location of TLR3, TLR7, TLR8,
isotypes specified by cytokines (Pone and Casali, and TLR9 may be optimal in allowing their sens-
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unpublished data). Therefore, TLR signaling ing of internalized nucleic acid MAMPs from
results in context-dependent modulation of both viruses and bacteria, while likely avoiding sensing
the magnitude and the fine structure of the B-cell host nucleic acids.202,209 While the dynamics of
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antibody responses. distribution and maturation of endosomal TLRs


TLR ligands may stimulate not only their is still a matter of active research,210 several reports
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cognate TLRs, but they may also happen to indicate that each endosomal TLR may not be
bind and cross-link the BCR in a small fraction located in exclusive endosomes, but rather they
of B cells, thereby activating an entirely different may be located to common endosomes hosting
stimulation pathway.203-206 In particular, TLR several different TLRs, where each TLR senses
ligands are thought to behave in two different its respective ligand as it is gradually exposed in
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ways: a TI-I (T-independent type I) and a TI-II a disintegrating microorganism.211,212


(T-independent type II) way.8,9,54,189,204,207 At Stimulation of the endosomal TLR9 by
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saturating doses, TI-I antigens, including LPS, unmethylated CpG DNA has been thought to
will bind all B cells via their TLR4, and therefore suppress class switching to IgG1 and IgE iso-
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virtually all B cells will be (polyclonally) activated types but promote class switching to IgG2a in
(mitogenic stimulation). At much lower LPS B cells activated by LPS or CD154 and IL-4,
concentrations, those fewer B cells whose BCR likely by regulating germline transcription.213
also happens to bind the O-saccharide component However, there has been a great variability in
of LPS with high affinity are thought to receive the data of different research groups, particu-
enough signals from both TLR4 (engaged by larly in the magnitude and the identity of the
the lipid A component of LPS) and BCR. TI-II TLR9-enhanced or -suppressed isotypes,213-218
ligands are highly repetitive antigens, such as the and the controversial involvement of T-bet (T
polysaccharides dextran and ficoll, which cross-link box expressed in T cells),213,219-221 a transcription

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12 Pone et al.

factor important for TH1 cell polarization. CpG cross-linking of BCR on B cells, as both are
stimulation was reported to suppress CSR to ITAM-bearing receptors. In another study, VSV
IgG1 and IgE induced by LPS or CD40 and (vesicular stomatitis virus) induced IFN-α in
the cytokine IL-4, and at the same time promote DCs upon its internalization to autophagosomes
CSR to the TH1 isotype IgG2a under these TH2 and simultaneous engagement of TLR7 by viral
conditions,213 suggesting that CpG stimulation RNA.231
could somehow divert the CSR machinery to In our studies, we have found that CpG stimu-
other target S regions, which is a role usually lation alone results in robust B-cell proliferation,
played by cytokines. but not in significant CSR, as indicated by the low
CpG can also modulate B-cell differentiation frequency of B cells expressing surface IgG, IgE,

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to plasmablasts/plasmacytes,86,222 thereby resulting or IgA measured by flow cytometry. However, in
in complex changes in the titer of isotype-switched the presence of BCR cross-linking (and appro-

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antibodies in culture supernatants, and therefore priate cytokines), CpG stimulation could lead to
complicating the monitoring of CSR. Further- robust CSR to all respective isotypes, as well as

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more, IFN-γ and TGF-β, two cytokines critical to plasma cell formation and antibody secretion.
for CSR to IgG2a and IgG2b, respectively, are Furthermore, contrary to previous reports,213,217,232
known to decrease B-cell proliferation and survival, our data indicate that TLR9 stimulation in the

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whereas CpG is a potent proliferation and survival absence of BCR cross-linking suppresses LPS- (or
stimulus, which adds additional complexity to the CD154-) and cytokine-induced CSR not only

