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Experimental Transplantation

Effects of Immunosuppressants on Induction of Regulatory Cells After Intratracheal Delivery of Alloantigen

Shibutani, Shintaro1; Inoue, Fumihiko1; Aramaki, Osamu2; Akiyama, Yoshinobu1; Matsumoto, Kenji1; Shimazu, Motohide1; Kitajima, Masaki1; Ikeda, Yoshifumi2; Shirasugi, Nozomu2,3; Niimi, Masanori2

Author Information
doi: 10.1097/01.TP.0000158023.21233.DE

Abstract

The immunosuppressive drugs that are currently administered after clinical transplantation achieve excellent results with respect to patient and allograft survival rates in the short term. However, long-term results with conventional immunosuppressive regimens have been somewhat disappointing, and the drugs that are used produce serious adverse effects, including nephrotoxicity, diabetes, neurotoxicity, and an increased risk of infection and cancer (1). Induction of tolerance to transplanted organs has not been achieved clinically, although in a small number of transplant recipients receiving conventional immunosuppressants, tolerance to the transplanted organ already seems to exist. In such patients, immunosuppressive therapy was discontinued for several reasons, including use of a weaning protocol in a group of liver-transplant recipients (2).

Immune regulation is one of the mechanisms of allograft tolerance (3). T-cell clones (4) and peripheral blood mononuclear cells (5) from transplant recipients with stable graft function were found to exhibit active immune mechanisms as regulatory cells in vitro and in an animal model of delayed-type hypersensitivity. These findings suggest that immune regulation may be involved in the acceptance of allografts in patients.

We previously reported that intratracheal delivery (ITD) of donor splenocytes in mice induced prolonged survival of fully allogeneic cardiac grafts, with generation of regulatory cells (6). Our ITD model is useful for examining the induction and function of regulatory cells because such cells are induced in only 7 days, without additional immunomodulatory treatment.

When conventional immunosuppressant agents are used to prevent graft rejection, it is important that they work synergistically with mechanisms that promote allograft tolerance, including induction of regulatory cells. However, the effects of these immunosuppressants on the interactions leading to induction of the regulatory mechanism remain unknown. In this study, we assessed the effect of various doses of conventional immunosuppressants (FK506, cyclosporine A [CsA], azathioprine [AZA], mycophenolate mofetil [MMF], and rapamycin [RAPA]) on induction of regulatory cells after ITD of alloantigen.

MATERIALS AND METHODS

Mice

Inbred male C57BL/6 (H2b), CBA (H2k), C3H (H2k), and BALB/c (H2d) mice were purchased from Sankyo Ltd. (Tokyo, Japan), housed in conventional facilities at the Biomedical Service Unit of Teikyo University, and used when between 8 and 12 weeks of ages in accordance with protocols for animal experimentation approved by the Animal Use and Care Committee of Teikyo University.

Conventional Immunosuppressants

FK506 (a gift of Fujisawa Pharmaceutical Co., Osaka, Japan) was sustained in normal saline for doses of 0.1, 0.3, 0.5, and 1.0 mg/kg of body weight. CsA (Novartis Pharma KK, Tokyo, Japan) was obtained from the Keio University Hospital pharmacy and diluted in saline for doses of 5, 10, or 25 mg/kg. These doses were chosen in the light of findings by Lagodzinski et al. (7), who observed that administration of 0.1 mg/kg per day of FK506 caused significant prolongation of survival of skin allografts in mice, an effect comparable to that provided by a dose of 3.5 mg/kg per day of CsA. MMF was donated in powder form by Roche Bioscience (Tokyo, Japan) and prepared daily by suspension in saline for doses of 20 and 40 mg/kg. AZA (a gift of GlaxoSmithKline KK, Tokyo, Japan) was sustained in normal saline for doses of 1.0 and 2.0 mg/kg. RAPA (Wyeth-Ayerst, Princeton, NJ) was obtained from the Emory University Hospital pharmacy and diluted in saline for doses of 0.2 and 0.4 mg/kg. All drugs were administered by intraperitoneal (IP) injection. The dosages of the immunosuppressants were first determined on the basis of the results of other experiments (7–16) and confirmed by a small number of survival data and interleukin (IL)-2 production in the mixed leukocyte culture (MLC) with regulatory population in our preliminary experiments.

Pretreatment with Intratracheal Delivery and Cardiac Transplantation

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 placed under general anesthesia, and the trachea was exposed by dissection of overlying muscles. Splenocytes (1×107 in 100 μL phosphate-buffered saline) from C57BL/6 donors were then injected into the trachea by using a 30-gauge needle and a 1-mL syringe (6). Immediately afterward, the skin was closed with a single layer of sutures. As controls, some naïve CBA mice underwent transplantation of a C57BL/6 heart without receiving ITD of splenocytes (no-treatment group), and some CBA mice were given ITD of syngeneic (CBA) or third-party (BALB/c) splenocytes before transplantation of a C57BL/6 heart (ITD-alone group).

