Critical role of B cells in the development of T cell tolerance to aeroallergens

Daphne C. Tsitoura1,2, V. Pete Yeung1, Rosemarie H. DeKruyff1 and Dale T. Umetsu1

1 Division of Immunology and Allergy, Department of Pediatrics, Stanford University, Stanford, CA 94305-5208, USA 2 Present address: Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10034, USA

Correspondence to: D. T. Umetsu; E-mail: umetsu{at}stanford.edu
Transmitting editor: L. Lanier


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Respiratory exposure to allergen induces the development of allergen-specific CD4+ T cell tolerance that effectively protects against the development of allergic-sensitization and Th2-biased immunity. The establishment of T cell unresponsiveness to aeroallergens is an active process preceded by a transient phase of T cell activation that requires T cell co-stimulation and is critically influenced by the antigen-presenting cell type. In this study we examined the role of B cells in the development of respiratory tolerance following intranasal (i.n.) exposure to a prototypic protein antigen. We found that respiratory exposure of BCR-transgenic (Tg) mice to minute quantities of cognate antigen effectively induced T cell unresponsiveness, indicating that antigen presentation by antigen-specific B cells greatly enhanced the development of respiratory tolerance. In contrast, respiratory T cell unresponsiveness could not be induced in B cell-deficient JHD mice exposed to i.n. antigen, although T cell tolerance developed in JHD mice reconstituted with B cells, suggesting that B cells are required for the induction of respiratory T cell tolerance. Respiratory exposure of BCR-Tg mice to cognate antigen induced activation of antigen-specific T cells and partial activation of antigen-specific B cells, as demonstrated by enhanced expression by B cells of class II MHC and B7 molecules but lack of antibody secretion. Our data indicate that B cells critically influence the immune response to inhaled allergens and are required for the development of allergen-specific T cell unresponsiveness induced by respiratory allergen.

Keywords: allergy, asthma, B lymphocyte, lung, mucosa, suppression, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The respiratory mucosa interfaces broadly with the environment and is continuously exposed to a wide variety of substances, including both innocuous antigens and pathogenic organisms. The immune system must discriminate constantly between inspired pathogens and non-replicating antigens, but when this homeostatic process is disrupted, aberrant immune responses against non-pathogenic environmental aeroallergens may develop, resulting in respiratory inflammation, culminating in tissue injury, allergy and asthma. While the exact immune mechanisms involved in the initiation and amplification of allergic inflammatory pathology are not fully understood, the development of allergen-specific Th2 cells producing IL-4, IL-5 and IL-13, but not IFN-{gamma}, plays a critical role in the pathogenesis of respiratory allergy and asthma (13).

Under normal circumstances, inhalation of protein antigens does not result in allergic Th2 responses, but rather in a state of antigen-specific T cell unresponsiveness, which in fact protects against Th2-biased immune responses (4,5). We and others demonstrated that this state of antigen-specific T cell unresponsiveness is associated with inhibition of antigen-specific T cell proliferation, with reduced Th1 and Th2 cytokine production, and immune deviation with minimal or no IgE synthesis (4,6,7). In addition, the establishment of CD4+ T cell tolerance to aeroallergens very effectively prevented the development of airway hyper-responsiveness and eosinophilic inflammation (5). The induction of systemic T cell unresponsiveness is preceded by a transient phase of T cell activation during which antigen-specific CD4+ T cells in the draining lymph nodes and lung compartment proliferate, and produce IL-4 and IL-5 (4,8). The initial expansion of antigen-specific T cells during mucosal tolerance induction is normally rapidly attenuated and the majority of the activated antigen-specific T cells are depleted. The small population of allergen-specific T cells that remain alive failed to respond on subsequent immunogenic challenges (4). The induction of tolerance to aeroallergens was selectively dependent on co-stimulation with CD86 (B7-2), but not CD80 (B7-1), similar to that observed for the development of allergic inflammatory responses (3).

The specific mechanisms that regulate the initial phase of Th cell reactivity following antigen inhalation and its subsequent down-regulation are poorly understood. The interaction of T cells with antigen-presenting cells (APC), such as respiratory tract dendritic cells (DC) (9) or macrophages (8), has a major influence, while the involvement of B cells has not been studied. Although it is clear that DC are the most competent APC of the immune system (10), several in vitro studies demonstrated that B cells are very effective in their capacity to activate naive CD4+ T cells (11,12). Primary Th cell responses can develop in mice lacking B cells (13,14). However, T cell priming in the complete absence of B cells results in responses of reduced magnitude (15,16) and inadequate help for antigen-specific antibody generation (17). B cells are particularly efficient in presenting protein antigens and sustaining effective interaction with the T cells in conditions where there are limiting numbers of DC or minimal amounts of antigen (13,18,19), and in enhancing Th2 responses (20,21).

