William M. Baldwin

Banff 07 Classification of Renal Allograft Pathology: Updates and Future Directions

The 9th Banff Conference on Allograft Pathology was held in La Coruna, Spain on June 23–29, 2007. A total of 235 pathologists, clinicians and scientists met to address unsolved issues in transplantation and adapt the Banff schema for renal allograft rejection in response to emerging data and technologies. The outcome of the consensus discussions on renal pathology is provided in this article. Major updates from the 2007 Banff Conference were: inclusion of peritubular capillaritis grading, C4d scoring, interpretation of C4d deposition without morphological evidence of active rejection, application of the Banff criteria to zero‐time and protocol biopsies and introduction of a new scoring for total interstitial inflammation (ti‐score). In addition, emerging research data led to the establishment of collaborative working groups addressing issues like isolated ‘v’ lesion and incorporation of omics‐technologies, paving the way for future combination of graft biopsy and molecular parameters within the Banff process.

Banff ’09 Meeting Report: Antibody Mediated Graft Deterioration and Implementation of Banff Working Groups

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v‐lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics‐technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.

Cytokines, adhesion molecules, and the pathogenesis of chronic rejection of rat renal allografts

Little is known of the host immune mechanisms responsible for initiation and progression of chronic rejection. We describe immunopathologic features associated with progressively deteriorating function of kidney allografts in the F344-to-Lewis rat strain combination, which differ at MHC and non-MHC loci. Initial rejection in untreated recipients was controlled by a brief course of CsA (5 mg/kg/day, for 10 days), resulting in >80% of recipients surviving up to a year despite declining renal function. In contrast to controls (isografts placed in untreated or CsA-treated Lewis rats), allografts from 12–16 weeks post-Tx showed segmental or global glomerulosclerosis, increasing tubular atrophy, interstitial fibrosis, and intimal proliferation leading ultimately to vascular occlusion. By flow cytometry, IgM and IgG alloantibodies peaked at 2–4 weeks, with a gradual decline to baseline thereafter. Immunohistology showed early and progressive deposition of IgM, IgG, C3, and fibrin in vessel walls and glomeruli. In addition, by 12 weeks, extensive infiltration by activated (IL-2R+) macrophages and CD4+ T cells were noted in glomeruli and blood vessels, in conjunction with staining for the cytokines TNF-α, IL-1, and IL-6. The persistent and dense intraglomerular expression of IL-6 was of particular interest, given its potent mitogenic effects for mesangial cells in vitro, and suggests a role for this cytokine as a mediator of mesangial expansion, advanced glomerular injury, and glomerulosclerosis in chronic rejection. Parallel timing of IL-6 and TNF-α expression was shown in serum samples by ELISA and bioassays. In vitro binding studies showed increased binding of naive host lymphocytes to allograft versus isografts, correlating with upregulation (peaking at week 16) of intercellular adhesion molecule-1 expression by graft endothelium. We conclude that cytokine production and upregulation of adhesion molecules occurring as part of a cellular immune response may be as important, to the etiology of chronic rejection as the hitherto widely emphasized antibody-mediated host responses.

The effect of soluble complement receptor type 1 on hyperacute xenograft rejection

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCRl) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n=13), hyperacute rejection was rapid, with a mean Xg survival of 17±4 min. Therapy with sCRl prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCRl (n=4) prolonged Xg survival to 64±29 min (not significant). Increasing the sCRl dose to 5.9 mg/kg (n=4) significantly delayed Xg rejection to 71±17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCRl, mean Xg survival was further prolonged to 189±36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCRl died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747±100 min, P-0.0004) in the remaining 6 recipients. sCRl administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95±1 and 96±1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCRl -treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCRl. These studies show that complement inhibition with sCRl significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.

B Cell Deficiency Confers Protection from Renal Ischemia Reperfusion Injury

Recent data have demonstrated a role for CD4+ cells in the pathogenesis of renal ischemia reperfusion injury (IRI). Identifying engagement of adaptive immune cells in IRI suggests that the other major cell of the adaptive immune response, B cells, may also mediate renal IRI. An established model of renal IRI was used: 30 min of renal pedicle clamping was followed by reperfusion in B cell-deficient (μMT) and wild-type mice. Renal function was significantly improved in μMT mice compared with wild-type mice at 24, 48, and 72 h postischemia. μMT mice also had significantly reduced tubular injury. Both groups of mice had similar renal phagocyte infiltration postischemia assessed by myeloperoxidase levels and similar levels of CD4+ T cell infiltration postischemia. Peritubular complement C3d staining was also similar in both groups. To identify the contribution of cellular vs soluble mechanism of action, serum transfer into μMT mice partially restored ischemic phenotype, but B cell transfers did not. These data are the first demonstration of a pathogenic role for B cells in ischemic acute renal failure, with a serum factor as a potential underlying mechanism of action.

