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The Journal of Immunology, 2003, 171: 5071-5076.
Copyright © 2003 by The American Association of Immunologists

CD8+ T Lymphocytes Regulating Th2 Pathology Escape Neonatal Tolerization1

Brigitte Adams*, Nathalie Nagy{dagger}, Frédéric Paulart*, Marie-Line Vanderhaeghen*, Michel Goldman* and Véronique Flamand2,*

* Laboratory of Experimental Immunology, Université Libre de Bruxelles, Brussels, Belgium; and {dagger} Department of Pathology, Erasme Hospital, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4+ T lymphocytes. We investigated the role of host CD8+ T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J x BALB/c)F1 spleen cells when CD8+ T cells were depleted by administration of anti-CD8 mAb or when {beta}2-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8+ T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8+ T cells were as efficient as control CD8+ T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in {beta}2-microglobulin-deficient mice. Likewise, CD8+ T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4+ T cells in vitro. The regulatory activity of CD8+ T cells depended on their secretion of IFN-{gamma} for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8+ T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4+ T cells. We conclude that regulatory CD8+ T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pioneer experiments by Billingham and colleagues (1, 2, 3) established that transplantation tolerance across MHC class I and class II barriers can be induced by neonatal inoculation of semiallogeneic spleen cells. Although intrathymic clonal deletion of donor-specific T cells was considered for several years as the dominant mechanism for neonatal tolerance (4), subsequent studies established that CD4+ T cells recognizing donor MHC class II Ags actually differentiate into Th2 cells in tolerant mice (5, 6, 7). The consequence of this Th2 response is the development of an immunopathological syndrome that includes IgE overproduction and hypereosinophilia (8, 9, 10).

As far as donor-specific CD8+ T cells are concerned, the inability of neonatally tolerant mice to generate effector cytotoxic T cell activity was well established (3, 11, 12, 13, 14) but the possible persistence of other CD8+ T cell functions in tolerant mice was less explored. Indeed, a recent study by Coudert et al. (9) demonstrated that the pathology triggered by neonatal injection of BALB/c mice with (C57BL/6 x BALB/c)F1 spleen cells was dramatically enhanced when both the donor and the recipient were {beta}2-microglobulin ({beta}2m)3 deficient (9), suggesting that MHC class I-CD8+ T cell interactions controlled the magnitude of the Th2 response. Herein, we first observed that when (A/J x BALB/c)F1 spleen cells are inoculated into BALB/c newborns, CD8+ T cell deficiency of the recipient is sufficient to enhance Th2 pathology. This model was therefore chosen to investigate the influence of CD8+ T cells on a Th2-type pathology induced at birth. We demonstrated that regulatory CD8+ T cells controlling Th2 responses are already functional in neonates and escape tolerance induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

BALB/c (H-2d), A/J (H-2k), and C57BL/6 (H-2b) were purchased from Harlan CPB (Zeist, The Netherlands) and along with (A/J x BALB/c)F1 hybrids were housed and bred in the animal facility of the Erasme Hospital (Brussels, Belgium). BALB/c {beta}2-microglobulin-deficient mice ({beta}2m-/-) were kindly provided by Dr. J.-C. Guéry (Institut National de la Santé et de la Recherche Médicale, Toulouse, France). BALB/c IFN-{gamma}-deficient mice (IFN-{gamma}-/-) were generously obtained from Dr. P. Matthijs (Rega Institute, University of Leuven, Leuven, Belgium).

In vivo treatments

Neonatal tolerance was induced in BALB/c mice by injection into the retro-orbital vein of 107 (A/J x BALB/c)F1 hybrid spleen cells within the first 24 h of life.

For experiments requiring CD8+ T cell depletion in vivo, recipients were injected with ascites preparation of rat IgG2a mAb to mouse CD8 (clone H35-17.2). Ab concentration in ascites was determined by rat Ig isotype-specific ELISA (Lo-Imex; University of Louvain, Brussels, Belgium). Newborns received an i.p. injection of 50 µg of H35-17.2 mAb on day 1 or 2 of life (day +1 after the cell infusion), day 5, and day 12 and 500 µg every week thereafter. Depletion of CD8+ T cells was determined on lymph node (LN) cells by flow immunocytometry on day of sacrifice. Treated animals always contained <1.0% CD8+ T cells.

