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Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

Background: It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection., Methods: In the present study, we searched for markers that would exclusively designate these high-avidity CTL., Results: FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression., Conclusion: Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.

Background: To determine mechanisms that trigger graft vascular disease (GVD) after heart transplantation, we studied parameters that reflect both early and late intragraft allogeneic reactions., Method: With reverse transcriptase-polymerase chain reaction analysis, mRNA expression of interleukin-2 (IL-2), interleukin-4, interleukin-6, interleukin-10, interferon-gamma, platelet-derived growth factor-alpha, and transforming growth factor-beta was measured in endomyocardial biopsy (EMB) specimens obtained from 34 recipients during the first acute rejection episode (n = 29) or at a comparable time after transplantation for patients without rejection (n = 5) and at time of assessment of GVD by coronary angiography at 1 year (n = 34)., Results: At the time of assessment of GVD, mRNA expression of IL-2, interleukin-4, and interleukin-6 were barely detectable, whereas messenger coding for interferon-gamma, interleukin-10, transforming growth factor-beta, and platelet-derived growth factor-alpha genes were constitutively expressed. Moreover, intragraft mRNA patterns of cytokines and growth factors between patients with GVD (n = 17) or without GVD (n = 17) were comparable. In contrast, during the first acute rejection episode a completely different pattern was found. Development of GVD was associated with IL-2 mRNA expression and not with the other cytokines analyzed. IL-2 mRNA was present in 77% of rejection EMB specimens obtained from patients with GVD versus 33% of the EMB specimens obtained from patients without GVD (p = 0.03) and not detectable in EMB specimens obtained from patients with no rejection. Also nonimmunologic risk factors such as longer ischemia time (median 193 vs 141 minutes; p = 0.002) and higher donor age (median 32 vs 23 years; p = 0.02) were associated with GVD. But no relation was found between these nonimmunologic risk factors and IL-2-positive acute rejections., Conclusions: Nonspecific risk factors and IL-2-positive rejections may independently trigger GVD after clinical heart transplantation.

Background: Anti-interleukin (IL)-2 receptor (IL-2R) antibodies have been used as rejection prophylaxis after organ transplantation. Despite this induction treatment, acute rejections may occur. We wondered whether these rejections developed via the IL-2/IL-2R pathway., Methods: In a prospective trial using BT563, a murine IgG1 anti-IL-2R antibody, for rejection prophylaxis after heart transplantation, 20 patients were treated in combination with cyclosporine from the day of transplantation (group A). As a control group, 31 patients were also treated with BT563, but in these patients, cyclosporine treatment was initiated on day 3 (group B)., Results: Three patients from group A and two patients from group B died in the first postoperative month (of causes not related to acute rejection) and were left out of the analysis of rejection incidence. Freedom from acute rejection at 1 week after transplantation in group A (14/17; 82%) was lower than in group B (16/29; 55%), although the difference did not reach statistical significance. There was no difference in either the number of acute rejection episodes at 12 weeks or the required rejection treatments between groups A and B. Infectious complications were evenly distributed in both groups. Immunohistochemistry showed that during acute rejection, in the presence of circulating BT563, IL-2R-bearing cells were present in only one of five rejection biopsies (20%), whereas these cells were often present (6/8, or 75%) in rejections occurring in the absence of BT563. The presence of BT563 was associated with a similar difference in the mRNA expression of IL-2 (2/5 vs. 6/8)., Conclusions: Apparently, despite adequate blockade of the IL-2/IL-2R pathway, patients may develop acute rejection, reflecting the redundancy of the cytokine network. The ever-present IL-15 may well be a candidate for overtaking the role of IL-2.

