WOFIE augments the immunosuppressive potency of FK-506

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WOFIE augments the immunosuppressive potency of FK-506 B Dresskea, N Zavazavab, D-S Huanga, X Lina, B Kremera and F Ftindrich” aDepartment of General and Thoracic Surgery and bInstitute of Immunology, University of Eel, Eel Received 6 October 1998; accepted 15 October 1998

Abstrack Bidirectional recognition of donor- and recipient-derived immunocompetent

cells has been proven to play a pivotal role for the induction of long-term unresponsiveness to allogeneic grafts. This study investigated the fate of heterotopic heart grafts with respect to the timing of subtherapeutic doses of FK-506 and with respect to the time point and type of donor antigen application, leaving space for mutual adaptation of alloreactive lymphocytes, designated as the ‘WOFIE-concept’ (window of opportunity for immunological engagement), originally described by R Caine. Methods. Heterotropic heart transplantation was performed using male DA (RTl?) donor and LEW (RTl.‘) recipient rats in the following groups (n = 6). IX-506 was applied intramuscularly (i.m.) using doses of 2 mgikg x body weight per day. Donor antigen application was performed either by DA blood transfusion, 2 ml intravenously (i.v.), or by i.v. transfusion of 5 x 10’ DA splenocytes. (i) LEW --f LEW, untreated; (ii) DA + LEW, untreated; (iii) DA + LEW, FK-506 days 0, 4-7; (iv) DA + LEW, FK-506 as group (iii) plus 2 ml of DA blood 6 h post-TX; (v) same as group (iv) but DA blood transfusion 24 h post-Tx, (vi) DA -+ LEW, F&506 as group (iii) plus DA splenocytes 6 h post-TX; (vii) same as group (vi) but DA splenocyte transfusion 24 h post-TX, (viii) DA -+ LEW, FK-506 days O-4 and (ix) DA --f LEW, FK-506 as group (viii) plus DA blood 6 h post-Tx. Immunohistochemical stainings (ARAAR-method) of the aUografts and flow cytometric analysis of recipient spleens were performed electively 3,7 and 14 days after organ reperfusion. Results. The mean graft survival differed significantly between groups and comprised (mean + SD days): (i) >lOO, (ii) 6.5 + 1.0, (iii) 31.6 f 12.1, (iv) 44.8 + 10.1, (v) 29.8 +: 14.2, (vi) 27.2 f 4.7, (vii) 14.6 2 4.2, 17.5 f 4.2, (viii) 17.5 2 4.2 and (ix) 18.8 f 2.8 days. Prolongation of graft survival and long-term unresponsiveness (group iv) revealed a substantially different pattern of graft infiltration. Conclusions. Effective treatment with unspecific immunosuppressants like FK-506 can be substantially improved if (i) mutual antigen recognition between donor and recipient immunocompetent cells is warranted, (ii) donor-derived blood-borne antigens are given immediately after graft reperfusion, and (iii) the type of inoculated donor antigen has a strong impact on graft survival as splenocytes which contain a large population of professional antigen-presenting cells failed to prolong graft survival after interrupted FK-506 treatment.

Address for correspondence: B Dresske, Department of General and Thoracic Surgery, University of Kiel, Arnold Heller Strasse 7, 24105 Kiel, Germany. 0 Amold

