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Interleukin-10 but not Transforming Growth Factor-β is Essential for Generation and Suppressor Function of Regulatory Cells Induced by Intratracheal Delivery of Alloantigen

Aramaki, Osamu1,2; Inoue, Fumihiko1,3; Takayama, Tadatoshi2; Shimazu, Motohide3; Kitajima, Masaki3; Ikeda, Yoshifumi1; Okumura, Ko4; Yagita, Hideo4; Shirasugi, Nozomu1,5; Niimi, Masanori1

doi: 10.1097/01.TP.0000153151.16350.53
Experimental Transplantation
Free

Background. We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-β in induction and effector phases of the regulatory cells.

Methods. CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-β monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-β mAb was administered weekly into the secondary recipients after the adoptive transfer.

Results. Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-β mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-β mAb did not abrogate suppressor function of the regulatory cells (MST 53 days).

Conclusion. IL-10 but not TGF-β was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.

1 Department of Surgery, Teikyo University, Tokyo, Japan.

2 Department of Digestive Surgery, Nihon University, Tokyo, Japan.

3 Department of Surgery, Keio University, Tokyo, Japan.

4 Department of Immunology, Juntendo University, Tokyo, Japan.

This work is supported partially by Grant Research on Cerebro- and Cardiovascular Disorder by Mitsubishi Pharma Research Foundation.

5 Address correspondence to: Nozomu Shirasugi, Department of Surgery, Teikyo University, 2–11–1 Kaga, Itabashi-ku, Tokyo 173–8605, Japan. E-mail: mniimi@med.teikyo-u.ac.jp.

Received 24 March 2004. Accepted 3 November 2004.

Transplantation tolerance is mediated by central or peripheral mechanisms. Several mechanisms of peripheral tolerance have been proposed, including T-cell anergy, T-cell deletion, and active immune suppression. Recently, among those, the active suppression by regulatory cells has been shown to be important for maintenance of transplantation tolerance (1, 2).

Presentation of soluble antigens to the respiratory and intestinal immune systems typically induces antigen-specific unresponsiveness (3). Oral administration of antigen is a classical method to induce antigen-specific peripheral tolerance and regulatory cells (4), termed as oral tolerance. Oral tolerance has been exploited to treat autoimmune diseases in animal models and humans (4) and has been also used in transplantation (5). The immune system of the respiratory mucosa has a microenvironment similar to that associated with oral tolerance (6). However, induction of tolerance and regulatory cells by means of the respiratory mucosa has been described only in a few studies of autoimmune (7) and allergic diseases (8).

Immunosuppressive cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β are powerful down-regulators of immune responses. Both IL-10 and TGF-β have been implicated in the induction and function of regulatory cells (4, 9). Blockade of IL-10 either by administration of a neutralizing monoclonal antibody (mAb) specific for IL-10 or by use of IL–10-deficient mice abrogated the function of regulatory cells to prevent colitis (10) and to prevent allograft rejection in a transplantation model (11). Neutralizing mAbs specific for TGF-β blocked immunosuppressive function of regulatory cells to prevent colitis in a mouse colitis model (12, 13). Moreover, it has been shown that IL-10 and TGF-β play critical roles in the induction of IL–10-producing type-1 regulatory T (Tr1) cells and TGF–β-producing type-3 helper T (Th3) cells, which are associated with mucosal tolerance (4, 14).

We have previously reported that intratracheal delivery of donor splenocytes induced donor-specific regulatory cells that afforded prolonged cardiac allograft survival in mice (15). In this study, we investigated the involvement of IL-10 and TGF-β in the generation and function of these regulatory cells by using neutralizing anti-IL-10 and anti-TGF-β mAbs.

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MATERIALS AND METHODS

Mice

Inbred male C57BL/10 (H-2b), CBA (H-2k), and BALB/c (H-2d) mice at 8 to 12 weeks of age were purchased from Sankyo Ltd. (Tokyo, Japan), housed in conventional facilities in the Biomedical Service Unit of Teikyo University, and used in accordance with protocols for animal experimentation approved by the Animal Care and Use Committee of Teikyo University.

