Kearney Annual Review

IY33CH16-Kearney ARI 13 January 2015 12:31
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Natural Antibody Repertoires:
Development and Functional
Role in Inhibiting Allergic
Airway Disease
John F. Kearney, Preeyam Patel, Emily K. Stefanov,
and R. Glenn King
Department of Microbiology, University of Alabama at Birmingham, Birmingham,
Alabama 35294; email: jfk@uab.edu
Annu. Rev. Immunol. 2015. 33:16.1–16.30
The Annual Review of Immunology is online at
immunol.annualreviews.org
This article’s doi:
10.1146/annurev-immunol-032713-120140
Copyright c 2015 by Annual Reviews.
All rights reserved
Keywords
B cells, allergy, neonatal immunity, hygiene hypothesis, chitin, asthma
Abstract
In this review we discuss the effects of microbial exposure on the B cell reper-
toire. Neonatal exposure to conserved bacterial carbohydrates and phospho-
lipids permanently reprograms the natural antibody repertoire directed to-
ward these antigens by clonal expansion, alterations in clonal dominance,
and increased serum antibody levels. These epitopes are present not only in
bacterial cell walls, but also in common environmental allergens. Neonatal
immunization with bacteria polysaccharide vaccines results in attenuated al-
lergic airway responses to fungi-, house dust mite-, and cockroach-associated
allergens in mouse models. The similarities between mouse and human natu-
ral antibody repertoires suggest that reduced microbial exposure in children
may have the opposite effect, providing a potential mechanistic explanation
for the hygiene hypothesis. We propose that understanding the effects of
childhood infections on the natural antibody repertoire and the mechanisms
of antibody-mediated immunoregulation observed in allergy models will lead
to the development of prevention/interventional strategies for treatment of
allergic asthma.
16.1

nAbs: natural
antibodies
INTRODUCTION
The hygiene hypothesis, in its various iterations, proposes that the escalating incidence of allergic
and autoimmune disease in industrialized countries is a consequence of increasingly sanitary living
conditions (see sidebar Hygiene Hypothesis). Hygienic practices result in less exposure of the
neonatal developing immune system to environmental microbes and parasites (1–3). It is generally
assumed that this decline in pathogen exposure disrupts the balance between Th1- and Th2-type
immune responses, promoting a bias for the Th2 responses that ultimately drive the development
of atopic and autoimmune diseases in susceptible children. Whereas B cells, which give rise to IgE
antibodies (Abs), are clearly important in atopy and allergy, little attention has been paid to the
effects of early microbial exposure on the B cell repertoire during the initiation of these diseases.
Early antigen experience has been shown to affect (a) the development of B cell subsets, (b) the
content of the expressed natural antibody (nAb) repertoire, and (c) B cell interactions with other
elements of the immune system. In this review, we describe experiments in mouse models that
document the effects of deliberate neonatal microbial exposure with selected bacterial vaccines on
the B cell compartment and circulating nAbs that have a strong and persistent dampening effect
on the development of allergic airway disease (Figure 1).
Although the origin and function of nAbs have been the subject of age-old discussions (4), these
topics, and the very definition of nAbs, remain controversial. One hallmark of nAbs is that they
can be found in germ- and antigen-free mice, observations which suggest that their production
may be driven, at least in part, by self-antigens (5). However, it is also clear that exposure to mi-
croorganisms, either intentionally or environmentally-derived, results in long-lasting effects on
the clonal diversity of these Abs, their circulating levels, and ultimately their biological functions
which include first line defense as well as immunoregulatory activities. It is often stated incor-
rectly that most nAbs are polyreactive and are therefore nonspecific. Before we continue with this
review, it is important to clarify and correct this viewpoint. In many instances, the assays used
to determine binding properties of nAbs have been variants of solid-phase ELISA-type assays.
These assays, unless used appropriately, may detect very low-avidity binding of Abs to unrelated
molecules. In contrast, some nAbs bind, with exquisite specificity, to defined oligosaccharides that
are expressed as shared epitopes of glycan structures present in microbial cell wall components as
well as glycosylated mammalian molecules. These Abs are “polyreactive” only in the sense that
HYGIENE HYPOTHESIS
Originally proposed by Strachan (1) in 1989, the hygiene hypothesis was introduced as an explanation for the inverse
correlation between the number of older siblings of school-age, British children and the occurrence of hay fever, as
reported in a large longitudinal study. Strachan postulated that smaller family sizes and higher sanitation standards
reduce the transmission of pathogens among children, resulting in an increased incidence of atopic disease. A variety
of preventive measures against infection, including immunizations, antibiotics, clean water sources, and safer foods,
have reduced microbial exposure of individuals within developed nations. Collectively these improvements in living
conditions have resulted in a remarkable decline in viral, bacterial, and helminth infections, especially among
children. Numerous epidemiological studies have demonstrated that these lifestyle changes are associated with a
reduction in infections and a concurrent emergence of allergic and autoimmune diseases. Currently, a variety of
factors interpreted to reflect early microbial exposure are associated with the risk of developing atopic disease. These
factors include delivery method, birth order, early day care attendance, family size, socioeconomic status, presence
of family pets, and properties of the commensal gut microflora.
16.2 Kearney et al.

Microbial
exposure
Atopic/
autoimmune
disease
Atopic/
autoimmune
disease
Alterations in
clonal frequencies
Elevated levels
of antibodies
TimeTime
Expansion
of repertoire
Figure 1
Early exposure to environmental polysaccharide antigens results in long-lasting changes in the immune system, including alterations in
the polysaccharide-reactive B cell repertoire, increased serum titers of antigen-specific antibodies, and increased frequencies of
antigen-specific B cells in circulation. Our experimental models show that these changes correlate with a decrease in allergic airway
disease. Based on these observations, we hypothesize that neonatal modulation of the B cell repertoire may have a global impact on
atopic and autoimmune diseases in general.
they bind the identical epitope on a variety of molecular entities and, as discussed later, these Abs
are dually capable of providing protection to specific infections (6, 7) (see sidebar Natural Antibod-
ies). Medical interventions to prevent microbial infections among children residing in developed
countries have reduced the carriage rate and resulting mortality of many opportunistic microbes.
However, 98% of the 3.9 million children that die annually are in developing countries (8). These
children suffer from a high rate of bloodstream infections which, in addition to gram-negative
enteric infections, include infections of gram-positive organisms, such as Streptococcus pneumoniae
NATURAL ANTIBODIES
The observation that certain Ab reactivity occurs in the sera of most animals despite the absence of any deliberate
immunization has been the subject of considerable speculation.
The antigenic targets of some well-characterized nAbs are shared by pathogens and host. These epitopes may
exist constitutively or represent neoepitopes that result from altered glycosylation of host proteins or oxidation of
host constituents. Targeting of these endogenous epitopes, which are usually sequestered from immunosurveillance,
provides beneficial housekeeping functions. It is also clear these Abs participate in immune responses to pathogenic
organisms; thus, the natural repertoire is thought to be evolutionarily conserved and of dual function. Examples
in this review are (a) Abs to GlcNAc epitopes expressed on multiple O-GlcNAcylated intracellular proteins and
bacterial polysaccharides and (b) phosphorylcholine epitopes on host-oxidized low-density lipids and bacteria and
host parasites. Although it is obvious that nAbs are generated in the absence of immunization, in the world we
live in, exposure to potential Ab-inducing agents is unavoidable and likely influences most aspects of immunity,
including homeostatic Ab production. In this review we describe several instances in which neonatal exposure to
bacteria-derived products alters the repertoire and titer of nAbs. Thus, even this most innate component of the
adaptive immune system can be plastic and responsive to environmental circumstance.
www.annualreviews.org • Antibody Modulation of Allergic Disease 16.3

