Markus Wahrmann

Immunoadsorption in severe C4d-positive acute kidney allograft rejection: a randomized controlled trial.

Antibody‐mediated rejection (AMR) frequently causes refractory graft dysfunction. This randomized controlled trial was designed to evaluate whether immunoadsorption (IA) is effective in the treatment of severe C4d‐positive AMR. Ten out of 756 kidney allograft recipients were included. Patients were randomly assigned to IA with protein A (N = 5) or no such treatment (N = 5) with the option of IA rescue after 3 weeks. Enrolled recipients were subjected to tacrolimus conversion and, if indicated, ‘anti‐cellular’ treatment. All IA‐treated patients responded to treatment. One death unrelated to IA occurred after successful reversal of rejection. Four control subjects remained dialysis‐dependent. With the exception of one patient who developed graft necrosis, non‐responders were subjected to rescue IA, however, without success. Because of a high graft loss rate in the control group the study was terminated after a first interim analysis. Even though limited by small patient numbers, this trial suggests efficiency of IA in reversing severe AMR.

Posttransplant HLA Alloreactivity in Stable Kidney Transplant Recipients—Incidences and Impact on Long-Term Allograft Outcomes

Humoral alloreactivity is well established to predict adverse allograft outcomes. However, in some recipients, alloantibodies may also occur in the absence of graft dysfunction. We evaluated if and how often complement‐ and noncomplement‐fixing alloantibodies are detectable in stable recipients and whether, in this context, they affect long‐term outcomes. Sera obtained from 164 kidney transplant recipients at 2, 6 and 12 months were evaluated by FlowPRA screening and single‐antigen testing for detection of IgG‐ or C4d‐fixing HLA panel reactivity and donor‐specific antibodies (DSA). Applying stringent criteria, we selected 34 patients with an uneventful 1‐year course (no graft dysfunction or rejection) and excellent graft function at 12 months [estimated glomerular filtration rate (eGFR) ≥60 mL/min and proteinuria ≤0.5 g/24 h]. Nine (27%) and 5 (15%) of these recipients tested positive by [IgG] and [C4d]FlowPRA screening, respectively. In five cases, DSA were identified. Frequencies of positive test results and DSA binding intensities were not significantly lower than those documented for patients who did not fulfill the above criteria. In recipients with an excellent 1‐year course, FlowPRA reactivity was not associated with lower eGFR or increased protein excretion during 68‐month median follow‐up. Our results suggest cautious interpretation of antibody monitoring in patients with normal‐functioning grafts.

Flow cytometry based detection of HLA alloantibody mediated classical complement activation

Complement-dependent cytotoxicity (CDC) panel reactive antibody (PRA) testing is used to assess recipient presensitization and post-transplant alloantibody formation in transplant recipients. However, CDC test results can be affected by false-positive reactions brought about by autoantibodies or antilymphocyte reagents. As an alternative to the CDC-PRA assay, detection of HLA alloantibodies using HLA antigen-coated microbeads (FlowPRA test) was recently established. FlowPRA testing, however, does not distinguish between (presumably more harmful) complement-fixing and noncomplement-fixing alloantibodies. In this study, we established a novel assay allowing flow cytometric detection of HLA alloantibody dependent classical complement activation using the FlowPRA test. For the detection of complement activation, FlowPRA beads were incubated with sera from highly sensitized dialysis patients (CDC-PRA reactivity >60%) and then stained for C4 (C4d, C4c) and C3 (C3d, C3c) fragments, as well as C1q deposition using indirect immunofluorescence. We demonstrate alloantibody induced induction of C4 fragment, and in parallel C1q deposition to HLA class I or class II beads. As shown by immunoblotting, C4 staining was not due to the presence of preformed C4 fragment-IgG/M complexes. Indeed, C4 fragment deposition in our in vitro system was demonstrated to result from de novo complement activation. First, inactivation of C4 by treatment of sera with methylamine, which inhibits cleavage of the internal thioester, completely abolished C4 fragment deposition. Second, C4 fragment deposition was not observed in the evaluation of C4-free immunoadsorption eluates obtained from highly sensitized dialysis patients. After supplementation with complement, however, eluates induced C4 deposition. Deposition of C4 split products and C1q was temperature-dependent with maximum binding after incubation at 4 degrees C for 60 min. In contrast, maximum C3 fragment deposition was found at 37 degrees C. At this temperature, C3 deposition occurred in an alloantibody and C4-independent fashion, presumably as a result of alternative complement activation. In summary, we describe a novel cell-independent and easy-to-perform PRA test that permits flow cytometry based detection of alloantibody induced classical complement activation. Future studies will have to evaluate its possible relevance as an alternative to CDC-PRA testing in clinical transplantation.