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Introduction Bidirectional recognition of donor- and recipient-derived immunocompetent cells has been proven to play a pivotal role for the induction of long-term unresponsiveness to allogeneic grafts. Additional transfusion of donor blood- or bone marrowborne cells has been demonstrated to augment anti-host immunogenic&y and was associated with improved graft survival. In the 196Os, before the introduction of cyclosporine A, Morris and his colleagues were able to show that previous blood transfusions to dialysis patients improved graft survival rates of renal transplants.’ They were among the first to suggest that deliberately exposing patients to alloantigen before transplantation may have a beneficial effect on graft outcome. A similar effect had been reported in experimental systems by Billingham et al. who reported about prolonged graft survival after pretreating the recipient with donor strain cells.’ Further studies from the Netherlands revealed that the’use of human leucocyte antigen (HLA)-matched blood transfusions had a beneficial effect. By matching the blood donor and the recipient for at least one HLA-DR antigen, graft survival could be improved over that obtained in recipients who received transfusions from DR-mismatched donors.3’4 The patients who received DR-matched transfusions were found to have a reduced frequency of cytotoxic cells against those MHC class I antigens which carried a haplotype mismatch between the blood donor and the recipient.4 However, with the introduction of cyclosporine A and the subsequent development of further immunosuppressive drugs and biological agents the effect of prior blood transfusions in clinical renal transplantation became less clear. Conversely, it is now argued that the current therapeutic use of unspecific immunosuppression impairs the crucial process of mutual engagement between immunocompetent donor and recipient cells. In line of this thinking, Sir R Calne developed the hypothesis that a window of opportunity for immunological engagement’ (WOFIE) warrants improved immunological interaction between cells of donor- and host-derived immunogenicity.5,6His concept envisages a short-term interruption of 48 h during the initial postoperative period of inductive immunosuppression. In an experimental setting of kidney transplantation between fully MHC-disparant outbred pigs, initial interruption of short-term (six doses) cyclosporine A based immunosuppression enhanced graft survival substantially. Additional transfusion of donor spleen leucocytes, 6 h after kidney grafting and within the time frame of the immunosuppressive window extended the mean graft survival even more. Interestingly, prior irradiation of donor spleen leucocytes spared immunogenic@ of these cells and resulted in acute graft rejection. This state of long-term unresponsiveness without the need of continuous immunosuppression was referred to as ‘operational tolerance’ as it did not extend to donor second-set skin grafts. Tolerance was also incomplete by in vitro criteria with strong alloreactivity to donor stimulator cells in mixed lymphocyte cultures. Nevertheless, this concept is of crucial importance as it reveals an opportunity to reduce the necessary dose of immunosuppressive drugs and to spare additional administration of steroids.

Objective The following study was conducted to investigate the fate of heterotopic heart allografts with respect to the timing of subtherTransplant Immunology 1998; 6: 243-249

apeutic doses of FK-506 and with respect to the type of donor antigen application. We postulated prolonged graft survival after application of the WOFIE concept which leaves space for mutual engagement of alloreactive lymphocytes and after arbitrary augmentation of the donor antigenicity with blood- or spleenderived transfusions from donor animals.

Material and methods Animals and transplantation procedures

Donor hearts of&12-week-old adult DA (RTla) male rats were transplanted heterotopically into the abdomen of age-matched male LEW (RTl.‘) recipient rats according to the technique described by Ono and Lindsey.’ Graft function was assessed on a daily basis by palpitation of the allografts through the abdominal wall. The time point of rejection was deemed complete when palpable ventricular contractions ceased. Recipient immunosuppression was achieved by a short course (five doses) of FK-506,2 mgikg x body weight, per day, intramuscularly (i.m.). Donor antigen application was either performed by donor (DA) blood transfusion, 2 ml, or by transfusion of DA splenocytes, intravenously into the tail vein. Splenocyte suspensions were prepared from donor spleens by gentle disaggregation of chopped tissue and administered by a slow intravenous injection of approximately 5 x lo7 splenocytes/ 2 ml saline. The experimental protocol included nine experimental groups (n = 6) as follows: (i) LEW + LEW, untreated; (ii) DA + LEW, untreated; (iii) DA + LEW, FK-506 at days 0, 4,5, 6 and 7; (iv) DA + LEW, FK-506 at days 0,4,5,6 and 7 and DA blood transfusion 6 h post-TX; (v) DA + LEW, FK-506 at days 0,4,5,6 and 7 and DA blood transfusion 24 h post-Tx, (vi) DA + LEW, FK-506 at days 0,4,5,6 and 7 and DA splenocyte transfusion 6 h post-TX; (vii) DA 4 LEW, FK-506 at days 0, 4, 5, 6 and 7 and DA splenocyte transfusion 24 h post-TX; (viii) DA + LEW, FK-506 at days 0, 1,2,3, and 4; (ix) DA + LEW, FK-506 at days 0, 1, 2, 3 and 4 and DA blood transfusion 6 h post-TX. In addition four animals of groups (iii) and (viii) were sacrificed electively on days 3,7, and 14 post-TX for histological and immunohistochemical evaluation and for flow cytometric analysis. Histology

