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B7/CTLA4 pathway is essential for generating regulatory cells after intratracheal delivery of alloantigen in mice1

Akiyama, Yoshinobu2; Shirasugi, Nozomu3; Uchida, Norio2; Matsumoto, Kenji2; Kitajima, Masaki2; Bashuda, Hisashi4; Yagita, Hideo4; Okumura, Ko4; Aramaki, Osamu3; Niimi, Masanori3 5

Immunobiology
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Background.  The mechanism of hyporesponsiveness induced by intratracheal (IT) delivery of alloantigen was examined and its effect on cardiac graft survival was assessed in studies in mice.

Methods.  In CBA (H2k) mice, donor splenocytes were given by IT delivery 7 days before transplantation of a C57BL/10 (H2b) heart. To determine whether regulatory cells were involved in hyporesponsiveness, splenocytes from mice given IT delivery of alloantigen and antibodies for B7–1, B7–2, or CTLA4 were adoptively transferred to naïve secondary recipients 7 days after delivery; those recipients underwent heart transplantation the same day. Effects on cell proliferation and cytokine production of splenocytes from mice given IT delivery of alloantigen were examined in mixed leukocyte cultures (MLC).

Results.  Cardiac graft survival was significantly prolonged in mice given IT delivery of alloantigen (median survival time [MST], 81 days); those given syngeneic splenocytes rejected grafts acutely (MST, 7 days;P <0.05). Adoptive transfer of splenocytes also significantly prolonged survival of cardiac grafts in secondary recipients (MST, 62 days). When B7–1, B7–2, or CTLA4 antibody was combined with IT delivery of alloantigen in the first recipient, all grafts were rejected within 14 days in second recipients after adoptive transfer. In mixed leukocyte cultures, splenocytes from these mice did not respond to alloantigen and production of interleukin-4 and interleukin-10 was increased.

Conclusions.  Donor splenocytes delivered IT induced hyporesponsiveness and regulatory cells in our animal model, and such induction was dependent on B7–1, B7–2, and CTLA4 signals.

2 Department of Surgery, Keio University School of Medicine, Tokyo, Japan.

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

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

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

1 This work was supported by the Research Fund of Mitsukoshi Health and Welfare Foundation.

Received 20 November 2001.

Revision Requested 10 December 2001.

Accepted 13 March 2002.

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INTRODUCTION

Donor-specific transfusion is known to induce donor-specific hyporesponsiveness over the histocompatibility barrier in clinical and experimental transplantation (1). Proposed mechanisms for this hyporesponsiveness include clonal deletion or inactivation (2), induction of suppresser cells (3,4), induction of blocking alloantibodies (5), or a combination of these factors. Hyporesponsiveness can also be induced by oral delivery of alloantigen: use of the phenomenon of oral tolerance is well established in treating autoimmune diseases in animal models and humans (6) and in transplantation (7,8). It was reported that oral administration of allogeneic cells produced antigen-specific prevention of sensitization by skin grafts and down-regulation of alloimmune responses in cardiac allografts, and those multiple mechanisms, including deletion, anergy, and active suppression, are involved in mediating oral tolerance (6). In contrast, induction of tolerance by means of the respiratory mucosa has been described in only a few studies, most of which addressed autoimmune or allergy diseases (9,10). Previously, however, we found that intratracheal (IT) delivery of alloantigen-induced hyporesponsiveness to fully allogeneic cardiac grafts in mice (11). We examined whether the hyporesponsiveness induced by such delivery of alloantigen involved immune regulation in response to alloantigen.

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MATERIALS AND METHODS

Mice

Male C57BL/10 (H2b), CBA (H2k), and BALB/c (H2d) mice were purchased from Sankyo (Tokyo, Japan). They were housed in standard facilities at the Biomedical Service Unit of Teikyo University and used between the ages of 8 and 12 weeks, in accordance with university guidelines for animal use and care.