GAS: group A
streptococcus/
Streptococcus
pyogenes
PS: polysaccharide
PC:
phosphorylcholine
GlcNAc: N-acetyl-d-
glucosamine
(9), Streptococcus pyogenes [group A streptococcus (GAS)] (10), and Streptococcus agalactiae [group B
streptococcus (GBS)] (11).
The Ab response to bacterial polysaccharides (PS) is often pauciclonal in mice and humans.
In the case of α-1,3-glucan Abs induced by the gram-negative, enteric, opportunistic pathogen
Enterobacter cloacae, a single heavy chain gene encodes this specificity (12, 13) and is absent in
some strains of mice, rendering them incapable of mounting this response (14). By contrast, Abs
to phosphorylcholine (PC), a phospholipid expressed on the C-polysaccharide of S. pneumoniae
(pneumococcus), display a highly conserved specificity across organisms and are produced by
one of the earliest B cell clones (B1a) (15) that arise during mouse development (16). Like those
reactive with PC, Abs toward N-acetyl-D-glucosamine (GlcNAc), the immunodominant structure
on the group A carbohydrate (GAC) of GAS, are ubiquitous in mammals. Antiglycan Abs can be
exquisitely specific; for example, monoclonal antisialyllacto-N-tetraose-reactive Abs derived from
a hybridoma generated following multiple immunizations with GBS1b differentiate between the
PS of GBS1a and GBS1b serotypes, which differ by a single glycosidic linkage (17). Some of these
Abs, including those reactive to pneumococcus and GBS, are sufficient to provide protection from
lethal challenge with the virulent bacteria against which they were raised.
In preliminary studies we found that many monoclonal Abs raised against bacteria PS react
with both the conidia and the hyphae of Aspergillus fumigatus (Figure 2). It was these observations
that led us to investigate whether anti-PS Abs engage other common environmental allergens,
modulate the immune response to inhaled particulates, and influence the induction of allergic
airway diseases. In this context, we review the ontogeny and functional activities of B cells and Abs
specific for conserved antigens on a group of clinically relevant organisms and consider how the
immune response to them can be harnessed to combat the development of allergic airway disease
sialyllacto-N-tetraose
GlcNAc
α-1,3-glucan
a b
Figure 2
Reactivity of monoclonal antibodies (mAbs) raised against bacterial antigens to Aspergillus fumigatus.
(a) A. fumigatus conidia were incubated on poly-L-lysine-coated slides for 9 h in 10% fetal calf serum and
RPMI 1640 medium at 37◦C. The resulting germlings were fixed in 95% ethanol for 30 min and stained
with mAbs to α-1,3-glucan ( green), sialyllacto-N-tetraose (red ), and N-acetyl-D-glucosamine (GlcNAc)
(blue). (b) Phase contrast of the same field with arrows showing conidia remnants. Images taken at 1,000×
original magnification.
16.4 Kearney et al.

(Figure 1). In the following sections, we discuss the clonal development of B cell responses to
bacterial exposure for several antigen specificities, and we end by reviewing protective effects of
anti-PS nAbs in mouse models of allergic airway disease.
B CELL ONTOGENY AND NATURAL ANTIBODY
B Cell Subsets
The source of natural IgM is controversial as it is unclear if only certain B cell subsets give rise to
nAb secreting cells, and whether these cells reside in particular niches of lymphoid tissues. B cell
populations in mice are divided into B1, marginal zone (MZ), and follicular (FO) B cell subsets
by phenotype, microanatomical localization, and function (18). B1 and MZ B cells are thought
to be the major sources source of nIgM (see sidebar Natural Antibodies). MZ B cells, juxtaposed
with metallophilic and marginal sinus-associated macrophages, initiate rapid production of Ab,
predominantly IgM, against blood-borne particulate antigens and give rise to plasmablasts and
long-lived IgM-producing plasma cells that can reside in both the spleen and bone marrow (13,
19). Likewise, B1 cells, enriched in the peritoneal and pleural body cavities, contain precursors
for natural serum IgM (20), and mucosal secretory IgA production (21). There are differences
in this context however, between antigen-specific IgA generated in response to viral or bacterial
infections in the gut and the homeostatic production of IgA in response to the resident microbiota
(22–24). The body cavities are not normally sites of Ab production, however IgM-secreting cells
derived from the B1 compartment are found in multiple locations including the bone marrow (25)
(Reynolds, Kuraoka and Kelsoe, submitted) and spleen (26, 27). B1 cells may have a larger role in
modulating local immune responses at sites of inflammation.
Neonatal Response to T-Independent Antigens
Through decades of study, much is known regarding the involvement of various BCR-associated
molecules, transcription factors, and fate-determination molecules such as Notch, in the devel-
opment and maturation of MZ, FO, and B1 B cells. There are very few studies, however, that
investigate the influence of neonatal antigen exposure on the establishment of PS-specific B cells
within these subsets. Neonates are thought to respond poorly, if at all, to vaccination with T-
independent antigens.
The differential production of Ab in response to immunization observed between neonates and
adults may result from the immaturity of spleen and bone marrow microenvironments necessary to
support long-term Ab-forming cell differentiation and survival. Deficiencies in bone marrow stro-
mal support (28), and in particular the poor production of a proliferation-inducing ligand (APRIL)
(29) during the neonatal period, may explain why early exposure to bacterial vaccines induces little
detectable serum Ab. Although neonatal mice produce little circulating Ab to PS immunization,
they do in fact respond: We have observed highly significant changes within the emerging
antigen-specific B cell clonal repertoire following vaccination (30). In addition, neonatal priming
with bacterial vaccines produces dramatic changes in the magnitude of the subsequent responses
in adult animals. As described in later sections, neonatal vaccination enriches the frequencies of
PS-specific cells within innate-like B cell subsets and in turn enables a rapid Ab response of greater
magnitude upon secondary exposure by immunization or infection with the PS-expressing organ-
ism. Interestingly, it was recently shown by Ig heavy chain sequence analysis that IgM-producing
(unlike IgG-producing) plasma cells from adult mouse bone marrow express Ig genes that are
entirely nonmutated and mostly germ line derived (31). This observation suggests that most

bone marrow–associated IgM-secreting plasma cells are not germinal center–derived and could
conceivably be generated by neonatal immunization or antigen exposure if bone marrow niches
were more receptive for early seeding of T-independent, antigen-activated plasmablasts (29).
Collectively, these results show that although neonatal exposure to the TI-antigens described
above generates only relatively modest increases in serum nAb, it fundamentally and permanently
alters the clonal diversity and frequency of B cells that respond to these conserved antigens in
the adult. These effects greatly influence the Ab response to subsequent vaccination or infection
and are preserved for the life of the mouse. As we will discuss in the next section, the described
repertoire changes to these and perhaps other bacterially derived glycans may have a dramatic
effect on the development of allergic airway disease.
Functional Constraints of Neonatal B Cell Responses to Polysaccharides
There is much evidence that in humans and mice rapid B cell production of Abs to bacterial
pathogens is essential for protection from infection and prevention of blood-borne dissemination.
It is highly significant, yet paradoxical, that during early life (in mice up to 3 weeks of age, in
humans up to 1.5 to 2 years) infants respond poorly, if at all, to defined polysaccharide antigens
such as the PC epitopes on C-polysaccharide of S. pneumoniae (7) or GlcNAc epitopes on the
GAS cell wall polysaccharide (32–34). Individuals within these age groups are more susceptible to
infection, particularly by gram-positive encapsulated bacteria.
Both the genetic and the environmental factors influencing the emerging B cell repertoire,
particularly those clones specific for bacterial PS cell wall antigens, are poorly understood. Factors
involved in the delayed appearance of PS-reactive Ab include (a) constraints in Ig gene expression
in the developmental B cell program during fetal and neonatal life (35–38), (b) the immature state
of lymphoid tissue development and function (39, 40), and (c) the presence of passively transferred
maternal Ab (41–43). Thus, the development of a B cell repertoire capable of providing innate-
like Ab-mediated protection or that can be stimulated by vaccination results from the complex
interplay of genetics and the environment, including the timing of antigen exposure (44). The
use of PS-protein conjugate vaccines has largely alleviated the burden of infections in infants,
but because their vaccine-mediated immunity wanes, the development of additional strategies is
necessary to provide sustained protection. In addition, the emergence of new capsular serotypes
has further complicated vaccine development. Long-term protection after immunization against
encapsulated bacteria is generally thought to depend on the persistence of functional Abs and
immunological memory. However, the anamnestic response that we consider classic memory
takes up to four days to develop and may be slow relative to the rapid invasion of encapsulated
bacteria (45–47). It is during this period that innate-like B cells, which produce long-lasting nAbs
or rapid Ab responses at sites of bacterial interdiction, such as the splenic MZ and mucosal sites,
may be more important than classic B cell memory for protection against certain pathogens (48).
Windows of Opportunity for Generating Long-Lived B Cell Clones
It is clear from mouse and limited human studies that Abs binding bacterial PS often are oligo-
clonal, are germ line encoded, and bind to conserved PS epitopes that are shared by multiple
environmental organisms. In the adult, some B cell clones result from developmental programs
that are active early in life (49, 50). In multiple examples, interference with these development
processes during the neonatal period results in long-lasting changes in the expressed adult B cell
repertoire (14, 49, 51). Bacterial infection in humans early in life, although less studied, may
render an individual unresponsive to later deliberate vaccination against the same bacteria (52–
54). Additionally, as observed in attempts to vaccinate against Haemophilus influenzae, although
16.6 Kearney et al.