Seven days after the ITD pretreatment in the CBA recipients, a cardiac graft from a C57BL/6 mouse was transplanted into the abdomen of each recipient by using a microsurgical technique (17). The allografts were monitored by daily palpation. Graft rejection was defined as complete cessation of graft contraction and confirmed by direct visualization and histologic assessment of the graft.

Adoptive Transfer Study

To assess the development of regulatory cells, we conducted an adoptive transfer study. CBA mice (primary recipients) were treated with ITD of C57BL/6 splenocytes. Seven days later, instead of cardiac transplantation, the primary recipients were sacrificed and the splenocytes were harvested for adoptive transfer. Then, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary CBA recipients and underwent transplantation of a heart from a C57BL/6 mouse the same day as the adoptive transfer. Some primary recipients were also given daily IP injections of FK506 (0.1, 0.3, 0.5, or 1.0 mg/kg), CsA (5, 10, or 25 mg/kg), MMF (20 or 40 mg/kg), AZA (1.0 or 2.0 mg/kg), or RAPA (0.2 or 0.4 mg/kg) from the time of the ITD of splenocytes until the day of the adoptive transfer. All mice survived the ITD of splenocytes, drug administration, or both, without any adverse effects. As controls, some secondary recipients received adoptive transfer of splenocytes from primary recipients given ITD of CBA or BALB/c splenocytes before undergoing transplantation of a C57BL/6 heart.

In the adoptive transfer study of CD4+ cells and CD11c+ cells, CD4+ cells and CD11c+ cells were purified from pooled splenocytes that were harvested from the pretreated CBA mice with ITD 7 days before by positive selection using MACS CD4 Microbeads (Miltenyi Biotec Inc., Auborn, CA; purity >98%) and MACS CD11c Microbeads (Miltenyi Biotec Inc.; purity >96%), respectively. Naïve CBA mice were intravenously injected with the CD4+ cells (2×107) or CD11c+ cells (2×106) and then received a heart transplant from a C57BL/6 donor the same day.

Adoptive Transfer Study in Different Strain Combination

To further elucidate the effects of the immunosuppressants on hyporesponsiveness by the immune regulation, we used different strain combinations, C3H mice as donors and C57BL/6 mice as recipients, and performed the adoptive transfer study in our mouse ITD model.

C57BL/6 mice (primary recipients) were pretreated with ITD of 1×107 C3H splenocytes. Seven days later, naïve C57BL/6 mice (secondary recipients) received adoptive transfer of 5×107 splenocytes from the primary C57BL/6 recipients pretreated with ITD and then underwent transplantation of C3H cardiac grafts the same day. Some primary recipients were also given daily IP injections of RAPA (0.4 mg/kg) and/or MMF (40 mg/kg) from the time of the ITD of splenocytes until the day of the adoptive transfer.

Enzyme-Linked Immunosorbent Assay for Cytokines Production

The IL-2 level in supernatant of MLC was determined by enzyme-linked immunosorbent assays (ELISA). CBA mice were given ITD of C57BL/6 splenocytes (1×107) on day −7 with or without IP administration of FK506, CsA, MMF, AZA, or RAPA daily from day −7 to 0. On day 0, the mice were sacrificed and their splenocytes were used as the responder cells. Splenocytes from naive C57BL/6 (allogeneic) mice were treated with 100 μg/mL mitomycin C (Kyowa Hakko, Osaka, Japan) for 30 min at 37°C and then used as the stimulator cells. The responder cells (2.5×106 cells/mL) were co-cultured with the stimulator cells (1×107 cells/mL) in Roswell Park Memorial Institute 1640 medium containing 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37°C in 96-well, U-bottomed tissue-culture plates (Iwaki Scitech Division, Tokyo, Japan) for 4 days. The IL-2 level in supernatant of the MLC was determined by ELISA. The capture monoclonal antibody (JES6–1A12), detection monoclonal antibody (JES6–5H4), and recombinant standard for IL-2 were obtained from BD Pharmingen (San Diego, CA).

CFSE Staining for the Regulatory Population

CBA mice were given ITD of C57BL/6 splenocytes (1×107) on day −7 with or without IP administration of FK506 (0.1 or 1.0 mg) daily from day −7 to 0. On day 0, the pretreated mice were sacrificed and their splenocytes were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (1 μm, Molecular Probes, Eugene, OR), and 5×107 of the labeled splenocytes were adoptively transferred into naïve CBA mice. Three days later, splenocytes were harvested from the CBA mice with adoptive transfer to determine the amount of CFSE+ population by flow cytometry.

Timing of Adoptive Transfer in FK506 Experiments

To examine whether FK506 delays development of regulatory cells by ITD of alloantigen, the timing of adoptive transfer was changed. CBA mice were given ITD of C57BL/6 splenocytes (1×107) with IP administration of a high dose of FK506 (1.0 mg/kg) daily for 7 days. Fourteen or 28 days after ITD pretreatment, the pretreated mice were sacrificed and 5×107 of the splenocytes were adoptively transferred into naïve CBA mice that underwent transplantation of a C57BL/6 heart the same day.

