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(Circulation. 1995;92:205-211.)
© 1995 American Heart Association, Inc.

Articles

Cardiac Allograft Vasculopathy

Association With Cell-Mediated but Not Humoral Alloimmunity to Donor-Specific Vascular Endothelium

Jeffrey D. Hosenpud, MD; John P. Everett, MD; Tony E. Morris, PhD; Kimberly A. Mauck, BS; Gary D. Shipley, PhD; Cynthia R. Wagner, PhD

From the Immunobiology Research Laboratory of the Oregon Cardiac Transplant Program; Departments of Medicine and Cell Biology and Anatomy, Oregon Health Sciences University; and Immunology Research, Portland Veterans Affairs Medical Center, Portland, Ore.

Correspondence to Jeffrey D. Hosenpud, MD, Division of Cardiology, Medical College of Wisconsin, 8700 West Wisconsin Ave, Milwaukee, WI 53226.

	Abstract

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Background Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease responsible for the majority of late deaths after cardiac transplantation. Although most consider this complication a manifestation of chronic allograft rejection, it has not been established whether this disease is a consequence of humoral or cell-mediated alloreactivity.

Methods and Results Human aortic endothelial cells (HAECs) were isolated from donor aortas obtained at the time of organ acquisition for 52 cardiac allograft recipients. Serum and peripheral blood mononuclear cells were obtained from these 52 allograft recipients at several time points during the first year after transplantation. Lymphocyte proliferation (LP) in response to donor-specific HAECs and alloantibody binding to interferon-–treated donor-specific HAECs were performed and correlated with clinical parameters, including HLA matching, acute cellular rejection, and coronary artery disease on surveillance angiography. Ten of the 52 patients studied had angiographic or autopsy evidence of coronary artery disease in the first posttransplantation year (CAV+ group). The CAV+ group had higher LP responses to their donor HAECs at 1 week, 3 months, and 6 months after transplantation compared with the CAV- group (1 week: 1439±222 versus 824±141 counts per minute [cpm], P=.026; 3 months: 1282±388 versus 884±94 cpm, P=.07; 6 months: 2504±635 versus 1540±209 cpm, P=.036; CAV+ versus CAV-, respectively). Only 8 of the 52 patients had donor-specific alloantibodies, and there was no relation between antibody presence and CAV. Other clinical parameters that correlated with CAV included the level of HLA-DR mismatch and the presence of late acute rejection.

Conclusions CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.

Key Words: transplantation • atherosclerosis • immune system • rejection

	Introduction

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Cardiac allograft vasculopathy (CAV), a rapidly developing and rapidly progressing obliterative vascular disease occurring in the transplanted heart, is the major cause of late death and repeat transplantation.^{1 2} This same phenomenon is also present in renal, lung, and liver allografts and has been designated chronic rejection, obliterative bronchiolitis, and vanishing bile duct syndrome, respectively.^{3 4 5} That these phenomena clearly represent a chronic alloimmunologic response to the transplanted organ is almost universally accepted. What is less clear are the mechanisms responsible for producing these changes. There has been substantial debate in the literature regarding whether the humoral or cellular arm of the immune system is important in the development of CAV. Several pieces of evidence have led investigators away from considering cell-mediated immunity important in CAV. First, despite the presence in animal models of a cellular inflammatory response to allograft vasculature,^{6 7 8 9 10 11} the same cannot be identified in human explant or autopsy specimens. More recent studies have appreciated a more subtle cellular infiltration into the vascular neointima.^{12 13 14 15 16} Second, the incidence of acute parenchymal rejection, clearly a cell-mediated process, markedly declines during the first months after transplantation,¹⁷ and only rarely have studies found a correlation between the incidence and severity of acute rejection and the subsequent development of CAV.¹⁸ Third, in the renal transplant literature, which has reported a high incidence of alloantibody formation (due primarily to multiple transplants in a given recipient and the use of sensitizing pretransplantation transfusion therapy), there was a documented association between high titers of alloantibody and poor prognosis and graft loss.^{19 20 21} Several studies have, therefore, attempted to measure and correlate the presence of alloantibodies to CAV and ultimate prognosis.^{22 23 24 25} A primary criticism of these prior investigations is that the targets used to detect antibody either were not of donor origin and therefore nonspecific²⁵ or did not use the likely immunological target for CAV, the vascular endothelial cell.^{23 24} Accordingly, the present study was designed to investigate both cell-mediated and humoral immunity to donor-specific vascular endothelium in a cohort of cardiac allograft recipients and to correlate these in vitro investigations with the development of angiographically documented CAV.