TdT: terminal
deoxynucleotidyl
transferase
Idiotype:
immunoglobulin
variable region marker
to identify B cell clones
memory resulting from vaccination is sufficient to generate a large Ab response upon infection,
vaccinated individuals do not have significant protection compared with unvaccinated individu-
als (vaccine failure) (55). It is also clear that the age at which a vaccine is administered is key in
achieving sustained Ab protection. The Ab unresponsiveness following the vaccination of infants
with DTaP (diphtheria, tetanus, and pertussis) is commonly attributed to the immaturity of the
neonatal immune system, but it is more likely due to vaccine interference with development of
appropriate B cell clones (29, 53, 56).
Interclonal Competition in the Developing Innate-Like B Cell Repertoire
Vaccine interference associated with conjugate vaccines has been attributed to carrier effects (57),
and the phenomenon of interclonal competition for space in the developing innate-like B cell
repertoire has not been well studied in humans. In mice, this space clearly exists. When mice
heterozygous for one of the two possible IgM allotypes are suppressed by an antiallotype Ab
during early development, the nonsuppressed, allotype-bearing B cells dominate the B1 niche for
the life of the animal. Thus, deletion of these clones during the neonatal period imprints the B cell
repertoire for the life of the mouse (58, 59). Importantly, we found that early B cell developmental
programs are critical for the establishment of certain Ab clonotypes. Premature induction of N-
additions by transgenic induction of the N-region-altering enzyme terminal deoxynucleotidyl
transferase (TdT) in fetal life permanently prevents the formation of the dominant PC-specific,
germ line–encoded, TEPC15 (T15) idiotype–bearing B cell clones. Although these mice produced
anti-PC Abs, they were not protected against pneumococcal infection (49). Similarly, the genetic
ablation of TdT resulted in an increase in the T15 idiotype–bearing Ab in comparison with levels
in wild-type mice. These data suggest a temporal window for germ line–encoded rearrangements
to occur in developing B cell progenitors and become represented in the adult repertoire (50). In
addition to conferring protection from pneumococcus, anti-PC Abs are reactive toward oxidized
lipids, including low-density lipoproteins, and they may be protective against the development of
atherosclerosis in models of this disease (60). By contrast, development of the dominant clone in the
BALB/c response to α-1,3-glucan, which is expressed widely in bacteria and fungi, requires TdT
activity. A critical aspartic acid residue within the VD-junction is encoded through N-additions;
TdT-deficient mice demonstrate greatly reduced responses to α-1,3-glucan and production of
the normally dominant J558-like B cell clones, which are normally recruited into the B1b and MZ
subsets (13, 19, 51, 61).
Many PS-specific Abs are encoded by germ line sequences, and somatic mutations do not
necessarilycorrelatewithenhancedfunction.InthecaseofAbsderivedfromhybridomasgenerated
following hyperimmunization with PS-protein carriers, long-lived IgG-switched clones harboring
V gene somatic mutations bind less effectively to the bacterial PS-associated epitopes (62, 63)
in comparison with germ line sequence–bearing Abs induced by bacterial immunization. Thus,
although PS-conjugates induce high frequencies of isotype-switched Abs and may recruit a more
diverse pool of B cells into the response, there is very little information about how many of these
clones actually produce protective Ab in mouse models of infection or in human PS-conjugate-
vaccinated humans.
Neonatal Antigen Exposure and the Subsequent Magnitude
and Clonal Diversity of the Adult Repertoire
These studies in mice, and limited studies in humans, collectively emphasize that neonatal ex-
posure to bacterial PS affects the production, quality, and quantity of Abs to PS antigens upon

IgM ANTIBODIES
Of the Ig isotypes, IgM or IgM-like molecules are the first to appear evolutionarily, ontogenetically, and in host
adaptive immune responses. The conservation of these molecules throughout the evolution of adaptive immune
systems is indicative of their role in host survival. IgM Abs exhibit very high avidity, effectively fix complement, and
are rapidly generated by B cell subsets and thus represent a powerful response to pathogen challenge. The production
of IgM through the expression of germ line–encoded Ig genes effectively couples a conserved evolutionary memory
with highly efficient effector functions without the need for somatic hypermutation normally associated with T
cell–driven germinal centers. IgM Abs constitute a large portion of nAbs, however little attention has been paid to
their role in immune responses or how their production can be manipulated to the host’s advantage. The inordinate
focus on the dogma that high affinity IgG responses is the goal of immunization and that so-called sticky, low-affinity
IgM Abs should be avoided is the primary reason for this dearth of information. Hopefully, recent findings such as
IgM memory cells in humans will lead to more focus on the functions of this first-line component of the adaptive
immune response.
reimmunization or challenge with these organisms in adulthood. It is also clear that the develop-
ment of a stable B cell repertoire is highly dependent on the timing of exposure to these organisms,
the nature of the particular antigens associated with these organisms, and clonal competition for
available space in the emerging B lymphocyte pool. Vaccinology has been dominated by the tra-
ditional view that a certain level of IgG Ab, if attained, will provide successful protection (64).
However, IgM Abs are not usually quantified, and not all Abs are equal with respect to the pro-
tection they confer. Only a few studies have attempted to define and preferentially induce B cell
clones that produce the most protective Abs (65–70) (see sidebar IgM Antibodies).
How neonatal exposure to antigen alters the clonal frequency, subset distribution, and
function of PS-specific B cells later in adulthood has not been adequately studied in humans. In
the next sections, we discuss evidence that there is a critical window during development when
the B cell subset and clonal repertoire are plastic and can be manipulated (22, 58, 71, 72). An
in-depth analysis of this critical developmental window may lead to appropriate interventions
to optimize vaccine strategies and maximize protection. Moreover, the probable introduction
of additional childhood vaccine regimens and alterations in the neonatal vaccination schedule
make it imperative that we understand the effects of vaccine interference on subsequent immune
responses to further vaccination or to infection resulting from multiple vaccinations, neonatal
chronic infections, or coinfection. Despite obvious differences, many aspects of neonatal immu-
nity are strikingly conserved between mice and humans. We share similar B cell lymphoid tissue
structure developmental programs, pauciclonality of anti-PS responses, and delayed responses to
PS antigens leading to increased neonatal susceptibility to bacterial infections. These similarities
validate the use of mouse models for these studies and suggest the observations in the following
sections are pertinent to human infant humoral responses against infection and during vaccination.
Historically, successful vaccines have induced Ab-based immunity. Identification of antigens
inducing protective Abs has been important in the development of candidate antigenic targets for
inclusion in conjugate or conventional vaccines. When new vaccines become available, studies will
need to focus on the mechanisms by which they influence the B cell repertoire and Ab production.
Past and ongoing studies of the neonatal response to well-characterized epitopes expressed on a
multimember panel of microbes have shown uniformly that early exposure to vaccines derived
from bacteria has long-lasting effects on the adult clonal repertoire. In addition, the magnitude of
the Ab response following reexposure is drastically increased. Thus, decisions in the present day
16.8 Kearney et al.

