Reepithelialized Orthotopic Tracheal Allografts Expand... : Transplantation (original) (raw)
Extensive defects of the tracheal airway represent a significant clinical dilemma. Tracheal transplantation may provide a solution for life-threatening defects of the airway after stenosis or carcinoma. Unfortunately, similar to solid-organ transplantation, tracheal allografts are susceptible to cell-mediated rejection. Tracheal allograft rejection is characterized by infiltration of CD4+ and CD8+ T cells into the graft resulting in severe damage to the epithelium, loss of the cilia, and edema of the graft (1–3).
The majority of experimental work in this area has been performed with the heterotopic tracheal transplant model. In this model, an acute rejection response is characterized by fibrosis and tracheal lumen obliteration (4–6). The recent introduction of the orthotopic tracheal transplant model has provided investigators with a more clinically relevant model. Orthotopic tracheal allograft rejection is manifested by progressive dyspnea and stridor caused by inflammation and fibrosis of the small airways and cellular infiltration of the lamina propria (5); however, complete luminal airway obliteration does not occur. This discrepancy has not been fully elucidated; however, recent evidence suggests that recipient-derived reepithelialization of the tracheal allograft prevents luminal obliteration in the orthotopic model. In contrast, in the heterotopic model, the allograft fails to reepithelialize and therefore undergoes luminal obliteration.
Two characteristics make tracheal grafts unique. First, the main targets of tracheal rejection, the major histocompatibility complex (MHC) class I-expressing epithelial cells (7) lining the lumen of the tracheal allograft, can be replaced by recipient epithelium after transplantation. Although immunosuppression facilitates the process of allograft reepithelialization by impacting the quality and kinetics of the process, reepithelialization will occur in nonimmunosuppressed recipients (8,9). The second unique characteristic of tracheal grafts is that this tissue has been shown to be a rich source of dendritic cells (DCs) (10). Because donor DCs represent the primary stimulus for direct presentation of donor alloantigens (11), the trachea may provide a particularly strong immune stimulus. In addition, DCs have been shown to function in the maintenance of self-tolerance (12). Therefore it is imperative to investigate tracheal transplantation in animal models before initiating human studies.
Our group previously demonstrated that immunocompetent mice do not undergo acute rejection after the cessation of cyclosporine A (CsA) treatment after reepithelialization (13). In addition, these allograft recipients demonstrate normal trachea with 86.5% ± 6.7% ciliated epithelium. The failure of acute rejection to ensue after withdrawal of immunosuppression raises several questions: (1) Does reepithelialization protect against chronic rejection of the trachea? (2) After reepithelialization, will the animals receiving allografts demonstrate immune sensitization against donor MHC antigens? (3) Do allograft recipients contain any donor elements that can impact the host immune response? This study analyzes animals that received tracheal allografts, long-term immunosuppression, and subsequent withdrawal of immunosuppression for evidence of chronic rejection and donor-specific immune memory response.
MATERIALS AND METHODS
Mice
Experimental Design
Age-matched mice were randomly assigned to six experimental groups. BALB/c donor tracheal segments (five tracheal rings) were orthotopically transplanted into class I and class II MHC-mismatched allogeneic C57BL/6 recipients (20 g) (Taconic Farms, Germantown, NY), as previously described (3). The recipients in groups 1 and 2 were not immunosuppressed, and splenocytes and the allograft segments were harvested 10 and 100 days after transplantation, respectively. Group 3 consisted of tracheal isografts. In group 4, tracheal allografts were treated with 10 mg/kg per day intraperitoneal CsA (Sandimmune, Novartis, Basel, Switzerland) for 50 days starting 1 day before transplantation to allow for complete allograft reepithelialization. On day 50, immunosuppression was withdrawn and mouse splenocytes were harvested 10 days later. Groups 5 and 6 were composed of BALB/c donor tracheal segments that were orthotopically transplanted into allogeneic C57BL/6 recipients that were immunosuppressed for 50 days. One hundred days after the initial transplant, the graft segments were removed and then heterotopically transplanted into BALB/c (group 5) or C57BL/6 recipients (group 6), respectively.
Transplantation of Tracheal Grafts
The tracheal grafting procedure was performed as previously described. Briefly, subcutaneous ketamine (50 mg/kg) and xylazine (10 mg/kg) (KA) anesthesia was administered preoperatively. With the use of an operating microscope (Wild M651, Wild Leitz, Willodale, Ontario), under KA anesthesia, the donor mouse tracheal segment was exposed through an anterior midline neck incision. A five-ring circumferential tracheal segment was excised and placed into a glass dish with cooled physiologic saline. The recipient mouse, under KA anesthesia, was prepared with the use of an operating microscope. The donor tracheal graft was orthotopically secured with 10-0 nylon interrupted transtracheal sutures. Heterotopic recipients underwent a midline laparotomy incision followed by implantation of the donor trachea segment into the omentum. The recipient animals were placed under a warming lamp and monitored for 3 hr.