	Methods

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Patient Inclusion Criteria and Division Into Study Groups
The present study was reviewed by the Institutional Review Board of the Oregon Health Sciences University. At the time of organ donation, after consent was received for organ donation for "any purpose authorized by law," a segment of ascending and transverse aorta was obtained from donors destined to provide hearts to patients waiting for cardiac transplantation at the Oregon Cardiac Transplant Program. All potential adult cardiac allograft recipients were asked to participate in this study, which required multiple blood specimens to be drawn, and all gave consent. Actual inclusion of cardiac transplant recipients into the study cohort required the following: (1) successful isolation of endothelial cells from the aortic segment obtained from the donor providing the heart to that given recipient, (2) survival of the recipient for at least 6 months with collection of blood samples through this time period, and (3) completion of an early (within 2 weeks of transplantation) coronary angiogram as a baseline and either a follow-up angiogram at the 1-year anniversary or direct examination of the coronary arteries on autopsy. From October 1990 through June 1993, 52 patients met the above criteria, and they form the study cohort.

Patients were divided into two groups: those with and those without CAV (CAV+ and CAV-, respectively), based on the 1-year posttransplantation angiogram or autopsy findings typical of CAV.^{26 27} All 1-year angiograms were compared directly with the baseline angiograms by clinicians who were blind to the results of the in vitro data. The 1-year angiogram was considered positive for CAV if there were any changes from baseline, including diffuse narrowing, luminal irregularities, distal vessel pruning, or frank stenoses, all findings previously well described as being consistent with CAV.²⁷

Human Aortic Endothelial Cell Isolation and Culture
Human aortic endothelial cells (HAECs) were isolated from segments of thoracic aorta of heart donors and maintained as individual isolates, as previously described.^{28 29 30} Briefly, segments of human ascending aorta were obtained during organ donation after consent was obtained for the donation of any and all tissues. HAECs were isolated by incubating the aortic tissue in a solution of 0.1% collagenase (CLS/type 1, 152 U/mg; Worthington Biochemical Corp) in RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 25 mmol/L HEPES, 24 mmol/L sodium bicarbonate, pH 7.2 (GIBCO), and incubated at 37°C for 1 hour. After the incubation, the HAECs were gently swabbed from the luminal surface of the aorta. The purity of the HAEC cultures was determined by their uniform immunofluorescent staining with antibodies to factor VIII–related antigen (A082, DAKO Corp)³¹ and their uptake of fluorescent-labeled acetylated LDL (DiI-Ac-LDL, Biomedical Technologies, Inc).³² The HAECs were cultured in tissue culture flasks pretreated with human fibronectin (4 µg/cm²; Collaborative Research Inc) in endothelial growth medium (EGM-UV, Clonetics Corp) supplemented with an additional 3% of fetal calf serum (Tissue Culture Biologicals) and were at passages 3 through 5 for these studies.

Preparation of Allograft Recipient Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMCs) were isolated as previously described³⁰ from cardiac allograft recipients' blood samples collected 7, 45, 90, 120, 180, and 360 days after transplantation. After isolation by density gradient centrifugation using Ficoll-Hypaque (Pharmacia Fine Chemicals), the PBMCs were cryopreserved.