concerning vaccines and immunization schedules may have dramatic and unexpected epidemio-
logic effects for decades.
B CELL ONTOGENY AND NATURAL ANTIBODY
RESPONSES TO BACTERIA
B Cells Responding to N-Acetyl-D-Glucosamine–Bearing Streptococcus pyogenes
The response to GlcNAc, the immunodominant component of the cell wall PS of GAS, has
been extensively characterized in mice. GAC is a high-molecular-weight polymer consisting of
a helical rhamnose core structure decorated with exposed GlcNAc moieties. In addition to this
structure, the cell wall of GAS contains large amounts of peptidoglycan, a biopolymer composed
of alternating units of GlcNAc and N-acetyl-muramic acid (MurNAc). This structure greatly
contributes to the speciﬁcity of Ab responses following infection or immunization with GAS to
these immunodominant GlcNAc residues. Neonatal immunization with GlcNAc-bearing GAS
produces long-lasting imprinting effects on the clonality of the GlcNAc repertoire that manifest
in the immune responses of these animals as adults. In mice neonatally immunized with GAS
14 days after birth, the resting levels of anti-GlcNAc IgM are substantially boosted (R. King
and J. New, unpublished observations). Moreover, these elevated titers are maintained through
adulthood, and likely for the life of the animal. As can be seen in Figure 3, sera of aged mice, in
this case 16 months old, contained >100 μg/mL of GlcNAc-inhibitable Ab to GAC. Abs reactive
PBS
Mannose
GalNAc
GlcNAc
OD405
8 weeks
16 months
Day 14 GAS
or PBS i.p.
Examine anti-GAS
antibody in serum
8 weeks 16 months
a
b c
20
PBS GAS PBS GAS
40
0 0
0.5
1.0
1.5
60
80
100
120
140
PG-PSIgMinserum(μg/mL)
Figure 3
(a) Neonatal immunization with Streptococcus pyogenes induces the production of long-lasting IgM antibodies
speciﬁc for GlcNAc. C57BL/6 mice were immunized at day 14 after birth with S. pyogenes and bled at 8 weeks
and at 16 months, without further deliberate immunization. (b) Amount of anti-GlcNAc IgM antibody in
serum was measured at 8 weeks and at 16 months. (c) GlcNAc, but not its enantiomer, GalNAc, greatly
inhibited the binding of serum from mice at 16 months of age to group A carbohydrate. Abbreviations:
GalNAc, N-acetyl-D-galactosamine; GAS, group A streptococcus; GlcNAc, N-acetyl-D-glucosamine; i.p.,
intraperitoneal; OD, optical density; PBS, phosphate-buffered saline; PG-PS, peptidoglycan polysaccharide.

GACGACGAC
IgMIgMIgM
Naive
Neonatally
immunized
Neonatally
immunized
Neonatally immunized
0.03 0.06
4 VH genes
81%
Other
29%
Other
19%
J606
62%
J606
35%
S107
9%
S107
46%
Naive
a
b
c
VH family identity
–102
0
102
103
104
0 103 104 105 0 103 104 105
Naive
Figure 4
Neonatal immunization with Streptococcus pyogenes alters the heavy-chain usage of the adult B cell repertoire
specific for GlcNAc. (a) Mice were neonatally immunized with S. pyogenes at day 14 after birth. In
comparison with mice neonatally treated with PBS, these mice had increased GAC-specific B cells in their
spleen. (b) The heavy chains of single-sorted B cells were sequenced to reveal both shared and unique
sequences between these two groups of mice. (c) A breakdown of the unique VH sequences found in the mice
neonatally exposed to either S. pyogenes or PBS demonstrates a dramatic switch in the frequencies of the
predominant clones as the result of early exposure to group A streptococcus. Abbreviations: GAC, group A
carbohydrate; GlcNAc, N-acetyl-D-glucosamine; PBS, phosphate-buffered saline.
to this antigen are less clonally restricted than those generated in response to PC or α-1,3-glucan;
multiple idiotype and sequence analyses in these mice suggest GlcNAc reactivity is encoded by
two VH genes that can differentially pair with one of four κ light chain genes or a λ light chain
gene (73). When Ig gene expression from single GlcNAc-binding sorted B cells from neonatally
primed mice is compared with that of PBS-treated mice, dramatic shifts in Ig VH gene usage can
be observed, as shown in Figure 4.
B Cell Response to Phosphorylcholine-Bearing Pneumococcus
Immunization or infection of mice and humans with unencapsulated or wild-type encapsulated
pneumococcus induces Abs to PC moieties expressed on the cell wall–associated PS and teichoic
acids. Analyses of B cell clones during ontogeny show that the earliest PC-specific B cells are not
detected until 4–5 days of age in mice (Figure 5a) and predominantly comprise non-T15 clones
(Figure 5b). With time the ratio of PC-reactive clones rapidly inverts, so that by 6 weeks of age the
16.10 Kearney et al.

2 3 4 6 7 8 9 10 12 21 44
0
20
40
60
80
100
Age (days)
PercentageofPC-binding
Bcells
T15+
T15–
PC-reactive B cells
VH S107
Immunized adultsImmunized neonates
Vк 22
Vк 24
Vк 22
Vк 8
2 3 4 6 7 8 9 10 12 21 44
0
2
4
6
8
10
Age (days)
Anti-PCfrequency(×10-6) a
b
dc
3 12
0
200
400
600
Age at primary immunization (days)
Anti-PCIgM(μg/mL)
Challenge as an adult
Anti-PC
T15 VH
T15 Id
Figure 5
Ontogeny and immune manipulation of phosphorylcholine (PC)-specific B cell clones: (a) PC-specific B cells increase in frequency
beginning at 4 days of age. Early in life, these B cell clones are mostly non–T15 idiotype. (b) With time, T15 idiotype clones expressing
the canonical heavy-chain rearrangement become the dominant clones in the adult. Mice exposed to pneumococcus at day 3 of life and
reimmunized as adults have increased circulating anti-PC IgM antibodies; however, these antibodies are mostly non–T15 idiotype. In
contrast, mice exposed to pneumococcus at day 12 of life and reimmunized as adults do not have as many circulating anti-PC IgM
antibodies. (c) However, a majority of these antibodies are of the T15 idiotype. (d ) Mice neonatally immunized with pneumococcus and
reimmunized as adults present with an altered clonal repertoire, in contrast to mice first immunized only as adults. Data modified from
Reference 71.
T15 clonotype dominates the adult anti-PC B cell response. Neonatal immunization during the pe-
riod of non-T15 clonal dominance allows what would have been low-frequency clones to predom-
inate the adult repertoire (Figure 5c). This results in a large increase in the magnitude of the anti-
PC response following adult rechallenge and permanently alters this clonal ratio (Figure 5c,d).
Detailed genetic analysis of anti-PC hybridomas derived from this experimental system identified
an altered frequency of light chain usage among these clones (71). The T15 idiotype, generated
by the pairing of the VH S107 Ig heavy chain with the Vκ 22 light chain, is replaced by clones
using predominantly Vκ 8 and Vκ 24 in mice challenged with S. pneumoniae vaccine as neonates
(Figure 5d ). In this case, early exposure to the heat-killed, whole-cell pneumococcus vaccine
primes for a greatly enhanced anti-PC Ab response, resulting in a >40-fold increase in the
frequency of PC-binding B cells detectable in the adult (P. Patel, J.F. Kearney, manuscript in
preparation). However, generation of Ab following reexposure occurs mostly as a result of the

expansion of alternative non-T15 clones. Consequently, serum Ab resulting from secondary
vaccine challenge is much less protective in a model of pneumococcal challenge in comparison
with mice in which the T15 idiotype–bearing Ab dominates the PC-reactive B cell pool (71).
These findings are similar to those discussed above involving the forced ectopic transgenic
expression of TdT, which results in fewer germ line–encoded Abs that are not protective against
pneumococcal infection (49), as well as results from experiments involving the administration of
anti-idiotype Abs at birth, which ablate the dominant T15 Ab response (7).
B Cells Responding to α-1,3-Glucan–Bearing Enterobacter
Similarly to the PC-reactive B cell clones, B cells in mice that respond to T-independent antigens
containing α-1,3-glucan epitopes do not appear until one week after birth but increase in number
with age (74) (Figure 6a). Ab responses to α-1,3-glucan epitopes are restricted to mice that possess
the IgHa locus and the appropriate VHJ558-3 gene. These responses are largely dominated by
B cell clones expressing the J558.3VH heavy chain paired with the λ1 light chain (Figure 6c).
Sequence analysis of B cell clones reactive with α-1,3-glucan revealed that the antigen receptor
gene for J558 idiotype–bearing B cell clones recruited into this response predominantly encodes
an arginine-tyrosine sequence at the VD junction (61). These clones are distinct from those that
appear early in ontogeny, at which time another anti-α-1,3-glucan B cell clone, M104, which
constitutes a minor population in the adult repertoire, is dominant (Figure 6b). The M104 and
J558
M104
Day 14Day 7 Day 21
Predominant anti-α-1,3-glucan IgVH
M104
J558
YD
RD
J558.3
(1–99)
D segment
(100–101)
JH.1
(102–117)
8 14 18 24 30
0
20
40
60
80
100
PercentageId+
J558M104E
8 14 18 24 30
0
1
2
3
4
5
Age (days) Age (days)
α-1,3-glucanfrequency(×10–7)
a b
c d
10%
56.3% 73.9%
Primary immunization
Idiotype profile of immune sera
α-1,3-glucan-reactive B cells
Figure 6
Ontogeny and immune manipulation of α-1,3-glucan-specific B cell clones: (a) α-1,3-glucan-specific B cells increase in frequency
beginning at 7–8 days of age. In neonates, these B cell clones are mostly M104E idiotypic. (b) With time, J558 clones expressing the
heavy chain rearrangement depicted in panel c become the dominant clones in the adult. (d ) Mice intraperitoneally immunized with
α-1,3-glucan at 7, 14, or 21 days after birth and rechallenged as adults present with alterations in their clonal repertoire. Data modified
from References 72 and 74.

