Functional Fc gamma receptor gene polymorphisms and donor-specific antibody-triggered microcirculation inflammation.

Fc-dependent effector mechanisms may contribute to antibody-mediated rejection (ABMR), and distinct gene polymorphisms modifying the function of Fc gamma receptors (FcγRs) may influence the capability of donor-specific antibodies (DSAs) to trigger inflammation. To evaluate the relevance of functional FcγR variants in late ABMR, 85 DSA-positive kidney allograft recipients, who were recruited upon antibody screening of 741 prevalent patients, were genotyped for polymorphisms in FcγRIIA (FCGR2A-H/R131 ; rs1801274), FcγRIIIA (FCGR3A-V/F158 ; rs396991), and FcγRIIIB (FCGR3B-neutrophil antigen 1 ([NA1]/NA2; rs35139848). Individuals with high-affinity FCGR3A-V158 alleles (V/V158 or V/F158 ) showed a higher rate (and extent) of peritubular capillaritis (ptc) in protocol biopsies than homozygous carriers of the lower-affinity allele (ptc score ≥1: 53.6% vs 25.9%; P = .018). Associations were independent of C1q-binding to DSA or capillary C4d. In parallel, there was a trend toward increased macrophage- and injury-repair response-associated transcript subsets. Kidney function over 24 months, however, was not different. In support of a functional role of FcγRIIIA polymorphism, NK92 cells expressing FCGR3A-V158 produced >2 times as much interferon gamma upon incubation with HLA antibody-coated cells as those expressing FCGR3A-F158 . FcγRIIA and FcγRIIIB polymorphisms were not associated with allograft morphology. Our data suggest that the presence of high-affinity FcγRIIIA variants may favor DSA-triggered microcirculation inflammation.

Anti-C1s monoclonal antibody BIVV009 in late antibody-mediated kidney allograft rejection-results from a first-in-patient phase 1 trial.

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.

Effect of the Anti-C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody-Induced Complement Activation-A Preclinical In Vitro Study.

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 µg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients.

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.

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) >or=60 mL/min and proteinuria

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.

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.

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.

Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection.

BACKGROUND: Capillary deposition of complement split product C4d has been suggested to be a valuable marker for humoral rejection. In this retrospective study we evaluated the clinical impact of C4d deposition in renal allografts with special emphasis on associations between C4d staining patterns and histological features of acute rejection. METHODS: One hundred and two allograft biopsies obtained from 61 kidney transplants (1-532 days after transplantation; median 14 days) were examined by immunohistochemistry on routine paraffin sections using a novel anti-C4d polyclonal antibody (C4dpAb). RESULTS: Fourty-two of 102 biopsies showed endothelial C4d deposits in peritubular capillaries (PTC). Histopathological analysis revealed a significantly lower frequency of positive C4d staining in biopsies with rather than in those without acute cellular rejection defined by the Banff grading schema (P<0.01). For clinical evaluation, patients were classified according to C4d staining in allografts (C4d(PTC) positive in at least one biopsy, n=31 vs C4d(PTC) negative in all biopsies, n=30). C4d(PTC) positive patients had significantly higher serum creatinine levels than C4d negative patients. Even in the absence of morphological evidence for rejection, differences in serum creatinine levels between C4d(PTC) positive and negative recipients were significant (6 months: 2.01+/-0.75 vs 1.41+/-0.27 mg/dl; 12 months: 1.95+/-0.60 vs 1.36+/- 0.34 mg/dl; 18 months: 1.98+/-0.50 vs 1.47+/-0.31 mg/dl; P<0.05). All patients with rejection resistant to conventional therapy (n=4) were in the C4d(PTC) positive subgroup. All recipients with panel reactive antibodies (PRA) >50% (n=8) were C4d(PTC) positive. CONCLUSIONS: Our data indicate that endothelial C4d deposition is associated with inferior graft outcome. We provide evidence that this immunohistochemical finding and its clinical impact are not associated with morphological signs of cellular rejection.

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor.

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.