Association Between HLA-G Expression and C4d Staining in Cardiac Transplantation

Abstract

After heart transplantation, allograft survival is often limited by rejection and vasculopathy. Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex-I protein expressed primarily by fetal cytotrophoblasts during early pregnancy, and it plays an essential role in maintaining maternal-fetal immune tolerance (1). Previous studies have demonstrated that HLA-G expression postheart transplant seems to confer protection against acute cellular rejection and cardiac allograft vasculopathy (2,3), possibly through inhibition of immune responses directed against the allograft. However, the relationship between HLA-G expression and antibody-mediated rejection remains unknown. Indeed, humoral rejection posttransplant has been linked to poor clinical outcomes (4). Therefore, this preliminary investigation was designed to assess the association between soluble HLA-G (sHLA-G) expression and C4d staining in heart transplant patients, because this is a routinely used technique in characterizing antibody-mediated rejection (5). Patients who had undergone heart transplantation at our center were enrolled in this study, which was approved by the research ethics board. Blood samples were collected in EDTA-containing tubes during routine endomyocardial biopsy procedures, stored at 4°C, and centrifuged for 20 min. Plasma was isolated, flash frozen, and stored at −80°C within 4 hr of blood collection until further analysis. Soluble HLA-G levels were detected by an ELISA specific for all HLA-G isoforms and quantified according to a purified protein standard curve, as previously described (6). All patients were followed up after approximately 6 months to monitor changes in HLA-G levels. Clinically significant acute cellular rejection episodes (International Society for Heart and Lung Transplantation grade ≥2R) and C4d expression within 1 year of HLA-G measurement were assessed by a cardiac pathologist blinded to the clinical information and HLA-G status of study patients. This investigation included 50 patients (40 men and 10 women) with a mean age of 48±12 years (range 23–64 years), who received a heart transplant between 1986 and 2008. Among study patients, 34 were HLA-G+ (≥100 ng/mL), whereas 16 were HLA-G− (<100 ng/mL). Six-month follow-up revealed no changes in HLA-G status among all patients. All patients had normal left ventricular function by echocardiogram at the time of HLA-G determination. There were no significant differences in demographic characteristics, pretransplant conditions, time of HLA-G measurement posttransplant, or immunosuppressive therapy between groups (Table 1). However, only 13% of HLA-G+ patients suffered clinically significant acute cellular rejection (International Society for Heart and Lung Transplantation grade ≥2R) compared with 63% HLA-G− patients (P<0.01), 1 year before and after HLA-G determination. Interestingly, the prevalence of patients who had biopsies with C4d deposition was also significantly lower in the HLA-G+ (13%) versus the HLA-G− (38%) group (P=0.03), within 1 year of the HLA-G measurement.TABLE 1: Characteristics of HLA-G+ and HLA-G− patientsEndomyocardial biopsy procedures for acute rejection episodes and coronary angiography or intravascular ultrasound for graft vasculopathy are used to assess the status of the allograft. Molecular and genetic markers may represent new ways to monitor the graft, which could minimize the potential complications of such invasive techniques. Our findings illustrate that sHLA-G expression seems protective against clinically significant acute cellular rejection. For the first time, we also show a negative association between sHLA-G levels and C4d staining, suggesting inhibition of the humoral response. Evidence indicates that myocardial HLA-G expression postheart transplant protects against coronary artery disease (2). We have also previously demonstrated that myocardial HLA-G expression reliably indicates a low risk of acute cellular rejection in patients from our center (3). This study suggests that sHLA-G expression is negatively linked to C4d staining in addition to acute cellular rejection. Because both forms of rejection contribute to graft vascular disease, sHLA-G expression could aid in identifying the immunologic risk of heart transplant recipients. Hence, further investigation between HLA-G expression and humoral rejection may be warranted through larger studies. Rohit Sheshgiri Vivek Rao Amelia Mociornita Heather J. Ross Delgado H. Delgado Heart Transplant Program Peter Munk Cardiac Centre Toronto General Hospital University Health Network Toronto, Canada