J558 idiotype antigen receptors differ slightly in the amino acid sequence within the heavy chain
VD junction. Immunization of neonates with a heat-killed or paraformaldehyde-fixed bacterial
vaccine preparation bearing α-1,3-glucan residues reverses the relative abundance of these anti-
α-1,3-glucan Abs following rechallenge as adults (Figure 6d ). Additionally, the magnitude of
this effect is age dependent, such that the relative abundance of the M104 Ab, normally a minor
constituent of this response, is inversely proportional to the age of animals at primary exposure
to the vaccine. Thus, similar to the previously discussed response to PC, early exposure to α-1,3-
glucan permanently alters the antigen-specific B cell repertoire both by increasing the frequency
of anti-α-1,3-glucan B cells and by fixing the relative frequencies of idiotypes to that present at
the time of immunization.
The mechanisms of these dramatic effects of early antigen exposure on the expressed antigen-
specific repertoire are not clear, but they may result from the differential affinity of B cell receptors
expressed by nascent clones. In the case of PC, the affinity of the germ line–encoded T15 clone
to the PC hapten is 10 times higher than that of Ab derived from other minor anti-PC clones.
Similarly, in the α-1,3-glucan system, equilibrium dialysis demonstrated that there is a 100-fold
difference in affinity for small α-1,3-glucan oligosaccharides between the J558 and the germ line–
encoded M104E Ab, with the J558 idiotype Ab exhibiting the largest affinity (Ka = 1.4 × 105
versus 1.3 × 103
). The early appearance of M104E occurs in the absence of N-additions and gen-
erally produces YD amino acids, whereas the J558 VD junction requires an N-addition (61). This
most likely contributes to its increased affinity. This phenomenon is further evidence of interclonal
competition. In a nonimmunized neonate, the J558 clone eventually dominates, possibly through
selection by low-dose exposure to environmental organisms expressing α-1,3-glucan epitopes.
However, early exposure to a relatively high dose of α-1,3-glucan-bearing antigen during the pe-
riod when M104E and J558 are at similar frequencies can result in an alteration in clonal dominance
in the adult Ab response (30). The reduction in competition for antigen between cells expressing
these two receptors for the same antigen allows both clones to enter the circulating repertoire.
There is clear evidence that nAbs to the epitope expressed by bacterial PS discussed in
this section are detectable at low levels in most strains of mice. We have shown that in these
three model systems, early exposure to antigen can reprogram clonal precursor frequency such
that (a) there is an increased frequency of antigen-specific B cells that respond to rechallenge,
(b) clonal frequencies of canonical antigen receptors within the antigen-specific B cell repertoire
are permanently and dramatically altered, and (c) nAb levels are increased for the life of the animal.
Human Natural Antibodies to GlcNAc, PC, and α-1,3-Glucans
The development of glycan-coated microchips has facilitated studies of the reactivity of normal
human sera to defined glycan epitopes. Although the sera of most humans contain common anti-
PS specificities, there is considerable individual variance in the patterns of glycan binding. In some
individuals, “holes” exist in reactivity (75), which may reflect the inability of individual subjects to
produce certain subsets of nAbs. nAbs to GlcNAc, PC, and a variety of glucans can be detected
in most human sera, but as in mice, they are at low or undetectable levels in neonates and have
been shown to be pauciclonal (76). Figure 7 is an adaption of retrospective data compiled by Gray
et al. (34), in which anti-PC Ab levels in sera from healthy infants, carriers, and those diagnosed
with pneumococcal infections were plotted against age. As expected, levels of detectable anti-PC
IgM Ab increase with age, but analysis of individual patients revealed that many infants, in some
cases less than 1 year old, can develop substantial levels of IgM anti-PC Abs, ranging from 30
to >1,500 μg/mL. Such high levels of anti-PC Abs were often associated with S. pneumoniae
otitis media infections and occasionally associated, depending on serotypes, with pneumococcal

0
1 2 4 6 8 12 2418 36 48
100
200
300
400
500
Age (months)
Antiphosphorylcholine(μg/mL)
Figure 7
Antiphosphorylcholine IgM antibodies measured in children from ages 1 month to 4 years: A total of 115
sera from 30 individual children with a history of exposure to Streptococcus pneumoniae, ranging from carriage
to overt infection, and who were treated at the University of Alabama at Birmingham were assayed for
antiphosphorylcholine antibodies. Data adapted from Reference 34.
carriage. Thus, it is clear that an infection with a live organism in humans does indeed induce
a strong Ab response at an early age. In this small study, no follow-up analysis was undertaken
to assess the long-term maintenance of anti-PC levels. In contrast to these infected individuals,
some infants did not have detectable anti-PC Abs by 4 years of age. Whether the delay in the
production of Abs of this specificity resulted from the absence of infection, the differential use of
antibiotics, or the failure of individuals within this cohort to produce Abs in response to infection
was not pursued.
Like the humoral response to PC, the response to GAS in humans is well characterized and
pauciclonal (77). Investigations into the ontogeny of Ab responses to GAS during the neonatal
period showed that anti-GAC Ab concentrations were highly variable among individuals but
increased until 7–17 years of age to levels maintained through adulthood. GAS is a prominent
human pathogen, and approximately 5% of healthy individuals are carriers. Children under 1 year
of age are typically immune to infection owing to maternal Abs; however, infection of children
with GAS results in elevated serum anti-GAC Abs, particularly in those between 2 and 6 years of
age (Figure 8). In adults, both IgM and IgG Abs are detectable, but it has not been determined
which isotype predominates in infants less than 3 years old (78). As in mice, Abs to GAC are
predominantly directed toward GlcNAc, as determined by inhibition assays using monomeric
GlcNAc (Figure 3).
These studies show that the production of nAbs to PC and GlcNAc in humans is similar to that
observed during ontogeny in mice and that infection with pneumococcus or GAS substantially
increases the levels of anti-PS Abs targeting these organisms. The human studies cited here reveal
the potential of neonatal infections to (a) alter the clonal profiles of B cells responding to these
antigens in the adult, (b) reprogram nAb levels, and (c) substantially boost anti-PC and anti-GlcNAc
responses. Thus, reprogramming of the nAb repertoire may be implemented to generate long-
lived protective nAbs, an innovative approach to overcoming the fleeting nature of protective Abs
induced by neonatal vaccination with PS-conjugates (54).

