The clinical significance of class II anti-human leukocyte antigen (HLA) antibodies has been well established in renal transplantation (1), but their role in heart transplantation (HTX) is unclear. Previous studies have shown that HTX recipients who have anti-HLA donor-specific antibodies (DSAs) are at an increased risk for early acute rejection and have a lower graft survival (2, 3). Newly introduced solid-phase array techniques can distinguish between HLA type and class I and II antibodies. These tests allow for simultaneous, sensitive, and specific detection of multiple anti-HLA antibodies, and the results are actively being used for recipient and donor selection (4). These improved techniques have been rapidly incorporated in clinical practice despite the significance of the different types and levels of HLA antibodies detected (class I vs. class II) being controversial in HTX. The role of DSA in cardiac allograft vasculopathy (CAV), which remains the leading cause of late morbidity and mortality in HTX recipients (5–7), is unclear. The aim of this study was therefore to evaluate the potential role of such alloantibodies on the progression of CAV as assessed by serial three-dimensional intravascular ultrasound (IVUS) examination and coronary angiography (8).
Baseline patient characteristics (Table 1) shows the demographic, clinical, and laboratory characteristics of patients in the DSA I+, DSA II+, and DSA− groups. Because our aim was to evaluate the impact of each DSA type by itself, we have excluded the two patients with both DSA I+ and DSA II+ from the analysis. Donor-specific antibody specificity and peak and total mean fluorescence intensity (MFI) values for each patient are shown in Table S1 in the Supplemental Digital Content (see Table S1, SDC,http://links.lww.com/TP/A726). There were no differences between the groups in recipient age, sex, donor age, conventional atherosclerosis risk factors, medical therapy, and primary or secondary immunosuppression. Trough levels of calcineurin inhibitor (CNI) and sirolimus (SRL) were within the therapeutic range in all the patients. History of ischemic cardiomyopathy was lower in the DSA I+ group compared to the other two groups. Flow cytometric crossmatches were performed in 17 patients in the DSA− group, in 3 patients in the DSA I+ group, and in 10 patients in the DSA II+ group. None of the patients in the DSA− and DSA I+ groups, whereas 6 of 10 patients in the DSA II+ group, had a positive flow cytometric crossmatch (P=0.0003). Three patients in the DSA II+ group, who had a positive flow cytometric crossmatch, were treated with plasmapheresis immediately after transplantation. Because only a small number of subjects had class I DSA, the relationship between DSA I and CAV could not be investigated; thus, analysis was performed only for the class II DSA.
Three-Dimensional IVUS Findings
Table 2 outlines the three-dimensional IVUS findings in the DSA− and DSA II+ patients. There was significant progression of plaque index (PI) from baseline to the first follow-up IVUS examination in the DSA II+ group (+0.04 [0.03] mm3, P=0.01), but there was no progression of PI observed in the DSA− group (−0.003 [0.07] mm3, P=0.8). The progression in PI in the DSA II+ group likely occurred because of a combination of a trend for accelerated progres sion in plaque volume and vessel constriction, a change measured from baseline to the first follow-up IVUS. The rate of progression in PI was thus significantly greater in the DSA II+ group when compared to the DSA− group (+13.8% [12%] vs. −7.9% [37%], respectively, P=0.02; Fig. 1) despite the baseline PI being higher in the DSA− group. The same trend for increased rate of PI progression was also observed in those patients (n=6) who had low DSA II value (peak MFI<2000) when compared to the DSA− patients (+13.4% [19%] vs. −7.9% [37%], respectively). Similarly, PI progression seemed to occur to the same extent in those DSA II+ HTX recipients who had a FCXM that was negative (n=5) compared to the 19 DSA− recipients in which FCXM was performed and negative (+12.5% [16%] vs. −15.2% [43%], respectively). There were no significant differences in the baseline volumetric data or the change in the different parameters by three-dimensional IVUS between the DSA I+ and the DSA− group (P>0.2 for all parameters).
The primary aim of our study was to evaluate the potential role of DSA on the progression of CAV. A cutoff value of anti-HLA antibodies greater than 300 MFI was used because this value has been validated in our laboratory to be above the “background noise” level to define the positivity for DSA. However, the significance of the various DSA MFI values is unknown. Many transplant programs use a cutoff of MFI greater than 1000 for recipient and donor selection; therefore, we have also performed a separate analysis using a cutoff of MFI>1000 and we present it in Table 3.
Using the higher DSA values, the results were similar. The rate of progression in PI was significantly greater in the DSA II+ group when compared to that in the DSA− group (+12.3% [7%] vs. −7.9% [18%], respectively, P=0.05). There was a strong trend for progression of PI from baseline to the first follow-up IVUS examination in the DSA II+ group (+0.03 [0.04] mm3, P=0.08), but there was no progression of PI observed in the DSA− group (−0.003 [0.07] mm3, P=0.8).
