Antibody-mediated Renal Allograft Rejection Research Articles

Histopathology and outcome of acute humoral rejection in renal allografts

Our purpose was to see if histopathologic features of acute antibody-mediated rejection (AMR) in renal allografts have prognostic value; and to compare two-year graft survival with and without additional therapy with plasmapheresis and intravenous immunoglobulin (IVIG). We reviewed renal allograft biopsies taken within the first 6 months after transplant from patients with C4d positive AMR, performed between January 2000 to December 2005 (n=57). We formed two groups: Group 1: biopsied between 2003 and 2005 (n=26), when C4d staining was routinely performed and option for plasmapheresis and IVIG was available; Group 2: biopsied between 2000 and 2002 (n=31), retrospectively found to be C4d positive. Patients whose biopsies showed cortical necrosis or arterial fibrinoid necrosis had early graft loss. Other histopathologic features did not statistically correlate with graft loss. Overall, additional plasmapheresis/IVIG treatment did not show convincing improvement in graft survival or function at 2 years post-transplant, but all six patients with thrombotic microangiopathy (TMA) who received plasmapheresis/IVIG had functioning grafts at two-year follow-up.

Proteasome Inhibitor-Based Primary Therapy for Antibody-Mediated Renal Allograft Rejection

Rapid and complete elimination of donor-specific anti-human leukocyte antigen antibodies (DSA) during antibody-mediated rejection (AMR) is rarely achieved with traditional antihumoral therapies. Proteasome inhibitor-based therapy has been shown to effectively treat refractory AMR, but its use as a primary therapy for AMR has not been presented. Our initial experience with proteasome inhibition as a first-line therapy for AMR is presented. Adult kidney transplant recipients with AMR, diagnosed by Banff criteria, received a bortezomib-based regimen as the primary therapy. Bortezomib therapy was administered per package insert with plasmapheresis performed immediately before each bortezomib dose, and a single rituximab dose (375 mg/m2) given with the first bortezomib dose. DSA were quantitated using single-antigen beads on a Luminex platform. Two patients underwent bortezomib-based therapy for acute AMR occurring within the first 2 weeks after transplantation. High DSA levels and positive C4d staining of peritubular or glomerular capillaries were present at the time of diagnosis. Both patients experienced prompt AMR reversal and elimination of detectable DSA within 14 days of bortezomib-based therapy. Renal function remains excellent with normal urinary protein excretion at 5 and 6 months after AMR diagnosis. One patient experienced a repeated elevation of DSA (including two new human leukocyte antigen specificities) 2 months after initial bortezomib therapy, but without C4d deposition or histologic evidence of AMR. Retreatment with bortezomib provided prompt, complete, and durable DSA elimination. Proteasome inhibitor-based combination therapy provides a potential means for rapid DSA elimination in early acute AMR in renal transplant recipients.

Antibody-mediated rejection of renal allograft in combined liver-kidney transplant

Liver transplantation is performed based on ABO blood type compatibility without dependence on crossmatch results. Combined liver-kidney transplantation (CLKT) is similarly performed without dependence of crossmatch results as the liver is thought to confer protection to the renal allograft against alloantibody. We report a case of CLKT in a sensitized patient with antibody-mediated rejection (AMR) of the renal allograft. AMR was confirmed with C4d staining and serial monitoring of donor-specific antibody (DSA). Despite intensive therapy directed against AMR and the presence of the liver allograft, the patient demonstrated increasing titers of alloantibody, never demonstrated adequate renal function, and ultimately expired after two months. This result demonstrates the potential for AMR of the renal allograft in sensitized recipients of CLKT.

Mechanisms of complement activation, C4d deposition, and their contribution to the pathogenesis of antibody-mediated rejection.