Lymphocyte Proliferation Assays
PBMC proliferation in response to donor-specific HAECs were performed as previously described³⁰ at three time points after transplantation: 1 week, 3 months, and 6 months. Briefly, PBMCs isolated from individual allograft recipients were added to the 96-well plates of irradiated (2000 rad, Cs¹³⁷) donor-specific HAECs at a ratio of 10 lymphocytes per 1 HAEC (3x10⁵ PBMCs:3x10⁴ HAEC). The PBMC and HAEC co-culture medium consisted of RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 25 mmol/L HEPES, 24 mmol/L sodium bicarbonate, pH 7.2, and 15% human AB serum (Normlcera-Plus, North American Biologicals, Inc). Controls included HAECs cultured alone and PBMCs cultured alone. Quadruplicate wells were set up for each variable. Incorporation of ³H-thymidine into co-cultures was determined after 96 hours of coincubation by the addition of 5 µCi/mL of ³H-thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear-Dupont) to each well for 20 hours. The PBMCs were then harvested onto glass-fiber filter paper using a semiautomated cell-harvesting apparatus (Skatron). The filter pads were dried, and relative ³H-thymidine incorporation was determined by liquid scintillation counting. Proliferation was expressed as the mean counts per minute (cpm) of PBMCs in co-culture minus the mean cpm of PBMCs alone.

Measurement of Donor-Specific Alloantibodies
The presence of alloantibody in the recipient serum capable of binding to the donor's HAECs was determined by flow cytometry as previously described²⁸ at two time points after transplantation: 6 weeks and 1 year. The 6-week time point was chosen based on prior literature demonstrating peak antibody detection at 1 to 2 months after transplantation.²⁵ The 1-year period was chosen based on the premise that if alloantibodies were a significant etiological factor in this form of chronic ongoing rejection, shown to progress year to year after transplantation,³³ alloantibodies should continue to be present at this time point. HAECs treated with recombinant human interferon- (rhIFN-; Collaborative Biomedical Technologies Inc; 500 U/mL for 96 hours) to upregulate MHC class I and class II antigens were harvested with 0.05% trypsin and 0.53 mmol/L EDTA in Hanks' balanced salt solution (GIBCO). Cells were incubated with undiluted recipient serum, negative control serum generated by pooling serum from AB+ blood group donors demonstrated to have undetectable anti-HLA antibodies by flow cytometry (Normlcera-Plus; North American Biologicals, Inc), or positive control serum pooled from 30 renal allograft recipients with high titers of antibodies to a broad range of HLA determinants. After 30 minutes (25°C), HAECs were washed (twice) and then stained with fluorescein-conjugated goat anti-human F(ab')₂ second antibody (Jackson ImmunoResearch Laboratories, Inc) for 30 minutes at 4°C. The samples were fixed with paraformaldehyde (0.1%) and then analyzed by flow cytometry with a Becton Dickinson flow cytometer (FACScan, Becton Dickinson). A minimum of 5000 cells per sample were analyzed. A bitmap/gate was set from the 90-degree light scatter versus forward angle light scatter (FALS) histogram to exclude any dead cells or debris from the analysis. The cells were analyzed using a logarithmic amplifier to determine the percentage of stained cells and their mean fluorescence intensity.

To determine the sensitivity of this assay, we studied multiple titrations of the positive control pooled serum containing high titers of alloantibodies in a standard complement-dependent cytotoxic assay with a panel of lymphocytes containing a broad range of HLA phenotypes as target cells (panel reactive assay [PRA]) and compared them with the flow cytometric analysis of antibody detection as above, but in this case a pool of blood group O+ HAECs were used representing a wide range of HLA antigens rather than the donor-specific HAECs as target cells. Fig 1 demonstrates this comparison. The flow cytometric antibody-binding assay was positive at between fourfold and eightfold greater dilution than was antibody detection by the complement-dependent cytotoxicity assay (PRA).

