generation of high affinity ige antibodies

7/29/2019 Generation of High Affinity IgE Antibodies

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Article

The Rockeeller University Press $30.00

J. Exp. Med. Vol. 209 No. 2 353-364

www.jem.org/cgi/doi/10.1084/jem.20111941

353

Antibodies beong to ve casses (IgM, IgD,

IgG, IgE, and IgA) depending on the constant

regions o the heavy chain (CH) they use. Cass

switch recombination (CSR) changes the CH

rom C to a downstream CH (Cs, C, orC) by DNA recombination and ooping-outo the intervening sequences (Sesing, 2006).

The irreversibe DNA rearrangement in the

CH region occurs between two switch (S) re-

gions, which ie upstream o each CH region

(except C). CSR is initiated by activation-induced (cytidine) deaminase (AID), which is

aso essentia or the introduction o somatic

hypermutations in the variabe regions o the

BCR (Muramatsu et a., 2000).

IgE is the most stringenty reguated secreted

Ig, with the owest serum concentration and

the shortest ha-ie o about 2 d or ess (Tadaet a., 1975; Stone et a., 2010). In contrast,

ce-bound IgE can remain or weeks on the

surace o ces bearing Fc receptors. Mast ces(as we as basophis) are prearmed with IgE

through FcRI moecues binding to the Cregion o IgE. Cross-inking o IgE moecues

by mutivaent antigen resuts in the activation

o mast ces. Upon activation, mast ces degran-

uate, reeasing histamine and other mediators

that diate bood vesses, initiate inammation,

and recruit other eector ces (Kinet, 1999;

Krat and Kinet, 2007; MacGashan, 2008).The afnity o IgE moecues or their anti-

gens is crucia in determining the ate o IgE-

decorated ces. It is possibe that mast ces

bearing IgE moecues with high afnity or the

antigens coud be degranuated in the presence

o just minute amounts o antigens. In contrast,

the high eves o antigen required to cross-ink

and degranuate mast ces covered by ow

afnity IgE moecues may not be physioogi-

cay achievabe. Mast ces are important com-

ponents o the homeostasis o tissues, and, in

such roe, they are benecia. A series o papers

have concusivey shown that IgE antibodiesare capabe o increasing surviva o growth

actordeprived mast ces (Asai et a., 2001).

There have been no studies comparing the

behavior o mast ces covered by IgE antibod-

ies that dispay ow versus high afnity binding

CORRESPONDENCEJuan J. Laaille:[email protected]

Abbreviations used: AID,

activation-induced (cytidine)deaminase; CSR, cass switch

recombination; GC, germina

center; HA, hemaggutinin;

HIES, hyper IgE recurrent

inection syndrome; hMT,

human metaothionein IIA;

NP, Nitropheny; PCA, passive

cutaneous anaphyaxis; qPCR,

quantitative PCR; TBmc,

TB monocona.

M.A. Curotto de Laaies present address is Singapore

Immunoogy Network, Singapore 138648.

Sequential class switching is required or

the generation o high afnity IgE antibodies

Huizhong Xiong,1,2

Jayashree Dolpady,1

Matthias Wabl,5

Maria A. Curotto de Laaille,1 and Juan J. Laaille1,3,4

1Molecular Pathogenesis Program, Helen L. and Martin S. Kimmel Center or Biology and Medicine at the Skirball Instituteo Biomolecular Medicine, 2The Sackler Institute o Graduate Biomedical Sciences, 3Department o Pathology;and 4Department o Medicine; New York University School o Medicine, New York, NY 10016

5Department o Microbiology and Immunology, University o Caliornia, San Francisco, San Francisco, CA 94143

IgE antibodies with high afnity or their antigens can be stably cross-linked at low con-centrations by trace amounts o antigen, whereas IgE antibodies with low afnity bind

their antigens weakly. In this study, we fnd that there are two distinct pathways to gener-ate high and low afnity IgE. High afnity IgE is generated through sequential classswitching () in which an intermediary IgG phase is necessary or the afnity

maturation o the IgE response, where the IgE inherits somatic hypermutations and highafnity rom the IgG1 phase. In contrast, low afnity IgE is generated through direct class

switching () and is much less mutated. Mice defcient in IgG1 production cannotproduce high afnity IgE, even ater repeated immunizations. We demonstrate that a small

amount o high afnity IgE can cause anaphylaxis and is pathogenic. Low afnity IgEcompetes with high afnity IgE or binding to Fc receptors and prevents anaphylaxis and

is thus benefcial.

2012 Xiong et al. This article is distributed under the terms o an AttributionNoncommercialShare AlikeNo Mirror Sites license or the rst six months aterthe publication date (see http://www.rupress.org/terms). Ater six months it isavailable under a Creative Commons License (AttributionNoncommercialShareAlike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Published January 16, 2012

http://jem.rupress.org/content/suppl/2012/01/12/jem.20111941.DC1.htmlSupplemental Material can be found at:


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354 Sequential switching produces high anity IgE | Xiong et al.

is aways indicative o sequentia switching. In our assay, ater

we ran a PCR over the S-S junctions, the products weresubjected to quantitative PCR (qPCR) on the S1 repeats.

