INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Currently, lung transplantation is considered the treatment of choice for end-stage diseases including primary pulmonary hypertension, pulmonary fibrosis, end-stage chronic obstructive lung disease, cystic fibrosis, and Eisenmenger's syndrome due to correctable cardiac lesions. Because of its recent arrival in the clinical arena, lung transplantation has enjoyed the most rapid growth in the field of solid organ transplantation during the past decade. The number of lung transplant procedures in the United States has increased exponentially from 18 cases in 1988 to 6,479 cases in 1996 (1, 2). With refinements in surgical techniques, patient selection, and perioperative care, the short-term survival in lung transplantation has improved markedly. In a very select group of low-risk patients with parenchymal lung disease, the 1-yr survival approaches 90% (3). However, in lung transplantation, as in any other solid organ transplantation, two major limitations currently exist: rejection, and complications secondary to the use of nonspecific immunosuppressive agents to prevent rejection. As a result, the intermediate and long-term graft survival is still poor. As of 1996, the overall 1- and 5-yr actuarial survival for lung allografts was 69% and 43%, respectively (2). Chronic rejection remains the number one cause of late graft loss. A better means to achieve graft acceptance is obviously needed.

Donor-specific transplantation tolerance is the state in which the host permanently accepts the transplanted organ without requiring antirejection drugs so that the infection, malignancy, and end organ toxicities associated with the use of nonspecific immunosuppression can be avoided (4). The induction of donor-specific transplantation tolerance, in which the immune system of the recipient is reeducated to see the donor as part of self, has been achieved across major histocompatibility complex (MHC) differences in rodents using mixed hematopoietic chimerism (5, 6). Fully allogeneic chimeras, in which the donor bone marrow totally replaces the recipient's marrow, exhibit donor-specific tolerance, but are prone to graft-versus-host disease (GVHD) (7) and are immunoincompetent to fight infection owing to a lack of the necessary antigen presenting cells for a full primary immune response (4, 8, 9). Mixed (donor/host) hematopoietic chimeras, which have both host- and donor-derived cells of multiple lineages in the peripheral blood, on the other hand, exhibit donor-specific transplantation tolerance, but are resistant to GVHD, and exhibit superior immunocompetence (10, 11). Mixed hematopoietic chimerism has been shown to be associated with tolerance to skin (5), islet cell (12), and cardiac grafts (13). However, donor-specific tolerance to lung allografts has never been reported. It has been demonstrated that different organs from the same donor are perceived differently by the recipient's immune system (14), with organs such as skin, lung, and gut being rejected more vigorously than organs such as liver, heart, and kidney (17). Furthermore, as with the liver and the gut, the lung carries with it a large number of passenger leukocytes in its bronchus-associated lymphoid tissues. Transplanting the lung allograft will adoptively transfer a large number of immunocompetent, donor-derived leukocytes that are alloreactive to the recipient. The risk for developing GVHD in a lung recipient who receives no immunosuppression, and whose immune system is tolerant to the donor (the mixed hematopoietic chimera) is unknown. In this study we investigated whether mixed hematopoietic chimerism induced donor-specific tolerance to lung allografts, and whether GVHD would develop in mixed chimeras after lung transplantation. We report herein, for the first time, that mixed hematopoietic chimerism induces donor-specific tolerance to rat lung allografts without causing GVHD.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Four- to eight-week-old MHC and minor antigen mismatched Fisher (F344; RT1.A^l ), Wistar Furth (WF; RT1.A^u), and ACI (RT1.A^a) male rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were housed in a specific pathogen-free facility at the University of Pittsburgh Biomedical Science Tower. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society of Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

Mixed Allogeneic Chimeras (F344 + WF  WF, ACI + F344  F344)

