Ann Thorac Surg 2003;75:257-263
© 2003 The Society of Thoracic Surgeons
Original article: general thoracic
Intrathymic inoculation of donor bone marrow induces long-term acceptance of lung allografts
Sen Li, MDa,
Louis B. Louis, IV, MDa,
Nobuyoshi Kawaharada, MD, PhDb,
Samuel A. Yousem, MDc,
Si M. Pham, MDa*
a Division of Cardiothoracic Surgery, University of Miami School of Medicine, Miami, Florida,, USA
b Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
c Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
Accepted for publication August 9, 2002.
* Address reprint requests to Dr Pham, Division of Cardiothoracic Surgery, University of Miami School of Medicine, Highland Professional Building, 5th Floor, 1801 NW 9th Ave, Miami, FL 33136, USA.
e-mail: spham{at}med.miami.edu
Presented at the Thirty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28–30, 2002.
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Abstract
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BACKGROUND: We investigated whether intrathymic inoculation of donor bone marrow at the time of transplantation induced long-term acceptance of lung allografts.
METHODS: Four- to-six-week-old August Copenhagen Irish (ACI) and Wistar Furth (WF) rats were used as donors and recipients, respectively. After being inoculated intrathymically with either donor-specific (ACI) or third-party (F344) bone marrow (2.0 x 107 cells/lobe), the recipient (WF) animal received a left lung transplant from an ACI donor. A short course of tacrolimus (1 mg/kg per day for 5 days) was administered. Animals were sacrificed at timed intervals after transplantation, and rejection was graded on a scale of 0 (none) to 4 (severe).
RESULTS: At 28 days, animals receiving donor-specific bone marrow have lower (p < 0.01) median rejection grade (MRG = 0.25; n = 6) than those receiving third-party bone marrow (MRG = 3; n = 6) and controls (no bone marrow; MRG = 2.5; n = 6). Animals receiving intrathymic donor bone marrow accepted lung allografts up to 380 days with minimal rejection (MRG = 2; n = 6). Long-term lung recipients also accepted a challenging donor-specific heart graft (n = 4) for more than 150 days. In mixed lymphocyte reaction assays, T lymphocytes of WF recipients that had received intrathymic bone marrow (from ACI donor) exhibited low response (similar to self antigens) to donor (ACI) cells, but reacted strongly (five times higher) to third-party (F344) cells.
CONCLUSIONS: Intrathymic inoculation of donor bone marrow at the time of transplantation along with a short course of tacrolimus induces long-term acceptance of lung allografts in rats. This simple approach of tolerance induction may have clinical application.
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Introduction
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One of the major limitations in lung transplantation, as in other solid organ transplantation, is the reliance on nonspecific, systemic immunosuppressive therapy to achieve graft acceptance. Even with the use of powerful immunosuppressant agents, rejection is still a major problem in lung transplant patients. It has been reported that 93% of lung recipients have at least one episode of acute rejection within the first year of transplantation [1]. Furthermore, obliterative bronchiolitis, a form of chronic rejection that carries up to 90% mortality, develops in up to 50% of lung transplant recipients two years after transplantation. As a result, lung allografts have the worst survival rate (40% at 6 years) [1] compared with other organ grafts. Improved means to achieve graft acceptance is desperately needed in clinical lung transplantation.
Donor-specific transplantation tolerance is a state in which the host permanently accepts the transplantedorgan without antirejection drugs, yet the host is able to retain its immunocompetence so that infection, malignancy, and end-organ toxicities related to nonspecific immunosuppression can be avoided. Although antigen (Ag)-specific tolerance had been achieved more than a quarter of a century ago by intrathymic inoculation of either soluble or cellular antigens [2–4], recent renewed interest in tolerance induction through intrathymic inoculation of donor antigens has been stimulated by the report by Posselt and colleagues [5] in which donor-specific tolerance to pancreatic islets was achieved in rodents by intrathymic inoculation of isolated pancreatic islets. Numerous studies have since confirmed this phenomenon of donor-specific tolerance for islet, heart, small bowel, and liver allografts in rodents by intrathymic inoculation of donor cellular antigens before transplantation [6–8]. Extension of this observation to the use of intrathymic inoculation of major histocompatibility (MHC) class II peptides [9], genetically engineered MHC class I-expressing cells [10], and extracts of T cells [11] or splenocytes [12] have also confirmed initial observations by Staples and coworkers [3] that intrathymic inoculation of soluble Ag induces Ag-specific tolerance. However, in those studies, the prospective recipients were pre-injected with donor antigens and antilymphocytic serum days or weeks before the actual organ transplantation, limiting the clinical application of this mode of tolerance induction. Recently, intrathymic inoculation of cellular and soluble antigens either at the time of or after organ transplantation have been demonstrated to induce donor-specific tolerance for cardiac and islet allografts in rats [13, 14]. However, tolerance induction for lung allografts through intrathymic inoculation with donor antigens has never been reported.