For neonatal CD8+ T cell transfer experiments, CD8+ T cells were positively selected from pooled LN using anti-CD8 mAb-coupled magnetic beads with the MACS system (Miltenyi Biotec, Paris, France) according to the manufacturer’s instructions. Purity >93% was routinely assessed by flow cytometry analysis. Briefly, 1 x 106 CD8+ T cells were injected i.v. along with the F1 spleen cells into {beta}2m-/- BALB/c newborns. B cell chimerism in LN was assessed by flow cytometry using double staining with FITC-conjugated anti-donor MHC class II mAb (anti-I-Ak) and biotinylated anti-CD45R/B220 mAb plus PE-conjugated avidin (BD PharMingen, San Diego, CA).

Mixed lymphocyte culture

MLC were prepared in complete RPMI 1640 culture medium (BioWhittaker, Petit-Rechain, Belgium) supplemented with 20 mM HEPES, 2 mM glutamine, 1 mM nonessential amino acids, 5% heat-inactivated FCS, and 10-5 M 2-ME.

Cells (2.5 x 106/well) from axillary, brachial, inguinal, and mesenteric LN from 4-wk-old BALB/c mice were stimulated with irradiated (2000 rad) syngeneic BALB/c, allogeneic A/J, or third-party C57BL/6 spleen cells (2.5 x 106/well) in 1 ml of culture medium in 48-well flat-bottom plates (Greiner Labotechnik, Frickhausen, Germany). For experiments testing the allorecognition, 20 µg/ml purified anti-H-2Kk mAb (AF3-12.1; BD PharMingen) was added to MLC at a ratio 1:1 of CD4+ and CD8+ T cells. Cultures were kept at 37°C in a 5% CO2 atmosphere and supernatants were harvested after 72 h and analyzed for the presence of cytokines (see below).

CD4+ or CD8+ T cell purifications were realized with LN cells from 4-wk-old wild-type or IFN-{gamma}-/- BALB/c mice that were first passed over a nylon wool column; the nonadherent fraction was further purified by complement-mediated lysis as previously described (15).

In experiments using neonatal-purified CD8+ T cells, total LN and spleens were collected from <=3-day-old mice and pooled. CD8+ T cells were purified with the anti-CD8 mAb-coupled magnetic beads MACS system (Miltenyi Biotec). Purity of isolated cell suspensions was assessed by immunocytofluorometry (>90%).

For IFN-{gamma} neutralization, IgG2a rat anti-mouse IFN-{gamma} mAb (clone 37895.11; R&D Systems, Oxon, U.K.) was added to the culture at 25 µg/ml. Purified mouse IgG (Sigma-Aldrich, St. Louis, MO) were used in control cultures.

CTL assay

Effector cells were generated in MLC. Five 106 LN cells/well were cultured with irradiated (2000 rad) allogeneic spleen cells (5 x 106/well) for 5 days in 24-well flat-bottom plates. Target cells were prepared by incubation of 1 x 106 spleen cells with 30 µg/ml LPS (serotype 0111:B4, Sigma-Aldrich) for CTL assay using {beta}2m-/- BALB/c effectors and with 4 µg/ml Con A (Sigma-Aldrich) for CTL assay using wild-type BALB/c effectors in 2 ml of complete medium for 3 days and pulsed overnight with 5 µCi [3H]thymidine (ICN Pharmaceuticals, Asse-Relegem, Belgium). Effector cells were harvested and plated at various E:T ratios in 96-well round-bottom plates containing 5 x 103 radiolabeled target cells for 4 h, and residual radioactivity was then measured through a Top Count scintillation counter. The percentage of specific lysis was calculated according to the formula: percent specific lysis = (spontaneous cpm count - total lysis cpm count)/(spontaneous cpm count - experimental cpm count) x 100.

Dosage of cytokines and IgE levels

The concentration of IFN-{gamma}, IL-4, IL-5, and IL-13 in supernatants was assessed by ELISA using an available ELISA kit (Duoset; R&D Systems) for IFN-{gamma}, IL-4, and IL-13 and Opt EIA set (BD PharMingen) for IL-5. Serum IL-5 and IL-13 levels were assessed using an Opt EIA set (BD PharMingen) for IL-5 and a Quantikine M immunoassay (R&D Systems) for IL-13. The lower limit of detection in the assays was 15 pg/ml.