Mixed allogeneic chimeras were prepared as previously described (13). Recipients were conditioned with 1,100 cGy from a cesium 137 source (Gamma-cell; Nordion, ON, Canada). Bone marrow was harvested from the long bones of donors by flushing with media 199 (Gibco, Grand Island, NY) containing 2 µg/ml gentamicin (minimum essential medium [MEM]), using a 22-gauge needle. The marrow was gently resuspended using an 18-gauge needle and filtered through sterile nylon mesh. Bone marrow cells were washed (1,000 rpm for 10 min), resuspended, and counted before antibody and complement treatment for T-cell depletion. Rat bone marrow (20 × 10⁶ cells/ml) was T-cell-depleted using a 1:1,000 dilution of mouse anti-rat CD3 monoclonal antibody (mAb) (IF4; mouse IgM; Serotec, Bioproducts for Science, Indianapolis, IN) in MEM at 37° C for 30 min. Cells were washed, treated twice with rabbit complement (prepared from retired breeder rabbits and titered for use in our laboratory) at 37° C for 30 min, and washed twice before resuspension in MEM. Adequacy of T-cell depletion (TCD) was confirmed by flow cytometric analysis using anti- T-cell receptor-fluorescein isothiocyanate (TCR-FITC) mAb (R73; mouse IgG1; Serotec) and rat anti-mouse IgM FITC (R6-60.2; rat IgG 2a; Pharmingen, San Diego, CA), the secondary (anti-isotypic) antibody for CD3. CD3 and TCR mAbs were prescreened to ensure that cross-blocking did not occur. Mixed allogeneic chimeras were reconstituted with 1.0 × 10⁷ TCD syngeneic bone marrow cells and 15.0 × 10⁷ TCD allogeneic bone marrow cells. Cell concentration was adjusted so that total volume of injectate was 2 ml. Recipients were reconstituted within 4 to 6 h of lethal irradiation via penile vein injection using a 27-gauge needle.

Characterization of Chimeras by Flow Cytometric Analysis

Recipients were characterized for engraftment using flow cytometry to determine the percentage of peripheral blood lymphocytes (PBL) bearing F344, WF, or ACI MHC Class I cell surface markers, as described previously (13). Briefly, PBL were collected into heparinized plastic serum vials, diluted with 200 µl of MEM, and layered over lymphocyte separation media (Organon Teknika, Durham, NC) at room temperature. After centrifugation for 30 min at 400 × g, the lymphocyte layer was aspirated from the interface and washed in MEM. Lymphocytes were incubated for 45 min at 4° C with anti-F344, anti-WF, or anti-ACI, Class I biotinylated mAbs, a generous gift from Drs. Heinz W. Kunz and T. J. Gill, III (18), prior to counterstaining with a streptavidin-conjugated fluorescein antibody (SA FITC; Pharmingen). Flow cytometric analysis of stained lymphocytes was performed on a FACSORT (Becton Dickinson, Mountain View, CA) flow cytometer. Positive staining was determined on a log scale based on the inflection point where staining of a control negative population is minimized while retaining staining of the maximal numbers of positive cells.

Assessment Of GVHD

Chimeras were evaluated for evidence of GVHD on a daily basis for the first month following reconstitution and weekly thereafter. The diagnosis of GVHD was based on the previously described criteria, which consists of diffuse erythema (particularly of the ear), hyperkeratosis of the foot pads, dermatitis, unkempt appearance, weight loss, or diarrhea (19). The diagnosis of GVHD was confirmed by the histologic analysis of skin, tongue, liver, and small intestine at the time of necropsy using hematoxylin-eosin staining.

Mixed Lymphocyte Reaction (MLR) Assay

MLR assays were performed as described previously (20). Briefly, lymph nodes were harvested, diced, and crushed with a glass stopper to release lymphocytes. Isolated cells were lysed with ammonium chloride, potassium bicarbonate (ACK)-buffered solution, washed, and resuspended in Dulbecco's modified Eagle medium (DMEM) (Gibco) supplemented with 0.75% fresh normal rat serum, 0.09 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.05 mM 2-mercaptoethanol, and 1 mM N^G-mono-methyl-L-arginine (NMA). Responders (4 × 10⁵) were stimulated with 4 × 10⁵ irradiated (20 Gy) stimulators in a total of 200 µl of media. Cultures were incubated at 37° C in 10% CO₂, pulsed on the fourth day with 1 µCi of ³(H) thymidine (New England Nuclear, Boston, MA), harvested on the fifth day with an automated harvester (PHD cell harvester; Technology Inc., Cambridge, MA), and counted in a beta scintillation counter (Beckman, Palo Alto, CA). Results were expressed as counts per minute (cpm) ± SEM and as stimulation indices. The stimulation index (SI) is the ratio of the cpm generated in response to a given stimulator over the baseline cpm generated in response to the host (13).