Skin Grafts
Mice that were nonimmunosuppressed for 100 days (group 2) (n=4), treated with CsA for 50 days, and then withdrawn for 50 days (group 4) (n=4), or naïve C57BL/6 mice (n=3) received BALB/c skin grafts on the dorsal thoracic wall under the administration of subcutaneous KA anesthesia. The animals were monitored by daily visual inspection for rejection of the grafts. Rejection was scored by total loss of viable tissue.
Cytotoxic T-Lymphocyte Assays
Spleens from the transplanted mice were harvested on the days indicated, and cells were isolated and restimulated for 5 days before assaying for cytotoxic T-lymphocyte (CTL) function. In brief, cells were harvested from the spleens, and a single cell suspension was made by teasing the spleens with forceps in a Petri dish containing Roswell Park Memorial Institute (Gibco, Carlsbad, CA) medium. The cells were pelleted, and red blood cells were lysed with 5 mL cold TrisNH4Cl buffer for approximately 3 min. The cells were then washed, counted, and cultured for 5 days in complete T-cell medium, at 3.75×106 cells/0.5 mL in 24-well plates (Costar), with 3.75×106/0.5 mL γ-irradiated stimulator cells (2,500 rad) in a total volume of 1 mL/well. Stimulator cells included BALB/c, C57BL/6, or third-party C3H splenocytes. On day 5, 2×106 target cells were labeled for 1 hr in at 37°C with 0.1 mCi 51Chromium (NEN, Boston, MA), washed, and subsequently cultured with the effector cells in 96-well V-bottom plates (Costar) at various effector to target ratios (100:1, 50:1, 25:1, 12.5:1). P815 (H-2d) target cells were used. Maximum and spontaneous release were measured from target cells incubated with 0.5% NP-40 or medium alone, respectively. After incubation for 4 hr at 37°C, 7% CO2, the cells were pelleted, and 100 μL of the supernatants were harvested and counted in a γ-counter (Wallac) for 51Cr-release. Cytotoxicity is measured by the percentage of 51Cr released from the target cells. The formula used for calculating the percentage of cytotoxicity is as follows:
Mixed Lymphocyte Reaction
Spleens were harvested from the eight transplant groups and then cultured at 2×105 cells/well with 2×105 cells/well γ-irradiated stimulator cells (2,500 rad), that is, BALB/c, C57BL/6, or third-party C3H at a 1:1 responder:stimulator ratio in round-bottom 96-well plates (Costar). These cells were incubated at 37°C with 7% CO2 for 72 hr in complete T-cell medium; 0.5 μCi/well [3H]-thymidine (ICN, Costa Mesa, CA) was added for the last 18 hr of culture, and the cells were harvested onto Unifilters (Packard, Meriden, CT), and incorporation of [3H]-thymidine into DNA was determined in a liquid scintillation counter (Wallac). Percentage of proliferation was calculated by assessing third-party (C3H) stimulated cells as 100% proliferation.
Enzyme-Linked Immunosorbent Assay
The concentration of interferon (IFN)-γ in the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) (Duo Set, R&D Systems, Minneapolis, MN). Supernatants were collected at various times (days 2 and 3) from the restimulated cultures containing both effector cells and γ-irradiated stimulator cells. Microtiter plates (Immulon) were coated with anti-IFN-γ antibody at 4 μg/mL and incubated overnight at 4°C. Plates were then blocked with 1% phosphate-buffered saline (PBS) and bovine serum albumin for a minimum of 2 hr at room temperature. After blocking, recombinant mouse IFN-γ was added to the plates starting with 10 ng/mL and serially diluted in PBS at 1:2 for a standard curve. All dilutions were performed in triplicates; 50 μL of triplicates of culture supernatants were added to the plate and incubated for 2 hr at room temperature. Plates were washed in PBS, and biotinylated goat anti-mouse IFN-γ antibodies were added at 400 ng/mL for 1 hr at room temperature. Plates were washed with PBS. Streptavidin-horseradish peroxidase was added at a 1:200 dilution in 1% PBS and bovine serum albumin. Plates were washed and developed with horseradish peroxidase substrate reagent, and 2N H2SO4 was added to stop the reaction. Samples were then read at 450 nm on a spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). A dose-response curve with purified IFN-γ was determined in each assay, and the results were used to calculate the absolute concentration of cytokines by the optical density of the sample in the linear portion of the dose-response curve.