Flow Cytometric Analysis

Single cell suspensions of spleen were analyzed by staining with fluorochrome-conjugated antibodies (anti-CD4 [Caltag Laboratories, Burlingame, CA], anti-CD25 [Pharmingen], or immunoglobulin isotype controls [Pharmingen]), after red blood cell lysis with a Trizma base ammonium chloride solution. In some experiments, CD11c+ cells were purified from the spleen in the pretreated CBA mice with ITD 7 days before, or the spleen in the untreated CBA mice with allograft transplantation 7 days before, by positive selection using MACS CD11c Microbeads, and stained with fluorochrome-conjugated antibodies (anti-CD11c, anti-I-Ek, anti-CD80, anti-CD86, anti-CD40, or immunoglobulin isotype controls [Pharmingen]). Stained cells were analyzed using Cellquest software on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA).

Statistical Analysis

Survival times of cardiac allografts in groups of mice were compared by using the Mann-Whitney U test. All statistical analyses were performed with StatView SE+ Graphics software (Abacus Concepts, Cary, NC). A P value less than 0.05 was considered to represent statistical significance. Other data, in the experiments of ELISA and flow cytometry, were compared by using the unpaired Student t test.

RESULTS

Pretreatment with Intratracheal Delivery of Donor Splenocytes and Adoptive Transfer

Naive CBA recipients rejected C57BL/6 cardiac grafts acutely (survival time 7, 7, 8, 9, 11, 14, and 14 days, n=7; median survival time [MST] 9 days). In CBA mice given C57BL/6 splenocytes by ITD 7 days before transplantation, all grafts survived for more than 28 days (survival time 28, 70, 81, >100, and >100 days, n=5; MST 81 days, P<0.01 compared with no-treatment group). CBA mice given ITD of CBA or BALB/c splenocytes before cardiac transplantation acutely rejected C57BL/6 grafts (survival time 7, 7, 9, 11, and 13 days, in five mice that received ITD of CBA splenocytes; 7, 8, 9, 10, and 12 days, in five mice that received ITD of BALB/c splenocytes; MST in both groups, 9 days, not significant [NS] compared with no-treatment group).

In the adoptive transfer study, secondary CBA recipients given an intravenous injection of splenocytes from primary CBA recipients that received ITD of C57BL/6 splenocytes demonstrated prolonged survival of C57BL/6 cardiac grafts (MST 50 days; Fig. 1, P<0.005 compared with no-treatment group). In contrast, secondary CBA recipients given adoptive transfer of splenocytes from primary CBA recipients that received ITD of CBA or BALB/c splenocytes acutely rejected C57BL/6 grafts (survival time 7, 8, 9, 13, and 16 days, in five mice with adoptive transfer from the mice with ITD of CBA splenocytes; 7, 9, 9, 11, and 15 days, in five mice with adoptive transfer from the mice with ITD of BALB/c splenocytes; MST in both groups, 9 days, P<0.01 for each group compared with the group with adoptive transfer from the mice given ITD of C57BL/6 splenocytes). These results showed that ITD of alloantigen induced donor-specific regulatory cells in our model.

FIGURE 1.
FIGURE 1.:
Results of adoptive transfer study in which primary CBA recipients were treated with intratracheal delivery (ITD) of C57BL/6 splenocytes alone or in combination with daily intraperitoneal (IP) administration of FK506, according to the doses of FK506 given. Seven days after the pretreatment of the primary recipients, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary recipients, and C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day (MST, median survival time; **, not significant; ##, P<0.01 compared with the ITD-alone group).

When CD4+ cells were purified from the pretreated CBA mice with ITD 7 days before and 2×107 of the CD4+ cells were adoptively transferred into naïve CBA recipients (secondary recipients), which received transplantation of C57BL/6 hearts the same day, the secondary recipients demonstrated remarkable prolongation of the cardiac allograft survival (survival time >60 days × 5). In contrast, adoptive transfer of CD4+ cells from naïve mice did not induce prolongation of allograft survival (survival time 8, 11, 12, 14, 14, and 24 days; MST 13 days). When CD11c+ cells were purified from the pretreated CBA mice with ITD 7 days before and 2×106 of the CD11c+ cells were adoptively transferred into naïve CBA recipients (secondary recipients), followed by cardiac transplantation of C57BL/6 hearts the same day into the secondary recipients, cardiac allograft survival was also prolonged (survival time 26, 35, 42, and >50 days × 7; MST >50 days), compared with that in secondary recipients with adoptive transfer of naïve CD11c+ cells (7, 7, 12, and 12 days; MST 9.5 days). These data suggest that the regulatory population consisted of not only CD4+ T cells but also dendritic cells (DCs) in our ITD model.