Histological examination of endomyocardial biopsy (EMB) is the main technique for rejection surveillance after heart transplantation. This technique is elaborate, inconvenient for the patient, and not without complications. We prospectively analyzed whether the measurement of C-reactive protein (CRP), an acute phase protein that quickly rises when there is inflammation, can serve as a marker for immunological quiescence and as an indicator for withholding EMB. During a 6-month period, CRP was measured in all patients referred for EMB as part of the routine follow-up after heart transplantation. Acute rejection in patients with a follow-up of more than 1 year was rare (1/76). In the majority of cases, EMB was taken within the 1-year post-transplantation (170/246 = 69%). In 71/170 biopsies (42%), CRP was < or = 1; in the other 99/170 (58%), CRP was > or = 2. When CRP was < or = 1, acute rejection was diagnosed in 12/70 cases (17%). In contrast, acute rejection was found in 28/99 cases (28%) with CRP > or = 2 (P = 0.1). Although CRP is elevated more often in the presence of acute rejection, its sensitivity does not allow CRP to replace the routine performance of EMB for monitoring rejection after heart transplantation. We did, however, find a prognostic significance with regard to the effect of rejection treatment: in all acute rejections with a CRP < or = 3 (n = 11), steroids were effective.

We used reverse transcriptase-polymerase chain reaction analysis to study the effects of anti-rejection prophylaxis with an anti-interleukin (IL)-2 receptor (IL-2R) monoclonal antibody (BT563) on the allogeneic process by analyzing intragraft IL-2, IL-4, and IL-15 mRNA expression. Analysis showed an association between rejection and intragraft IL-2 mRNA and IL-4 mRNA transcription, whereas IL-15 was constitutively expressed: IL-2, 62% (8/13) during rejection versus 23% (8/35) during immunological quiescence (P < 0.01); IL-4, 69% versus 23% (P < 0.01). BT563 therapy influenced the intragraft mRNA expression of IL-2 and IL-4 but not of IL-15. In endomyocardial biopsies (EMB) showing rejection, mRNA expression of IL-2 was detectable in 40% (2/5) during BT563 treatment versus 75% (6/8) in the absence of BT563; for IL-4, 23% versus 88%, respectively. In contrast, IL-15 mRNA transcription was not affected. Quantitative analysis in rejection EMB showed comparable IL-15 mRNA levels during and after BT563 treatment. This study demonstrates that therapeutic intervention within the IL-2-dependent T-cell activation cascade does not completely prevent rejection. Other cytokines, such as IL-15, may participate in IL-2-independent rejections.

Background: Treatment policy of acute rejection after heart transplantation has been changed after adopting the ISHLT endomyocardial biopsy grading system in 1991., Objective: To determine the effect of this policy change on clinical outcome after transplantation., Methods: The outcome of 147 patients who had a transplant before (early group, median follow up 96 months) and 114 patients who had a transplant after (late group, median follow up 41 months) the introduction of the ISHLT biopsy grading system was studied retrospectively. Initially "moderate rejection" according to Billingham's conventional criteria was treated. From January 1991 grade 3A and higher was considered to require intensification of immunosuppression., Results: There were some differences between the two groups: recipients (50 v 44 years) as well as donors (28 v 24 years) were older in the "late group" and more patients of this group received early anti-T cell prophylaxis (92% v 56%). Despite more extensive use of early prophylaxis more rejection episodes were diagnosed (2.4 v 1.4) and considerably more courses of rejection treatment were instituted in the late compared with the early group (3.2 v 1.5). There were no deaths because of rejection in the late group, however, more infections occurred within the first year (mean 1.8 v 1.4) and more non-skin malignancies within the first 41 months were diagnosed (8 of 57 v 6 of 147, 95% CIs of difference includes 0). The incidence of graft vascular disease in the late group has been comparable to the early group until now., Conclusion: The interpretation of the ISHLT grading system resulted in lowering of the threshold for the diagnosis of rejection thereby increasing the number of rejections and subsequently the immunosuppressive load and its complications.