Complement split products have emerged as useful markers of antibody-mediated rejection in solid organ transplants. One split product, C4d, is now widely accepted as a marker for antibody-mediated rejection in renal and cardiac allografts. This review summarizes the rationale for the use of C4d as a marker of antibody-mediated rejection, along with the clinical evidence supporting its use in the clinical diagnosis of antibody-mediated rejection. Antibody-independent mechanisms by which C4d can be activated by the classical and lectin pathways of complement activation are also identified. Finally, mechanisms by which complement activation stimulates effector cells (neutrophils, monocytes, macrophages, platelets, and B and T lymphocytes) as well as target cells (endothelial cells) are discussed in relation to antibody-mediated allograft rejection.

Successful Treatment of Chronic Antibody-Mediated Rejection With IVIG and Rituximab in Pediatric Renal Transplant Recipients

Chronic antibody-mediated rejection (CAMR) of renal allografts has recently been recognized as a defined nosologic entity. The outcome of CAMR is poor; there is no established treatment protocol for this condition. We therefore initiated a pilot study on treatment of CAMR with an antihumoral regimen consisting of high-dose intravenous immunoglobulin (IVIG) and the chimeric anti-CD20 antibody rituximab. Six pediatric renal transplant recipients with CAMR received four weekly doses of IVIG (1 g/kg body weight per dose), followed by a single dose of rituximab (375 mg/m2 body surface area) 1 week after the last IVIG infusion. Renal allograft biopsies were evaluated using the Banff '05 classification. Human leukocyte antigen-specific antibodies were detected by panel-reactive lymphocytotoxicity and solid phase ELISA assays. Median glomerular filtration rate during 6 months before intervention dropped by 25 (range, 11-26) mL/min/1.73 m2 (P<0.05) and increased in response to antihumoral therapy by 21 (-14 to +30) 6 months (P<0.05) and by 19 (-14 to +23) mL/min/1.73 m2 12 months (P=0.063) after start of treatment. Glomerular filtration rate improved or stabilized in 4 patients; the two nonresponders had the highest degree of transplant glomerulopathy, the highest degree of C4d deposition in peritubular capillaries and pronounced interstitial inflammation. The treatment regimen was well tolerated. This pilot study demonstrates that CAMR in pediatric renal transplant recipients can be treated successfully and safely with a combination of IVIG and rituximab. This observation should encourage more extensive studies to evaluate this new treatment strategy.

Complement C4d and pathologic diagnosis of antibody-mediated renal allograft rejection

同种异体肾移植排异无非是以T细胞介导的细胞性排异和以抗体介导的体液性排异两种形式.在组织学上虽然对肾移植急性细胞性排异已有较好的认识,但对抗体介导的体液排异由于病变不典型,缺乏足够的认识和共识[1].近10年来,补体片段C4d在移植肾毛细血管上沉积与抗体介导排异的相关性研究备受瞩目[2-4],2007年发表的Banff分类也将C4d免疫阳性染色作为诊断急、慢性抗体介导排异的标准之一[5].我们就移植肾抗体介导排异、补体片段C4d及C4d在排异诊断中的意义做一综述如下。

Anti-glutathione S-transferase T1 antibody-mediated rejection in C4d-positive renal allograft recipients

Chronic humoral rejection is a progressive form of graft injury, with defined diagnostic criteria, the crucial one being the evidence of circulating anti-donor antibodies. These antibodies are mainly directed against human leucocyte antigens (HLA), but other targets have also been described. We previously reported that antibodies against the Glutathione S-transferase T1 (GSTT1) enzyme appear in recipients without the GSTT1 gene who receive a graft from a GSTT1-positive donor. The primary aim of this study was to analyse the role of GSTT1 in cases of antibody-mediated rejection (AMR) in the absence of anti-HLA antibodies. A second objective was to describe the distribution of the GSTT1 enzyme in the human kidney. Four renal biopsies from four renal transplanted patients with declined renal function and circulating anti-donor GSTT1 antibodies were studied for C4d deposits in sections of paraffin-embedded tissue samples. Anti-donor-specific HLA and MICA antibody detection was done with the Luminex platform and anti-GSTT1 antibodies were tested by indirect immunofluorescence on rat tissues and ELISA assay. DNA of the patients was extracted for GSTT1 genotyping. Four patients with the GSTT1 donor/recipient mismatch developed anti-GSTT1 antibodies 32, 42, 48 and 60 months after the transplant. One patient also had donor-specific anti-HLA antibodies. Their biopsies showed pathologic lesions compatible with chronic antibody-mediated rejection (CAMR), along with positive C4d deposition in peritubular capillaries in three of them, being no valuable in the other case. This is the first study reporting an association between the appearance of chronic antibody-mediated renal allograft rejection and the occurrence of de novo production of anti-GSTT1 antibodies, in the absence of anti-HLA donor-specific antibodies. This fact suggests a potential role of the GSTT1 system in anti-graft immune response.