View larger version (31K): [in this window] [in a new window]   Figure 1. Bar graph of sensitivity of the standard complement-dependent lymphocytotoxic assay (referred to as the panel reactive assay [PRA]) compared with flow cytometric analysis of antibody binding to pooled human aortic endothelial cells. Comparison uses serum pooled from renal transplant candidates with high titers of alloantibodies. Flow cytometry is able to detect antibody in this pooled serum at fourfold to eightfold greater dilution compared with PRA.

Clinical and Immunological Parameters/Data Analysis
The presence or absence of CAV was also related to a variety of clinical and immunological parameters, including recipient age, recipient sex, donor age, underlying cardiac disease, average (during the first 6 months during a tapering schedule) and maintenance (at 12 months) doses of immunosuppressive agents, early (during the first 3 months) and late (at 6 to 12 months) acute rejection, and HLA matching. Acute rejection was diagnosed by surveillance endomyocardial biopsies (approximately 20 in the first year). A rejection episode for the purposes of the present study was defined as any biopsy of International Society for Heart and Lung Transplantation grade³⁴ of 2 or greater necessitating an augmentation of immunosuppression. Cytomegalovirus (CMV) infection was defined to be present only if virus was cultured on surveillance buffy coat and urine cultures obtained monthly for the first 6 months as part of this center's standard clinical protocol. Primary and secondary CMV infections were defined as culture positivity occurring in those patients who had negative and positive CMV serology, respectively, before transplantation. Continuous clinical variables were compared between CAV+ and CAV- groups with t tests. Categorical variables were compared using ² analysis. All clinical variables were then subjected to a multivariate ANOVA using Hotelling's analysis. In vitro data were compared between CAV+ and CAV- patients with t tests. Significance was considered present at a value of P<.05.

	Results

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Fifty-two patients met the criteria for inclusion into the study protocol during the enrollment period. Of these, 51 survived at least 1 year to undergo routine surveillance coronary angiography. One patient had a sudden death episode 7 months after transplantation and was resuscitated but died soon afterward in the hospital; autopsy determined there to be extensive CAV. A total of 10 patients had CAV by the prospective criteria on angiography or autopsy, whereas 42 patients had no evidence of CAV by angiogram. Table 1 presents clinical demographics of the study group. There were no significant differences between those with and without CAV; however, donor age tended to be slightly greater in the CAV+ group. There was 1 patient in each group who entered the study having undergone a second transplantation. In the CAV+ group, the patient underwent prophylactic repeat transplantation 5 days after the first transplantation because the patient accidently received an ABO blood group–mismatched heart. In the CAV- group, the patient underwent repeat transplantation for CAV in the first heart.

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

Table 2 demonstrates the clinical factors that relate to immunological activity. There were no differences in average doses of immunosuppressive agents in the first 6 months (calculated by averaging the drug dose at each of the first 6-month anniversaries) after transplantation. Immunosuppressive doses routinely start high and are tapered to maintenance levels by the end of 6 months after transplantation. With multivariate analysis, there were no differences in any of the variables studied; however, 7 of 52 cases lacked complete data for all variables and thus were eliminated. With univariate analysis, there was, however, a statistically significantly lower maintenance dose of both azathioprine and prednisone at the 12-month time period in the CAV+ group.

View this table: [in this window] [in a new window]   Table 2. Patient Immunological Parameters

The highest incidence of acute rejection from all studies, including our data, occurs during the first 3 months after transplantation.^{17 35 37} In this cohort, there were no differences in the average rejection incidence in the first 3 months after transplantation between the CAV+ and CAV- groups. With univariate analysis, there was a slightly but statistically significant increased rejection from 6 to 12 months after cardiac transplantation in the CAV+ group. But as can be seen, the overall incidence has fallen substantially in both groups comparing the two time periods.

HLA-A, -B, and -DR (serology) phenotypes were available for all donors and recipients. There were no differences in the numbers of HLA-A and -B locus mismatches (of four possible, one -A and one -B for each allele) between CAV+ and CAV- groups. There was a significant increase in the number of -DR mismatches (of two possible, one for each allele) in the CAV+ group compared with the CAV- group with univariate analysis.