Because the majority of class II DSAs were against DQ, it was possible that the association was caused by DQ rather than by class II antibodies. We repeated the analysis with only patients with DQ antibodies (see Table S2, SDC, http://links.lww.com/TP/A726). The results demonstrate a similar trend, but the difference in the rate of progression in PI was not significant.
DSA and Angiographic CAV
There were 36 (71%) of 51 patients who had grade I CAV or greater during a median follow-up period of 3.7 years (range, 2.2–5.2 years). Kaplan-Meier curves show considerable difference in the presence of angiographic CAV significantly affecting the DSA II+ group as compared to DSA− patients at 1 year (42.5% [18%] vs. 19.6% [7%]), 2 years (57.2% [18%] vs. 36.2% [9%]), and 4 years (100.0% [0%] vs. 64.2% [9%], P=0.05). There were seven patients (14%) who had grade II CAV or greater during the median follow-up period of 3.7 years. There was a clear trend for an increased rate of developing grade II CAV or greater in DSA II+ patients compared to DSA− patients at 1 year (16.6% [15%] vs. 3.3% [3%]) and 4 years (58.3% [30%] vs. 18.2% [8%]).
This is the first preliminary study that demonstrates a significant association of class II DSA with progression of CAV as detected by three-dimensional IVUS and coronary angiography early after HTX. Therefore, preformed class II DSA may be a risk factor for accelerated CAV after HTX. Because only a few patients had class I DSA, the relationship between DSA I and CAV could not be investigated.
Major histocompatibility complex class II antigens are constitutively expressed in human capillary and venous endothelium, including that of coronary arteries, and could be a target for alloimmune responses (9, 10). Donor-specific antibodies may cause direct endothelial cell damage by complement activation or by targeting natural killer cells and macrophages. Donor-specific antibodies have also been shown to activate endothelial exocytosis of granules that contain prothrombotic mediators, resulting in exaggerated mitosis and inflammation of the vessel (11). Whether such responses as a result of class II DSA play a role in a “smoldering” inflammatory response, contributing to accelerated plaque progression, remains speculative and will have to be assessed in further studies.
This study could have important implications for clinical practice in HTX. First, we suggest that class II DSA be considered as an important component of the overall assessment of immunologic risk in HTX recipients. Second, HTX in the presence of demonstrable DSA against class II (even in lower MFIs) may necessitate close posttransplant monitoring for CAV. Third, the role of a virtual crossmatch by excluding donors with unacceptable antigens to which recipients have DSA, especially of class II type, in minimizing the development of CAV needs to be further explored. Newer interventions that may attenuate the formation or levels of DSA such as rituximab may also affect the development of subsequent CAV (12).
Before the advent of sensitive techniques for detecting anti-HLA antibodies, risk factors of CAV were considered to be more traditional risk factors of atherosclerosis such as hyperlipidemia, diabetes, donor age, ischemic time, and others (6, 13). The important role of immune factors in CAV emerges from more recent studies (13, 14), suggesting that both adaptive (cellular and humoral) and innate immune responses result in the progression of CAV.
The introduction of solid phase–based techniques for HLA antibody analysis has enabled specific and sensitive determination of the presence of donor-specific HLA class I and class II (immunoglobulin [Ig] G) antibodies in transplant recipients. There are an increasing number of transplant centers making determinations of transplant eligibility and donor suitability based on the results obtained by these solid-phase arrays. However, the impact of DSA detected by these sensitive techniques on long-term posttransplant outcomes such as graft vasculopathy is unclear. Most published studies in this field have focused on renal transplant recipients (1, 15, 16) and have shown that DSAs against HLA class II antigens are associated with an increased risk of developing transplant glomerulopathy and antibody mediated rejection (AMR). There is paucity of data on the role of HLA class II antibodies on HTX outcomes (17, 18). Although previous animal and clinical trials have implicated the role of AMR on the progression of CAV (19–23), DSAs may not necessarily be associated with AMR, and this finding may have been related to the sensitivity of the tests used to detect DSA (24).
Flow cytometry crossmatch methods are more sensitive than the cytotoxic assays, and a positive flow crossmatch has been shown to be associated with AMR and graft survival (18). However, prospective flow crossmatches are not readily available to make decisions regarding HTX feasibility. In this study, no significant difference in mortality or in the incidence of AMR was observed between patients with or without DSA. The lack of difference may be because of the small number of patients assessed and the limited duration of follow-up, emphasizing the need for larger observational studies. Furthermore, most of our patients had low DSA values, and perhaps higher DSA values are required for the development of AMR.