With regard to T cell tolerance, it was initially believed that small, resting B cells could function as potent tolerizing APC (22,23), since T cell inactivation was thought to result from TCR stimulation in the absence of co-stimulatory signals (24). More recent studies using in vivo models demonstrated that B cells were not essential for the establishment of peripheral T cell unresponsiveness to soluble peptides, superantigens and repeated doses of protein administered i.v. or orally (2527). However, it is possible that in these studies, the outcome of T cell–antigen interactions was influenced more by the dose and localization of the antigen than an absolute requirement for B cells (28) or by contamination with IgA+ B cells that have been shown to be present in B cell-deficient µMT mice (29).

In this study we examined the role of B cells in the development of peripheral T cell tolerance following respiratory exposure to protein antigen. We analyzed the effect of primary exposure to intranasal (i.n.) ovalbumin (OVA) on the subsequent development of OVA-specific immune responses in BALB/c mice, in BCR-transgenic (Tg) mice and in mice genetically depleted of B cells (JHD mice), which unlike µMT mice do not contain contaminating IgA+ B cells (29). We found that B cells greatly influenced the induction of respiratory tolerance, and thus, in the presence of high numbers of antigen-specific B cells, respiratory exposure to minute quantities of cognate antigen induced CD4+ T cell unresponsiveness. On the contrary, treatment of B cell-deficient JHD mice with i.n. antigen failed to result in down-regulation of subsequent antigen-specific T cell responses. The lack of i.n. tolerance induction in the absence of B cells was not due to lack of antigen presentation by other APC, since normal proliferative responses to OVA were induced by i.p. immunization of JHD mice. Exposure to i.n. antigen was associated with partial activation of the antigen-specific B cells (increased expression of cell surface activation markers, MHC class II and B7 molecules), but not with antibody production, suggesting that further signals were required for isotype switch and Ig synthesis. These studies indicate that B cells play a critical role in the immune response to inhaled allergens and are required for the development of allergen-specific T cell unresponsiveness induced by respiratory allergen.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Wild-type BALB/c mice were obtained from the Jackson Laboratory (Bar Harbor, ME). MD4 mice Tg for hen egg lysozyme (HEL)-specific IgM and IgD expressed in B cells (30) were backcrossed to the BALB/c strain (31). After seven generations, mice that screened positive for I-Ed were selected and interbred. B cell-deficient JHD mice (32) were obtained from Dr D. Huszar (GenPharm International, Mountain View, CA) and were backcrossed to the BALB/c strain for seven generations. JHD mice were maintained on 1% sulfatrim pediatric suspension (Butler, Hayward, CA) for 4 days/week. Homozygous B cell-deficient mice were generated by interbreeding and identified by PCR specific for the JH region (33). All mice were housed in pathogen-free conditions at the laboratory animal facilities of Stanford University, in accordance with the guidelines of National Institutes of Health. Mice used for experiments were sex and age matched.

Materials
HEL and OVA were obtained from Sigma (St Louis, MO). A HEL–OVA conjugate was prepared as previously described (31). All cells were cultured in DMEM (Sigma) supplemented with 10% FCS (Gemini Bioproducts, Calabasas, CA), 2 mM glutamine, 20 µg/ml gentamicin and 5 x 10–5 M 2-mercaptoethanol.

Induction of tolerance to i.n. antigen
Mice lightly anesthetized with methoxuflurane received i.n. 30 µl of PBS containing OVA or HEL–OVA (grade V; Sigma) on 3 consecutive days. Control mice received i.n. PBS. Ten days later the mice were immunized i.p. with 10 µg of OVA in 2 mg of aluminum hydroxide (alum) in a volume of 0.5 ml.

Bone marrow transplantation
To reconstitute the B cell repertoire in JHD B-cell deficient mice, bone marrow (BM) cells were obtained from naive BALB/c mice. BM was flushed from femurs and tibias of donor mice with PBS/10% FCS and passed through sterile mesh filters to obtain single-cell suspensions. The BM cells were depleted of the red blood cells, washed and resuspended in normal saline. Cells (8–10 x 106) were injected i.v. in age/sex-matched, lightly irradiated (350 rad) JHD mice. Control irradiated JHD mice received BM cells from naive JHD mice. The reconstitution of B cells in the recipients was confirmed by examining the number of CD19+ cells in the periphery 3 weeks after the BM transfer.

CD4+ T cell purification
Purified CD4+ T cells were prepared from spleen cells as previously described (31). Briefly, cells were passed over goat anti-mouse IgG- and IgM-coated (Jackson ImmunoResearch, West Grove, PA) plates followed by two treatments with antibodies against MHC class II, CD8, and HSA plus baby rabbit C (Pel-Freez, Brown Deer, WI).