0.5–1 2–3 4–6 7–17 ≥18 0.5–1 2–3 4–6 7–17 ≥18
0
20
40
60
80
Age (years)
Anti-GACantibody(μg/mL)
Recent GAS infectionNo report of GAS infection
Figure 8
Anti-GAC antibodies from 120 healthy and 57 GAS-infected children and adults. Healthy subjects were
aged 12 months to ≥18 years, and the infected subjects were aged 2 to ≥18 years. Infected children were
confirmed to have pharyngitis caused by bacitracin-susceptible β-hemolytic streptococci. Data adapted from
Reference 78. Abbreviations: GAC, group A carbohydrate; GAS, group A streptococcus.
NEONATALLY EXPANDED B CELLS AND ALLERGIC DISEASE
Rethinking the Proposed Th1/Th2 Mechanism
Allergy is the third most common chronic disease among children younger than 18 years (79).
Allergic diseases occur in susceptible individuals who encounter environmental stimuli that
promote sensitization to inhaled antigens. It is clear that many human atopic diseases are initiated
neonatally and are harbingers of more severe IgE-mediated disorders, including allergic asthma,
later in life. Atopic diseases, including allergic asthma, are driven by the priming of Th2 effector
cells and the production of allergen-specific IgE by B cells in response to environmental antigens.
Allergen engagement through IgE, bound by FcεR on basophils and mast cells, results in the
release of soluble mediators, including histamine, cysteinyl leukotrienes, and cytokines. These
reactions have immediate effects on bronchial constriction and mucus production and delayed
effects driven by the activity of recruited leukocytes. Because the initiating process of atopic
disease requires sensitization and the production of allergen-specific IgE, understanding the
mechanisms leading to this adaptive immune response is paramount to our understanding of the
development of allergic asthma and may ultimately provide mechanistic clues into the role of
early microbial exposure in modulating this disease. Whereas considerable research efforts into
the allergic cascade have led to therapeutic strategies to alleviate the symptoms of allergies and
asthma, little progress has been made toward developing strategies for prevention of early disease.
The earliest mechanistic explanations of the hygiene hypothesis were centered on the Th1/Th2
polarization of T helper cells. Because Th1- and Th2-polarized responses involve feedback mech-
anisms that were thought to inhibit the development of one another, a simple explanation of the
hygiene hypothesis was that in the absence of early inflammatory stimulation, CD4 T cell differ-
entiation defaults toward a Th2 response and results in atopic reactions and ultimately allergic
disease. Although Th2 responses are clearly involved in the development of allergic reactions,
several lines of evidence suggest that a systemic Th2 polarization, as a result of the reduced Th1-
driven responses, is an inadequate explanation. Studies in mice have demonstrated that the adoptive
transfer of Th1- or Th2-polarized antigen-specific T cells in a model of ovalbumin-induced air-
way hyperresponsiveness (AHR) not only failed to prevent airway hyperactivity, but also induced

Treg: regulatory
T cell
airway inflammation (80). Induction of Th1 responses by respiratory syncytial virus and influenza
A infections has been shown to exacerbate, instead of alleviate, asthma (81). In addition, reduction
in Th2 polarization following the pharmacological eradication of parasitic infections is ineffec-
tive in alleviating allergic symptoms. In some cases, parasitic infections can exacerbate asthmatic
symptoms (82). There is evidence that parasite infections that occur during childhood are more
successful at preventing the development of allergic disease in comparison with those that occur
in adults, possibly after the development of allergic disease (83). Although these data are both
conflicting and controversial, one striking exception is the observation of a strong inverse corre-
lation in humans between the childhood disease scarlet fever (systemic S. pyogenes infection) (84)
and adulthood asthma (85) (see sidebar Hygiene Hypothesis).
In the time since the hygiene hypothesis was introduced it has become clear that the Th1/Th2
imbalance is an oversimplified explanation of the mechanisms involved in controlling allergy
development. There appears to be stronger evidence that an imbalance between the Th2/Treg
cell repertoires early in life contributes greatly to the development of allergic disease (26, 83, 86).
The presence of Tregs in cord blood, in the periphery, and locally in the lung is inversely correlated
with the development of allergic disease (87, 88). Most important, Tregs have been shown to be
able to suppress both Th1 and Th2 cytokine production (89). There is a direct correlation between
early microbial exposure and the generation of a robust Treg response, resulting from epigenetic
changes that occur in utero, as a consequence of maternal microbial exposure, or perinatally,
with the acquisition of a diverse gut microbiome (86, 90). Our understanding of the mechanisms
by which early microbial exposure imprints the immune system to influence the development of
allergic disease is incomplete.
Rethinking the Role of Antibodies and B Cells in Allergic Disease
Whereas many of the mechanisms explaining the hygiene hypothesis have been centered around
altered T cell responses early in life, little attention has been paid to the effects of early microbial
exposure on the B cell repertoire prior to the initiation of these diseases. The role of Abs and B cells
in models of allergic disease appears to be dependent on the allergen and route of sensitization.
IgE is clearly a central component in allergic asthma, and passive systemic administration of anti-
IgE Ab has been used to ameliorate asthmatic symptoms in humans and in mice (91). Although
allergen-specific Abs passively administered (92, 93) or systemically induced Abs prior to and
during sensitization or challenge have been shown to be protective in most models, systemic Abs
can also exacerbate allergic airway disease (94, 95). It is also clear that B cell responses to allergen-
bearing particulates have significantly different immunological outcomes from B cell responses to
purified or soluble allergens in the lung (96). B cells have also been shown to have a dispensable
role in the induction of ovalbumin-dependent AHR (97); however, they have also been implicated
in the induction of T cell tolerance to inhaled soluble allergens (98). Little is known regarding the
contribution of non-IgE Abs and B cells in the development of allergies and asthma in humans.
Cross-Reactivity of Polysaccharide-Specific Antibodies
The allergens humans are naturally exposed to rarely, if ever, take the form of a single purified
protein. Rather, potential T cell–sensitizing allergens are cargo associated with innate immunity–
activating particulates that are composed of chitin, glucans, PC, etc. (99) (see sidebar Innate Re-
ceptors). Inhalation of these innate-receptor-activating complexes triggers the influx of leukocytes
into mucosal and lumenal compartments and results in the sensitization of allergenic lymphocytes
responsive to the allergen cargo. This, in turn, triggers the cascades of cellular interactions and

INNATE RECEPTORS
Recognition of polysaccharide ligands expressed by pathogens, potential allergens, and self by innate receptors is a
primary step in the recognition of the antigen. Engagement of these receptors will initiate inflammatory responses
that clear potential pathogens but can also initiate allergic and autoimmune reactions. Anti-PS IgM Abs against
these ligands have the potential to block or alter these ligand-receptor interactions. These include CD206, TLR2,
RegIIIg, and DC-SIGN for GlcNAc and its polymer chitin and CD36, Dectin-1, and Dectin-2 for β-1,3 glucans;
PAFR and CD36 for phosphorylcholine. New receptors and ligands are still being identified, but it is clear that
these polysaccharides are important, as engagement of innate receptors by these carbohydrate ligands leads to the
release of cytokines and chemokines. Certain pathogens also use the receptors to invade nonphagocytic cells and
evade immune detection. The modulation of these interactions offers an extension of the effects of early bacterial
exposure put forth in the hygiene hypothesis and the potential roles the IgM Abs to these conserved polysaccharide
structure in ameliorating allergic and autoimmune diseases.
cytokine networks, leading to inflammation, airway mucus production, remodeling, and subse-
quent obstruction of airways. It is important to consider, in the context of the hygiene hypothesis,
that chitin, glucans, and PC are expressed on a wide spectrum of environmental organisms, in-
cluding microbes and parasites. In addition to a decrease in bacterial infections, worm infections
have largely disappeared in children in industrialized countries. As stated previously, infections by
some of these organisms are known to prevent or alleviate common childhood allergies and other
immune-related diseases. Although there are multiple hypotheses regarding the involvement of
parasites in the development of allergy, protection from the development of allergic reactions is
generally attributed to immunomodulatory effects of these infections (100). It is of great interest
to this discussion that many nematodes express large amounts of PC-bearing molecules, as seen in
Figure 9a,b. The integuments of Heligmosomoides polygyrus contain large amounts of chitin (not
shown) and PC (Figure 9b). Deliberate infection of mice with the intestinal nematode H. polygyrus
or Strongyloides stercoralis induces the production of large amounts of anti-GlcNAc (Figure 9c,d)
and anti-PC Abs (not shown). Thus, early nematode infestation has the potential to affect the
development of nAbs reactive with these epitopes, providing an alternative or additional mecha-
nism for the protection against allergic diseases observed in individuals infected by some parasites.
Neonatal exposure to bacterial or helminth infections likely alters the clonal diversity and levels of
some nAbs, including specificities demonstrated to provide protection against diverse pathogenic
organisms (101) that may be relevant to the development of allergic disease.
Mouse Models of Allergic Airway Disease
The immune responses induced in allergy models appear to vary depending on the allergen
and route of sensitization. Although many mouse allergy studies use alum-based models with
purified allergens, proteins, or surrogate-sensitizing antigens administered intraperitoneally (102),
sensitizationsviaairwaysorskinaremorerelevanttohumandisease.Moreover,alum-basedmodels
have proven ineffective for the evaluation of interventional procedures involving the development
of allergic disease in humans. Additionally, allergy occurs by long-term incremental exposure to
sensitizing allergens; for these reasons we have developed models replicating natural respiratory
sensitization and allergic challenge with clinically relevant allergens to examine the effects of
neonatal immunization and the role of nAbs in the induction of allergic airway disease.