Lung Transplantation

Mixed chimeras were transplanted 4 to 6 wk after bone marrow reconstitution, when flow cytometric analysis of peripheral blood had been performed to determine chimerism. Donor-specific (F344 or ACI) and third-party left lung allografts were transplanted by the cuff technique as previously described (21). Graft cold ischemic time (at 4° C in heparinized lactated Ringer's solution) ranged from 20 to 40 min. No immunosuppressive agents were administered post-transplant. Graft rejection was monitored by chest radiograph (CXR) every 2 to 3 d for the first 2 wk and then monthly thereafter. If an infiltrate was present by CXR, the animal was killed, the allograft excised, and examined grossly for evidence of technical failure, and histologically for rejection or infection.

Histologic Analysis of Rejection

Rejection was graded blindly by a lung transplant pathologist (S.A.Y.), using a previously reported scoring system (22). Briefly, the grading scale is as follows: Grade 0no cellular infiltrates. Grade 1perivascular infiltrates of small round and transformed large lymphoid cells and immunoblasts and endotheliolitis (endothelial cell hypertrophy and epithelioid changes). Grade 2perivascular mononuclear infiltrates with spread into adjacent alveolar septae and prominent alveolar macrophages. Grade 3perivascular mononuclear infiltrate with peribronchiolar inflammation, lymphocyte epidermotropism, epithelial cell necrosis, diffuse interstitial pneumonitis with alveolar pneumocyte necrosis, hyaline membranes, and intra-alveolar accumulation of neutrophils and macrophages. Grade 4vascular thrombosis with massive intra-alveolar hemorrhage and infarction, diffuse interstitial pneumonia with diffuse alveolar damage.

Cytokine mRNA Measurement

RNA extraction. Lung tissue samples were homogenized and total RNA was isolated with RNAzol (Biotecx Laboratories, Inc., Houston, TX) according to the protocol of Chomczynski and Sacchi (23). The RNA was quantitated spectrophotometrically and stored in precipitated form at 80° C.

Reverse transcription. Total RNA (2.5 µg) was used for first-strand complementary DNA (cDNA) synthesis in 10 µl of 250 µM Tris-HCl, 375 mM KCl, 50 mM dithiothreitol (DTT) (Gibco BRL, Bethesda, MD), 15 mM MgCl₂, 250 µg/ml actinomycin D (Sigma, St. Louis, MO), 20 U/ml mouse Moloney leukemia virus reverse transcriptase (Gibco BRL), 50 µg/ml oligo dT (Boehringer Mannheim, Indianapolis, IN), 10 mM deoxyribonucleoside triphosphate (dNTP) (Boehringer Mannheim), and human placental ribonuclease (RNAse) inhibitor (Gibco BRL) for 60 min at 37° C.

³²P-end-labeled polymerase chain reaction. The equivalent of 300 ng of transcribed cDNA was amplified using radiolabeled reverse transcriptase-polymerase chain reaction (RT-PCR). -actin and cytokine-specific primers (interleukin-2 [IL-2], IL-4, IL-6, IL-10, interferon-gamma [IFN-]) were designed according to the specifications of Innis and have been reported elsewhere (24, 25). In brief, the sense primer was terminally labeled with ³²P using T4 polynucleotide kinase (USB, Cleveland, OH) and gamma-radioactive phosphorus-deoxyadenosine triphosphate (-³²P-dATP). Unincorporated nucleotides were removed by separation with a Sephadex G-50 (Sigma) spin column. Radiolabeled sense primer was added to unlabeled sense and antisense primer to make a stock primer mix. Amplification was done in 50 µl under the following conditions: 1.5 mM MgCl₂, 10 mM Tris-HCl, 50 mM KCl, 1.25 mM dNTP (Boehringer Mannheim), and 1.25 U Taq Polymerase (Perkin-Elmer, Norwalk, CT); 30-cycle amplification was performed for semiquantitation with ³²P-labeled primers and 40 cycles for cold PCR. Cycling temperatures were: melting at 94° C for 1 min, annealing at 57° C for 2 min, and extension at 72° C for 3 min. RT-PCR of all samples in one comparative study was done simultaneously and with the same stock reagents to ensure similar biochemical conditions. Radiolabeled PCR products were separated in 5% polyacrylamide gels, dried, and quantitated by a BetaScope radioanalytical scanner and autoradiography. -actin was amplified to assess the integrity and equal loading of RNA. Oligonucleotide primers and probes used were listed as follows:

IL-2 upstream 5'-ACG CTT GTC CTC CTT GTC AAC-3' 401 bp

IL-2 downstream 5'-CAG ATG GCT ATC CAT CTC CTC-3'

IL-4 upstream 5'-GAC TCC ATG CAC CGA GAT GTT-3' 206 bp

IL-4 downstream 5'-TTC TCA GTG AGT TCA GAC CGC-3'

IL-6 upstream 5'-ACA GCG ATG ATG CAC TGT CAG-3' 337 bp

IL-6 downstream 5'-ATG GTC TTG GTC CTT AGC CAC-3'

IL-10 upstream 5'-GAC CAG CAA AGG CCA TTC CAT CCG GGG-3' 518 bp

IL-10 downstream 5'-GTC CTG CAG TCC AGT AGA TGC CGG GTG-3'

IFN- upstream 5'-CAA GGC ACA CTC ATT GAA AGA-3' 297 bp

IFN- downstream 5'-CTC GAA CTT GGC GAT GCT CAT-3'.

Experimental Design

Syngeneic (Group A) and allogeneic (Groups B and C) control animals were naive recipients that received syngeneic and allogeneic lung grafts, respectively. Recipient animals in these groups did not receive bone marrow or radiation treatment. Groups D and E (donor-specific chimeras) were chimeric animals (F344 + WF  WF and ACI + F344  F344, respectively) that received donor-specific lung allografts. Group F (third-party chimera) consisted of chimeric animals (F344 + WF  WF) that received a third-party (ACI) lung allograft. Group G (radiation control) consisted of animals (WF) that received lethal radiation (1,100 cGy) and a mixture of donor (F344) and recipient (WF) bone marrow, as those in Groups D, E, and F, but failed to engraft donor bone marrow, and reconstituted with 100% host (WF) bone marrow. These animals were transplanted with donor-specific allografts (F344) and served as controls for the effects of lethal radiation and bone marrow reconstitution.

Statistical Analysis

Cytokine messenger RNA (mRNA) concentrations were expressed as mean ± standard error of the mean and comparisons performed using t test. Histologic scores were compared between groups using the Mann-Whitney U test. Differences were considered significant when p < 0.05. All statistical analyses were performed using a StatView 4.5 program software (Abacus Concepts, Inc., Berkeley, CA) and Statistica software (1991 Statsoft, Inc., Tulsa, OK).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of Mixed Allogeneic Chimeras

Engraftment in mixed allogeneic rat chimeras was evaluated by PBL typing using biotinylated mAbs and flow cytometric analysis 28 d after bone marrow reconstitution. Mixed chimerism was defined as at least 1% of donor lymphocytes in the peripheral blood as determined by flow cytometry. Chimerism below this level is not reliably detected by flow cytometry. Eighty-five percent of rats that received CD3-depleted bone marrow engrafted as mixed chimeras, consistent with our previously reported experience (13). Levels of PBL chimerism in animals that engrafted ranged from 5 to 97% (Table 1), and persisted up to the time of killing (> 300 d). The chimerism was multilineage, consisting of T cells, B cells, macrophages, and granulocytes of both donor and recipient origins, suggesting that the pluripotent hematopoietic stem cell had engrafted (13). None of the chimeras exhibited clinical or histologic evidence of GVHD before or after lung transplantation.

In Vivo Evidence of Donor-specific Tolerance: Long-Term Acceptance of Lung Grafts

After lung transplantation, animals were killed at predetermined time intervals or when there was opacification of the transplanted lung on chest radiograph to assess the degree of rejection by histology. Rejection grades are summarized in Table 1. Donor-specific lung allografts were permanently accepted by mixed chimeric rats with no to minimal rejection (grade  2) up to 317 d after transplantation (> 60 d: 11 animals; > 100 d: 5 animals; > 150 d: 3 animals; > 300 d: 3 animals; Groups D and E). The donor-specific tolerance achieved in this model was not dependent on selected rat strain combinations, because the ACI to F344 strain combination (Group E) yielded similar results as the F344 to WF combination (Group D). Chimeras with 5% donor cell engraftment were as tolerant to donor-specific lung allografts as those with 97% donor chimerism (Groups D and E). Rat chimeras rejected MHC-disparate third-party lung allografts in less than 10 d (Group F), with a time course similar to unmanipulated controls (Groups B and C), indicating that immunocompetence was preserved in these chimeras. Donor-specific tolerance for lung allografts was not present in rats that did not engraft with donor bone marrow (Group G), suggesting that donor bone marrow engraftment (or the presence of hematopoietic stem cell macrochimerism) is vital for the induction of tolerance in this model.