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Antibodies

Neutralizing anti-mouse IL-10 (JES5–2A5, rat immunoglobulin [Ig]G1) and anti-mouse TGF-β (1D11, rat IgG1) mAbs were prepared from the hybridomas obtained from ATCC (Manassas, VA) by protein G affinity chromatography. Control rat IgG was purchased from Sigma Chemical Co. (Tokyo, Japan).

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Intratracheal Delivery of Alloantigen

Splenocytes were used as the source of alloantigen. Single-cell suspensions were depleted of erythrocytes by hypotonic lysis with water for 5 seconds. CBA mice were placed under general anesthesia, the trachea was exposed by dissection of the overlying muscles, and 1×107 C57BL/10 splenocytes in 100 μL of phosphate-buffered saline were injected into the trachea by using a 30-gauge needle and 1 mL syringe. Immediately afterward, the skin was closed with single-layer sutures. All mice survived the intratracheal delivery pretreatment, and no adverse effects of the treatment were observed during the observation period.

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Heart Transplantation

A fully vascularized heterotopic heart graft from C57BL/10 or BALB/c (third-party) mouse was transplanted into the abdomen of untreated or pretreated CBA mice using microsurgical techniques (16). The allografts were monitored by daily palpation. Cardiac graft rejection was defined as complete cessation of graft contraction and was confirmed by direct visualization and histologic assessment of the graft.

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Adoptive Transfer Experiments

Splenocytes were prepared from CBA mice that had been given the intratracheal delivery of 1×107 C57BL/10 splenocytes 7 days before. Naive CBA mice (secondary recipients) were intravenously injected the splenocytes (5×107) and then received a transplant with a heart from C57BL/10 or BALB/c mouse on the same day.

In an adoptive transfer study of CD4+ cells, CD4+ cells were purified from pooled splenocytes that were harvested from the pretreated CBA mice with intratracheal delivery 7 days before by positive selection using MACS CD4 Microbeads (Miltenyi Biotec Inc., Auborn, CA; purity > 98%). Naive CBA mice (secondary recipients) were intravenously injected the CD4+ cells (2×107) and then received a transplant with a heart from C57BL/10 mouse on the same day.

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Administration of Neutralizing mAbs in the Induction Phase

CBA mice (primary recipients) were given the intratracheal delivery of C57BL/10 splenocytes (1×107) on day −7 and intraperitoneal administration of anti-IL-10 mAb, anti-TGF-β mAb, or control IgG (1 mg/mouse) daily from day −7 to −5. The dosage and schedule of mAbs was determined to be effective based on results in other experiment (17–20). Their splenocytes were subjected to the adoptive transfer on day 0 as described above.

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Administration of Neutralizing mAbs in the Effector Phase

Splenocytes were prepared from CBA mice (primary recipients) that had been given the intratracheal delivery of C57BL/10 splenocytes (1×107) 7 days before and adoptively transferred into naive CBA mice (secondary recipients) as described above. The secondary recipients were given C57BL/10 cardiac allograft on the same day (day 0) and intraperitoneal administration of anti-IL-10 mAb, anti-TGF-β mAb, or control IgG (1 mg/mouse) on day 0, 7, and 14 (17–20).

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Direct Effect of Neutralizing Abs on Allograft Rejection

To examine direct effect of mAbs and control IgG on allograft rejection, naive CBA mice receiving transplants with a C57BL/10 heart were treated with intraperitoneal administration of neutralizing mAb specific for either IL-10 or TGF-β or control IgG (1 mg/mouse daily on day 0–2) (17–20).

