Review article

Immune mechanisms of allergen-specific sublingual

immunotherapy

Allergen-specific immunotherapy has been used inhumans for almost a century with the aim to redirectinappropriate immune responses in atopic patients (1, 2).It has proven to be efficacious to treat type I allergies to avariety of allergens (3–8). While the parenteral (subcuta-neous) route of immunization is still a reference, localroutes (e.g. intranasal, oral) have been considered as analternative with mixed results, both in terms of efficacyand tolerance (9). Of note, a specific form of oraltolerance induction, i.e. sublingual immunotherapy(SLIT) is raising a lot of interest as a noninvasiveprocedure, as it has been shown to be efficacious,provided that high doses of allergen (i.e. 50–100-foldthe subcutaneous dose) are administered (10–25). In arecent meta-analysis, encompassing 22 clinical studiesevaluating SLIT in 979 patients with allergic rhinitis tohouse dust mite, pollens (from grass, parietaria, olive,ragweed, cupressus) and cat dander, it was concluded thatSLIT significantly reduces both symptoms and medica-tion requirements (26). Importantly, it is now widelyadmitted that SLIT is much safer than subcutaneousimmunotherapy (SCIT), with no evidence of anaphylacticshock recorded after more than 500 million doses admin-istered to humans (12, 17, 27, 28). Whereas SLIT has

been successfully used to treat allergic patients, ourunderstanding of the immunological mechanismsinvolved has been limited. We review herein recentscientific advances, which provide some clues on effec-tor/regulatory immune mechanisms elicited during suc-cessful allergen-specific immunotherapy in general, andSLIT in particular. Based on such improved biologicalfoundations, we comment on arising opportunities todesign second-generation sublingual allergy vaccinesrelying upon well-characterized recombinant allergens,capable of controlling T-cell polarization following vac-cine-mediated and subsequent natural exposure to theallergen.

Immunomodulation during allergen-specific immunotherapy

As summarized in Fig. 1, allergen-specific immunothera-py, whether it be SCIT or SLIT, is known to reduce bothimmediate as well as late-phase allergen-induced symp-toms, by acting both on humoral, as well as on cellularimmune mechanisms involved in allergic inflammation (4,29–33). Schematically, three categories of immunologicalchanges are induced by active immunotherapy, encompas-

Sublingual immunotherapy has been shown in some clinical studies to modulateallergen-specific antibody responses [with a decrease in the immunoglobulin E/immunoglobulin G4 (IgE/IgG4) ratio] and to reduce the recruitment and acti-vation of proinflammatory cells in target mucosa. Whereas a central paradigmfor successful immunotherapy has been to reorient the pattern of allergen-spe-cific T-cell responses in atopic patients from a T helper (Th)2 to Th1 profile,there is currently a growing interest in eliciting regulatory T cells, capable ofdownregulating both Th1 and Th2 responses through the production of inter-leukin (IL)-10 and/or transforming growth factor (TGF)-b. We discuss hereinimmune mechanisms involved during allergen-specific sublingual immuno-therapy (SLIT), in comparison with subcutaneous immunotherapy. DuringSLIT, the allergen is captured within the oral mucosa by Langerhans-likedendritic cells expressing high-affinity IgE receptors, producing IL-10 and TGF-b, and upregulating indoleamine dioxygenase (IDO), suggesting that such cellsare prone to induce tolerance. The oral mucosa contains limited number ofproinflammatory cells, such as mast cells, thereby explaining the well-establishedsafety profile of SLIT. In this context, second-generation vaccines based onrecombinant allergens in a native conformation formulated with adjuvants aredesigned to target Langerhans-like cells in the sublingual mucosa, with the aimto induce allergen-specific regulatory T cells. Importantly, such recombinantvaccines should facilitate the identification of biological markers of SLIT efficacyin humans.

P. Moingeon1, T. Batard1, R. Fadel1,F. Frati2, J. Sieber3, L. Van Overtvelt11Stallerg�nes, Antony, France; 2Stallergenes ItaliaS.r.l, Milan, Italy; 3Stallergenes GmbH & Co. KGGermany, Kamp-Lintfort, Germany

Key words: allergy vaccine; Langerhans cells;regulatory T lymphocyte; sublingual immunotherapy.

Philippe MoingeonResearch and DevelopmentStallergenes6 rue Alexis de Tocqueville92160 AntonyFrance

Accepted for publication 5 September 2005

Allergy 2006: 61: 151–165 Copyright � Blackwell Munksgaard 2006

ALLERGY

DOI: 10.1111/j.1398-9995.2006.01002.x

151

sing (i) modulation of allergen-specific antibody responses;(ii) reduction in recruitment and activation of proinflam-matory cells; and (iii) changes in the pattern of allergen-specific T-cell responses (Table 1). Although such biologi-cal changes are usually better documentedwith SCIT, thereare, in this regard, no clear-cut qualitative differencesbetween SCIT and SLIT, suggesting that immune mech-anisms at play are similar (Table 1).With respect to allergen-specific antibody responses,