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Preparation of Splenocytes and Delivery

Splenocytes were used as the source of alloantigen. Single-cell suspensions were depleted of erythrocytes by hypotonic lysis with water for 5 sec. CBA mice were given splenocytes from C57BL/10, CBA, or BALB/c mice by IT delivery. For IT delivery, general anesthesia was induced, the trachea was exposed by dissection of the overlying muscles, and 100 μL of phosphate-buffered saline with 1×107 splenocytes was injected into the trachea by using a 30-gauge needle and a 1-mL syringe. Immediately afterward, the skin was closed with single-layer sutures. For intravenous (IV) delivery, 100 μL of phosphate-buffered saline with 1×107 splenocytes was injected into the tail vein using a 30-gauge needle and a 1-mL-syringe.

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Heart transplantation

A cardiac graft from a C57BL/10 or BALB/c mouse was transplanted into the abdomen of a CBA mouse 7 days after IT delivery of alloantigen (Fig. 1a). After induction of anesthesia, fully vascularized heterotopic hearts were transplanted using microsurgical techniques (12). Graft function was monitored by palpation of the grafted hearts at least three times per week. Rejection was confirmed by electrocardiography, histologic examination, and direct visualization of the graft.

Figure 1

Figure 1

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Adoptive Transfer of Cells from Pretreated Mice into Naïve Mice

CBA mice were given IT delivery of splenocytes (1×107) from C57BL/10, CBA, or BALB/c mice, either alone or with intraperitoneal (IP) injection of 100 μg rat monoclonal antibody (mAb) specific for mouse B7–1 (1G10; PharMingen, San Diego, CA) (15), B7–2 (GL-1; PharMingen) (13), or CTLA4 (UC10–4F10–11; PharMingen) (14) or an isotype control antibody (R35–95, A19–3; PharMingen). Seven days later, splenocytes (5×107) from these pretreated CBA mice (primary recipients of splenocytes) were adoptively transferred by intravenous (IV) administration to naive CBA mice (secondary recipients). A cardiac graft from a C57BL/10 or BALB/c mouse was transplanted into each secondary recipient immediately after adoptive transfer (Fig. 1b).

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Mixed Leukocyte Cultures (MLC) for Studies of Cell Proliferation

A suspension of splenocytes (3×106) was resuspended in 2 mL complete medium with 100 μg/mL mitomycin C (MMC) (Kyowa Hakko, Osaka, Japan) and incubated for 30 min at 37°C. The complete medium was RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with HEPES (5 mM/L; Sigma, St Louis, MO), penicillin (100 μg/mL; Life Technologies), streptomycin (100 μg/mL; Life Technologies), 2-mercaptoethanol (50 μM/L; Sigma), and 10% fetal calf serum (Life Technologies). After the treatment, the cell suspension was washed three times. Cell viability after MMC treatment, as assessed by trypan blue (Cosmo Bio, Tokyo, Japan) exclusion test, was more than 90%.

Stimulator cells were prepared from C57BL/10 (allogeneic) splenocytes or CBA (syngeneic) splenocytes and treated with MMC. Responder cells were co-cultured with MMC-treated stimulator cells (1×106) in complete medium in a humidified 5% CO2 atmosphere (CH-16M; Hitachi, Tokyo, Japan) at 37°C in 96-well, flat-bottomed tissue-culture plates (Iwaki Scitech Division, Tokyo, Japan) for 3 to 6 days. Proliferation activity was assessed by using an enzyme-linked immunosorbent assay (ELISA) (Biotrak, version 2; Amersham, Little Chalfont, UK) (15) and an ELISA reader (EL ×800 Universal Microplate Reader; Bio-Tek Instruments, Highland Park, VT) at 450 nm. For the experimental groups of responders and stimulators, MMC-treated C57BL/10 splenocytes (1×106) were added to splenocytes (0.25×106) from either CBA mice given IT delivery of alloantigen or naïve CBA mice. To assess regulator function, splenocytes (0.25×106) from either CBA mice given IT delivery of alloantigen, or naive CBA mice were added to MMC-treated C57BL/10 splenocytes (1×106) in combination with naive CBA splenocytes (0.25×106).