Donor hearts were harvested selectively on days 3,7, and 14 or subsequent to the diagnosis of graft rejection. Isolated tissues were sliced into small pieces, either snap-frozen in liquid nitrogen and stored by -80°C or subjected to buffered formaldehyde, embedded in paraffin, cut at 4 pm, and stained with haematoxylin and eosin (H&E). Immunohistochemical analysis was performed by APAAPstaining according to the technique described by Cordell et al. using the monoclonal antibodies NKR-Pl 3.2.3 (mouse IgGl, natural killer cells), KiM2R (mouse IgGl, macrophages), KiTlR (mouse IgG2a, pan T-cells) and KiBlR (mouse, IgGl, B-cells).* Flow cytometric analysis For lymphocyte preparations spleens of recipient animals were excised, minced and pressed through a 60-gauge-mesh stainless steal screen into Hepes buffered RPM1 (HBSS) containing 1% heat-inactivated normal rat serum. Lymphocytes were obtained by centrifugation on FicollHypaque gradients (density = 1.077), at 3oOgfor 20 min, at 4°C.

WORE augments the immunosuppressive potency oj FK-506 245

After collection of the mononuclear cells from the gradient interface they were resuspended in RPM1 1640, supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 @ml steptomy&r, 5 x lr5 M fl-mercaptoethanol and 5% normal rat serum and washed three times. Flow cytometric analysis was performed on a FACSScan II @e&on Dickinson, CA, USA) using the following panel of directly fluoresceinated monoclonal antibodies: MRGGx-3 R-PE (mouse IgGl), CD8 R-PE (mouse IgGl), CD4 R-PE (mouse IgGl), CD3 R-PE (mouse IgGl) and NKR-P1/3.2.3 affinity purified F(ab’)s FITC conjugated mouse IgGl. After determining that the populations of interest resided in the region of low to intermediate forward and side scatter (lymphoid gate), in all subsequent experiments cells were gated on typical lymphocyte and mononuclear (NK cells) physical parameters. A total of 5000 to 10 000 events/sample were analysed using Consort 40 software (E&ton Dickinson). statisrtiea Animal survival rates were calculated according to the Kaplan-Meier life-table analysis using the Graphpad InStat statistics program, Version 2.01. Differences in organ survival rates between the various groups were assessed using the generalized Savage (MantelCox) log-rank test (p-value < 0.05). Deaths secondary to technical complications were censored. Reeults In the LEW-to-DA strain combination heart grafts in untreated recipients were rejected between postoperative days 6 and 7. The mean organ survival rate in this allogeneic strain combination comprised 6.5 + 1.0 days. Continuous immunosuppressive treatment with five doses of FK-506, 2 mgkg body weight per day, from day 0 until day 4 (group viii) prolonged graft survival to 17.4 -+ 4.2 days. A 72-h window’ of immunosuppression following an initial single dose at the day of transplantation (group iii) substantially enhanced allograft survival to 31.6 f 12.1 days (p < 0.05 versus group viii) (Figure 1). Interestingly, FK-506-treated animals did not show a sudden cease of ventricular contractions but gradual enlargement and induration of the cardiac graft accompanied by progressing bradycardia for a period of 5-15 days until contractions finally stopped. If 2 ml of DA (donor) blood were transfused 6 h after organ reperfusion into the tail vein of WOFIE-treated LEW recipients (group iv) a further delay of graft rejection was achieved. Rejection of the