SCIT often induces an initial increase in seric immuno-globulin E (IgE) levels, prior to a subsequent downregu-lation in the following months (34–39). In patients allergicto grass pollen, SCIT prevents the seasonal rise in IgEantibodies associated with natural exposure to allergens(37, 39). Moreover, successful SCIT protocols resulting inclinical improvement of patients often elicit allergen-specific IgG responses (mostly IgG1 and IgG4) and in afew reported cases, IgA responses (Table 1) (5, 40–44).Similarly, SLIT was shown to increase allergen-specificIgG4 levels comparedwith placebo (10, 11, 13),with amore

limited impact on specific IgE responses (18). A decrease inthe IgE/IgG4 ratio has been observed in a number of SLITstudies (11, 24, 45), with some exceptions (46). A meta-analysis of six SLIT studies with detailed analysis ofantibody responses concluded on a consistent increase inallergen-specific IgG4 levels (26). Such changes in the IgE/IgG4 ratio were found to correlate with a decrease in thelate-phase skin reaction to the allergen andwith the overallclinical efficacy of the vaccine in some studies (23, 24) butnot all (13, 18). In a recent phase I/II trial with grass pollentablets, SLIT was shown to elicit allergen-specific sericIgAs in a dose-dependent fashion (47) and a smallupregulation of IgA responses was also observed whenSLIT was used in house dust mite allergic patients (45).Altogether, allergen-specific IgG (and IgA) antibodiesinduced by immunotherapy are thought to contribute tothe positive clinical response through distinct and nonexclusive mechanisms: (i) these antibodies can competewith IgEs for binding to the allergen, thereby preventingboth basophil or mastocyte degranulation (38–40), as well

Figure 1. Humoral and cellular immune mechanisms involved in type I allergy and immunotherapy: an integrated view. Allergen-specific immunotherapy deals with the cause of type I allergies, by inducing long-term reorientation of an existing and inappropriateimmune response (associated with Th2 cytokine production, high IgE levels, activation of mast cells, basophils and eosinophils leadingto release of proinflammatory mediators such as histamine, eicosanoids and proteases). CD4+ T helper cells (e.g. Th1, Th2 or Tregulatory) stimulated following allergen exposure or desensitization are critical in controlling these various components of theimmune system through the production of distinct cytokines. IL-4 and IL-13 induce IgE class switching, IL-5 supports eosinophilrecruitment and activation, and IL-13 increases mucus production. Regulatory T cells inhibit both Th1 and Th2 responses through theproduction of IL-10 and TGFb. While atopic patients exhibit allergen-specific Th2 CD4+ cells, healthy people rather mount T regresponses when exposed to allergens. Thus, the purpose of allergen-specific immunotherapy is to restore tolerance by shifting T-cellresponses from Th2 to T Reg.

Moingeon et al.

152

Table1.

Modifications

ofimmunological

parametersduringallergen-specific

immunotherapy

(SCITandSLIT)inhumans

Parameters

Allergensused

Comments

Reference

Allergen-specific

antibody

responses(IgE,IgG1,IgG4,

IgA)

Venoms,pollens

(ragw

eed,birch,

grasses,parietaria,olive),dustmites

Used

as:

–naturalbiologicale

xtracts,allergoids

recombinant

protein(Bet

v1)

associated

with

anadjuvant

(e.g.

CaPO

4,Alum

,MPL)for

SCIT

–naturalbiologicale

xtracts,and

allergoids

withoutanyadjuvant

for

SLIT

Subcutaneous

immunotherapy

Immunoglobulin

isotypes:S

CITinducesan

initial

increase,followed

byasubsequent

decrease

ofspecificIgEs,aswellasan

increase

inallergen-specific

IgG1

andIgG4.

AnriseinIgG4/IgEratio

hasbeen

documentedwith

hypoallergenicformsof

recombinant

Betv1(peptidefra

gmentsandtrimer).Insomestudies,butnotall,

changesinaffinity

ofIgG4

antibodiesfortheallergen

duringimmunotherapy

have

been

show

n.AmonophosphoryllipidA(MPL)adjuvantedvaccinereducesseasonally

boostedIgEresponsesandinducesstrong

IgG1

andIgG4

responsesagainst

grass-pollenallergens.SCITelicits

allergen-specific

IgAresponsesdetectable

intheblood

5,34–40,

44,83

Functionalcharacterizationof

antibodies:IgG4

exhibitsomeblocking

activity.Insomebut

notallstudies,a

corre

lationwas

foundbetweenIgG(mostly

IgG4)concentrations

and

clinicaloutcom

es.IgG4antibodiesreduce

allergen

T-cellresponsesby

inhibiting

bindingof

allergen-IgEcomplexes

toantigen

presentingcells

40–44,48,4

9

Sublingualimmunotherapy

Immunoglobulin

isotypes:S

LITagainsthousedustmite

orgrasspollenincreasesspecific

IgEs

andIgG4,inassociationwith

animprovem

entof

therespiratoryfunction.

Several

studieswith

SLITreportinductionof

IgG4

responses,withoutanyincrease

ordecrease

ofIgEs

(i.e.ƒ

ofIgG4/IgEratio).Forexam

ple,SLITwith

aParietaria

judaicaextra

ctinchildrenreducesrhinitissymptom

sandboth

skin

andconjunctivalallergen

reactivity

inparallelw

ithincreasedspecificIgG4

seric

antibodies.In

contrast,preseasonalrush

SLITagainstP.judaicarhinoconjunctivitisor

SLITinasthmaticchildrensensitizedto

miteshadno

detectable

impact

onspecificIgEor

IgG4

levels,d

espite

improvem

ent

ofsymptom

anddrug

scores.A

phaseI/IIstudy

conductedwith

grasspollen(Phleum

pratense)tabletsdocumentedadose

dependentinductionof

allergen

specificIgG4

andIgAresponses.Ameta-analysisconductedon

sixSLITstudiesconcludedthat

the

combinedstandardizedmeandiffe

rences

(SMD)

betweentre

ated

patientsandcontrolsfor

changesinallergen-specific

IgEwas

0.22

(P¼

0.06),whereas

SMDwas

0.6(P<0.00001)

forallergen-specific

IgG4

10,11,14,23,24,26,

45,47

Recruitmentandactivationof

proinflammatorycells

(mast

cells,eosinophilsandbasophils)

Pollens

(ragw

eed,grasses,parietaria),

dustmites

Used

as:

–naturalbiologicale

xtracts,allergoids

associated

with

anadjuvant

(e.g.