In vitro proliferation assay
Lymph node or spleen cells were harvested, passed through a nylon mesh and cultured (5 x 105 cells/well) with or without OVA in 0.2 ml of complete DMEM. After 72 h, the cultures were pulsed with 0.25 µCi [3H]thymidine for 12–16 h and the incorporated radioactivity was measured in a Betaplate scintillation counter (MicroBeta Trilux; Wallac, Turku, Finland). In some experiments purified CD4+ T cells (3 x 105 cells/well) were cultured in vitro with or without OVA in the presence of irradiated APC (2 x 105 cells/well) obtained from the spleen of naive mice.

Flow cytometry
Anti-CD19–FITC, anti-CD19–phycoerythrin (PE), anti-B7.1–FITC, anti-B7.2–FITC and anti-CD69–FITC were purchased from PharMingen (San Diego, CA). Anti-MHC class II (MKD6) (ATCC, Rockville, MD) was purified by ammonium sulfate precipitation from ascites fluid and FITC conjugated. Spleen or lymph node cells were incubated with the appropriate antibodies for 30 min, and, after washing, were fixed with 1% paraformaldehyde and analyzed on a Becton Dickinson (Rutherford, NJ) FACScan. Analysis was performed on 10,000 collected events.

Measurement of OVA-specific Ig
Mice were bled and OVA-specific antibodies were measured using modified OVA-specific ELISAs. For the measurement of OVA-specific IgG1 and IgG2a, plates were coated overnight with 5 µg/ml OVA. After washing and blocking, serial dilutions of sera were added for 24 h. Subsequently, the plates were incubated with horseradish peroxidase-conjugated goat anti-IgG subclass-specific antibodies (Southern Biotechnology Associates, Birmingham, AL), washed and developed by adding o-phenylenediamine. The OD was determined at 492 nm. Anti-OVA IgG1 and IgG2a mAb 6C1 and 3A11 respectively were used as standards for the quantification of each IgG subclass. For the determination of OVA-specific IgE, the rat anti-mouse IgE mAb EM95 (5 µg/ml) was used to coat the plates overnight. After the samples were applied for 24 h, biotinylated OVA (10 µg/ml) was added for 2 h, followed by 1-h incubation with horseradish peroxidase-conjugated streptavidin (Southern Biotechnology Associates). Plates were developed with o-phenylenediamine substrate and the OD was determined at 492 nm. The standard for the OVA-specific IgE ELISA was sera from mice hyperimmunized with OVA that had been quantitated for IgE.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigen-specific B cells promote the induction of i.n. tolerance
To determine whether B cells play an important role in the development of tolerance to aeroallergens, we first examined the effect of respiratory exposure of mice bearing antigen-specific B cells to cognate antigen. For this purpose we used BALB/c mice expressing a transgene for the BCR specific for HEL (HEL-Tg mice) (31). Administration of minute quantities of the cognate antigen, HEL–OVA (10 µg x 3), i.n. to HEL-Tg mice resulted in the induction of OVA-specific T cell unresponsiveness, manifested by a significant reduction in the Th cell proliferative response that normally occurred after immunogenic challenge with OVA in alum i.p. (Fig. 1A). The reduction in proliferation of T cells from HEL-Tg mice was associated with a significant reduction in IL-4 (92 ± 1.8 to <5 pg/ml), IL-5 (5.5 ± 0.8 to <0.1 ng/ml), IL-13 (2.7 ± 0.1 to <0.7 ng/ml) and IFN-{gamma} (38 ± 1.2 to <0.18 ng/ml) production. The inhibitory effect of i.n. OVA was dependent on the interaction between cognate antigen with antigen-specific B cells, since i.n. exposure of HEL-Tg mice to the same low dose of the non-cognate antigen, OVA (10 µg x 3) rather than HEL–OVA, or exposure of wild-type BALB/c mice to similar doses of i.n. HEL–OVA, did not result in T cell tolerance (Fig. 1A). We previously showed that i.n. administration of high doses of OVA (100 µg x 3, i.n.) to BALB/c mice induced significant OVA-specific CD4+ T cell unresponsiveness, though administration of doses of OVA <=10 µg x 3 did not induce OVA-specific T cell unresponsiveness (4). Treatment of the HEL-Tg mice with the high dose of OVA (100 µg x 3) induced OVA-specific tolerance, indicating that antigen presentation of OVA by non-B cells was normal in the HEL-Tg mice. These studies indicate that minute quantities of cognate antigen, HEL–OVA (10 µg), presumably endocytosed by HEL-specific B cells via HEL-specific receptors, induced T cell tolerance, whereas minute quantities of non-cognate antigen, OVA, presumably taken up non-specifically by DC or polyclonal B cells, did not. These results therefore strongly implicate antigen-specific B cells as being critically important in the development of antigen-specific T cell unresponsiveness on respiratory exposure to antigen.



