Induction of tolerance to hind limb allografts in rats receiving cyclosporine A and antilymphocyte serum: effect of duration of the treatment

Abstract

Background.  

This study assessed the ability of antilymphocyte serum (ALS) and cyclosporine A (CsA) to induce tolerance for hind limb composite tissue allograft in rats without chronic immunosuppression.

Methods.  

Hind limb transplantations were performed in Lewis-Brown-Norway (LBN, RT1^l+n) and Lewis (LEW, RT1^l) rats. Treatment consisted of ALS only (0.4 mL/kg), CsA only (16 mg/kg), and a combination of CsA and ALS, and it was administered 12 hr before surgery at three different intervals (7, 14, and 21 days). Long-term survivors were tested for tolerance by standard skin grafting from the recipient (LEW), the donor (LBN), and the third party (ACI, RT1^a) 60 days after cessation of the treatment and by mixed lymphocyte reaction at 100 days. T-cell lines were analyzed with flow cytometry.

Results.  

Single use of ALS in all treatment intervals did not prolong allograft survival. Single use of CsA extended survival up to 23 days in the 21-day protocol group. CsA and ALS caused indefinite survival in two of six rats in the 14-day protocol and in all six rats in the 21-day protocol (>420 days). The six long-term survivors in the 21-day protocol accepted the skin grafts from the donor (LBN) and the recipient (LEW) and rejected third-party grafts (ACI). Tolerant animals showed a donor-specific hematopoietic chimerism of 35% to 42% in the peripheral blood. Mixed lymphocyte reaction assay demonstrated tolerance to the host and donor alloantigens and increased response to the third party.

Conclusions.  

Administration of CsA and ALS for 21 days induced donor-specific tolerance in the recipients of the rat hind limb composite tissue allografts. The mechanism of tolerance should be investigated further.

Composite tissue allografts (CTA) consist of bone, muscle, skin, nerve, and tendon and are excellent sources of reconstructive material for lost tissues resulting from trauma, burns, tumor surgery, and congenital defects. The first human hand transplantation was performed in 1998 (¹). Since then, CTA transplants have gained some popularity as a viable alternative source of reconstruction. However, CTA transplants are not routinely performed today because of the need for lifelong nonspecific immunosuppressive agents to prevent the immune system from rejecting the foreign tissue. These agents are associated with an increased rate of neoplasm and opportunistic infections (²). Moreover, because CTAs are immunogenic in nature, higher doses of immunosuppressive drugs are needed than with solid organ allografts (³). Even with excellent patient compliance, conventional immunosuppressive protocols are not sufficient to prevent chronic rejection. Thus, researchers have been actively looking for alternative methods that can induce lifelong tolerance using less toxic regimens.

One such method is tolerance—a state of specific unresponsiveness to donor antigens that is achieved without chronic immunosuppression. Tolerance has been induced in different transplant models, including CTAs, using combinations of low-dose radiation antilymphocyte serum (ALS), cyclosporine A (CsA), and other conventional immunosuppressants (^4–7). Treatment with ALS for a short period of time and infusion of donor bone marrow (BM)-derived cells has been shown to induce donor-specific tolerance in skin allografts in mice (^8,9). In subsequent studies, the combined use of ALS and BM-derived cells demonstrated a broad usefulness in various models (^10–12).

Recent studies in human kidney and liver allograft patients have revealed a reduction in the rates of acute rejection in patients treated with conventional immunosuppression and adjuvant BM transplantation (^13,14). The efficacy of conventional immunosuppression with an ALS and BM infusion regimen has been proven in various models. In this study, the usefulness of this combination with vascularized bone marrow transplantation (VBMT) was investigated. VBMT as an element of hind limb allografts used in this study has been considered as a constant source of donor-derived BM cells to the recipient (^15–17). Talmor et al. have shown that the combination of CsA and vascularized BM provides a means of inducing chimerism in lymphoid tissues in nonirradiated allograft recipients (¹⁸). In a study using mismatched mouse skin allografts, Hale et al. preconditioned the recipients with ALS and sirolimus and infused donor BM (¹⁹). They reported a stable multilineage hematopoietic chimerism with donor-specific tolerance without radiation. Also, ATG and FK506 have been used in the clinical setting to prolong the allograft survival in the recent hand allograft transplant (¹). In this study, the efficacy of combined CsA and ALS therapy was tested in a clinically relevant treatment schedule on the hind limb allograft transplants.