IgE serum levels were determined by sandwich ELISA using purified rat IgG1{kappa} mAb directed against the {epsilon} H chain of mouse Ig (clone LO-ME-3, Lo-Imex; University of Louvain) as capture mAb and purified biotinylated rat IgG2a{kappa} anti-{epsilon} H chain of mouse Ig (clone LO-ME-2) plus peroxidase-coupled avidin as detection. Serum IgE concentration was quantified from four titration points using standard curves generated with purified mouse IgE{kappa} (clone C38-2; BD PharMingen).

Histology

Histology was performed on LN and spleen. Tissue sections were stained with H&E after fixation in 10% neutral Formalin solution and paraffin embedding. Tissue eosinophilia was quantified by counting the number of eosinophils present in at least nine distinct high-power fields (0.0025 mm2) across the organ. The results were confirmed with the staining of cyanide-resistant eosinophil peroxidase activity (16). Histological analysis was performed by a pathologist unaware of the experimental groups.

Statistics

Group comparisons were made using a two-tailed nonparametric Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD8+ T cell deficiency enhances Th2 pathology induced by neonatal injection of semiallogeneic cells

The pathology induced by neonatal inoculation of semiallogeneic cells includes lymphoid hyperplasia, serum hyper-IgE, and eosinophilia, the latter changes being directly related to overproduction of IL-4 and IL-5 (8, 9, 10, 17, 18). These parameters were monitored to determine the influence of CD8+ T cell deficiency on the severity of the syndrome induced in BALB/c newborns by injection of (A/J x BALB/c)F1 spleen cells. As shown in Table I and Fig. 1, spleen enlargement, serum hyper-IgE, and eosinophil numbers in LN were significantly increased in mice depleted of CD8+ T cells by injection of anti-CD8 mAb. Similar findings were made in BALB/c {beta}2m-/- mice in which CD8+ T cell differentiation is hampered by the lack of MHC class I expression in the thymus (Table I and Fig. 1). The enhanced pathology in CD8+ T cell-deficient mice was associated with increased serum levels of IL-5 and IL-13 compared with neonatally immunized wild-type mice (Fig. 2). MLR performed with LN cells from neonatally injected mice as responders and donor-type spleen cells as stimulators indicated an enhanced production of IL-5 and a decreased production of IFN-{gamma} by donor-specific T cells of CD8+ T cell-depleted or {beta}2m-/- mice as compared with wild-type mice (Table II). Taken together, these data suggest that the Th2 cell activities in neonatally injected mice were controlled by CD8+ T cells.


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  		Table I. CD8+ T cell deficiency exacerbates Th2 pathology after neonatal injection of semiallogeneic spleen cells

 


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  		FIGURE 1. Eosinophil infiltrates in spleens after neonatal injection of semiallogeneic spleen cells. Eosinophil quantification made using cyanide-resistant eosinophil peroxidase activity staining on spleen tissue sections (original magnification, x100). Uninjected 4-wk-old BALB/c showed intact white pulp and red pulp and no eosinophil infiltration (A). Injection of F1 spleen cells in BALB/c mice induced eosinophil infiltration of red pulp, moderate enlargement of the organ size, and structure alteration (B). A significant enhancement of the pathological features was observed in neonatally injected CD8+ T cell-deficient mice (D and F). We observed major and diffuse eosinophil infiltration and total disruption of organ architecture in mice depleted with anti-CD8 mAb (D) as well as, though to a lesser extent, in {beta}2m-/- mice (F). C and E, Spleen of control anti-CD8 mAb-treated BALB/c mice and uninjected {beta}2m-/- mice. An impressive regression of the splenic eosinophilic infiltration was observed when CD8+ T cells from control (G) or neonatally immunized (H) mice were coinjected with the F1 cells at birth in {beta}2m-/- mice.

 


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  		FIGURE 2. Serum levels of IL-5 and IL-13 in neonatally injected mice. Four-week-old mice were bled and serum IL-5 and IL-13 cytokines levels were detected by ELISA. Uninjected groups from both wild-type and {beta}2m-/- mice ({square}) are compared with neonatally F1 cell-injected groups ({blacksquare}). Effect of transfer of purified CD8+ T cells from control (ctl) or F1 cell-injected mice in {beta}2m-/- newborns along with the F1 spleen cells is further compared with the neonatally injected group. **, p < 0.002 as compared with the F1 cell-injected {beta}2m-/- group. *, p < 0.05 as compared with the wild-type-injected group. Results are representative of two separate experiments; each experimental group is composed of at least eight mice. SC, spleen cells.