IB
overall antibody response. to TH1 but also to TH2 isotypes, and suppresses
plasma cell formation. However, in the presence
of BCR cross-linking, LPS and CpG, as well
C. BCR Signaling Synergizes With Tlr
TR as LPS and Pam3CKS4, synergized in inducing
Signaling CSR in a dose-dependent way (Pone and Casali,
unpublished observations). Similar synergistic
IS
TLR-induced CSR depends on the correct cellular and antagonistic effects have been reported for
trafficking and localization of the engaged TLRs, cytokine production by B cells43 and PBMCs233
as well as whether other signals, particularly those stimulated with specific pairwise combinations of
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from the BCR, are also activated.6,199-202,223,224 TLR TLR ligands.


ligands such as LPS or flagellin can elicit antibody CpG and other surface and endosomal TLR
responses directed against them, complicating the ligands, such as Pam3CSK4 and R848, might not
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delineation of the respective roles of BCR and display a TH1 vs TH2 bias in inducing B cells to
TLRs in anti-microbial antibody responses. B cells undergo CSR to specific Ig isotypes (like cytokines
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phagocytose antigen in a FcR- or BCR-dependent do), but like LPS, these may act as inducers of
way,225-228 efficiently process internalized antigenic NF-κB and AID, with the latter initiating CSR
proteins, as well as sense various MAMPs present to isotypes specified by cytokine-induced germline
in the phagocytosed pathogen. BCR and TLR9 transcription. The mechanism by which CpG
signaling synergize in the induction of NF-κB in antagonizes LPS- and CD40-induced CSR in
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B cells,199,223,229 and BCR signaling recruits TLR9- the absence of BCR cross-linking could be due to
containing endosomes to autophagosomes,199,223 sequestration of signaling adapters or due to signal
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possibly as a way of efficiently coordinating the paralysis. Another possibility could be that in the
sensing of nucleic acid TLR ligands present when absence of strong BCR cross-linking, CpG may
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a B cell internalizes pathogens. enter the cell via BCR-mediated uptake, setting
Signaling cross-talk between ITAM receptors off weak BCR signaling (low ITAM signaling),
and TLRs could modulate responses in immune which has been proposed to negatively regulate
cells.6,224 For example, DCs responded to ssRNA TLR and cytokine signaling in macrophages.6,224
by producing IFN-γ only if the viruses had been Conversely, during dual stimulation with BCR
previously opsonized with antibodies, which cross-linking and CpG (mimicking the internal-
cross-link Fc receptors on the DC membrane230; ization of microorganisms by BCRs that have
cross-linking of activating FcRs in DCs and high-affinity for their external epitopes), the strong
other cells can serve the same function as the ITAM signaling synergizes with TLR9 signaling,

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TLR and BCR Induction of CSR 13

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TR
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D
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FIGURE 3. The interplay of surface TLR4 and endosomal TLR9 in CSR. a, LPS induces CSR to isotypes, as
directed by cytokines. b, CpG by itself does not induce significant CSR; however, in the presence of BCR cross-
linking (indicated by a virus bound by BCR; experimentally it can be mimicked by reagents that cross-link the
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BCR polyclonally), it behaves similar to LPS in inducing CSR to the isotypes specified by cytokines. c, CpG sup-
presses LPS-induced CSR; however, CpG synergizes with LPS in a dose-dependent way in the presence of BCR
cross-linking (d).
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and results in differentiation, as hallmarked by by corresponding pathogenic MAMPs, and then


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CSR (Fig. 3). produce TH1 or TH2 cytokines that initiate the
Considering CpG as a general inducer of B induction of TH1 or TH2 cells, which then amplify
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cells to undergo CSR to isotypes directed by the the polarization and induce B-cell differentiation
different cytokines, without itself displaying an and CSR to specific TH1 or TH2 isotypes.13,26,239-241
intrinsic TH1 vs TH2 isotype bias, does not con-
Whether B cells, after directly sensing the type of
tradict the reported TH1 vs TH2 polarizing effect
of CpG in other innate immune cell types, and its pathogen via engagement of their TLRs by the
use as a predominantly TH1 vaccine adjuvant.234-238 corresponding pathogenic MAMPs, can undergo
It is likely that cells such as DCs and macrophages CSR to the most appropriate isotypes, as is the
sense the pathogen type present in an infection by case for LPS-induced CSR to IgG3, remains to
engagement of various PRRs (including TLRs) be determined.