cardiac allograft was postponed until 44.8 + 10.1 days @ < 0.03 versus group iii). The time point of donor blood injection was crucial as its administration at a later point of time (24 h, see group v) had no intluence on graft survival in comparison with WOFIE-treatment alone (Figure 2). ‘Bansfusion of DA blood without an initial immunosuppressive window (group ix) was not effective as graft survival comprised only 18.8 f 2.8 days (Figure 3). It is of note that transfusion of 5 x 10’donor splenocytes into the recipient’s tail vein within the ‘immunosuppressive window’ failed to prolong allograft survival. Conversely, application of unprocessed spleen cell suspensions containing the whole repertoire of immunocompetent spleen cells led to accelerated acute rejection in comparison to the animals which had received intermittent FK treatment (Figure 4). As already mentioned, timing of donor blood administration which augments the graft’s immunogenicity was crucial. Resides, the type of antigen and the type of antigen presenting cells included within the donorderived transfusion played an important role on the host’s alloreactive immune response. Thus, application of DA splenocyte suspensions 6 h (group vi) after transplantation decreased graft survival (mean organ survival rate 27.2 + 4.7 days in comparison to group iii animals. Moreover, splenocyte transfusion

-=-syngamkeenird +

FK-

+FK&xkruv,DAbbodehp.Tx +FK+vMnw,DAbkdWhp.Tx

Figure 2 Kaplan-Meier blots were generated and compared with log rank (Savage Mantel-Cox) analysis. Each group consisted of six animals. Additional transfusion of DA (donor) blood 6 h after organ reperhtsion in to the tail vein of ‘WOFIE’-treated LJZWrecipients led to postponed rejection of cardiac allografts until 44.8 & 10.1 days @ < 0.03 versus WOFfE treatment without donor antigen application). The time point of donor blood injection was crucial as its administration at a later point of time (24 h after reperfusion) had no effect on graft survival in comparison with WOFIE treatment alone.

-+FK,novvindaw,DAbbod6hp.Tx 1

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Kaplan-Meier blots were generated and compared with log rank (SavageMantel-x) analysis. Each group consisted of six animals. Interruption of FK-506 immunosuppression @K-window)for 72 h after a single dose of 2 mg/kg body weight at the day of transplantation subFipwe

1

stantialiy enhanced organ survival (31.6 2 12.1 days) compared to continuous FK-treatment (mean organ survival 17.4 2 4.2 days),p < 0.05.

Transplant Immunology 1998; 6: 243-249

Figure 3

Kaplan-Meier blots were generated and compared with log rank (Savage Mantel-Cox) analysis. Each group consisted of six anhnals. Additional application of 2 ml DA (donor) blood without an initial immunosuppressive window did not affect graft survival compared to continuous FK-506 immunosuppression from days 0 to 4 alone (median organ survival 18.8 -C2.8 days).

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FIow cytometric analyeis lb further delineate the mechanisms by which short-term inter-

Figure 4 Kaplan-Meier blots were generated and compared with log rank (Savage Mantel-Cox) analysis. Each group consisted of six animals. Transfusion of 5 x 10’ DA splenocytes into the recipients tail vein within the ‘immunosuppressive window’ failed to prolong allograft survival. Conversely application of unprocessed spleen cell suspensions led to accelerated acute rejection compared to WOFIE-treated animals. Timing of splenocyte application was of crucial importance. Transfusion at a later time (24 h post-TX) led to accelerated allograft rejection within 14.6 +- 4.2 days (p < 0.01 versus WOFIE-treatment alone) whereas administration of DA splenocytes 6 h post-Tx was less effective (median organ survival 27.2 + 4.7 days).