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Mixed Lymphocyte Culture

CBA mice were given intratracheal delivery of C57BL/10 splenocytes (1×107) on day −7 with or without intraperitoneal administration of anti-IL-10 mAb, or anti-TGF-β mAb, or control IgG (1 mg/mouse) daily from day −7 to −5. On day 0, the mice were killed, and their splenocytes were used as the responder. Splenocytes from naive C57BL/10 (allogeneic) or CBA (syngeneic) mice were treated with 100 μg/mL mitomycin C (MMC) (Kyowa Hakko, Osaka, Japan) for 30 minutes at 37°C and then used as the stimulator. The responder cells (2.5×106 cells/mL) were cocultured with the stimulator cells (10×106 cells/mL) in RPMI1640 medium containing 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37°C in 96-well, flat-bottomed tissue-culture plates (Iwaki Scitech Division, Tokyo, Japan) for 3 to 6 days.

To analyze regulatory T-cell function, anti-IL-10 mAb, anti-TGF-β mAb, or control IgG was added at the beginning of the culture. In other words, CBA mice were given intratracheal delivery of C57BL/10 splenocytes (1×107) on day −7. On day 0, the mice were killed and their splenocytes (2.5×106 cells/mL) were cocultured with MMC-treated C57BL/10 splenocytes (10×106 cells/mL) in the presence or absence of anti-IL-10 mAb, or anti-TGF-β mAb, or control IgG (50 μg/mL) for 4 days.

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Proliferation Assay

Proliferation was assessed by using an enzyme-linked immunosorbent assay (ELISA) for bromodeoxyuridine incorporation (Biotrak, version 2; Amersham, Little Chalfont, UK) (21) according to the manufacturer’s instruction.

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ELISA for Cytokine Production

IL-10 and TGF-β level in supernatant of the mixed lymphocyte culture (MLC) on day 4 were determined by ELISA. The capture mAb (JES5–2A5), detection mAb (JES5–16E3), and recombinant standard for IL-10 were obtained from BD PharMingen (San Diego, CA). TGF-β was measured by using TGF-β1 Immunoassay System (Genzyme-Techne, Minneapolis, MO) according to the manufacturer’s instruction.

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Histologic Analysis

Grafts were fixed in 5% neutral buffered formalin and embedded in paraffin by standard procedures. Tissue samples (5-μm thick) were sectioned with a microtome and stained with hematoxylin-eosin.

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Statistical analysis

Statistical analysis was performed using StatView SE + Graphic (Abacus Concepts Inc, Cary, NC). Graft survival data are indicated as median survival time (MST). Difference in MST between two groups was evaluated by Mann-Whitney U test. Proliferation and ELISA data are indicated as the mean±standard deviation (SD) of 7 replicates in one representative experiment. Differences between two groups were evaluated by Student’s t test.

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RESULTS

Induction of Donor-Specific Regulatory Cells by Intratracheal Delivery of Donor Splenocytes

As we previously reported (15), the intratracheal delivery of C57BL/10 splenocytes into CBA recipients 7 days before transplantation significantly prolonged the survival of C57BL/10 cardiac allografts (MST 65 days) as compared with acute rejection in the untreated recipients (MST 7 days) (Fig. 1A). This effect was donor-specific because BALB/c cardiac grafts were acutely rejected (MST 7 days) (Fig. 1A).

FIGURE 1.

FIGURE 1.

Adoptive transfer of splenocytes from the CBA mice that had been given the intratracheal delivery of C57BL/10 splenocytes 7 days before into naive CBA mice (secondary recipients) significantly prolonged the survival of C57BL/10 cardiac allografts in the secondary recipients (MST 68 days) as compared with adoptive transfer of splenocytes from untreated CBA mice (MST 12 days) (Fig. 1B). This effect was again donor-specific because BALB/c cardiac allografts were acutely rejected (MST 10 days) (Fig. 1B). These results indicated that donor-specific regulatory cells, which prolonged cardiac allograft survival, were generated by the intratracheal delivery of donor splenocytes 7 days before transplantation.