Isolation and Characterization of Tracheal Dendritic Cells
BALB/c tracheae were transplanted into C57BL/6 mice and treated with CsA for 50 days and withdrawn from treatment for 50 days (group 4). At day 100 the donor grafts and the control recipient C57BL/6 tracheae at the proximal and distal ends of the donor graft were harvested and treated with collagenase (Roche, Indianapolis, IN) for 1 hr at 37°C. The grafts were then washed in PBS and blocked for nonspecific binding with anti-mouse CD16/CD32 (Fc receptor block) (Pharmingen) and 1% normal mouse serum (Jackson Laboratories, Bar Harbor, ME) for 30 min at 4°C. The cells were then washed with PBS and stained with CD11c phycoerythrin (PE) (HL3), I-Ab fluorescein isothiocyanate (FITC) (AF6–120.1), or the respective mouse isotype controls, hamster IgG1 PE (G235–2356), and mouse IgG2a FITC (G155–178) (Pharmingen, all used at 1:200) for 30 min at 4°C. The cells were then washed three times and analyzed by flow cytometry (Beckman Coulter, Fullerton, CA).
Medium
The medium consisted of 500 mL Roswell Park Memorial Institute, 1 mM sodium pyruvate (Gibco), 10 mM non-essential amino acids (Gibco), 2 mM l-glutamine (Gibco), 100 μg/mL penicillin streptomycin (Gibco), 10% fetal calf serum (Hyclone, Logan, UT), and 50 μg/mL β-2-mercapto-ethanol (Sigma-Aldrich, St. Louis, MO).
Histology
Tracheal grafts were removed from the mice and placed in 10% buffered formalin for fixation. The grafts were serially sectioned at 7 microns and stained with hematoxylin-eosin (Sigma Aldrich) to assess the presence of lymphocytic infiltration into the lamina propria and cilia structure.
For evaluation of reepithelialization of tracheal allografts with recipient-derived epithelium, BALB/c tracheae were transplanted into C57BL/6 mice and treated with CsA for 50 days. At day 50 the grafts were harvested. Ten-micron–thick serial sections were cut from the grafts after transplantation for assessment of mucosal phenotype. C57BL/6 mouse epithelium was detected using biotin-conjugated mouse anti-mouse H-2Kb monoclonal antibody (AF6–88.5) at 1:200 (Pharmingen). BALB/c spleen and trachea were used as controls to assess for cross-reactivity of the antibodies. Endogenous peroxidase activity was blocked by incubating slides in 0.3% H2O2 solution in PBS for 10 min. Slides were then rinsed, and the primary antibody was applied to the tissue sections and left to incubate at room temperature for 2 hr. Slides were then washed with PBS three times, and streptavidin-horseradish peroxidase (Pharmingen) was applied and incubated for 30 min at room temperature. The slides were then washed in PBS three times, and DAB was applied and allowed to incubate for 5 min. After the slides were rinsed in water, counterstaining with hematoxylin solution was performed for 30 sec. The slides were then dehydrated with four changes of alcohol (95%-100%), and a coverslip was applied. Serial sections were evaluated individually and in sequence from proximal to distal.
Statistics
All data in this study are presented as the mean ± standard deviation. The Student t test was performed to compare control groups with experimental groups. Differences were considered significant at a P value less than 0.05.
RESULTS
Nonimmunosuppressed Animals Are Sensitized to BALB/c (H-2d) Haplotype (Groups 1 and 2)
Ten-day and 100-day nonimmunosuppressed C57BL/6 control animals demonstrated primed alloantigen responses against BALB/c spleen cells after receiving BALB/c tracheal allografts, as measured by both cytokine release assays (Fig. 1A and B) and proliferative responses (Fig. 2) in comparison with the naive responses observed toward third-party C3H-stimulated responders. In addition, CTL activity from the nonimmunosuppressed animals was significantly higher than in naïve C57BL/6 control animals (Fig. 3A and B). This priming was evident in IFN-γ, proliferative, and CTL responses both at day 10 (Figs. 1A, 2, and 3A) and day 100 (Figs. 1B, 2, and 3B) after the grafts had been implanted (groups 1 and 2) (*_P_≤0.05 in Figs. 1–8). Serial histologic sections from the 10-day nonimmunosuppressed mice show evidence of significant cellular infiltration into the lamina propria (Fig. 4A), which was previously shown to be primarily CD4/CD8+ T cells (1–3). After 100 days without immunosuppression, the animals display a fibrotic lamina propria and a flat architecture (Fig. 4B). In comparison, the control isografted mice display thick cilia and a well-organized epithelium with no cellular infiltration into the lamina propria (Fig. 4D).