CaPO

4,Alum

)for

SCIT

–naturalbiologicale

xtracts,allergoids

recombinant

protein(Phlp1,Betv1)

withoutanyadjuvant

forSLIT

Subcutaneous

immunotherapy

Impact

oncellrecruitment:SCITreducestherecruitmentinto

theskin

andmucosa(nose,lung)o

finflammatorycells

includingmastcells,eosinophils,b

asophils,n

eutro

phils

50–60

Impact

onproinflammatorymediators:a

decrease

inseric

eotaxinwas

foundto

beassociated

with

successfulIT.S

CITdecreasesthereleaseof

inflammatorymediators(e.g.h

istamine,

kinins,

PGD2)b

ybasophils

andmastcells

Sublingualimmunotherapy

Impact

oncellrecruitmentandactivation:Rush

SLITto

Parietaria

inducesareductionin

neutrophils

andeosinophils

recruitmentas

wella

sICAM

-1expression

aftereyeor

nasalchallenge,

incorre

lationwith

areductionin

both

symptom

anddrug

intake

scores.S

ublingual

challengewith

increasing

dosesof

recombinant

Phlp

1or

Betv1show

slim

itedreactogenicitywith

mastcells

63,65,66

Impact

onproinflammatorymediators:S

LITinchildrenwith

grasspollenallergydecreasesnasal

eosinophilcationicproteinlevelsandpreventsincrease

intryptasefollowingallergen-specific

challenge.In

childrenallergicto

D.pteronyssinus,SLITreducesseric

ECPandprolactin

levels

(thelatte

rlikelyassociated

with

areducedactivationof

Tlymphocytes).Along-te

rmimmunological

effect

ofSLITisto

reduce

residualsignsof

persistent

inflammation(decreaseinsolubleICAM

-1,in

mucosa,which

corre

lateswith

adecrease

inmetacholineresponsiveness)

61–65

Immune mechanisms of SLIT

153

Table1.

Continued

Parameters

Allergensused

Comments

Reference

T-cellresponsesandcytokine

patte

rns

Venoms/PLA2,dustmites,pollens

(ragw

eed/Am

ba1,grasses),cat

dander/Feld1

Used

as:

–naturalbiologicalextracts,

allergoids,p

urified

naturalallergen

(Amba1)or

peptides

associated

with

anadjuvant

(e.g.C

aPO4,A

lum,C

pGs)

forSC

IT–naturalbiologicalextracts,

withoutanyadjuvant

forSLIT

Subcutaneous

immunotherapy

Impacton

theTh1/Th2ratio:S

CITdownregulates

allergen-specific

Th2responses(bothin

termsof

cytokine

productionandproliferation)with

orwithoutstimulationof

allergen-

specificTh1cells.R

apidinvitro

allergen-inducedTh-2

lymphocyteapoptosishasbeen

observed

inonestudy.Increase

insignalinglymphocyticactivatingmolecules

(SLAM)

gene

expression

duringpollenIT.Insomestudies,Th2responsesareincreasedduringthe

titrationphase,

andtheTh2/Th1ratio

isonlysignificantlydecreasedduringthemaintenance

phase.Modificationof

Th2/Th1ratio

corre

lateswith

clinicalimprovem

ent,decrease

inlate-phase

skinreactions

andallergen-specific

IgElevels.A

ssociationbetweenallergens

(e.g.A

mba1)

andoligoCpGimmunostim

ulatorysequences,used

asamixor

asconjugate

vaccines

was

foundto

decrease

Th2andto

enhances

Th1responses

53,66–80,8

2,112

Modificationof

localT-cellresponses:d

ecreaseof

Th2cytokines(IL-4/IL-5/IL-3)a

ndincrease

ofTh1cytokines(IFNc,

IL-12),atlocallevels(e.g.intheskinfollowingintra

dermalgrasspollen

challenge).D

ecreaseinrecruitmentintarget

organs

ofTh2CD

4+Tcells

expressing

theCXCR1

receptor

81,112

Impacton

regulatory

Tcells:Inductionof

CD4+,C

D25+

Tregulatory

lymphocytes

andTr1IL-10

producingcells,intheblood.

Anincrease

inIL-10mRN

Aandproteinisdetected

inskinbiopsies

from

allergen

challenged

subjectsfollowingwaspvenomimmunotherapy.S

omeof

theseregulatory

Tcells

exhibitaCD

62L)

CCR5

+profile

suggestingacapacityto

migrate

toperipheraltissues.Early

orsustainedincrease

ofregulatory

cytokines(IL-10andTGF-b,respectively)isproposed

tocorre

late

with

successfulimmunotherapy.

83,84,90,112,148

Studiesconductedwith

peptides:studies

conductedwith

peptides

(Feld1,PLAA2)dem

onstrate

askew

ingof

theTh1/Th2ratio

towards

theformer,inductionof

SOCS

2andof

IL-10-producingcells.

Injections

ofsm

all(16

to17-mer)Feld1peptides

inhibitthecutaneouslate-phase

reactionto

whole

catdander,a

swella

sallergen-stim

ulated

productionby

PBMCs

ofboth

Th1(IFN-c)and

Th2(IL-4,

IL-13)cytokines,concom

itant

with

anincrease

inIL-10production

4,79,82

Sublingualimmunotherapy

Impacton

theTh1/Th2ratio:inadultswith

seasonalrhinoconjunctivitis,SLITwith

agrasspollen

extra

cthadno

detectableeffecton

sublingual

infiltra

tionby

CD3+

Tcells

orlocalproductionof

IL-12.Nostimulationof

T-cellproliferationor

cytokine

productioninchildrenallergicto

grass

pollen,

despite

apositiverescue

onmedicationuse.