During cardiac rejection we studied the kinetics of IL-2 and IL-4 mRNA and subsequent protein production by in vivo primed graft-infiltrating lymphocytes (GIL), using semiquantitative RT-PCR and ELISA. Following in vitro stimulation with either donor or third-party antigens, mRNA expression of IL-2 and IL-4 were already detectable 1-2 h after stimulation, while their protein production could be measured from 4 h onwards at least until 48 h. At both the mRNA and protein level, we measured a donor-specific signal for IL-2 and for IL-4 production (p = 0.02), while the relative donor-specific IL-2 mRNA level was significantly higher than the relative IL-4 mRNA level (p = 0.002). These observations suggest that after in vitro challenge with donor antigens, GIL obtained from rejecting cardiac allografts predominantly produce IL-2 mRNA and protein.

Background: To analyze the relevance of CD4-positive cytotoxic T-lymphocytes (CTL) in clinical heart rejection, we studied the frequency and avidity of donor human leukocyte antigen class II-specific CTL present within the graft during a rejection episode and during a period without rejection., Methods: For this analysis endomyocardial biopsies of heart transplant recipients were cultured to obtain graft-infiltrating lymphocytes (GIL). GIL cultures exhibiting donor class II-directed cytotoxicity were considered for this study. With limiting dilution analysis, the frequency of donor class II-specific CTL that had been primed by donor antigens in vivo (designated cCTL) was determined in GIL cultures established from endomyocardial biopsies taken during a rejection episode (n = 10) or during a period without rejection (n = 11). Addition of anti-CD4 to the limiting dilution analysis revealed the fraction of donor class II-specific cCTL having a high avidity for donor antigen., Results: During a rejection episode, 196 (median) donor class II-specific cCTL/106 GIL were present. In a period without rejection, the frequency of donor class II-specific cCTL was not significantly different (median = 330/10(6); p = 0.1). Addition of anti-CD4, however, revealed that donor class II-specific cCTL with a high avidity for donor antigen are predominant during a rejection episode (median = 100%) but are in minority during a period without rejection (median = 35%; p < 0.0001)., Conclusions: These results suggest that graft-infiltrating CD4+ CTL can mediate heart rejection provided they have a high avidity for donor antigen.

Background: We studied the effect of antirejection therapy on intragraft cytokine mRNA expression., Methods: Therapy consisted of three doses of 1 gm of intravenous methylprednisolone. We determined its effect on intragraft mRNA expression of immunoregulatory (interleukin-2, interleukin-4) and inflammatory cytokines (interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha), and the high-affinity interleukin-2 receptor (p55 chain) in endomyocardial biopsy specimens from cardiac allograft recipients., Results: By reverse-transcriptase polymerase chain reaction methods, we detected mRNA transcription for interleukin-2 in 56% of the pretreatment endomyocardial biopsy specimens (n = 16), for interleukin-4 in 31%, and for interleukin-6 in 56% of the specimens, and interleukin-2 receptor, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha were constitutively expressed. Individual cytokine mRNA profiles were not helpful in differentiating between rejections that proved to be methylprednisolone resistant (n = 9) or methylprednisolone sensitive (n = 7). After successful antirejection therapy, the overall intragraft mRNA expression was downregulated. None of the posttreatment endomyocardial biopsy specimens taken from six patients with methylprednisolone-sensitive rejections expressed the interleukin-2 gene, in contrast to 88% of the endomyocardial biopsy specimens obtained from eight patients with methylprednisolone-resistant rejections (p = 0.005). Moreover, intragraft interleukin-4 and interleukin-6 mRNA transcripts were hardly detectable (both 17%) in methylprednisolone-reversible rejections, but in ongoing rejections interleukin-4 mRNA expression was found in 62% (p = 0.14), and interleukin-6 was found in 88% of the endomyocardial biopsy specimens (p = 0.03). Semiquantitative analysis showed that the intragraft interleukin-2 receptor, interleukin-1 beta, and tumor necrosis factor-alpha mRNA levels were lower in posttreatment endomyocardial biopsy specimens from methylprednisolone-reversible rejections than in endomyocardial biopsy specimens from methylprednisolone-irreversible rejections (p = 0.03)., Conclusions: Our data suggest that the efficacy of antirejection therapy with methylprednisolone is reflected in intragraft cytokine mRNA profiles.

