Clinical Antibody-mediated Rejection Research Articles

Cardiac magnetic resonance T1 mapping pre and post contrast in heart transplant patients with clinical antibody-mediated rejection: a preliminary experience

Background Antibody-mediated rejection (AMR) is characterized by histopathological and immunophenotypic findings such as activated endothelial cells, intravascular macrophages and evidence of capillary C4d deposition. This inflammatory reaction could be followed by diffuse fibrosis. Cardiac magnetic resonance (CMR) with recently T1 mapping is a promising technique to identify diffuse myocardial fibrosis. The purpose of this study was to assess T1 mapping in patients with AMR. Methods Two patients with clinical AMR (histopathological and immunophenotypic findings, presence of donor-specific allo antibodies and allograft dysfunction) performed a CMR study one week (for the first patient) and 3 weeks (for the second patient) after the treatment of AMR (plasmapheresis, IV Immunoglobulins and Rituximab). Images were acquired on a 1.5 Tesla scanner (Siemens) including T1 mapping using a shortened modified look-locker inversion-recovery sequence and T2 mapping in a matched mid-ventricular short axis slice using a black- blood single shot fast spin echo pulse sequence. Segmental and global T1 values were measured before and 15 minutes after administration of 0.2 mmol/kg of Gadoteric acid and compared to our cohort of 17 controls. Results Mean non contrast T1 values were significantly higher in heart transplants patients compared to controls (1100 ± 5 ms vs 947 ± 29 ms, P < 0.001). Segmental T1 values were significantly higher in the 6 regions of interest compared to controls (P < 0.001 in all segments). Mean post contrast T1 values were not significantly different in patients and controls. Mean T2 value was higher in patients compared to controls (73 ± 13 vs 50 ± 4 ms), suggesting the presence of global edema. Conclusions Heart transplant patients with clinical antibodymediated rejection show a significant increased global and segmental non contrast T1 values suggesting the presence of diffuse myocardial fibrosis. Further studies are required to confirm these data. Funding

Predictive Value of HLA Antibody Characteristics in Progression of Subclinical Antibody Mediated Rejection in Heart Transplant Recipients

Purpose Donor specific antibody (DSA) is a risk factor for antibody-mediated rejection (AMR). Concomitant detection of C4d & C3d in heart biopsies correlates with DSA and allograft dysfunction. Detection of C4d only in surveillance biopsies may not be associated with allograft dysfunction. However, conversion from C4d only to C4d + C3d is noted in ~20% of cases (C4d converters). We studied DSA patterns in clinical (C4d+C3d+ & C4d converters) & subclinical (C4d+) AMR. Methods and Materials Of 1589 transplants, 610 were screened for AMR (last 6 yr). We studied DSA patterns in C4d+ asymptomatic patients, C4d+C3d+ biopsies symptomatic patients & patients who started as C4d+ & converted to C4d+C3d+. We also compared cPRA & crossmatches. Results Significant differences in DSA incidence, mean cPRA, & DSA mean MFI were noted across all groups with C4d+ patients having the lowest values ( table 1 ). DSA mean MFI was significantly higher in patients with clinical AMR ( table 1 ). C4d converters had significantly higher mean cPRA & DSA, driven by higher DQ DSA but not class I DSA compared to patients who remained C4d+ ( Table 2 ). Having any DSA, DQ DSA or cPRA≥50% at time of biopsy was associated with C4d conversion with high -ve predictive value (NPV) but low +ve predictive value (PPV). Conclusions Our data suggest that HLA antibody characteristics may be relevant in determining which subclinical C4d+ may not progress to clinical AMR & warrants investigation in larger cohorts. Table 1 Any DSA DSA Cl I DSA Cl II DSA Cl I +II mean ± SE cPRA mean ± SE MFI C4d+: n=34 19, 56% 9, 26% 12, 35% 2, 6% 44.8 ± 5.1 7398.5 ± 1525.1 C4d+C3d+: n=30 28, 93% 20, 67% 26, 87% 18, 60% 87.4 ± 5.4 12633.0 ± 1222.8 C4d conv: n=11 11, 100% 4, 36% 10, 91% 3, 27% 81.1 ± 8.9 8378.2 ± 1950.9 p .0003 .005 .022 Table 2 Any DSA DSA Cl I DSA Cl II DR DSA DQ DSA cPRA≥50 C4d+: n=34 19, 56% 9, 26% 12, 35% 3, 9% 11, 32% 16, 47% C4d conv: n=11 11, 100% 4, 36% 10, 91% 6, 55% 9, 82% 10, 91% p .008 .70 .002 .003 .004 .014 PPV .38 .30 .46 .67 .45 .39 NPV 1 .78 .96 .86 .92 .95

