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Control of functional T helper cell polarization by dendritic cells

Limao Vieira, P.

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Citation for published version (APA):Limao Vieira, P. (2002). Control of functional T helper cell polarization by dendritic cells.

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Download date: 10 Mar 2020

CHAPTERR 8

Glucocorticoid ss Transientl y Inhibi t thee Maturatio n of Dendriti c Cells butt Permanentel y Downregulat e

thee IL-12 Productio n

Estherr C. de Jong, Pedro L. Vieira, Joost H.N. Schuitemaker, Eddy A. Wierenga, PaweJJ Kalihski, Martien L. Kapsenberg

Glucocorticoidss (GC) are potent immunosuppressive agents that are widely used to treatt inflammatory diseases. Amongst their various effects, GC affect the function of dendriticc cells (DC). GC are applied topically on tissues where DC reside in an immaturee form (iDC), capable to take up antigen. Upon activation, iDC migrate to the drainingg lymph nodes while undergoing maturation into mature effector CD83+ DC (mDC),, which are able to efficiently activate naive Th cells but no longer sample antigen.. Recently, it has been demonstrated that GC inhibit cytokine production and the maturationn of DC. However, it is unclear whether these effects are permanent or transientt and can be overcome by subsequent reactivation. To this aim, monocyte-derivedd immature DC were induced to mature by LPS in the presence or absence of GC.. After removal of the GC by extensive washing, the cells were restimulated with the proinflammatoryy cytokines IL-1p7TNF-a and their maturation status and cytokine productionn upon CD40 ligation was analyzed. Our data show that inhibition of maturationn by GC could be overcome by a secondary maturation stimulus. However, thee downregulation of the production of bioactive IL-12p70 could not be reversed. This studyy may indicate that topical application of GC on tissues containing iDC may allow forr the migration of anti-inflammatory lL-12-deficient effector DC.

SubmittedSubmitted for publication

117 7

Chapterr 8

INTRODUCTION N

Glucocorticoidss (GC) are potent anti-inflammatory and immunosuppressive agents that

aree widely used in the treatment of inflammatory disorders, such as autoimmune and

allergicc diseases (1). The therapeutic effects of GC have been generally ascribed to an

inhibitoryy effect on Th cell proliferation due to the down regulation of IL-2 production and

signall transduction via the IL-2 receptor (2, 3). Recently, It has been demonstrated that

GCC also affect T cell activation via interference with early phosphorylation events

inducedd after TCR triggering (4). Moreover, GC inhibit the secretion of both type 1 Th

cellss cytokine IFN-7 as well as the type 2 cytokines, IL-4 and IL-5 (1, 5, 6). Previously,

alsoo the downregulatory effect of GC on cytokine production by monocytes,

macrophagess and dendritic cells (DC) was demonstrated (7-9).

DCC are regarded as professional antigen presenting cells as they have the unique

capacityy to stimulate naive T cells (10). DC, in their immature form (iDC), reside in

epitheliall and connective tissues, especially in the skin, lung and gut, and are very

efficientt in taking up antigen. Upon activation, iDC undergo maturation and most of

themm will migrate to the draining lymph nodes (11 -13). This maturation can be triggered

byy various signals including inflammatory cytokines, e.g. IL-1p/TNF-a, exposure to

microbiall products, such as LPS, DNA or dsRNA, and contact with T cells through

CD400 ligation. Maturing DC rapidly lose their antigen uptake capacity and, at the same

time,, gain expression of the costimulatory molecules CD80 and CD86. Consequently,

theyy not only acquire the capacity to stimulate naive T cells antigen-specifically (signal

1),, but also to efficiently costimulate them {signal 2) (13-15). Doing so, these

immunogenicc mature DC (mDC) may polarize (signal 3) Th celts into Th1 or Th2 celts

(16).. Depending on the conditions during their initial activation as sentinel DC, DC

mayy develop into Th1-promoting DC1 orTh2-promoting, IL-12 deficient DC2 (16,17).

Inn this respect, the production of bioactive IL-12p70 is an important factor as it is very

potentt in inducing Th1 responses (18).