 

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  		Table II. Cytokine production in MLR

 
CD8+ T cells from tolerant mice regulate CD4+ T cell-mediated cytotoxicity and the production of Th2 cytokines through IFN-{gamma}-dependent and -independent pathways

Before investigating the regulatory activity of CD8+ T cells of BALB/c mice neonatally inoculated with (A/J x BALB/c)F1 spleen cells, we first verified the tolerant status of these mice by assessing CTL activities of total LN T cells against donor-type (A/J) or third-party (C57BL/6) targets. In agreement with several previous studies (4, 12, 19, 20), neonatally injected mice were indeed unable to generate anti-donor CTL against donor-type cells while they developed normal levels of anti-third-party CTL activities (data not shown). We then cotransferred CD8+ T cells from control or neonatally tolerant wild-type BALB/c mice along with (A/J x BALB/c)F1 spleen cells in {beta}2m-/- newborn mice. The injection of CD8+ T cells from either control or tolerant mice dramatically reduced the overproduction of both IL-5 and IL-13 (Fig. 2) as well as the severity of Th2 pathology (Table I). Furthermore, donor-specific cytotoxicity, assessed on LPS blasts to facilitate the detection of CD4+ T cell-mediated cytotoxicity, was fully restored in {beta}2m-/- mice when CD8+ T cells from either control or tolerant mice were cotransferred with F1 spleen cells (Fig. 3). The percentage of donor B-220+/I-Ak+ B cells was assessed on 2-wk-old BALB/c {beta}2m-/- recipient LN after neonatal injection. It revealed that the coinjection of BALB/c CD8+ T cells with the (A/J x BALB/c)F1 spleen cells did not alter the microchimerism (0,87 ± 0.11% in F1 spleen cells injected {beta}2m-/- mice (n = 5) vs 0,79 ± 0.15% in coinjected BALB/c CD8+ T cells and F1 spleens cell {beta}2m-/- mice (n = 8)).



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  		FIGURE 3. Donor-specific cytotoxic activity in neonatally injected {beta}2m-/- mice. Anti-A/J or anti-C57BL/6 CTL activity was generated in a 5-day MLC using total LN cells from 4-wk-old uninjected ({circ}) or neonatally injected {beta}2m-/- mice with (A/J x BALB/c)F1 spleen cells alone ({triangledown}) or coadministered with CD8+ T cells isolated from either control ({square}) or tolerant ({diamond}) BALB/c mice. Results of at least five individual mice in each experimental group are expressed as percentages (mean ± SEM) of specific lysis at different E:T ratios. The results are representative of two independent experiments.

 
To further investigate the regulation of Th2 responses by CD8+ T cells, we added graded numbers of BALB/c CD8+ T cells to MLC prepared between CD4+ T cells from tolerant BALB/c mice and donor-type A/J stimulators. As shown in Fig. 4, CD8+ T cells from neonatally injected mice inhibited the production of IL-4, IL-5, and IL-13 by CD4+ T cells in a dose-dependent manner and were as efficient as control CD8+ T cells in doing so. The suppressive activity in this system was generated by the recipient CD8+ T cells that required recognition of donor alloantigens as it was not observed when donor type (A/J x BALB/c)F1 CD8+ T cells were added to the MLC (data not shown). This was confirmed by the observation that suppression of IL-4 production in this system decreased from 82 to 38% when anti-H-2Kk mAb was added to the MLC. Since IFN-{gamma} was found to mediate inhibition of Th2 responses by CD8+ T cells in other systems (15, 21, 22, 23), we repeated this MLC with CD8+ T cells from IFN-{gamma}-deficient mice. As shown in Fig. 4A, IFN-{gamma}-deficient CD8+ T cells lacked the ability to inhibit IL-5 synthesis but remained able to regulate both IL-4 and IL-13. We demonstrated that IFN-{gamma} contributes also to the regulatory activity of CD8+ T cells from tolerant mice as IFN-{gamma} neutralization with anti-IFN-{gamma} mAb abrogated the ability of CD8+ T cells from neonatally injected mice to down-regulate the IL-5 synthesis by alloreactive CD4+ T cells in vitro (Fig. 4B). This led us to assess the production of IFN-{gamma} by purified CD8+ T cells in MLC. Although purified CD8+ T cells from neonatally injected mice produced lower levels of IFN-{gamma} in response to allostimulation than control CD8+ T cells in most experiments, they were clearly stimulated by donor alloantigens to produce this cytokine (Table III).