We rst vaidated the method using our IgE hybridomas

and ve subcones rom one o them. PCR was perormed

using primers ocated at 5 o S and 3 o S. The size o thePCR-ampied S-S junctions was between 3 and 7 kb

(Fig. 1 B). There was a dominant band or each hybridoma.

The our hybridomas had dierent sized bands, and the ve

subcones had the same sized band as the origina hybridoma

(Fig. 1 B). 24 minipreps ran rom coned S-S junctions

to a given antigen; instead, afnity correations were per-

ormed by studying strong and weak cross-inking o FcR insevera aternative ways (Kaesniko et a., 2001; Gonzaez-

Espinosa et a., 2003; Yamasaki et a., 2004; Kohno et a.,

2005; Rivera et a., 2008). These studies showed that a high

eve o cross-inking o FcRI receptors resuts in mast cedegranuation as we as cytokine/chemokine secretion,

whereas a ow eve o cross-inking eads to cytokine and

chemokine secretion but no degranuation. However, so ar

there is no concusive evidence that the increasing afnity

o a given antigen or IgE can turn the unction o IgE rom

benecia to pathogenic.

At the moecuar eve, there are two paths to produce

IgE antibodies rom IgM (Fig. 1 A). One is the direct switch-

ing pathway, in which recombination occurs between S and

S; the other is the sequentia switching pathway, which is

a two-step process observed in both mice and humans. In

mice, S rst recombines with S1, and the hybrid switchregion undergoes a second recombination with S. At theend o sequentia switching, S1 remnants can be ound insome o the rearranged S-S junctions (Fig. 1 A). It has been

reported that in mice up to 30% o the recombined regions

contain S1 remnants (Siebenkotten et a., 1992; Mander et a.,1993). In human ces, the switch to in a ymphoid ce inewas preceded by an S-S recombination (Mis et a., 1992).

In B ymphocyte cutures rom mice harboring a deetion in

the IgM switch region, IgE+ ces coud be ound at neary

norma eves (70%), and they were derived rom CSR events

invoving S1 (Zhang et a., 2010).We previousy reported that sterie transcripts and

switched transcripts, which are generated beore and ater theswitch to C, respectivey, were abundanty ound in IgG1+

ces. Furthermore, puried IgG1+ ces coud switch to IgEin vitro and in vivo. In addition, afnity-enhancing somatic

mutations ound in the IgG1+ ces were aso ound in IgE+

B ces, athough with some deay (Erazo et a., 2007). We

proposed that sequentia cass switching through an IgG1

intermediate was a way to generate high afnity IgE antibod-

ies. The IgE ces were absent rom germina centers (GCs),

and we proposed that they inherited somatic mutations and

afnity maturation rom the preceding IgG1 phase.

Despite prior studies by mutipe aboratories (Yoshida

et a. [1990], Erazo et a. [2007], and reerences therein), it is

uncear whether sequentia switching to IgE through an IgG1

intermediate is an obigatory step to generate high afnity

IgE antibodies. In this study, we demonstrate that, indeed,high afnity IgE antibodies in the mouse are generated

through an IgG1 intermediate.

RESULTS

A simple assay to quantiy sequential DNA

switching in IgE+ cells

Because our hypothesis is that high afnity IgE antibodies are

generated through an IgG intermediate, we deveoped a PCR

assay to quantiy S remnants in the S-S junctions o IgE+ces. As schematized in Fig. 1 A, the nding o S1 remnants

Figure 1. A simple PCR assay to detect sequential switching to

IgE. (A) Schematic representation o direct and sequential switchingtogether with the chromosomal products. S1 remnants are always in-

dicative o sequentially switched S-S junctions. (B and C) Molecularcharacterization o the S-S junction rom IgE-producing hybridomas.(B) S-S PCR on the DNA o hybridomas SE1.3, Boettcher, 15.3, and b4.

(C) S1 qPCR on miniprepped S-S junctions. The abundance is calcu-lated as the relative transcription normalized to neomycin resistance gene

expression xed at 10,000.



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The second experimenta system is a we-studied system

o immunization o WT mice with the hapten Nitropheny

(NP) conjugated with carrier proteins. The resuts using both

experimenta systems were in agreement, as shown in the

oowing paragraphs.

