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(Circulation. 1998;97:1257-1263.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Assessment of the Contribution That Direct Allorecognition Makes to the Progression of Chronic Cardiac Transplant Rejection in Humans

Philip I. Hornick, BSc, FRCS; Philip D. Mason, PhD, MRCP; Magdi H. Yacoub, FRCS; Marlene L. Rose, PhD; Richard Batchelor, MD, FRCPath; ; Robert I. Lechler, PhD, FRCP, FRCPath

From the Departments of Immunology (P.I.H., P.D.M., R.B., R.I.L.) and Cardiothoracic Surgery (P.I.H.), Royal Postgraduate Medical School, Hammersmith Hospital, London, UK, and the Department of Cardiothoracic Surgery, Harefield Hospital (M.H.Y., M.L.R.), Middlesex, UK.

Correspondence to Philip Hornick, Department of Cardiothoracic Surgery, Royal Postgraduate Medical School, DuCane Rd, Hammersmith Hospital, London W12 ONN, UK.

	Abstract

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Background—Two populations of T cells contribute to allograft rejection. T cells with direct allospecificity are activated after recognition of intact MHC alloantigens displayed at the surface of donor passenger leukocytes carried within the graft. In contrast, T cells with indirect allospecificity recognize donor alloantigens as processed peptides associated with self (recipient)–MHC class II molecules. In small animal models of transplantation, direct pathway T cells dominate the acute rejection process and are rendered tolerant to the graft after the loss of donor passenger leukocytes. It has been argued that indirect pathway T cells contribute substantially to continual graft damage after passenger cell loss. The purpose of this study was to determine whether donor-specific tolerance could be detected in T cells with direct anti-donor allospecificity in human heart transplant recipients after prolonged graft residence.

Methods and Results—Alloreactive helper (HTLf) and cytotoxic (CTLf) T cells were enumerated by use of limiting dilution analysis. These assay systems were refined to make them specific for the direct pathway of allorecognition and more sensitive in the case of the HTLf assay. Recipient:anti-donor frequencies were generated in 10 long-term recipients of heart grafts with progressive chronic rejection and compared with those against equivalently HLA mismatched recipient:third-party controls. For HTLf, direct pathway donor-specific hyporesponsiveness was detected in 5 of the 10 recipients (HTLf <1:100 000). Of these 5 recipients, 4 also had low anti-donor CTLf (<1:100 000). In the 5th recipient, although the CTLf was >1:100 000, it was significantly lower than that estimated against the third-party control.

Conclusions—Donor-specific hyporesponsiveness is demonstrated in 50% of recipients in both the HTLf and CTLf compartments of the direct alloresponse. Direct allorecognition therefore appears unlikely to be responsible for the progression of chronic rejection, implicating indirect allorecognition as the predominant immunological driving force. Furthermore, these data have potential implications for graft outcome, adjustment of immunosuppression, and recipient monitoring.

Key Words: transplantation • immunology • rejection • immune system • lymphocytes

	Introduction

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Donor-specific immunological tolerance is the most desirable, although elusive, goal in clinical transplantation for two main reasons. The first is that protracted immunosuppressive treatment is associated with an increased susceptibility to both opportunistic infections and malignancy. The establishment of immunological tolerance to an allograft would obviate the need for their use. The second reason is that despite the impressive improvement in graft survival in the first year after transplantation, the attrition rate of organ allografts after the first year has not changed significantly over the past 2 decades because of the inexorable course of chronic rejection.

Chronic rejection of the cardiac allograft is manifest by the development of TxCAD.¹ Its morphological characteristics differ from conventional coronary artery disease, although in its most advanced form it may resemble conventional nontransplant coronary artery disease.² Despite improvements in early survival, the major cause of late death in recipients of cardiac allografts is TxCAD, accounting for 40% of all deaths or retransplantations that occur within 5 years of transplantation.³ The process is limited exclusively to the allograft, including venous structures, and its progression may be very rapid compared with conventional atheroma.^{2 4 5 6 7}

Both alloantigen-dependent and -independent events appear to influence this condition (reviewed in References 8 and 9^{8 9} ). However, tangible experimental evidence for an immune basis is indicated by data derived from small animal models of TxCAD,^{10 11 12} as well as an observed correlation with anti-endothelial antibodies and anti-HLA antibodies produced by the recipient.^{13 14 15 16} Immunological factors may well act in concert with antigen-independent processes, including traditional risk factors for atherogenesis. The relative contribution of nonimmune risk factors is controversial and depends on the series analyzed.^{4 5 6 17 18 19}

Given that there is strong evidence implicating immunological mechanisms in this long-term rejection process,^{10 11 12} effective strategies to induce donor-specific tolerance should extend the lifespan of allografts.