The aim of this study was to determine whether long-term acceptance of lung allografts could be achieved in MHC- and non-MHC-mismatched rats by intrathymic inoculation of donor bone marrow cells (ITBMC) at the time of lung transplantation.
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Material and methods
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Animals
Four- to six-week-old August Copenhagen Irish (ACI; RT1Aa), Wistar Furth (WF; RT1Au), and Fisher (F344; RT1Al) male rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were housed in a specific pathogen-free facility. All animals were treated in compliance with the "Principles of Laboratory Animal Care," formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 85-23, revised 1985).
Experimental design
The left lung was transplanted orthotopically as described previously [15]. At the time of lung transplantation, some animals received intrathymic injection of either bone marrow (study groups) or cell culture media (controls). After transplantation, some recipients received three intraperitoneal injections of antilymphocytic serum (ALS; Accurate Chemical Co, Westbury, NY; 1 mL/rat on days 0, 2, and 4), while others received five intramuscular injections of tacrolimus (FK506, Prograf; Fujisawa, Japan; 1 mg/kg, on days 0, 2, 4, 6, and 8). Graft rejection was monitored by chest radiograph every 2 to 3 days for the first 2 weeks and then monthly thereafter. Animals were killed at timed intervals, or when opacification of the transplanted lung was observed on chest radiograph. The allograft was retrieved and examined for evidence of technical failure, infection, or rejection. Rejection was graded on a scale of 0 (no rejection) to 4 (severe rejection) in a blinded fashion [15].
Bone marrow preparation and intrathymic inoculation
Bone marrow cells were harvested by flushing the long bones with Medium 199 (GIBCO BRL Products; Gaithersburg, MD). The bone marrow was then depleted of red blood cells by centrifugation on a Ficoll-Hypaque gradient, resuspended in Medium 199 at a concentration of 2.0 x 107 cells per 100 µL, and injected into each lobe (50 µL/lobe) of thymus through an upper median sternotomy, using a 30-gauge needle.
Mixed lymphocyte reaction assay
Mixed lymphocyte reaction assays were performed as previously described [16]. Briefly, lymphocytes isolated from cervical lymph nodes were resuspended in 1640 RPMI medium (Gibco) supplemented with 1 mmol/L sodium pyruvate, 2 mmol/L glutamine, 100 U/mL penicillin, 0.05 mmol/L 2-mercaptoethanol, and 0.5 mmol/L NG-mono-methyl-L-arginine (NMA). Responder cells (2 x 105) were stimulated with 2 x 105 irradiated stimulators in a total of 200 µL media. Cultures were incubated in 10% CO2 at 37°C and pulsed with 1 µCi [3H] thymidine (New England Nuclear, Boston, MA) on the fourth day. Twenty-four hours later cultures were harvested with an automated harvester and counted in a beta scintillation counter (Beckman, Palo Alto, CA). Results were expressed as counts per minute (cpm) ± standard error of the mean (SEM) and as a stimulation index. The stimulation index is the ratio of the cpm generated in response to a given stimulator over the base line cpm generated in response to the host.