Immunizing TBmc mutipe times at 3-wk intervas

with OVA-PEP1 enabed us to observe the gradua increase

o IgE afnity to PEP1 (Fig. 2 A). Ater each immunization,

we determined the serum titer against PEP1 and sorted

IgE+IgG1Fashigh ces rom the speen (not depicted). As the

number o immunizations o TBmc mice with OVA-PEP1

increased rom one time to our times, the PEP1-specic IgE

serum titer raised rom 50 to 4,000 (Fig. 2 A). IgE+IgG1Fasow

ces were aso sorted but did not contain ampiabe switch

regions (not depicted); they were positive or FcRI (notdepicted), indicating that they were IgE-coated ces. As we

reported previousy, a mid acid treatment is sufcient to dis-

odge IgE moecues rom CD23, the ow afnity IgE recep-

tor, but not rom the high afnity FcRI (Erazo et a., 2007).DNA was extracted to perorm S-S PCR. We ob-

served a smear o dierent sizes (not depicted). Interestingy,

as the number o immunizations increased, we observed

dominant S-S bands, which was not seen in other experi-menta modes such as the NP-immunized human metao-

thionein IIA (hMT) mice and the anti-IgDtreated WT mice

(see beow). We appied the aorementioned singe coony

qPCR assay to determine the S1 ratio. As we increased thenumber o immunizations, the S1 ratio went up rom 0.03

o each hybridoma indicated that a inserts were o the same

size, reassuring us that no major recombination events took

pace during the PCR and coning steps.

The second PCR step was a quantitative rea-time PCR

on S1 to target the characteristic 49-mer S1 consensussequences (Mowatt and Dunnick, 1986). S1 contains a ong(11.5 kb) region characterized by >120 imperecty repeated

49-mer sequences. Actua consensus S1 sequence repeats

perecty ony nine times, whereas the other repeats have

sma but compicated changes ike overaps and truncations.

qPCR detected the S1 remnant in one o the our hybrid-omas and a o its subcones. The other three hybridomas

were negative (Fig. 1 C). Both positive and negative qPCR

resuts were conrmed by DNA sequencing. Because it was

apparent that the IgE-producing hybridomas ony retained

one S-S aee, the S1 qPCR yieded a quantitativesigna o either >10,000 or 0, i.e., S1 was either present

or absent.

To evauate the requency o sequentiay switched IgE

ces within a popuation o ces during an immune response,

we dened an S1 ratio, which is the number o S1+

junctions among the tota number o S-S junctions. Eventhough not a sequentiay switched ces had S1 remnants(Fig. 1 A), as a popuation, the requency o S1+ ces stiprovided a comparative measure o the extent o sequentia

switching. The S1 ratio in hybridomas was either 0 or 1,

and an in vivo IgE response wi have a ratio anywhere

between 0 and 1.

Increased sequential switching to IgE correlates

with increased IgE afnity or antigen

For the present experiments on afnity maturation o the IgE

response, we used two experimenta systems. The TBmc sys-tem (Curotto de Laaie et a., 2001) consists o heavy and

ight chain inuenza hemaggutinin (HA)-specic Ig knockin

mice crossed with OVA-specic TCR transgenic mice

(D011.10) and RAG1 KO mice, a on a BALB/c genetic

background. The HA-specic knockin Ig genes were de-

rived rom the high afnity BCR 17-9 hybridoma (Schuze-

Gahmen et a., 1988), and as a consequence, the antibodies

are intrinsicay o high afnity or HA. Because o both the

absence o natura reguatory T ces (T reg ces) and the

BALB/c background, these mice dispay a high HA-specic

IgE response ater one immunization with the cross-inked

antigen OVA-HA. There is no increase in afnity-enhancing

mutations o the anti-HA response ater OVA-HA immu-nization (Erazo et a., 2007). To study afnity maturation, we

used a singe mutant HA peptide, PEP1, in which a pheny-

aanine residue repaced one o the tyrosine residues in HA.

Binding o the endogenous anti-HA antibodies to PEP is

1,000 times weaker than to HA (Pinia et a., 1993). In TB

monocona (TBmc) mice, immunization with OVA-PEP1

aows the tracking o afnity maturation both by the appear-

ance o PEP1-binding B ces (undetectabe i mice are not

immunized with PEP1) and PEP1 afnityenhancing muta-

tions in the Ig genes.

Figure 2. Sequential switching is correlated with increased IgE

afnity or antigen. (A) TBmc mice were immunized with 100 gOVA-PEP1 or one, two, and our times as indicated in the x axis. Serumwas taken or ELISA to determine the IgE anti-PEP1 titer, and IgE+ cells

were sorted to prepare DNA, S-S PCR, cloning, and S1 qPCR assay.Blue bars indicate the anti-PEP1 IgE titer (right y axis), and the red lineindicates the S1 ratio o S-S junctions (let y axis). Three mice

rom each group and at least 24 clones/mouse were randomly chosen orS1 qPCR. SD is indicated. (B) S1 ratio in A o each individual mouse by

single colony qPCR.



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o sequentia switching through IgG1. We sorted IgE+ ces

and ran the S-S PCR in the same way we did on TBmcmice. In contrast to the TBmc situation, dominant S-S

bands were not observed ater our immunizations in hMT

mice or the WT ittermates. We then cacuated the S1 ratiobased on 100 coned S-S junctions derived rom our WTmice and a simiar number o hMT mice. The S1 qPCR assay

showed that, depending on the mice, 1030% o WT S-Sjunctions had S1 remnants (Fig. 4 D). Interestingy, S-Sjunctions rom hMT mice not ony acked S1 remnants,which was expected, but contained no other intervening

S region (not depicted). Thus, in hMT mice, IgE antibodies

are excusivey generated through direct switching.