Pivotal Role of Complement-Fixing HLA Alloantibodies in Presensitized Kidney Allograft Recipients

Recipient presensitization represents a major risk factor for kidney allograft loss. Complement fixation may be a critical attribute of deleterious alloantibodies. We investigated clinical impact of complement‐fixing HLA presensitization employing [C4d]FlowPRA, a novel assay permitting selective detection of HLA panel reactive antibody (PRA)‐triggered C4 complement split product deposition. A cohort of 338 kidney transplants was evaluated for presensitization applying [C4d]FlowPRA together with [IgG]FlowPRA and complement‐dependent cytotoxicity (CDC)‐PRA. Analysis of HLA class I alloreactivities revealed a high incidence of C4d‐positive graft dysfunction in [IgG]FlowPRA(+)/[C4d]FlowPRA(+) and [IgG]FlowPRA(+)/[C4d]FlowPRA(−) recipients (23% and 22% vs. 3% in [IgG]FlowPRA(−) patients). Only patients with complement‐fixing HLA class I immunization had inferior graft survival [75% (3 years) vs. 91% and 89%, respectively (p = 0.036)]. Despite frequent finding of capillary C4d deposition (28%), complement‐fixing HLA class II immunization was not associated with inferior survival rates. This may have been due to reduction of clinical effects by intense immunosuppression in presensitized patients. Evaluating CDC, 29% of CDC‐PRA(+)/[C4d]FlowPRA(+) recipients had C4d‐positive graft dysfunction. For these patients 3‐year graft survival was worst, followed by CDC‐PRA(+)/[C4d]FlowPRA(−) and CDC‐PRA(−) patients (76% vs. 81% vs. 90%, p = 0.014). Results highlight a strong impact of complement‐fixing HLA presensitization. Discerning complement‐activating abilities of HLA alloantibodies, [C4d]FlowPRA may help identify recipients at particularly high risk for graft rejection and loss.

Risk factors for capillary C4d deposition in kidney allografts: evaluation of a large study cohort.

Background. Capillary deposition of the complement split product C4d has turned out to be a valuable marker of antibody-mediated rejection. The impact of pre- and posttransplant variables including particular immunosuppressive regimens on the frequency of C4d deposition has not yet been systematically investigated in a large multivariate analysis. Methods. In this retrospective study, the authors evaluated the incidence of C4d deposition in 388 kidney transplant recipients subjected to diagnostic biopsy within the first 6 months and analyzed the influence of potential confounders on the rate of C4d-positive graft dysfunction by applying multivariate logistic regression. Results. Sixty-six recipients (17%) developed linear C4d deposits in at least a quarter of peritubular capillaries, a finding associated with inferior 1-year allograft survival (73% vs. 88% in C4d-negative patients, P=0.0003). A 50% reduction in the odds of C4d-positive graft dysfunction was found if calcineurin inhibitor or mycophenolate mofetil (MMF) therapy was started 2 to 4 hr before transplantation when compared with initiation after surgery (adjusted odds ratio [OR], 0.5; P=0.03). No differences with respect to C4d staining results were found for the use of tacrolimus, MMF, or sirolimus, or for cyclosporine C2 monitoring. Retransplantation (OR, 3.6; P<0.001) and presensitization (OR, 3.1; P=0.002) turned out to be strong independent risk factors for C4d deposition. Conclusions. The authors’ results suggest a reduced risk of C4d-positive graft dysfunction for patients receiving immunosuppression before transplantation. Apart from first dose timing, no influence of particular immunosuppressive strategies on C4d staining results was found.

Effect of the proteasome inhibitor bortezomib on humoral immunity in two presensitized renal transplant candidates.

Background. Recipient presensitization represents a major hurdle to successful renal transplantation. Previous case series have suggested that the proteasome inhibitor bortezomib directly affects the alloantibody-secreting plasma cells in rejecting allograft recipients. However, the ability of this agent to desensitize nonimmunosuppressed transplant candidates before transplantation is currently unknown. Methods. In this analysis, two sensitized hemodialysis patients were selected to receive two subsequent bortezomib cycles. Bortezomib was given at 1.3 mg/m2 on days 1, 4, 8, and 11. Dexamethasone was added to the second cycle to enhance treatment efficiency. Serial immune monitoring included cytotoxic panel reactive antibody testing, Luminex single antigen testing for anti-human leukocyte antigen (HLA) IgG with or without C4d-fixing capability, and ABO antibody detection. Results. During a half-year follow-up period, cytotoxic panel reactive antibody decreased from 87% to 80% (patient 1) and 37% to 13% (patient 2). Patient 1 showed a 40% reduction in binding intensities of identified Luminex HLA single antigen reactivities and, in parallel, slight reductions in ABO blood group antibody and total immunoglobulin levels. In patient 2, bortezomib did not affect circulating antibody levels in a meaningful way. Both patients showed a more than 50% reduction in the levels of anti-HLA antibody-triggered C4d deposition to Luminex beads. Conclusion. Our initial experience suggests that, without additional immunosuppressive measures, bortezomib has modest effects on circulating antibodies against HLA or blood group antigens. The reduced levels of antibody-triggered complement fixation, however, imply potential clinical relevance of proteasome inhibition for recipient desensitization.