Effect of FK506 or Cyclosporine A on Induction of Regulatory Cells

When primary CBA recipients were given a daily dose of 0.1 mg/kg FK506 after ITD of C57BL/6 splenocytes, survival of C57BL/6 cardiac grafts in secondary CBA recipients was prolonged after adoptive transfer of splenocytes from the primary recipients (MST 55 days, P=NS compared with the ITD-alone group; Fig. 1). These results suggest that this dose of FK506 did not interfere with induction of regulatory cells after ITD of alloantigen. However, when primary recipients were given 0.3, 0.5, or 1.0 mg/kg FK506 or 5, 10, 25 mg/kg CsA after ITD of donor splenocytes, secondary recipients acutely rejected C57BL/6 grafts (MST 11, 7, and 7 days, respectively, in the three FK506 dosage groups; and 13, 7, and 7 days, respectively, in the CsA group, P<0.01 for each group compared with the ITD-alone group; Figs. 1 and 2). Thus, various high doses of FK506 and any dose of CsA abrogated induction of regulatory cells in our model.

FIGURE 2.
FIGURE 2.:
Results of adoptive transfer study in which primary CBA recipients were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of cyclosporine A (CsA), according to the doses of CsA given. Seven days after the pretreatment of the primary recipients, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary recipients, and C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day (MST, median survival time; ##P<0.01 compared with the ITD-alone group).

Effect of Azathioprine or Mycophenolate Mofetil on Induction of Regulatory Cells

When primary CBA recipients were given a daily dose of 1.0 or 2.0 mg/kg AZA after ITD of C57BL/6 splenocytes, all C57BL/6 cardiac grafts in secondary CBA recipients (both dosage groups) were rejected acutely (MST 13 and 10 days, respectively, P<0.01 compared with the ITD-alone group; Fig. 3). These results suggest that AZA abrogated induction of regulatory cells after ITD of alloantigen. On the other hand, when secondary CBA recipients were given adoptive transfer of splenocytes from primary recipients pretreated with ITD of alloantigen and 20 mg/kg MMF, the majority of cardiac grafts survived for more than 100 days (MST >100 days, P<0.05 compared with the ITD-alone group; Fig. 4). Moreover, when 40 mg/kg MMF was given to primary recipients along with ITD pretreatment, all grafts survived indefinitely (>100 days × 10), suggesting that MMF facilitated induction of regulatory cells after ITD of alloantigen.

FIGURE 3.
FIGURE 3.:
Results of adoptive transfer study in which primary CBA recipients were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of azathioprine (AZA), according to the doses of AZA given. Seven days later after the pretreatment of the primary recipients, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary recipients, and C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day (MST, median survival time; ##P<0.01 compared with ITD-alone group).
FIGURE 4.
FIGURE 4.:
Results of adoptive transfer study in which primary CBA recipients were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of mycophenolate mofetil (MMF), according to the doses of MMF given. Seven days later after the pretreatment of the primary recipients, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary recipients, and C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day (MST, median survival time; #P<0.05; ##P<0.01 compared with ITD-alone group).

Effect of Rapamycin on Induction of Regulatory Cells

When primary CBA recipients were given ITD of C57BL/6 splenocytes in combination with 0.2 mg/kg RAPA, secondary CBA recipients demonstrated prolonged survival of C57BL/6 cardiac grafts after adoptive transfer (MST 50 days, P=NS compared with the ITD-alone group; Fig. 5). When 0.4 mg/kg RAPA was given to primary recipients along with ITD treatment, graft survival was markedly prolonged (MST >100 days, P<0.05 compared with the ITD-alone group). These results suggest that high doses of RAPA (0.4 mg/kg) facilitate induction of regulatory cells after ITD of alloantigen.

FIGURE 5.
FIGURE 5.:
Results of adoptive transfer study in which primary CBA recipients were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of rapamycin (RAPA), according to the doses of RAPA given. Seven days later after the pretreatment of the primary recipients, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes (5×107 given by intravenous injection) from the primary recipients, and C57BL/6 cardiac grafts were transplanted into the secondary recipients the same day (MST, median survival time; **, not significant; #P<0.05, compared with ITD-alone group).