at a later point of time led to accelerated graft rejection within 14.6 + 4.2 days @ < 0.01 versus group iii). Histological evaluation of donor hearts was performed on postoperative days 3, 7, and 14. First signs of tissue damage, such as severe endocarditis, became evident in untreated LEW recipient rats as soon as 24 h after reperfusion. Animals of group viii which received continuous immunosuppression demonstrated first signs of acute rejection on postoperative day 3. These included endocarditis and myocytolysis within the subendocardial regions. On postoperative day 7, these allografts showed progressive tissue destruction, such as myocardial fibrosis and widespread infiltration of inflammatory cells. The application of the ‘immunosuppressive window’ postponed this process of graft rejection. Infiltration of immunocompetent cells started 7 days after transplantation, whereas myocytolysis and vascular lesions in allografts could be detected first on day 14 after reperfusion. Immunohistochemical

evaluation

One crucial point of interest related to this study focused on the characterization of graft infiltrating immunocompetent cells at the given time points 3, 7 and 14 days post-TX. The first infiltration of natural killer cells and macrophages in the allogeneic control (group ii) was observed already 3 h after organ reperfusion and progressively increased in the first 3 postoperative days whereas significant T and B cell infiltration started at a later point of time, namely 3 days post-Tx. Interestingly, at day 3 post% allografts of animals which received continuous IX-therapy (group viii) showed widespread infiltration of macrophages and natural killer cells which concentrate in the regions of early graft destruction: perivascular, in the endocardium and the subendocardial region (Figure 5). Conversely, the first significant infiltration of these cell populations representing the innate immune response could be observed in the window animals’ 14 days post-Tx. B cells and T cells were not detected in both groups during the first weeks following transplantation despite of severe signs of graft rejection in group viii.

Transplant Immunology 1998; 6: 243-249

ruption of the applied immunosuppressive therapy modified the immune response of LEW recipients, flow cytometric analysis of recipient splenocytes harvested on postoperative days 3, 7, and 14 was performed (Figure 6). The impact of an ‘immunosuppressive window’ was compared to LEW recipients who received continuous FK-506 treatment for the first five days following transplantation. Whereas there were no signlflcant differences concerning the distriiution of immunocompetent splenic cells in both groups compared to the syngeneic and allogeneic untreated controls at day 3 post-Tx, we found a significant increase of CD3+-cells seven days after transplantation in all animals with IX-506 treatment. Interestingly, there was a difference in the number of splenic CD4+ T-helper cells and cytotoxic CD8+ T-cells between LEW-recipients with continuous FK-therapy and those with intermittent immunosuppression. Interruption of immunosuppression resulted in a clear increase of T-helper cells over the observation period of 14 days. LEW major histocompatability complex (MHC) class II+ cells that comprise B lymphocytes, macrophages, monocytes, and dendritic cells were substantially decreased 7 and 14 days post-TX in the ‘window’animals. Conversely, natural killer ceUs as members of the innate immune response were substantially increased 7 and 14 days after grafting. However, continuous FK506 treatment decreased the NK-cell population in the spleen within the first postoperative week.

Discussion The effective application of donor cells of hematopoetic origin as ori ‘nally described by Medawar and colleagues dates back to 1953.PrBy then, it was assumed that neonatal tolerance was based on an all-or-none phenomenon in which intrathymic clonal deletion speciftcaUy obliterated the host’s immune response to donor antigens. In extension to these original experiments Streilein provided unequivocal evidence that the type of tolerogen (class I and/or II alloantigen differences) had a major influence on the resulting mechanisms of tolerance induction.g In this context, it became clear that antigen-application including class I antigen differences induce the clonal deletional type of tolerance, whereas injected cells which differed for MHC class II molecules stimulated suppressor like cells which could be adoptively transferred. These important studies are in line with our own findings which demonstrate that graft acceptance based on priming of recipients with donor antigens involves a multiplicity of mechanisms. It should be. that subsets of donor-derived blood-borne cells give rise to quite opposing immune responses within the same host. In rodents erythrocytes and platelets which express MHC class I but not class II molecules have been shown to be capable to induce unresponsiveness in vivo.loS1lCranston et al. using a renal allograft model found that B cells and CD4+ T-cells were effective to enhance graft s~rvival.~~ Moreover, the same type of antigen applied at various points of time resulted in profound differences of aUograft survival. A complication in this experimental setting involved the fact that all experiments were performed under the umbrella of a very potent immunosuppressive drug, notably FK-506, known to inhibit calcineurindependent transcription factors, such as NF KB and AP-1. These, in turn, are crucial intranuclear executioners for the appropriate promotor gene activation of interleukin 2 (IL-2), the major cytokine targeted by FK506.‘3 IL-2 itself stimulates cytotoxic

