1 Department of Molecular Cell Biology, VU University Medical Center, PO Box 7057,1007 MB Amsterdam, The Netherlands 2 Present address: Unilever Health Institute, 3133 AT Vlaardingen, The Netherlands 3 Present address: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
Correspondence to: J. N. Samsom; E-mail: jn.samsom.cell{at}med.vu.nl
Transmitting editor: Dr. J. Borst
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Abstract |
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Keywords: infectious tolerance, mucosal regulatory T cell, plasticity, Th1, Th2
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Introduction |
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In addition to these thymic-derived Tr cells, another subset of Tr cells is generated in the periphery in response to administration of soluble antigen via the mucosa (1). Recently, we observed that intrinsic characteristics of the nasal draining lymph nodes are critically important for the induction of tolerance via the nasal mucosa. A series of transplantation experiments revealed not only that the presence of cervical lymph nodes was necessary for nasal tolerance induction, but also that this function could not be assumed by peripheral lymph nodes when transplanted to this site (12). After nasal or oral tolerance induction, the Tr cells that can be isolated from the spleen and that are able to transfer tolerance, are CD4+ T cells (mucosal Tr cells), but not CD8+ T cells (13,14). More detailed knowledge of the phenotype of these mucosal Tr cells is lacking.
It is unclear what fate the transferred mucosal Tr cells undergo in the naive recipient. Do the transferred Tr cells expand by proliferation, establishing an exponentially increasing cohort of cells that suppress a subsequent sensitization and challenge? Or do these mucosal Tr cells transfer the regulatory capacity to a naive CD4+ T cell of the recipient that subsequently suppresses sensitization and challenge?
Furthermore, little is known about the plasticity of the Tr cells that are generated after mucosal tolerance induction. During polarization of naive T cells into effector Th1 and Th2 cells, their plasticity is lost. As a result, after three to four cell divisions, Th1 and Th2 cells are incapable of re-expressing alternative cytokines when re-stimulated under opposing conditions (15). Previously, we have established that application of ovalbumin (OVA) via the mucosa is effective in the suppression of highly polarized Th1 and Th2 immune responses. It is unclear whether, after adoptive transfer and subsequent Th1 or Th2 sensitization and challenge, the mucosal Tr cells are influenced by the polarized environment in the host. Do the Tr cells retain their suppressive capacity in the recipient or do these cells acquire an irreversible differentiation analogous to Th1 and Th2 cells?
In this study we have addressed three questions on the fate, plasticity and identity of mucosal Tr cells that are generated after nasal tolerance induction. We show that Tr cells, induced by nasal OVA application, mediate tolerance by passing the tolerizing capacities on to naive CD4+ T cells. Surprisingly, these mucosal Tr cells do not loose plasticity during their differentiation from naive CD4+ T cells, as demonstrated by the capacity to suppress naive T cells irrespective of initial or ongoing cytokine polarization of the immune response. Finally, we show that Tr cells reside in both CD25+ and CD25 subpopulations; however, only the CD25 Tr cells display antigen specificity.
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Methods |
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Induction of tolerance and subsequent antigen sensitization for delayed-type hypersensitivity (DTH) or allergic response
To induce tolerance, mice received 10 µl saline containing 100 µg OVA (type VII; Sigma, St Louis, MO) intranasally on each of 3 consecutive days. Control mice received 10 µl saline intranasally. Previously, we have described two models resulting in opposite cytokine-skewing, denoted as Th1 (lacking IgE) and Th2 [lacking IFN- (13)].