Interferon (IFN)-γ production in splenocytes from grafted mice. (A, B) IFN-γ was measured in splenocytes from mice that were nonimmunosuppressed or (C) immunosuppressed for 50 days with cyclosporine A (CsA), then with- drawn 50 days. (D) Nonimmunosuppressed isograft. All cultures were stimulated with BALB/c, C57BL/6, or C3H-irradiated splenocytes, and supernatants were collected on days 2 and 3. *_P_≤0.05 or **_P_≤0.005.
Proliferative responses in splenocytes from grafted mice. Percentage of proliferation of splenocytes from C57BL/6 mice transplanted with BALB/c trachea (groups 1–3) or C57BL/6 trachea (group 4). Data are reported as percentage of proliferation relative to the naïve response of the splenocytes from the retransplanted animal stimulated with C3H spleen cells that is set at 100%. Group 1: 10-day nonimmunosuppressed splenocytes. Group 2: 100-day nonimmunosuppressed splenocytes. Group 3: CsA-treated 50 days, splenocytes harvested at day 100. Group 4: splenocytes from isograft. All groups were restimulated with irradiated BALB/c, C57BL/6, or C3H splenocytes. *_P_≤0.05.
Cytotoxic T lymphocytes (CTLs) from splenocytes of grafted mice. (A, B) CTLs were measured in splenocytes from mice that were nonimmunosuppressed or (C) immunosuppressed for 50 days with CsA, then withdrawn 50 days. All cultures were restimulated with BALB/c irradiated splenocytes. Target cells used are P815 that are H-2d positive. *_P_≤0.05 or **_P_≤0.005.
Histologic staining of tracheal grafts. Serial histologic sections of orthotopically transplanted trachea that were (A) nonimmunosuppressed for 10 days, (B) nonimmunosuppressed for 100 days, (C) immunosuppressed with CsA for 50 days, then withdrawn 50 days, or (D) control isograft.
Histologic staining of a BALB/c tracheal graft orthotopically transplanted into C57BL/6 at day 50 (A) BALB/c trachea were used as controls to assess for cross-reactivity of the antibodies. (B) Graft is stained for recipient major histocompatibility complex (MHC) class I (H-2Kb) demonstrating the presence of recipient-derived C57BL/6 epithelium in the donor BALB/c graft (arrow).
IFN-γ and proliferation of splenocytes from heterotopically transplanted mice, and histology of the retransplanted tracheae (BALB/c → C57BL/6→BALB/c). (A) C57BL/6 mice received BALB/c tracheal allografts and were immunosuppressed 50 days with CsA, withdrawn for 50 days, and then had their tracheae removed to be subsequently retransplanted into BALB/c mice heterotopically for 14 days. Splenocytes from these animals were then co-cultured with irradiated BALB/c, C57BL/6, or C3H splenocytes. Supernatants were collected on day 2 of culture, and IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA). (B) Percentage of proliferation of recipient mice spleen cells after stimulation with BALB/c, C57BL/6, or third-party C3H splenocytes. Data are reported as percentage of proliferation relative to the naïve response of the splenocytes from the retransplanted animal stimulated with C3H spleen cells that is set at 100%. (C) Serial histologic sections demonstrate complete obliteration of the lumen on heterotopic retransplantation of the trachea into BALB/c mice. *_P_≤0.05 or **_P_≤0.005.
IFN-γ and proliferation of splenocytes from heterotopically transplanted mice, and histology of the retransplanted tracheae (BALB/c→C57BL/6→C57BL/6). (A) C57BL/6 mice received BALB/c tracheal allografts and were immunosuppressed 50 days with CsA, withdrawn for 50 days, and then had their tracheae removed to be subsequently retransplanted into C57BL/6 mice heterotopically for 14 days. Splenocytes from these animals were then co-cultured with irradiated BALB/c, C57BL/6, or C3H splenocytes. Supernatants were collected on day 2 of culture, and IFN-γ was measured by ELISA. (B) Percentage of proliferation of recipient mice spleen cells after stimulation with BALB/c, C57BL/6, or third-party C3H splenocytes. Data are reported as percentage of proliferation relative to the naïve response of the retransplanted splenocytes stimulated with C3H spleen cells that is set at 100%. (C) Serial histologic sections demonstrate no obliteration of the lumen on heterotopic retransplantation of the trachea into C57BL/6 mice, but instead remain patent. *_P_≤0.05 or **_P_≤0.005.
Flow cytometric analysis of recipient (I-Ab) graft-infiltrating dendritic cells (DCs). Graft DCs were stained with isotype controls (A) or recipient-specific MHC class II (I-Ab) fluorescein isothiocyanate (FITC) and CD11c phycoerythrin (PE) antibody (C). (B) As a positive control, recipient C57BL/6 trachea at the proximal and distal ends of the graft were harvested and stained with recipient-specific MHC class II (I-Ab) FITC and CD11c PE antibody.