Cancer in patients on dialysis and after renal transplantation

26. Jordan SC, Vo AA, Peng A et al. Intravenous gammaglobulin (IVIG): a novel approach to improve transplant rates and outcomes in highly HLA-sensitized patients [see comment]. Am J Transplant 2006; 6: 459–466 27. Haas M, Montgomery RA, Segev DL et al. Sub-clinical acute antibody-mediated rejection in positive crossmatch renal allografts. Am J Transplant 2007; 7: 576–585 28. Warren DS, Simpkins CE, Cooper M et al. Modulating Alloimmune responses with plasmapheresis and IVIG. Curr Drug Target Cardiovasc Hematol Disord 2005; 5: 214–222 29. Montgomery Robert A, Zachary Andrea A. Transplanting patients with a positive donor-specific crossmatch: a single center’s perspective. Pediatr Transplant 2004; 8: 535–542 30. Jordan Stanley C, Pescovitz Mark D. Presensitization: the problem and its management. Clin J Am Soc Nephrol 2006; 1: 421– 432 31. Stegall MD, Gloor J, Winters JL et al. A comparison of plasmapheresis versus high-dose IVIG desensitization in renal allograft recipients with high levels of donor specific alloantibody. Am J Transplant 2006; 6: 346–351 32. Zachary Andrea A, Montgomery Robert A, Leffell Mary S. Factors associated with and predictive of persistence of donor-specific antibody after treatment with plasmapheresis and intravenous immunoglobulin. Hum Immunol 2005; 66: 364–370 33. Zou y, Stastny P, Susal C et al. Antibodies against MICA antigens and kidney transplant rejection. N Eng J Med 2007; 357: 1293–1300 34. Mizutani K, Terasaki P, Bignon JD et al. Association of kidney transplant failure and antibodies against MICA. Hum Immunol 2006; 67: 683–691 35. Leffell Mary S, Montgomery Robert A, Zachary Andrea A. The changing role of antibody testing in transplantation. Clin Transpl 2005; 259–271

A retrospective review of the outcome of plasma exchange and aggressive medical therapy in antibody mediated rejection of renal allografts: A single center experience

Antibody-mediated rejection (AMR) has been recognized as a major cause of renal allograft loss. Protocols using plasma exchange (PE) to reverse rejection have mixed results. A retrospective chart review was performed to determine the clinical response to PE inpatients with AMR of renal allograft. A good response to treatment was defined as a decline in serum creatinine (SCr) to within 25% above the prerejection value or discontinuation of dialysis with a SCr <2 mg/dl within 3 months of discharge from the hospital and disappearance of donor-specific alloantibodies (DSA). Twenty-two patients, treated with PE for biopsy proven AMR with or without acute-cellular rejection (ACR), were included in the study. Sixty-four percent of patients had concurrent AMR and ACR. Fifty-two percent of all patients had a good response to antirejection therapy, whereas 63% of patients with only AMR and 46% of patients with both AMR and ACR had a good response. Good response to PE did not correlate with the number of plasma volumes exchanged (P = 0.09), but correlated with a shorter period from transplantation to the rejection episode (P = 0.002). Only a shorter interval between transplantation and the acute rejection episode correlated with a good response to PE.

