kearney annual review

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Natural Antibody Repertoires:Development and FunctionalRole in Inhibiting AllergicAirway DiseaseJohn F. Kearney, Preeyam Patel, Emily K. Stefanov,and R. Glenn KingDepartment of Microbiology, University of Alabama at Birmingham, Birmingham,Alabama 35294; email: [email protected]

Annu. Rev. Immunol. 2015. 33:16.1–16.30

The Annual Review of Immunology is online atimmunol.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 inbacterial cell walls, but also in common environmental allergens. Neonatalimmunization with bacteria polysaccharide vaccines results in attenuated al-lergic airway responses to fungi-, house dust mite-, and cockroach-associatedallergens in mouse models. The similarities between mouse and human natu-ral antibody repertoires suggest that reduced microbial exposure in childrenmay have the opposite effect, providing a potential mechanistic explanationfor the hygiene hypothesis. We propose that understanding the effects ofchildhood infections on the natural antibody repertoire and the mechanismsof antibody-mediated immunoregulation observed in allergy models will leadto the development of prevention/interventional strategies for treatment ofallergic asthma.

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nAbs: naturalantibodies

INTRODUCTION

The hygiene hypothesis, in its various iterations, proposes that the escalating incidence of allergicand autoimmune disease in industrialized countries is a consequence of increasingly sanitary livingconditions (see sidebar Hygiene Hypothesis). Hygienic practices result in less exposure of theneonatal developing immune system to environmental microbes and parasites (1–3). It is generallyassumed that this decline in pathogen exposure disrupts the balance between Th1- and Th2-typeimmune responses, promoting a bias for the Th2 responses that ultimately drive the developmentof atopic and autoimmune diseases in susceptible children. Whereas B cells, which give rise to IgEantibodies (Abs), are clearly important in atopy and allergy, little attention has been paid to theeffects 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) thecontent of the expressed natural antibody (nAb) repertoire, and (c) B cell interactions with otherelements of the immune system. In this review, we describe experiments in mouse models thatdocument the effects of deliberate neonatal microbial exposure with selected bacterial vaccines onthe B cell compartment and circulating nAbs that have a strong and persistent dampening effecton the development of allergic airway disease (Figure 1).

Although the origin and function of nAbs have been the subject of age-old discussions (4), thesetopics, and the very definition of nAbs, remain controversial. One hallmark of nAbs is that theycan be found in germ- and antigen-free mice, observations which suggest that their productionmay 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 onthe clonal diversity of these Abs, their circulating levels, and ultimately their biological functionswhich 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 thisreview, it is important to clarify and correct this viewpoint. In many instances, the assays usedto 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 unrelatedmolecules. In contrast, some nAbs bind, with exquisite specificity, to defined oligosaccharides thatare expressed as shared epitopes of glycan structures present in microbial cell wall components aswell 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 inversecorrelation between the number of older siblings of school-age, British children and the occurrence of hay fever, asreported in a large longitudinal study. Strachan postulated that smaller family sizes and higher sanitation standardsreduce the transmission of pathogens among children, resulting in an increased incidence of atopic disease. A varietyof 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 livingconditions have resulted in a remarkable decline in viral, bacterial, and helminth infections, especially amongchildren. Numerous epidemiological studies have demonstrated that these lifestyle changes are associated with areduction in infections and a concurrent emergence of allergic and autoimmune diseases. Currently, a variety offactors interpreted to reflect early microbial exposure are associated with the risk of developing atopic disease. Thesefactors include delivery method, birth order, early day care attendance, family size, socioeconomic status, presenceof family pets, and properties of the commensal gut microflora.

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Microbial

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Elevated levels of antibodies

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Figure 1Early exposure to environmental polysaccharide antigens results in long-lasting changes in the immune system, including alterations inthe polysaccharide-reactive B cell repertoire, increased serum titers of antigen-specific antibodies, and increased frequencies ofantigen-specific B cells in circulation. Our experimental models show that these changes correlate with a decrease in allergic airwaydisease. Based on these observations, we hypothesize that neonatal modulation of the B cell repertoire may have a global impact onatopic and autoimmune diseases in general.

they bind the identical epitope on a variety of molecular entities and, as discussed later, these Absare 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 developedcountries 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). Thesechildren suffer from a high rate of bloodstream infections which, in addition to gram-negativeenteric 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 deliberateimmunization has been the subject of considerable speculation.

The antigenic targets of some well-characterized nAbs are shared by pathogens and host. These epitopes mayexist constitutively or represent neoepitopes that result from altered glycosylation of host proteins or oxidation ofhost 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 pathogenicorganisms; thus, the natural repertoire is thought to be evolutionarily conserved and of dual function. Examplesin this review are (a) Abs to GlcNAc epitopes expressed on multiple O-GlcNAcylated intracellular proteins andbacterial polysaccharides and (b) phosphorylcholine epitopes on host-oxidized low-density lipids and bacteria andhost parasites. Although it is obvious that nAbs are generated in the absence of immunization, in the world welive 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 tobacteria-derived products alters the repertoire and titer of nAbs. Thus, even this most innate component of theadaptive immune system can be plastic and responsive to environmental circumstance.