In Vitro Detection of C4d‐Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody‐mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody‐triggered C4d deposition to HLA antigen‐coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86–0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50–0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83–1.05] vs. 0.76 [0.56–0.97] calculated for C4d‐fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement‐fixing donor‐specific alloreactivities failed to improve the predictive value of FlowPRA‐based serology. In conclusion, our results suggest that detection of complement‐fixing HLA panel reactivity could provide a specific tool for monitoring of C4d‐positive AMR.

Complement component C3 activation: the leading cause of the prozone phenomenon affecting HLA antibody detection on single-antigen beads.

Background Luminex-based anti-HLA IgG detection on single-antigen flow beads (SAFB) represents a valuable tool for characterization of allosensitization patterns. Assay interpretation, however, may be impeded by false-low test results caused by the prozone effect. Recent experimental data have related this artifact to direct blockade of IgG detection by complement component C1 as a possible candidate mechanism. Methods To dissect a causative role of C1 complex formation and subsequent steps of classical complement activation, native or modified sera obtained from transplant candidates with HLA class I sensitization were evaluated applying SAFB-based IgG, IgM, C1q, C1r, C1s, or C4 and C3 split product detection, respectively. Results Evaluating a total of 1,164 single-antigen reactions, serum dilution (1:10) revealed an 11% incidence of the prozone effect as defined by a greater than 100% increase in IgG mean fluorescence intensity. Prozoning was found to be related to the amount of antibody-triggered C1q, C4 or C3 split product deposition, and was eliminated by serum modifications affecting the integrity of the C1 complex (dithiotreitol, ethylenediaminetetraacetic acid, heat inactivation). Remarkably, the same effect was achieved without C1 disintegration, either by serum treatment with methylamine to block C4 and C3 split product binding or by cobra venom factor to trigger C3 consumption. Conclusions Our results reinforce a central role of C1 as a trigger of prozoning. However, we provide evidence that C1 may exert its effects only indirectly, namely via inducing C3 fragment deposition, which by coating of the bead surface may block the binding of IgG detection reagents.

Clinical relevance of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients

Donor‐specific alloantibodies (DSA), especially those fixing complement, may pose a particular immunologic risk to transplant recipients. To assess the clinical impact of C4d‐ or non‐C4d‐fixing (IgG) HLA sensitization, pretransplant sera obtained from 338 kidney allograft recipients prescreened by FlowPRA were retrospectively evaluated by Luminex single antigen (SA) testing using a novel fluorescent‐labeled anti‐C4d reagent for detection of antibody‐triggered C4d deposition in addition to IgG binding. Recipients with [IgG]DSA (n = 39) showed a substantially higher rate of C4d positive rejection (33%) than 16 patients with [IgG] non‐DSA (0%) or 283 antibody‐negative patients (4%, multivariate analysis excluding retransplantation because of high co‐linearity: P < 0.0001), and adversely affected 5‐year death‐censored graft survival (74% vs. 81% and 90%, respectively, multivariate model: P < 0.05). [C4d] DSA (n = 21) and [C4d] non‐DSA (n = 25) increased rates of C4d positive rejections to a similar extent (24% and 28% vs. 4% in recipients without C4d‐fixing reactivity; multivariate analysis: P ≤ 0.002) with a trend towards adverse 5‐year graft survival (76% and 76% vs. 90%; P ≤ 0.2). In conclusion, Luminex‐based characterization of HLA sensitization may be a useful strategy for risk stratification. Possibly as a result of intensified immunosuppression in presensitized recipients, identification of C4d‐fixing DSA was not associated with a further increase of rejection and graft loss rates.

Peritransplant immunoadsorption for positive crossmatch deceased donor kidney transplantation.

Various desensitization protocols were shown to enable successful living donor kidney transplantation across a positive complement‐dependent cytotoxicity crossmatch (CDCXM). Positive crossmatch transplantation, however, is less well established for deceased donor transplantation. We report a cohort of 68 deceased donor renal allograft recipients who, on the basis of broad sensitization (lymphocytotoxic panel reactivity ≥40%), were subjected to a protocol of peritransplant immunoadsorption (IA). Treatment consisted of a single session of immediate pretransplant IA (protein A) followed by posttransplant IA and antilymphocyte antibody therapy. Twenty‐one patients had a positive CDCXM, which could be rendered negative by pretransplant apheresis. Solid phase HLA antibody detection revealed preformed donor‐specific antibodies (DSA) in all 21 CDCXM‐positive and in 30 CDCXM‐negative recipients. At 5 years, overall graft survival, death‐censored graft survival and patient survival were 63%, 76% and 87%, respectively, without any differences between CDCXM‐positive, CDCXM‐negative/DSA‐positive and CDCXM‐negative/DSA‐negative recipients. Furthermore, groups did not differ regarding rates of antibody‐mediated rejection (24% vs. 30% vs. 24%, p = 0.84), cellular rejection (14% vs. 23% vs. 18%, p = 0.7) or allograft function (median 5‐year serum creatinine: 1.3 vs. 1.8 vs. 1.7 mg/dL, p = 0.62). Our results suggest that peritransplant IA is an effective strategy for rapid desensitization in deceased donor transplantation.