CMV has been associated with the development of CAV in a number of studies.^{38 39} In this cohort, however, we saw no association between total or any form of CMV infection and CAV.

Fig 2 demonstrates the recipient PBMC proliferative responses to the donor's HAECs at the three time points studied. Assays were successfully completed for 46, 47, and 51 of the 52 patients in the cohort at each of the time points. Reasons for incomplete data included inadequate cryopreservation of recipient PBMCs, culture contamination, and inadequate numbers of PBMCs collected to perform the assays. All 52 patients are represented in at least one of the time points. There were statistically significant increases in PBMC proliferative responses to donor HAECs in the CAV+ group compared with the CAV- group at both 1 week and 6 months and a borderline significant increase at the 3-month time point.

View larger version (43K): [in this window] [in a new window]   Figure 2. Bar graph of recipient peripheral blood mononuclear cell proliferation in response to exposure to donor-specific human aortic endothelial cells, which is statistically significantly greater in the group with cardiac allograft vasculopathy (CAV+) compared with the CAV- group at 1 week and 6 months and is borderline statistically greater at the 3-month time point.

To not miss any patient with potentially low levels of circulating alloantibodies, extremely liberal criteria were used to designate flow cytometric results as being positive. A positive result was considered to be present if recipient serum produced a mean shift of 10 or more channels above control serum for a given recipient serum/donor HAEC isolate pair. A borderline positive result was a mean shift of five to nine channels above control levels. Alloantibodies were present in 4 of the 52 patients early (3 at 6 weeks and 1 at 11 weeks) after transplantation, and an additional 3 patients had borderline positive results at 6 weeks. One additional patient had borderline positive results at 1 year. These data are presented in Table 3. Of the 4 positive patients, 2 had documented positive flow cross-matches with donor lymphocytes using serum obtained before transplantation (preformed antibodies), so only 2 patients had definitive de novo production of donor-specific alloantibody detected. By 1 year, no antibody could be detected in 4 of the 8 patients, and in the 1 patient who had extremely high levels of alloantibody at 6 weeks (patient 3 in Table 3), this level had declined substantially by 1 year. One patient had persistent levels of alloantibody at 11 weeks (6-week sample not available) and 1 year; one patient had borderline elevated antibody levels at both 6 weeks and 1 year; and the remaining patient had no antibody detected at 6 weeks but had borderline antibody levels at 1 year. Fig 3 demonstrates the flow histograms for this patient showing the antibody binding to donor-specific HAECs using control serum (top), recipient serum obtained at 6 weeks after transplantation (middle), and serum obtained at 1 year (bottom). Finally, there was no relation between the presence of donor-specific alloantibodies (both positive and borderline) and the development of CAV (Fig 4).

View this table: [in this window] [in a new window]   Table 3. Patients With Alloantibody Detected

View larger version (16K): [in this window] [in a new window]   Figure 3. Flow cytometry histograms of antibody binding using serum from the patient with the greatest amount of antibody detected in this series. This patient had preformed donor-specific antibodies before transplantation (see text). Despite the extremely high levels of antibody in the serum obtained at 6 weeks after transplantation (middle), antibody levels fell to almost baseline levels by 1 year (bottom). Top, The same donor human aortic endothelial cells exposed to blood group AB+ control serum containing no detectable alloantibody.