Acute rejection (AR) and graft vascular disease (GDV) are processes mediated, at least in part, by cellular processes. Therefore, we cultured graft-infiltrating lymphocytes (GIL) from endomyocardial biopsies (EMB) taken during the first year after transplantation, determined their phenotypic composition, and correlated it to AR and GVD. We observed more often GIL growth from EMB with AR than from non-rejection EMB (P = 0.02), but no difference was found between patients with and without GVD 1 year after transplantation. CD4+ cells were always more numerous than CD8+ cells, and no difference in phenotypic composition was found between AR and non-rejection EMB nor between EMB derived from patients with or without signs of GVD. In conclusion, AR is correlated with cell growth of EMB, but the development of GVD is not associated with AR, GIL growth from EMB, or their phenotypic composition.

Monitoring for the responses to alloantigens presented either by the direct or the indirect presentation pathway have been reported to be of clinical value after kidney transplantation. Amongst others, the level of these responses may be dependent on the immunosuppressive treatment. We studied both presentation routes in peripheral blood mononuclear cells (PBMC) of cardiac transplant patients, who experienced episodes of rejection, and related them to the in vivo cyclosporin A (CsA) levels in plasma. PBMC of the recipients were stimulated with irradiated donor cells to determine the direct presentation pathway. As a method for the activation of the immune response via the indirect pathway, PBMC were stimulated with tetanus toxoid. Both immune responses increased when CsA levels inadvertently decreased to inadequate concentrations and histological rejection was diagnosed. After clinical heart transplantation, CsA may prevent rejection by blocking both the direct and the indirect antigen presentation pathway.

Background: Anti-interleukin-2 receptor monoclonal antibodies have been used successfully in the prevention of rejection in cardiac allografts in several animal models., Methods: In an open randomized study murine monoclonal CD3 antibody and BT563, a murine anti-interleukin-2 receptor monoclonal antibody, were given as rejection prophylaxis during the first week after heart transplantation. Cyclosporine therapy was initiated at the third postoperative day., Results: In half the BT563-treated patients an early rejection was histologically shown at week 1, whereas heart transplant recipients treated with murine monoclonal CD3 antibody had a rejection incidence at week 1 of only 9%. During BT563 treatment CD25-positive cells (i.e., cells bearing the interleukin-2 receptor) were not detectable in peripheral blood. However, immunohistologic studies of endomyocardial biopsy specimens taken 1 week after transplantation showed the presence of CD25-positive cells within these specimens in 8 of 10 (80%) of patients with rejection. In patients without rejection CD25-positive cells were present in the biopsy specimens of only two of nine patients (22%). Reverse-transcriptase polymerase chain reaction studies on biopsy material showed the presence of messenger RNA for the interleukin-2 receptor in all and for interleukin-2 in three of five (60%) of biopsy specimens of rejecting grafts., Conclusions: Although CD25-positive cells were not detectable in peripheral blood during BT563 treatment, these cells were at the same time found to be present within 80% of the endomyocardial biopsy specimens from the rejecting grafts. By initiating cyclosporine treatment at day 0, the synergistic effect of combining cyclosporine and anti-interleukin-2 receptor monoclonal antibodies may result in a lower rejection incidence.