Mice were sensitized for a Th1 response as previously described (13). In short, 25 µl incomplete Freunds adjuvant (IFA; Difco, Detroit, MI) mixed with 25 µl saline containing 100 µg OVA was injected s.c. in the tail base 1 day after the last intranasal OVA or saline administration. Five days later, a challenge was given by injecting 20 µl saline, which contained 10 µg OVA, in the auricles of both ears. Directly before challenge, the initial thickness of the ear was determined with an engineers micrometer (Mitutoyo, Tokyo, Japan). DTH responses were expressed as the mean increase in ear thickness of both ears 24 h post-challenge, following subtraction of the mean ear thickness before challenge. In some experiments, mice were sensitized and challenged for a Th1 response using hen egg lysozyme (HEL; Sigma). The same amounts of HEL were used for sensitization and challenge as were used for sensitization and challenge with OVA. In all experiments the ear thickness was measured in a blinded fashion.
Sensitization for a Th2 response was performed as described previously (14). Briefly, on days 1 and 14 after the last intranasal OVA treatment, mice received an i.p. injection of 2 µg OVA dissolved in 100 µl saline mixed with 100 µl aluminum hydroxide rehydragel (AlOH; Reheis Chemical, Berkely Heights, NJ). Four days after the second injection, mice were challenged intragastrically with 200 µl saline containing 1 mg OVA. As a parameter for the Th2 response, IgE levels were measured. Blood was collected via a small incision in the tail vein at t = 2, 0, 2, 4 and 6 days with regard to the intragastric challenge, and stored as serum at 80°C until IgE levels were measured.
Measurement of serum IgE by ELISA
Both total and OVA-specific IgE serum levels were determined by using a modified capture ELISA as described elsewhere (16). In brief, Falcon Microtest III plates (Becton Dickinson, San Diego, CA) were coated overnight at 4°C with rat anti-mouse IgE [EM95 (16)]. As a positive control, anti-TNP IgE-3 (PharMingen, San Diego, CA) for total IgE or pooled serum from multiple OVA-boosted mice for OVA-specific IgE was used. Total IgE was detected with biotinylated rat anti-mouse IgE (PharMingen, San Diego, CA). For detection of OVA-specific IgE, digoxigenin-coupled OVA [coupled as described by van Halteren et al. (16)] was used. Sub sequently, for both total and OVA-specific IgE determination, either peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) or peroxidase-coupled sheep anti-digoxigenin Fab fragments (Boehringer, Mannheim, Germany) was added. Finally, the enzymatic reaction was developed by addition of o-phenylenediamine dihydrochloride (Sigma, St Louis, MO) in 0.1 M phosphate-citrate buffer, containing 0.015% hydrogen peroxide. Absorbance was measured at 490 nm and IgE concentrations were determined with reference to the standard curves.
Experiments with C57BL/6-Ly5.1 and C57BL/6-Ly5.2 mice
C57BL/6-Ly5.1+ mice were tolerized and sensitized for a DTH response as described. Seven days after challenge, spleens from tolerant and naive control mice were collected. Single-cell suspensions were obtained by mincing the spleens and straining them through 100-µm gauze. Erythrocytes were depleted from this cell suspension by incubation in a lysis buffer (150 mM NH4Cl and 1 mM NaHCO3, pH 7.4) for 5 min on ice. For partial purification of CD4+ T cells, the spleen cells were incubated for 30 min on ice with a B cell-specific rat mAb [clone 6B2, anti-B220; affinity purified from culture supernatant of hybridoma cells with Protein GSepharose (Pharmacia, Uppsala, Sweden)]. Next, the cells were washed and resuspended in HBSS (Biowhittaker, Verviers, Belgium) supplemented with 1% heat-inactivated FCS. Subsequently, sheep anti-rat IgG Dynabeads (Dynal, Oslo, Norway) were added in a 2:1 bead:cell ratio. After 30 min incubation on a rotator at 4°C, positively stained B cells were depleted using a magnetic particle concentrator (MPC; Dynal). The remaining cell population contained between 50 and 60% CD4+ T cells, as determined by flow cytometry (FACStar; Becton Dickinson). Previously, we have shown that the contaminating CD8+ T cells do not transfer tolerance (14). The enriched CD4+ T cells were