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GAS: group Astreptococcus/Streptococcuspyogenes

PS: polysaccharide

PC:phosphorylcholine

GlcNAc: N-acetyl-d-glucosamine

(9), Streptococcus pyogenes [group A streptococcus (GAS)] (10), and Streptococcus agalactiae [group Bstreptococcus (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 pathogenEnterobacter cloacae, a single heavy chain gene encodes this specificity (12, 13) and is absent insome strains of mice, rendering them incapable of mounting this response (14). By contrast, Absto phosphorylcholine (PC), a phospholipid expressed on the C-polysaccharide of S. pneumoniae(pneumococcus), display a highly conserved specificity across organisms and are produced byone of the earliest B cell clones (B1a) (15) that arise during mouse development (16). Like thosereactive with PC, Abs toward N-acetyl-D-glucosamine (GlcNAc), the immunodominant structureon the group A carbohydrate (GAC) of GAS, are ubiquitous in mammals. Antiglycan Abs can beexquisitely specific; for example, monoclonal antisialyllacto-N-tetraose-reactive Abs derived froma hybridoma generated following multiple immunizations with GBS1b differentiate between thePS of GBS1a and GBS1b serotypes, which differ by a single glycosidic linkage (17). Some of theseAbs, including those reactive to pneumococcus and GBS, are sufficient to provide protection fromlethal challenge with the virulent bacteria against which they were raised.

In preliminary studies we found that many monoclonal Abs raised against bacteria PS reactwith both the conidia and the hyphae of Aspergillus fumigatus (Figure 2). It was these observationsthat 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 allergicairway diseases. In this context, we review the ontogeny and functional activities of B cells and Absspecific for conserved antigens on a group of clinically relevant organisms and consider how theimmune response to them can be harnessed to combat the development of allergic airway disease

sialyllacto-N-tetraose

GlcNAc

α-1,3-glucan

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Figure 2Reactivity 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 andRPMI 1640 medium at 37◦C. The resulting germlings were fixed in 95% ethanol for 30 min and stainedwith 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.

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(Figure 1). In the following sections, we discuss the clonal development of B cell responses tobacterial exposure for several antigen specificities, and we end by reviewing protective effects ofanti-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 tonAb secreting cells, and whether these cells reside in particular niches of lymphoid tissues. B cellpopulations in mice are divided into B1, marginal zone (MZ), and follicular (FO) B cell subsetsby phenotype, microanatomical localization, and function (18). B1 and MZ B cells are thoughtto be the major sources source of nIgM (see sidebar Natural Antibodies). MZ B cells, juxtaposedwith metallophilic and marginal sinus-associated macrophages, initiate rapid production of Ab,predominantly IgM, against blood-borne particulate antigens and give rise to plasmablasts andlong-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 precursorsfor natural serum IgM (20), and mucosal secretory IgA production (21). There are differencesin this context however, between antigen-specific IgA generated in response to viral or bacterialinfections 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 cellsderived 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 inmodulating 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-associatedmolecules, 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, thatinvestigate the influence of neonatal antigen exposure on the establishment of PS-specific B cellswithin 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 andadults may result from the immaturity of spleen and bone marrow microenvironments necessary tosupport 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 littledetectable 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 emergingantigen-specific B cell clonal repertoire following vaccination (30). In addition, neonatal primingwith bacterial vaccines produces dramatic changes in the magnitude of the subsequent responsesin adult animals. As described in later sections, neonatal vaccination enriches the frequencies ofPS-specific cells within innate-like B cell subsets and in turn enables a rapid Ab response of greatermagnitude 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 areentirely 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 couldconceivably be generated by neonatal immunization or antigen exposure if bone marrow nicheswere more receptive for early seeding of T-independent, antigen-activated plasmablasts (29).

Collectively, these results show that although neonatal exposure to the TI-antigens describedabove generates only relatively modest increases in serum nAb, it fundamentally and permanentlyalters the clonal diversity and frequency of B cells that respond to these conserved antigens inthe adult. These effects greatly influence the Ab response to subsequent vaccination or infectionand are preserved for the life of the mouse. As we will discuss in the next section, the describedrepertoire changes to these and perhaps other bacterially derived glycans may have a dramaticeffect 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 bacterialpathogens 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, inhumans up to 1.5 to 2 years) infants respond poorly, if at all, to defined polysaccharide antigenssuch as the PC epitopes on C-polysaccharide of S. pneumoniae (7) or GlcNAc epitopes on theGAS cell wall polysaccharide (32–34). Individuals within these age groups are more susceptible toinfection, 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. Factorsinvolved in the delayed appearance of PS-reactive Ab include (a) constraints in Ig gene expressionin the developmental B cell program during fetal and neonatal life (35–38), (b) the immature stateof lymphoid tissue development and function (39, 40), and (c) the presence of passively transferredmaternal 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 complexinterplay of genetics and the environment, including the timing of antigen exposure (44). Theuse 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 isnecessary to provide sustained protection. In addition, the emergence of new capsular serotypeshas further complicated vaccine development. Long-term protection after immunization againstencapsulated bacteria is generally thought to depend on the persistence of functional Abs andimmunological memory. However, the anamnestic response that we consider classic memorytakes up to four days to develop and may be slow relative to the rapid invasion of encapsulatedbacteria (45–47). It is during this period that innate-like B cells, which produce long-lasting nAbsor 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 multipleenvironmental organisms. In the adult, some B cell clones result from developmental programsthat are active early in life (49, 50). In multiple examples, interference with these developmentprocesses during the neonatal period results in long-lasting changes in the expressed adult B cellrepertoire (14, 49, 51). Bacterial infection in humans early in life, although less studied, mayrender an individual unresponsive to later deliberate vaccination against the same bacteria (52–54). Additionally, as observed in attempts to vaccinate against Haemophilus influenzae, although