Antibody-Mediated Renal Allograft Rejection

Alloantibodies to HLA class I or II and other antigens expressed by endothelium cause a variety of effects on renal transplants, ranging from acute to chronic rejection, and even apparent graft acceptance (accommodation). Recognition of these conditions and appropriate therapy requires demonstration of C4d in biopsies, commonly confirmed by tests for circulating alloantibody. Substantial practical experience by pathologists in the interpretation and pitfalls of C4d stains are reviewed along with considerations of the clinical significance and pathologic mechanisms of the different effects of antibody on the endothelium of the renal allograft. Clinical trials will be needed to ascertain the optimal treatment for the newly appreciated conditions chronic humoral rejection and accommodation.

Subclinical Acute Antibody-Mediated Rejection in Positive Crossmatch Renal Allografts

Subclinical antibody-mediated rejection (AMR) has been described in renal allograft recipients with stable serum creatinine (SCr), however whether this leads to development of chronic allograft nephropathy (CAN) remains unknown. We retrospectively reviewed data from 83 patients who received HLA-incompatible renal allografts following desensitization to remove donor-specific antibodies (DSA). Ten patients had an allograft biopsy showing subclinical AMR [stable SCr, neutrophil margination in peritubular capillaries (PTC), diffuse PTC C4d, positive DSA] during the first year post-transplantation; 3 patients were treated with plasmapheresis and intravenous immunoglobulin. Three patients had a subsequent rise in SCr and an associated biopsy with AMR; 5 others showed diagnostic or possible subclinical AMR on a later protocol biopsy. One graft was lost, while remaining patients have normal or mildly elevated SCr 8-45 months post-transplantation. However, the mean increase in CAN score (cg + ci + ct + cv) from those biopsies showing subclinical AMR to follow-up biopsies 335 +/- 248 (SD) days later was significantly greater (3.5 +/- 2.5 versus 1.0 +/- 2.0, p = 0.01) than that in 24 recipients of HLA-incompatible grafts with no AMR over a similar interval (360 +/- 117 days), suggesting that subclinical AMR may contribute to development of CAN.

Putative Antibody-Mediated Rejection With C4d Deposition in HLA-Identical, ABO-Compatible Renal Allografts

We sought evidence for non-MHC antibody-mediated rejection in renal allografts by a systematic study of rejected HLA-identical sibling renal allografts. Among 162 recipients of HLA-identical, ABO-compatible sibling donor kidneys transplanted at the Massachusetts General Hospital from 1964 to 2005, we identified 15 grafts that were lost from rejection and two additional grafts with reversible acute rejection, which provided 30 samples for study. All samples were stained for C4d by immunofluorescence in frozen tissue ( n = 7) or by immunohistochemistry in paraffin embedded tissues ( n = 10). We found that two of 17 grafts had positive C4d staining of peritubular capillaries. Histology revealed acute antibody-mediated rejection in one and acute cellular rejection type 1 in the other. Both grafts were matched at HLA-A, B, and C loci and had a nonreactive mixed lymphocyte response. Genotyping and serological analysis were not available. Compared with a published series, C4d+ irreversible rejection was more common in HLA nonidentical than HLA-identical grafts (75% vs 6.7%, respectively, P < .002). We conclude that antibody-mediated rejection, presumably due to non-MHC antigens other than ABO-blood groups does occur, but infrequently. This may account for some of the HLA antibody negative cases that develop antibody-mediated rejection.

Antibody-Mediated Rejection in Renal Allografts

The past 15 years have seen major advances in the understanding of the effects of anti-donor antibodies on renal allografts at various stages after transplantation. These advances have been due in large part to pathologic examination of both early and late renal allograft biopsies, including both routine histologic evaluation and immunohistology to detect complement split products. As pathologists have become increasingly adept at diagnosing antibody-mediated rejection (AMR) on allograft biopsies, substantial progress has been made in the treatment of AMR and in successful renal transplantation in recipients with pre-existing antibodies against donor blood group (ABO) and/or major histocompatibility (HLA) antigens. This article reviews the pathologic features of hyperacute, acute, and chronic AMR, including some newer findings impacting diagnosis and outcomes, and differences in the implications of similar pathologic findings in ABO- versus HLA-incompatible renal allografts.