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

Antibody‐mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant.

Antibody to human leukocyte antigen triggers endothelial exocytosis.

Although antibodies to HLA play a role in the pathogenesis of diseases processes such as rejection of transplanted organs, the precise mechanisms by which antibodies cause tissue injury are not completely understood. We hypothesized that antibodies to host tissues cause inflammation in part by activating endothelial exocytosis of granules that contain prothrombotic mediators such as von Willebrand Factor (VWF) and proinflammatory mediators such as P-selectin. To test this hypothesis, we treated human endothelial cells with murine monoclonal antibody W6/32 to HLA class I and then measured exocytosis by the release of VWF and the externalization of P-selectin. Antibody to HLA activates endothelial exocytosis in a dose-dependent manner over time. The biologically active complement split product, C5a, adds a slight but significant increase to antibody induction of exocytosis. Antibody to HLA alone or with C5a did not damage the cells. Cross-linking of HLA appears to play a role in the ability of antibody to activate exocytosis, because the W6/32 monovalent Fab fragment did not activate VWF release, but the bivalent F(ab′)2 was effective in triggering exocytosis. To explore the in vivo effects of antibody upon graft injury, we infused W6/32 F(ab′)2 antibody to human HLA into severe combined immunodeficient/beige mice that had been transplanted with human skin grafts. Antibody to HLA activated exocytosis and inflammation in human skin grafts. Our data show that antibody to host antigens can activate human endothelial cell exocytosis and leukocyte trafficking. By triggering vascular inflammation, antibody activation of exocytosis may play a role in transplant rejection.

Complement deposition in early cardiac transplant biopsies is associated with ischemic injury and subsequent rejection episodes.

BACKGROUND Prolonged warm or cold ischemia is associated with poor survival of cardiac transplants, and ischemic changes in early posttransplantation endomyocardial biopsies correlate with the later development of chronic rejection. In animal models, tissue ischemia has been shown to activate complement. METHODS To determine whether ischemic changes in endomyocardial biopsies were associated with complement deposition, biopsies obtained 1-3 weeks after transplantation from 33 patients were evaluated immunohistologically for C4d and C3d deposition as well as for IgM, IgG, and IgA. The histological changes associated with ischemic injury were scored independently, using previously reported criteria without knowledge of the immunohistochemical results. RESULTS Diffuse capillary and pericapillary deposition of C4d or C3d were detected in endomyocardial biopsies of 14 of the 33 patients. The majority of biopsies (79%) with C4d or C3d deposits had histological evidence of ischemic injury, including eight of the nine biopsies containing both C4d and C3d deposition. In contrast, only 8 of 18 (45%) of the biopsies without C4d or C3d deposition had ischemic injury. Only trace amounts of IgM and no IgG or IgA were demonstrable in the biopsies. Only 2 of the 14 biopsies with C4d or C3d deposition had evidence of moderate acute rejection, whereas 5 of the 18 biopsies without C4d or C3d deposition had moderate acute rejection. However, C4d and C3d deposition did correlate with repeated acute rejection episodes on subsequent biopsies. CONCLUSIONS Thus, ischemic changes are associated with the activation of complement. Complement activation may in turn promote tissue injury and provide a potential target for future treatment.

Antibody and Complement in Transplant Vasculopathy

Advances in immunosuppression have decreased the incidence of acute rejection, but the development of vasculopathy in the coronary arteries of transplants continues to limit the survival of cardiac allografts. Transplant vasculopathy has also been referred to as accelerated graft arteriosclerosis because it has features of arteriosclerosis, but it is limited to the graft and develops over a period of months to years. Although the pathological features of transplant vasculopathy are well defined, the causative mechanisms are not completely understood. This review focuses on the mechanisms by which antibody and complement can cause or contribute to coronary vasculopathy in cardiac transplants. Antibodies and complement can have independent effects, but the combination of antibodies and complement with inflammatory cells has greater pathogenic potential for the endothelial and smooth muscle cells of the coronary arteries. For example, stimulation through receptors for IgG or complement split products can activate macrophages, but stimulation through combinations of these receptors generates synergistic results. Together, antibodies and complement efficiently integrate the activation of endothelial cells, platelets, and macrophages, which are 3 of the primary components in the pathogenesis of transplant vasculopathy. Recent findings indicate that antibodies and complement produced within the transplant may contribute to vascular pathology in some transplants. Acute rejection caused by antibodies and complement has been treated by combinations of plasmapheresis, intravenous gamma-globulin and monoclonal antibodies to CD20 on B lymphocytes. The effect of these treatment modalities on the development of coronary vasculopathy is unknown.