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Fig. 1. Presence of antigen-specific B cells promotes the induction of tolerance to aeroallergens. (A) BALB/c and HEL-Tg mice were exposed to PBS, OVA (100µg x 3) i.n. or HEL–OVA conjugate (10 µg x 3) i.n. and subsequently immunized with OVA in alum i.p. 10 days later. (B) HEL-Tg mice were exposed on 3 consecutive days to i.n. PBS or increasing concentrations of HEL–OVA prior to immunization with OVA in alum i.p. Spleen cells were harvested from the mice 5 days after the i.p. immunization and assayed for in vitro proliferation in response to increasing concentration of OVA. Results are expressed as mean c.p.m. ± SD of triplicate cultures.

 
To further confirm that antigen-specific B cells facilitate the induction of i.n. tolerance to minute quantities of inhaled antigen, we exposed HEL-Tg mice i.n. to a range of HEL–OVA concentrations from 0.01 to 10 µg and subsequently immunized the mice with OVA in alum i.p. Splenic T cells from these mice exposed to as low as 0.01 µg i.n. HEL–OVA proliferated poorly when stimulated in vitro with OVA (Fig. 1B). In contrast, the induction of tolerance with non-cognate OVA antigen rather than HEL–OVA in either BALB/c or HEL-Tg mice required a much higher dose of OVA (100 µg x 3). These findings indicated that the enhanced capture and presentation by the HEL-specific B cells of cognate antigen greatly facilitated the development of respiratory T cell tolerance to the inhaled cognate antigen.

Ineffective induction of i.n. tolerance in B cell deficient mice
We next determined whether the development of antigen-specific T cell unresponsiveness required the presence of B cells by examining the effect of i.n. exposure to OVA in JHD mice. The JHD mice are genetically depleted of B cells by targeted deletion of the JH region of the IgM locus, and such mice lack both B cells and serum Ig (32). We previously demonstrated that immunization of JHD mice with antigen in alum primes JHD Th cells for antigen-specific proliferation analogous to that observed in wild-type BALB/c mice (17). However, Fig. 2 shows that i.n. exposure of JHD mice to OVA (100 µg x 3) failed to induce OVA-specific tolerance, such that after the i.p. immunization with OVA in alum, CD4+ T cells from these mice proliferated vigorously on in vitro re-stimulation with OVA. In contrast, i.n. exposure of BALB/c mice to OVA (100 µg x 3) induced OVA-specific T cell tolerance, manifest by significantly reduced CD4+ T cell proliferative responses to OVA. The cells from BALB/c mice initially treated with PBS produced IL-4 (98 ± 2 pg/ml) and IFN-{gamma} (30 ± 1 ng/ml), while BALB/c mice treated with i.n. OVA produced undetectable levels of IL-4 and IFN-{gamma}. In contrast, cells from JHD mice, whether treated with i.n. PBS or with i.n. OVA, produced high levels of IL-4 (67 ± 2 and 60 ± 2 pg/ml) and IFN-{gamma} (26 ± 1 and 25.5 ± 0.9 ng/ml), indicating that T cell tolerance in wild-type BALB/c, but not in JHD, mice was associated with a reduction in IL-4 production. These results strongly suggested that B cells were essential for the development of tolerance to inhaled allergens.



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Fig. 2. B cells are required for the induction of i.n. tolerance. BALB/c and JHD mice were exposed to i.n. PBS or OVA (100 µg) on 3 consecutive days and immunized i.p. with OVA (10 µg) in alum 10 days later. Spleen cells were harvested after 5 days and assayed for proliferation in response to increasing concentrations of OVA in vitro. Results are expressed as mean c.p.m. ± SD of triplicate cultures.

 
To further establish that the failure to induce i.n. tolerance in JHD mice was due to the absence of B cells, we examined the induction of i.n. tolerance in JHD mice reconstituted with B cells. Because B cell-deficient mice such as JHD and µMT mice tend to reject purified B cells that are adoptively transferred into them, we developed a system in which lightly irradiated JHD mice received 8 x 106 BM cells i.v. from antigenically naive, syngeneic BALB/c donors. Three weeks after the BM transplantation, when donor B cells were shown to reach a stable persistent level in the recipient mice (34), spleen and mesenteric lymph node cells were harvested and evaluated for the presence of CD19+ B cells by flow cytometry. Figure 3(A) shows that no B cells were detected in the lymphoid organs of JHD mice, as has been recently shown (29), while the percentage of CD19+ cells obtained from reconstituted JHD mice was almost equal to that of the spleen or lymph nodes of wild-type BALB/c mice, indicating that the transferred B cells expanded and repopulated the lymphoid tissues. The successful reconstitution of the B cell repertoire in JHD mice was also confirmed by the presence of germinal centers in the spleen and mesenteric lymph nodes of BM recipients that were immunized with OVA in alum i.p. 3 weeks after the transplantation (data not shown). Furthermore, we found that the i.p. immunization induced increased levels of OVA-specific IgM and IgG in the BM reconstituted JHD mice (data not shown). Control JHD mice that received BM cells from JHD donors did not develop germinal centers, or splenic B cells (data not shown), which excluded a non-specific effect of irradiation and BM cell transfer as a cause for germinal center development.