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14 Pone et al.

While the role of TLRs in autoimmunity is antibodies during early infection when pathogen
beyond the scope of this review, the reader is referred loads are relatively low and T cell help is not
to several recent reviews on the subject.172,242-249 yet fully available. LPS stimulation of mouse
Briefly, it appears that under conditions in which B cells in vitro results in IgM and IgG3 antibody
TLRs sense host ligands that resemble MAMPs, production. The first antibodies to be produced
such as phospholipids that resemble LPS and could in vivo against an antigen encountered for the
engage TLR4, or self RNA or DNA that could first time are IgM, which can contain the patho-
engage TLR3, TLR7/8, and TLR9, autoreactive B gen in the first hours to days of infection. Studies
cells are activated to produce autoantibodies.250-253 in mice selectively deficient in secreted IgM,254
This could be particularly important in the genera- suggest that the initial T-independent production

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tion of anti-DNA class-switched autoantibodies, of IgM serves not only to contain pathogens, but
which are the main cause of systemic lupus ery- by forming complexes with pathogens, serves as

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thematosus. an autocrine factor for continued local B-cell
proliferation via IgM-mediated multimeriza-

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tion of soluble antigen that can now efficiently
IV. TLR- AND BCR-INDUCED B-CELL cross-link the BCR. In the absence of timely
ANTIBODY RESPONSES AGAINST T-cell help, other cell types, such as follicular

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MICROBIAL PATHOGENS dendritic cells, may provide a level of stimulation
sufficient for B-cell activation and early IgM,255

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A. TLR Signaling in T-Independent B-Cell and likely IgG, secretion.
Differentiation TR
1. TLR Signaling During the Initial Phase 2. TLR Signaling During B-Cell
of B-Cell Differentiation Differentiation in the Absence
of T-Cell Help
IS
TLRs were likely one of the first immune recep-
tors that recognized conserved molecular pat- Even though optimal CSR requires T-cell help,
terns of pathogens.4 A recent report described some CSR does occur without T-cell help fol-
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the rearrangement of lamprey antigen receptors, lowing challenge with viral, bacterial, and fungal
where an APOBEC-related enzyme was found pathogens (Table 4). While the titers of class-
to deaminate the gene segments encoding these switched antibodies in the absence of T-cell help
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receptors, leading to their rearrangement and thus are usually much lower than the optimal titers
diversification to a limited number of configura- produced in the presence of normal T-cell help,
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tions.32 Another report on lamprey immunity in several cases the titers are enough to confer
revealed the presence of B- and T-like cells, which some protection against infection. Class-switched
sense pathogens via membrane-expressed VLRs, antibodies can be generated against invading
with the B-like cells progressing through blast pathogens without T-cell help because the enzyme
formation and secreting soluble antigen-specific AID can be induced in part by the conserved
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VLRs.36 Thus, it is possible that TLRs, VLRs, or homeodomain transcription factor HoxC4145,256,257
related primordial PRRs were the original antigen in a T-independent fashion.17,54,176,177,189,192,258-260
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receptors, and during evolution were replaced by CSR that occurs in the absence of TH cells261
the newer and more and versatile BCRs, which is particularly applicable to antiviral antibody
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are based on the Ig domain fold. TLRs continued responses.258,262 Immunization of T-cell-deficient
to provide information regarding the presence mice with polyoma virus resulted in the produc-
and type of pathogen to innate immune cells tion of virus-specific class switched IgG, although
and B cells. in lower titers compared with wild-type mice,
TLR-mediated differentiation of B cells may whereas immunization with either a viral antigen
be a critical component of T-independent, but or VLPs (virus-like particles) did not result in IgG
early and effective B-cell clonal amplification and production, possibly due to the presence of dual
differentiation leading to the production of class TLR and BCR engagement in the case of whole
switched, potentially hypermutated, protective virus and lack of TLR engagement in the case of