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Production of Cytokines

ELISA was performed to detect interleukin (IL)-2, -4, -10, and interferon (INF)-γ in culture medium on day 4 of MLC. The IL-10 capture mAb (JES5–2A5), detection mAb (JES5–16E3), and recombinant standard were from PharMingen (16). The capture and detection mAbs for IL-2 (JES6–1A12 and JES6–5H4, respectively), IL-4 (BVD-1D11 and BVD-24G2), and INF-γ (R4–6A2 and XMG1.2) were from Caltag Laboratories (Burlingame, CA) (17). Recombinant standards for IL-2, IL-4, and INF-γ were from PeproTech (London, UK). Absorbance was read at 405 nm by using an ELISA reader (EL ×800 Universal Microplate Reader).

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Statistical Analysis

Cardiac allograft survival in groups of mice was compared with Mann-Whitney U tests, and results in MLC were compared with unpaired Student’s t tests. All statistical analyses were done with StatView SE + Graphic software (Abacus Concepts, Cary, NC). A P value less than 0.05 was considered to represent statistical significance.

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RESULTS

Prolonged Survival of Fully Allogeneic Cardiac Grafts in Mice Pretreated with IT Delivery of Alloantigen

Naïve CBA mice showed acute rejection of cardiac grafts from C57BL/10 mice (median survival time [MST] ±SD, 7±1.0 days;Fig. 2a). When CBA mice were pretreated with donor C57BL/10 splenocytes given by IT delivery, graft survival increased significantly (MST±SD, 81±46.5 days;P <0.05). Intratracheal administration of phosphate-buffered saline (i.e., no treatment) alone did not increase graft survival (MST±SD, 7±3.8 days). Also, IV injection of C57BL/10 splenocytes was significantly less effective in prolonging graft survival than IT delivery (MST±SD, 14±14.2 and 81±46.5 days, respectively). The effect of IT pretreatment was donor-specific because IT administration of BALB/c splenocytes did not increase survival of C57BL/10 cardiac allografts (MST±SD, 7±3.2 days;Fig. 2b). CBA mice that were pretreated with IT delivery of C57BL/10 splenocytes rejected BALB/c cardiac grafts.

Figure 2

Figure 2

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Prolonged Survival of Cardiac Allografts after Adoptive Transfer of Splenocytes from Mice Pretreated with IT Delivery of Alloantigen

To determine whether regulatory cells were involved in the hyporesponsiveness to alloantigen, naïve CBA mice (secondary recipients) were given splenocytes from CBA mice given IT delivery of alloantigen (primary recipients) in an adoptive transfer. When primary recipients were given C57BL/10 splenocytes, secondary recipients accepted C57BL/10 but not BALB/c cardiac grafts (MST±SD, 62±25.9 and 7±1.0 days, respectively;Fig. 3a). When primary recipients were given CBA and BALB/c splenocytes, secondary recipients had acute rejection of C57BL/10 grafts (MST±SD, 7±1.0 and 7±0.7 days, respectively). Thus, alloantigen-generated regulatory cells were induced by IT delivery of alloantigen in primary recipients.

Figure 3

Figure 3

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Graft Survival after Adoptive Transfer from Mice Coadministration of B7–1, B7–2, or CTLA4 mAb

To assess the role of costimulatory pathways in generating regulatory cells in our model, IT delivery of alloantigen was combined with IP injection of mAb specific for B7–1, B7–2, or CTLA4. When primary recipients of alloantigen were also given anti-B7–1, B7–2, or CTLA4 mAbs, all grafts in secondary recipients (those receiving adoptive transfer of splenocytes) were rejected within 20 days (MST±SD, 13±2.3, 13±7.3, and 13±4.5 days, respectively;Fig. 3b). However, when primary recipients were given no antibody or isotype control antibody, graft survival in secondary recipients was significantly prolonged (MST±SD, 62±25.9 and 55±32.8 days, respectively). These findings show that blockade of costimulatory pathways by mAbs abrogated induction of regulatory cells in our model.