Cell-mediated lymphocytoxicity
Cell-mediated lymphocytoxicity assays were performed as described previously [17]. Briefly, the cytotoxic activity of lymphocytes (effector cells) harvested from cervical lymph nodes of naive WF rats (control) and lung allograft recipients (10 months after transplantation) was measured in 51chromium (Cr)-release assays to determine their ability to lyse target cells. Effector cells were prepared by coculturing lymphocytes obtained from control and experimental animals with 
-irradiated (2,000 cGy) donor lymphocytes for 5 days. Subsequently, the viable cells were enriched by passing the cells over a Ficoll-Hypaque gradient. Target cells were prepared by stimulating lymphocytes (from cervical lymph nodes) obtained from donor and third-party rats with Concanavalin (Con) A for 5 days, and then labeled with 51Cr. Various numbers of effector cells were cultured with 1 x 104 51Cr-labeled target cells in each well of a 96-well round-bottomed plate to give effector-to-target ratios of 100:1 to 1:1. Each set of cultures was performed in triplicate. The plates were centrifuged (at 200g) for 2 minutes and then incubated at 37°C for 4 hours in a humidified, 5% CO2 incubator. Spontaneous release of 51Cr was measured by incubating the target cell with 100 µL complete media alone, while targets were treated with 100 µL 2% sodium dodecyl sulfate solution, obtaining the maximal release. Plates were centrifuged (at 200g) for 10 minutes after incubation, and 100 µL supernatant harvested from each well was subjected to radioactivity determination in a gamma counter. The percent lysis for each concentration of effector cells was calculated as follows: [(experimental-spontaneous release)/(maximum release-spontaneous release)]x100.
Statistical analysis
Statistical analyses were performed using Statistica Software (version 5.5, Statsoft, Inc, Tulsa, OK). Histologic scores were compared between groups using the Mann-Whitney U test.
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Results
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Effect of intrathymic inoculation of bone marrow on lung allograft survival
Rejection grades are summarized in Table 1.
Unmodified WF recipients acutely rejected ACI lung allografts within 7 days (group A). Intrathymic inoculation of donor-specific bone marrow without tacrolimus treatment did not improve graft acceptance (group B), while a short course of tacrolimus concurrently with nonspecific thymic manipulation (intrathymic injection of Medium 199) resulted in only modest prolongation of graft acceptance (group C). In contrast, intrathymic inoculation of donor specific bone marrow and a short course of tacrolimus resulted in long-term acceptance of donor-specific (groups D and E) but not third-party (group F) lung allografts. Furthermore, the effect of intrathymic bone marrow injection with a course of tacrolimus in prolonging the acceptance of lung allografts is not limited to certain rat strain combinations, because both ACI to WF and F344 to ACI rat strain combinations yielded similar results (groups D and E). Although these grafts were accepted long-term, they have more leukocyte infiltration (as assessed by the rejection grades) than syngeneic controls (groups D versus I). A short course of ALS in the postoperative period did not improve acceptance of lung allografts in WF recipients that received intrathymic donor-specific bone marrow (groups G and H).
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Table 1. Rejection of Lung Allografts in Recipients Treated With Intrathymic Inoculation of Donor Bone Marrow Cells
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Figure 1
depicts the histology of a normal rat lung, a lung allograft in a WF recipient that had received intrathymic inoculation of either donor-specific (at 360 days after transplantation), or third-party (at 28 days) bone marrow. The third-party graft has severe acute cellular rejection with perivascular and interstitial infiltrates (grade 3), whereas the donor-specific graft appears histologically normal.
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Fig 1. Histology of donor-specific and third-party lung allografts. (A) Normal rat lung. (B) Donor-specific lung allograft at 360 days after transplantation displayed only scattered inflammatory cells, but essentially preservation of normal architecture. (C) Third-party left lung allograft 28 days after transplantation had severe acute cellular rejection with perivascular and interstitial infiltrates.
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Effect of extrathymic injection of donor bone marrow cells on the survival of lung allografts
To determine the role of the thymus in this model of donor bone marrow infusion, we examined whether extrathymic injection of donor bone marrow by either intravenous or intraperitoneal routes would yield similar results to the intrathymic route. WF recipients receiving a similar course of tacrolimus after lung transplantation from ACI donors were administered 2 x 107 ACI bone marrow cells either intraperitoneally or intravenously at the same time of lung transplantation. Lung allograft survival was not significantly prolonged through these extrathymic routes of bone marrow administration (Table 2).