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TLR and BCR Induction of CSR 15

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

Pathogen/Antigen Mouse Model Ig Produced Reference


Polyoma virus TCRβ–/– x δ–/– IgM, IgG 316
B cell transfer in SCID IgM, IgG 317
CR2–/– B cell transfer in IgM, IgG
SCID 318

VSV TCRα–/–; TCRβ–/– IgM, IgG2a 319


Rotavirus TCRαβ–/– x γδ–/– IgM, IgG, IgA 320
TCRαβ–/– IgA 321

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LCMV, VSV, Pichinde virus CD154–/– IgM, IgG2a 322
Influenza virus CD4 –/– IgM, IgG1, IgG2b, IgG3 323

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CD40–/–; MHCII–/– IgM, IgG 324
Ehrlichia muris CD4–/–; CD8–/–; MHCII–/– IgM, IgG1, IgG2c, IgG2b, IgG3 325

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Ehrlichia chafeensis CD4 –/– IgG1, IgG2a, IgG2b, IgG3 326
Borrelia burgdorferi TCRβ–/–; TCRβ–/– x δ–/– IgM, IgG3 327

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Bacillus anthracis TCRα –/– IgG 328
Yersinia enterocolitica nude IgG 329

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Citrobacter rodetium CD4–/– IgM, IgG1, IgG2c, IgG2b, IgG3, IgA 330
Porphyromonas gingivalis TCRα –/–
TR 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; TCRβ ; –/– IgM, IgG1, IgG2a, IgG2b, IgG3 335
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MHCI–/–; MHCII–/–
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antigen alone or VLP immunization.263 In other do not peak until several days after their initial
studies, natural IgA antibodies were produced in activation. IgM production, in addition to provid-
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response to TLR ligands of commensal gut bac- ing some early and direct protection by targeting
teria,264 natural IgE antibodies were generated to pathogenic epitopes, also serves in the activation
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activate innate immune mast cells without T-cell of complement, which then results in microbe
help,265 and IgG2a antibodies were produced out- coating with C3d, which binds the CD21/CD35
side GC follicles three days after immunization.266 receptors on the B-cell surface and enhances B-cell
Considering the low-level CSR that occurs in the activation.267 The antibody-pathogen complexes
absence of T-cell help, it is possible that this can are drained in secondary lymphoid organs, where
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have functional significance in helping to contain they are destroyed by professional phagocytes such
the low pathogen loads during the early stages of as macrophages, and in the process stimulate the
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infection when T-cell help has not yet peaked198,267 T-dependent GC reaction.99
(Fig. 4). Direct TLR stimulation of B cells during
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the GC reaction may help to sustain and shape


the processes of antibody affinity maturation by
B. TLR Signaling May Play Roles During acting as a reporter of pathogen load and infec-
the T-Dependent Phase of B-cell tion conditions.10,20,49,175,191 For example, initial
Differentiation TLR-mediated activation of B cells leads to the
up-regulation of co-stimulatory CD80/CD86 and
Initial IgM and IgG production by B cells is likely MHC-II receptors, thereby priming B cells for
due to their activation by various TLRs and the more abundant and efficient interactions with T
BCR, since antigen-specific T helper cell numbers cells and DCs.268 T-cell help alone may not be

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16 Pone et al.