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(a)

Figure 5 APAAlQtainingwith the monoclonal antibody KiM2R (macrophages), x250. Figure 5(a) shows a cardiac allograft of an animal that received continuous FK-therapy 3 days after organ reperfusion with widespread infiltration of macrophages in the endocardium and the subendocardial region. Conversely there was only minimal macrophage infiltration in allografts of J_EW-recipients that received intermittent FK-506 immunosuppression (Figure 5b). There were similar findings concerning allograft NK cell frequency. B and T cells did not titrate the grafts in the first 2 weeks.

lymphocyte response of both, the innate and the cellular immune effector arm~.~~~~Here, we provide clear evidence that the ‘window of opportunity’ strongly affected the frequency and the distribution ofimmunocompetent cells found in spleens from cardiac allografted LEW rats. Long-term graft acceptance clearly correlated with a profound increase of CD4+-cells and concomittent recruitment of MHC class II+- (0x3+-) cells. These cells whose specific immunoregulatory role along application of the WOFIE-concept still has to be characterized in more detail are endowed with the capability to pick up antigens, to proteolyse them, and to present them as peptides within the binding groves of their MHC class II molecules.” However, presentation of nonself-peptides in the context of host-restricted MHC molecules (now designated as indirect presentation) will only stimuTransplant Immunology 1998; 6: 243-249

late Tcell proliferation if soxalled ‘second-signals’ are provided appropriately. 1419Without ‘professional’ second-signals T-cells are rendered anergic and fail to respond to the recognized nonself-antigen epitopes. in this context, only after administration of nonprofessional APCs (donor blood) the engagement of immunocompetent cells favoured graft acceptance. The transfer of donor splenocytes, known to consist of high numbers of professional APCs, failed to silence the host’s immurie response. Applying the WOFIE concept resulted in signiGcantly lower numbers of NK-cells and macrophages in the graft within the 6rst 3 postoperative days. Thus even initial mutual engagement between donor and recipientderived immunocompetent cells was warranted, there was less graft infiltration of these effector cells. A possible interpretation of these results must take into account

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that the initial proliferative stimuli pmvided by the ‘window of opportunity’ were turned down efktively after the second-set administration of FK-506.The fact that combination of WOF’IE together with simultaneous blood transfusions extended graft survival significantly (in annparison to blood transfusions without WOFE) emphasize the importance of WOFIE to silence potentially alloreactive m otbenvk sensitized. In amsiatency with currently debated mechanisms of T-cell cross-talk, we postulate a shift from the TIN- to the TH2-type of cytokine release from CD4+-cells which were found h recipient spleens in greater

numbers and progmkvely increasing during the first 2 weeks after transplantatiorl of cardiac allografts. In summary, simultaneous enhancement of donor antigenicity by the use of donor blood substantially suppressed the alloreactive immune response if applied within the appropriate time frame after transplantation. For a more comprehensive level of diiion of these tidings qtokine patterns and in vibr~assays of the cyto~oxicand proliferative profiles from alloreactive lymphocytes will be the subjects of forthcoming experiments.