Long-term survival of heart transplant recipients is limited by the development of transplant coronary artery disease (TCAD). We analysed whether the development of TCAD is correlated with the incidence of acute rejection episodes, with the formation of anti-HLA antibodies or with the composition and function of T lymphocyte cultures derived from endomyocardial biopsies. TCAD was assessed by visual analysis of annually performed coronary angiograms and defined as the presence of all vascular changes, including minor wall irregularities. One year after transplantation, 31 of the 77 patients studied had TCAD (40%). The median age and mean number of HLA mismatches in patients with or without TCAD were highly comparable. The patient groups did not differ in incidence of acute rejection episodes, nor in percentage of endomyocardial biopsies yielding T cell cultures. At 1 year after transplantation, lymphocyte cultures from 18/31 TCAD+ patients (58%) and 27/46 TCAD- patients (57%) were analysed. The TCAD+ patients had, compared with the TCAD- patients, a higher median percentage of CD8+ T cells (71% versus 25%, P = 0.06) and a lower median percentage of CD4+ T cells (4% versus 40%, P = 0.04). Similar differences were found in a longitudinal analysis of the culture results of endomyocardial biopsies (EMBs) obtained during the first year. The cytotoxic reactivity of the cultures against donor HLA class I or class II antigens was comparable in the two groups, although a difference in recognition of heart specific antigens remains possible. The fact that EMB-derived cultures from TCAD+ and TCAD- patients differed in T cell phenotype populations gives some support to the hypothesis that cellular immunological processes are involved in the development of TCAD. However, while the median values differed, the overlap of the percentages of CD8+ cells in cultures from TCAD- and TCAD+ patients shows that other factors besides CD8+ T cells also play a role.

The immune response to an allograft is regulated by cytokines produced by cells infiltrating the allograft. However, the immunopathogenesis of allograft rejection is not completely understood. To investigate the role of cytokines after clinical heart transplantation, we analysed the expression of cytokine genes in sequentially taken endomyocardial biopsies (EMB) by using the reverse transcriptase-polymerase chain reaction (RT-PCR). We analysed 44 EMB from 11 recipients: 21 EMB before or during rejection, and 23 EMB without histological evidence of acute rejection. A strong correlation was found between IL-2 gene expression and histologically proved rejection (16/21 versus 1/23 without rejection, P < 0.001; chi 2 test). Also, expression of IL-4 and IL-6 genes was more often found in EMB during rejection than in EMB without signs of rejection (IL-4, 62% versus 35%; and IL-6, 81% versus 39%, respectively). No relation with rejection or with immunological quiescence was observed for the presence of IL-10 gene transcripts. IL-10, but also IL-6 mRNA were detectable in donor heart tissue before transplantation (9/10). In contrast, IL-2 and IL-4 gene transcripts were absent in these samples. These differences could not be explained by the presence or absence of T cells, since the gene for the constant region of the beta-chain (C beta) of the T cell receptor (TCR) not only was expressed in post-transplant EMB but also in pre-transplant donor heart tissue. Our results provide strong evidence that the immunoregulatory cytokines IL-2, IL-4 and IL-6 are important local regulators in the graft during acute rejection. The role of IL-10 in the immunologic response to the transplanted organ needs further investigation.

The value of pulsed-wave transmitral Doppler for the diagnosis of moderate acute rejection was examined in a total of 347 Doppler recordings obtained in 32 consecutive cardiac allograft recipients. Serial Doppler examinations (median, 11 per patient; range, 1 to 23) were performed simultaneously with endomyocardial biopsies from the first week after heart transplantation to a follow-up of 186 days (median; range, 10 to 395 days after transplantation). Pulsed-wave transmitral Doppler did not allow noninvasive diagnosis of moderate acute rejection in individual patients. Peak filling rate normalized for mitral stroke volume, early diastolic velocity, and mean diastolic velocity were significantly increased, whereas diastolic filling period was decreased during moderate acute rejection compared to other biopsy classes. The wide overlap of measurements in individual recipients with or without rejection may be due, however, to a variety of hemodynamic factors after transplantation affecting diastolic function, which are superimposed on the restrictive left ventricular filling pattern caused by persistent mild acute rejection and left ventricular hypertrophy. These hemodynamic factors include pulmonary hypertension, perioperative ischemia, reperfusion injury, and changes in both blood pressure and loading conditions caused by hypertension and its treatment. Differences between studies with regard to the detection of moderate acute rejection by transmitral Doppler may be caused by chance, because most studies were relatively small. Differences in methods, patient selection, duration of follow-up, prevalence of hypertension and left ventricular hypertrophy, and differences in antihypertensive drug regimens may also play a role. Furthermore differences in the incidence of mild acute rejection, its treatment, and the type of maintenance immunosuppressive regimen used may have influenced the outcome of these studies considerably.