Survival of challenging cardiac allografts in recipients with long-surviving lung allografts
To test the robustness of donor-specific tolerance in this model, we performed heterotopic heart transplantation using donor-specific hearts to recipients that had received intrathymic bone marrow with long-surviving lung allografts. Challenging cardiac allografts were transplanted into the abdomen of recipient rats 150 days after lung transplantation. All donor-specific challenging cardiac allografts (n = 4) survived for more than 150 days after transplantation in recipients that had long-surviving lung grafts. At the time of sacrifice, all heart grafts had strong contractility and no histologic evidence of cellular rejection or allograft vasculopathy (Fig 2).
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Fig 2. Donor-specific (August Copenhagen Irish [ACI] rat) heart 150 days after transplantation into a Wistar Furth (WF) rat recipient that had received an ACI lung allograft and intrathymic ACI bone marrow 100 days before receiving the challenging heart. At the time of sacrifice, the heart had strong contractility. There was no rejection or allograft vasculopathy in the cardiac graft.
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In vitro evidence of donor-specific tolerance in long-term lung recipients
Mixed lymphocyte reaction assay
To assess the function of T-helper cells from recipients with long-surviving lung grafts, we performed mixed lymphocyte reaction assays. Lymphocytes harvested from the lymph nodes of either naïve WF or WF recipients of intrathymic ACI bone marrow with a long-surviving ACI lung allograft (10 months) were stimulated with -irradiated WF, ACI, or F344 lymphocytes. Lymphocytes of the long-term lung allograft recipients were unresponsive to host (WF) and donor-type (ACI) alloantigens but responded strongly to third-party (F344) alloantigens (Fig 3).
Cell-mediated lympholysis assay
Cell-mediated lympholysis assays were performed to assess the function of T-cell effector cells in recipients with long-surviving lung grafts. As shown in Figure 4,
lymphocytes obtained from naive WF rats were capable of killing donor-type (ACI) lymphoblasts. In contrast, lymphocytes from recipients that had received donor-specific bone marrow and lung transplants 10 months earlier were unresponsive to donor targets.
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Fig 4. Cell-mediated lymphocytotoxicity activity of against donor (ACI) target cells by 51chromium (Cr)-release assay. Cell-mediated lymphocytotoxicity activity of lymphocytes from naive Wistar Furth (WF) ( ) and WF recipient that had received ACI lung allograft and intrathymic ACI bone marrow 300 days earlier (•). Mean percentage of specific cell lysis of triplicate wells in a single experiment is shown. Spontaneous release of 51Cr was less than 15% of maximum release.
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These data suggest that specific unresponsiveness in animals with intrathymic injection of donor bone marrow combined with a short course of tacrolimus is due to either deletion or anergy of both helper and cytotoxic T-lymphocytes to donor alloantigens.
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Comment
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In this study we have demonstrated that donor-specific tolerance for lung allografts from MHC- and minor-mismatched, high-responder rat strain combinations (ACI donors to WF recipients) could be achieved by ITBMC. Furthermore, donor-specific tolerance is achievable by concurrent intrathymic inoculation of donor bone marrow and lung transplantation, making this mode of tolerance induction clinically relevant. Another important finding of clinical importance is that the use of a calcineurin inhibitor during the perioperative period did not interfere with the induction of tolerance in this model. Perico and associates [18] reported that the immunosuppressive therapy abrogates unresponsiveness to renal allografts induced by thymic inoculation of donor antigens. However, several authors were able to achieve donor-specific unresponsiveness by using a short course of tacrolimus and cyclosporine immediately after thymic inoculation of donor antigen and heart transplantation in rats [13, 19]. Matsuura and associates [19] reported that donor- and organ-specific unresponsiveness to Brown Norway heart allografts was achieved in Lewis rats by giving ITBMC and immunosuppressive therapy with either ALS or tacrolimus at the time of transplantation. Tacrolimus (1 mg/kg, days 0, 2, 4, 6, 8) prolonged survival in both the tacrolimus + ITBMC (MST > 140 days) and the tacrolimus-alone (MST > 140 days) groups. However, Brown Norway skin grafting provoked the rejection of long-surviving Brown Norway heart grafts in the tacrolimus-alone group, but did not do so in either the ALS + ITBMC or the tacrolimus + ITBMC groups. Klatter and associates [13] demonstrated that tolerance induction for cardiac allografts could be achieved by simultaneous cardiac transplantation and intrathymic injection of donor splenocytes and treatment with antilymphocytic serum in high-responder rat strain combinations, provided that low doses of cyclosporine were administered perioperatively. In the present study, tacrolimus (1 mg/kg, days 0, 2, 4, 6, 8) did not significantly prolong lung allograft survival. However the combination of a short course of tacrolimus and ITBMC results in long-term acceptance of lung allografts in high responder rat strain combinations (ACI to WF and F344 to ACI). The fact that these long-term accepted grafts have more leukocyte infiltration (as assessed by the rejection grades) than the syngeneic control (groups D versus I, Table 1) suggests that there is low-grade immunologic response to the lung allograft in this model.