Adoptive Transfer Study in Different Strain Combination

C3H allografts were acutely rejected in secondary C57BL/6 recipients with adoptive transfer of naïve C57BL/6 splenocytes in control group, (survival time 8, 8, 8, 9, 10, and 10 days, MST=8.5 days, n=6). In contrast, when secondary recipients received adoptive transfer of splenocytes from the primary C57BL/6 recipients pretreated with ITD alone, the recipients demonstrated modest prolongation of C3H graft survival (survival time 10, 11, 12, 13, and 18 days, MST=12 days, n=5, P<0.05 compared with that in the control group). When ITD treatment was combined with 0.4 mg/kg RAPA in the primary recipients, C3H allograft survival in the secondary recipients with adoptive transfer was further prolonged compared with that in the recipients with adoptive transfer from the mice pretreated with ITD alone (survival time 15, 16, 19, 25, and 27 days, MST=19 days, n=5, P<0.05). When ITD treatment was combined with 40 mg/kg MMF in the primary recipients, C3H allograft survival in the secondary recipients was also significantly prolonged (survival time 14, 15, 19, 19, and 20 days, MST=19 days, n=5, P<0.05 compared with that in the recipients with adoptive transfer from the mice pretreated with ITD alone). Moreover, when ITD treatment was combined with both 0.4 mg/kg RAPA and 40 mg/kg MMF in the primary recipients, C3H allograft survival in the secondary recipients was also significantly prolonged (survival time 16, 18, 25, 32, 33 days, MST=25 days, n=5, P<0.05 compared with that in the recipients with adoptive transfer from the mice pretreated with ITD alone). These data suggest that the effects of RAPA and MMF to facilitate hyporesponsiveness induced by the regulatory cells were also applicable in different strain combination in our mouse ITD model.

Mechanism for the Effects of FK506 on Induction of Regulatory Cells: Interleukin-2 Production from the Regulatory Cells

When responder cells were harvested from the CBA mice given ITD of C57BL/6 splenocytes in combination with 0.1 mg/kg FK506, IL-2 production in allo-MLC did not change significantly compared with that from CBA mice treated with ITD alone (Fig. 6A) (0.204±0.03 μg/mL vs. 0.204±0.035 μg/mL, n=4, respectively, P=NS). In contrast, when responder cells were harvested from the CBA mice given ITD of C57BL/6 splenocytes in combination with 1.0 mg/kg FK506, IL-2 production was significantly decreased (Fig. 6A) (0.022±0.013, n=4, P<0.05 compared with that in the ITD-alone group). These data suggest that the decrease in the level of IL-2 may be associated with abrogation of inducing regulatory cells.

FIGURE 6.
FIGURE 6.:
(A) Results of interleukin (IL)-2 production in the supernatant of mixed leukocyte culture (MLC). CBA mice were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of FK506 (0.1 mg/kg or 1.0 mg/kg). Seven days later after the pretreatment, the mice were sacrificed and their splenocytes were used as the responder cells. Splenocytes from naive C57BL/6 (allogeneic) mice were treated with 100 μg/mL mitomycin C for 30 min at 37°C and then used as the stimulator cells. The responder cells (2.5×106 cells/mL) were co-cultured with the stimulator cells (1×107 cells/mL) in Roswell Park Memorial Institute 1640 medium containing 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37°C in 96-well, U-bottomed tissue-culture plates for 4 days. IL-2 level in supernatant of the MLC was determined by enzyme-linked immunosorbent assay (ELISA). Data are indicated as the mean±standard deviation (SD) of six replicates in one representative experiment. We performed three separate experiments and achieved similar results. NS, not significant; #P<0.05, compared with ITD-alone group. (B, C) Results of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled regulatory population in the spleen after adoptive transfer from the pretreated mice with ITD. Splenocytes were harvested from the pretreated mice with ITD in the presence or absence of FK506 (0.1 mg/kg or 1.0 mg/kg), labeled with CFSE (1 μm), and adoptively transferred into the naïve CBA mice (secondary recipients). Three days later, the number of CFSE+ cells, including the regulatory cell population, in the spleens of secondary recipients was determined by flow cytometry. On flow cytometry, live cells were gated and 30,000 were counted for analysis. Data are indicated as the mean±SD (n=5). ##P<0.01, compared with ITD-alone group. (D) Results of CD4+CD25+ cells in the regulatory population. CBA mice were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of RAPA (0.4 mg/kg). Seven days later after the pretreatment, the mice were sacrificed and their splenocytes were stained with fluorochrome-conjugated anti-CD4 and anti-CD25; then the number of CD4+CD25+ cells in each group was analyzed by flow cytometry. The number of CD4+CD25+ cells in naïve splenocytes was also analyzed as control. On flow cytometry, live cells were gated and 30,000 were counted for analysis. Data are indicated as the mean±SD (n=5). #P<0.05, compared with ITD-alone group; *P<0.05, compared with naïve control group. (E) Results of expression of cell surface molecules on dendritic cells (DCs) in the regulatory population. CBA mice were treated with ITD of C57BL/6 splenocytes alone or in combination with daily IP administration of MMF (40 mg/kg). Seven days later after the pretreatment, the mice were sacrificed and CD11c+ cells were purified from the spleen in the pretreated CBA mice by positive selection using MACS CD11c Microbeads (Miltenyi Biotec Inc., Auborn, CA) and stained with fluorochrome-conjugated antibodies (anti-CD11c, anti-I-Ek, anti-CD80, anti-CD86, anti-CD40, or immunoglobulin isotype controls). DCs harvested from the spleen in the CBA recipients with C57BL/6 allografting 7 days before (DCs with allo-stimulation) were also stained as positive control. On flow cytometry, CD11c+ cells were gated and 50,000 were counted for analysis. Isotype controls (line histogram); data (filled histogram). Data are from one representative experiment. We performed three separate experiments and achieved similar results.