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FIGURE 4. Model on the role of endosomal TLRs in B-cell antibody responses. a, B cells in the secondary lymphoid
organs can be directly activated by viruses that cross-link the BCR. This then leads to their phagocytosis, double-
membrane autophagosome formation, and trafficking of the autophagosomes to TLR-containing endosomes. If
any present viral TLR ligands are sensed by TLR3, TLR7/8, or TLR9, an appropriate response is then mounted
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by the B cells, which includes their differentiation (and in some cells, T-independent CSR), the up-regulation of
receptors that interact with TH cells and DCs, thus enhancing the GC reaction (b). The result of T-independent (a)
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and T-dependent (b) B cell differentiation is the formation of antigen-specific plasma cells, and memory B cells,
which can be quickly differentiated to plasma cells upon subsequent re-infection (c).
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sufficient for optimal antibody responses, with cells, and DCs, eventually giving rise to plasma
direct TLR stimulation of B cells contributing and memory B cells, whose Ig genes encode
to optimal antibody titers and CSR,49 though antigen-specific, high-affinity, and class-switched
this issue remains unclear.128,129,175,191 During antibodies.199,223,269
the GC reaction, TLR signals in B cells are Immunization with CpG and hepatitis antigen
likely integrated with signals from the BCR, T resulted in higher antibody affinities than immu-

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TLR and BCR Induction of CSR 17

nization with antigen alone.270 When TLR ligands injection, also contain mycobacterial components
in a RSV (respiratory syncytial virus) vaccine were (which include various TLR ligands) in order to
inactive, the resulting antibodies were low-affinity elicit robust immune responses to soluble protein
due to impaired SHM and were not protective.271 antigens.7,10,175,273,274 Since the discovery of the
These reports suggest that TLRs play a role in crucial adjuvanting properties of TLR and other
SHM, since their engagement contributes to the PRR ligands, there has justifiably been an explo-
generation of high-affinity antibodies, though the sion in the application of defined adjuvants for
molecular and cellular mechanisms responsible use in vaccine development.274-297
have not yet been elucidated. The immune system, All current clinical vaccines elicit protective
including B cells, need to continually recognize

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antibodies,87,88 frequently also eliciting the mobili-
and bind pathogens in order to ultimately clear zation of other immune cells, including NK cells,
them. In particular, nucleic acid TLR ligands may

O
cytotoxic T cells, TH cells, DCs, macrophages,
be important signals for SHM, because they would
and so forth.274 As mentioned previously, several
signal the presence of replicating, and potentially

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studies have reported that CpG or antigen-
hypermutating, microorganisms.272
CpG fusion immunization in humans and mice
result in higher antibody titers and/or antibody

U
C. TLR and BCR Signaling in Vaccine affinities for antigen and/or longer-lasting protec-
tion,19,234,235,269,270,298-303 possibly due to enhanced

IB
Research
CSR and SHM and expansion of high-affinity
1. Vaccine Adjuvants TR memory B cells. The reader may refer to a recent
review for an extensive list of examples where
Well before the discovery of the TLRs, vac- prior CpG administration has resulted in some
cinologists empirically used adjuvants such as protection before challenge with pathogens.304 A
Freund’s complete adjuvant, Freund’s incomplete comprehensive list of current clinical and experi-
IS
adjuvant, and Ribbi’s adjuvant, which, in addi- mental vaccines that target T-independent bacte-
tion to containing oils for forming depots upon rial antigens has also been recently reviewed.305
D
R
FO
T
O
N

FIGURE 5. The design features of modular vaccines. a, Virus-mimicking biodegradable particles with favorable
pharmacokinetics, which by themselves are not immunogenic, are used as scaffolds for the incorporation of pro-
tein antigens from one or several pathogens. Initial screens determine the most immunogenically suitable protein
antigens (brown triangles) to use. In addition, combinations of one or more natural or synthetic TLR ligands are
included, usually mimicking their occurrence in the actual pathogen(s). b, Particles mimicking bacteria are coated
with bacterial protein antigens as well as TLR ligands characteristic of each bacterium. These composite vaccines
(a, b) activate both innate immune cells as well as B lymphocytes, which will differentiate to plasma cells that
secrete antigen-specific antibodies, as well as to memory B cells that can be activated upon potential future
re-infection. c, Legend indicates the protein antigens and the various TLR ligands used in modular vaccines.

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18 Pone et al.