Somatic mutations that enhance afnity to NP

are not present in IgE genes rom IgG1-defcient mice

In the C57BL/6 (B6) mouse strain, the repertoire o B ces

responding to NP has been extensivey studied (Cumano and

Rajewsky, 1986; Furukawa et a., 1999; Weiser et a., 2011).

It was determined that VH186.2 was preerentiay used in the

anti-NP response, paired with IgL1.

The maturation o the NP-specic response incudes two

dierent mutation paths: in one o them, the rst junctiona

residue in the CDR3 is Y99. In those B ces, afnity to NP

is greaty increased by a W33L mutation. Additiona mutations

do not urther enhance the afnity or NP in the Y99-type

ces. The second path invoves heavy chains with a junc-

tiona CDR3 residue G99, encoded by N nuceotides. In this

case, high afnity is achieved through the accumuation

o severa mutations, mainy K59R, G57D, S66N, and other

contributors, and aows or much higher afnities (Furukawa

et a., 1999; Weiser et a., 2011). However, there is a timing

trade-o: it has been determined that the Y99/W33L typeo ces appears reativey eary during the anti-NP response,

whereas the G99/K59R type o ces appears ate, but the

atter type takes over upon secondary immunization

as it accumuates additiona afnity-enhancing mu-

tations (Jacob et a., 1993; Takahashi et a., 2001;

Murakami et a., 2010; Nishimura et a., 2011).

In contrast to the B6 strain, knowedge on the

moecuar basis o afnity maturation o the NP re-

sponse in BALB/c mice is scanty. Because o the act

that B6 mice make poor IgE responses, our work was

perormed in hMT mice (and ittermates) backcrossed

norma eves o tota IgE (Jung et a., 1994), indicating that

IgE does not necessariy derive rom sequentia switching.

However, we propose that the IgE afnity maturation in

hMT mice is impaired.

We immunized hMT and their WT ittermates with

NP19KLH our times at 3-wk intervas and coected serum

to assess the NP-specic antibody responses. Pates were

coated with NP4BSA and NP23BSA. NP23 is a highy conju-

gated moecue that binds both ow and high afnity anti-

bodies, whereas NP4 has a ow degree o cross-inking and

binds ony high afnity antibodies. A higher NP4/NP23 ratio

indicates a higher afnity to NP (Herzenberg et a., 1980;

Aen et a., 1988; Smith et a., 1994; Chen et a., 2000; Shimizu

et a., 2003; OConnor et a., 2006). IgE antibodies rom

hMT mice had a much ower NP4/NP23 ratio than IgE anti-

bodies rom WT mice (Fig. 4 A, et). Absoute absorbance

o NP23-bound IgE was simiar between hMT and WT mice,

but the binding o NP4 was much weaker in hMT IgE than

in WT IgE (Fig. 4 A, right). As expected, tota IgE eves

were simiar in hMT and WT (Fig. 4 B), indicating that the

ow NP4/NP23 ratio in hMT mice was not caused by the

deciency o IgE production but rather by the afnity matu-

ration o the IgE response. We appied the same ELISA tech-

nique to study the IgG2a and IgG1 responses in the same

mice. IgG2a constitutes a good contro because the S2a

region is, ike S, ocated downstream S1, and it is at 5 nextto S. Thus, IgG2a is a good isotype to identiy whether anynonspecic eect on IgE coud be taking pace. hMT mice

did not have any IgG1, whereas IgG1 o high afnity was

readiy detected in WT mice (Fig. 4 C, et). The afnity

maturation o the IgG2a response was the same in hMT and

in WT mice (Fig. 4 C, right), indicating that IgG2a afnity

maturation occurs independent o1 sequentia switching.We sought to demonstrate that the high afnity NP-

specic IgE response was accompanied by moecuar remnants

Figure 4. IgE afnity is impaired in IgG1-defcient mice.(A) WT and hMT mice were immunized with NP19KLH our times.

Serum was analyzed by ELISA with NP4- and NP23-coated plates.(let) NP4/NP23 ratio. (right) Absolute absorbance value. Errorbars indicate SEM. Each dot represents one mouse. (B) Same

sera used in A were subjected to total IgE ELISA. (C) IgG1 andIgG2a ELISA with NP4/NP23 plates. (D) S1 ratio in WT and hMTmice using the S1 qPCR assay. Each dot represents one animal

(our animals per group). The number o S1+ junctions in eachmouse is shown in the right panel, divided by the total number

o S-S sequences. (AD) Horizontal bars indicate the mean.



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the same mice (Fig. 5 B), consistent with the observation in

TBmc anti-PEP1 response (Erazo et a., 2007). In contrast

with IgE, the requency o mutations in IgG2a VDJ genes

was simiar between hMT and WT mice (Fig. 5 B). Note

that the reduction in the requency o mutations is ess

pronounced than the reduction in afnity, indicating that

the seection o high afnity mutants is more aected than

the overa eve o mutations.