Whenn GC are used as topical drugs for the treatment of inflammatory diseases, they

normallyy are applied on tissues containing iDC as well as mDC as a result from the

inflammation.. At these sites, GC will exert direct effects on the iDC cytokine production,

especiallyy IL-12p70, as we and others described before (7, 8,19). On the other hand, it

hass been demonstrated that GC inhibit the maturation of iDC into mDC, which may

inhibitt the recruitment of naive Th cells (20-22). However, it is unknown whether this

118 8

GCC inhibit DC maturation transiently but IL-12 production permanently

inhibitionn of maturation and subsequent initiation of the immune response is permanent

orr that, after withdrawal of the GC, the remaining DC regain the capacity to develop into

genuinee mature effector DC that will migrate to the draining lymph nodes and prime

naivee Th cells. Therefore, we investigated to what extent the inhibition of maturation and

IL-122 production by GC is transient or can be overcome by the reactivation of the DC.

Too this aim, human monocyte-derived iDC were matured in the absence or presence of

clobetasol-17-propionatee (CP). After removal of the GC by extensive washing, the DC

receivedd a second boost of maturation factors and after 24 h were analyzed for their

expressionn of cell surface molecules, and their capacity to produce bioactive IL-12p70.

Thee present data show that GC inhibit the maturation of DC but this can be reversed

afterr a secondary maturation signal. Nevertheless, the obtained GC-treated mature,

effectorr DC do not produce bioactive IL-12p70 and are therefore, DC2-type cells that

inducee the development of Th2 cells from naive precursors.

MATERIALSS AND METHODS

Cytokines ,, antigen s and reagent s

Humann rGM-CSF (sp. act. 1.11x107 U/mg) was a gift from Schering-Plough, Uden, The

Netherlands.. Human r!FN-y(sp. act. 8x107 U/mg) was a gift from Dr P.H. van der Meide

(Utrechtt University, Utrecht, The Netherlands). Human rlL-4 (sp. act. 1x108 U/mg) and

humann rTNFa (sp. act 108 U/mg) were obtained from PBH (Hannover, Germany).

Humann r1L-1(5 (sp. act 5x107 U/mg) was obtained from Boehringer Mannheim

(Mannheim,, Germany). LPS (Difco, Detroit, Ml) was used at a final concentration of 100

ng/ml.. The GC clobetasol-17-propionate (CP; Sigma) and hydrocortisone (HC; Sigma)

weree kept as stock solutions of 10"2 M in 95% ethanol, stored at , and diluted into

completee culture medium just prior to use. In all experiments, a 1:104 final dilution of

95%% ethanol was included as a vehicle control.

Inn vitr o generatio n of iDC fro m periphera l bloo d monocyte s

Venouss blood from healthy donors was collected by venipuncture in sodium-heparin

containingg tubes (VT100H; Venoject, Terumo Europe, Leuven, Belgium). PBMC were

isolatedd by density centrifugation on Lymphoprep (Nycomed, Torshov, Norway).

Subsequently,, PBMC were centrifuged on a Percoll (Pharmacia, Uppsala, Sweden)

gradient,, consisting of three density layers (1.076, 1.059 and 1.045 g/ml). The light

119 9

Chapterr 8

densityy fraction floating on the middle layer, containing predominantly monocytes, was

collected,, washed and seeded in 24-well culture plates (Costar, Cambridge, MA) at a

densityy of 5x105 celts/well. After 60 min of incubation at , non-adherent cells were

removedd and adherent cells were cultured in Iscove's modified Dulbecco's medium

(IMDM;; Life Technologies Ltd., Paisley, UK) containingg gentamycin {86 jig/ml; Duchefa,

Haarlem,, The Netherlands) and 1% FCS (Hyclone, Logan, UT), supplemented with

GM-CSFF (500 U/ml) and IL-4 (250 U/ml) to obtain iDC as described elsewhere (35). At

dayy 3, the culture medium including the supplements, was refreshed. At day 6,

CD1a+CD14"" iDC were ready for use.

Inductio nn of maturatio n of iDC in the absenc e or presenc e of GC

Att day 6 the maturation of iDC was induced by the addition of either rtL-1[3/rTNF-a, or

byy LPS. After 48 h, fully mature CD1a+ CD83+ mDC were obtained. To study the effect

off GC on the maturation, CP was added together with the maturation factors in a

concentrationn range from 10"6 to 10"10 M, whereas HC was used at concentrations of

lO^too 10'8 M. To study the reversibility of the inhibition of the maturation of DC by GC,

iDCC were matured with LPS in the absence or presence of GC. After 48 h cells were

thoroughlyy washed to remove LPS and GC and cultured in IMDM/1% FCS containing

IL-44 and GM-CSF alone for 24 h. Subsequently, the maturation was induced by the

additionn for 24 h of IL-1 p7TNF-a after which, at day 10, the expression of CD83 and the

productionn of IL-12p70 was determined.