Mechanism for the Effects of FK506 on Induction of Regulatory Cells: CFSE-Labeled Population in the Secondary Recipients

When the CFSE-labeled splenocytes were adoptively transferred from the pretreated CBA mice with ITD alone, 7.78% of CFSE+ cells were detected in the spleen of the CBA mice with adoptive transfer (Fig. 6B and C) (n=5). When 0.1 mg/kg of FK506 was combined with ITD pretreatment, the level of detected CFSE+ cells did not change significantly (7.83%, n=5, P=NS compared with that from the mice with ITD alone). On the other hand, when harvested from the CBA mice given ITD of C57BL/6 splenocytes in combination with 1.0 mg/kg FK506, the level of detected CFSE+ cells was decreased compared with that from CBA mice treated with ITD alone (0.652%, n=5, P<0.01).

Mechanism for the Effects of FK506 on Induction of Regulatory Cells: Timing of the Adoptive Transfer

To examine whether FK506 delays development of regulatory cells by ITD of alloantigen, the timing of adoptive transfer was changed. When the adoptive transfer was performed 14 or 28 days after pretreatment with ITD plus a high dose of FK506 (1.0 mg/kg), graft survival was not prolonged (survival time 11, 11, 11, 15, and 17 days, in five mice with adoptive transfer 14 days after pretreatment with ITD; 15, 17, 22, 24, and 27 days, in five mice with adoptive transfer 28 days after pretreatment with ITD; MST 11 days and 22 days, respectively, P<0.01 for each group compared with MST [50 days] in the ITD-alone group). These data suggest that a high dose of FK506 did abrogate hyporesponsiveness induced by the regulatory cells even when the timing of the adoptive transfer was delayed.

Mechanism for the Effects of Rapamycin on Induction of Regulatory Cells: The Number of CD4+ CD25+ Cells

The number of CD4+ CD25+ cells was determined by flow cytometric analysis in (1) naïve splenocytes, (2) the regulatory population harvested from the spleen of the pretreated mice with ITD alone 7 days before, and (3) the regulatory population harvested from the pretreated mice with ITD plus RAPA (0.4 mg/kg) (Fig. 6D).

Naïve splenocytes contained 8.43±1.08×105 of CD4+ CD25+ cells (n=5). On the other hand, the regulatory population that was induced by ITD alone contained 10.2±1.05×105 of CD4+ CD25+ cells (n=5, P<0.05 compared with that in naïve splenocytes). Moreover, the number of CD4+ CD25+ cells in the regulatory population was further increased when RAPA was combined with the pretreatment of ITD (12.5±1.05×105 cells, n=5, P<0.05 compared with that in the pretreated mice with ITD alone). These data indicate that the regulatory population by ITD contained an increased number of CD4+ CD25+ cells compared with naïve splenocytes, and that RAPA induced a further increase in the number of CD4+ CD25+ cells compared with that in the regulatory population by ITD alone. These data suggest that one of the mechanisms for the effect of RAPA (0.4 mg/kg) on facilitating the immune regulation resulted from the increase in the number of CD4+ CD25+ cells.

Mechanism for the Effects of Mycophenolate Mofetil on Induction of Regulatory Cells: Expression of Cell Surface Molecules on Dendritic Cells

To investigate the mechanisms for the effect of MMF on induction of regulatory cells, we examined expression of cell surface molecules on DCs in the regulatory population by flow cytometry. Expression of MHC class II molecule (I-Ek), CD80, CD86, and CD40 was determined by flow cytometric analysis (1) on DCs in the regulatory population that was harvested from the spleen of the pretreated CBA mice with ITD alone 7 days before; (2) on DCs in the regulatory population that was harvested from the spleen of the pretreated CBA mice with ITD plus MMF (40 mg/kg); or (3) on DCs harvested from the spleen in the CBA recipients with C57BL/6 allografting 7 days before (allo-stimulated DCs, for positive control). The expression of I-Ek, CD80, CD86, and CD40 was up-regulated on allo-stimulated DCs (Fig. 6E). DCs that were harvested from the regulatory population in the pretreated mice with ITD alone showed a lower expression of CD80 and CD86. Furthermore, in the presence of MMF along with the ITD pretreatment, expression of CD86 was further decreased, and expression of CD40 was modestly decreased, compared with that on the allo-stimulated DCs or on the DCs from the pretreated mice with ITD alone. The regulatory population in our ITD model may contain not only CD4+ cells but also CD11c+ DCs. These data suggest that DCs in the regulatory population induced by ITD pretreatment have tolerogenic phenotypes with lower expression of costimulatory molecules and that MMF strengthens the effect of the regulatory DCs to facilitate hyporesponsiveness in our ITD model.