washed, resuspended in saline and 25 x 106 cells were transferred to naive C57BL/6-Ly5.2+ acceptor mice by i.v. injection via the lateral tail vein. Upon transfer, the acceptor mice were sensitized for a Th1 response. Seven days after challenge, the spleens were collected from these C57BL/6-Ly5.2+ tolerant mice and partially purified CD4+ T cells were isolated from single-cell suspensions. To determine whether the CD4+Ly5.2+ T cells had also become suppressive, highly purified CD4+ T cells were used. These were prepared by incubation of the enriched CD4+ T cell population with biotinylated anti-Ly5.1 antibody (clone A20; affinity purified from culture supernatant of hybridoma cells with Protein GSepharose and biotinylated according to the manufacturers instruction), FITC-conjugated anti-Ly5.2 antibody (clone AL-1; affinity purified from culture supernatant of hybridoma cells with Protein GSepharose and labeled with FITC according to the manufacturers instructions) and phycoerythrin (PE)-conjugated anti-CD4 antibody (GK1.5; PharMingen) for 30 min on ice. After washing, CD4+Ly5.2+Ly5.1 T cells were sorted by flow cytometry. The cells were washed, resuspended in saline and 1 x 106 CD4+ T cells were transferred to naive acceptor mice by i.v. injection via the lateral tail vein.
Transfer experiments with BALB/c mice
For transfer of tolerance, donor BALB/c mice were tolerized and sensitized for a Th1 response or a Th2 response as described. Spleens of Th1-tolerant mice were collected and pooled 7 days post-challenge, whereas spleens of Th2-tolerant animals were collected at 4 days post-challenge. As a control, spleens from naive BALB/c mice were taken and partially purified CD4+ T cells were isolated from single-cell suspensions as described above.
For transfer of purified CD4+CD25+ T cells and CD4+CD25 T cells, the enriched CD4+ T cells were stained with FITC-conjugated anti-CD4 antibody (clone GK1.5, affinity purified from culture supernatant of hybridoma cells with Protein GSepharose and FITC-labeled according to the manufacturers instructions), PE-conjugated anti-CD25 antibody (clone PC61, anti-IL2R; PharMingen) and biotinylated anti-MHC class II antibody (clone M5/114, affinity purified from culture supernatant of hybridoma cells with Protein GSepharose) for 30 min on ice. After washing, CD4+CD25+ and CD4+CD25 T cells were sorted (FACSVantage; Becton Dickinson). The CD4+CD25+ T cell population was approximately 90% pure (the remaining 10% consisted of 6% CD4CD25 cells and 4% CD4+CD25 cells) and the purity of the CD4+CD25 cell population was
98% as determined by re-analysis. After washing, the cells were resuspended in saline and 105 cells were transferred to naive acceptor mice by i.v. injection via the lateral tail vein.
When CD4+CD25+ T cells and CD4+CD25 T cells were purified using MACS microbeads, the enriched CD4+ T cells were incubated with biotinylated anti-CD25 antibody (clone PC61; PharMingen) for 15 min on ice, followed by anti-biotin immunomagnetic beads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 min at 4°C. Magnetic separation was performed with an MS column according to the suggested protocol. The CD4+CD25+ T cell population was 80% pure (the remaining 20% consisted of 14% CD4CD25 cells and 6% CD4+CD25 cells) and the purity of the CD4+CD25 cell population was
86% (the remaining 14% consisted mainly of CD4CD25 cells) as determined by re-analysis. After washing, the cells were resuspended in saline and 105 cells were transferred to naive acceptor mice by i.v. injection via the lateral tail vein.
To determine whether dendritic cells (DC) were capable of transferring tolerance, purified splenic DC were used. DC were isolated from a suspension of spleen cells using anti-CD11c immunomagnetic beads (MACS) according to the manufacturers instructions. This enriched DC population was then incubated with FITC-conjugated anti-MHC class II antibody (clone M5/114) and PE-conjugated anti-CD11c antibody (clone HL3; PharMingen) for 30 min on ice. Further purified CD11c+ MHC class II+ DC were obtained by flow sorting. After washing, cells were resuspended in saline and the numbers indicated were transferred to naive acceptor mice by i.v. injection via the lateral tail vein.