The mechanism by which tacrolimus and ITBMC induce donor-specific tolerance for lung allografts while the combination of ALS and ITBMC does not, remains to be elucidated. Several authors reported that ALS plus intrathymic inoculation of donor cells failed to induce tolerance for heart allografts in high responder rat strain combinations. Shen and coworkers [20], using ALS and intrathymic inoculation of donor splenocytes to induce tolerance for cardiac allografts, reported that complete graft tolerance was not seen in strain combinations that included major and minor histocompatibility complex incompatibilities. Dark agouti (DA) cardiac graft survival in Piebald–viral–glaxo (PVG) recipients (full MHC and non-MHC incompatibility) was 50.6 days, while PVG.RT1a graft survival in Piebald–viral–glaxo (PVG) hosts (full MHC incompatibility) was 165.8 days and in PVG.R1 recipients (partial MHC incompatibility) was 163.8 days. Antilymphocytic serum has been shown to deplete peripheral mature T cells and accelerate the traffic of stem cells through the thymus without penetrating the intact thymus [21–23]. It is possible that by not penetrating the thymus, ALS does not delete already matured thymic T cells that are ready to emigrate from the thymus at the time of intrathymic inoculation of donor antigens.
Tacrolimus, on the other hand, is a T-cell specific immunosuppressant that prevents the activation of mature T cells by inhibition of cytokine gene expression (especially interleukin-2). In addition, tacrolimus has been demonstrated to cause reversible medullary atrophy of the thymus and reduction of mature thymocytes in the thymus [24, 25]. Pugh-Humphreys and colleagues [25] investigated the influence of tacrolimus (1 mg/kg per day for 7 days) on the rat thymus and reported that the medullary compartment was reduced with a concomitant decrease in MHC class I- and MHC class II-positive cells and in CD37+ (mature medullary) thymocytes. Flow cytometric analysis of thymocytes showed that tacrolimus induced increases in bright, Thy-1.1+ cells and in numbers of CD4+ and CD8+ thymocytes, while CD37+ cells were less numerous than in controls. These tacrolimus-induced abnormalities were reversed within 14 days of drug withdrawal. These findings suggest that tacrolimus reversibly disrupts the thymic microenvironment and may interfere with the function or maturation of T lymphocytes. Of significance is the fact that tacrolimus significantly reduces the number of mature thymocytes in the thymus. This fact suggests that tacrolimus, but not ALS, reduces these mature thymocytes, which have not been exposed to donor antigens during their ontogeny, and thus are not tolerant at the time of intrathymic inoculation of donor antigens. Tacrolimus may, therefore, be able to induce donor-specific tolerance for lung allografts, whereas ALS does not.
The current study also confirms the important role of the thymus in this model because intraperitoneal or intravenous administration of bone marrow did not yield long-term graft survival, although the intrathymic route did. One of the limitations of this study is that the animals used were young animals (4- to 6-week-old rats) with functional thymuses. Because the thymus becomes atrophic with age, the effectiveness of this mode of tolerance induction in older animals is unknown.
In summary, we have demonstrated that donor-specific tolerance for lung allografts could be achieved by intrathymic inoculation of donor bone marrow at the time of lung transplantation in combination with a short course of tacrolimus in major- and minor MHC-mismatched rats. These findings have potential clinical implication because it is logistically possible to adapt this protocol to induce long-term acceptance to organ allografts in clinical transplantation, especially in pediatric patients.
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Acknowledgments
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Supported in part by the American Heart Association (SL, SMP) and the American Lung Association (SMP).
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Discussion
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DR G. ALEXANDER PATTERSON (St. Louis, MO): I really enjoyed that report. It was nicely presented and very nicely illustrated.