MATERIALS AND METHODS

Animals

Animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The institution’s animal care facility is accredited by the American Association for the Accreditation of Laboratory Animal Care.

Inbred 6- to 8-week-old male rats weighing 150 to 175 g were used (Harlan Sprague-Dawley, Indianapolis, IN). Lewis rats (LEW, RT1^l) served as the recipients of limb allografts from Lewis-Brown Norway rats (LBN, RT1^l+n). For evaluation of tolerance, skin grafts were taken from LEW, LBN, and ACI (RT1^a) rats. Intraperitoneal pentobarbital anesthesia (40 mg/kg) was administered to the rats, and surgical procedures were performed by two surgeons using standard microsurgical techniques under the operative microscope (Zeiss OP-MI 6-SD, Carl Zeiss, Goettingen, Germany).

Experimental Groups

Transplant groups and the number of transplants performed in each group are summarized in Table 1. In all treatment groups, CsA (Novartis, East Hanover, NJ) and ALS (Accurate, Westbury, NY) were administered 12 hr before surgery for three different treatment intervals (7, 14, and 21 days). CsA 16 mg/kg was administered subcutaneously every day for the first week and was tapered to a half-dose during the second and third weeks. ALS 0.4 mL was administered intraperitoneally every day during the first week, every other day during the second week, and twice a week during the third week. The ALS was prepared from rabbit anti-rat lymphocyte cells that were obtained from animals with no history or clinical signs of infectious diseases (in compliance with Food and Drug Administration procedure). Modified colorimetric test microtiter assay was used to determine the activity exhibiting more than 80% cytotoxicity on rat lymphocytes in its 1:800 diluted form.

Table 1: Table 1. Treatment groups, number of allograft transplants, and allograft survival time

Transplantation Technique

Limb transplantations were performed as described previously (²⁰). Briefly, a circumferential skin incision was made in the proximal one third of the right limb. The femoral artery and vein were dissected, clamped, and cut proximal to the superficial epigastric artery. The femoral nerve was dissected and cut 1 cm distal to the inguinal ligament. The biceps femoris muscle was transected to expose the sciatic nerve. The nerve was then transected proximal to its bifurcation. The limb was amputated at the mid-femoral level. The donor was also prepared in a similar way. The limb was again amputated at the mid-femoral level. The donor limb was attached to the recipient limb by a 20-gauge intramedullary pin and a simple cerclage wire. All large muscle groups were sutured in juxtaposition. The iliac vessels of the donor and femoral vessels of the recipient were anastomosed using a standard end-to-end microsurgical anastomosis technique under an operating microscope with 10/0 sutures. The femoral and sciatic nerves were repaired using a conventional epineural technique with four 10.0 sutures.

Flow Cytometric Analysis

Phenotype analysis of mononuclear cells was performed according to the manufacturer protocol (Becton Dickinson, San Diego, CA) with minor modifications. Anticoagulated whole peripheral blood samples (50 μL) were collected from each group of LEW recipients. The samples were incubated for 20 to 30 min in the dark at room temperature with a 5 μL mixture of mouse anti-rat monoclonal antibodies conjugated with the fluorescein isothiocyanate (FITC) or phycoerythrin (PE) against NKR-P1^-FITC (Clone 3.2.3)/CD8a^-PE (Clone OX8); CD4^-FITC (Clone OX35)/CD8a^-PE; and CD90^-FITC (Clone OX7)/CD3^-PE (Clone OX33). Red blood cells were lysed with FACS lysing solution (Becton Dickinson), and the samples were washed twice in the washing buffer (2% bovine serum albumin, 0.1% NaN₃/phosphate-buffered saline [PBS]) fixed with 1% phosphonoformic acid solution and kept covered at 4⁰C until analysis by flow cytometry (FACS Scan, Becton Dickinson).