Figure 1 depicts the chest radiographs of a third-party left lung allograft at 10 d (left panel ) and of a donor-specific left lung graft at > 100 d after transplantation (right panel ). The donor-specific graft was radiographically normal whereas the third-party graft had complete opacification, indicative of severe rejection. Figure 2 shows the histology of a normal rat lung (A), a third-party lung graft at 10 d (B), and a donor-specific graft at 104 d (C ) after transplantation. The third-party graft has severe acute cellular rejection with perivascular and interstitial infiltrates (Grade 3) while the donor-specific graft appears histologically normal.

View larger version (98K): [in this window] [in a new window]   Figure 1.   Chest radiographs of two rat chimeras after left lung transplantation. (Left panel ) Third-party left lung allograft 10 d after transplantation. The presence of severe infiltrates, volume loss, and near total "white out" is indicative of severe rejection. (Right panel ) Donor-specific allograft 150 d after transplantation with a clear left lung field indicating graft acceptance and preserved function. The linear intense opacifications in the left lung field represent the cuffs used for vascular anastomoses.

View larger version (87K): [in this window] [in a new window]   Figure 2.   Rat lung histology. (A) Normal rat lung. (B) Third-party left lung allograft 10 d after transplantation, showing severe acute cellular rejection (grade 3) with perivascular and interstitial infiltrates. (C ) Donor-specific lung allograft at 104 d after transplantation displaying only scattered inflammatory cells, but essentially preservation of normal architecture.

In Vitro Evidence of Donor-specific Tolerance

Mixed lymphocyte reaction (MLR). Mixed allogeneic rat chimeras (F344 + WF  WF) that had accepted the donor-specific lung graft (F344) for more than 60 d were assessed for donor-specific tolerance in vitro using an MLR assay directed against self (WF), donor-specific (F344) and third-party (ACI) antigens. Lymphocytes harvested from the lymph nodes of chimeras were functionally tolerant to host and donor-strain alloantigens, yet competent to respond to third-party alloantigens (Figure 3).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Mixed lymphocyte reaction. Lymphocytes from naive F344 rat, naive WF rat, and mixed allogeneic chimeras (F344 + WF  WF) were cocultured with irradiated recipient (WF), donor (F344), and third-party (ACI) splenocytes in MLR assay. Values are shown as mean ± SEM of triplicate cultures in a 1:1 responder to stimulator ratio. The stimulation index (SI) is shown above each bar.

Intragraft cytokine gene expression. To compare the intragraft cytokine gene expression, selected syngeneic (WF to WF), and donor-specific (F344) lung grafts were harvested at 30 and 60 d after transplantation. Snap-frozen lung tissues were analyzed for cytokine mRNA by semiquantitative RT-PCR. The mRNA profiles for T-helper cell type 1 (Th1; IFN-, IL-2) and Th2 (IL-4, IL-6, and IL-10) cytokines in the syngeneic grafts and donor-specific grafts that had been transplanted into mixed chimera were identical (Figure 4). These data suggest that there is no alloimmune reactivity to the donor-specific graft not only at the histologic but also at the molecular levels.