Next we anayzed the typica mutations that coner higher

binding afnity to NP in mice o BALB/c background. In

IgE genes, the K59R mutation was ound ony in WT IgE

but not hMT IgE (Fig. 5 A). In contrast, the K59R mutation

had a simiar requency in IgG2a rom both WT and hMT

mice. About ha o the K59R IgE cones in WT mice had

the additiona G57D and/or S66T/N mutations (Fig. 5 A).

Finay, we aso studied the J and D segment usage in

these cones. In C57BL/6 mice, the eary NP-specic B ces

(Y99 type) have highy enhanced usage o the D16.1-JH2

segments, and even the ate-arising NP-specic B ces (G99

type) predominanty use the D16.1 segment (Furukawa

et a., 1999). In the BALB/c background mice, there was no

strict preerence or the usage o certain JH (not depicted) or

DH (Tabe S2) segments; however, there was a preerence

o JH3 in NP+IgG1+ ces, whereas NPIgG1+ ces used

more the JH4 segment (P = 0.0001; not depicted). Thus, JH3

usage correates with higher afnity or NP in the BALB/c

background. In accordance with this, the JH usage in tota

IgE+ ces rom hMT mice showed preerence or JH4 over

JH3, whereas the higher afnity IgE+ ces rom WT mice

showed preerence or JH3 over JH4 (P = 0.0001; not de-

picted). In summary, these data strongy support the mode in

which high afnity IgE is generated through sequentia

switching invoving S1.

onto the BALB/c background. Our characterization o the

NP-specic response in mice o BALB/c background in-

dicated that the preerred VH gene was homoogous to

VH186.2 and that the IgL chain aso dominated the NPresponse in BALB/c mice (not depicted). We immunized

BALB/c background mice with NP19KLH, sorted IgG1+

ces that did or did not bind NP-PE (not depicted), and

sequenced the VDJ segments. Interestingy, the Y99/W33L

path was not ound on >300 DNA sequences rom NP+

B ces, at any time point ranging rom day 7 to day 80

ater immunization (Fig. 5 A). Instead, acquisition o NP

binding was accompanied by an increase in the requency

o the K59R mutation, which in approximatey ha o the

cases had G57D and/or S66T/N mutations. G99 was

ound ony in some o these antibodies, but a the junc-

tions contained N nuceotides (Tabe S2). Thus, the path-

way that coners the highest NP binding afnity in B6 mice

is the one that operates in BALB/c mice.

With this knowedge in hand, we sought to anayze the

afnity-enhancing mutations in IgE, IgG2a, and IgG1 in WT

and hMT mice immunized with NP19KLH. We ampied

VH1.1 sequences (homoogous to the B6 VH186.2) in IgE+,

IgG2a+, and IgG1+ ces. We then studied the mutations in

96 DNA sequences or IgE, 82 sequences or IgG2a, and 62

sequences or IgG1 (the atter ony rom WT mice). The IgE

VDJ repertoire in hMT mice had signicanty ewer muta-

tions than WT mice (Fig. 5, B and C). Ony 8% o the IgE

cDNAs rom hMT mice had 2024 nuceotide mutations,

and no cDNA had 2529 mutations. In contrast, in WT

mice, 13% and 17% o the IgE VDJ regions contained 2024

and 2529 mutations, respectivey. The percentage o se-

quences with high requency o mutations in IgE genes rom

WT mice was sighty ower than that o IgG1 genes rom

Figure 5. Mutations in the VDJ region o IgG1-defcient mice and their WT littermates. (A) Total splenocytes rom the same hMT and WT litter-mate mice used in Fig. 4 A were prepared or cDNA. PCR was perormed using a common VH1.1 orward primer and C and C2a reverse primers. NP

high

and NPlow IgG1+ cells were sorted by staining with NP-PE. cDNA was prepared, and VH1.1- C1 PCR was perormed. Ater sequencing, the NP anityenhancing mutations were tabulated. (B and C) Total nucleotide mutation analysis o the same sequences described in A (B) and the analysis o total

mutation requency (C). Error bars indicate SEM.

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Antigen-driven high afnity but not low afnity IgE

causes anaphylaxis

The dierentia pathoogica unction o IgE antibodies

with high versus ow afnity or their antigens has been

proposed but not uy expored. Anaphyaxis is a ie-

threatening reaction caused by antigen-induced cross-

inking o IgE moecues bound to mast ces. In this study,

we used a oca passive cutaneous anaphyaxis (PCA) assay

in the ears to quantiy the eect o high and ow afnity

IgE antibodies in vivo. The amount o IgE, rom IgG- and

IgA-depeted serum, was normaized in a cases. First, mice

were injected in the ears with anti-HA IgE, oowed by

cross-inked HA (OVA-HA) together with Evans bue by

systemic route. This combination induced strong extrav-

asation o Evans bue into the ears (Fig. 7 A, et). However,

there was virtuay no Evans bue eakage when injection

o the same anti-HA IgE antibodies was oowed by ow

afnity antigen OVA-PEP1 (Fig. 7 A, midde et). As indi-

cated above (Erazo et a., 2007), HA-specic antibodies are

1,000-od ess reactive to OVA-PEP1 than to OVA-HA.