For assessment of chimerism in peripheral blood, mononuclear cell combinations of conjugated mouse anti-rat RT1^l-FITC (Lewis major histocompatibility complex [MHC] class I, Clone B5, Becton Dickinson Pharmingen, San Diego, CA) and RT1^n-FITC (Brown Norway MHC class I, Clone MCA-156, Serotec, Oxford, UK) with CD4^-PE or CD8a^-PE were used. For RT1ⁿ staining, purified anti-rat CD-32 (FcγII Block Receptor) antibody (1:10) was added to block the Fc-mediated adherence of the antibodies. After 3 to 4 min of preincubation, the samples were further incubated with 5 μL of RT1ⁿ for 30 min in 4^oC and then incubated with goat anti-mouse IgG-FITC conjugated rat absorbed antibody (Serotec). Next, samples were washed twice in the washing buffer and stained with CD4^-PE or CD8^-PE conjugated mouse anti-rat monoclonal antibody as described previously. The control panel included isotype IgG₁-FITC/IgG₂-PE control-matched antibodies or PBS incubated samples or both. Analyses were performed on 1×10⁴ mononuclear cells using FACS Scan and CellQuest)BD Biosciences, Bedford, MA) software.

Skin Grafting

For in vivo testing of the donor-specific tolerance and immunocompetence, skin grafts were taken from the recipient (LEW), donor (LBN), and third party (ACI) rats and placed on the long-term survivors 60 days after the treatment was stopped. The standard skin grafting procedure described by Billingham and Brent was used (²¹). Full-thickness skin grafts 16 mm in diameter were taken. Graft beds were prepared by excising 18-mm circles on the lateral dorsal thoracic walls of the recipients. Care was taken to remove panniculus carnosus from the grafted skin. Both sides of the thoracic wall were used for allogeneic grafts, and the mid sternum was used for the syngenic grafts. All grafts were separated by a 10-mm skin bridge. A standard compressive dressing and adhesive bandage were used for 7 days. The grafts were evaluated on a daily basis after transplantation for signs of rejection. Rejection was defined as the destruction of more than 80% of the graft.

Mixed Lymphocyte Reaction Assay

Long-term limb allograft survivors (n=6) receiving CsA and ALS for 21 days were tested for alloantigen reactivity by mixed lymphocyte reaction (MLR) responsiveness at 100 days posttransplant. Responder T-cell suspensions were prepared from the peripheral blood. Lymphocytes were freshly isolated using histoplaque (Sigma, St. Louis, MO), and the buffy coat layer was washed using PBS. The cells were resuspended in complete medium, RPMI (Gibco, Gaithersburg, MD) supplemented with 10% of fetal calf serum (Gibco), 2 mM L-glutamine, 5×10⁻⁵ M 2-beta mercaptoethanol, 10 mM HEPES, penicillin (100 U/mL), and streptomycin (100 U/mL) (Gibco); 2×10⁵ cells were delivered in triplicate to the wells of a 96-well round bottom tissue culture plates. Stimulator cells were prepared from spleens of the naïve syngenic (LEW), semi-allogenic (LBN), and third party (ACI) rats. Splenocytes were isolated gently by passing/mincing each spleen through a sterile mesh bag with complete RPMI medium. The splenocyte suspensions were treated with Tris-ammonium chloride for 5 min at room temperature and then washed in complete medium. The cells were inactivated by mitomycin C (Sigma) for 30 min at 37°C and then washed in complete medium. Splenocytes (0.5×10⁶ and 0.25×10⁶ cells) were resuspended in complete medium and incubated for 72 hr with the responder cells at 37°C and 5% CO₂ in the air. After 72 hr, cultures were pulsed with 1 μCi [³H] thymidine. After 12 to 18 hr incubation at 37°C and 5% CO₂ in the air, the cultures were harvested onto the fiber filter mats, and the amount of ³H incorporation was determined by a beta counter. The stimulation index (SI) was calculated by dividing the mean counts per minute from responders against host (LEW), donor (LBN), or third party (ACI) by mean background counts per minute.

Statistical Analysis

Statistical significance was determined with a two-tailed Student t test for comparison of means with unequal variances. A P value of less than 0.05 was considered statistically significant.