Polyclonal Rabbit Antithymocyte Globulin Triggers B-Cell and Plasma Cell Apoptosis by Multiple Pathways

Polyclonal antithymocyte globulin (ATG) is widely used as an anti-T-cell agent for induction and treatment of acute cellular rejection in solid organ transplantation. The authors recently demonstrated that rabbit (r) ATG can be used in combination with plasmapheresis to effectively treat antibody-mediated renal allograft rejection. This observation suggested that rATG may have anti-B cell activity. The authors tested the complement-independent, apoptosis-inducing properties of rATG on CD27- naive B cells, CD40 ligand-activated B cells, and plasma cells in vitro by annexin V staining, subdiploid DNA content, caspase activation, and loss of mitochondrial membrane polarity. Potential surface targets for rATG were assayed by competitive inhibition of monoclonal antibody binding. Rabbit ATG strongly induced apoptosis in vitro against naive, activated B cells and bone marrow resident plasma cells at clinically relevant concentrations (1-100 ng/mL). The authors found rATG activity against numerous B-cell surface proteins and observed that crosslinking of CD30, CD38, CD95, CD80, and HLA-DR likely accounts for this activity. F(ab)2 fragments of rATG showed 90% of the activity of the intact molecule, suggesting participation of the Fc fragment. Inhibition of caspase- and cathepsin-dependent apoptotic pathways partially inhibits rATG-induced B-cell apoptosis. Immunohistochemical staining of pediatric thymi demonstrated the presence of CD20+ B cells and CD138+ plasma cells within the thymic parenchyma, which accounts for the anti-B-cell activity in rATG. Polyclonal rATG induces complement-independent apoptosis of naive, activated, and plasma B cells. This effect appears to involve the caspase- and cathepsin-mediated apoptosis pathways.

C4d deposition in lung allografts is associated with circulating anti-HLA alloantibody

The complement activation demonstrated by vascular C4d deposition is used to diagnose antibody-mediated rejection (AMR) in renal allografts, but remains controversial in lung transplantation (LTX). Methods C4d deposition was assessed by immunohistochemistry in 192 lung transplant biopsies from 32 patients. ELISA analysis was performed on 415 serum samples in those 32 temporally and rejection-grade matched LTX patients; 16 patients developed HLA-Ab, while the other 16 patients remained negative. The specificity of C4d staining was further compared in 18 additional LTX patients without HLA-Ab or acute cellular rejection (ACR), but in the presence of CMV-pneumonitis or reperfusion injury. Results Specific subendothelial C4d deposition was seen in 5 of 16 (31%) patients with HLA-Ab and was absent in 16 patients without HLA-Ab ( p < 0.05). All patients with specific C4d deposition exhibited donor-specific HLA-Ab. There were 13 patients with bronchiolitis obliterans syndrome in the group of 16 HLA-Ab positive patients, versus 2/16 in ELISA-negative patients ( p < 0.005). One of 7 patients with CMV pneumonitis and 2 of 11 patients with reperfusion injury also showed C4d positivity (not statistically significant). Conclusions In this study, specific subendothelial C4d deposition was a marker for the involvement of HLA-Ab in lung allograft rejection. The patchy nature, low sensitivity, and specificity of C4d staining might limit clinical use in protocol biopsies. However, in patients with decreasing pulmonary function, refractory ACR and/or HLA-Ab, specific C4d deposition may serve as a marker of coexistent AMR.