α-GlcNAcIgM(μg/mL) 100μ 100μ100μ
Time (days) Time (days)
Initial 14 28
100
101
102
103
104
P = 0.001
Initial 30 60 90
0
25
50
75
100
P = 0.002
c d
aa
PCPC
b
Figure 9
The parasitic nematode Heligmosomoides polygyrus contains phosphorylcholine (PC), and roundworm
infections elicit strong N-acetyl-D-glucosamine (GlcNAc)-specific responses. (a) Cryosections of an adult
H. polygyrus worm were stained with (b) anti-PC IgM antibodies ( green). Mice were immunized one to three
times with (c) Strongyloides or (d ) H. polygyrus, and the levels of anti-GlcNAc IgM in the serum were
determined.
Antibody-Mediated Dampening of Aspergillus fumigatus–Induced
Allergic Airway Disease
The A. fumigatus asthma model is highly relevant to human disease, as a high proportion of
people with asthma suffer from allergy to this organism (103). A major component of A. fumigatus
cell walls is chitin, which is the second most abundant PS in nature and is found in crustacean
and insect exoskeletons and parasitic nematodes (104, 105). As GlcNAc-containing polymers and
glycoproteins are found in bacterial cell walls, common allergens, and host tissues, we hypothesized
that Abs directed to this moiety may be the link between infection with organisms expressing
GlcNAc epitopes, including GAS, and the reduced incidence of allergic airway disease.
In addition to Abs against GlcNAc, which bind chitin particles <10 μm, multiple monoclonal
Abs raised against bacteria with defined epitope specificities, including PC and α-1,3-glucan,
react with conidia and hyphae of A. fumigatus (Figure 2, Table 1). The staining profiles of
these monoclonal Abs are strikingly different, likely a result of the differential distribution and
accessibility of the PS moieties within the dynamic fungal cell wall. The reactivity of these PS-
specific Abs with conidia and hyphae of A. fumigatus led us to investigate whether monoclonal Abs
of these well-defined specificities or bacterial PS vaccines would influence immune processes in
the lung, affecting the initiation or progression of allergic airway disease induced by sensitization
to this organism.
Administration of anti-GlcNAc Abs reduced the uptake of A. fumigatus by macrophages
and dendritic cells in vivo (106). Abs passively administered directly into the lungs of mice,
or those persisting systemically following intravenous administration, reduced the recruitment

Table 1 Comprehensive list of monoclonal antibodies and their defined epitope specificities on
Streptococcus and Enterobacter pathogens
Antibody Isotype Epitope Epitope found on
HGAC-78, CTD 110 IgM GlcNAc S. pyogenes
RL-2 IgG1 GlcNAc S. pyogenes
HGAC-39 IgG3 GlcNAc S. pyogenes
1–21, Dex 7, A16 IgM α-1,3-glucan E. cloacae
8–21 IgG2b α-1,3-glucan E. cloacae
7D7-2 IgG3 α-1,3-glucan E. cloacae
P5-1, J558, P5-1 IgA α-1,3-glucan E. cloacae
SMB19 IgM Sialyllactose-N-tetraose S. agalactiae
BH8, M603, M511 IgM Phosphorylcholine S. pneumoniae
S107 IgA Phosphorylcholine S. pneumoniae
of macrophages, dendritic cells, eosinophils, basophils, and other lymphocytes into the lung
parenchyma and the bronchoalveolar space of mice sensitized to A. fumigatus in a pulmonary
allergy model. In addition to these effects, passive introduction of anti-PS Abs correlates with
a marked reduction in multiple allergy-associated cytokines within the lung. A similar effect is
observed in vitro, where the inclusion of PS-specific Abs in cultures of bone marrow–derived
macrophages and A. fumigatus preparations blocks cytokine production and decreases the expres-
sion of cellular markers of activation.
Thus, the modulation of the adaptive immune response to A. fumigatus–derived epitopes is due
to the reduced activation and antigen acquisition or processing resulting in diminished presenta-
tion of A. fumigatus–derived antigens by dendritic cells (E. Stefanov and J. Kearney, manuscript
in preparation). This effect results in reduced A. fumigatus–specific CD4+
T cell activation and
cytokine production in both in vivo and in vitro systems. In vivo, the culmination of these effects
is an abrogation of A. fumigatus–specific CD4+
T cell memory formation. Thus, passive immu-
nization of mice with PS-specific monoclonal Abs or active immunization with bacterial vaccine
preparations modulates the allergic response to seemingly unrelated fungal allergens. Moreover,
neonatal immunization with GAS primes the adult Ab response, as described earlier in this re-
view, and dampens the immune response to purified chitin and the induction of allergic responses
to intratracheal administration of live A. fumigatus conidia. Importantly, the protection against
sensitization to A. fumigatus following neonatal vaccination is remarkably long lived. As depicted
in Figure 10, neonatal exposure to GAS results in protection against the manifestations of an
inflammatory allergic response to A. fumigatus for 4.5 months following a single immunization.
Antibody-Mediated Dampening of House Dust Mite–Induced
Allergic Airway Disease
Given the data described above, we have begun to expand this line of investigation to other
environmental antigens commonly associated with allergic asthma. House dust mites (HDM),
like A. fumigatus, are ubiquitous environmental and household allergens. Approximately 30% of
individuals that exhibit respiratory allergies demonstrate reactivity to HDM (107).
HDM preparations contain numerous allergen-bearing structures that share epitopes with
microbes. HDM exoskeletons are composed of chitin, and allergen-bearing fecal pellets are
encased in a chitinous mesh. In addition, we and others have shown that monoclonal anti-PC

aa
bb
cc
Figure 10
Mice neonatally immunized with Streptococcus pyogenes are protected from the development of allergic
pathology following a model of Aspergillus fumigatus–induced allergic airway disease: At the conclusion of the
A. fumigatus–induced allergic airway disease model (4.5 months after neonatal exposure), sections of
paraffin-embedded lungs were stained with hematoxylin and eosin. Mice neonatally immunized with
(a) S. pyogenes present with significantly fewer cellular infiltrates surrounding their airways in comparison
with (b) mice neonatally immunized with Streptococcus agalactiae or (c) mice treated with phosphate-buffered
saline. Images taken at 160× original magnification.
Abs bind either intact HDM, as shown in Figure 11, or, perhaps more important, micron-sized
particulates derived from HDM (108, 109; P. Patel and J. Kearney, manuscript in preparation).
As in the A. fumigatus allergy model, passive administration of anti-PC Abs or immunization
of neonates with pneumococcal vaccines attenuates the sensitization and allergic response to
HDM. There is a dramatic reduction in the relative numbers of inflammatory cells infiltrating
the bronchoalveolar space of mice exposed to pneumococcus early in life compared with those
treated with PBS (Figure 12). These findings (P. Patel and J. Kearney, manuscript in preparation)
are similar to the dampening effect of neonatal GAS immunization on the allergic response to
A. fumigatus (106). Although we have shown antigen specificity is important for this protective
effect, adult mice immunized with pneumococcus, despite producing high levels of anti-PC Ab,
do not benefit from the same level of protection as those exposed to pneumococcus as neonates.
This observation suggests that the generation of serum Abs in response to immunization is not

a b
Isotope control Antiphosphorylcholine IgA
Egg
Egg
Superior
Inferior
Figure 11
Whole-bodied dust mites display phosphorylcholine (PC) epitopes: Unmilled whole-bodied
Dermatophagoides pteronyssinus mites were cytospun at 1,000 rpm for 5 min before being stained with either
(a) isotype or (b) anti-PC IgA antibodies. Anti-PC antibodies bind both the exoskeletons and the eggs of the
insects. Images taken at 160× original magnification.
sufficient to confer protection from sensitization to inhaled allergens. The effect we observe
in this model may result from the expansion of the innate-like B cell compartment following
neonatal immunization. Perhaps the enhanced frequency of B cells of these PS specificities allows
for recruitment of sufficient numbers of these cells into mucosal sites during the earliest phases
of allergic inflammation, where they differentiate to provide protective Ab.
Naive mouseb
NeutrophilsNeutrophils
Alv macsAlv macs
a Mouse with early pneumococcal exposure
Alv macsAlv macs
NeutrophilsNeutrophils
Figure 12
Early pneumococcal exposure results in decreased infiltration of neutrophils into the bronchoalveolar space
following exposure to house dust mite. Bronchoalveolar lavage fluid was collected from mice that were
neonatally exposed to pneumococcus or phosphate-buffered saline (PBS) following subjection to a house
dust mite allergy model. The same volume of cells from each lavage was cytospun at 1,000 rpm for 5 min
before being stained with a modified Wright stain (Sigma). (a) Mice exposed to pneumococcus early in life
present with a smaller number of neutrophils infiltrating the bronchoalveolar space in comparison with
(b) mice neonatally immunized with PBS. Images taken at 630× original magnification. Abbreviation:
Alv macs, alveolar macrophages.