The principal aim of the present study was to determine whether the presence of DSA before HTX is associated with accelerated progression of CAV as detected by three-dimensional IVUS. Three-dimensional IVUS is presently considered the gold standard for the evaluation of CAV (25–27) and quantifies both intimal thickening and changes in external elastic membrane area, a process of arterial remodeling. This is especially important considering that several studies have emphasized that lumen loss in CAV may be caused not only by intimal thickening and plaque progression but also by changes in external elastic membrane area (27–29). Our study showed a trend toward accelerated vessel volume constriction despite a trend toward higher plaque progression in patients with class II DSA in the first year after transplantation, culminating in significant PI progression in the DSA II+ group. This suggests that the mechanism of early lumen loss in these patients may be due to a combination of accelerated rate of intimal hyperplasia and adverse vessel remodeling (28). The presence of CAV assessed by subsequent routine coronary angiography validates our IVUS findings. Interestingly, CAV occurs irrespective whether patients had low or high class II DSA values and thus may question the strategy of transplant centers in using higher anti-HLA antibody values as a cutoff to list unacceptable antigens for HTX recipients if the goal is to prevent CAV.
Patients were not typed for HLA-DPB1, although DP antibodies can be important; hence, we may have underestimated the influence of class II DSAs.
In our preliminary study, HTX recipients with pretransplantation class II DSA compared to DSA− recipients have an accelerated rate of coronary plaque progression as assessed by three-dimensional IVUS and increased development of angiographic CAV. Larger, multicenter observational studies will be required to validate these observations.
MATERIALS AND METHODS
The study was a nonrandomized, retrospective, single-center study approved by the Mayo Clinic institutional review board. From January 2006 to December 2009, 104 patients underwent transplantation in our institution, from which we identified 40 HTX recipients in whom anti-HLA antibodies were determined before transplantation using single-antigen bead (SAB) assay (One Lambda, Inc, Canoga Park, CA; Luminex platform [Luminex Corp, Austin, TX]) and had baseline and a 1-year follow-up IVUS study as part of a standard surveillance protocol. An additional 11 randomly chosen patients, who underwent transplantation between January 2004 and January 2006, had sera available for SAB assay, and had baseline and 1-year follow-up IVUS study performed, were included in the study. Primary immunosuppressive agents (CNI or SRL) and secondary immunosuppressive agents mycophenolate mofetil (MMF) and azathioprine (AZA), and the dose of prednisone was not modified based on the presence or absence of DSA. All HTX recipients received induction therapy with antithymocyte globulin (ATG), as part of a standard induction protocol. Immunosuppression was managed and dosed as previously described (26). Three patients in the DSA II+ group, who had a positive flow cytometric crossmatch, were treated with plasmapheresis immediately after transplantation. Routine endomyocardial biopsies were performed weekly for 6 weeks after transplantation, beginning a week after completing induction therapy, every 2 weeks from 6 weeks to 3 months, monthly from 3 to 6 months, and then at 3-month intervals until the end of the second year as well as 10 to 15 days after any biopsy specimens that showed a rejection of grade 2R or higher. Total rejection score was calculated for each patient. Each biopsy result was graded and assigned based on the International Society for Heart and Lung Transplantation (ISHLT) R grading as follows: 0R = 0, 1R = 1, 2R = 2, and 3R = 3. Total rejection score was calculated by dividing the cumulative scores for all the biopsies by the total number of biopsy specimens taken until the time of the last IVUS evaluation. Any rejection score was calculated as 0R = 0, 1R = 1, 2R = 1, and 3R = 1 and represented the total number of rejections, regardless of severity, experienced by that patient during the same period normalized for the total number of biopsy specimens.
Coronary Angiography and IVUS Examination and Analysis
Cardiac allograft vasculopathy was categorized using recent ISHLT guidelines (8).
Intravascular ultrasound was performed at baseline (0.30 years [range, 0.17–1.15 years] after transplantation) and 0.93 years (range, 0.83–1.05 years) after the baseline examination.