Cyclosporine A-Treated Animals Show No Evidence of Chronic Rejection 50 Days After Cyclosporine A Withdrawal (Group 4)
Fifty days after the withdrawal of 50-day CsA treatment, tracheae were removed from the animals and examined histologically for evidence of rejection. The serial histologic sections of the tracheae of the animals displayed ciliated columnar cells lining the lumen (Fig. 4C). The lamina propria of the tracheae from the CsA-treated animals were not undergoing fibroproliferative responses and were not infiltrated with recipient-derived T cells. Therefore, there was no evidence of chronic rejection occurring in these animals, even up to 50 days after withdrawal of CsA.
Proliferative and Cytokine Responses from Mice Immunosuppressed 50 Days Display No Evidence of Priming to Donor Alloantigens (Group 4)
Spleen cells from animals 50 days after cessation of CsA were restimulated in vitro using irradiated donor (BALB/c), recipient (C57BL/6), or third-party (C3H) spleen cells to determine whether the animals that underwent transplantation demonstrated primed responses to donor alloantigens. Splenocytes from animals 50 days after cessation of 50 days of CsA treatment (group 4) secreted IFN-γ and proliferated equivalently whether stimulated with donor (BALB/c) or third-party stimulators (C3H) (Figs. 1C and 2, respectively). This suggests that the CsA treatment in these animals abrogated the priming leading to naive cytokine and proliferative responses to donor alloantigens.
Cyclosporine A-Treated Animals Show Evidence of Priming of Anti-Donor Cytotoxic T Lymphocytes (Group 4)
In contrast with the lack of priming as determined by cytokine release and proliferation, splenocytes from CsA-treated mice demonstrated enhanced killing against donor target cells in comparison with naïve C57BL/6 splenocytes when stimulated with BALB/c-irradiated splenocytes in a chromium release assay. The CsA-treated animals demonstrated up to a 3.7-fold increase in specific lysis (Fig. 3C) as opposed to naïve C57BL/6 mice when stimulated with BALB/c splenocytes. This indicates that the CsA-treatment did not prevent the expansion of recipient anti-donor CTLs in these animals.
Heterotopic Retransplantations: BALB/c→C57BL/6→BALB/c and BALB/c→C57BL/6→C57BL/6 (Groups 5 and 6)
As previously discussed in the Introduction, the grafts fully regenerate recipient epithelium on orthotopic transplantation. As can be seen in Figure 5, BALB/c trachea transplanted into C57BL/6 recipients have reepithelialized with C57BL/6 epithelium by day 50 on CsA treatment, and these mice fail to demonstrate evidence of chronic rejection after withdrawal of CsA treatment. To assess the immunogenicity of the reepithelialized grafts, the tracheal allografts from the C57BL/6 recipients were removed and heterotopically retransplanted into either BALB/c or C57BL/6 recipients. Fourteen days later, the retransplanted heterotopic grafts were removed and histologically examined for cellular infiltration indicative of rejection. In addition, the spleens of these mice were also harvested at day 14 and restimulated in vitro with donor (BALB/c), recipient (C57BL/6), or third-party (C3H) irradiated spleen cells. The cultures were monitored for proliferation and IFN-γ secretion.
Heterotopic Transplants BALB/c→C57BL/6→BALB/c (Group 5)
C57BL/6 mice that received a BALB/c allograft were treated with CsA for 50 days, withdrawn from CsA treatment, and sacrificed at day 100 (group 4). The grafts from these mice were then heterotopically retransplanted into an allogeneic recipient (BALB/c→C57BL/6→BALB/c) (group 5). Fourteen days after this heterotopic retransplantation of the graft, spleen cells from the recipient animals (group 5) displayed enhanced IFN-γ release (Fig. 6A) and enhanced proliferative responses (Fig. 6B) on in vitro restimulation with C57BL/6 splenocytes. Serial histologic sections of the heterotopically transplanted tracheae from the CsA-treated mice (BALB/c→C57BL/6→BALB/c) demonstrate complete obliteration of the tracheal lumen (Fig. 6C) indicative of allograft rejection.
Heterotopic Transplants BALB/c→C57BL/6→C57BL/6 (Group 6)
C57BL/6 mice received a BALB/c allograft for 50 days under CsA treatment, and subsequently 50 days after the CsA cessation (group 4), their grafts were removed and heterotopically retransplanted into isogenic recipients (BALB/c→C57BL/6→C57BL/6) (group 6). No rejection was observed in these animals. In vitro restimulation of spleen cells from these animals 14 days after heterotopic retransplantation demonstrated a profound reduction of IFN-γ release (Fig. 7A) and proliferative response (Fig. 7B) to the original donor alloantigens (H-2d) in comparison with cells stimulated with third-party C3H splenocytes. In addition, serial sections of the heterotopically transplanted tracheae from the immunosuppressed animals demonstrate no cellular obliteration of the lumen, indicating no rejection in these animals (Fig. 7C).