Proliferation assays
Lymph nodes were isolated from tolerant and non-tolerant mice 1 week after ear challenge. Single-cell suspensions were cultured with 0.5 mg/ml OVA in U-bottomed 96-well plates at 5 x 106 cells/ml in DMEM (Gibco, Life Technologies, Breda, The Netherlands) supplemented with 10% heat-inactivated FCS (Biowhittaker), 50 U/ml sodium penicillin G (Biowhittaker), 50 µg/ml streptomycin (Biowhittaker), 2 mM L-glutamine (Biowhittaker) and 50 µM ß-mercaptoethanol (Merck, Darmstadt, Germany). After 4 days, the cultures were pulsed with 0.8 µCi/well [3H]thymidine (Amersham, Roozendaal, The Netherlands) and harvested 16 h later. Results are expressed as mean values of triplicates.
Statistics
For ear swelling responses, the mean increase in ear thickness of both ears was determined for each mouse per group. For Th2 responses, data are expressed as mean IgE levels ± SD per group. Groups consist of seven mice, unless otherwise indicated. Groups were compared using an ANOVA followed by the TukeyKramer multiple comparisons test for ear swelling responses. For serum IgE levels, a MANOVA was used. For proliferation assays, data are expressed as mean values of triplicates. Groups were compared to the control group using an unpaired t-test. P < 0.05 was considered significant.
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Results |
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To follow the fate of the tolerizing Tr cells, mice with an allelic difference in Ly5 (CD45) were used which enabled us to discriminate between donor and recipient cells. Donor C57BL/6-Ly5.1 mice received an intranasal OVA instillation on 3 consecutive days. One day after the last nasal intranasal OVA administration, mice were sensitized for a DTH response by an injection of OVA/IFA s.c. Five days later, mice were challenged by an injection of OVA in the auricles of both ears. Nasal OVA treatment led to tolerance as only marginal ear swelling was measured (mean increase in ear thickness was 5.25 ± 0.54 compared to 10.58 ± 2.75 x 102 mm in control mice treated nasally with saline). From these tolerant C57BL/6-Ly5.1 mice splenic CD4+ T cells were purified and transferred into naive C57BL/6-Ly5.2 recipient mice (transfer I, Fig. 1A). These C57BL/6-Ly5.2 recipient mice were then sensitized and challenged with OVA to confirm that they had become tolerant after this cell transfer (mean increase in ear thickness was 5.43 ± 0.73 versus control 9.83 ± 1.66 x 102 mm). To rule out the possibility that a non-responsive T cell population was induced, simply due to the regimen of sensitization and challenge, splenic CD4+ T cells from mice that had received saline intranasally were adoptively transferred to naive recipients. Transfer of CD4+ T cells from these OVA-primed mice did not inhibit the DTH response in the recipient mice (mean increase in ear thickness was 10.88 ± 1.62 versus control 11.81 ± 2.49 x 102 mm), indicating that these cells remain responsive to antigenic stimulation.
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As shown in Fig. 1(B), the Ly5.2+CD4+ T cells of the recipient of transfer I are able to suppress the DTH response in naive recipients (group A), indicating that they have become Tr cells. Recipients of both Ly5.1+ and Ly5.2+CD4+ T cells (group B) were also tolerant, as ear swelling responses were similar to those in recipients of Ly5.2+CD4+ T cells, but significantly different from recipients of naive CD4+ T cells (group C). Moreover, it should be noted that at the time of isolation of CD4+ cells for transfer II, the spleens of the C57BL/6-Ly5.2+ mice contained only a minor fraction of CD4+ T cells (<0.5% of total CD4+ T cells) of the original donor Ly5.1 allotype.