Adecrease

inseric

IL-13andT-cell

proliferativeresponses(associatedwith

aneffect

onsymptom

s)hasbeen

observed

withoutany

detectable

change

inTh1/Th2ratio,inchildrentre

ated

with

D.pteronyssinusextra

cts

23,46,86

Impacton

regulatory

Tcells:Inapreliminaryreport,

inductionof

circulatingIL-10producing

peripheralT

cells

inhousedustmite

allergicpatients

87

Moingeon et al.

154

as allergen capture and presentation to T lymphocytes byFceRI+ and CD23+ antigen-presenting cells (APCs) (48,49); and (ii) such antibodies may act as blocking antibodiesby engaging low-affinity Fc receptors for immunoglobulins(e.g. FccRII) expressed by B lymphocytes, basophils, ormast cells. As FccRII receptors contain immunoreceptortyrosine-based inhibitorymotifs (ITIM), they transduce, asa consequence, negative signals preventing cellular activa-tion and release of soluble proinflammatory mediatorsfollowing co-aggregation with FCeRI receptors (42, 43).In a number of studies, SCIT was shown to inhibit both

the recruitment and activation in mucosa of proinflamma-tory cells involved in the allergic reaction (50–60). Forexample, successful SCIT has been associated with adecrease in the recruitment of mast cells, basophils andeosinophils in the skin, nose, eye and bronchial mucosa,following provocation or natural exposure to allergens(Table 1). Similarly, SLIT prevented the recruitment ofeosinophils in the eye or in the nose after allergen challenge(61–65). SLIT with grass pollen extracts was shown todecrease local or systemic levels of eosinophil cationicprotein (ECP), without any increase in tryptase (61, 62).Similarly, rush SLIT to parietaria reduced the recruitmentof neutrophils and eosinophils to the nasal mucosa as wellas the expression of the intercellular adhesion molecule-1(ICAM-1) (66).In the context of an emerging integrated picture of the

physiology of immune responses, the aforementionedchanges in immune parameters are assumed to be a directconsequence of an impact of immunotherapy on CD4+ T-cell responses (Fig. 1). It is well known that allergicpatients usually mount strong allergen-specific CD4+

T-cell responses of the T helper (Th)2 type, characterizedby the secretion of high amounts of interleukin (IL)-4, IL-5 and IL-13 cytokines (30, 37, 67). In this regard, a centralgoal for immunotherapy has been to reorient allergen-specific T-cell responses in atopic patients from a Th2 toTh1 profile [the latter being rather associated with theproduction of interferon (IFN)-c and IL-12 cytokines](53, 68–80). A number of studies have indeed correlatedsuccessful SCIT with the induction of Th1 responses and/or the decrease of Th2 cytokine production (thus, it is theTh1/Th2 balance which appears critical). Interestingly, ina recent SCIT study, the Th2/Th1 ratio was found toincrease during the titration phase, prior to decreasingduring the maintenance phase, in parallel with late-phaseskin reactivity and allergen-specific IgE levels (70). Inseveral SCIT studies with grass pollen, a switch from aTh2 to a Th1 profile was not consistently observed at asystemic level, but was rather detected locally (i.e. withinthe nasal mucosa or the skin) (53, 69, 81). This observa-tion emphasizes the importance of documenting immunechanges not only in peripheral blood, but also locally intarget organs. More recently, it has been shown that SCITinduces a new subset of CD4+ Th cells, called regulatoryT cells, which exhibit a capacity to downregulate both Th1and Th2 responses through the production of IL-10 and/

or transforming growth factor (TGF)-b (see below) (50,82–85). There is less evidence on the impact of SLIT onT-cell responses. In several studies conducted in childrenor adults with seasonal allergic rhinoconjunctivitis tograss pollen, no significant effect of SLIT on T-cellfunctions (i.e. cytokine production, proliferation) wasobserved (23, 46). SLIT does not induce any detectablechanges in the numbers of dendritic cells (DCs) nor Tlymphocytes in the epithelium or lamina propria of theoral mucosa (14). Immunization through the sublingualroute was nevertheless shown in other studies to decreasethe production of the Th2 cytokine IL-13 and theproliferation of PBMCs from patients allergic to housedust mite (86, 87). As of today, there is still no firmevidence that SLIT can induce regulatory T cells. Apreliminary study suggests that SLIT increases IL-10production in peripheral blood mononuclear cells(PBMCs) from house dust mite (HDM) allergic patientsfollowing in vitro stimulation with Dermatophagoidesfarinae antigens, but also with recall antigens (e.g.Candidaalbicans) or PHA, when compared with untreated allergicpatients (88). That some IL-10-secreting T cells are notallergen-specific raises the possibility of a bystanderimmunosuppressive effect of SLIT. Of note, high-doseSLIT regimens with ovalbumin in mice induces ova-specific T cells producing TGF-b in the spleen of sensitizedanimals (L. Van Overtvelt, P. Moingeon, unpublishedresults). Further studies are needed to document unam-biguously regulatory T cells induction during SLIT.