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Cell Proliferation

In MLC, maximum proliferation of CBA splenocytes against MMC-treated C57BL/10 splenocytes occurred on day 4. Proliferation of responder splenocytes from CBA mice given IT delivery of C57BL/10 antigen was significantly less than that of responder splenocytes from naïve CBA mice (Fig. 4a). When splenocytes from CBA mice given IT delivery of C57BL/10 antigen were added to a mixed culture of naïve CBA splenocytes and MMC-treated C57BL/10 splenocytes, the response was significantly suppressed compared with that occurring when naïve CBA splenocytes were added (Fig. 4b).

Figure 4

Figure 4

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Production of Cytokines

ELISA analyses found that when splenocytes from CBA mice given IT delivery of C57BL/10 antigen were used as responders, production of IL-4 and IL-10 was significantly higher and production of IL-2 was significantly lower than when splenocytes from naïve CBA mice were used (Fig. 5). There was no difference between the two groups in production of INF-γ.

Figure 5

Figure 5

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DISCUSSION

In this study, IT delivery of C57BL/10 donor splenocytes induced prolonged survival of C57BL/10 heart graft and generated regulatory cells in CBA recipient. This IT delivery of donor splenocytes also induced hyporesponsiveness to cardiac graft in other strain combination (C3H heart to C57BL/6 recipient, MST=40 days). Our protocol could also induce modest prolonged survival of skin grafts (MST=12 days) compared with skin graft in naive mice (MST=8 days). Moreover, the protocol induced modest prolongation of xenogeneic heart graft from SD rat in CBA recipient (MST=11 days) compared with xenogeneic heart graft in naive mice (MST=9 days).

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Induction of Hyporesponsiveness and Regulatory Cells by IT Delivery of Alloantigen

It was previously reported that alloimmune response is regulated by lymphocytes that induce allograft tolerance (18). We confirmed that IT delivery of alloantigen generated regulatory cells to alloantigen and induced hyporesponsiveness to allografts. To our knowledge, this is the first report of generation of regulatory cells in mice without transplantation. Previously, regulatory cells were transferred from animals with long-term grafts to naïve syngeneic secondary recipients in many models of operational tolerance. This led to development of the idea that regulation may be a fundamental component of the mechanisms responsible for inducing and maintaining tolerance to alloantigen. However, induction of regulatory cells in those models required more than 100 days after grafting (19,20). In our model, adoptive transfer of cells from mice given IT delivery of donor splenocytes and did not undergo transplantation resulted in hyporesponsiveness in naïve secondary recipients in only 7 days. It is unclear why such a short period after administration of donor splenocytes was sufficient to generate regulatory cells.

The respiratory tract contains bronchus-associated lymphoid tissue, which is similar to gut-associated lymphoid tissue (GALT). GALT was found to play an important role in the mucosal immune system (20,21), and several studies showed that GALT functions as an immunologic apparatus for inducing oral tolerance (22). However, our previous studies (11) showed that cells delivered into the trachea were lysed and likely presented as peptides associated with recipient major histocompatibility complex molecules (i.e., the indirect pathway) (11). Thus, it is probable that bronchus-associated lymphoid tissue functions as a special microenvironment to induce regulatory cells and hyporesponsiveness to allografts by means of the indirect pathway. In terms of phenotype of the regulatory population, when the transferred splenocytes from pretreated with IT delivery of donor splenocytes were depleted of CD25 positive cells by negative selection, the CD25 negative population could not induce prolonged survival of cardiac grafts (MST=8 days) (23).