View larger version (26K): [in this window] [in a new window]   Figure 4.   Cytokine mRNA levels (normalized to those of -actin) in lung allografts at 30 and 60 d after transplantation. mRNA levels were measured in lung tissue by radiolabeled RT-PCR. Quantification of radioactive counts was performed with a Betascope radioanalytical scanner. Each data point represents the mean ± SE of at least eight animals. There was no difference in Th1 and Th2 cytokine profiles (t test) between the syngeneic grafts and donor-specific grafts that had been transplanted into mixed chimeras. Notice the difference in the scale of the coordinates for each cytokine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Over the past decade the short-term survival of lung recipients has dramatically improved. One-year survival rate has approached 90% in a very select group of low-risk patients (3). However, little progress has been made to alter the incidence and prevalence of acute and chronic rejection in lung transplant recipients. Under the current immunosuppressive regimens, 93% of lung recipients have at least one episode of acute rejection within 1 yr after transplantation, and up to 50% of lung transplant recipients develop obliterative bronchiolitis (OB) 2 yr after transplantation (26, 27). OB is a form of chronic rejection that carries up to 90% mortality for which there is no effective treatment but retransplantation. A unique feature of the lung allograft is that it is in direct contact with the ambient organisms, making it extremely susceptible to a variety of bacterial, viral, and fungal infections. The use of nonspecific immunosuppressants exacerbates this problem. Infection, therefore, is the most common cause of death in lung transplant recipients (28). In addition, the incidence of posttransplant lymphoproliferative disease is highest in lung transplant recipients, probably because of the higher level of immunosuppression required to maintain graft survival (29). Recipients of lung allografts are, therefore, more affected by the chronic use of nonspecific immunosuppressants, and the inability to prevent rejection than recipients of other organs. Improved means to induce long-term graft acceptance is desperately needed in clinical lung transplantation.

Our data indicate that mixed hematopoietic chimerism induces and maintains donor-specific tolerance for lung allografts in MHC, and minor histocompatibility disparate rat strain combinations without the need for chemical immunosuppression. Furthermore, these mixed chimeras do not develop GVHD, and are immunocompetent as demonstrated in vivo by their ability to reject vigorously third-party grafts, and in vitro by their strong reactivity toward third-party alloantigens in the MLR assays. Our data also clearly demonstrate that a level of donor chimerism detectable by flow cytometry, which has a threshold of 0.5 to 1%, is sufficient for the induction of systemic donor-specific tolerance in this model. If mixed chimerism was not detectable by flow cytometry, donor-specific tolerance could not be induced. However, after a minimal level of donor hematopoietic chimerism is achieved, the degree of chimerism (ranging between 5% and 97% of donor cells in peripheral blood) does not appear to affect the development or degree of tolerance.

Although mixed hematopoietic chimerism has been associated with donor-specific tolerance for a variety of solid organ and cellular grafts (5, 12, 13, 30), our data demonstrate, for the first time, that mixed hematopoietic chimerism induces donor-specific tolerance to lung allografts, an organ that is highly antigenic partly due to the large number of passenger leukocytes residing in the bronchus-associated lymphoid tissues (31). The tolerance to donor-specific lung allografts was demonstrated not only by histology and MLR, but also by intragraft Th1 and Th2 cytokine profiles. Mixed hematopoietic chimerism, therefore, induces robust tolerance to lung allografts.

The results of the current study lend further support to the concept that mixed hematopoietic chimerism induces transplantation tolerance. However, the clinical application of this concept to induce tolerance awaits the ability to develop nonlethal conditioning techniques to achieve mixed hematopoietic chimerism. A great body of evidence accumulated during the past decade suggests that mixed hematopoietic chimerism and tolerance induction could be achieved at less than lethal doses of irradiation. After Cobbold and coworkers reported that engraftment of MHC-disparate marrow could be achieved using a combination of depleting and nondepleting mAbs plus radiation (32), Sharabi and Sachs modified this nonlethal mouse model of mixed hematopoietic chimerism by adding 700 cGy of thymic irradiation to the depleting anti-CD4 and anti-CD8 mAbs, and reduced the total-body irradiation (TBI) to 300 cGy (33). More recently, durable multilineage mixed hematopoietic chimerism and donor-specific transplantation tolerance was achieved across fully mismatched MHC plus minor antigen barriers at substantially lower doses of radiation in mice by the administration of a single dose of cyclophosphamide 2 d after bone marrow transplantation (34). Translations of these strategies to large animal models are under way and have shown promising results (35).

In summary, we have demonstrated that permanent donor-specific tolerance can be achieved for lung allografts in rats through mixed hematopoietic stem cell chimerism. Excellent long-term survival was achieved without histologic or clinical evidence of GVHD or rejection. Most importantly, recipients were immunocompetent to respond to MHC disparate third-party antigens in vivo and in vitro. Chimerism may provide a viable clinical approach to achieve graft acceptance in pulmonary transplantation. The development of a nonmyeloablative/nonlethal approach to achieve mixed hematopoietic chimerism will be critical in permitting the application of this concept to clinical solid organ transplantation.