Two pathways contribute to allorecognition of HLA-mismatched tissues (reviewed in References 20 through 22^{20 21 22} ). The "direct" alloresponse involves recognition of intact donor HLA antigens on the surface of donor cells by recipient T lymphocytes. Donor, bone marrow–derived APCs that are transplanted with the graft play a major role in triggering recipient T cells with direct anti-donor allospecificity. The strength of this response is accounted for by the uniquely high precursor frequency of T cells with specificity for allogeneic MHC molecules. Its vigor that appears to violate the rules of self–MHC restriction is driven primarily by antigenic mimicry.^{23 24} Direct helper and cytotoxic T cell recipient: anti-donor HTLf/CTLf frequencies are on the order of 1:10³ to 10⁴ and always <1:10⁵ for four to six HLA antigen mismatched donor and recipient pairs.^{25 26} On the basis of the results of experimental models of transplantation, this pathway is most active during the first few weeks after grafting but becomes less potent once the donor-derived APCs have left the graft.^{27 28} Indeed, in the rodent, once the donor APCs are eliminated, the graft appears to lose its immunogenicity in several strain combinations and is not rejected.^{29 30} Furthermore, the presentation of donor alloantigens by the parenchymal cells of the graft appears to favor the induction of donor-specific tolerance.³¹ The second pathway, the "indirect" alloresponse, involves the presentation of donor alloantigens that are shed from the cells of the graft by recipient APCs to recipient T cells. This requires the internalization and processing of the alloantigens that are then recognized in peptidic form bound to recipient HLA class II molecules. This is the same means by which T cells recognize conventional protein antigens. It has been argued that this pathway contributes to later, more chronic forms of rejection.²⁷ Although firm evidence for such a role in the context of chronic rejection is still lacking in human transplant recipients, its importance in graft rejection has been demonstrated in rodent models^{32 33} and recently in humans demonstrating acute cardiac allograft rejection³⁴

The purpose of this study was to determine whether donor-specific tolerance could be detected in T cells with direct anti-donor allospecificity in human heart transplant recipients after prolonged graft residence. Recipients with evidence of progressive TxCAD were selected. The recipient:anti-donor response was quantified by use of an improved technique of limiting dilution analysis which specifically estimates frequencies of recipient helper (HTLf) and cytotoxic (CTLf) T cells with direct allospecificity for donor cells.

	Methods

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Recipients
Ten heart transplant recipients (patients A through J) receiving maintenance cyclosporine and azathioprine immunosuppression were investigated, all of whom had developed chronic TxCAD in their first posttransplant year. The length of graft residence at the time of alloreactive T-cell frequency estimation varied from 1 to 7 years. Chronic rejection was diagnosed by use of standardized angiographic criteria.⁵ Its progression was demonstrated from the time of these studies in all recipients by angiography performed on at least one additional occasion. No recipients received donor bone marrow or donor-specific transfusions. No recipients were diabetic or hypertensive, and none had any evidence of previous cytomegalovirus infection (clinical evidence and detection of early antigen fluorescent foci by the Deaff test). For all recipients, posttransplant hyperlipidemia was treated and corrected.

HLA Typing
All recipients and donors were typed by use of conventional serological methods,^{35 36} and from April 1993, HLA-DR typing was performed by polymerase chain reaction amplification with sequence-specific primers.^{37 38}

HTLf and CTLf Limiting Dilution Assays
Methods to estimate HTLf and CTLf have been previously described.^{39 40} The stimulator cells were derived from donor splenic tissue that was cryopreserved at the time of the organ harvest; the responder T cells were prepared from recipient's PBMCs.

We further refined these assay systems to make them specific for the direct pathway of allorecognition and more sensitive in the case of the HTLf assay by eliminating the confounding effects of extraneous IL-2 production: Specificity for the direct pathway of allorecognition was achieved by depletion of APCs from the responder cell population, thus removing any possibility of self-restricted alloantigen (indirect) presentation. Previous reports using non–APC-depleted responder cells cannot negate and have not accounted for a contribution to the estimated frequency by self-restricted alloantigen recognition.