When IgE afnity to PEP1 increased through afnity

maturation caused by repeated immunizations, these IgE

antibodies dispayed the capabiity to cause Evans bue

extravasation upon OVA-PEP1 chaenge (Fig. 7 A, midde

right). As expected, unconjugated monovaent soube

PEP1 coud not cross-ink anti-PEP1 IgE and did not cause

the PCA (Fig. 7 A, right). The resuts were conrmed by

spectrophotometric determination o the eaked dye upon

cutting and dissoving the aected and contro ears (Fig. 7 B).

Next, we titrated the amount o injected IgE in the ears.

In seria diution experiments, very diuted anti-

HA IgE (1:100 diution) caused a strong PCA

reaction by OVA-HA, and the same happenedin the case o anti-PEP1 IgE with OVA-PEP1.

However, in the presence o OVA-PEP1, anti-

HA IgE did not cause anaphyaxis, even when

injected undiuted (Fig. 7 C). To iustrate the

importance o afnity maturation o IgE in

anaphyaxis, we studied IgE obtained ater

Direct switching to IgE upon antigen-independent

B cell stimulation

Ater conrming the importance o sequentia cass switch-

ing to IgE or the afnity maturation o two dierent anti-

gen-driven T cedependent responses, we investigated

the roe o sequentia switching to a known poycona

stimuator o IgE and IgG1 production, anti-IgD serum

(Finkeman et a., 1989). We injected the WT BALB/c

with goat anti-IgD antibodies and, as a contro, with one

NP19KLH immunization. Anti-IgDtreated mice produced

a substantia IgE response (Fig. 6 A, et). However, the

IgE+Fashigh raction contained


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IgG1 phase. Our data showed that afnity-enhancing mu-

tations in VDJ segments rom IgE cDNAs but not rom the

IgG2a cDNA, whose C region ies next to C, were greaty

reduced in IgG1-decient mice. Thus, afnity maturation

o IgE can occur through a GC IgG1 phase. Whether these

IgG1+ ces that switch to IgE+ ces are immediate post-GC

ces, memory ces, or ces committed to become pasma-

basts remains the subject o uture studies. We previousy

showed that GC IgG1 ces can express sterie as we as

switched transcripts, but doube expressing IgG1+/IgE+ces produced in homozygous knockin TBmc mice do not

have a GC phenotype (Erazo et a., 2007).

It has been known or a ong time that the dose and re-

quency o immunizations have an impact on the amount

and ratio o IgE and IgG1 antibodies (Vaz et a., 1971). It is

ikey that at higher doses, T reg ces can be recruited to the

response and suppress the IgE response (Curotto de Laaie

et a., 2001).

We previousy reported that IgE ces were absent rom

GCs during Nippostrongylus brasiliensis inection as we as

during immunization o TBmc with cross-inked OVA-HA

(Erazo et a., 2007). In act, the evidence that GCs are not

avorabe structures or IgE+ ces is mounting. Bc6-decient

zero, one, two, and our immunizations with OVA-PEP1,

using normaized amount o tota IgE. Ony IgE rom mice

immunized our times was capabe o causing the u anaphy-

actic reaction (Fig. 7 D), which was conrmed by spectro-

photometric measurement (Fig. 7 E).

We next perormed PCA competition experiments be-

tween high and ow afnity IgE and aso IgM and IgG1 anti-

bodies. Our experiments showed that when injected with

high afnity anti-PEP1 IgE, ony anti-HA IgE coud eiminate

the OVA-PEP1induced anaphyaxis reaction, in a dose-

dependent manner. Neither IgM nor IgG1 coud reduce the

OVA-PEP1mediated anaphyactic reaction (Fig. 7, F and G).

It is thus cear that the potentia or causing pathoogy o ow

afnity and high afnity IgE antibodies is very dierent. In

summary, the afnity maturation o the IgE response requires

an IgG intermediate, and the consequence o this process is the

generation o antibodies capabe o eiciting anaphyaxis.

DISCUSSION

Sequentia switching was discovered >20 yr ago(Yoshida et a., 1990). However, previous studies did not in-

vestigate whether S remnants represented transient markerso an instant sequentia switching event or there was a ceuar

Figure 7. Anaphylaxis caused by IgE o high

afnity to its antigen can be competed by IgE

o low afnity or the same antigen. TBmc micewere immunized with OVA-HA or OVA-PEP1 twotimes, and sera were collected and depleted o IgG

and IgA. IgE concentration was measured by ELISA.25 ng o total IgE was injected intradermally into

the ear o recipient BALB/c mice. 24 h later, 50 gOVA-HA, OVA-PEP1, or unconjugated PEP1 wasinjected intravenously together with 1% Evans

blue. (A and B) 30 min later, mice were sacriced,and the extent o Evans blue leakage was deter-mined either visually (A) or spectrophotometri-

cally (B; three mice per group). (C) Beore injectinginto the ears, sera were diluted as indicated.