I have a couple of questions. I don’t know if you heard Dr Chiu’s lecture yesterday, but it was intriguing work that has a lot of similarity to this, the idea of a bone marrow stem cell being able to create function in another organ, a heart specifically without any evidence of rejection subsequently. So I have two questions. First of all, what do you need the thymus for, in other words, if you just did the injection of the cells alone? Do they need to be injected intrathymically? The work that Dr Chiu presented yesterday suggested that maybe you don’t need to do that.
Secondly, what are the cells that are accomplishing this? Did you inject whole bone marrow, or did you select certain cells from bone marrow?
DR LOUIS: Thank you for your comments. We actually looked at other routes of injecting the donor bone marrow. Animals that received bone marrow either intravenously or intraperitoneally at the time of transplant rejected their lung grafts at a rate that was similar to controls.
As for the bone marrow itself, the only thing we did was to deplete the bone marrow of red blood cells prior to transplant. Our goal was to expose the recipient of the lung graft to donor MHC antigens at the time of transplantation.
DR THOMAS M. EGAN (Chapel Hill, NC): That was a very nice presentation and your results are clear, but my concern is that human thymuses don’t do the same things that rodent thymuses do in the sense that by the time we’re adults, most of the gland in a human is involuted and probably this type of approach would be less likely to work in a human. The idea is interesting. How do you propose to test it in humans?
DR LOUIS: Thank you very much. Recently, Dr Pham was involved in a study with Dr Webber from the Children’s Hospital at the University of Pittsburgh, who conducted a trial of intrathymic inoculation of donor bone marrow in pediatric heart transplant recipients. It is encouraging to note that recipients of donor bone marrow had less acute rejection episodes in the first 6, 12, and 24 months after transplant. In addition, their freedom from rejection after 12 months was significantly better than control patients, who received no bone marrow.
It is indeed a problem that the human thymus regresses by our the mid-20s. However, French and associates (French RA, Broussard SR, Meier WA, et al. Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution. Endocrinology 2002;143:690–9) recently demonstrated that the thymus in aging rodents could be reconstituted with growth hormone. This finding holds promise when combined with the observations made by Jamieson and colleagues (Jamieson BD, Douek DC, Killian S, et al. Generation of functional thymocytes in the human adult. Immunity 1999;10:569–75), who reported that thymopoesis was observed in adults up to 56 years of age. However, until these findings are translated into human clinical trials, the beneficiaries of this technique will probably be pediatric patients.
DR SAQIB MASROOR (Miami, FL): I have a comment about the earlier question concerning whether other cells can induce tolerance and how it compares to the work in Dr Chiu’s lab in which bone marrow cells are being used to repopulate myocardium. Mark Hardy at Columbia, and some others in heart and other transplant models, have used these bone marrow cells and gone down to extracting the cell membranes and then solubilizing those and actually gone to a molecular level. They have injected soluble antigens from the cell membranes of these cells in the thymus to induce tolerance. This phenomenon is different from what is being done in Dr Chiu’s lab of repopulating adult myocardium. These bone marrow cells are only being used as a source of antigen to induce tolerance to that specific antigen rather than repopulating an organ.
DR MICHAEL S. MULLIGAN (Seattle, WA): The clinical study that you mentioned also assessed lung transplant patients, and unfortunately there was no difference in survival, no difference in acute rejection events, but the incidence of biopsy-proven bronchiolitis obliterans dropped from 38% to 5% in that limited clinical trial. It is unclear why there was no apparent effect on acute rejection. It is possible that the intrathymic injection provides for more rapid establishment of central clonal deletion as opposed to peripheral clonal deletion of donor-specific T cells. I don’t know. Regardless, one might be able to not only enhance the effects of intrathymic injections but make peripheral blood injections more efficacious by at the same time providing costimulatory blockade either against the CD28 or CD40 pathways, and those experiments would be worth trying.
DR LOUIS: Absolutely. The illustration of thymic tolerance that I presented was very diagrammatic and we did not actually tease out the mechanism itself. The mechanism of tolerance seen here probably involves both clonal deletion and the development of anergy.
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References
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Abstract
Introduction
= WF; 
= ACI; 
= F344. (CPM = count per minute.)