The key to maximizing sensitivity and specificity of the HTLf assay is to eliminate extraneous sources of IL-2 production so that the only stimulus to IL-2 release is the direct anti-donor alloresponse of the IL-2–producing T cells in the recipient. We identified three sources of extraneous IL-2 production. Autologous mixed lymphocyte reactions may occur within the responder population and within the irradiated stimulator population. "Back presentation" by responder APCs to T helper cells resident within the donor population provides an additional potential source of extraneous IL-2. Such "extraneous" IL-2 production may thus alter the estimation HTLf. Our solution has been to use monoclonal antibodies to deplete the responder population of antigen presenting cells and the stimulator population of CD4⁺ and CD8⁺ T cells. These cellular interactions no longer occur, and unwanted IL-2 production is markedly reduced. These modifications have led to significant differences in T helper cell frequencies compared with the results obtained with unseparated responder and stimulator cell populations. Such maneuvers are clearly essential in the detection of donor-specific tolerance.⁴¹

Preparation of Responder and Stimulator Cells
Preparation of Recipient (Responder) PBMCs
Peripheral blood was obtained from heart graft recipients. Mononuclear cells were isolated by density gradient centrifugation on Lymphoprep 1.077 g/mL (Nycomed Pharma AS). After extensive washing, responder cells (10⁷ cells per 1 mL) were resuspended in a freezing mixture comprising serum from AB donors and 30% dimethyl sulfoxide (Analar BDK). AB serum was added at 1:3 volumes to a give a final concentration of 7.5% dimethyl sulfoxide and 75% AB serum. Samples were subsequently stored in liquid nitrogen until required.

Preparation of Stimulator Spleen Cells
Portions of spleen were obtained during organ retrieval. Single-cell suspensions were released by injecting cold sterile RPMI into the splenic material with a syringe; mononuclear cells were then enriched on Lymphoprep gradients and cryopreserved as above.

Culture Medium for HTLf Assays
The assay culture medium for all HTLf assays consisted of RPMI 1640, supplemented with sodium bicarbonate (0.24% final concentration), L-glutamine (2 mmol/L), penicillin (50 IU/mL), streptomycin (50 µg/mL), sodium pyruvate (1 mmol/L) (all from Flow Laboratories), and 5% AB serum.

Antibody-Mediated Depletion
Stimulator Cells
Cryopreserved spleen cells were thawed rapidly and washed twice in RPMI 1640. CD4⁺ and CD8⁺ T lymphocyte depletion was carried out with anti–CD4⁺- and anti–CD8⁺-coated immunomagnetic beads (Dynabeads, Dynal AS). Immunomagnetic beads are at a concentration of 1.4x10⁸/mL. Beads were added to spleen cells at a ratio of 2:1, assuming that 70% of the stimulator cell population were CD4⁺ and that 30% of the stimulator cell population were CD8⁺. Immunomagnetic cell separations were performed at 4°C. Stimulator cells were gently mixed for 45 to 60 minutes. A magnet was then applied to the outside wall of the test tube to collect the bound cells and free beads as per the manufacturer's instructions. The stimulator cell suspension was then subjected to a further two rounds of depletion, this time at a ratio of 1:1.

Responder Cells
Cryopreserved responder cells were thawed rapidly and washed twice in RPMI 1640. Adherent cells were removed from PBMCs by incubation for 1 hour at 37°C on tissue culture–grade Petri dishes (Greiner). PBMCs were incubated on ice for 30 minutes with an antibody cocktail consisting of mouse anti-human monoclonal antibodies against HLA-DR, CD14, CD19, CD56, CD16, and CD33 (Becton Dickinson). The cells were then washed with cold RPMI and incubated with goat anti-mouse antibody microbeads (Miltenyl Biotec GmbH). Depletion was achieved by running the cell suspension and microbeads through a MiniMacs separation column applied to a magnet (Miltenyl Biotec GmbH). Eluted cells were then used for subsequent experiments.

Flow Cytometry
To determine the efficiency of depletion, cells were stained with directly conjugated anti-CD3-FITC and anti-DR-PE antibodies (Simultest, Becton Dickinson) and then washed and fixed with 1% paraformaldehyde in PBS. Cells were subsequently analyzed by use of an EXCEL flow cytometer (Coulter Electronics). In all experiments, T cell–depleted stimulators contained 4% CD3⁺ cells, and HLA class II–depleted responders contained 1% DR⁺ cells.