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  		FIGURE 4. CD8+ T cells from neonatally injected mice regulate the Th2 cytokines production in vitro. A, Increasing numbers (Nb) of control wild-type BALB/c CD8+ T cells (•), CD8+ T cells from neonatally injected wild-type BALB/c mice ({circ}) or CD8+ T cells from uninjected IFN-{gamma}-/- BALB/c mice ({triangleup}) were added to purified BALB/c CD4+ T cells in a MLC containing allogeneic A/J-irradiated spleen cells. IL-4, IL-5, and IL-13 were measured after 72 h of culture. B, Anti-IFN-{gamma} mAb ({blacktriangleup}) or isotype control mAb ({triangleup}) were added to a MLC containing purified CD4+ and CD8+ T cells from neonatally injected BALB/c mice. IL-5 levels were determined by ELISA after 72 h of culture. The results are representative of two independent experiments.

 

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  		Table III. Production of IFN-{gamma} by alloreactive CD8+ T cells in tolerant mice

 
Regulatory CD8+ T cells are functional in early life

We determined whether the regulatory function of CD8+ T cells was indeed exerted in the neonatal period. For this purpose, we first analyzed the influence of CD8+ T cells purified from LN of 3-day-old mice on the production of Th2 cytokines by tolerant adult CD4+ T cells in vitro. As shown in Fig. 5, neonatal CD8+ T cells were perfectly able to down-regulate the production of IL-4, IL-5, and IL-13 when added to MLC. Interestingly, CD8+ T cells from mice injected on the day of birth with F1 cells appeared more efficient than those from naive mice in this assay, suggesting that they could have been primed in vivo. In line with their inhibitory action on IL-5 synthesis, we found that purified CD8+ T cells from newborn mice were able to secrete IFN-{gamma} upon allostimulation (Table IV) although the level of IFN-{gamma} produced was about one-third of the level measured when adult CD8+ T cells were used as responders (data not shown).



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  		FIGURE 5. CD8+ T cells from newborn mice are able to regulate the production of Th2 cytokines. Increasing numbers (Nb) of purified CD8+ T cells from 3-day-old BALB/c uninjected (•) or F1 cell neonatally injected ({circ}) mice were cultured with purified CD4+ T cells from 4-wk-old neonatally injected BALB/c mice and irradiated A/J allogeneic spleen cells. Cytokine production was measured by ELISA on the supernatants after 72 h of culture. The results are representative of two independent experiments.

 

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  		Table IV. Production of IFN-{gamma} by alloreactive neonatal CD8+ T cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been well established in several experimental models that CD8+ T cells inhibit Th2 responses in adult animals (15, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Both direct interactions between CD4+ T cells and CD8+ T cells and indirect mechanisms involving modifications of APCs by CD8+ T cells were shown to be involved. Indeed, CD8+ T cells recognizing peptides derived from the TCR V{beta} region in the context of the nonclassical MHC class Ib Qa1 molecule down-regulated Th2 activities of CD4+ T cells expressing the corresponding TCR (22). More recently, murine CD8+ T cells were found to induce IL-12 synthesis by dendritic cells, resulting in the production of IFN-{gamma} by CD4+ T cells and thereby in inhibition of IL-4 and IgE synthesis (23). Similar interactions between CD8+ T cells and dendritic cells were also demonstrated using human cells in vitro (31). In the latter model, the involvement of CD8+ T cell-derived IFN-{gamma} was established. This contrasts with the observations made in two different murine models where CD8+ T cells from IFN-{gamma}-/- mice efficiently down-regulated Th2 responses in vivo (23, 24), suggesting that they acted by producing other Th1-polarizing factors such as CCR5 ligands like macrophage-inflammatory protein 1{alpha} or macrophage-inflammatory protein 1{beta} (32) or by killing CD4+ T cells or APCs. Herein, we found that CD8+ T cells from neonatally injected mice that do not develop antidonor cytotoxicity were as efficient as control CD8+ T cells in controlling Th2 responses and that the regulation of IL-5 but not of IL-4 or IL-13 required the production of IFN-{gamma} by CD8+ T cells. One can speculate that when CD8+ T cells do not operate through cytotoxicity, suppression of IL-5 synthesis involves direct IFN-{gamma}-mediated interactions with CD4+ T cells. The inhibition of IL-4 and IL-13 production would then depend on the induction of IL-12 synthesis by dendritic cells, a phenomenon previously shown not to require the production of IFN-{gamma} by CD8+ T cells (23).