The method for performing IVUS has been described elsewhere (26, 30). Briefly, IVUS was performed during routine coronary angiography after intracoronary administration of 100 to 200 μg of nitroglycerin. Mechanical pullback (0.5 mm/s) was performed from the mid to distal left anterior descending coronary artery to the left main coronary artery with a 20-MHz, 2.9 French, monorail, electronic Eagle Eye Gold IVUS imaging catheter (Volcano Therapeutics, Inc, Rancho Cordova, CA) and a dedicated IVUS scanner (Volcano Therapeutics). The IVUS images were stored on a CD-ROM for later offline three-dimensional volumetric IVUS analysis. Offline volumetric analysis of IVUS data was performed (echo Plaque 2, version 2.5; INDECSystems, Inc, Santa Clara, CA) by operators who were unaware of group assignment. The Simpson rule for volumetric measurement was used. Proximal and mid left anterior descending coronary artery regions were defined for the interrogated artery. Starting with the first complete vascular ring distal to the bifurcation with the left circumflex artery lumen, plaque and vessel volumes were analyzed. Each measured volume was normalized to the examined segment length (mm3/mm) to compensate for differences in examined vessel segment length. A PI was calculated as follows: (PV/vessel volume), where PV is plaque volume. Changes in PV, lumen volume, and vessel volume or PI were defined as last follow-up minus baseline volume measures value and as percent change. The semiautomated contour detection of both the lumen and the media-adventitia interface was performed at intervals of either 16 or 32 frames, depending on the heterogeneity of the image. All other measurements were carried out automatically. Border detection was corrected manually in all frames after automatic border detection. An example of an IVUS study defining each of the measurements is shown in Figure 2.
Donor HLA Class I and Class II Typing
Low-resolution class I HLA-A, B, Cw, and class II HLA-DRB1, DQB1, typing was performed using the LABType SSO (One Lambda), which uses sequence-specific oligonucleotide probes bound to fluorescently coded microspheres using the LABScan 100 flow analyzer (Luminex). The assignment of the HLA typing is based on the reaction pattern compared to patterns associated with published HLA gene sequences using the HLA-Visual software (One Lambda).
SAB Analysis of Alloantibody
Pretransplantation sera were screened for HLA-Abs using a panel of up to 100 different color-coded beads each coated with purified single HLA class I and class II antigens (LABScreen Single Antigen Beads; One Lambda) using Luminex-based technology. Donor typing performed was at low to medium resolution, and serologic equivalents were reported. In cases with more than one allele, if only one bead was positive, whereas the other was negative, based on the low- to medium-resolution typing and considering the common well-defined allele, the bead that would correspond to the donor type was considered as DSA. For DQ, because the beads have different DQA1 and DQB1 combinations, in cases where DQA1 donor typing was available, beads representing the donor DQA1-DQB1 combination were considered to define the DSA. In cases where DQA1 typing was not available, the common DRB1-DQA1-DQB1 haplotypes were considered to define a positive DSA. For example, if the donor typed as DR17-DQ2, DQA1*05-DQB1*02:01 bead was considered as the DSA. However, in this study, we did not find any DSA with only DQA1 specificity. One to eight dilutions were performed on highly sensitized individuals to rule out interfering substances. In addition, commercial products were used when the negative control bead had a very high MFI value. The results were expressed as MFI value for each HLA-Abs, which corresponds with the strength of the antibody: the higher the MFI, the stronger the reaction. Donor-specific antibodies were defined as HLA-Abs to the HLA antigens shared by the donor and were defined at serological equivalent levels. An MFI value greater than 300 was defined as a positive result for the presence of DSA. The highest level of MFI for HLA class I and II DSA was then used to classify patients by SAB DSA level.
Flow Cytometric Crossmatch
Three-color FCXM was performed for 60% (31/51) of patients before transplantation (FACSCalibur, BD Biosciences, San Jose, CA). Alloantibody binding was assessed using fluorescein isothiocyanate–conjugated F(ab)′2 goat anti-human IgG serum by way of indirect immunofluorescence, and three fluorescence parameters (CD3, CD19) were used to identify T and B cells. The interpretation of the FCXM was compared by directly comparing the fluorescence intensity of the donor T and B lymphocytes after treatment with patient serum to the intensity of donor cells after treatment with a negative control serum using a 1024 scale. A median channel fluorescence shift greater than 52 and greater than 106 was interpreted as positive for T cells and B cells, respectively.
Continuous parameters were presented as means (SD) and compared using the Student t test. Ordinal data were presented by median and first and third quartiles and were compared using the exact nonparametric Wilcoxon rank sum test. Differences from baseline to the follow-up IVUS examinations were compared by use of a paired t test. Intravascular ultrasound values at end of follow-up between groups were compared by analysis of covariance, with the baseline value of the term included in the analysis as a covariate. Categorical data were compared between groups using the chi-square or the Fisher exact test whenever the expected value in one of the cells was less than 5. The time to onset of CAV distributions was calculated from the time of transplant according to the Kaplan-Meier method and compared by means of the log-rank test. Time to onset of CAV was defined as the time of diagnosis of ISHLT CAV grade 1 (mild) categorized using the recent ISHLT guidelines. All P values were two-sided, and values less than 0.05 were considered to indicate statistical significance. All data were analyzed with the JMP System software version 8.0 (SAS Institute, Inc, Cary, NC).
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