Maintenance of the IL-2–Dependent Indicator Cell Line, CTLL-2
The continued proliferation of the murine cytotoxic T lymphoblastic line CTLL-2 (European Collection of Animal Cell Cultures, Salisbury, UK) is dependent on the presence of human or murine IL-2 or murine IL-4.^{42 43} The CTLL-2 cells do not proliferate in response to human (unlike mouse) IL-4. The fact that the mouse IL-4 receptor does not bind human IL-4 was also reported by Mosmann et al,⁴³ and we have confirmed this using CTLL-2 cells. The line was maintained in culture medium with the addition of human recombinant IL-2 (10 U/mL, Boehringer) and 10% FCS. The cells were cultured in 25-cm² flasks (Costar) and were subcultured every 3 days. Before use in a limiting dilution assay, the CTLL-2 cells were washed twice and cultured overnight in normal culture medium but without recombinant IL-2. We added 1x10³ cells to all wells of each assay.

HTLf Assay
The design of the HTLf assay was as described previously.³⁹ After the depletion processes outlined above, the stimulator and responder cells were resuspended in RPMI with 5% AB serum. Graded numbers (0.03125x10⁴, 0.0625x10⁴, 0.125x10⁴, 0.25x10⁴, 0.5x10⁴, 1x10⁴, 2x10⁴, 3x10⁴, 4x10⁴, and 5x10⁴) of responder cells in 50 µL were added to 24 replicate wells of U-bottom 96-well microtiter plates (Flow Laboratories). In some cases, fewer responder dilutions were used because of a limitation in stimulator (donor) material. Stimulator cells were gamma-irradiated with 35Gy by use of a ¹³⁷cesium source (Gammacell 1000, Atomic Energy of Canada Ltd), and 5x10⁴ cells were added in 100 µL to each of the wells. Plates were incubated at 37°C in 5% CO₂ and 95% air for 72 hours. After incubation, the plates were gamma-irradiated with 25Gy (8-MeV linear accelerator, Philips MEL). The presence or absence of IL-2 production in each well was assessed by adding 1x10³ CTLL-2 cells in 25 µL of medium. Adding the IL-2–responsive cells directly to the wells has been shown to be more sensitive than adding the IL-2–responsive cells to previously removed supernatant.⁴⁴ Eight hours later, 1 µCi of tritiated thymidine (³H-TdR) (Amersham International plc) in 25 µL of medium was added to each well. After a further 16-hour incubation, the cells were harvested onto glass-fiber filter mats, and the³H-TdR incorporation by CTLL-2 was assessed by liquid scintillation spectrophotometry (1205 Betaplate, Pharmacia Wallac).

CTLf Assay
Cultures were set up and incubated as described above. On days 3 and 6, recombinant IL-2 was added to give a final concentration in the wells of 5 U/mL on each occasion. On day 10 of culture, 1x10^{4 51}Cr-labeled phytohemagglutinin–activated cells prepared from the original donor spleen cells were added to the cultures as target cells. The supernatants were harvested 4 hours later, and the⁵¹Cr released was measured in a gamma counter.

Control wells for the calculation of background activity consisted of 24 wells containing irradiated stimulator cells alone. Wells were classified positive for IL-2 production or cytotoxic activity, if ³H-TdR incorporation or ⁵¹Cr release exceeded the mean plus 3 SD of these control wells, respectively. Other control wells for both HTLf and CTLf contained responder cells in medium alone (negative control).

Statistical Analysis
Frequencies of alloreactive Th cells were calculated by use of a maximum likelihood statistical program with GLIM software (NAG Ltd) on the basis of the method of Finney.⁴⁵ The proportion of negative wells at each sample size of responder cells is linearly related to the frequency of responder cells according to the Poisson distribution: -log_eP_neg=fX, where P_neg is the proportion of negative wells, f is the frequency of responder cells, and X is the number of responder cells per well. The 95% confidence limits of the frequencies and ² estimates of probability were calculated. From the ² values and degrees of freedom (the number of responder dilutions minus one), probability estimates of the data conforming to "single-hit" kinetics may be calculated. Assays with values of P.05 are likely to conform to single-hit kinetics, ie, that a single cell type, the alloreactive IL-2–producing Th cell, is limiting. Assays with P<.05 that may not conform to single-hit kinetics were discarded. Frequencies are regarded as different if their 95% confidence limits (2 SD) do not overlap.