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TdT: terminaldeoxynucleotidyltransferase

Idiotype:immunoglobulinvariable region markerto 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 inachieving sustained Ab protection. The Ab unresponsiveness following the vaccination of infantswith DTaP (diphtheria, tetanus, and pertussis) is commonly attributed to the immaturity of theneonatal immune system, but it is more likely due to vaccine interference with development ofappropriate 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 cellrepertoire has not been well studied in humans. In mice, this space clearly exists. When miceheterozygous for one of the two possible IgM allotypes are suppressed by an antiallotype Abduring early development, the nonsuppressed, allotype-bearing B cells dominate the B1 niche forthe life of the animal. Thus, deletion of these clones during the neonatal period imprints the B cellrepertoire for the life of the mouse (58, 59). Importantly, we found that early B cell developmentalprograms are critical for the establishment of certain Ab clonotypes. Premature induction of N-additions by transgenic induction of the N-region-altering enzyme terminal deoxynucleotidyltransferase (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 producedanti-PC Abs, they were not protected against pneumococcal infection (49). Similarly, the geneticablation of TdT resulted in an increase in the T15 idiotype–bearing Ab in comparison with levelsin wild-type mice. These data suggest a temporal window for germ line–encoded rearrangementsto occur in developing B cell progenitors and become represented in the adult repertoire (50). Inaddition to conferring protection from pneumococcus, anti-PC Abs are reactive toward oxidizedlipids, including low-density lipoproteins, and they may be protective against the development ofatherosclerosis in models of this disease (60). By contrast, development of the dominant clone in theBALB/c response to α-1,3-glucan, which is expressed widely in bacteria and fungi, requires TdTactivity. 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 ofthe normally dominant J558-like B cell clones, which are normally recruited into the B1b and MZsubsets (13, 19, 51, 61).

Many PS-specific Abs are encoded by germ line sequences, and somatic mutations do notnecessarily correlate with enhanced function. In the case of Abs derived from hybridomas generatedfollowing hyperimmunization with PS-protein carriers, long-lived IgG-switched clones harboringV 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 morediverse pool of B cells into the response, there is very little information about how many of theseclones actually produce protective Ab in mouse models of infection or in human PS-conjugate-vaccinated humans.

Neonatal Antigen Exposure and the Subsequent Magnitudeand 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 hostadaptive immune responses. The conservation of these molecules throughout the evolution of adaptive immunesystems is indicative of their role in host survival. IgM Abs exhibit very high avidity, effectively fix complement, andare rapidly generated by B cell subsets and thus represent a powerful response to pathogen challenge. The productionof IgM through the expression of germ line–encoded Ig genes effectively couples a conserved evolutionary memorywith highly efficient effector functions without the need for somatic hypermutation normally associated with Tcell–driven germinal centers. IgM Abs constitute a large portion of nAbs, however little attention has been paid totheir role in immune responses or how their production can be manipulated to the host’s advantage. The inordinatefocus on the dogma that high affinity IgG responses is the goal of immunization and that so-called sticky, low-affinityIgM Abs should be avoided is the primary reason for this dearth of information. Hopefully, recent findings such asIgM memory cells in humans will lead to more focus on the functions of this first-line component of the adaptiveimmune 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 foravailable 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 cellclones that produce the most protective Abs (65–70) (see sidebar IgM Antibodies).

How neonatal exposure to antigen alters the clonal frequency, subset distribution, andfunction of PS-specific B cells later in adulthood has not been adequately studied in humans. Inthe next sections, we discuss evidence that there is a critical window during development whenthe B cell subset and clonal repertoire are plastic and can be manipulated (22, 58, 71, 72). Anin-depth analysis of this critical developmental window may lead to appropriate interventionsto optimize vaccine strategies and maximize protection. Moreover, the probable introductionof additional childhood vaccine regimens and alterations in the neonatal vaccination schedulemake it imperative that we understand the effects of vaccine interference on subsequent immuneresponses to further vaccination or to infection resulting from multiple vaccinations, neonatalchronic 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 tissuestructure developmental programs, pauciclonality of anti-PS responses, and delayed responses toPS antigens leading to increased neonatal susceptibility to bacterial infections. These similaritiesvalidate the use of mouse models for these studies and suggest the observations in the followingsections are pertinent to human infant humoral responses against infection and during vaccination.

Historically, successful vaccines have induced Ab-based immunity. Identification of antigensinducing protective Abs has been important in the development of candidate antigenic targets forinclusion in conjugate or conventional vaccines. When new vaccines become available, studies willneed 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 amultimember panel of microbes have shown uniformly that early exposure to vaccines derivedfrom bacteria has long-lasting effects on the adult clonal repertoire. In addition, the magnitude ofthe Ab response following reexposure is drastically increased. Thus, decisions in the present day

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concerning vaccines and immunization schedules may have dramatic and unexpected epidemio-logic effects for decades.