Recipient Dendritic Cells Infiltrate the Donor Tracheal Allograft
Graft-derived DCs were isolated and characterized by flow cytometric analysis. The donor graft was harvested at day 100 and stained with CD11c PE and I-Ab FITC. Recipient DCs positive for CD11c PE and I-Ab FITC were observed in the BALB/c grafted segment. This suggests that recipient I-Ab DCs infiltrate the BALB/c tracheal allografts and that these cells can be observed at day 100 after transplantation (Fig. 8C) compared with the isotype controls (Fig. 8A). As a positive control, the recipient (H-2b) tracheal tissue at the proximal and distal ends of the graft were also harvested and stained for CD11c and I-Ab. As expected, these recipient (H-2b) positive controls display intratracheal DCs that are CD11c and I-Ab positive (Fig. 8B).
Mice Reject Skin Grafts with Similar Kinetics
Mice that were nonimmunosuppressed for 100 days (group 2), immunosuppressed 50 days then withdrawn for 50 days (group 4), or naïve C57BL/6 mice that received BALB/c skin grafts all rejected the grafts with similar kinetics. All skin grafts were rejected by day 11 (data not shown).
DISCUSSION
Tracheal transplantation holds great promise as a reconstructive option for extensive tracheal airway defects; however, the side effects of long-term immunosuppression represent a deterrent. Recent work suggests that tracheal allografts undergo progressive reepithelialization with recipient-derived epithelium when orthotopically transplanted (1–3), and that this recipient-derived isogenic epithelium is critical for the prevention of obliteration of the tracheal allograft (1) with recipient cells (14). In previous studies we showed that 50 days of CsA treatment leads to a rapid and complete reepithelialization of the trachea, and that acute graft rejection is prevented in these animals after cessation of immunosuppression (13). This suggests that clinical tracheal transplantation may be accomplished after induction of immunosuppression without the need for long-term treatment.
In our present study, allograft recipients were immunosuppressed for 50 days and then withdrawn from immunosuppression for 50 days (group 4). One hundred days after the initial transplant, and 50 days after immunosuppression withdrawal, the allografts were assessed for evidence of chronic rejection both immunologically and histologically. Restimulation of these recipient splenocytes in vitro with donor BALB/c spleen cells failed to show any signs of immune sensitization when measured by proliferation or by the release of IFN-γ 2 to 3 days after the initiation of the cultures. Histologically, the tracheae showed dense ciliated epithelium without evidence of T-cell infiltration. When cells from these cultures were tested in chromium release assays against donor P815 (H-2d) target cells 5 days after initiation, a significant increase in CTLs were detected in comparison with naïve C57BL/6 animals stimulated with BALB/c stimulators. This observation suggests that the CsA treatment, although effective in preventing rejection, seems to allow an expansion of allogeneic memory CD8+ T cells, yet these T cells fail to attack the graft. To address the functionality of these CTLs, BALB/c skin grafts were transplanted onto C57BL/6 mice from group 2 (nonimmunosuppressed mice), group 4 (immunosuppressed 50 days, withdrawn 50 days), or naïve C57BL/6 mice. The BALB/c donor skin grafts rejected with similar kinetics in all groups. This would suggest that the tracheal grafts in group 4 are essentially being protected by the regenerated recipient epithelium from functionally active CTLs. Graft rejection has been shown to be a two-step process in which antigen-presenting cells such as DCs activate naïve T cells in the draining lymph nodes. The T cells then migrate from the draining lymph nodes to the site of the graft where they recognize the antigenic MHC molecules and cause rejection of the graft. Therefore, if the graft lacks antigen, the T cells may not recognize or attack the graft. It has been demonstrated that in the absence of antigen targets in a graft, alloantigen T cells do not induce rejection (15,16). Our observation that secondary BALB/c skin grafts undergo rejection, whereas the tracheal grafts do not in the immunosuppressed mice 50 days after withdrawal, supports this hypothesis.