(D and E) Similar PCA assay was perormed usingsera rom TBmc mice immunized with OVA-PEP1one, two, or our times. The PEP1-specic IgE titer

is shown above the photos. A total o 25 ng IgE wasinjected per ear. 24 h later, recipient BALB/c micewere injected intravenously with OVA-PEP1 and

Evans blue. The ears are shown in D, and Evansblue was measured spectrophotometrically in E.

(F) 6 ng o high anity anti-PEP1 IgE was mixedwith 100 times more PBS, anti-HA IgE, IgG1, or IgMto perorm the PCA assay mediated by OVA-PEP1.

The extent o Evans blue leakage was determinedvisually. (G) Quantication o Evans blue leakageby spectrophotometry in the ears injected with 1 ,

20, 100, and 500 times more IgM, IgG, or low anityIgE (three mice each group). (B, C, E, and G) Error

bars indicate SEM.



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JEM Vol. 209, No. 2

Article

361

(Aguado et a., 2002). It thus appears that during some T ce

ymphopenic disorders, IgE switching resembes much o

what we showed here or directy switched antibodies.

A nonexcusive aternative is that natura IgE antibodies

may be the resut o switching to IgE at the immature B ce

deveopmenta stage. It has recenty been shown that im-

mature B ces in bone marrow and speen switch to IgE

in a direct CSR, whereas the mature B ces have apropensity to switch via an IgG1 intermediate (Wesemann

et a., 2011). In contrast to natura IgE generation, conven-

tiona IgE responses, which invove afnity maturation, are

generated through a dierent mechanism, invoving a key

IgG phase.

Natura IgE antibodies may be invoved in conditions

such as hyper IgE recurrent inection syndrome (HIES),

WiskottAdrich syndrome, Omenn syndrome, and DiGeorge

syndrome. The combination o immunodeciency and Th2

bias sometimes resuts in the production o extremey high

eves o IgE (50,000 U/m) without, however, aergic

symptoms such as rhinitis, asthma, urticaria, and anaphyaxis

(Grimbacher et a., 2005; Ozcan et a., 2008; Minegishi, 2009;

Freeman and Hoand, 2010). I a these IgEs had under-

gone afnity maturation to bind environmenta antigens with

high afnity, these patients woud have severe aergic re-

actions upon minima exposure to antigen. Thus, it is ikey

that hyper IgE responses in HIES patients are o ow afnity to

environmenta antigens and are directy switched , ikenatura IgE responses. Compared with HIES patients, chi-

dren with aergic asthma have ower eves o IgE; however,

these IgEs showed hypermutations and afnity maturation

(Kerze et a., 2010).

IgE responses have traditionay been considered impor-

tant in the cearance o some heminthes, which inducea massive IgE production caused by very efcient Th2 di-

erentiation induced by parasite components. Interestingy,

athough some o the IgE antibodies are specic or the he-

minthes (Jarrett and Haig, 1976; Pochanke et a., 2007), the

vast majority o the IgE antibodies do not react to the

parasites. Evoutionariy, or the parasite, it coud resut

in a deayed rejection, as ow afnity IgE competes with

parasite-specic IgE or FcRI receptors and cross-inking ohost eector ces woud be perturbed (Pritchard, 1993). For

the host, heminth nonspecic IgE binding to mast ces

woud reduce immunopathoogy and prevent serious sys-

temic aergic reactions.

It has been ong known that recombinant Fc ragments andsynthetic peptides rom C can inhibit antigen-specic IgE reac-tions when present at 200- or 1,000-od excess, respectivey

(Geha et a., 1985; Burt and Stanworth, 1987; Nio et a., 1992).

We observed inhibition o high afnity IgE-mediated anaphy-

axis by the addition o intact IgE o ow afnity or the antigen,

athough compete inhibition o anaphyaxis required arge

moar excess. It ceary demonstrates the benecia and patho-

genic unctions o ow and high afnity IgE, respectivey.

Besides the eect on basophi and mast ce degranuation,

IgE antibodies have aso been shown to aciitate antigen

mice have severey impaired GC ormation and no afnity

maturation, but they harbor an increased number o IgE+

ces (Ye et a., 1997). Simiary, deciency o Dock8 (dedica-

tor o cytokinesis 8) caused unstabe GCs in mice (Randa

et a., 2009) and hyper IgE syndrome in humans (Engehardt

et a., 2009; Zhang et a., 2009). IL-21 is a critica cytokine

produced by TFH ces; simiary to Bc6 and Dock8, IL21R-

decient mice have higher IgE and ower IgG1 eve (Ozaki

et a., 2002). Thus, the incompatibiity between IgE and GCs

can be expained by our sequentia switching mode, in which

IgG1+ intermediate ces undergo the GC phase.

