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.
MATERIALS AND METHODS
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.
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.
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.
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).
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).
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).
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.
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.
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.
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.
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).
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.
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).
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).
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).
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.
1. Reinsmoen NL, Matas AJ, Donaldson K, et al. Impact of transfusions and acute rejection on posttransplantation donor antigen-specific responses in two study populations. Cooperative Clinical Trial in Transplantation Research Group. Transplantation 1999; 67: 697.
2. Hadley GA, Anderson CB, Mohanakumar T. Selective loss of functional antidonor cytolytic T cell precursors following donor-specific blood transfusions in long-term renal allograft recipients. Transplantation 1992; 54: 333.
3. Lenhard V, Massen G, Seifert P, et al. Characterization of transfusion-induced suppressor cells in prospective kidney allograft recipients. Transplant Proc 1982; 14 (2): 329.
4. Bean MA, Mickelson E, Yanagida J, et al. Suppressed antidonor MLR responses in renal transplant candidates conditioned with donor-specific transfusions that carry the recipient’s noninherited maternal HLA haplotype. Transplantation 1990; 49: 382.
5. Phelan DL, Rodey GE, Anderson CB. The development and specificity of antiidiotypic antibodies in renal transplant recipients receiving single-donor blood transfusions. Transplantation 1989; 48: 57.
6. Weiner HL. Oral tolerance. Proc Natl Acad Sci USA 1994; 91: 10762.
7. Sayegh MH, Zhang ZJ, Hancock WW, et al. Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered antigen. Transplantation 1992; 53: 163.
8. Hancock WW, Sayegh MH, Kwok CA, et al. Oral, but not intravenous, alloantigen prevents accelerated allograft rejection by selective intragraft Th2 cell activation. Transplantation 1993; 55: 1112.
9. Kreutzer B, Laliotou B, Cheng YF, et al. Nasal administration of retinal antigens maintains immunosuppression of uveoretinitis in cyclosporin-A-treated Lewis rats: future treatment of endogenous posterior uveoretinitis. Eye 1997; 11: 445.
10. Lowrey JL, Savage ND, Palliser D, et al. Induction of tolerance via the respiratory mucosa. Int Arch Allergy Immunol 1998; 116: 93.
11. Shirasugi N, Ikeda Y, Akiyama Y, et al. Induction of hyporesponsiveness to fully allogeneic cardiac grafts by intratracheal delivery of alloantigen. Transplantation 2001; 71: 561.
12. Niimi M, Hara M, Bushell A, et al. Results of heart transplantation. In: Timmermann W, Gassel H-J, Ulrich K, eds. Organ transplantation in rats and mice: Microsurgical techniques and immunological principles. Berlin, Springer, 1998: 637.
13. Bashuda H, Seino K, Kano M, et al. Specific acceptance of cardiac allografts after treatment with antibodies to CD80 and CD86 in mice. Transplant Proc 1996; 28: 1039.
14. Walunas TL, Lenschow DJ, Bakker CY, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1994; 1: 405.
15. Perros P, Weightman DR. Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine. Cell Prolif 1991; 24: 517.
16. Sander B, Hoiden I, Andersson U, et al. Similar frequencies and kinetics of cytokine producing cells in murine peripheral blood and spleen. Cytokine detection by immunoassay and intracellular immunostaining. J Immunol Methods 1993; 166: 201.
17. Abrams JS, Roncarolo MG, Yssel H, et al. Strategies of anti- cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev 1992; 127: 5.
18. Bushell A, Niimi M, Morris PJ, et al. Evidence for immune regulation in the induction of transplantation tolerance: A conditional but limited role for IL-4. J Immunol 1999; 162: 1359.
19. Ito H, Hamano K, Gohra H, et al. Role of microchimerism on long-term graft survival after donor-specific transfusion in a rat heart transplantation model. Transplant Proc 1998; 30: 3862.
20. Matsuura Y, Matsuoka T, Fuse Y. Ultrastructural and immunohistochemical studies on the ontogenic development of bronchus-associated lymphoid tissue (BALT) in the rat: special reference to follicular dendritic cells. Eur Respir J 1992; 5: 824.
21. Tango M, Suzuki E, Gejyo F, et al. The presence of specialized epithelial cells on the bronchus-associated lymphoid tissue (BALT) in the mouse. Arch Histol Cytol 2000; 63: 81.
22. Faria AM, Weiner HL. Oral tolerance: Mechanisms and therapeutic applications. Adv Immunol 1999; 73: 153.
23. Akiyama Y, Shirasugi N, Niimi M, et al. CD25+
regulatory cells generated by intratracheal delivery of alloantigen. Transplant Proc (in press).
24. Zheng XX, Markees TG, Hancock WW, et al. CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment. J Immunol 1999; 162: 4983.
25. Bolling S, Lin H, Wei R, Turka L. Preventing allograft rejection with CTLA4Ig: effect of donor-specific transfusion route or timing. J Heart Lung Transplant 1996; 15: 928.
26. Judge TA, Wu Z, Zheng XG, et al. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term survival. J Immunol 1999; 162: 1947.
27. Samoilova EB, Horton JL, Zhang H, et al. CTLA-4 is required for the induction of high dose oral tolerance. Int Immunol 1998; 10: 491.
28. Salomon B, Lenschow DJ, Rhee L, et al. B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 2000; 12: 431.
29. Takahashi T, Tagami T, Tamazaki S, et al. Immunologic self-tolerance maintained by CD25+
regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med 2000; 192: 303.
30. Cottrez F, Hurst SD, Coffman R, et al. T regulatory cells 1 inhibit a Th2-specific response in vivo. J Immunol 2000; 165 (9): 4848.
31. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol 2001; 166: 3789.