B CELL ONTOGENY AND NATURAL ANTIBODYRESPONSES 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, hasbeen extensively characterized in mice. GAC is a high-molecular-weight polymer consisting ofa helical rhamnose core structure decorated with exposed GlcNAc moieties. In addition to thisstructure, the cell wall of GAS contains large amounts of peptidoglycan, a biopolymer composedof alternating units of GlcNAc and N-acetyl-muramic acid (MurNAc). This structure greatlycontributes to the specificity of Ab responses following infection or immunization with GAS tothese immunodominant GlcNAc residues. Neonatal immunization with GlcNAc-bearing GASproduces long-lasting imprinting effects on the clonality of the GlcNAc repertoire that manifestin the immune responses of these animals as adults. In mice neonatally immunized with GAS14 days after birth, the resting levels of anti-GlcNAc IgM are substantially boosted (R. Kingand J. New, unpublished observations). Moreover, these elevated titers are maintained throughadulthood, and likely for the life of the animal. As can be seen in Figure 3, sera of aged mice, inthis case 16 months old, contained >100 μg/mL of GlcNAc-inhibitable Ab to GAC. Abs reactive

PBSMannose GalNAc GlcNAc

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Figure 3(a) Neonatal immunization with Streptococcus pyogenes induces the production of long-lasting IgM antibodiesspecific for GlcNAc. C57BL/6 mice were immunized at day 14 after birth with S. pyogenes and bled at 8 weeksand at 16 months, without further deliberate immunization. (b) Amount of anti-GlcNAc IgM antibody inserum was measured at 8 weeks and at 16 months. (c) GlcNAc, but not its enantiomer, GalNAc, greatlyinhibited 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.



GAC GAC GAC

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Figure 4Neonatal immunization with Streptococcus pyogenes alters the heavy-chain usage of the adult B cell repertoirespecific for GlcNAc. (a) Mice were neonatally immunized with S. pyogenes at day 14 after birth. Incomparison with mice neonatally treated with PBS, these mice had increased GAC-specific B cells in theirspleen. (b) The heavy chains of single-sorted B cells were sequenced to reveal both shared and uniquesequences between these two groups of mice. (c) A breakdown of the unique VH sequences found in the miceneonatally exposed to either S. pyogenes or PBS demonstrates a dramatic switch in the frequencies of thepredominant clones as the result of early exposure to group A streptococcus. Abbreviations: GAC, group Acarbohydrate; 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 bytwo VH genes that can differentially pair with one of four κ light chain genes or a λ light chaingene (73). When Ig gene expression from single GlcNAc-binding sorted B cells from neonatallyprimed mice is compared with that of PBS-treated mice, dramatic shifts in Ig VH gene usage canbe 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 encapsulatedpneumococcus induces Abs to PC moieties expressed on the cell wall–associated PS and teichoicacids. Analyses of B cell clones during ontogeny show that the earliest PC-specific B cells are notdetected 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

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Figure 5Ontogeny and immune manipulation of phosphorylcholine (PC)-specific B cell clones: (a) PC-specific B cells increase in frequencybeginning at 4 days of age. Early in life, these B cell clones are mostly non–T15 idiotype. (b) With time, T15 idiotype clones expressingthe canonical heavy-chain rearrangement become the dominant clones in the adult. Mice exposed to pneumococcus at day 3 of life andreimmunized as adults have increased circulating anti-PC IgM antibodies; however, these antibodies are mostly non–T15 idiotype. Incontrast, mice exposed to pneumococcus at day 12 of life and reimmunized as adults do not have as many circulating anti-PC IgMantibodies. (c) However, a majority of these antibodies are of the T15 idiotype. (d ) Mice neonatally immunized with pneumococcus andreimmunized as adults present with an altered clonal repertoire, in contrast to mice first immunized only as adults. Data modified fromReference 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 identifiedan altered frequency of light chain usage among these clones (71). The T15 idiotype, generatedby the pairing of the VH S107 Ig heavy chain with the Vκ 22 light chain, is replaced by clonesusing 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 vaccineprimes for a greatly enhanced anti-PC Ab response, resulting in a >40-fold increase in thefrequency of PC-binding B cells detectable in the adult (P. Patel, J.F. Kearney, manuscript inpreparation). However, generation of Ab following reexposure occurs mostly as a result of the



expansion of alternative non-T15 clones. Consequently, serum Ab resulting from secondaryvaccine challenge is much less protective in a model of pneumococcal challenge in comparisonwith 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 transgenicexpression of TdT, which results in fewer germ line–encoded Abs that are not protective againstpneumococcal infection (49), as well as results from experiments involving the administration ofanti-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 antigenscontaining α-1,3-glucan epitopes do not appear until one week after birth but increase in numberwith age (74) (Figure 6a). Ab responses to α-1,3-glucan epitopes are restricted to mice that possessthe IgHa locus and the appropriate VHJ558-3 gene. These responses are largely dominated byB 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 receptorgene for J558 idiotype–bearing B cell clones recruited into this response predominantly encodesan arginine-tyrosine sequence at the VD junction (61). These clones are distinct from those thatappear early in ontogeny, at which time another anti-α-1,3-glucan B cell clone, M104, whichconstitutes a minor population in the adult repertoire, is dominant (Figure 6b). The M104 and