2. New Generation Modular Vaccines V. CONCLUSIONS

Based on the promise of defined TLR agonists The synergy of BCR and TLR signaling criti-
for use in vaccines, a new generation of vaccines is cally regulates B-cell antibody responses, and in
currently being developed. These vaccines employ particular CSR. BCR cross-linking facilitates the
particles made of biodegradable materials, lipid encounter of endosomes containing TLR9, and
vesicles, or self-assembling protein subunits, that in likely other TLRs, to autophagosomes as a way
addition to containing protein antigen, are infused of surveilling the presence of nucleic acid TLR
with internal or external TLR ligands192,306-312 (Fig. ligands during an infection. BCR stimulation
5). This approach allows for modular, systematic,

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provides information on the affinity of antigen
and controlled testing of the best combinations of for antibody, and the spacing and topology of
protein antigens from the pathogens and natural

O
antigenic arrays on the pathogen, whereas TLR
or synthetic TLR agonists, taking into account
stimulation may instruct B cells on the nature
the dual TLR and BCR induction of B-cell dif-
of the pathogen, e.g., the presence of a Gram+ve

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ferentiation that was described in the previous
section. Generally, a rigid array of a minimum or Gram-ve bacterium or of an RNA or DNA
of 15 to 20 repeating antigens induces optimal virus. It would be particularly useful for B cells

U
BCR signaling.192 to sense multiple TLR signals from a pathogen
W hile modular vaccine design is still under in a combinatorial fashion, thereby leading to a

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development, as a general approach, the particle more appropriate and specific response.
and the TLR ligand combinations used should The dual presence of BCR and TLRs on B cells
mimic the pathogen as closely as possible; for allows them to respond to a greater variety and
example, it may not be beneficial to use flagellin
TR combination of signals than previously thought,
to develop modular viral vaccines because it does including traditionally innate signals, thereby
not naturally occur in viruses. The TLR ligands allowing for fine-tuned responses that best deal
IS
can be LPS, Pam3CSK4, flagellin, and so forth, with the pathogenic threat. The interplay and
which should be incorporated on the outside of synergy between BCR, TLRs, and T-dependent
the vaccine particle, and CpG, ssRNA, dsRNA, signals is also proving to be both challenging and
D

or agonists such as imiquimod or R848, which rewarding. The ultimate task remains to predict
should be best incorporated inside the particle. B-cell behavior during an infection in vivo based
Future studies may, however, report beneficial, on studies of responses to defined combinations
R

synergistic combinations of TLR ligands that are of stimuli under different conditions in vitro and
not normally found in the same pathogen, which in vivo. Knowledge of fundamental B-cell signals
FO

may apply especially in the cases of co-infection and typical responses would be beneficial for the
by two or more pathogens.
design of therapies for immune deficiency or
Taking this approach one step further, recent
autoimmunity, and for the development of effec-
studies have explored the feasibility of develop-
tive vaccines.
ing broad-spectrum vaccines, where immunity
T

is provided against more than one pathogenic


strain, or against several related or even unrelated
O

pathogens.313,314 In this case, antigens from several ACKNOWLEDGMENTS


different pathogens are incorporated in the same
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particle or separately in mixtures of different par- Owing to space limitations, we could cite only a
ticles, with TLR ligands typical of each pathogen fraction of the papers relevant to the topics dis-
also incorporated in the particles. In principle, cussed in this review article. We apologize to the
this approach should allow for mobilization of other authors of publications that were not cited.
B cells that produce antibodies specific for each We would like to thank members of the Casali
pathogen. Issues remaining to be resolved relate laboratory for invaluable discussions and critical
to the safety and efficacy of CpG and other TLR reading of this manuscript. This work was sup-
agonists in eliciting broad-spectrum immunity to ported by NIH grants AI 045011, AI 0790705,
emerging and re-emerging pathogens.313,315 and AI 060573 to P.C.

Critical Reviews™ in Immunology

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TLR and BCR Induction of CSR 19

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