Proteasome Inhibitor Therapy for Antibody-Mediated Lung Transplant Rejection

Purpose Antibody mediated rejection (AMR) is a significant challenge after lung transplantation and no consensus exists on the optimal therapeutic approach. Bortezomib may serve as a treatment option by eradicating the antibody-producing plasma cells. We report clinical experience with bortezomib for AMR after lung transplantation. Methods and Materials Five lung transplant recipients with clinical AMR and donor-specific antibodies (DSA) (defined as MFI > 2000) were treated with bortezomib (1.3 mg/m 2 ). Serial measurements of DSA were conducted by single antigen bead on Luminex, OneLambda. The immunodominant DSA (iDSA) was assigned to the DSA with the highest MFI value at the time of diagnosis. Results In this cohort, AMR developed a median of 10.5 months after transplant; bortezomib treatment resulted in elimination of iDSA in 2 patients (1, 2), and a 50% reduction without clearance in another (3). The remaining two patients (4, 5) had persistent DSA without decline in MFI. Despite iDSA reduction in patients 1, 2, and 3, this did not translate to a meaningful clinical benefit (absence of resolution of acute AMR in patient 1). During therapy, one patient experienced grade IV thrombocytopenia; otherwise, side effects were mild and transient. Prior to AMR, all patients had normal graft function; however, all 3 patients surviving to hospital discharge developed chronic, severe graft dysfunction. Two of the 5 patients are still alive 132 (2) and 230 (5) days after treatment for AMR. Conclusions Although these preliminary results do not support a strong clinical or immunological response to bortezomib, we cannot exclude a prescribing bias for use in more severe cases of AMR. Bortezomib treatment for AMR warrants further investigation to fully elucidate its potential benefit. iDSAMFI at DiagnosisMFI at last follow-upResolution of acute AMRPatient 1DQ64516

Prevention and treatment of alloantibody-mediated kidney transplant rejection

Antibody-mediated rejection (AMR), which is commonly caused by preformed and/or de novo HLA alloantibodies, has evolved as a leading cause of early and late kidney allograft injury. In recent years, effective treatment strategies have been established to counteract the deleterious effects of humoral alloreactivity. One major therapeutic challenge is the barrier of a positive pretransplant lymphocytotoxic crossmatch. Several apheresis- and/or IVIG-based protocols have been shown to enable successful crossmatch conversion, including a strategy of peritransplant immunoadsorption for rapid crossmatch conversion immediately before deceased donor transplantation. While such protocols may increase transplant rates and allow for acceptable graft survival, at least in the short-term, it has become evident that, despite intense treatment, many patients still experience clinical or subclinical AMR. This reinforces the need for innovative strategies, such as complementary allocation programs to improve transplant outcomes. For acute AMR, various studies have suggested efficiency of plasmapheresis- or immunoadsorption-based protocols. There is, however, no established treatment for chronic AMR and the development of strategies to reverse or at least halt chronic active rejection remains a big challenge. Major improvements can be expected from studies evaluating innovative therapeutic concepts, such as proteasome inhibition or complement blocking agents.