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Fig. 3. BM transplantation reconstitutes the B cell repertoire in JHD mice. Spleen cells from (A) untreated JHD mice, (B) JHD mice that received BM cells from nave BALB/c donors, (C) JHD mice that received BM cells from JHD donors and (D) untreated BALB/c mice were stained with anti-CD19–FITC, and analyzed by flow cytometry. Shaded histograms represent cells stained with isotype control mAb. The results are representative of six experiments.

 
Successful reconstitution of the B cells in JHD mice restored the capacity of the mice for i.n. tolerance induction. Thus, mice treated with i.n. OVA then immunized i.p. with OVA in alum developed OVA-specific T cell tolerance, as indicated by the failure of T cells from these mice to proliferate in vitro on stimulation with increasing concentrations of OVA (Fig. 4). In contrast, T cells from reconstituted JHD mice pre-treated with PBS proliferated vigorously. T cells from mice deficient for B cells (JHD or JHD recipients of JHD B cells) proliferated vigorously whether on not they received i.n. OVA or PBS. Therefore, reconstitution of the B cells in JHD mice restored the missing element that was required for the induction of T cell unresponsiveness following exposure to i.n. antigen, supporting the observation that B cells have a critical role in regulation of respiratory tolerance.



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Fig. 4. Reconstitution of JHD mice with B cells restores the induction of i.n. tolerance. JHD mice received BM cells from BALB/c (B -> J) or from JHD (J -> J) mice. The recipients were then exposed i.n. to PBS or OVA (100 µg) on 3 consecutive days and immunized i.p. with OVA in alum 10 days later. Spleens were harvested after 5 days, and splenic CD4+ cells were purified and cultured with irradiated APC (2 x 105 cells/well) obtained from the spleens of naive mice. The in vitro proliferative response to increasing concentrations of OVA was assessed. Results are expressed as mean c.p.m. ± SD of triplicate cultures.

 
Absence of B cells interferes with the initial phase of T cell activation after exposure to i.n. antigen
The development of peripheral T cell tolerance after exposure to i.n. antigen is preceded by a transient phase of antigen-specific T cell activation, during which the T cells are capable of proliferating and secreting cytokines (4). To examine how the presence and absence of antigen-specific B cells affected this initial phase of CD4+ T cell activation, HEL-Tg, BALB/c or JHD mice were exposed to i.n. HEL–OVA or OVA respectively. Two days after the last administration of antigen or PBS, T cells from the bronchial lymph nodes of these mice were harvested and re-stimulated in vitro with OVA. As expected, T cells from HEL-Tg mice treated with i.n. HEL–OVA responded vigorously (though transiently), as did cells from control wild-type BALB/c mice exposed to i.n. OVA (Fig. 5), indicating that T cells destined to be ‘tolerized’ were activated and responsive to antigen stimulation at this early time point. On the contrary, T cells from JHD mice pre-exposed to i.n. OVA failed to proliferate (Fig. 5), suggesting that the lack of B cells impeded the initial transient T cell activation that precedes the development of T cell tolerance. T cells from control mice that were treated with i.n. PBS also did not proliferate. Cytokine production by these cultured T cells reflected T cell proliferation. Thus T cells from HEL-Tg and BALB/c mice pre-exposed to i.n. antigen secreted IL-4, IL-5 and IFN-{gamma} at this early time point of T cell expansion (4), while T cells from JHD mice pre-exposed to i.n. OVA produced no detectable cytokines (data not shown). Taken together, these results indicated that B cells greatly influenced the outcome of the initial interaction of the i.n. administered antigen with T cells and this initial interaction determined the subsequent development of systemic tolerance.



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Fig. 5. Failure of T cell activation following exposure to i.n. antigen in JHD mice. BALB/c and JHD mice were exposed i.n. to OVA (100 µg x 3), while HEL-Tg mice were exposed i.n. to HEL–OVA conjugate (10 µg x 3). After 48 h bronchial lymph node cells were harvested and cultured in vitro with increasing concentrations of OVA. The proliferation in response to OVA was assessed after 72 h. Results are expressed as mean c.p.m. [3H]thymidine incorporated ± SD of triplicate cultures.