J558

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Predominant anti-α-1,3-glucan IgVH

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56.3% 73.9%

Primary immunization

Idiotype profile of immune sera

α-1,3-glucan-reactive B cells

Figure 6Ontogeny and immune manipulation of α-1,3-glucan-specific B cell clones: (a) α-1,3-glucan-specific B cells increase in frequencybeginning at 7–8 days of age. In neonates, these B cell clones are mostly M104E idiotypic. (b) With time, J558 clones expressing theheavy chain rearrangement depicted in panel c become the dominant clones in the adult. (d ) Mice intraperitoneally immunized withα-1,3-glucan at 7, 14, or 21 days after birth and rechallenged as adults present with alterations in their clonal repertoire. Data modifiedfrom References 72 and 74.



J558 idiotype antigen receptors differ slightly in the amino acid sequence within the heavy chainVD junction. Immunization of neonates with a heat-killed or paraformaldehyde-fixed bacterialvaccine 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 ofthis effect is age dependent, such that the relative abundance of the M104 Ab, normally a minorconstituent of this response, is inversely proportional to the age of animals at primary exposureto 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 frequencyof anti-α-1,3-glucan B cells and by fixing the relative frequencies of idiotypes to that present atthe 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 receptorsexpressed by nascent clones. In the case of PC, the affinity of the germ line–encoded T15 cloneto 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-folddifference 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). Thismost likely contributes to its increased affinity. This phenomenon is further evidence of interclonalcompetition. In a nonimmunized neonate, the J558 clone eventually dominates, possibly throughselection 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 dominancein the adult Ab response (30). The reduction in competition for antigen between cells expressingthese 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 inthis section are detectable at low levels in most strains of mice. We have shown that in thesethree model systems, early exposure to antigen can reprogram clonal precursor frequency suchthat (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 repertoireare 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 normalhuman 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 someindividuals, “holes” exist in reactivity (75), which may reflect the inability of individual subjects toproduce certain subsets of nAbs. nAbs to GlcNAc, PC, and a variety of glucans can be detectedin most human sera, but as in mice, they are at low or undetectable levels in neonates and havebeen shown to be pauciclonal (76). Figure 7 is an adaption of retrospective data compiled by Grayet al. (34), in which anti-PC Ab levels in sera from healthy infants, carriers, and those diagnosedwith pneumococcal infections were plotted against age. As expected, levels of detectable anti-PCIgM Ab increase with age, but analysis of individual patients revealed that many infants, in somecases less than 1 year old, can develop substantial levels of IgM anti-PC Abs, ranging from 30to >1,500 μg/mL. Such high levels of anti-PC Abs were often associated with S. pneumoniaeotitis media infections and occasionally associated, depending on serotypes, with pneumococcal



01 2 4 6 8 12 2418 36 48

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tip

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Figure 7Antiphosphorylcholine IgM antibodies measured in children from ages 1 month to 4 years: A total of 115sera from 30 individual children with a history of exposure to Streptococcus pneumoniae, ranging from carriageto overt infection, and who were treated at the University of Alabama at Birmingham were assayed forantiphosphorylcholine antibodies. Data adapted from Reference 34.

carriage. Thus, it is clear that an infection with a live organism in humans does indeed inducea strong Ab response at an early age. In this small study, no follow-up analysis was undertakento 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 theproduction of Abs of this specificity resulted from the absence of infection, the differential use ofantibiotics, or the failure of individuals within this cohort to produce Abs in response to infectionwas not pursued.

Like the humoral response to PC, the response to GAS in humans is well characterized andpauciclonal (77). Investigations into the ontogeny of Ab responses to GAS during the neonatalperiod showed that anti-GAC Ab concentrations were highly variable among individuals butincreased until 7–17 years of age to levels maintained through adulthood. GAS is a prominenthuman pathogen, and approximately 5% of healthy individuals are carriers. Children under 1 yearof age are typically immune to infection owing to maternal Abs; however, infection of childrenwith GAS results in elevated serum anti-GAC Abs, particularly in those between 2 and 6 years ofage (Figure 8). In adults, both IgM and IgG Abs are detectable, but it has not been determinedwhich isotype predominates in infants less than 3 years old (78). As in mice, Abs to GAC arepredominantly directed toward GlcNAc, as determined by inhibition assays using monomericGlcNAc (Figure 3).