322 Donor Specific Antibodies (DSA): Can They Predict C4d Staining and Antibody Mediated Rejection (AMR)?

Purpose: The utility of DSA in surveillance for AMR in pediatric heart transplant recipients is controversial. We hypothesize that DSA is predictive of C4d positive biopsy and clinical AMR. Methods and Materials: The records of 59 recipients (median age at transplant 1.9 yrs, range 0.04-17.6 yrs) transplanted between 10/2005 and 10/2010 were retrospectively reviewed for all DSA performed within 24 hours of endomyocardial biopsy (EMB; n 155.) DSA were determined by single antigen solid phase assay. Using a receiver-operating characteristic plot (AUC 0.79), we set our threshold for a DSA to be a median fluorescence intensity (MFI) of 6000. C4d was defined as 25% of endothelial cells stained by immunohistochemical methods in formalinfixed EMB samples. We also examined the association between patients with DSA and the presence of hemodynamically compromising AMR (grade O, C4d positive EMB). Results: The median duration of follow up was 2.7 yrs with an average of 2.6 paired biopsy-DSA determinations per patient. A total of 11 patients had DSA , 6 had AMR, and 11 biopsies were C4d . DSA correlated with C4d (p 0.0001) with a negative predictive value of 96%, positive predictive value of 53%, specificity of 93% and sensitivity of 63%. An association was also found between DSA and AMR (p 0.0005): 5/6 patients with AMR were DSA while 6 other DSA patients never presented with AMR. Conclusions: Using a DSA threshold of 6000 MFI, a high specificity and negative predictive value for C4d staining was observed. DSA was also associated with the presence of AMR. This retrospective review of DSA in pediatric heart recipients justifies a prospective analysis of the predictive value of DSA-based surveillance for AMR.

31 Value of Immunoperoxidase Staining of C3d in the Diagnosis of Antibody Mediated Rejection in Heart Transplant Recipients

Purpose: According to a recent consensus conference on antibody-mediated rejection (AMR), AMR should be defined solely by pathologic criteria. The presence of donor specific antibody (DSA) was not mandated in the AMR definition as DSA were not always detected during AMR episodes. This may be due to DSA being adhered to the donor heart. It is believed that the detection of DSA during AMR episodes might lead to worse outcome. We reviewed our heart transplant patients with AMR to assess this hypothesis. Methods and Materials: We evaluated 53 patients status post heart transplant from 2003 to 2010 with treated AMR within the first year post transplant. Of these, 14 patients had concomitant circulating DSA and 39 patients did not. Outcomes were compared for subsequent 3-year survival, freedom from cardiac allograft vasculopathy (CAV, stenosis 30%), and freedom from non-fatal major adverse cardiac events (NF-MACE: myocardial infarction, heart failure, coronary stenting, need for defibrillator, stroke, and new peripheral vascular disease). Results: Patients who developed treated AMR with and without circulating DSA were found to have similar subsequent 3-year survival (71% vs. 82%, p 0.37), subsequent 3-year freedom from CAV (80% vs. 86%, p 0.62), and freedom from NF-MACE (100% vs. 92%, p 0.29). There was no difference in the incidence of hemodynamic compromise rejection between groups (7% vs. 8%, p 0.96). Conclusions: The finding of DSA in conjunction with pathology confirmed AMR does not appear to impact 3-year outcome after heart transplantation. Further studies to assess strength and other characteristics of DSA during AMR episodes may shed light to define its role in clinical AMR.