Regulatory T cells and allergy vaccines

Although both anergy and T-cell depletion are known tocontribute to the establishment of peripheral toleranceagainst environmental antigens, it is now broadly admit-ted that antigen-specific T-cell populations with suppres-sive/regulatory function play a key role in controllingimmune responses to both self- and nonself-antigens (89–95). These cells, termed regulatory T cells, are heteroge-neous, and include both: (i) naturally occurringCD4+CD25+ T cells and (ii) cells induced in theperiphery following antigen exposure (e.g. Tr1 cells,Th3 cells, and CD8+ regulatory T cells). These varioussubsets of regulatory T cells can be distinguished on thebasis of their surface markers and the pattern of cytokinesthey produce (Table 2). Antigen/allergen presenting DCsplay a critical role in the induction of T-cell-mediatedtolerance, in that immature DCs in the absence ofproinflammatory signals and possibly subsets of special-ized DCs can both support the differentiation of regula-tory T cells (96, 97). Regulatory T cells are usually anergicwith a low spontaneous proliferation rate, and are highlydependent on exogenous IL-2. They can downregulateboth Th1 and Th2 immune responses against viruses,bacteria, parasites and allergens (89–92), either by directcell–cell contact (involving PD1, membrane-bound

Immune mechanisms of SLIT

155

TGF-b or CTLA4 molecules) or through the productionof immunosuppressive cytokines such as TGF-b (Th3cells), or IL-10 (Tr1 cells) (Table 2) (90–92, 98, 99).Naturally occurring CD4+CD25+ regulatory T cells

represent 5–10% of peripheral CD4+ T cells in healthymice and humans. They are mainly thymus-derived, butcan also be induced in the periphery following sustainedantigen or TGF-b stimulation of CD4+CD25)-naive Tcells (99). These cells express the forkhead/winged helixtranscription factor Fox p3, which appears to play a keyrole in supporting their regulatory function (100).Type 1 regulatory (Tr1) T cells have a low-proliferative

capacity, produce high levels of IL-10 and TGF-b andlow levels of IL-2 and IL-4. Tr1 cells can be generated invitro by stimulating naive CD4+ T cells in the presence ofIL-10, with/without IFN-a (101), vitamin D3 plus dex-amethasone (102) or a combination of anti-CD46 plusanti-CD3 antibodies (103). CD4+CD25+ regulatory Tcells expressing the a4b7 integrin can also convert naiveCD4+ T cells into Tr1 cells (104).T helper type 3 (Th3) cells which produce TGF-b, IL-4

and IL-10 are induced following oral administration ofthe antigen (105). In vitro, CD4+CD25+ regulatory Tcells expressing the a4b1 integrin have been shown toconvert naive CD4+ T cells into Th3 cells (104).As of today, there is a growing evidence supporting the

role of regulatory T cells in controlling the developmentof asthma and allergic disease in a variety of models

(Fig. 2), although it is not clear yet which of the variousregulatory T cell subsets are the most important in thisregard (85, 92, 94). A revised version of the hygienehypothesis proposes that a limited exposure to infectiouspathogens during infancy, most particularly telluricmycobacteria and parasites, may prevent the establish-ment of not only a Th1, but also a T reg repertoire,thereby explaining in part the observed increase inprevalence of allergies in developed countries (106).Several studies document an association between atopyand a deficit in T reg functions. For example, childrenborn with a dysfunctional Fox p3 gene exhibit a deficit inCD4+CD25+ regulatory T cells and develop severeautoimmune diseases often associated with eczema,elevated IgE levels, eosinophilia and food allergy [thepolyendocrinopathy, enteropathy, and X-linked inherit-ance (IPEX) syndrome] (107). Moreover, for at leastsome atopic subjects with active disease, the suppressiveactivity of CD4+CD25+ regulatory T cells is significantlydecreased in vitro when compared with nonatopic indi-viduals, potentially explaining the loss of toleranceagainst allergens (108). In patients allergic to birchpollen, a functional deficit has also been observed inCD4+CD25+ regulatory T cells, which appears to peakduring the pollen season, and impacts the capacity ofsuch cells to inhibit Th2, but not Th1 responses (109).Moreover, DCs from children with allergic rhinitis can beimpaired in their capacity to produce IL-10 (110).

Table 2. Characteristics of naturally occurring and induced CD4+ and CD8+ regulatory T cells

Naturally occurring TR cellsCD4+CD25+

Peripherally induced TR cellsTr1 and CD4+CD25+/)

Peripherally induced TR cellsCD4+ Th3 CD8+ T Reg

Surface markersCD25 +++ ) to ++ ++ +GITR ++ ) ?ICOS ++ ++ ?CTLA-4 +++ + ++ )Other markers CD103, LAG3, CD122, neurophilin1 CD122, T1-ST2Foxp3 ++ ) (+/) after activation) ?CD45 RB low + + + +CD45 RO + + +

Cytokine secretedIL-10 +/) ++++ +/) ++TGF-b +/) ++ ++++ +/)IL-4 ) +/) +/)IL-5 ) ++ ?IL-2 ) +/) )IFN-c +/) ++ +/)

Cell contact (CTLA-4, PD1)IL-10, TGF-b (in vitro)

IL-10, TGF-bCell contact (CTLA-4, PD1) TGF-b IL-10, TGF-b

Suppressive mechanism(s)Differentiation/inductionfactor(s)

Immature or tolerogenicDCs

TGF-b

Immature or tolerogenicDCs

IL-10, IFN-aIL-4, anti-CD3/antiCD46, VitD3/dexamethasone, a4b7+TR

Immature or tolerogenicDCs

TGF-b, IL-4, IL-10, a4b1+TR

Immature or tolerogenicDCs?

Induction by allergen-specificimmunotherapy

++ (SCIT) +++ (SCIT, possibly SLIT) +++ (oral IT), likely SLIT -swallow Unknown

Moingeon et al.