 
Exposure to i.n. antigen results in B cell activation, but not antibody synthesis
Antigen capture though BCR and presentation to T cells has been associated with B cell activation (35). To determine if this occurred in HEL-Tg mice, we examined bronchial lymph node cells from HEL-Tg mice 24 h after treatment with i.n. HEL–OVA or PBS for the expression of various activation markers. Analysis with flow cytometry revealed an increase in the cell size and enhanced expression of CD19, CD69, CD80, CD86, CD40 and MKD6 (MHC class II) on B cells from HEL–OVA pre-treated mice compared to B cells from PBS-treated animals (Fig. 6). Increased levels of activation markers were also observed on the surface of B cells from bronchial lymph nodes of BALB/c mice exposed to i.n. OVA, although the changes were less marked since only a small fraction of such B cells would be activated by OVA (data not shown). These results indicate that antigen-specific B cells become activated after exposure to i.n. antigen, consistent with the idea that antigen-specific B cells play an important role in the induction of T cell tolerance following exposure to i.n. antigen.



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Fig. 6. Exposure of HEL-Tg mice to i.n. antigen results in B cell activation and expression of cell surface activation markers. HEL-Tg mice were exposed i.n. to PBS (solid line) or HEL–OVA conjugate (10 µg) (shaded histogram). After 24 h bronchial lymph node cells were harvested, stained with anti-CD19, anti-B7.1, anti-B7.2 and MKD6 (anti-MHC class II), and analyzed by flow cytometry and for cell size by forward light scatter. The results are representative of four experiments using four to six mice per group.

 
Although B cells were activated 24 h after exposure to i.n. OVA, Ig class switch and production of antigen-specific antibodies did not occur. In these experiments we used normal BALB/c rather than HEL-Tg mice to examine isotype switch, since B cells in the HEL-Tg mice cannot undergo isotype switching. OVA-specific IgG and IgE were not detected in sera of mice exposed to i.n. OVA alone (without subsequent immunization with OVA in alum) (Fig. 7A and B). Antigen-specific B cells, however, could be induced to secrete OVA-specific IgG, as well as IgE antibody by treatment of BALB/c mice with i.n. OVA plus LPS, an immunization regimen that prevents T cell tolerance and induces OVA-specific effector Th cells (Fig. 7A and B). Mice that were pre-treated with i.n. OVA without LPS and then immunized with OVA in alum i.p. were capable of generating significant amounts of OVA-specific IgG, but IgE antibody production was greatly reduced compared to mice pretreated with i.n. PBS (Fig. 7C and D), indicating the development of split tolerance. Therefore, we believe that exposure to i.n. antigen partially activated B cells, which then enhanced the subsequent development of antigen-specific T cell tolerance. However, further differentiation of the B cells into antigen-specific Ig-producing cells required additional stimulation from Th cells.



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Fig. 7. Production of OVA-specific Ig in mice following exposure to i.n. OVA. Intranasal antigen without subsequent immunization with OVA in alum does not induce Ig production. BALB/c mice were exposed i.n. to PBS, OVA (100 µg) or OVA (100 µg) + LPS on 3 consecutive days. Two weeks later the mice were bled, and the levels of OVA-specific IgG (A) and IgE (B) in the sera were quantitated by isotype-specific ELISA. Exposure to i.n. antigen prior to immunization with OVA in alum prevents IgE, but not IgG production. BALB/c mice were first exposed i.n. to PBS or OVA (100 µg x 3). Ten days later the mice were immunized i.p. with OVA in alum and after an additional 2 weeks sera from the mice were examined for OVA-specific IgG (C) and IgE (D) by ELISA. Results are expressed as the mean ± SD of triplicate determinations.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we examined the role of B cells in the induction of peripheral T cell tolerance to inhaled allergens, which is of critical importance for protection against respiratory allergy and asthma. B cells have been thought to play an important, although not obligatory, role as APC in the development of primary CD4+ T cell responses, but their involvement in the induction of immunological tolerance remains controversial. Our results indicate that antigen-specific B cells are essential for down-regulation of T cell reactivity to aeroallergens and prevention of allergic sensitization. In particular, we found that in the presence of high numbers of antigen-specific B cells, exposure to minute quantities of i.n. allergen resulted in very effective suppression of the specific Th cell responses, while in the absence of B cells, respiratory tolerance could not be induced. Exposure to i.n. antigen was associated with rapid partial activation of the antigen-specific B cells, characterized by increased expression of MHC class II and co-stimulatory molecules, and increased cell size, but not with antibody production. Subsequent immunogenic stimulation resulted in enhanced production of antigen-specific IgG, without parallel production of allergen-specific IgE (a form of immune deviation), but resulted in antigen-specific CD4+ T cell unresponsiveness.