Mouse with early
Enterobacter exposure Naive mouseba
Figure 13
In comparison with mice exposed to phosphate-buffered saline (PBS), mice neonatally exposed to
α-1,3-glucan-bearing Enterobacter produce less mucin in their bronchioles, following a cockroach allergy
model. At the conclusion of the cockroach allergy model, paraffin-embedded lungs were stained with
periodic acid–Schiff stain. (a) Mice neonatally immunized with α-1,3-glucan-bearing Enterobacter present
with significantly fewer mucin-producing goblet cells (magenta) in their bronchioles in comparison with
(b) mice neonatally immunized with PBS. Image taken at 400× original magnification.
Antibody-Mediated Dampening of Cockroach-Induced Allergic Airway Disease
Similar to HDM, chitin is a major component of cockroach exoskeletons and allergen-bearing
fecal pellets. We recently discovered that cockroach particulates, although mostly associated
with plant and fungal epitopes, contain α-1,3-glucan moieties, as detected by monoclonal Abs
(Table 1). As a model for early bacterial exposure, mice were neonatally immunized with
α-1,3-glucan-bearing E. cloacae before being subjected to a cockroach allergy model. Similarly
to the A. fumigatus and HDM allergy models, early exposure to E. cloacae provides significant
protection from allergic challenge with cockroach allergen (P. Patel and J. Kearney, manuscript
in preparation). As can be seen in Figure 13, there is a dramatic reduction in the number of
mucin-producing cells in the bronchioles of mice neonatally exposed to E. cloacae compared with
those that were treated with PBS following allergic exposure to cockroach.
Proposed Mechanisms of Neonatally Induced Protection
Against Allergic Airway Inflammation
It is of great interest that in each of these models there is a strong dependence on early microbial
exposure to dampen the development of allergic disease. Mice immunized with bacterial vac-
cines as adults are not protected in their complementary model of allergic disease, in contrast to
mice neonatally immunized. A variety of studies have demonstrated that both humans and mice
exposed to microbes early in life have an increased number of Tregs that suppress allergic dis-
ease. However, in our systems, passively administered Ab provides the same protective effects as
neonatal immunization. Although it is clear neonatal microbial exposure may have a multitude of

immunological consequences, within the context of the described models, specific IgM appears
sufficient to prevent sensitization and allergic responses. Collectively these observations suggest
that changes within the developing B cell compartment resulting from neonatal exposure to these
PS are responsible for the protective effects in that they alter the clonality and abundance of nAbs
within the lung environment during sensitization or allergenic challenge.
A simple explanation is that Abs of these specificities, when present at sufficient concentra-
tions, act as blocking Abs. By engaging epitopes common to multiple allergens, including GlcNac
(chitin), PC, and glucans, nAbs may abrogate the recognition of these moieties with innate re-
ceptors. Numerous innate receptors are involved in the initial recognition of allergenic particles
and promote immune activation that results in sensitization. This idea is supported by the ob-
servation that neonatal immunization with GAS, or passive transfer of anti-GlcNAc Abs, reduces
the number of A. fumigatus conidia engulfed by subsets of dendritic cells in the lungs of mice. It
is also possible that particles complexed with IgM are rapidly cleared from the mucosal surface,
resulting in reduced exposure to TLR ligands, proteolytic enzymes, or other inflammatory signals
implicated in the initial response to inhaled allergens. Alternatively, the presence of IgM com-
plexes may result in alterations in allergen processing, thereby diminishing the canonical allergic
response. Experimental systems to distinguish between these potential mechanisms are currently
being developed.
Regardless of the mechanisms, neonatal immunization has long-lasting and profound effects
on the development of allergic responses in these models, each of which represents a single, well-
defined specificity. It is important to consider, in the context of the hygiene hypothesis, that all of
these specificities are present on multiple microbes that are unlikely to be encountered in isolation.
Thus, the effects of neonatal immunization we have described are likely to occur not only for these
specificities but also for others in response to natural infection, many of which may influence the
development of atopic diseases.
SUMMARY AND CONCLUSIONS
Collectively, these data support the concept that IgM nAbs are protective against the induc-
tion of allergic airway disease induced by A. fumigatus, HDM, and cockroaches by blocking
microorganism–innate receptor interactions. B cells responding in the early stages of life to con-
served antigens associated with the cell walls of common environmental microbes can directly
affect the levels of nAbs reactive with these organisms. The Abs produced by these B cells modu-
late the uptake and subsequent processing and presentation of allergenic epitopes of substructures
borne by these and other environmental antigens. Thus, there are long-term effects of neonatal
exposure to environmental microorganisms on the development of hypersensitivity and allergic
airway disease in later life. Our studies show that significant protection against airway sensiti-
zation is dependent on the timing of induction and the antigenic targets of these Abs. In fact,
immunizing an adult mouse prior to initiating a sensitization protocol fails to dampen the allergic
response in the A. fumigatus model. These findings in mice concur with observations in humans
that the possibility of reversing the disease declines with time after onset (110). Currently, there
are no preventive regimens to abrogate the development of atopic disease initiated neonatally. By
contrast, our findings suggest that alternatives to conventional allergen immunotherapy could be
instituted at early stages in at-risk children, which could be effective in treating and potentially
abrogating the development of atopic diseases. As we have shown, microorganism-associated anti-
gens can induce potentially protective Abs during early life. We propose that an understanding
of the mechanisms associated with Ab-mediated immunoregulation will lead to the development
of preventive or interventional strategies to treat allergic diseases. Identifying the antigens that

induce optimal Ab-mediated protection against T cell hyperactivity may help us explain why cer-
tain parasitic infections have a similar ameliorating effect on asthma (111) and may possibly be
a step toward more rationale for the proposed use of probiotic therapy in treating atopic disease
(112). Additionally, early selection for these conserved antigen-bearing microbes in the gut might
contribute to induction of Tregs early in life, potentially dampening the development of allergic
disease in adulthood. In addition, these studies can broaden the suggested protective mechanisms
conferred by early microbial exposure against other autoimmune diseases frequently discussed
in the context of the hygiene hypothesis, such as type 1 diabetes, multiple sclerosis, and Crohn
disease, which are increasing in incidence in industrialized societies (84).
SUMMARY POINTS
1. Neonatal B cells responsive to T-independent antigens develop in an ordered program.
2. The B cell response to T-independent antigens can be reprogrammed by neonatal antigen
exposure.
3. There is a limited time for windows of opportunity after birth for this reprogramming
and generation of long-lived B cell clones, which results in permanent changes in the
subsequent magnitude and clonal diversity of the adult repertoire.
4. Antibodies to conserved antigens on bacteria that commonly cause infections in humans
have the potential to react with other organisms, including fungi and insects, associated
with allergies.
5. Neonatal exposure but not adult exposure to bacterial vaccines induces antibodies that
dampen the allergic airway response to fungal and insect allergies.
FUTURE ISSUES
1. Is there evidence of reprogramming of the natural antibody repertoire in humans?
2. Are levels of natural antibodies of certain specificities in humans correlated with atopy
or allergic symptoms?
3. What benefit does clonal heterogeneity afford in the prevention of allergic airway disease
in mice?
4. Does reprogramming the natural antibody repertoire in mice affect the development of
other atopic diseases, such as food allergy?
5. Does neonatal immunization influence the development of or induce other cellular
subsets?
6. How can these observations effectively translate into the development of preventive
therapeutics?
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We wish to acknowledge the technical assistance of Lisa Jia and Jeffrey Sides, the scientific con-
tributions of past and present members of the Kearney lab to the concepts discussed in this
manuscript, and the expert, patient editing of this manuscript by Stewart New and Dr. Denise
Kaminski. This work was supported by National Institutes of Health Grants RO1AI14782-36
and RO1AI100005-02. J.F.K. was a recipient of a Senior Investigator Award from the American
Asthma Foundation.
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