156

Interestingly, allergen-specific IL-10-secreting Tr1 cellsare highly represented in healthy individuals in compar-ison with allergen-specific IL-4-secreting Th2 cells, sug-gesting that regulatory T cells are predominant duringnatural immune responses to environmental allergens innonatopic donors (111). Natural Tr1 responses are alsoinvolved in establishing phospholipase A2 tolerance inbeekeepers exposed to multiple stings (112, 113). Incontrast, this pattern of T-cell responses is reversed inallergic individuals with a heavy skewing towards Th2relative to T reg responses (111, 114). A functional role ofregulatory T cells has been demonstrated in vivo in mice,on the basis of adoptive transfer experiments showingthat allergen-specific CD4+CD25+ cells or Tr1 clonesinhibit allergen-induced Th2 responses and IgE produc-tion, as well as airway eosinophilia (90–92).Regulatory T lymphocytes can control an established

allergic response via distinct mechanisms (Fig. 2): IL-10and TGF-b decrease IgE production and enhance IgG4and IgA production, respectively. Both cytokines lowerthe release of proinflammatory mediators by downregu-lating IgE-dependent activation of basophils and mast

cells and by decreasing survival and activation of eosin-ophils. IL-10 and TGF-b also inhibit the production ofTh2 cytokines such as IL-4 and IL-5 (50, 90, 113). Inaddition, regulatory T cells exhibit a direct inhibitoryeffect on Th1 and Th2 T cells, through cell–cell contact,or by decreasing the antigen presenting function of DCs.

Recently, several successful immunotherapy studiesconducted through the subcutaneous route in patientswith either grass pollen, house dust mite or venom allergyhave shown an induction of various types of regulatory Tcells including CD4+CD25+ regulatory T cells (84, 90,115) or IL-10-producing CD4+CD25) Fox p3-Tr1 cells(82, 83). Importantly, the suppressive activity of regula-tory T cells induced in the course of immunotherapyappears to be allergen specific (83). Collectively, thesestudies suggest the importance of stimulating allergen-specific T regs during immunotherapy. They also provethat it is feasible to elicit regulatory T cells from a pool ofCD4+CD25)-naive T-cell progenitors in allergic patients,some of which may present a deficit in their T regfunction and repertoire. Early IL-10 production (withindays) and sustained TGF-b levels (over a year after

Figure 2. Regulatory T-cell dynamics in allergy. Regulatory T cells producing IL-10 and/or TGF-b are induced not only in atopicpatients by successful immunotherapy, but also during natural allergen exposure in healthy people. As per the hygiene hypothesis,limited exposure to bacteria and parasites in developed countries may result in a poor establishment of a T reg repertoire duringchildhood, thereby contributing to an increase in the frequency of allergies. Certain atopic patients have a deficit in their T regfunction, although several SCIT studies have shown that it is possible to induce allergen-specific regulatory T cells in atopic patients.Regulatory T cells can control and regulate all effector mechanisms activated during allergy and Th2 responses through the productionof IL-10/TGF-b and/or cell–cell contact. IL-10 is a potent suppressor of total and allergen-specific IgEs, whereas it induces anantibody isotype switch towards IgG4. TGF-b also decreases IgE production and induces immunoglobulin isotype switch towardsIgA. IL-10 and TGF-b act directly or indirectly on human airways to decrease both mucus production and airway hyper-reactivity.

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immunization) have been proposed as potential biologicalcorrelates for successful immunotherapy (90).

Oral mucosa and immune responses

SLIT and induction of peripheral tolerance

Sublingual immunotherapy takes advantage of an import-ant physiological mechanism (i.e. oral tolerance), whichhas been evolutionarily conserved to ensure immunetolerance to various antigenic stimuli from the environ-ment, especially from food and commensal bacteria (116).During SLIT, as for immunization at any mucosal surface,the allergen is captured locally (i.e. within the oral mucosa)by Langerhans-like DCs following either phagocytosis,macropinocytosis or receptor-mediated endocytosis. Sub-sequent to allergen capture, DCs mature and migrate toproximal draining lymph nodes (e.g. submaxillary, super-ficial cervical and internal jugular), as a consequence ofchanges in expression of surface receptors (e.g. the CCR7chemokine receptor) involved in adhesion and trafficking(Fig. 3) (117, 118). Those lymph nodes represent special-ized microenvironments favoring the induction of mucosaltolerance through the production of blocking IgG anti-bodies (IgG2b inmice) and the induction of T lymphocyteswith suppressive function (119, 120). Importantly, themagnitude of CD4+T-cell responses elicited within lymphnodes is directly proportional to the number of allergen-carrying DCs that migrate to lymph nodes, which clearlyrepresents a limiting step (121). Eventually, as a conse-quence of the circulation of allergen-specific activatedeffector T cells throughout the body and the persistence ofmemory cells, a local (i.e. sublingual) administration of theallergen during desensitization results in both systemic andmucosal protective immune responses.Dendritic cells in the sublingual mucosa exhibit mor-

phological characteristics of Langerhans cells, includingthe presence of intracytoplasmic Birbeck granules (122).Interestingly, Langerhans-like cells from the oral mucosaconstitutively express both low- (CD23) and high-(FCeRI) affinity receptors for IgEs, which may facilitateIgE-mediated allergen capture in atopic individuals (122).Perhaps, more importantly, upon engagement of such IgEreceptors, oral Langerhans-like cells produce IL-10, TGF-b and upregulate indoleamine 2-dioxygenase (IDO), arate-limiting enzyme-metabolizing tryptophan, therebyresulting in a decrease in T-cell proliferation (122, 123).As discussed above, there is still no formal evidence of Treg induction via the sublingual route. Nevertheless, on thebasis of its aforementioned characteristics, the immunesystem in the oral mucosa appears prone to induce activetolerance mechanisms against allergens and antigens fromthe environment. Consistent with this, there is preliminaryevidence that SLIT elicits IL-10-producing T cells inhumans (88) and antigen-specific TGF-b+ T cells inmurine (P. Moingeon, T. Batard, R. Fadel, F. Frati,J. Sieber, L. VanOvertvelt, unpublished results). The latter

observation is consistent with the fact that in a SLIT-swallow protocol, allergen exposure directly to the gutimmune system is expected to stimulate a Th3 response(105).