Recent studies have shown that APC can mediate T cell suppression, either upon cross talk with anergic T cells, or via the secretion of IL-10 and/or TGF-ß (17,18). To exclude the possibility that the results obtained from both T cell transfers I and II were due to contaminating DC, adoptive transfers were performed with purified splenic DC from OVA-tolerized mice. In our hands, transfer of 5 x 104 DC, a number which is at least 5-fold excess of the possibly contaminating DC in both transferred T cell populations, did not inhibit the DTH response in recipient mice (mean increase in ear thickness was 8.45 ± 1.15 versus control 9.56 ± 1.15 x 102 mm).
Together, these results clearly demonstrate that Tr cells, when transferred to naive recipients, induce the CD4+ T cells of the recipient to differentiate into Tr cells.
Transferred CD4+ Tr cells retain their suppressive capacity in a polarized environment
In the experiments described above, mice were challenged to develop a DTH response, a type of stimulus that is biased towards inducing Th1 cells. Previously, we have shown in an allergy model that nasal OVA application can effectively tolerize polarized Th2 responses, as measured by the suppressive effect on both total and OVA-specific IgE production (16). Since upon differentiation Th1 and Th2 cells undergo irreversible lineage commitment, we questioned whether after defined sensitization and challenge Tr cells would also lose their plasticity. To test this, we assessed whether Tr cells from a Th1-biased animal would be able to suppress the sensitization of both Th1 and Th2 responses, and whether this also applied to Tr cells from a Th2-biased animal.
To induce tolerance, mice received a nasal OVA application on each of 3 consecutive days. Thereafter, mice were divided in two groups. One group was sensitized for a DTH response by an injection of OVA/IFA s.c. (Th1-type response). Five days later, mice were challenged by an injection of OVA in the auricles of both ears. It was confirmed that nasal OVA-treated mice were tolerant (mean increase in ear thickness was 4.03 ± 0.66 x 102 mm), as ear swelling responses were reduced in comparison to saline-treated mice, which displayed significantly enhanced ear swelling responses (mean increase in ear thickness was 9.38 ± 1.27 x 102 mm). The other group was sensitized for an allergic response by two i.p. injections of OVA in AlOH with a 2-week interval (Th2-type response). Four days after the last i.p. injection, mice were challenged for IgE by an intragastric injection of OVA. In the sera from mice treated with OVA intranasally significantly lower IgE levels were detected (2687.99 ± 753.64 ng/ml total IgE) than in sera from saline treated mice (8805.25 ± 588.32 ng/ml total IgE, on day 4 after challenge), indicating tolerance. From the spleens from Th1-tolerant and Th2-tolerant mice, CD4+ Tr cells were purified and transferred into naive recipients (Th1 and Th2 acceptors respectively). Control mice received naive splenic CD4+ T cells. Upon transfer, Th1 acceptors, Th2 acceptors and control mice were divided in two groups; half of the mice were sensitized for a Th1 response, whereas the other half of the mice were sensitized for a Th2 response. As depicted in Fig. 2, the acceptor mice that were sensitized for a Th1 response showed a similar reduction in DTH response irrespective whether the transferred CD4+ Tr cells were derived from Th1- or Th2-tolerant animals.
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Identification of subsets of mucosal CD4+ Tr cells
Until now, it has been unclear whether the regulatory activity of mucosal Tr cells is limited to a specific subset of CD4+ splenocytes. Flow cytometric analysis of CD4+ Tr splenocytes and control CD4+ splenocytes from a non-tolerized donor did not yield any significant phenotypic differences. Since the detection limit may be too high to distinguish a discriminating marker of a Tr subset and in view of the vast amount of data demonstrating a role for CD4+CD25+ T cells in immune regulation (5), we determined whether the mucosal CD4+ Tr cells are CD25+ or CD25. Therefore, mice were tolerized by a nasal OVA instillation on 3 consecutive days. These mice were then sensitized for a Th1 response by an injection of OVA/IFA s.c. and challenged by an injection of OVA in the auricles of both ears to confirm tolerance (mean increase in ear thickness was 4.7 ± 0.48 versus control 9.56 ± 0.72 x 102 mm).