Acute and Hyperacute Humoral Rejection in Kidney Allograft Recipients Treated with Anti-Human Thymocyte Antibodies

Equine and rabbit antihuman thymocyte globulins (ATGs) have been used in renal transplantation for prevention and treatment of acute rejection. We now report that hyperacute and acute antibody-mediated rejection of renal allografts occurred in three newly transplanted patients who received ATG for induction therapy. Antibody studies performed using complement-dependent cytotoxicity, flow cytometry, enzyme-linked immunosorbent assay, and Luminex yielded negative results for antilymphocytic and antiendothelial cell antibodies in the pretransplant sera obtained from these patients. ATG treatment was initiated at the time of transplantation. One of the patients experienced hyperacute rejection and required transplant nephrectomy within 24 h of transplantation. The other two patients developed acute antibody-mediated rejection within 14 days after transplantation. None of the patients developed antihuman leukocyte antigen antibodies when humoral rejection occurred. However, xenoantibodies that strongly bound to human lymphocytes and, importantly, to activated endothelial cells, were identified in the sera obtained at the time of humoral rejection. Hence, our results strongly implicate ATG in the induction of antibody-mediated rejection of kidney allografts. Flow cytometry testing of ATG reactivity to endothelial cells may be useful in identifying and discarding the ATG lots containing xenoantibodies that can bind to activated endothelial cells of the transplant.

Acute Humoral Rejection of Human Lung Allografts and Elevation of C4d in Bronchoalveolar Lavage Fluid

Antibody-mediated rejection is well established for renal allografts but remains controversial for lung allografts. Cardinal features of antibody-mediated rejection in renal allografts include antibodies to donor human leukocyte antigen (HLA) and evidence for antibody action, such as complement activation demonstrated by C4d deposition. We report a lung allograft recipient with circulating antibodies to donor HLA who failed treatment for acute cellular rejection but responded to therapy for humoral rejection. To address the second criteria for antibody-mediated rejection, we determined whether complement activation could be detected by measuring C4d in bronchoalveolar lavage fluid (BALF) by ELISA. Airway allergen challenge of asthmatics activates the complement pathway; therefore, we used BALF from asthmatics pre- and post-allergen challenge to measure C4d. These controls demonstrated that ELISA could detect increases in C4d after allergen challenge. BALF from the index patient had elevated C4d concomitant with graft dysfunction and anti-donor HLA in the absence of infection. Analysis of BALF from 25 additional lung allograft recipients showed that C4d concentrations >100 ng/mL were correlated with anti-HLA antibodies (p = 0.006), but were also observed with infection and in asyptomatic patients. The findings support the occurrence of anti-HLA-mediated lung allograft rejection and suggest that C4d measurement in BALF may be useful in diagnosis.

Antibody-Mediated Rejection of Human Renal Allografts: An Electron Microscopic Study of Peritubular Capillaries

The role of humoral rejection in acute and chronic rejection of human renal allografts other than in hyperacute rejection has not been well established, and its importance may be underestimated. Recently, a specific histological pattern of antibody-mediated rejection of renal allografts has been recognized. The antigens targeted by this mode of rejection are not well defined but are likely located on the endothelium of small vessels (arterioles and glomerular and peritubular capillaries). In both cellular and humoral rejection, the microvasculature of transplanted organs appears to be a main target of injury. This study describes the ultrastructural changes of peritubular capillaries, over a period of up to 8 months, in 14 biopsy specimens obtained from 5 renal allograft recipients diagnosed with “pure” antibody-mediated rejection. In peritubular capillaries, there is progression of injury from necrosis of endothelial cells with lifting and denudation of basement membrane to complete disappearance of capillaries. Acutely, acute tubular necrosis is a constant finding. At 2 to 3 months posttransplantation, the remaining capillaries are dilated, misshapen, and distorted, and are surrounded by a reduplicated and thickened basement membrane. These changes are associated with increased interstitial fibrosis and tubular atrophy, comparable to a sort of renal “asphyxial” death. The author concludes that in “pure” antibody-mediated rejection, the endothelium of peritubular capillaries is a main target of injury. The potential role of antibody-mediated rejection in acute and chronic rejection of renal allografts needs to be explored further.