Endothelitis in cardiac allograft biopsy specimens: Possible relationship to antibody-mediated rejection

The incidence of endothelitis in cardiac transplants, and the relationship to clinical symptoms and humoral rejection, is unclear. Recently, the finding of intravascular macrophages has been found to represent antibody-mediated rejection. This study investigated the role of intravascular T lymphocytes in antibody-mediated rejection. A total of 819 sequential biopsy specimens from 93 cardiac allograft recipients were prospectively studied. Rejection was graded according to International Society for Heart and Lung Transplantation (ISHLT) criteria and inflammatory infiltrates characterized by immunohistochemical staining for CD3, CD4, CD8, CD68, and CD20. Endothelitis was defined as lymphocyte and macrophage infiltrates within arteriolar, capillary, or venular walls, with endothelial swelling, in contrast to perivascular inflammation of cellular rejection. Complement C4d was identified in capillary walls by immunofluorescent staining and immunohistochemical staining on paraffin sections. Endothelitis was identified in 27 specimens (3%) from 14 patients (15%). ISHLT rejection grades were 0 in 6 specimens, 1R in 20 (1A in 8; 1B in 12), and 2R (3A) in 1. In all cases, there were admixtures of macrophages and T lymphocytes. Inflammation was most prominent in venules. C4d was localized in 12 of the 27 specimens (44%). C4d was localized in 31 of 796 specimens without endothelitis (p < 0.001). The endothelial infiltrates were CD3, CD4, CD8, and CD68+. Twelve of 14 patients had > 0 panel reactive antibodies (PRA), 9 were above 10%, and 8 were above 25%; 5 patients were treated for clinical antibody-mediated rejection, and 4 had possible cardiac allograft vasculopathy by ultrasound imaging (mean follow-up, 40 months). Endothelitis is present in more than 10% of heart transplant recipients and is associated with complement deposition on biopsy samples. Approximately 33% of patients have clinical evidence of humoral rejection. The eventual risk for developing graft vascular disease remains undetermined.

Diagnosis of antibody-mediated rejection in cardiac transplantation: a call for standardization

Currently clinical antibody-mediated rejection (AMR) requires demonstration of histopathologic changes, presence of donor-specific allo-antibodies and allograft dysfunction to establish the diagnosis. Pathology practice patterns in the immunopathologic evaluation and interpretation of cardiac transplant biopsies vary. Specific recommendations for post-transplant allo-antibody monitoring are lacking. Recently, the occurrence of surveillance biopsies being positive for immunopathologic markers of AMR without concomitant graft dysfunction is increasingly recognized. This review focuses on issues of standardization in the diagnosis of AMR and the need for updated criteria. Concomitant use of C4d and C3d proved to be strongly predictive of the presence of circulating allo-antibodies and allograft dysfunction in heart transplant recipients. Asymptomatic patients with histopathologic evidence of AMR in their biopsies appear to have increased risk for cardiac allograft vasculopathy and cardiovascular mortality. The role of nonhuman leukocyte antigen antibodies in AMR has not been adequately addressed. A consensus on the frequency of AMR screening, antibody panels, interpretation and reporting of stains will enhance standardization of the diagnosis of AMR. Further studies are needed to define asymptomatic or subclinical AMR and to determine the long-term outcome of pathologic evidence of complement activation without allograft dysfunction.

ABO Antibody Titer and Risk of Antibody‐Mediated Rejection in ABO‐Incompatible Renal Transplantation

Therapeutic plasma exchange (TPE) preconditioning with immunosuppressive therapy reduces ABO antibody titers, permitting engraftment of ABO‐incompatible (ABO‐I) kidney transplants. The posttransplant predictive role of ABO antibody titers for antibody‐mediated rejection (AMR) is unknown. This retrospective study evaluated 46 individuals who received TPE to permit ABO‐I kidney transplantation. ABO antibody titers were performed using donor‐type indicator red cells. Seven individuals (15.2%) experienced clinical or subclinical AMR. There was no significant difference between recipient blood group, number of pretransplant TPE and baseline titer between those with and without AMR. At 1–2 weeks posttransplant the median titer was 64 (range 4 – 512) among individuals with AMR and 16 (range 2 – 256) among individuals without AMR. Total agglutination reactivity score was significantly higher among individuals with AMR (p = 0.046). The risk of AMR was significantly higher among individuals with an elevated posttransplant titer of ≥64 (p = 0.006). The sensitivity of an elevated posttransplant titer was 57.1% with a specificity of 79.5%. The positive predictive value was 33.3% and the negative predictive value was 91.2%. Most individuals with AMR have an elevated titer, however, the positive predictive value of a high titer for AMR is poor.