Analysi ss of expressio n of cel l surfac e molecule s by flow cytometr y

Mousee anti-human mAbs against the following molecules were used: CD1a (OKT6;

Orthoo Diagnostic System, Beerse, Belgium), CD80 and CD86 (B7-24 and 1G10; kindly

providedd by Innogenetics N.V., Ghent, Belgium), MR (15-2) a gift from Dr F. Noorman,

Gaubiuss Laboratory, TNO Prevention and Health, Leiden, The Netherlands), CD83

(Immunotech,, Marseille, France). Bound mAb were detected by FITC-conjugated goat

anti-mousee mAb (Jackson Immunoresearch Laboratories Inc., West Grove, PA).

IL-12p700 productio n by DC

CD1a++ DC (4x104 ceils/well) were stimulated with mouse CD40L-expressing mouse

plasmacytomaa cells (J558-CD40L; a kind gift from Dr P. Lane, University of

120 0

GCC inhibit DC maturation transiently but IL-12 production permanently

Birmingham,, Birmingham, UK), 4x104 cells/well in the presence of rhulFN-? (1000

U/ml),, in 96-well flat-bottom culture plates (Costar) in IMDM containing 10% FCS in a

finalfinal volume of 200 uJ. Supernatants were harvested after 24 h and stored at C until

thee levels of IL-12p70 were measured by ELISA.

Cytokin ee measurement s

Measurementss of IL-12p70 levels in the culture supernatants were performed by

specificc solid-phase sandwich ELISA as described previously. The limit of detection of

thesee ELISA is 3 pg/ml.

Statistica ll analysi s

Dataa were analyzed for statistical significance (GraphPad, InStat TM, Version 2.02)

usingg ANOVA. Individual comparisons were performed using the Student's Mest A P

valuee <0.05 was taken as the level of significance.

RESULTS S

GCC inhibi t the maturatio n and capacit y to produc e IL-12p70 of huma n

monocyte-derive dd iDC

Too study the effects of CP and hydrocortisone (HC) on the maturation and capacity to

producee IL-12p70 of sentinel iDC, monocyte-derived iDC were cultured for 48 h with the

proinflammatoryy cytokines lL-1|5/TNF-a in the absence or presence of several

concentrationss of CP or HC. In the absence of GC the expression of mDC marker,

CD83,, as well as the costimulatory molecules CD80 and CD86 were upregulated,

whereass the expression of the mannose receptor (MR) was lost. The addition of CP or,

too a lesser extent, HC inhibited the full maturation of iDC in part of the cells, as shown

byy the impaired upregulation of the expression of CD83, CD80 and CD86, as well as

thee inhibition of the down regulation of the MR (Fig. 1A). The inhibition of the maturation

byy GC is clearly dose-dependent, CP being active at much lower concentrations as

comparedd to HC (Fig. 1B).

Previously,, we have reported that GC strongly inhibit the production of bioactive

IL-12p700 by iDC both in a direct fashion and after pre-incubation (19). Here, we studied

thee effect of the presence of GC during the maturation of DC on the ability to produce

!L-12p700 upon subsequent restimulation with CD40L as a mimic for the activation in the

121 1

Chapterr 8

lymphh nodes by naive Th cells. In control conditions, mDC stimulated with CD40L

producee considerably less IL-12p70 as compared to iDC stimulated similarly with

CD40L.. The presence of CP during maturation, however, further inhibited the IL-12p70

productionn up to approximately 70% at 10"8 M (Fig. 1C), whereas the presence of HC

affectedd the IL-12p70 only slightly at the highest concentration tested (10"6 M). These

effectss are probably the net result of two opposite actions of GC. On the one hand GC

stronglyy inhibit the IL-12p70 production during pretreatment (19). On the other hand GC

partiallyy inhibit the maturation of DC, which will result in a less pronounced decrease of

thee IL-12 p70 levels in the mDC, as iDC produce considerably more IL-12p70.