In parallel, we found that transfer of CD8+ T cells restored donor-specific cytotoxic activities in neonatally injected mice. This was observed in an in vitro assay using LPS blasts as targets to allow detection of CD4+ T cell cytotoxic activities in vitro. The fact that chimerism developed despite these cytotoxic activities suggest that they do not operate in vivo where CD8+ T cell-mediated cytotoxicity would be required for efficient rejection of F1 cells.

Our observation that CD8+ T cells from neonatally tolerant mice are able to suppress the response to donor alloantigens is consistent with a previous report demonstrating that anti-donor CD8+ T cells persist after neonatal induction of transplantation tolerance but are suppressed by CD4+ T cells with regulatory properties (33). Because we found that anti-donor CD4+ Th2 activities are themselves inhibited by CD8+ T cells, one can conclude that CD4+ and CD8+ T cell responses against donor alloantigens are indeed cross-regulated in allotolerant mice. CD4+ T cells appear to dominate this cross-talk since neonatally tolerant wild-type mice eventually develop an allospecific Th2 response that is amplified in {beta}2m-/- mice.

An important finding made in the course of this study is the demonstration that CD8+ T cells able to down-regulate Th2 responses are already functional at birth. Indeed, it is well established that CD4+ T cell responses induced in early life are Th2 biased (34, 35, 36), especially when secondary responses are considered (37). Several factors were previously shown to promote this Th2 bias, including the intrinsic property of newborn CD4+ T cells to produce high levels of IL-4 and their deficient up-regulation of CD40 ligand (CD154) as well as APC defects (5, 34, 38, 39, 40, 41, 42). It was previously shown that the Th2 bias of CD4+ T cells is not absolute since Th1 responses could be induced in newborn mice after immunization with dendritic cells (43), rIL-12 (44), CD40 ligation (40), CFA (45), live virus (46, 47), DNA vaccines (48, 49), or CpG nucleotides (50). Likewise, Th1 responses were elicited in human newborns by bacillus Calmette-Guérin vaccination (51). Since CD8+ T cell responses to the immunogen were demonstrated in several of these settings, it is plausible that CD8+ T cells were involved in the down-regulation of Th2 activities. This suggested mechanism might be relevant to the "hygiene hypothesis" which proposes that the current rise in the incidence of allergic diseases is related to decreased exposure to certain pathogens in early life (52). On the basis of our findings, we suggest that infectious or pharmacological agents promoting CD8+ T cell activities in early life might indeed reduce the risk of developing Th2 sensitization and associated atopic disorders.


   Acknowledgments
 
We thank Dr. Alain Le Moine for his critical reading of this manuscript and Aurore de Lavareille for her collaboration in this work.


   Footnotes
 
1 This work was supported by the Fonds National de la Recherche Médicale of Belgium, a Pôle d’Attraction Inter-Universitaire-Service Fédéraux des Affaires Scientifiques, Techniques et Culturelles of Belgium, the Commission of the European Union, the Actions de Recherche Concertées-Direction Générale de l’Enseignement Non Obligatoire et de la Recherche Scientifique-Ministcre de l’Education, de la Recherche et de la Formation of the French Community of Belgium. B.A. was funded by the Belgian Kid’s Foundation. V.F. is a research associate at the Fonds National de la Recherche Scientifique of Belgium. Back

2 Address correspondence and reprint requests to Dr. Véronique Flamand, Laboratory of Experimental Immunology, Université Libre de Bruxelles, Route de Lennik, 808, Brussels B-1070, Belgium. E-mail address: vflamand{at}ulb.ac.be Back

3 Abbreviations used in this paper: {beta}2m, {beta}2-microglobulin; LN, lymph node. Back

Received for publication December 4, 2002. Accepted for publication September 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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