In vivo-activated, committed donor-specific cytotoxic lymphocytes (cCTL) can be propagated and expanded from endomyocardial biopsies (EMB) in IL-2-enriched medium especially during an acute rejection episode. We report here our efforts to detect these cCTL by the same technique in peripheral blood at the moment of rejection and when no rejection was diagnosed. During or just before rejection, significantly less frequent (P less than 0.01) donor reactive cCTL were found in PBL samples (two out of 20) than in the simultaneously taken EMB samples (13 out of 19). Donor B-LCL and/or third-party B-LCL were lysed by 15 PBL samples. Inhibition studies revealed that this lysis was due to LAK-like cytotoxicity. The results show that peripheral blood does not reflect intra-graft events, which is probably the reason for the irreproducible results of diagnosis of rejection by monitoring immunological parameters in the peripheral blood.

Chronic rejection (CR) is a major problem in long-term survival in heart transplantation. We analysed whether the occurrence of CR correlates with the incidence of acute rejections (AR) or with characteristics of endomyocardial biopsy-derived cell cultures. CR was diagnosed by annual angiography and defined as all coronary vascular changes. One year after transplantation 24 of the 63 patients had CR (38%). The incidence of AR in CR+ and CR- patients was comparable. The patients in both groups had similar individual median percentages of EMB-yielding cell cultures. During the first year the CR- patients had more cultures in which at least 60% of the cells were CD4+ T cells (50% vs 37%, P = 0.05), due to a stronger CD4 predominance in the first 6 months. In the second year the CD4 predominance in the patients diagnosed as CR+ after 1 year tended to be higher (P = 0.08). The patients had comparable percentages of cultures predominated by CD8+ T cells, gammadelta T cells or NK cells, irrespective of the time interval. These results might indicate that CD4+ T lymphocytes play a dual role in the aetiology of CR.

Recent studies comparing the effects of induction therapy with polyclonal antilymphocyte globulins (ALG) or with monoclonal T-cell-specific antibodies are not unanimous. Therefore, 55 heart recipients were allocated to either 7-day courses of polyclonal ALG (n = 28) or of monoclonal OKT3 (n = 27). Additionally, azathioprine and low dose steroids were given. There were no severe side effects after OKT3; the course of ALG, however, had to be discontinued in 20 patients because of extensive flares. No differences between the two groups were found in freedom from rejection or in the incidence of infection. The 1- and 2-year survival was 96% in both groups. Although monoclonal and polyclonal induction therapies are equally effective for rejection prophylaxis, OKT3 may be preferred because of a lack of important side effects. However, the fact that a shorter course of ALG is equally effective may be in favour of ALG.

In a consecutive series of 146 kidney transplant recipients treated with cyclosporin A a strong correlation between matching for the HLA-A, HLA-B, and HLA-DR loci specificities and outcome of the grafts was observed in male recipients with non-O blood groups. Such a beneficial effect of matching was not found in female patients or male patients with blood group O. In these patients survival of the grafts at one year was good irrespective of the number of HLA-A, B, and DR mismatches. Also in 47 male heart transplant recipients immune responsiveness against mismatched HLA antigens was related to blood group. A significantly higher incidence of rejection episodes was observed in male patients with non-O blood groups (n = 32) than in those with blood group O (n = 15). Matching for HLA-DR reduced the number of acute rejection episodes in male patients with non-O blood. These findings may help explain the controversial reports about the importance of HLA matching in organ transplantation. Furthermore, as most candidates for heart transplantation are male and not of blood group O, the higher incidence of graft rejection in these patients underscores the need for an exchange strategy of donor hearts.