As the number o immunizations increased, the requency

o S-S junctions that contain S1 remnants increased, inparae with an increase in IgE afnity. It is remarkabe

that during the course o immune responses we did not see

S remnants other than S1 in S-S junctions. The reason

or the strong S1 dominance is that sterie transcripts romI1 and I promoters share some o the triggers, such as IL-4/STAT6, which coud turn on S1 and S in the same ce,whereas other sterie transcripts such as I2a or I2b woudremain sient. I this is correct, in other IgE responses in

which IgG1 is not activated, we may possiby nd remnants

other than S1 in S-S junctions.We did not nd evidence o the contribution o the

memory IgM+ B ces to the high afnity IgE response (Dogan

et a., 2009; Tomayko et a., 2010; Pape et a., 2011), as we

ound amost no afnity maturation in the absence o IgG1.

However, it is possibe that memory IgM+ B ces are prone

to undergo switching to IgE via IgG1 rather than through the

direct switching route.

In B6 mice, the high afnity to NP rst comes rom a com-

bination o VH186.2 and IgL 1. O the two paths that urther

increase VH186.2 afnity or NP, the rst path (Y99/W33L)does not have N nuceotides in the VD junctions, whereas the

second path (G99/K59R) uses N nuceotides. In BALB/c mice,

we ound NP-binding ces were aso highy enriched in IgL,and the K59R with other afnity-enhancing mutations were

ound. Interestingy, there were many N nuceotides in these

junctions, even though they did not necessariy encode G99.

These observations show that the mechanism used by BALB/c

mice to produce high afnity anti-NP IgE is very simiar to

the second path in B6 mice, which can be time consuming, but

end up with a higher afnity than the rst path.

IgE antibodies are spontaneousy increased in mice with

T ce immunodeciencies such as nu/nu, CD4/, MHC

cass II/; these IgEs have been reerred to as natura IgEantibodies (McCoy et a., 2006). Like the conventiona IgE

antibodies, natura IgE antibodies are dependent on T ces,

CD40CD40L interactions, and IL-4; however, organized

ymphoid structures such as GC are not required, and natu-

ra IgE antibodies do not accumuate afnity-enhancing

somatic hypermutations (McCoy et a., 2006). Importanty,

ymphopenia-induced proieration in T cedecient

mice induces T ces that deveop predominanty aong

the Th2 pathway (Miner et a., 2007). The high IgE ound

in LatY136F knockin mice coud have a simiar genesis



10/12


DNA sequence analysis. For VDJ sequencing, tota RNA extraction and

cDNA synthesis were perormed by standard procedures. PCR reactions

were perormed with high deity ExTaq Poymerase (Takara Bio Inc.) and

the common orward primer VH1.1 in combination with primers specic

or IgE, IgG1, or IgG2a constant regions (Erazo et a., 2007). For switch

region sequencing, PCR-ampied junctions were coned into TA vector

and miniprepped. A the sequences were perormed by Macrogen Corp.

Point mutations in S1 regions with ength o 0.1 ater

background subtraction.

Flow cytometry. Beore staining or FACS anaysis, singe-ce suspensions

rom speen were treated or 1 min with ice-cod acid buer (0.085 M NaC,

0.005 M KC, 0.01 M EDTA, and 0.05 M NaAcetate, pH 4) to remove

extrinsic IgE antibodies noncovaenty bound to CD23. The sampes were

then neutraized with a arge voume o ce cuture medium and washed

twice beore staining. The oowing reagents were used: FITCanti-IgG1(A85-1), biotinanti-IgE (R35-72), PerCPanti-B220 (RA3-6B2), PE

anti-Fas (jo2), PE-Cy7anti-Fas (jo2), NP-PE (Biosearch), and APC-

streptavidin (BD).

S1 qPCR. Ces were sorted by a MoFo cytometer. DNA was extracted

rom the ces with ysis buer where proteinase K at 50 g/m was

reshy added. The oowing primers were used to ampiy S-S junctions:

SF, 5-TTGGGGAAGGGAAAATAAA-3; and SR, 5-TAGGGCT-

GTTGGTCATAGAT-3. Ampied junctions were coned into TA vector

(Invitrogen) and miniprepped. qPCR was perormed using the oow-

ing S1 primers: S1F, 5-AGCCAGGACAGGTGGAAGT-3; and S1R,

5-CTGCTCTGCCTGGGTCA-3.

http://dx.doi.org/10.1126/science.1069057http://dx.doi.org/10.1016/S1074-7613(01)00157-1http://dx.doi.org/10.1016/S1074-7613(01)00157-1http://dx.doi.org/10.1159/000232441http://dx.doi.org/10.1159/000232441http://dx.doi.org/10.1002/eji.1830170323http://dx.doi.org/10.1002/eji.1830170323http://dx.doi.org/10.1159/000232441http://dx.doi.org/10.1159/000232441http://dx.doi.org/10.1016/S1074-7613(01)00157-1http://dx.doi.org/10.1016/S1074-7613(01)00157-1http://dx.doi.org/10.1126/science.1069057


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