E

8’” % l

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Figure6 Each bar representsthe mean k SD of antigen-positivecells determinedby flowcytometryof recipientsplenocytes(four anitnal&roup) at various time pointspost-TX(3,7 and 14 days after grafting), As cardiac grafts were rejected within 6-7 days in the allogeoeic control no flow cytometric analysis was performed 14 days post-Pi in this group. Whereas there were no differences of the distribution of immunocompeteot cells 3 days after grafting between the v&ous groups, there was a significant increase of CD3+-ceils 7 days after transplantation in ail animals with F’K-506treatment (a). But numbers of CI34+ T-helperand CDS+cytotoxic T-cells differed between the LEW-recipients with continuous FK-therapy and those with intermittent immunosuppressioo. Interruption of immuoosuppression resulted in a clear increase of T-helper cells over the observation period of 14 days (b). LEW MHC class II+ cells were substantially decreased 7 and 14 days post-% in the ‘window’-animals (d). Conversely, natural killer cells were increased 7 and 14 days after grafting. Continuous FK-506 treatment decreased the NK-cell population in the spleen within the first postoperative week (e). Tmnsplant hmunolog~

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WOFIEaugments

References 1 Morris PJ, Ting A, Stocker JW Leucocyte antigens in renal transplantation. The paradox of blood transfusion in renal transplantation. hfed JAust 1968,2: 1088-90. 2 Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature 1953; 12:603-606. 3 Lagaaij EL, Hemremamr IPH, Ruigrok MB et al. Effect of oneHLA-DR-antigen matched and completely HLA-DR mismatched blood transfusions on survival of heart and kidney allografts. N Engl J Med 1989; 321: 701-705. 4 van Bvuyver E, Mooijaart IUD, ten Berge IJM et al. Pretransplantation blood transfusion revisited. N Engl J Med 1991; 325: 1210-13. 5 Calne RY, Watson CJE, Brons IGM et al. ‘lblerance of porcine renal allografts induced by donor spleen cells and seven days treatment with cyclosporine. Tmnspkantation 1994; 57: 1433-35. Calne R. WOFIE hypothesis. Some thoughts on an approach toward allograft tolerance. 7innspl Proc 1996; 28: 1152. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J i%omc Cardiinwc Swg 1968,57: 225-29. Cordell JL, Falini B, Erber WN et al. Immunoenxymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (ARM complexes). JH&ochem Cytochem 1984; 32: 219-29. 9 Streilein Jw. Neonatal tolerance of H-2 alloantigens: procuring graft acceptance the old-fashioned way. YZPanspkzntation 1991; 52: 1. 10 Wood KJ, Morris PJ. The blood transfusion effect: Suppression of renal allograft rejection in the rat using purified blood components. Tmnspl Pmc 1985; 17: 2419-20.

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11 Wood KJ, Evins J, Morris PJ. Suppression renal allograft rejection in the rat by class I antigen on purified erythrocytes. Tmnsplantation 1985; 39: 56-62. 12 Cranston D, Wood KJ, Morris PJ. Pretreatment with lymphocyte subpopulations and renal allograft survival in the rat. Panspkmtation 1986,43: l309-13. 13 Braxelton TR, Morris RE. Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK 506), sirolimus (rapamycin), mycophenolat mofetil and leflunomide. Curr Opin Immunol1996; 8: 710-20. 14 Morgan DA, Ruscetti FW, Gallo RC. Selective in vitro growth of T lymphocytes from normal human bone marrows. Science 1976; 1007-1008. 15 Hermann F, Camristra SA, Lindemamr A et al. Functional consequences of monocyte IL-2 receptor expression. Induction of IL-@ secretion by IFN and IL-2. J Zmmunol1989; 142: 139-45. 16 Mule JJ, Shu S, Rosenberg SA. The anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in vivo. J Zmmwwll985; Ws: 646-52. 17 Weiss S, Bogen B. MHC class II-restricted presentation of intracellular antigen. Cell 1991; 64: 767-76. 18 Shoskes DA, Wood KJ. Indirect presentation of MHC antigens in transplantation. Zmmwwl Today 1994; 15: 32-38. 19 Sayegh MH, Wats&urger B, Carpenter CB. Mechanisms of T cell recognition of alloantigen. The role of peptides. Tmnsplantation 1994: 57: 1295-302.