There is considerable evidence that B cells are capable of inducing both priming and anergy in T cells. It was initially proposed that the activation state of the B cells determines the outcome of B–T cell interactions. Thus, it was believed that resting B cells induce T cell unresponsiveness, while activated B cells promote T cell expansion and differentiation toward an effector phenotype (15,22,23). The recent demonstration that the development of T cell unresponsiveness following antigen stimulation under tolerogenic conditions is an active process requiring co-stimulation, however, challenges this notion (4,36,37). In our model of tolerance to aeroallergens we found that the presence and activation of B cells after exposure to i.n. antigen was essential for the establishment of CD4+ T cell unresponsiveness. Antigen inhalation led to B cell activation, presumably by direct Ig–receptor cross-linking and resulted in increased expression of MHC class II and B7 molecules. The up-regulation of accessory molecules on the surface of B cells may provide the necessary threshold of co-stimulatory signals for activation and acquisition of a tolerogenic phenotype by the CD4+ T cells. B7-2-mediated signaling in particular is of critical importance for the development of T cell tolerance to allergens (4,38).

Alternatively, the activation of B cells may be essential for the generation and maintenance in mucosal lymphoid tissues of IL-4 and IL-10 (27), which may enhance IL-10 production in DC. We recently showed that respiratory exposure to antigen is associated with a burst of IL-10 production in DC (39), which can directly induce T cell tolerance, possibly by inducing the development of regulatory T cells (37). While the abrogation of i.n. tolerance in B cell-depleted (JHD) mice confirmed the important role of B cells in the development of this phenomenon, we do not suggest that B cells are the only APC required for the induction of i.n. T cell tolerance. DC are considered the most potent APC of the immune system and have the potential to present antigen to T cells in a priming, as well as in a tolerogenic, fashion (10,37). Furthermore, there is evidence that DC in the respiratory tract are key regulators in the initiation and establishment of chronic allergen-specific Th2-mediated lung inflammation (40). However, in the context of respiratory tolerance, B cells may interact with DC (39) in a way that enhances the tolerogenic capacity of DC in the respiratory tract, e.g. through the production of IL10 (27), or perhaps by providing the scaffolding or the lymphoid architecture for B cell–DC–T cell interactions that induce tolerance to occur. Regardless of the mechanism, our results using BCR-Tg and B cell-deficient mice demonstrated that antigen-specific B cell activation following exposure to i.n. antigen, even at very low concentrations, significantly promoted the induction of respiratory tolerance and that the development of respiratory T cell tolerance requires the presence of B lymphocytes.

Antigen presentation by B cells may be critical at conditions of limited allergen exposure in non-inflammatory environments (13,18,19). Some investigators have proposed that uptake and presentation of soluble protein antigens in secondary lymphoid organs by APC other than B cells is extremely limited, while the opposite occurs with peptide fragments (41,42). B cells may provide signals necessary for effective expansion, ‘tolerogenic differentiation’ of the antigen-specific CD4+ T cells. Exposure to i.n. antigen of normal and BCR-Tg mice, but not B cell-deficient mice, was accompanied by a short period of antigen-specific T cell proliferation, and Th1 and Th2 cytokine production. The absence of T cell proliferation in cultures of lymphoid cells from JHD mice (Fig. 5) may reflect inadequate in vivo expansion of OVA-specific T cells, due to deficient antigen presentation to T cells. It is possible that optimal magnification of this initial phase of T cell reactivity is an essential step for generation of high enough frequencies of tolerized T cells that mediate the development of antigen-specific T cell unresponsiveness and DC or macrophages do not provide enough signals to support it under normal circumstances. In this regard, it was recently demonstrated that the development of tolerance to an antigen expressed in the pancreatic islets was dependent on the amount of antigen expressed and on the degree of antigen-specific T cell proliferation in the pancreatic lymph nodes before the establishment of unresponsiveness (43).

We have previously shown that the initial phase of CD4+ T cell activation after exposure to i.n. OVA is followed by clonal deletion of the majority of OVA-specific CD4+ T cells and functional inactivation of the small surviving population (4). It is possible that in JHD mice, exposure to i.n. OVA resulted in a limited degree of T cell activation, which was inadequate for extensive deletion or anergy of the antigen-specific Th cells, most of which appeared to remain intact to respond to secondary immunization in a way analogous to that observed in naïve mice. The interaction of CD40 on B cells with its counter-receptor on T cells might deliver some of the signals required for the amplification of the initial phase of CD4+ T cell reactivity after antigen inhalation. CD40–CD40 ligand interaction has been shown to be necessary for the development of tolerance in the gut mucosa (44). B cell-dependent T cell activation has also been considered critical for the development of CD4+ T cell memory during the primary encounter with antigen (45,46). In the complete absence of B cells, an effective memory pool of Th cells is not generated and this step appears to be independent of the acquisition of characteristics of effector phenotype (17,46). Mucosally induced anergic CD4+ T cells may have also undergone differentiation into a memory-like state (47).