Pharmacokinetics and pharmacodynamics of the sublingual route

During SLIT, the vaccine is kept under the tongue for1–2 min and then swallowed (as per the sublingual/

Figure 3. Oral mucosa as an immunocompetent site. Macro-molecules such as allergens exhibit a limited direct absorptionthrough the oral mucosa. This mucosa is rich in Langerhans-likedendritic cells expressing constitutively high-affinity receptorsfor IgE (FCeRI). Following engagement of such receptors, DCsproduce IL-10 and TGF-b and upregulate IDO. These cells arespecialized in antigen or allergen capture and are instrumental instimulating T lymphocytes following migration to regionallymph nodes. Such lymph nodes, including submaxillary,superficial cervical and internal jugular lymph nodes represent afavorable environment for IgG responses (blocking antibodiesof the murine IgG2b isotype) as well as for the stimulation of Tcells with suppressive functions. For those reasons combined,immune responses elicited following oral immunization arebiased towards tolerance induction. The oral mucosa containsfew or no mast cells, eosinophils and basophils, and the integrityof the lamina propria is likely key in limiting contact of allergenswith such cells in submucosal tissues or in the blood, therebyexplaining the excellent safety profile of SLIT.

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swallow procedure). When the vaccine is immediatelyswallowed, clinical efficacy is substantially decreased(22, 124). On the basis of human studies demonstratingthat a limited therapeutic effect is observed when thevaccine is spat out, it has been proposed that sublingual-swallow immunotherapy is more efficient than sublin-gual-spit because it enhances the duration of contact ofthe allergen with the oral mucosa (124). Whereasimmunomodulation is initiated within minutes followingcontact with the oral mucosa, allergens absorbed in thegastrointestinal tract also probably contribute to theefficacy of SLIT.The tissue under the tongue is highly vascularized, with

blood vessels draining directly into the jugular vein. As aconsequence, small synthetic drugs administered sublin-gually are quickly absorbed and enter the bloodstreamwithout passing through the intestine and the liver (125,126). For example, the sublingual route is commonly usedin humans to administer the vasodilator nitroglycerin(glyceryl trinitrate), as a treatment for angina pectoris,leading to a plasmatic peak within 5 min with an overallbioavailability of approximately 70% (125). Similarly,sublingual administration of the opioid analgesic bupr-enorphine yields a bioavailability of up to 55% throughthe sublingual route, in comparison with 15% obtainedafter direct ingestion (126). In contrast with theseobservations made with small synthetic molecules, bio-distribution studies in humans demonstrated that there isno significant direct absorption of peptides or proteins –including allergens – through the sublingual mucosa(127–130). Following administration of the radiolabeledParietaria judaica pollen allergen (Par j 1), either as anorosoluble tablet or a solution kept in the mouth for1–1.5 or 20–30 min, respectively, no direct absorptioninto the blood was detected (129, 130). Only afterswallowing and contact with the gut mucosa, the radio-labeled Par j 1 allergen got distributed rapidly in theblood, with a plasmatic peak detectable within 2 h (129).Such biodistribution studies also suggested that signifi-cant amounts (e.g. 20% of the administered dose) of theallergen can persist for 2 h on the sublingual mucosalsurface, even though patients were allowed to rinse theirmouth extensively (129, 130). Consistent with this obser-vation, a limited proteolytic activity against allergens isfound in saliva, whereas in contrast exposure to gastro-intestinal fluids results in complete allergen degradation(131–133).As for SCIT, conventional SLIT protocols rely upon a

build-up phase (with a gradual increase in the dosingduring 4–6 weeks) and a maintenance phase (withadministration of the maximum dose one to three timesa week over several years). In the course of suchimmunization schemes, it has been proposed that bothdownregulation of proinflammory cells and upregulationof blocking IgG antibodies as well as IL-10 productionoccur quickly (i.e. within days) following immunotherapy(16). In contrast, a detectable impact on Th1 and Th2

responses and adaptive immunity is rather thought tooccur within months (16). Whereas immunological chan-ges can be detected shortly following immunotherapy, arecent study has suggested that SLIT is efficacious onsymptoms and medication after at least 1 year of therapy,with maximum benefit being observed only after 2 years(134). It should be emphasized, however, that several rushand ultrarush protocols for SLIT have demonstrated thatit can induce a decrease in skin reactivity and a readilydetectable clinical benefit within weeks or even days (16,66, 135). Whereas little is known on the duration ofSLIT-induced immunomodulation, several independentstudies have suggested that SLIT in children withrhinoconjunctivitis to either grass pollen or HDM,prevents asthma as well as new sensitizations (20, 136,137). Given that in some of these studies, a follow-up ofup to 10 years after the initiation of SLIT was performed,it is likely that immune memory mechanisms are estab-lished following SLIT.

Why high doses for SLIT?

It is well established that SLIT requires more allergen(at least 50–100 times) than SCIT to reach the samelevel of efficacy (15). One explanation for this is thatcurrent immunotherapy protocols rely upon an adjuvant(e.g. calcium phosphate or aluminum hydroxide) forsubcutaneous but not sublingual administration.Although it has been suggested that reaching a thresholdcumulative dose of allergen is important for a successfulSLIT, it is also possible that current vaccines do nottarget immune cells efficiently, most particularly DCs.Thus, high-dose regimens likely facilitate capture ofsufficient amounts of allergens by sentinel DCs withinthe oral mucosa, which clearly represents a critical stepto induce a strong and long-lasting T-cell response (121).Interestingly, numerous studies suggest that low or highdoses of antigens exhibit a radically distinct effect on Tlymphocyte stimulation, with low doses inducing Th2responses, whereas higher doses of antigen ratherstimulating Th1 CD4+ T cells (138, 139). Moreover,exposure to high doses of allergen during immunothera-py may promote the trafficking of Th1 over Th2 cells bymodulating surface expression of adhesion moleculesand chemokine receptors selectively on those cells (140).The influence of antigen dosage on regulatory T cells ispresently unclear: initial studies suggested that smalldoses of antigen administered orally were efficient ateliciting IL-10+ TGF-b+ Th3 cells in the gut (105).However, regulatory T cells are poorly reactive withantigen in vitro, raising the hypothesis that a strongantigenic stimulation may be necessary to induce pro-liferation and activation of such T cells. Importantly,high doses of antigen will provide a strong initial burstof T lymphocyte stimulation, leading to the establish-ment of T-cell-mediated immune memory and long-termtolerance (141).