B B CD833 CD80 CD86 MR

LL k L LL

kk k k k kk k k L

125 5

iDC C

IL-ip/TNF-a a

IL-ip/TNF-a a ++ CP

IL-1p/TNF-a a ++ HC

Q Q O O 'o o c c 0 0 '55 5

X X 0) )

5? ?

755 "

50 0

25 5 CP P

HC C

10-99 10"8 CP

10-77 10"6 HC

GCC concentratio n (M)

iDC C

mDC C

CPIO- 'M M

CP10-10M M

HCIO^M M

HCIO^M M

CC 3

<uu E

00 400 800 1200 1600 2000

IL-12p70(pg/ml ) )

Figur ee 1. GC inhibi t the maturatio n and the productio n of IL-12p70 of DC in a dose-dependen t fashion .. (A) Maturation of iDC was induced with ll_-1p* (10 ng/ml)fTNF-a (50 ng/ml) in the absence or presencee of CP (10'8 M) or HC (10'6 M). After 48 h, the cells were analyzed by FACS for the expression of CD83,, the costimulatory molecules CD80 and CD86, and the MR. (B) The presence of CP (10' -10" M) andd HC (lO^-IO-6 M) inhibited the maturation dose-dependently as determined by CD83 expression. (C) Afterr maturation in the absence or presence of GC, DC were washed and stimulated with CD40L-transfectedd J558 cell line in the presence of IFN-y (1000 U/ml). After 24 h, supernatants were harvested andd the concentration of IL-12p70 in the supernatant was determined by ELISA. Data were analyzed for statisticall significance by Student's Mest. *F<0.05, "P<0.01. Data are from one representative experimentt of five.

122 2

GCC inhibit DC maturation transiently but IL-12 production permanently

Thee capacity to produce TNF-a and IL-6 upon restimulation with CD40L was also

reducedd in DC matured in the presence of GC as compared to control mDC, although

too a lesser extent, by approximately 50-40% (data not shown).

Thee inhibitio n of maturatio n by GC is transien t and can be reverte d by

reactivatio nn wit h maturatio n factor s (IL-1p7TNF-a)

DCC reside at sites that are constantly exposed to incoming pathogens. As it is known

thatt the presence of GC inhibit the maturation of sentinel iDC, we wondered whether

thiss inhibition, and therefore the initiation of the immune response, was permanent or

couldd be overcome by subsequent activation of the DC. To this aim, iDC were activated

withh LPS in the absence or presence of CP, and thoroughly washed after 48 h, rested

forr another 24 h and subsequently activated with IL-1p7TNF-a. As is clearly shown in

Fig.. 2 the CP-induced inhibition of maturation as measured by CD83 expression could

bee overcome by subsequent activation in the absence of CP. When LPS was used to

reactivatee the DC that were previously activated with IL-1 p7TNF-a in the presence of CP

noo restoration of the maturation was observed (data not shown).

immature e

b'' ' o ' ' 0 ' ' 0 ' -.o* CDB3 3

LPS S

Figur ee 2. Inhibitio n of maturatio n by GC is reversible .. Upon resuming the maturation, GC pretreatedd DC become mature effector DC. Maturationn was induced with LPS (100 ng/ml) in the absencee or presence of CP (10"8 M). After 48 h, the cellss were thoroughly washed to remove the GC. Afterr 24 h of resting in IMDM/1% FCS/GM-CSF, maturationn factors (IL-1p7TNF-a; 10 ng/ml and 50 ng/ml),, were added to resume the maturation. After anotherr 24 h cells were harvested, washed and analyzedd for their expression of CD83.

LPSS + CP

—— No reactivation

~~ Reactivatio n wit h IL-1 p/TNF-a

123 3

Chapte rr 8

Primary y stimulation n

none e none e none e

LPS S LPS S LPS S

LPS+CP P LPS+CP P LPS+CP P

// Secondary stimulation n

// none /MF F // MF+IFN-Y

// none /MF F / M F + I F N - Y Y

// none /MF F / M F + I F N - Y Y

10 101 102 103 104

IL-12p70(pg/ml) )

Figur ee 3. The inhibitio n of the productio n of IL-12p70 by GC is not restore d upo n resume d maturation .. Maturation was induced in the absence or presence of CP and resumed as described above inn the absence or presence of IFN-y (1000 U/ml). The cells were harvested, washed and stimulated with thee CD40L-transfected J558 cell line. After 24 h, supematants were harvested and the concentration of IL-12p700 in the supernatant was determined by ELISA. Data were analyzed for statistical significance by Student'ss Mest. *P<0.05, **P<0.01. Data represent one out of five experiments with similar results.