RESULTS

Hind Limb Allograft Survival

Table 1 shows transplant survival time in the allograft and isograft controls and in the three treatment groups (7, 14, and 21 days). All isograft controls (n=6) survived indefinitely. In the allograft control group (n=9), the first signs of rejection (e.g., erythema of the skin) started on the seventh day. Combined ALS and CsA treatment extended the allograft survival for all three treatment durations compared with the use of ALS or CsA alone (P <0.05 for both comparisons). Animals in the 21-day protocol (n=6) uniformly accepted the hind limb allografts (420 days). Under the 14-day protocol, only two of the six limb recipients demonstrated indefinite survival (>360 days).

Flow Cytometric Analysis of In Vivo Depletion

The levels of CD3, CD4, CD8, CD90, and NK cells were similar at day 7 in all treatment groups, and no statistically significant differences were found among the groups (Fig. 1). At days 14 and 21, all receptor levels gradually increased in the recipients of the 7-day and 14-day protocols. Long-lasting depletion until posttransplant day 35 was observed only in the 21-day protocol (P <0.05).

Figure 1: Percentage of T cells positive for CD3, CD4, CD8, CD90, and NK receptors in the rats that received combined cyclosporine A (CsA) and antilymphocyte serum (ALS).

Determination of Donor-Specific Chimerism

Evaluation of donor-specific chimerism in lymphoid cells that were isolated from the peripheral blood of the long-term survivors showed stable donor-specific chimerism ranging from 37% to 42% (RT1^n-IgG-FITC single positive cells). Examination of double color RT1^n-IgG-FITC/CD4^-PE- and RT1^n-IgG-FITC/CD8^-PE-stained peripheral lymphocytes revealed 3.4% and 12.8% of double positive CD4 and CD8 T-cell subpopulations, respectively (Fig. 2, A1 and A2). The differences between the total number of donor-specific RT1^n-IgG-FITC single positive chimeric cells and the sum of RT1^n-IgG-FITC/CD4^-PE and RT1^n-IgG-FITC /CD8^-PE double positive chimeric T cells were due to the existence of another peripheral blood cell subpopulation that possessed chimeric phenotype (B-cells, monocytes, granulocytes). These cells were not determined by flow cytometry assessment. Flow cytometric analysis of the mononuclear cells isolated from the peripheral blood of naïve LBN (F1) positive control demonstrated 31% to 35% of single positive RT1^n-IgG-FITC cells, 9.4% of double positive RT1^n-IgG-FITC/CD4^-PE cells, and 5.3% of RT1^n-IgG-FITC/CD8^-PE cells (Fig. 2, B1 and B2). No single RT1^n-IgG-FITC and double RT1^n-IgG-FITC/CD4^-PE or RT1^n-IgG-FITC/CD8^-PE positive cells were found in the naïve Lewis controls (Fig. 2, C1 and C2).

Figure 2: Flow cytometric analysis donor-specific chimerism in lymphocytes collected from the peripheral blood of the long-term survivors (420 days). Freshly isolated mononuclear cells were stained with the monoclonal mouse anti-rat RT1^n-IgG-FITC and CD4^-PE or CD8^-PE antibodies. Flow cytometric dot plot representation of the single positive (RT1^n-IgG-FITC) cells and chimeric double positive CD4/RT1^n-IgG-FITC and CD8/RT1^n-IgG-FITC T cells isolated from the peripheral blood of Lewis (LEW) recipient (Fig. 1, A1 and A2), naïve Lewis-Brown-Norway (LBN) positive control (Fig. 1, B1 and B2), and naïve Lewis negative control (Fig. 1, C1 and C2).

Donor-Specific Tolerance In Vitro: Mixed Lymphocyte Reaction Reactivity

All six recipients under the 21-day protocol showed increased responsiveness to the third party (ACI) challenge at posttransplant day 100. The SI revealed that only 25% of the cells from the naïve animals were reactive against ACI antigens, whereas 81% were reactive in the long-term survivors (P <0.05). This increased response is probably related to the presensitization of the allograft recipients during the skin grafting procedure from the ACI donors. Responsiveness to the donor antigen (LBN), however, was significantly reduced in all recipients. The SI showed a suppressed response in long-term survivors against LBN antigens (SI ranged from 7% in naïve recipients to 4% in tolerant recipients, P <0.05) (Fig. 3).

Figure 3: Lymphocytes from long-term survivors in 21 days combined protocol were cocultured with irradiated recipient (LEW), donor (LBN), and third-party (ACI) alloantigens in mixed lymphocyte reaction (MLR) assay. Values are mean ± SD of triplicate cultures in a 1:1 responder to stimulator ratio. Stimulation index (SI, number of responders/total number of cells × 100) (above each bar).