These studies show that the production of nAbs to PC and GlcNAc in humans is similar to thatobserved during ontogeny in mice and that infection with pneumococcus or GAS substantiallyincreases the levels of anti-PS Abs targeting these organisms. The human studies cited here revealthe potential of neonatal infections to (a) alter the clonal profiles of B cells responding to theseantigens in the adult, (b) reprogram nAb levels, and (c) substantially boost anti-PC and anti-GlcNAcresponses. 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 Absinduced 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 ≥180

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An

ti-G

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an

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od

y (

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)Recent GAS infectionNo report of GAS infection

Figure 8Anti-GAC antibodies from 120 healthy and 57 GAS-infected children and adults. Healthy subjects wereaged 12 months to ≥18 years, and the infected subjects were aged 2 to ≥18 years. Infected children wereconfirmed to have pharyngitis caused by bacitracin-susceptible β-hemolytic streptococci. Data adapted fromReference 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 thatpromote sensitization to inhaled antigens. It is clear that many human atopic diseases are initiatedneonatally 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 effectorcells 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 therelease of soluble mediators, including histamine, cysteinyl leukotrienes, and cytokines. Thesereactions have immediate effects on bronchial constriction and mucus production and delayedeffects driven by the activity of recruited leukocytes. Because the initiating process of atopicdisease requires sensitization and the production of allergen-specific IgE, understanding themechanisms leading to this adaptive immune response is paramount to our understanding of thedevelopment of allergic asthma and may ultimately provide mechanistic clues into the role ofearly microbial exposure in modulating this disease. Whereas considerable research efforts intothe allergic cascade have led to therapeutic strategies to alleviate the symptoms of allergies andasthma, 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/Th2polarization 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 thehygiene 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 allergicdisease. 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 adoptivetransfer 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: regulatoryT cell

airway inflammation (80). Induction of Th1 responses by respiratory syncytial virus and influenzaA infections has been shown to exacerbate, instead of alleviate, asthma (81). In addition, reductionin Th2 polarization following the pharmacological eradication of parasitic infections is ineffec-tive in alleviating allergic symptoms. In some cases, parasitic infections can exacerbate asthmaticsymptoms (82). There is evidence that parasite infections that occur during childhood are moresuccessful at preventing the development of allergic disease in comparison with those that occurin adults, possibly after the development of allergic disease (83). Although these data are bothconflicting 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/Th2imbalance is an oversimplified explanation of the mechanisms involved in controlling allergydevelopment. There appears to be stronger evidence that an imbalance between the Th2/Tregcell 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 correlatedwith the development of allergic disease (87, 88). Most important, Tregs have been shown to beable to suppress both Th1 and Th2 cytokine production (89). There is a direct correlation betweenearly microbial exposure and the generation of a robust Treg response, resulting from epigeneticchanges 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 mechanismsby which early microbial exposure imprints the immune system to influence the development ofallergic 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 aroundaltered T cell responses early in life, little attention has been paid to the effects of early microbialexposure on the B cell repertoire prior to the initiation of these diseases. The role of Abs and B cellsin 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). Althoughallergen-specific Abs passively administered (92, 93) or systemically induced Abs prior to andduring sensitization or challenge have been shown to be protective in most models, systemic Abscan 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 topurified or soluble allergens in the lung (96). B cells have also been shown to have a dispensablerole in the induction of ovalbumin-dependent AHR (97); however, they have also been implicatedin the induction of T cell tolerance to inhaled soluble allergens (98). Little is known regarding thecontribution 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 purifiedprotein. 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 leukocytesinto mucosal and lumenal compartments and results in the sensitization of allergenic lymphocytesresponsive 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 aprimary step in the recognition of the antigen. Engagement of these receptors will initiate inflammatory responsesthat clear potential pathogens but can also initiate allergic and autoimmune reactions. Anti-PS IgM Abs againstthese 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 thatthese polysaccharides are important, as engagement of innate receptors by these carbohydrate ligands leads to therelease of cytokines and chemokines. Certain pathogens also use the receptors to invade nonphagocytic cells andevade immune detection. The modulation of these interactions offers an extension of the effects of early bacterialexposure put forth in the hygiene hypothesis and the potential roles the IgM Abs to these conserved polysaccharidestructure 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 infectionshave largely disappeared in children in industrialized countries. As stated previously, infections bysome of these organisms are known to prevent or alleviate common childhood allergies and otherimmune-related diseases. Although there are multiple hypotheses regarding the involvement ofparasites in the development of allergy, protection from the development of allergic reactions isgenerally attributed to immunomodulatory effects of these infections (100). It is of great interestto this discussion that many nematodes express large amounts of PC-bearing molecules, as seen inFigure 9a,b. The integuments of Heligmosomoides polygyrus contain large amounts of chitin (notshown) and PC (Figure 9b). Deliberate infection of mice with the intestinal nematode H. polygyrusor 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 thedevelopment 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 ofsome nAbs, including specificities demonstrated to provide protection against diverse pathogenicorganisms (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 allergenand route of sensitization. Although many mouse allergy studies use alum-based models withpurified allergens, proteins, or surrogate-sensitizing antigens administered intraperitoneally (102),sensitizations via airways or skin are more relevant to human disease. Moreover, alum-based modelshave proven ineffective for the evaluation of interventional procedures involving the developmentof allergic disease in humans. Additionally, allergy occurs by long-term incremental exposure tosensitizing allergens; for these reasons we have developed models replicating natural respiratorysensitization and allergic challenge with clinically relevant allergens to examine the effects ofneonatal immunization and the role of nAbs in the induction of allergic airway disease.