Study Design
Recipient (responder):anti-donor HTL and CTL frequencies were compared with those generated between responder T cells and third-party donor (stimulator) splenic APCs with equivalent HLA mismatch. An additional control was included to establish the immunogenicity of the donor spleen cells; 24 replicate cultures were set up containing 5x10⁴ donor spleen cells and 5x10⁴ third-party responder T cells. HTLf and CTLf assays involving spleen cells that failed to give rise to 24 of 24 positive wells with the third-party responder T cells were discarded.

	Results

Top Abstract Introduction Methods Results Discussion References

 
All the recipients included in this study had angiographic evidence of progressive chronic rejection as manifested by TxCAD. Furthermore, all the heart grafts were mismatched for between four and six HLA-A, -B, and -DR alleles. The third-party spleen cells used as a control for each recipient were selected to have the same degree of HLA mismatch as the recipient's donor cells.

In all recipients, both CTLf and HTLf were measured against stored donor cells and against a third party population of cryopreserved spleen cells. These data are presented in graphical form in Figs 1 and 2, and the actual numerical frequencies, together with 95% confidence intervals, are shown in the Table. In past analyses, we have taken the figure of 1:100 000 as the cutoff between high (>1:100 000) and low (<1:100 000) frequencies. Indeed, in several independent studies, HTL and CTL frequencies <1:100 000 were never observed in the face of four to six HLA antigen mismatches^{25 26} when responder cells from normal healthy individuals were used. Accordingly, we chose to define donor-specific hyporesponsiveness as being present if the recipient:anti-donor frequency was <1:100 000 and if the frequency measured against the donor was a log-order of magnitude less than the recipient: anti–third-party frequency. On this basis, donor-specific hyporesponsiveness was detected in 5 of the 10 recipients. The HTLf in the other five recipients were >1:100 000. Of the 5 recipients with low HTLf, 4 also had low anti-donor CTLf (<1:100 000), and in 2 of these, the CTLf (D and E) was actually below the limits of sensitivity of the assay. In the 5th recipient (recipient H), although the CTLf was >1:100 000, it was nonetheless significantly lower than that estimated against the third-party spleen cells.

View larger version (17K): [in this window] [in a new window]   Figure 1. IL-2–producing T helper cell frequencies are shown depicting the recipient:anti-donor () and recipient:anti-third party () direct alloresponse. For recipients D, E, H, I, and J, donor antigen-specific hyporesponsiveness is demonstrated as the recipient:anti-donor frequencies are less than 1/100 000 and a log order of magnitude lower than the respective recipient anti–third-party frequency.

View larger version (16K): [in this window] [in a new window]   Figure 2. Cytotoxic T-cell frequencies are shown depicting the recipient:anti-donor () and recipient:anti-third party () direct alloresponse. For recipients D, E, I, and J, donor antigen-specific hyporesponsiveness is again demonstrated. Although not <1/100 000, patient H demonstrates a statistically significant lower recipient:anti-donor frequency compared with the respective anti–third-party frequency.

View this table: [in this window] [in a new window]   Table 1.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The specific coronary artery vasculopathy is the manifestation of chronic rejection of the cardiac allograft (TxCAD). It can, in many respects, be regarded as an inevitable consequence of the transplantation process. Although both antigen- and non–antigen-dependent mechanisms play a role in its evolution, immune mechanisms undoubtedly make a significant contribution to its incidence and progression.

In this context, three specific points merit discussion. First, donor-specific direct pathway T-cell hyporesponsiveness occurs in the context of clinical cardiac transplantation in a substantial proportion of recipients. Furthermore, this hyporesponsiveness occurred in both the CTLf and HTLf compartments of the recipient anti-donor T cell repertoire. Although related observations have been made,^{46 47 48 49} no previous studies have reported specific direct pathway hyporesponsiveness in both HTLf and CTLf demonstrated by limiting dilution analysis and using donor material.