Efficacy of Induction Therapy with ATG and Intravenous Immunoglobulins in Patients with Low‐Level Donor‐Specific HLA‐Antibodies

Low-level donor-specific HLA-antibodies (HLA-DSA) (i.e. detectable by single-antigen flow beads, but negative by complement-dependent cytotoxicity crossmatch) represent a risk factor for early allograft rejection. The short-term efficacy of an induction regimen consisting of polyclonal anti-T-lymphocyte globulin (ATG) and intravenous immunoglobulins (IvIg) in patients with low-level HLA-DSA is unknown. In this study, we compared 67 patients with low-level HLA-DSA not having received ATG/IvIg induction (historic control) with 37 patients, who received ATG/IvIg induction. The two groups were equal regarding retransplants, HLA-matches, number and class of HLA-DSA. The overall incidence of clinical/subclinical antibody-mediated rejection (AMR) was lower in the ATG/IvIg than in the historic control group (38% vs. 55%; p = 0.03). This was driven by a significantly lower rate of clinical AMR (11% vs. 46%; p = 0.0002). Clinical T-cell-mediated rejection (TCR) was significantly lower in the ATG/IvIg than in the historic control group (0% vs. 50%; p < 0.0001). Within the first year, allograft loss due to AMR occurred in 7.5% in the historic control and in 0% in the ATG/IvIg group. We conclude that in patients with low-level HLA-DSA, ATG/IvIg induction significantly reduces TCR and the severity of AMR, but the high rate of subclinical AMR suggests an insufficient control of the humoral immune response.

196: Pattern of C4d staining is important in cardiac transplantation

Purpose: Staining for the complement split product, C4d has been used to detect possible antibody mediated rejection (AMR) in cardiac transplantation. Staining with immunohistochemical (IHC) techniques has been associated with clinical AMR. However, until now there have been only qualitative descriptions of C4d staining. We analyzed C4d staining to describe the pattern of staining with correlation to cardiac filling pressures.

Histologic Findings of Antibody-Mediated Rejection in ABO Blood-Group-Incompatible Living-Donor Kidney Transplantation

The purpose of this study was to characterize the histology of antibody-mediated rejection (AMR) in ABO blood-group-incompatible (ABOI) kidney transplants as well as on protocol biopsies performed at the time of stable allograft function. Between 5/99 and 1/02, we performed 32 ABOI kidney transplants (13 A2, 19 non-A2 blood-group living donors). Nineteen biopsies were performed for allograft dysfunction, and 127 protocol biopsies were performed 0, 3, 7, 14, 28 days and 3 and 12 months post transplant. Twenty-five of 32 patients have functioning allografts (mean 585 days post transplant). Nine of 32 (28%) developed clinical AMR. Biopsy revealed glomerular thrombi (78%), mesangiolysis (78%), peritubular capillary C4d staining (56%) and neutrophil infiltration (67%), interstitial hemorrhage and necrosis (56%) and arteriolar thrombi (33%). Subclinical AMR was diagnosed by protocol biopsies in four patients. Findings consisted of glomerular thrombi (100%), mesangiolysis (25%), and C4d staining (100%). In late protocol biopsies performed 214–420 days post transplant, mild mesangiolysis was seen in 2/17 (11.7%), and C4d immunostaining was detected in 3/12 (25%). AMR is characterized by glomerular thrombi, mesangiolysis, peritubular capillary neutrophil infiltration interstitial hemorrhage, necrosis, and C4d deposition. Glomerular thrombi appear early in AMR and may appear prior to graft dysfunction.