α-G

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Figure 9The parasitic nematode Heligmosomoides polygyrus contains phosphorylcholine (PC), and roundworminfections elicit strong N-acetyl-D-glucosamine (GlcNAc)-specific responses. (a) Cryosections of an adultH. polygyrus worm were stained with (b) anti-PC IgM antibodies ( green). Mice were immunized one to threetimes with (c) Strongyloides or (d ) H. polygyrus, and the levels of anti-GlcNAc IgM in the serum weredetermined.

Antibody-Mediated Dampening of Aspergillus fumigatus–InducedAllergic Airway Disease

The A. fumigatus asthma model is highly relevant to human disease, as a high proportion ofpeople with asthma suffer from allergy to this organism (103). A major component of A. fumigatuscell walls is chitin, which is the second most abundant PS in nature and is found in crustaceanand insect exoskeletons and parasitic nematodes (104, 105). As GlcNAc-containing polymers andglycoproteins are found in bacterial cell walls, common allergens, and host tissues, we hypothesizedthat Abs directed to this moiety may be the link between infection with organisms expressingGlcNAc epitopes, including GAS, and the reduced incidence of allergic airway disease.

In addition to Abs against GlcNAc, which bind chitin particles <10 μm, multiple monoclonalAbs 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 ofthese monoclonal Abs are strikingly different, likely a result of the differential distribution andaccessibility 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 Absof these well-defined specificities or bacterial PS vaccines would influence immune processes inthe lung, affecting the initiation or progression of allergic airway disease induced by sensitizationto this organism.

Administration of anti-GlcNAc Abs reduced the uptake of A. fumigatus by macrophagesand 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 onStreptococcus and Enterobacter pathogens

Antibody Isotype Epitope Epitope found onHGAC-78, CTD 110 IgM GlcNAc S. pyogenesRL-2 IgG1 GlcNAc S. pyogenesHGAC-39 IgG3 GlcNAc S. pyogenes1–21, Dex 7, A16 IgM α-1,3-glucan E. cloacae8–21 IgG2b α-1,3-glucan E. cloacae7D7-2 IgG3 α-1,3-glucan E. cloacaeP5-1, J558, P5-1 IgA α-1,3-glucan E. cloacaeSMB19 IgM Sialyllactose-N-tetraose S. agalactiaeBH8, M603, M511 IgM Phosphorylcholine S. pneumoniaeS107 IgA Phosphorylcholine S. pneumoniae

of macrophages, dendritic cells, eosinophils, basophils, and other lymphocytes into the lungparenchyma and the bronchoalveolar space of mice sensitized to A. fumigatus in a pulmonaryallergy model. In addition to these effects, passive introduction of anti-PS Abs correlates witha marked reduction in multiple allergy-associated cytokines within the lung. A similar effect isobserved in vitro, where the inclusion of PS-specific Abs in cultures of bone marrow–derivedmacrophages 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 dueto 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, manuscriptin preparation). This effect results in reduced A. fumigatus–specific CD4+ T cell activation andcytokine production in both in vivo and in vitro systems. In vivo, the culmination of these effectsis 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 vaccinepreparations 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 responsesto intratracheal administration of live A. fumigatus conidia. Importantly, the protection againstsensitization to A. fumigatus following neonatal vaccination is remarkably long lived. As depictedin Figure 10, neonatal exposure to GAS results in protection against the manifestations of aninflammatory allergic response to A. fumigatus for 4.5 months following a single immunization.

Antibody-Mediated Dampening of House Dust Mite–InducedAllergic Airway Disease

Given the data described above, we have begun to expand this line of investigation to otherenvironmental antigens commonly associated with allergic asthma. House dust mites (HDM),like A. fumigatus, are ubiquitous environmental and household allergens. Approximately 30% ofindividuals that exhibit respiratory allergies demonstrate reactivity to HDM (107).

HDM preparations contain numerous allergen-bearing structures that share epitopes withmicrobes. HDM exoskeletons are composed of chitin, and allergen-bearing fecal pellets areencased in a chitinous mesh. In addition, we and others have shown that monoclonal anti-PC



a a

b b

c c

Figure 10Mice neonatally immunized with Streptococcus pyogenes are protected from the development of allergicpathology following a model of Aspergillus fumigatus–induced allergic airway disease: At the conclusion of theA. fumigatus–induced allergic airway disease model (4.5 months after neonatal exposure), sections ofparaffin-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 comparisonwith (b) mice neonatally immunized with Streptococcus agalactiae or (c) mice treated with phosphate-bufferedsaline. Images taken at 160× original magnification.

Abs bind either intact HDM, as shown in Figure 11, or, perhaps more important, micron-sizedparticulates 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 immunizationof neonates with pneumococcal vaccines attenuates the sensitization and allergic response toHDM. There is a dramatic reduction in the relative numbers of inflammatory cells infiltratingthe bronchoalveolar space of mice exposed to pneumococcus early in life compared with thosetreated 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 toA. fumigatus (106). Although we have shown antigen specificity is important for this protectiveeffect, 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 11Whole-bodied dust mites display phosphorylcholine (PC) epitopes: Unmilled whole-bodiedDermatophagoides 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 theinsects. Images taken at 160× original magnification.

sufficient to confer protection from sensitization to inhaled allergens. The effect we observein this model may result from the expansion of the innate-like B cell compartment followingneonatal immunization. Perhaps the enhanced frequency of B cells of these PS specificities allowsfor recruitment of sufficient numbers of these cells into mucosal sites during the earliest phasesof allergic inflammation, where they differentiate to provide protective Ab.