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Need for Costimulatory Signal to Induce Regulatory Cells

Robust activation of naïve T cells to alloantigen requires two signals: through the T-cell receptor and costimulatory molecules. The B7/CD28 pathway is one of the most important costimulatory pathways. B7–1 and B7–2 molecules on antigen-presenting cells deliver a costimulatory signal by interacting with CD28 expressed on resting T cells (24). It was previously reported that CTLA4 immunoglobulin, which blocks both B7–1 and B7–2, can be used to induce transplantation tolerance to allografts (25). However, other data suggested that negative signals of CTLA4 have a key role in induction of tolerance and that CD28 is the dominant T-cell costimulatory pathway for most in vivo immune responses, including rejection of transplants (14). Only limited information on the role of the CTLA4 pathway during an alloimmune response is available, and there is none on its role in alloresponse in the context of CD28 blockade. Given the suggested requirement for CTLA4 engagement to induce anergy (14), this pathway might be required for the induction or maintenance phases of transplant tolerance (26).

In this study, we examined the importance of CTLA4-mediated signals in long-term allograft survival. To assess the role of negative signals through CTLA4, we used anti-CTLA4 mAb, which acts as a blocking mAb that enhances T-cell responses in vivo. Our finding that blockade of B7–1/CTLA4 and B7–2/CTLA4 by anti-B7–1 and anti-B7–2 mAbs abrogated long-term graft survival after IT delivery of alloantigen suggested that the B7–1/B7–2 pathway may be directly involved in induction of hyporesponsiveness (11). Our results using anti-CTLA4 mAb suggest that interaction between B7 and CTLA4 provides a crucial CD28-independent negative signal that presumably induces regulatory cells. Several previous investigations demonstrated that CTLA4 signal was necessary for induction of tolerance (27,28) and of regulatory cells in autoimmune disease (28,29). We showed clearly that the B7/CTLA4 pathway was also essential for induction of regulatory cells and subsequent hyporesponsiveness in organ transplantation. Furthermore, CD28-deficient mice (on C57BL/6 background) could be induced hyporesponsiveness to fully allogeneic C3H cardiac graft by intratracheal delivery of donor splenocytes (MST=50 days) and this effect was abrogated by administration of CTLA4 immunoglobulin (MST=12 days). Moreover, the effect was also abrogated when the pretreatment was combined with anti-CD86 antibody (MST=11 days). Data demonstrate that induction of hyporesponsiveness by IT delivery of donor splenocytes may be independent with the B7–2/CD28 pathway and that the CTLA4 signal may play an important role in it.

Regarding other costimulatory pathways, our preliminary data demonstrated that anti-CD40 ligand antibody (MR1) augmented the ability of intratracheal delivery of donor splenocytes to induce indefinite survival (>100 days) of C57BL/10 cardiac grafts in CBA recipients. Moreover, IT delivery of donor splenocytes combined with MR1 induced prolonged survival of C57BL/6 skin grafts in CBA mice (MST=25 days).

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Role of Cytokines in Hyporesponsiveness Induced by IT Delivery of Alloantigen

Our studies in MLC showed that IT delivery of alloantigen suppressed proliferation of splenocytes from CBA mice against C57BL/10 antigen. Moreover, when splenocytes from naïve CBA mice were co-cultured with splenocytes from CBA mice given IT delivery of alloantigen, cell proliferation was markedly suppressed. These data indicate the existence of regulatory cells induced by IT delivery of alloantigen. Furthermore, examination of cytokines in MLC showed that production of IL-10 and IL-4 was higher in splenocytes from mice treated with IT delivery of alloantigen than in controls. IL-10, which is the anti-inflammatory properties of cytokines, was shown previously to inhibit antigen-induced proliferation and cytokine synthesis by T cells (30). Moreover, it was reported that IL-10 is required for functioning of regulatory cells, which inhibit graft rejection in vivo (31). Our results suggest that suppression of IL-2 and induction of IL-4 and IL-10 contribute to the immune regulation and hyporesponsiveness induced by IT delivery.

In conclusion, we found that IT delivery of donor splenocytes significantly increased survival of fully mismatched cardiac allografts in mice and that this effect was donor specific. The hyporesponsiveness resulting from IT delivery of alloantigen was dependent on active suppression by regulatory cells, and both B7–1/CTLA4 and B7–2/CTLA4 signals were necessary for induction of regulatory cells in our model. Production of both IL-4 and IL-10 was up-regulated in our in vitro assay.

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