One week after challenge, splenic CD4+ Tr cells from the Th1-tolerant mice were sorted into CD25+ (90% pure) and CD25 (98% pure) fractions, and subsequently transferred to naive acceptor mice. As a control, splenic CD4+ T cells were from naive, non-OVA-exposed mice were also transferred to naive recipients. The acceptor mice were then sensitized and challenged for a Th1 response with either OVA or an unrelated antigen HEL. It was observed that the CD4+CD25+ T cells from tolerant mice were able to suppress both the OVA- and HEL-specific DTH responses, whereas the CD4+ T cells from non-tolerized donors failed to induce tolerance to either antigen in the recipient (Fig. 4). In contrast to the CD4+CD25+ subset, the CD4+CD25 T cells only suppressed the DTH response against OVA, but not the DTH response against HEL. This demonstrates that suppression by the CD4+CD25 population requires the antigen used for tolerization, whereas suppression by the CD4+CD25+ T cells is not specific for the applied antigen.
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Discussion |
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We report that in mice, application of OVA via the nasal mucosa induces CD4+ Tr cells in the spleen that mediate tolerance by transferring their regulatory capacity to naive T cells in vivo. These Tr cells retain their plasticity in polarized microenvironments, as demonstrated by the finding that mucosal Tr cells recovered from a Th2-biased mouse are capable of suppressing both Th1 and Th2 responses, and with equal potency as mucosal Tr cells recovered from a Th1-biased mouse. Moreover, both CD4+CD25+and CD4+ CD25 subsets exerted regulatory activity. However, only the CD4+CD25 suppressed responses specific for the antigen used for tolerization.
Our observation that mucosal Tr cells transfer their regulatory activity to naive T cells in a naive recipient resembles properties of Tr cells that were described in tissue transplantation studies (19). In these studies, tolerance induced to skin grafts using non-depleting anti-T cell mAb was reported as being infectious. It is unclear why mucosal Tr cells transfer their function instead of expanding exponentially. However, the characteristic of poor division has been observed for many cells with suppressor activity. For example, naturally existing CD25+ Tr have been reported to be anergic to stimulation via the TCR, but to proliferate and even lose their tolerogenic capacity upon addition of exogenous IL-2 (20,21).
The cellular interactions that regulate the transfer of the suppressive activity from a Tr cell to a naive T cell may comprise a sequence of events. Initially, after i.v. transfer and subsequent sensitization of the acceptor mouse with OVA, the Tr cell may encounter an APC that presents OVA in the vicinity of a naive CD4+ T cell. Upon recognition of antigen in the context of an APC the Tr cell will be activated and may regulate the naive T cell via multiple pathways. Regulation may involve direct signaling, e.g. via Notch, that has previously been suggested to block the differentiation of precursor T cells into both Th1 and Th2 cells (22). Furthermore, since T cells also express Notch ligands, the suppressed T cells may signal to adjacent naive T cells to further spread the block in differentiation and may thus render cells competent for subsequent regulation steps (23). Other regulatory mechanisms may be provided by the secretion of anti-inflammatory cytokines, such as IL-10 and TGF-ß by either the Tr cell or the APC (10,11,18,24). However, it has been shown that IL-10 does not play a role in nasal tolerance, since administration of anti-IL-10 mAb either during the induction or during sensitization does not restore DTH responses in nasally tolerized mice (13). A recent in vitro study using human naturally existing CD25+CD4+ Tr cells elegantly showed that these Tr cells need cellcell interaction for suppression and transfer of tolerogenic potencies, whereas the induced secondary CD4+ suppressor T cells do not (25). Instead, these cells inhibit via soluble mediators like TGF-ß. Whether these mechanisms are also operating in our in vivo model is unclear. However, the CD25 Tr subset may suppress differently than the CD25+ Tr population, since we observed that the former is antigen specific, whereas the latter is not. In addition, down-regulation of the response may involve surface molecules such as CTLA-4 and PD-1 (8,26). The contribution of each of these pathways is the subject of current investigation.