B B 150 0

oo 1 0 0 - 0

o o l --Q. .

CM M 500 -

150 0

100 0

50 0

T T —O O

1\\ ° _1_ _

vehiclee 1010 10 "9 10 "8 10

clobetasol-17-propionat ee (M)

vehiclee 108 10 "7 106

hydrocortison ee (M)

Figur ee 4. CD40L-induce d IL-12p70 productio n by matur e DC is less susceptibl e to modulatio n byy GC than that of immatur e DC. DC were matured with IL-1p7TNF-a (open symbols) or kept immaturee closed symbols). After washing both cell population were stimulated with the CD40L-transfectedd J558 cell line in the absence and presence of various concentrations of CP (A) or HC (B). Afterr 24 h supematants were harvested and the IL-12p70 production was measured by ELISA. Data weree analyzed for statistical significance by Student's Mest. *P<0.05, " P O . 0 1 . Data represent one out of fivee experiments with similar results.

124 4

GCC inhibit DC maturation transiently but IL-12 production permanently

Thee inhibitio n of the capacit y to produc e IL-12p70 is not restore d upon

resume dd maturatio n

IL-12p700 is a major inducer of Th1 responses and we and others have shown that

GCC are potent suppressors of the capacity to produce IL-12, both in iDC (19) as well

ass during maturation (Fig 1C). Therefore, we questioned whether the capacity to

producee bioactive lL-12p70 was also restored by reactivation of GC-arrested DC.

Thee presence of CP strongly downregulated the production of IL-12p70 upon CD40

ligationn (Fig 3.), and this capacity was not restored upon resumed maturation. GC-

treatedd DC produced minimal amounts (around the detection level for the ELISA) of

IL-12p70.. Even the presence of IFN-7, a potent enhancer of IL-12p70 production,

couldd not further upregulate these levels of IL-12p70 (Fig 3).

Matur ee DC have a reduce d responsivenes s to GC

Whenn an epithelial tissue is inflamed, as is the case in for instance atopic dermatitis or

psoriasis,, this epithelium will not only contain immature DC but also mature effector DC.

Thereforee we studied the effect of GC on mature DC. As is reported before, immature

DCC are very susceptible to the inhibitory effects of GC on the IL-12 production upon

CD400 ligation (Fig 4). In the presence of CP a dose-dependent inhibition of maximally

90%% was observed, whereas in the presence of HC approximately 60% inhibition was

observed.. However, the ability of DC to produce IL-12 was only mildly affected by GC

afterr the maturation of DC induced by both LPS and IL-1(3n~NF-a. Although the potent

GCC CP dose-dependently downregulated the IL-12 production to maximally 35% (65%

inhibition)) of the amount observed in control-treated DC, only a minor reduction in

lL-12p700 production of approximately 20% was observed in the presence of HC.

DISCUSSION N

Wee and others have previously demonstrated that GC affect DC in various ways in both

mann and mouse. Not only sentinel DC that reside in peripheral tissues as antigen-

trappingg DC are inhibited in their cytokine production but also their maturation into T cell

stimulatoryy effector DC is inhibited. The present study, however, shows that the GC-

inducedd inhibition of maturation into effector DC can be overcome with a second

activationn after withdrawal of the GC. This indicates that the effect of GC on DC-

maturationn is transient and GC-treated DC can become effector DC. However, once the

125 5

Chapterr 8

capacityy to produce IL-12 is downregulated by GC, this cannot be restored. Even the

presencee of the potent IL-12 enhancer IFN-7 at the time of stimulation with CD40L (to

mimicc the interaction with T cells) did not enhance the IL-12 production by GC-treated

effectorr DC.

Thee total loss of the capacity to produce bioactive IL-12p70 suggest that these

effectorr DC have become DC2 that promote the development of Th2 cells from naive

precursors.. This is in line with previous observations that memory CD4+ Th cells

stimulatedd with GC-treated human monocytes (7) or murine macrophages (8), show a

decreasedd production of IFN-7, due to a decreased production of IL-12 by these antigen

presentingg cells.