DISCUSSION

This study confirmed that ITD of donor splenocytes prolonged survival of fully allogeneic cardiac grafts and generated donor-specific regulatory cells in mice. Induction of regulatory cells is one of the mechanisms by which allograft tolerance is produced (3). In many models of operational tolerance, regulatory cells can be transferred from animals with long-surviving grafts to naive syngeneic secondary recipients (18). However, in models other than ours, induction of regulatory cells required more than 100 days after grafting. In our model, adoptive transfer of cells from primary recipient mice that underwent ITD of donor splenocytes, but not transplantation, produced hyporesponsiveness in naive secondary recipients of allografts by 7 days after pretreatment of the primary recipients. This suggests that regulatory cells were induced in only 7 days in our model. We do not know why this happened so quickly, but we speculate that the microenvironment of the respiratory tract may have a special capacity for generating such cells. Results in our ITD model showed that adoptive transfer of not only CD4+ cells but also CD11c+ DCs induced remarkable prolongation of cardiac allograft survival in secondary recipients. These data indicated that the regulatory population consisted of not only CD4+ T cells but also DCs. Therefore, in the presence of alloantigen to T cells, DCs in the trachea presumably may have a special function with respect to induction of hyporesponsiveness to alloantigen.

With recent advances in the field of immunology, promising new therapies have arisen that could possibly eliminate lifelong drug therapy and promote indefinite acceptance of donor tissue (19). However, attempts to achieve induction of tolerance in clinical transplant recipients would require immunosuppressive coverage, at least initially, to mitigate the risk of rejection because the variables for reliable tolerance induction in humans have not been established. Nonspecific immunosuppressive agents commonly used to prevent T-cell–mediated acute rejection include calcineurin inhibitors (FK506 or CsA) (20), antiproliferative agents (AZA or MMF) (21), and inhibitors of growth-factor signal transduction (RAPA) (22). In this study, we examined the effects of these drugs on induction of regulatory cells in our murine ITD model.

In adoptive transfer studies, we found that high doses of FK506 or any dose of CsA given to primary recipients resulted in acute rejection of cardiac grafts in secondary recipients. It was previously shown that blockade of both signals 1 and 2 of T-cell activation by CsA and costimulation blockade prevents activation-induced cell death of alloreactive T cells, as well as allograft tolerance (11). These results, together with the data from the current study, indicate that under certain circumstances, global immunosuppression, which completely blocks T-cell activation, may perturb the development of regulatory cells and tolerance. In previous investigations, we showed that many signals, including CD80, CD86, CTLA4, CD70, OX40, and CD153, are required for independent generation of regulatory cells (23,24). Therefore, some immunosuppressant agents may regulate one of these important pathways.

On the other hand, a dose of 0.1 mg/kg FK506 did not abrogate prolonged graft survival in secondary recipients after adoptive transfer in our model. Thus, a low dose of this agent may not completely block cell proliferation, and a small population of cells that proliferate can divide for many generations. It might be suggested that the 0.1 mg/kg dose of FK506 was too low to induce hyporesponsiveness, but this could not have been the case because all C57BL/6 cardiac grafts in CBA recipients survived indefinitely when this dose was administered by IP injection daily (graft survival, >100 days × 6, n=6). Consistent with these results is a previous study that found that the MST of heterotopic cardiac grafts in rats increased from 6 to 9 days after baseline administration of 0.04 mg/kg per day FK506 (25).

The exact mechanisms of the effects of different doses of FK506 on induction of regulatory cells still remain unknown, although one possibility is that the agent changes the trafficking pattern of regulatory cells. When the mice were pretreated with ITD and a high dose of FK506 (1.0 mg/kg), the level of CFSE+ cells was lower than that in mice given adoptive transfer of cells from the pretreated mice with ITD alone (Fig. 6B and C, P<0.05). In contrast, when the mice were pretreated with ITD and a low dose of FK506 (0.1 mg/kg), the level of CFSE+ cells did not change compared with that in mice given adoptive transfer of cells from the pretreated mice with ITD alone. Consistent with our data, two reports showed the effect of FK506 on the function of lymphocyte migration. Tsuzuki et al. (26) reported that FK506 inhibits very late antigen-4/vascular cell adhesion molecule-1–mediated migration of lymphocytes in vitro and in vivo. Cristillo et al. (27) reported that FK506 attenuated mRNA transcription of IP-10, one of the important chemokines that has been implicated as a crucial mediator of T-cell migration. Taken together, these data suggest that one possibility of the mechanisms is that FK506 may affect the trafficking pattern of the regulatory population to abrogate hyporesponsiveness by regulatory cells.