Our findings demonstrate alloantigen primed CD8+ T cells, yet at the same time, we observed naïve proliferative and IFN-γ responses in the CsA-treated animals. There are several possible explanations for these results. CsA is a widely used drug in transplantation, and its mechanism of action involves the inhibition of the phosphatase calcineurin. On T-cell activation, calcineurin dephosphorylates nuclear factor of activated T cells, resulting in its translocation to the nucleus where it mediates transcription of genes (17–21) encoding proteins such as interleukin (IL)-2, IL-4, and CD40 ligand (22). Several studies have documented that CsA treatment is more efficient in suppressing CD4+ T cells than CD8+ T cells (23,24). As a result, expansion of the CD8+ T cells may not be efficiently blocked and may be mediated by the cytokine IL-15. IL-15 is a cytokine that has been shown to induce naïve CD8+ T cells, but not CD4+ T cells, to up-regulate the CD69 activation marker (25). Therefore, although CsA-treatment abrogates the IL-2 response, the CD8+ T cells may have been activated in the presence of IL-15, but the CD4+ T-cells may not have been activated. Furthermore, it has been demonstrated that IL-2 is not necessary for the in vivo generation of CD8+ memory T cells (26), and that in vitro culture of antigen-activated CD8+ T cells with IL-15 stimulated their development into memory-like cells that are able to mediate rapid recall responses (27,28). Irrespective of these findings, it is our observation that the CD8+ T cells failed to attack the graft. Even though the CD8+ effector T cells are functional, they may be unable to attack the graft as a result of a complete loss of donor MHC-bearing epithelial cells there.
When 100-day reepithelialized tracheae were retransplanted heterotopically into allogeneic (BALB/c→C57BL/6→BALB/c), or isogenic (BALB/c→C57BL/6→C57BL/6) mice, the allogeneic grafts were rejected and the isogenic grafts were not rejected. The reepithelialized BALB/c graft harvested from the primary recipient, then retransplanted orthotopically into a C57BL/6 mouse (BALB/c→C57BL/6→C57BL/6), failed to undergo obliteration, a manifestation of rejection. This suggests that the graft has largely become a C57BL/6 graft. Indeed, flow cytometric analysis of DCs present in the graft of the immunosuppressed mice 50 days after withdrawal of CsA treatment for 50 days (group 4) before heterotopic retransplantation expresses recipient I-Ab MHC class II on CD11c positive cells. This demonstrates that the graft has been infiltrated with recipient C57BL/6 DCs, as may be expected in a steady turnover state. Surprisingly though, there are seemingly more infiltrating recipient C57BL/6 DCs in the graft (4.11%) than in the control C57BL/6 recipient trachea (2.03%). The reason for the increased numbers of infiltrating DCs in the graft is not clear. Together, the infiltration of the I-Ab-positive recipient DCs, reepithelialization of the BALB/c graft with C57BL/6 epithelium, and obliteration of the graft when retransplanted heterotopically into BALB/c mice (BALB/c→C57BL/6→BALB/c) all indicate that the graft has largely become C57BL/6 (H-2b). However, the graft may exist as a chimera containing elements of both recipient C57BL/6 and donor BALB/c, because responses toward BALB/c antigens were reduced when splenocytes from heterotopically transplanted C57BL/6 mice (BALB/c→C57BL/6→C57BL/6) were restimulated with BALB/c splenocytes, compared with third-party C3H-stimulated splenocytes. This would indicate that the reepithelialized graft must still contain some donor elements that imprinted the recipient rendering it incapable of specifically responding to a BALB/c primary sensitization on heterotopic retransplantation into the C57BL/6 mouse. In addition, the infiltrating recipient (I-Ab positive) DCs would explain the primed responses toward C57BL/6 on heterotopic transplantation of the tracheae into BALB/c (BALB/c→C57BL/6→BALB/c), that is, the C57BL/6 DCs present in the graft are most likely initiating the alloresponse in the BALB/c mice.
These data argue the following: (1) After reepithelialization, tracheal allografts fail to demonstrate evidence of chronic rejection 50 days after cessation of immunosuppression; (2) immunosuppression with CsA may show selectivity in its ability to prevent immune sensitization to different T-cell populations, and (3) even 100 days after transplantation, elements of the original graft may exist because they can impact the secondary recipient specifically reducing immunity.
ACKNOWLEDGMENTS
The authors thank S. Czelusniak for helpful editing of the article; J. Liu for his excellent surgical techniques in performing the transplant surgery; and C. Lopez and B. Moltedo for expert help and advice in flow cytometry.
Experimental groups
REFERENCES
1. Neuringer IP, Mannon RB, Coffman TM, et al. Immune cells in a mouse airway model of obliterative bronchiolitis. Am J Respir Cell Mol Biol 1998; 19: 379.
2. Ikonen TS, Brazelton TR, Berry GJ, et al. Epithelial re-growth is associated with inhibition of obliterative airway disease in orthotopic tracheal allografts in non-immunosuppressed rats. Transplantation 2000; 70: 857.
3. Genden EM, Boros P, Liu J, et al. Orthotopic tracheal transplantation in the murine model. Transplantation 2002; 73(9): 1420.