These experiments do not reveal the mechanism for this tolerance. Induction of anergy or deletion are both potential mechanisms. The mechanisms of this allograft-induced hyporesponsiveness are therefore not clear, although suppression would seem unlikely in that limiting dilution analysis plots in which the number of seeded responder cells is plotted in a semilogarithmic fashion against the fraction of nonresponding cultures always yielded straight lines, confirming single-hit kinetics. We consider that they are likely to reflect the consequence of alloantigen recognition on the surface of donor cells that are incapable of providing the molecular interactions that T cells need to become fully activated.³¹ On the basis of experimental models of transplantation, it was first proposed that the activation of CD4⁺ and CD8⁺ T cells requires the simultaneous receipt, of two distinct signals.⁵⁰ The first signal results from the ligation of the T-cell antigen receptor. It has become clear in recent years that the second signal is provided by key "costimulatory" molecules, such as B7.1 (CD80) and B7.2 (CD86), that are selectively expressed by specialized, bone marrow–derived APCs.^{51 52 53} More importantly, the receipt of signal 1 owing to cognate recognition of antigen in the absence of signal 2 is not a neutral event but leads to the T cell being rendered refractory to further stimulation (anergy).⁵⁴ It is well known that the inflammatory response that commonly accompanies allografing leads to the induction of HLA class II molecule expression by a variety of tissue cells, including endothelial cells and fibroblasts in the heart and epithelial cells in the kidney. However, these cells lack expression of key costimulatory molecules and may therefore lead to the induction of nonresponsiveness in allospecific T cells.

The second point is that the progression of chronic rejection, as manifested by TxCAD, is unlikely to be mediated by or dependent on a persistently high frequency of T cells with direct anti-donor allospecificity. All the recipients included in this study had clear evidence of progressive TxCAD. There were no detectable differences in terms of the number of prior acute rejection episodes, donor ischemic time, donor:recipient HLA mismatch, and cytomegalovirus status, which might conceivably contribute to hyporesponsiveness, compared with the rest of the study group. Not only did the recipients have TxCAD, but the time of onset of disease (within 1 year of transplantation) was no different from that of the recipients with no evidence of hyporesponsiveness. The detection of hyporesponsiveness in recipient T cells with direct anti-donor allospecificity in the face of progressive TxCAD indicates that the direct pathway alloresponse is not responsible.

The final point that merits discussion is an inference, namely that strategies designed to promote tolerance induction in transplant recipients will need to take into account T cells with indirect allospecificity. It is clear from these data that chronic rejection can still proceed despite profound alloreactive hyporesponsiveness in direct pathway T cells. While the relative contribution of antigen- and non–antigen-dependent factors in the progression of chronic rejection remains unclear, perhaps the most plausible explanation for its inexorable progression is that it continues to be driven by T cells with indirect allospecificity^{20 32 33} with some contribution from non–antigen-dependent risk factors, eg, lipids, according to the model of Ross.⁵⁵ If this is the case, this response will be continually stimulated by a continual supply of specialized recipient APCs. Furthermore the graft itself will be unable to promote tolerance to itself in the way that it can for T cells with direct allospecificity. Based on these considerations, the induction of tolerance in T cells with indirect allospecificity remains one of the outstanding challenges in the biology of allotransplantation.

One of the unresolved questions that arises from these data is why donor-specific hyporesponsiveness occurred in some, but not in other, recipients. There are several possible ways to account for these data; however, the explanation that we favor is that the residual T cells with direct anti-donor specificity in recipients with long-standing allografts are those of low affinity and that the high-affinity clones have been rendered nonresponsive in all recipients.

These data provide encouragement that direct pathway donor-specific tolerance is achievable in clinical transplantation without the need for specific tolerizing protocols. We additionally demonstrate, in contrast to other investigators,^{46 48} that the attainment of donor-specific hyporeactivity is not necessarily associated with stable graft function, as evidenced by the angiographic progression of TxCAD.

Objective and quantifiable evidence of a diminution in T cells with direct alloreactivity may have relevance for the reduction of immunosuppresive regimens; however, until the activity and immunosuppressive susceptibility of indirect alloreactive mechanisms have been assessed in the context of chronic rejection, such a reduction would seem potentially perilous. These data offer the challenge of devising strategies to inhibit the more persistent, indirect pathway of allo-sensitization.

	Selected Abbreviations and Acronyms

 

APC = antigen-presenting cell CTLf = cytotoxic T cells HTLf = helper T cells IL = interleukin PBMC = peripheral blood mononuclear cell TxCAD = transplant-associated coronary artery disease

	Acknowledgments

 
This work was supported by grants received by P. Hornick from the British Heart Foundation and the Royal College of Surgeons of England.

Received July 18, 1997; revision received November 25, 1997; accepted December 1, 1997.

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