Previous studies demonstrated that B cells were not required for the induction of tolerance to superantigens, peptides and i.p., i.v. or orally administered soluble proteins (2527,48). This may be due to differential effects of the various treatments on the initial activation of antigen-specific T cells. The nature, dose, way of release of the antigen as well as the local milieu during the initial encounter with the T cells influence qualitatively and quantitatively the ensuing T cell response (28). Moreover, it is worth mentioning that in the previous studies examining the role of B cells in tolerance, µMT B cell-deficient mice were used. These mice have a targeted deletion of the µ region, but are reported to have low levels of {kappa} chain rearrangements (49). More recently, µMT, but not JHD, mice have been shown to contain IgA+ B cells in Peyer’s patches and ileum and develop high levels of serum IgA (29). These IgA+ B cells in µMT mice (27) may have participated in tolerance induction and perhaps explain the development of mucosal tolerance in µMT mice, but not in JHD mice. Together these results indicate that mucosal tolerance cannot occur in the complete absence of B cells and that antigen uptake by mucosal B cells enhances the development of T cell tolerance.

In our model, respiratory antigen exposure resulted in activation of antigen-specific B cells, but not in antibody generation (unless mice were subsequently immunized with OVA in alum). This finding implies that a deficiency existed at the level of DNA rearrangements between the Ig switch regions and that further signals were required for the induction of Ig synthesis. It is possible that the co-stimulation provided by the activated CD4+ T cells was not sufficient or was not sustained enough for the B cells to undergo isotype switch. Alternatively, stimulation from soluble factors may be necessary for the B cells to progress to Ig synthesis. However, it is clear that B cell tolerance was not induced following allergen inhalation, since the antigen-specific B cells were not eliminated or anergized, but on the contrary they responded to subsequent immunogenic challenge with increased secretion of antigen-specific IgG1 and IgG2. Expansion of the IgM-producing B cells in response to i.n. antigen, despite the lack of full maturation to plasma cells, may explain the fact that the levels of specific IgG after the i.p. immunization were much higher in mice pre-exposed to i.n. antigen than in previously naive mice. A two-phase model for complete B cell activation has been proposed (50). In particular, after antigen stimulation there is an initial T cell-independent phase of limited clonal B cell expansion and secretion of IgM which is followed by complete B cell maturation, with isotype switch and affinity maturation, when adequate T cell help is present (50,51). IgE synthesis was not induced in mice exposed to i.n. antigen even after the i.p. immunization indicating that there were differential requirements for switch recombination to the {gamma}1 and {epsilon} loci. We suggest, therefore, that in the B cell compartment, i.n. tolerance results in a form of immune deviation that blocks IgE, but not IgG1, synthesis. This goes along with the fact that the requirements for IgE synthesis are distinct from those for IgG1 synthesis, and involve the presence of IL-4, IL-13 and CD40 activation (52).

In conclusion, our results strongly suggest that B cells play a critical role in the induction of tolerance to aeroallergens. We propose that during respiratory exposure to antigen, in the absence of inflammatory signals, antigen-specific B cells take up antigen and amplify antigen presentation by tolerance-inducing DC. Under normal circumstances the number of antigen-specific B cells is extremely limited; however, these B cells must provide important signals since in their absence respiratory tolerance does not occur. These findings are important in the design of new vaccination strategies for allergic disease and asthma that induce allergen-specific T cell tolerance which hinders the survival of allergen-specific Th2 cells. For example, strategies that promote preferential antigen up-take by the B cells may more effectively achieve the development of peripheral tolerance. Further studies of the mechanisms of i.n. tolerance induction will greatly enhance the development of practical therapies for the prevention and treatment of respiratory allergy and asthma.


    Acknowledgements
 
We thank Chris Goodnow for providing the MD4 HEL Ig-Tg mice and Dennis Huszar for providing the JHD mice. Supported by the NIH Public Health Service grants AI24571 and AI26322, and a fellowship from the California Lung Association.


    Abbreviations
 
APC—antigen-presenting cell

BM—bone marrow

DC—dendritic cells

HEL—hen egg lysozyme

i.n.—intranasally

OVA—ovalbumin

PE—phycoerythrin

Tg—transgenic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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