View larger version (18K): [in this window] [in a new window]   Figure 4. Pie charts showing no relation between the presence of alloantibody and the development of cardiac allograft vasculopathy (CAV) as assessed by angiography or autopsy.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data presented define a prospective cohort of cardiac allograft recipients of whom 19% developed chronic rejection (CAV); in the 19%, indexes of cell-mediated alloreactivity to appropriate vascular targets were significantly higher than in those without CAV. Also, alloantibody detection was extremely low for the entire group, suggesting that the humoral arm of the immune system likely plays little or no role in CAV. In addition to the in vitro data presented, several aspects of the patients' characteristics and courses support the role of cell-mediated immunity, including a greater HLA-DR mismatch (the target for CD4+ T lymphocytes), a higher incidence of late acute rejection, and lower maintenance doses of prednisone and azathioprine. A reasonable hypothesis for interpreting these data would be that the standard practice of tapering immunosuppression based on declining parenchymal rejection will potentially allow patients with the worst histocompatibility matching to escape levels of immunosuppression adequate to suppress cell-mediated alloresponses. This escape is potentially subtle enough to not result in frank parenchymal rejection in most cases (the incidence of acute rejection in the 6- to 12-month period is still quite low) but is adequate to allow chronic T-cell–mediated vascular attack and injury or activation. We have previously reported that allogenic lymphocytes are capable of activating vascular endothelium, resulting in upregulation of mesenchymal growth factor production.³⁰ A further extension of the proposed hypothesis would be that the chronic cell-mediated alloresponse results in coronary endothelial cell activation and production of growth factors such as platelet-derived growth factor or basic fibroblast growth factor, resulting in stimulation of smooth muscle cell migration and proliferation.

Alternatively, it is conceivable that the differences seen in acute parenchymal rejection and chronic vascular rejection, manifest in this case as CAV, represent differences in lymphocyte–target cell interactions. As early as the late 1970s, Liburd et al⁴⁰ demonstrated reduced donor-specific cell-mediated lympholysis over time in renal allograft recipients. These data were confirmed in later studies by Mohanokumar and colleagues.^{41 42} The target cells used in these studies were donor-derived lymphocytes or lymphocyte lines sharing HLA antigens with the recipient's donor. The mechanisms responsible for this donor-specific accommodation have been ascribed to suppressor cells,⁴³ anti-idiotypic antibodies,⁴⁴ anti-idiotypic T cells,⁴⁵ and Veto cells.⁴⁶ In any case, this response to donor-relevant lymphocytes appears to parallel the incidence of acute parenchymal rejection.

In a previous study involving a subset of the current cohort, we demonstrated that in contrast to the response seen to donor-derived or relevant lymphocytes, the response to donor-specific endothelial cells actually increased over time, coincident with decreased doses of chronically administered immunosuppressive agents.⁴⁷ The explanation for this dichotomy between target cells from the same donor is not clear. It is interesting to note that activation of T cells by lymphocytes and macrophages requires an interaction between CD28 on the T cell and B7 on the target cell.⁴⁸ Human endothelial cells do not express B7, and therefore, an alternative ligand interaction such as CD2-LFA-3 as proposed by Savage and colleagues⁴⁹ may provide this co-stimulation. It is conceivable that secondary signals derived from these alternative ligand interactions differ in their long-term responses and susceptibility to chronic immunosuppression and the various phenomenology proposed for allograft accommodation.

The low prevalence of recipient antibodies directed against donor endothelial cells in the present study contrasts with that of other investigators, who, using microlymphocytotoxic panels, have reported prevalences of anti-HLA antibodies ranging from 10%²⁴ to 82%^{25 50} in their cardiac allograft recipients. The significance of these antibodies is unclear. Although several authors found an association between antibodies detected by PRA and reduced graft survival,^{24 50 51} others reported no such relation.²⁵ Because these antibodies are detected by screening against standard HLA reference panels, their specificity to the recipient's donor is unknown. Rather than being a primary agent of graft rejection, these antibodies may just be a secondary marker of increased donor alloreactivity against the donor graft.

The low prevalence of antibody binding in the present study may be a failure of our assay to detect antibodies that are bound to soluble donor HLA antigens found in recipients' sera⁵² or to anti-idiotype antibodies produced by the recipients.⁵² The former scenario is unlikely, because in recipients identified with soluble HLA antigens, at least 56% of them died within several years of transplantation⁵² ; thus, the release of donor antigens into the circulation heralded a severely reduced graft survival rate. In contrast, the presence of donor-directed antibodies in our population was neither favorably nor adversely associated with acute or chronic rejection. The presence of anti-idiotype antibodies binding to the donor-directed antibodies in our recipients cannot be excluded. However, the authors describing these antibodies in cardiac allograft recipients examined only a small subset of patients, so the prevalence is unknown.