When CD4+ cells were purified from the pretreated CBA mice with intratracheal delivery 7 days before and 2×107 of the CD4+ cells were adoptively transferred into naive CBA recipients that received transplantation of C57BL/10 hearts immediately afterward, allograft survival was remarkably prolonged (survival time 61, 61, 68, 69, >100 days; MST 68 days). On the other hand, adoptive transfer of CD4+ cells from naive CBA mice did not induce prolongation of graft survival (survival time, 8, 8, 11, 14, 21 days; MST 11 days). These data indicate that CD4+ cells might be one of the regulatory populations in our intratracheal delivery model.

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Effect of Anti-IL-10 and Anti-TGF-β mAb Treatment on the Induction of Regulatory Cells

To address the possible contribution of IL-10 and TGF-β to the induction of donor-specific regulatory cells, we administered neutralizing anti-IL-10 or anti-TGF-β mAb for 3 consecutive days after the intratracheal delivery of C57BL/10 splenocytes into CBA mice (primary recipients). Seven days after the intratracheal delivery, their splenocytes were adoptively transferred into naive CBA mice (secondary recipients), which were then challenged with C57BL/10 cardiac allografts to assess the development of donor-specific regulatory cells in the primary recipients. As shown in Figure 2, the anti-IL-10 mAb treatment abrogated the development of regulatory cells in the primary recipients because the cardiac allograft survival in the secondary recipients was significantly shortened (MST 20 days) as compared with the control IgG treatment (MST 79 days). In contrast, the anti-TGF-β mAb treatment in the primary recipients did not significantly affect the cardiac-allograft survival in the secondary recipients (MST 88 days). Administration of either anti-IL-10 or anti-TGF-β mAb without the intratracheal delivery of C57BL/10 splenocytes in the primary recipients did not significantly affect the acute rejection in the secondary recipients (Fig. 6). These results indicated that IL-10, but not TGF-β, was essential for the induction of donor-specific regulatory cells by the intratracheal delivery of donor splenocytes.

FIGURE 2.

FIGURE 2.

FIGURE 6.

FIGURE 6.

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Effect of Anti-IL-10 and Anti-TGF-β mAb Treatment on the Suppressor Function of Regulatory Cells

We next examined the possible contribution of IL-10 and TGF-β to the suppressor function of regulatory cells by administering neutralizing anti-IL-10 or anti- TGF-β mAb into the secondary recipients. As shown in Figure 3, the anti-IL-10 mAb treatment significantly shortened the cardiac-allograft survival in the secondary recipients (MST 27 days) as compared with the control IgG treatment (MST 58 days). In contrast, the anti-TGF-β mAb treatment did not show a significant effect (MST 53 days). Administration of either anti-IL-10 or anti-TGF-β mAb with or without adoptive transfer of splenocytes from untreated CBA mice did not significantly affect the acute rejection in the secondary recipients (data not shown). These results indicated that IL-10, but not TGF-β, was essential for the suppressor function of the regulatory cells induced by the intratracheal delivery of donor splenocytes.

FIGURE 3.

FIGURE 3.

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Effect of Anti-IL-10 and Anti-TGF-β mAb Treatment on the Development of IL-10 Producing Regulatory Cells

We next examined the effect of neutralizing anti-IL-10 and anti-TGF-β mAb treatment on the development of the regulatory cells and their IL-10 and TGF-β production after the intratracheal delivery of donor splenocytes. In MLC, maximal proliferation of CBA splenocytes against MMC-treated C57BL/10 splenocytes occurred on day 4. As we previously reported (15), the intratracheal delivery of C57BL/10 splenocytes into CBA mice induced proliferative hyporesponsiveness to the donor alloantigen upon restimulation in vitro. This induction of hyporesponsiveness was abrogated by the anti-IL-10 mAb treatment but not by the anti-TGF-β mAb treatment(Fig. 4A). Figure 4B shows that the intratracheal delivery of C57BL/10 splenocytes induced IL–10-producing regulatory cells, as indicated by the markedly enhanced production of IL-10 upon restimulation in vitro. This enhanced production of IL-10 in vitro was significantly inhibited by the anti-IL-10 mAb treatment but not by the anti-TGF-β mAb treatment in vivo (Fig. 4B). In contrast, the TGF-β production upon in vitro restimulation was not significantly affected by the intratracheal delivery of C57BL/10 splenocytes on the anti-IL-10 mAb treatment (Fig. 4C). Anti-TGF-β mAb treatment in vivo inhibited the TGF-β production upon restimulation in vitro (Fig. 4C). These data suggest that the administered anti-TGF-β mAb worked efficiently in vivo, although the dosage may not be saturating. Nevertheless, it did not affect the induction or function of the regulatory cells (Figs. 2 and 3). These results indicated that IL-10, but not TGF-β, played a critical role in the induction of the hyporesponsiveness and IL–10-producing regulatory cells by the intratracheal delivery of donor splenocytes.