Basedd on in vitro experiments we have recently proposed the concept that the type

off activation and the type of tissue in which the immature, sentinel DC resides,

determiness the development in Th1-promoting type 1 DC (DC1) or a Th2-promoting

DC22 (16,17). In this respect, PGE2 not only enhances the maturation of DC (23,24) but

itt also induces the development of highly immunogenic mDC that produce very tow

levelss of IL-12 and promote the development of Th2 cells from naive Th ceils (DC2)

(24).. On the other hand, IFN-7 promotes the development of highly stimulatory DC that

producee high levels of IL-12 and promote the development of Th1 cells (25). These

subtypess of myeloid mDC are fully committed, since their IL-12 production profiles are

stablee and resistant to further modulation (24, 25). Moreover, also different types of

pathogenss induce the development of polarized DC1 or DC2 effector cells from

sentinel,, immature DC. Pathogens associated with type 1 responses in vivo such as

intracellularr bacteria or viruses induce the development of DC1 whereas toxin from

extracellularr bacteria and helminth extracts induce the development of DC2 in vitro (17).

Thee present study demonstrates that also pharmacological compounds may

contributee to the development of effector DC1 or DC2 cells. Several studies have

indicatedd potent effects of pharmacological compounds on DC function, not only GC

(19-22,26)) but also vitamin D (27), p2-agonists (28) and many more. To our knowledge

itt was, however, never determined what happens after withdrawal of the compound and

whetherr all functions of the DC are permanently inhibited. In case of GC, part of the

inhibitoryy action could be reversed.

Thee observation that only the inhibition of maturation can be restored but not the

capacityy to produce IL-12 could be explained by differences in signal transduction

126 6

GCC inhibit DC maturation transiently but IL-12 production permanently

routes.. It has been demonstrated by Rescignio et al. (29) that survival and maturation of

DCC are regulated by different pathways, e.g. ERK regulates survival whereas NF-KB is

responsiblee for maturation. NF-KB is a family of transcription factors consisting of 5

members,, RelA (or p65), RelB, cRel, p50 and p52. The IL-12 promoter region contains

aa NF-KB half site (30). It is tempting to speculate that GC transiently inhibit NF-KB and

thereforee inhibit the maturation whereas a different pathway involved in regulation of

cytokinee production is irreversibly shot down. The latter could also be due to a effect on

AP-1,, which has been demonstrated to be affected by GC in T cells, and has recently

beenn demonstrated to be present and active in the IL-12p40 promoter (31).

Previously,, we and others suggested that due to the inhibited induction of

maturationn and upregulation of costimulatory molecules GC-treated DC may induce

IL-10-secretingg T cells and therefore be tolerogenic DC3. In addition, it has been

demonstratedd that repetitive activation of Th cells with immature DC induces high

levelss of IL-10 upon restimulation. As GC inhibit the maturation only transiently, GC-

treatedd DC do induce proper Th cells responses without the induction of IL-10 (data

nott shown) and cannot, therefore, be designated "tolerogenic".

GC-treatedd effector DC are anti-inflammatory, due to their reduced production of IL-

12,, TNF-a and IL-6. This suggests that the anti-inflammatory effects of GC are not only

duee to a direct inhibition of T cell functions via inhibition of the expression of IL-2 and

thee IL-2R, but also via inhibition of the DC function. The induced Th2 profile may be

responsiblee for the enhanced levels of IgE antibodies as has been reported in both

allergicc and non-allergic individuals using GC in long-term therapeutic strategies

(32-34).. Moreover, the GC-treated DC may hamper the initiation of the appropriate

immunee response as observed in patients using GC for a longer period of time. These

patientss have frequent bacterial infections and difficulty in clearing these infections.

Although,, this may originate in part from a direct downregutatory effect of GC on T cells,

thee present study suggests that this may also follow from the effects of GC on DC,

alteringg their role in the mounting the proper immune response e.g. a Th1 response.

Acknowledgment s s Thiss work was financially supported by the Fundacao para a Ciência e a Tecnologia, Lisbon,, Portugal, grant PRAXIS XXI/BD/9195/96 (to P.L.V.).

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