Rather than investigating anti-HLA antibodies, another group retrospectively investigated antiendothelial antibodies produced by cardiac allograft recipients, using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.⁵³ Peptide-specific antiendothelium antibodies were found in 15 of 21 patients who developed CAV within 2 years of transplantation but in only 1 of 20 who did not develop CAV. Thus, the presence of antibody was strongly associated with the development of CAV, which is in contrast to our results. Although Dunn et al⁵³ confirmed the ability of these antibodies to bind coronary endothelium by positive immunofluorescent staining of recipient serum on frozen sections of coronary vessels, they could not demonstrate binding to the endothelium-lined microvasculature of donor atrial tissue. Because CAV is characterized by diffuse involvement of the allograft vasculature^{54 55} rather than being confined to the coronary epicardial vessels, the relevance of these antibodies as an etiological agent of CAV, especially given their initial identification in pooled human umbilical vein endothelial cells, is unclear. An equally plausible hypothesis is that they are a marker of endothelial cell damage secondary to this process.

Several aspects of the present study require comment. First, the endothelial cells used in these assays were derived from ascending aorta. Although there is reasonable evidence to suggest that the entire allograft vasculature is involved in the chronic rejection process as previously noted,^{54 55} it is conceivable that differences in endothelial cells cultured from these two beds might result in different outcomes. Second, the effector cell population consisted of PBMCs rather than purified lymphocytes or lymphocyte subsets due to limitations in the amount of blood available for study from each recipient. LP is largely a manifestation of the CD4+ lymphocyte subset,¹⁶ whereas induction of MHC class II antigens on resting endothelial cells is mediated primarily by the CD8+ lymphocyte subset, although other subsets such as NK cells can also accomplish this response.⁵⁶ Therefore, it is likely that we are measuring phenomena induced by multiple populations of cells. Whether this is a true limitation is unclear, given that this mixed cellular response is more apt to be representative of in vivo phenomena than responses generated using individual lymphocyte subsets.

Finally, despite washing, the PBMCs have been subjected to and likely continue to be influenced by the in vivo environment containing cyclosporine, prednisone, and azathioprine from which they were isolated. It is very possible that our results are, in fact, influenced by these agents. Incorporation of ³H-thymidine into activated (HAEC-exposed) PBMCs, given the relatively low counts, could have been influenced by immunosuppression; however, incorporation levels based on cpm are not substantially different from those reported by other investigators performing mixed lymphocyte/endothelium proliferative assays.^{57 58} In contrast to other investigators,^{30 59} we did not use 5-fluorodeoxyuridine to inhibit constitutive thymidine synthesis in our HAEC cultures out of concern of complicating the interpretation with potential interactions between this agent and the other immunosuppressive agents. Despite these concerns, it is important to emphasize that our in vitro data are likely to be mimicking the actual in vivo activity in these allograft recipients who are treated with varying doses of immunosuppressive agents.

This investigation does not provide a mechanism for the development of cardiac allograft vasculopathy, only an association. It is conceivable that the recipient cell-mediated responses to donor-specific vascular endothelium do not represent primary events but rather secondary phenomenon after initial damage from some undefined immunological event. Nevertheless, this is the first prospective investigation using reasonably appropriate and patient-specific immunological effectors and targets in a large patient cohort that demonstrates a clear association between the cellular arm of the immune system and the ultimate development of chronic rejection.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grant RO1-HL-43369, the Department of Veterans Affairs, and the American Heart Association. The authors express appreciation to Susan Fogg for her expertise in cell isolation and culture, MaryAnn Head for her assistance in the flow cytometry studies, and the Division of Cardiopulmonary Surgery and the Pacific Northwest Transplant Bank for obtaining donor tissue.

Received November 2, 1994; revision received January 9, 1995; accepted January 14, 1995.

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