FIGURE 4.

FIGURE 4.

FIGURE 4.

FIGURE 4.

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Effect of Anti-IL-10 and Anti-TGF-β mAb on the Hyporesponsiveness to Donor Alloantigen

We next examined the effect of neutralizing anti-IL-10 or anti-TGF-β mAb treatment on the hyporesponsiveness to donor alloantigen upon restimulation in vitro. As shown in Figure 5, the addition of anti-IL-10 mAb, but not anti-TGF-β mAb, was primarily responsible for the hyporesponsiveness to donor alloantigen induced by the intratracheal delivery of donor splenocytes.

FIGURE 5.

FIGURE 5.

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Direct Effect of Neutralizing Abs on Allograft Rejection

Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (MST 7 days) (Fig. 6). When CBA mice were given control IgG at the time of transplantation of a C57BL/10 heart, these mice rejected C57BL/10 cardiac grafts acutely (MST 7 days) (Fig. 6). CBA mice given either anti-IL-10 mAb or anti-TGF-β mAb at the time of transplantation of a C57BL/10 heart showed modest graft prolongation (MST 13 and 15 days, respectively), although anti-TGF-β mAb induced significant prolongation (P<0.05 compared with that in the control IgG-treated group).

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DISCUSSION

In our model, intratracheal delivery of C57BL/10 splenocytes induced prolonged survival of C57BL/10 heart graft in CBA mice. This effect was donor-specific and could be adoptively transferred into naive secondary recipients, indicating that donor-specific regulatory cells were generated in the primary recipients by the intratracheal delivery of donor splenocytes. We also found that intratracheal delivery of donor splenocytes suppressed proliferative response and increased IL-10 production upon restimulation with donor alloantigen in vitro. We have examined the contribution of IL-10 and TGF-β to the induction and effector phases of the regulatory cells and obtained the following conclusions: (1) IL-10, but not TGF-β, was essential for the induction of regulatory cells; (2) IL-10, but not TGF-β, was essential for the suppressor function of regulatory cells; and (3) IL-10, but not TGF-β, was responsible for the proliferative hyporesponsiveness.

Recently, several subsets of regulatory T cells have been characterized in both mice and humans, including Tr1 cells, Th3 cells, and CD25+ CD4+ Tr cells. Tr1 cells produce a high level of IL-10 and are involved in down-regulation of inflammatory immune responses (14). IL-10 is also a critical factor for the development of Tr1 cells. Th3 cells secret TGF-β and are involved in oral tolerance (4). The development and the suppressor function of Th3 require TGF-β. Some CD25+ CD4+ Tr cells also produce IL-10 and TGF-β, but their suppressor activity is cell-contact dependent and mainly mediated cell-surface TGF-β and cytotoxic T-lymphocyte antigen 4 (22).

Our present findings of the IL-10 dependence for induction and suppressor function suggest that the regulatory cells induced by intratracheal delivery of donor splenocytes presumably may consist of Tr1 cells. This is consistent with previous reports indicating that delivery of soluble antigen to the respiratory tract induced antigen-specific regulatory T cells in an IL–10-dependent manner (9). It has been suggested that IL–10-producing pulmonary dendritic cells (DC) play a critical role in the process. Likewise, IL–10-producing DC cross-presenting the intratracheally delivered donor alloantigen may play a critical role in the induction of donor-specific Tr1 cells in our model.