In Vivo Evidence of Donor-Specific Tolerance: Skin Grafting

All six rats under the 21-day protocol of CsA and ALS uniformly accepted skin allografts from the donor (LBN) and the recipient (LEW) but rejected those from the third party (ACI). However, the two long-term survivors under the 14-day protocol rejected both the LBN and ACI skin grafts.

DISCUSSION

In this study, combined use of ALS and CsA induced donor-specific tolerance in hind limb allografts. We found that only the 21-day protocol was successful in inducing tolerance in all recipients. This confirms the importance of eliminating mature T cells from the periphery until the hematopoietic reconstitution takes place. Flow cytometry showed that more than 90% of T-cell lines were depleted at day 7, and that they gradually repopulated by day 63. These results indicate that using ALS and CsA to delete T cells in an adult animal can revert the immune system back to an immature developmental state. The T-cell component of the immune system then redevelops in the presence of CTA antigens, and those cells reactive against them are deleted or rendered unresponsive. Failure to achieve tolerance in the 7-day and 14-day protocols was probably related to the incomplete elimination of mature T cells from the periphery.

The engraftment of donor BM cells in the recipient’s hematopoietic organs as a method to induce transplant tolerance requires the ablation of the host’s immune system to create the “space” for engraftment. This ablation is performed by the use of various degrees of irradiation with or without additional immunosuppression (^22,23). Studies with mixed allogeneic chimerism involve total body or lymphoid irradiation followed by the reconstitution with a mixture of host and donor T-cell depleted BM (^24,25). Although this approach is less toxic than lethal irradiation, radiation is still involved as an integral part of the host conditioning, which limits its use in humans. In an attempt to identify less-toxic regimes, investigators tried to replace irradiation with chemical methods of ablation.

The study performed by Hale et al. was the first to show that cytoreduction and ablation using ALS, sirolimus, and donor-specific BM infusion could induce tolerance and chimerism in a mismatched murine skin allograft model (¹⁹). In their study, the engraftment of the donor BM cells was encouraged by host conditioning, which was achieved with two methods. First, the use of broad-spectrum immunosuppression (sirolimus) prevented immediate graft rejection. Second, the use of ALS created the space needed for engraftment. These two components are similar to the combination regimen (ALS and CsA) that we started 12 hr before transplantation.

Our approach to BM transplantation differs significantly from the cellular bone marrow transplantation (CBMT) models. The rat hind limb allograft represents a VBMT model, which provides a constant flow of donor-specific progenitor cells (^26,27). It has been suggested that hematopoietic reconstitution is faster in VBMT than in CBMT (¹⁷). This is probably related to the stromal microenvironment, which is transplanted as a part of the vascularized BM. It has been shown that stromal cells transplanted in the form of femoral bone fragments prevent graft failure in a lethally irradiated mouse model (²⁸). Stromal cells are essential for BM-derived cells to proliferate and differentiate into hematopoietic progenitors. Transplantation of a donor-derived BM graft under the kidney capsule along with BM cells has been shown to facilitate reconstitution of the host with marrow cells of donor origin (²⁹). We have previously investigated whether the BM cells transplanted in the form of tibial bone fragments and engrafted into the recipient’s BM cavity (the tibia) are functionally capable of engrafting, replicating, and inducing tolerance. We found that 6% to 10% chimerism can be achieved after such a transplantation, and that it can extend skin allograft survival up to 60 days across a semiallogeneic MHC barrier under a 7-day protocol of CsA and anti-T-cell receptor mAb treatment (unpublished data). Therefore, VBMT along with its microenvironment would seem to be a better source of donor-origin hematopoietic cells than a CBMT. On the other hand, a VBMT in the form of a hind limb allograft can cause graft versus host disease (³⁰). We did not expect such a finding in the current study because of the semiallogeneic mismatch between the recipient (LEW) and the donor (LBN).

In conclusion, this study demonstrates the successful induction of tolerance in CTAs under a 21-day protocol of CsA and ALS. Studies are designed to further investigate the mechanism of tolerance in this model and in transplant recipients of fully mismatched allografts.