Another possibility is that FK506 delays development of regulatory cells. However, this is unlikely because graft survival in secondary recipients was not prolonged when adoptive transfer was performed 14 or 28 days after pretreatment with ITD plus a high dose of FK506 (MST=11 days, in five mice with adoptive transfer 14 days after pretreatment with ITD; MST=28 days, in five mice with adoptive transfer 28 days after pretreatment with ITD, P<0.01 for each group compared with the ITD-alone group).

Moreover, two reports recently described that IL-2 was essential for expansion and homeostasis of regulatory T cells (28–30). In the present study, a high dose of FK506 (1.0 mg/kg) inhibited IL-2 production that was observed in MLC with regulatory cells induced by ITD alone. However, a low dose of FK506 (0.1 mg/kg) did not. Taken together, the differential effect of FK506 on hyporesponsiveness by the regulatory cells may be caused by regulating IL-2 production. Our findings suggest that when FK506 or CsA is introduced into a clinical organ-transplantation regimen, low doses of FK506 may be more useful than CsA for inducing and maintaining the regulatory mechanism.

The antiproliferative agents MMF and AZA prevent expansion of alloactivated T-cell and B-cell clones. AZA has been used as an immunosuppressive agent since the 1960s. In recent years, some reports have suggested that MMF provides better immunosuppression than AZA in patients who have undergone renal transplantation (31–34). Furthermore, MMF may also have a direct beneficial effect on kidney function (35). In the current study, MMF and AZA showed opposite effects on induction of regulatory cells after ITD of alloantigen: AZA abrogated such induction, whereas MMF facilitated it. The mechanisms for this difference are unknown. MMF has been shown to affect DCs directly by inhibiting costimulatory molecule expansion and production of IL-12 (36). In addition, a previous study (37) showed that treatment with 1α,25-dihydroxyvitamin D3 and MMF induces tolerance with an increased frequency of regulatory T cells. In that investigation, administration of both agents induced DCs with a tolerogenic phenotype, with down-regulated expression of CD40, CD80, and CD86 costimulatory molecules. These findings, along with our results showing that the regulatory population in our studies may contain DCs and CD4+ cells and the data from the current investigation (Fig. 6E), support the idea that treatment with MMF facilitates induction of regulatory cells in our model.

RAPA is a new drug approved for treatment of patients who have undergone organ transplantation. Because CsA and FK506 have nephrotoxic effects, RAPA is being tested alone and in combination with these agents in attempts to decrease the incidence of kidney damage and subsequent graft rejection in transplant recipients (22). The mechanism of action of RAPA is fundamentally different from that of other immunosuppressants: It inhibits cell-cycle progression and probably induces T-cell apoptosis. Costimulation blockade and treatment with RAPA, but not with CsA, was found to be critical in facilitating tolerance to skin grafts in mice, presumably by inducing unresponsiveness in alloreactive T cells after activation-induced cell death (11). In a study in adult rats, Tian et al. (38) demonstrated that treatment with RAPA increased the percentage of CD4+CD25+ T cells in the periphery, and this action may contribute to the immunosuppressive effect of the agent. In the current study, ITD pretreatment increased the number of CD4+CD25+ cells in the regulatory population, and the presence of RAPA (0.4 mg/kg) with ITD pretreatment further increased the number of CD4+CD25+ cells, which may result in the facilitation of hyporesponsiveness induced by ITD of alloantigen.

To further elucidate the effects of the immunosuppressants on hyporesponsiveness by the immune regulation, we used a different strain combination, C3H mice as donors and C57BL/6 mice as recipients, and performed adoptive transfer study in our mouse ITD model. Pretreatment with ITD of C3H splenocytes also induced hyporesponsiveness to C3H cardiac allografts in C57BL/6 recipients. Moreover, when ITD was combined with RAPA (0.4 mg/kg) and/or MMF (40 mg/kg), further prolongation of allograft survival was also observed in this strain combination, compared with that with ITD alone. These data suggest that the effects of RAPA and MMF to facilitate hyporesponsiveness induced by the regulatory cells were also applicable in different strain combination in our mouse ITD model.

A variety of immunosuppressive drugs are currently used to inhibit allograft rejection in clinical transplantation (19). However, these agents have failed to induce long-term acceptance of allografts, and rejection, especially chronic rejection, remains an important problem. Induction of transplantation tolerance by generation of regulatory cells might be one strategy for achieving permanent acceptance of allografts in clinical transplantation without incurring serious toxicity from treatment regimens. Our findings suggest that when such a strategy is developed, an immunosuppressive regimen that includes MMF and high doses of RAPA may be useful in facilitating immune regulation and inhibiting rejection. Low doses of FK506 or RAPA may be useful over the long term because they did not interfere with immune regulation in our model. In contrast, CsA, AZA, and high doses of FK506 may not be good choices for a posttransplantation regimen because they abrogated induction of immune regulation.

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Keywords:

Regulatory cells; Immunosuppressants; Trachea; Cardiac grafts; Adoptive transfer; Mice

© 2005 Lippincott Williams & Wilkins, Inc.