Alternatively, some donor antigen-presenting cells (APC), including DC, in the intratracheally delivered splenocytes may acquire the pulmonary DC-like tolerogenic property in the respiratory mucosal environment, which then induce Tr1 cells by direct presentation in an IL–10-dependent manner. Another possibility is that donor T cells in the intratracheally delivered splenocytes may first develop into Tr1 cells in response to IL–10-producing pulmonary DC and then migrate to the spleen where they inactivate host APC in an IL–10-dependent manner. However, these are not the cases because intratracheally delivered splenocytes stained with fluorescent dye (carboxyfluorescein succinimidyl ester) were not detected at all either in paratracheal lymph nodes or in the spleen (23). Furthermore, intratracheal delivery of donor Kb peptide also induced the regulatory cells and subsequent hyporesponsiveness to fully allogeneic cardiac graft (24). These data suggest that the intratracheally delivered splenocytes were destroyed and that the degraded donor peptide might be presented by way of the indirect pathway.

Our model of intratracheal delivery is very unique and useful for investigating the roles of regulatory cells because the regulatory cells were induced by intratracheal delivery of alloantigen alone without transplantation in only 7 days. Therefore, we can clearly examine mechanisms on the induction phase and suppressor function individually. Primary CBA recipients (i.e., after intratracheal delivery of alloantigen without transplantation) are investigated to examine the mechanism on induction phase. Secondary CBA recipients (i.e., after adoptive transfer and grafting heart) are investigated to examine the mechanism on suppressor function. In our study, the roles of IL-10 and TGF-β either in induction of the regulatory cells or for their suppressor function were determined in our model, combined with adoptive transfer system. To determine the roles of these cytokines in the induction of the regulatory cells, the CBA mice with intratracheal delivery, but not the transplant recipients, were given either anti-IL-10 mAb or anti-TGF-β mAb. Seven days later, splenocytes from these pretreated CBA mice were adoptively transferred into naive CBA recipients that underwent transplantation of cardiac allograft the same day of adoptive transfer. Adoptive transfer of the splenocytes from the mice pretreated with intratracheal delivery concomitant with anti-IL-10 mAb could not induce prolongation of allograft survival compared with that from the mice pretreated with intratracheal delivery alone. These data suggest that IL-10 is necessary for induction of regulatory cells. On the other hand, to determine the role of these cytokines for suppressor function of the regulatory cells, naive CBA recipients of allograft transplantation received either anti-IL-10 mAb or anti-TGF-β mAb treatment at the same time of the adoptive transfer of the splenocytes from the CBA mice pretreated with intratracheal delivery of alloantigen 7 days before (i.e., the splenocytes containing the regulatory cells). Administration of anti-IL-10 mAb abrogated prolongation of allograft survival that was induced by adoptive transfer of the regulatory cells. These data suggest that anti-IL-10 mAb also affected the suppressor function of the regulatory cells in the naive recipients.

The mice were given 1 mg of anti-TGF-β mAb intraperitoneally in this study, on the basis of results of other experiments (19, 20). Our data suggest that anti-TGF-β mAb treatment did not affect the induction or suppressor function of the regulatory cells (Figs. 2 and 3). It might be possible that this dose of anti-TGF-β mAb is not completely effective in neutralizing TGF-β in vivo. However, anti-TGF-β mAb in this dose in vivo presumably inhibited the TGF-β production upon restimulation in MLC (Fig. 4C). Furthermore, treatment with anti-TGF-β mAb at this dose induced significant prolongation of C57BL/10 allograft survival in CBA recipients (Fig. 6). These data suggest that the administered anti-TGF-β mAb at this dose worked efficiently in vivo in our model, although it may not have been saturating. In summary, our data suggest that IL-10, but not TGF-β, was required for generation and suppressor function of the regulatory cells by intratracheal delivery of alloantigen in our model.

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Keywords:

Hyporesponsiveness; Mucosal tolerance; Mouse; Cardiac transplantation; Adoptive transfer

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