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SLIT safety profile

On the basis of extensive clinical experience, it is nowfirmlyestablished that SLIT is very well tolerated both in adultsand young children (22, 26, 27). Side effects are usuallylocal, encompassing oral itching, swelling, and irritation,and occur usually within minutes, thus, likely implying anIgE-mediated reaction. As inflammatory markers such astryptase orECPare not increased locally during SLIT, IgE-mediated reactions occurring in the mouth followingsublingual exposure to the vaccine are usually ratherlimited (62). Allergen-challenge experiments performed inhumans with recombinant allergens (e.g. rPhl p 1, rBet v 1)have confirmed that the sublingual mucosa is at least 10times less reactive than the nasal mucosa or the skin (142).This is likely explained by the fact that only limitednumbers of proinflammatory cells (such as mast cells) arepresent in the oral mucosa. Thus, in contrast to SCIT,allergens exposed to the oral mucosa are not in directcontact with basophils from the blood or mast cells intissues.While SLIT efficacy clearly correlates with allergen

dosage, high-dose regimens do not appear to enhancedramatically the frequency of systemic or local adverseevents, in contrast to SCIT (28). Importantly, high dosesof allergens have been shown in vitro to be less efficientthan low doses in inducing the release of proinflamma-tory mediators by mast cells (143). In a phase Idose-finding study with sublingual grass pollen tablets,high-dose regimens were very well tolerated provided thata short-dose escalation was conducted prior to reachingthe maintenance dose (144).

Implications for the development of second-generationsublingual vaccines

Based on our current understanding of immune mecha-nisms underlying allergen-specific immune responses,optimizing vaccines to control the type of T lymphocytes,and thus the pattern of cytokines induced during naturalexposure to the allergen or vaccination, is critical. Second-generation vaccines could possibly be more efficacious ifspecifically designed to elicit a strong allergen-specificregulatory T-cell response without exacerbation of the Th2allergic response, thereby limiting adverse reactions. Forthis, the sublingual route appearsmostly appropriate giventhat FCeRI+ IL-10/TGF-b-producing sentinel DCs pre-sent in the oral mucosa are likely to be prone to inducetolerance, as opposed to immunostimulation.While the first generation of sublingual vaccines

currently used is based on natural biological extracts,new vaccines are being developed which rely uponselected recombinant allergens (145, 146). There is, as oftoday, considerable interest in developing hypoallergenicversions of target allergens in which IgE-binding epitopes

have been knocked down by side-directed mutagenesis ordisruption of the three-dimensional structure of themolecule (145, 146). Such vaccines exhibit a reducedallergenic activity while retaining a capacity to elicitblocking IgG antibodies and T-cell recognition. Whilethis approach looks promising for vaccines administeredparenterally, a more appropriate strategy for SLIT israther to rely upon recombinant allergens presented in themost native conformation (147), to allow IgE-mediatedtargeting and capture of allergens by FCeRI+ DCs in theoral mucosa of atopic patients.

Strategies based on biological or synthetic adjuvantsand formulations to improve allergen presentation to oralLangerhans cells should be investigated to enhance SLITefficacy, reduce allergen dosing, and simplify immuniza-tion schemes. T reg adjuvants specially designed for thesublingual route may (i) target the allergen toLangerhans-like cells; (ii) trigger the production of IL-10 and/or TGF-b by antigen-presenting cells whileenhancing their capacity to migrate to local lymph nodesto ensure T cell priming; and (iii) facilitate the polariza-tion of T-cell responses towards regulatory T cells byengaging surface receptors such as ICOS, CD46 orspecific Toll-like receptors (e.g. TLR4).

Among many advantages, such molecularly definedvaccines will facilitate the identification of immunologicalcorrelates of clinical efficacy. Such biological surrogatemarkers would be extremely useful to ease the develop-ment of future vaccines, most particularly with the long-term goal of making vaccines tailored to patient-specificallergen sensitization patterns determined on the basis ofcomponent-resolved diagnostic. Importantly, whereas inthe past the immunological follow-up of immunotherapytrials focused on humoral (IgE/IgG4) responses, toolsand methods should rather be developed to monitor indetail allergen-specific CD4+ T-cell responses [e.g. usinghuman leukocyte antigen (HLA) class II-peptide solubletetramers, quantitative Elispot measurement of Th1/Th2/T reg cytokine-producing T cells, real-time PCR analysisof early events in T-cell polarization, etc.].

Altogether a better understanding of immune mecha-nisms involved in allergen-specific immunotherapy, com-bined with the power of molecular engineering andinnovative antigen delivery systems should offer theopportunity to rationally design second-generationrecombinant vaccines specifically tailored for the sublin-gual route. The development of such second-generationvaccines should help to confirm (or not) current workinghypotheses, to improve vaccine efficacy, and to evaluatesimpler administration schemes.

Acknowledgments

The authors wish to thank Danielle Michel for excellent secretarialassistance and Patrick Buchoux for help in preparing the graphics.

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