Surprisingly, the microenvironment in which the mucosal Tr cells spread their tolerance to naive cells does not affect the plasticity of the response. The potency to suppress Th1 and Th2 responses did not differ between Tr cells derived from a Th1-biased mouse and a Th2-biased mouse. Although these data suggest that Tr cells retain their plasticity, it cannot be excluded that within the Tr population Th1- or Th2-biased Tr cells exist. This broad down-regulatory activity is very encouraging when we consider the potential use of mucosal Tr cell induction for the treatment of immunological disorders that are characterized by highly polarized cytokine profiles. Further more, since previous and ongoing cytokine responses do not alter the suppressive capacity of the Tr cell, this observation indirectly underlines the tight regulation with which the Tr cell exerts its suppressive function.
To unravel the intrinsic properties of mucosal Tr cells, it is of key importance to determine their phenotype and to assess their relationship with other types of Tr cells that have been described. In this study, we demonstrate that both splenic CD4+CD25+ and CD4+CD25 T cell subsets isolated from mice that were tolerized intranasally with OVA were equally capable of suppressing a systemic challenge to OVA. However, only the CD4+CD25 T cell subset shows antigen specificity.
Our observation that the CD4+CD25+ T cell population suppresses responses to the unrelated antigen HEL is in accordance with the current literature and suggests that these cells may represent a peripheral version of the thymus-derived CD4+CD25+ Tr cells (6,27). However, if the CD4+CD25+ Tr subset would largely be constituted of thymus-derived Tr cells, it could be expected that the CD4+CD25+ subset from naive mice would also suppress the DTH response. Since the latter subset failed to induce tolerance in the recipient mice at the same ratios, it may imply that the CD4+CD25+ Tr cells from nasally tolerized mice are indeed mucosally induced, as has been suggested for low-dose oral tolerance induction (28,29). The absence of suppressive activity by the CD25+ Tr subset from naive mice could be explained by the fact that it may depend on the ratio of suppressor and effector cells.
We are the first to report that CD4+CD25 T cells induced by nasal antigen application suppress systemic challenge in an antigen-specific manner. It is questioned where these T cells are generated. We have previously shown that the cervical lymph nodes are essential for the induction of nasal tolerance (12) and therefore we hypothesize that the application of OVA via the nasal mucosa causes naive T cells to differentiate into CD4+CD25 Tr cells under the influence of a specific microenvironment present only in these lymph nodes. Moreover, once mucosal Tr cells have been generated, other naive CD4+ T cells may acquire their suppressive capacity in non-mucosal tissue via the mechanism of infectious tolerance. Whether the CD4+CD25 T cells are related to the recently described naturally existing CD4+CD25+ Tr subset is unclear. To our knowledge, there is no evidence that the thymus exports any CD4+ T cells pre-committed to immune regulation within the CD4+CD25 subset (30).
In sum, we report that in nasal tolerance, Tr cells are operating that transfer their tolerogenic potencies to naive T cells. Moreover, we identified a CD4+CD25 Tr subset that is antigen specific, but exerts its suppressive function regardless of initial or ongoing cytokine polarization.
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Acknowledgements |
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Abbreviations |
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CTLAcytotoxic T lymphocyte-associated antigen
DCdendritic cell
DTHdelayed-type hypersensitivity
IFAincomplete Freunds adjuvant
HELhen egg lysozyme
OVAovalbumin
PEphycoerythrin
TGFtransforming growth factor
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References |
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