4. Hertz MI, Jessurun J, King MB, et al. Reproduction of the obliterative bronchiolitis lesion after heterotopic transplantation of mouse airways. Am J Pathol 1993; 142: 1945.
5. Boehler A, Chamberlain D, Kesten S, et al. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation 1997; 64: 311.
6. Adams BF, Brazelton T, Berry G, et al. The role of respiratory epithelium in a rat model of obliterative airway disease. Transplantation 2000; 69: 661.
7. Richards DM, Dalheimer ST, Hertz MI, et al. Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease. J Immunol 2003; 171: 6919.
8. Genden EM, Iskander A, Bromberg JS, et al. The kinetics and pattern of tracheal allograft re-epithelialization. Am J Respir Cell Mol Biol. 2003; 28: 673.
9. Genden EM, Iskander AJ, Bromberg JS, et al. Orthotopic tracheal allografts undergo reepithelialization with recipient-derived epithelium. Arch Otolaryngol Head Neck Surg 2003; 129: 118.
10. Holt PG, Schon-Hegrad MA, Oliver G. MHC-class II antigen-bearing dendritic cells in pulmonary tissues of the rat. Regulation of antigen presentation activity by endogenous macrophage populations. J Exp Med 1998; 167: 262.
11. Gould DS, Auchincloss H Jr. Direct and indirect recognition: the role of MHC antigens in graft rejection. Immunol Today 1999; 20: 77.
12. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003; 21: 685.
13. Genden EM, Govindaraj S, Chaboki H, et al. Re-epithelialization of orthotopic tracheal allografts prevents rejection after withdrawal of immunosuppression. annals of otology, rhinology and laryngology. Ann Otol Rhinol Laryngol 2005; 114: 279.
14. Jordan JL, Hurley CL, Lee TDG, et al. Recipient cells form the proliferative lesion in the rat heterotopic tracheal allograft model of obliterative airway disease. J Heart Lung Transplant 2003; 22: 357.
15. Chen Y, Demir Y, Valujskikh, et al. Antigen location contributes to the pathological features of a transplanted heart. Am J of Pathol 2004; 164: 1407.
16. Grazia TJ, Pietra BA, Johnson ZA, et al. A two-step model of acute CD4 T-cell mediated cardiac allograft rejection. J Immunol 2004; 172: 7451.
17. Flanagan WM, Corthesy B, Bram RJ, et al. Nuclear association of a T-cell transcription factor blocked by FK506 and cyclosporine A. Nature 1991; 352: 803.
18. Northrop JP, Ho SN, Chen L, et al. NF-AT components define a family of transcription factors targeted in T-cell activation. Nature 1994; 369: 497.
19. Shaw KT, Ho AM, Raghavan A, et al. Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells. Proc Natl Acad Sci U S A 1995; 92: 11205.
20. Loh C, Carew JA, Kim J, et al. T-cell receptor stimulation elicits an early phase of activation and a later phase of deactivation of the transcription factor NFAT1. Mol Cell Biol 1996; 16: 3945.
21. Timmerman LA, Clipstone NA, Ho SN, et al. Rapid shuttling of NF-AT in discrimination of Ca2+ signals and immunosuppression. Nature 1996; 383: 837.
22. Rao A, Luo C, Hogan PG. Transcription factors of the NF-AT family: regulation and function. Annu Rev Immunol 1997; 15: 707.
23. Hu H, Yinchen D, Feng P, et al. Effect of immunosuppressants on T-cell subsets observed in vivo using carboxy-fluorescein diacetate succinimidyl ester labeling. Transplantation 2003; 75: 1075.
24. Koenen HJPM, Michielsen ECHJ, Verstappen J, et al. Superior T-cell suppression by rapamycin and FK506 over rapamycin and cyclosporine a because of abrogated cytotoxic T-lymphocyte induction, impaired memory responses, and persistent apoptosis. Transplantation 2003; 75: 1581.
25. Kanegane H, Tosato G. Activation of naive and memory T cells by interleukin-15. Blood 1996; 88: 230.
26. Dai Z, Konieczny BT, Lakkis FG. The dual role of IL-2 in the generation and maintenance of CD8+ memory T cells. J Immunol 2000; 165: 3031.
27. Manjunath N, Shankar P, Wan J, et al. Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes. J Clin Invest 2001; 108: 871.
28. Weninger W, Crowley MA, Manjunath N, et al. Migratory properties of naive, effector, and memory CD8 (+) T cells. J Exp Med 2001; 194: 953.
Keywords:
Transplantation immunology and immunobiology; T-cell activation; Immunosuppression
© 2005 Lippincott Williams & Wilkins, Inc.