Naive mouseb

NeutrophilsNeutrophilsAlv macsAlv macs

a Mouse with early pneumococcal exposure

Alv macsAlv macsNeutrophilsNeutrophils

Figure 12Early pneumococcal exposure results in decreased infiltration of neutrophils into the bronchoalveolar spacefollowing exposure to house dust mite. Bronchoalveolar lavage fluid was collected from mice that wereneonatally exposed to pneumococcus or phosphate-buffered saline (PBS) following subjection to a housedust mite allergy model. The same volume of cells from each lavage was cytospun at 1,000 rpm for 5 minbefore being stained with a modified Wright stain (Sigma). (a) Mice exposed to pneumococcus early in lifepresent 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 earlyEnterobacter exposure Naive mouseba

Figure 13In comparison with mice exposed to phosphate-buffered saline (PBS), mice neonatally exposed toα-1,3-glucan-bearing Enterobacter produce less mucin in their bronchioles, following a cockroach allergymodel. At the conclusion of the cockroach allergy model, paraffin-embedded lungs were stained withperiodic acid–Schiff stain. (a) Mice neonatally immunized with α-1,3-glucan-bearing Enterobacter presentwith 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-bearingfecal pellets. We recently discovered that cockroach particulates, although mostly associatedwith 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 withα-1,3-glucan-bearing E. cloacae before being subjected to a cockroach allergy model. Similarlyto the A. fumigatus and HDM allergy models, early exposure to E. cloacae provides significantprotection from allergic challenge with cockroach allergen (P. Patel and J. Kearney, manuscriptin preparation). As can be seen in Figure 13, there is a dramatic reduction in the number ofmucin-producing cells in the bronchioles of mice neonatally exposed to E. cloacae compared withthose that were treated with PBS following allergic exposure to cockroach.

Proposed Mechanisms of Neonatally Induced ProtectionAgainst Allergic Airway Inflammation

It is of great interest that in each of these models there is a strong dependence on early microbialexposure 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 tomice neonatally immunized. A variety of studies have demonstrated that both humans and miceexposed 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 asneonatal immunization. Although it is clear neonatal microbial exposure may have a multitude of



immunological consequences, within the context of the described models, specific IgM appearssufficient to prevent sensitization and allergic responses. Collectively these observations suggestthat changes within the developing B cell compartment resulting from neonatal exposure to thesePS are responsible for the protective effects in that they alter the clonality and abundance of nAbswithin 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 particlesand 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, reducesthe number of A. fumigatus conidia engulfed by subsets of dendritic cells in the lungs of mice. Itis 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 signalsimplicated 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 allergicresponse. Experimental systems to distinguish between these potential mechanisms are currentlybeing developed.

Regardless of the mechanisms, neonatal immunization has long-lasting and profound effectson 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 ofthese 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 thesespecificities but also for others in response to natural infection, many of which may influence thedevelopment 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 blockingmicroorganism–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 directlyaffect 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 substructuresborne by these and other environmental antigens. Thus, there are long-term effects of neonatalexposure to environmental microorganisms on the development of hypersensitivity and allergicairway 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 allergicresponse in the A. fumigatus model. These findings in mice concur with observations in humansthat the possibility of reversing the disease declines with time after onset (110). Currently, thereare no preventive regimens to abrogate the development of atopic disease initiated neonatally. Bycontrast, our findings suggest that alternatives to conventional allergen immunotherapy could beinstituted at early stages in at-risk children, which could be effective in treating and potentiallyabrogating 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 understandingof the mechanisms associated with Ab-mediated immunoregulation will lead to the developmentof 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 bea 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 mightcontribute to induction of Tregs early in life, potentially dampening the development of allergicdisease in adulthood. In addition, these studies can broaden the suggested protective mechanismsconferred by early microbial exposure against other autoimmune diseases frequently discussedin the context of the hygiene hypothesis, such as type 1 diabetes, multiple sclerosis, and Crohndisease, 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 antigenexposure.

3. There is a limited time for windows of opportunity after birth for this reprogrammingand generation of long-lived B cell clones, which results in permanent changes in thesubsequent magnitude and clonal diversity of the adult repertoire.

4. Antibodies to conserved antigens on bacteria that commonly cause infections in humanshave the potential to react with other organisms, including fungi and insects, associatedwith allergies.

5. Neonatal exposure but not adult exposure to bacterial vaccines induces antibodies thatdampen 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 atopyor allergic symptoms?

3. What benefit does clonal heterogeneity afford in the prevention of allergic airway diseasein mice?

4. Does reprogramming the natural antibody repertoire in mice affect the development ofother atopic diseases, such as food allergy?

5. Does neonatal immunization influence the development of or induce other cellularsubsets?

6. How can these observations effectively translate into the development of preventivetherapeutics?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight 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 thismanuscript, and the expert, patient editing of this manuscript by Stewart New and Dr. DeniseKaminski. This work was supported by National Institutes of Health Grants RO1AI14782-36and RO1AI100005-02. J.F.K. was a recipient of a Senior Investigator Award from the AmericanAsthma Foundation.

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