C4d Staining Research Articles

Bortezomib for Management of Acute Antibody Mediated Rejection Post-Liver Transplantation

Introduction: Acute antibody mediated rejection (AMR) post liver transplaniont (LT) is uncommon and characterized by histopathologic features on liver biopsy, positive c4d stain, and elevated human leucocyte antigen (HLA) donor-specific antibodies (DSA). There is a paucity of literature on the use of Bortezomib for acute AMR in LT. Herein, we present our experience with Bortezomib for management of acute AMR post LT. Methods: Patients who underwent LT and developed acute AMR at a single center from January, 2014 to present were reviewed. Those treated with Bortezomib were included. Results: Two patients developed acute AMR post LT and both patients received Bortezomib for management of acute AMR. Patient 1: 62-year-old female who underwent LT for cirrhosis secondary to hepatitis C. 124 days after LT, patient developed abnormal liver function tests (LFT). Liver biopsy demonstrated acute on chronic rejection and c4d stain was positive. DSA was very high for HLA class II antibodies. DSA remained elevated after pulse steroids. Patient underwent 6 cycles of plasmapheresis (PP) and IVIG with persistently elevated DSA. 145 days after LT, she began Bortezomib (1.3mg/m2 IV) and PP. She received 6 cycles of therapy. DSA 6 days after completion of therapy showed trace HLA class II DSA. Liver biopsy performed 6 days after completion of therapy showed changes of chronic rejection however no cellular infiltrate and C4d stain was negative. Patient 2: 18-year-old male who underwent LT for chronic rejection in a liver graft. 2 days after LT, HLA class II DSA was elevated. Liver biopsy demonstrated a mixed inflammatory infiltrate and c4d stain was positive. He received pulse dose steroids and repeat DSA was elevated. 8 days after OLT, patient received Bortezomib (1.3mg/m2 IV) and PP. He received 4 cycles of therapy. 9 days after last dose of Bortezomib, liver biopsy showed decreased, yet persistent, inflammatory infiltration and c4d stain was positive. Class II DSA remains persistently elevated. Conclusion: Bortezomib appears to have a role in the management of acute AMR in LT recipients. While patient #2 continued to have persistent AMR, he only received 4 cycles of Bortezomib and PP as compared to 6 cycles for patient #1. The literature on the use of Bortezomib in this setting is limited to a single case series of 3 patients. While additional research is required, our study suggests that Bortezomib may be considered as salvage therapy in select patients with acute AMR.

Acute Liver Rejection After Exposure to Nivolumab in Treatment of Metastatic Hepatocellular Carcinoma

72 year old man with an orthotropic liver transplantation for hepatitis C with hepatocellular carcinoma (HCC) in 2012, complicated by metastatic HCC with a solitary lung lesion in 2015. Patient was initially treated with stereotactic body radiation with progression of disease in 2017 despite systemic therapy with sorafenib and regorafenib. Due to limited treatment options for progressive lung metastasis and excellent liver graft function, treatment with nivolumab was initiated. On routine monitoring, liver tests became elevated ten weeks after exposure to nivolumab with suspicion for either acute rejection or immune mediated hepatitis. Empiric treatment with oral steroids and an increased tacrolimus trough level to 5 ng/mL from 2-3 ng/ml failed to improve liver tests. A liver biopsy revealed lymphomonocytic infiltrate with endothelitis consistent with moderate acute rejection with Rejection Activity Index of 5. C4D staining was negative. Intravenous immunoglobulin 1 g/kg and methylprednisone 500 mg were employed without a durable response. Thymoglobulin 1.5 mg/kg/day with cumulative dose 6 mg/kg was started for steroid-resistant rejection with good response. Patient's liver enzymes trended down and normalized with addition of mycophenolate mofetil to tacrolimus to maintain remission. Recurrence rate of HCC is approximately 10% in five years in post-transplant population where transplant was performed due to HCC. Despite promising data in advanced HCC of immune checkpoint inhibitors (CPI) like nivolumab, an anti-PD-1 checkpoint inhibitor, these agents have not been studied in liver transplant recipients due to possibility of rejection and fatal liver failure. Current data regarding acute liver rejection caused by CPIs is scant and heterogenous. Most of the fatal rejections are found in the pediatrics population with aggressive fibrolamellar HCC and acute rejection happened within one week. There is a single case report of complete cancer remission with pembrolizumab, for metastatic HCC after live transplant. Here we report a case of acute liver rejection ten weeks after exposure to nivolumab that was resistant to steroids and IVIG. In this case, aggressive second line treatment with thymoglobulin was proven to be effective in the treatment of acute rejection. Factors that contribute to acute graft rejection with CPI use remain unknown and there is a need to develop regimens that optimize tumor response and minimize the risk of allograft rejection.2337_A Figure 1. Alanine aminotransferase/serum glutamic pyruvic transaminase trend2337_B Figure 2. Total bilirubin trend

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.

Molecular Assessment of C4d-Positive Renal Transplant Biopsies Without Evidence of Rejection

IntroductionImmunohistochemical staining for C4d in peritubular capillaries has been part of antibody-mediated rejection (AbMR) definition in the Banff Classification for Allograft Pathology since 2003. However, it has limited sensitivity and specificity, therefore the clinical significance of C4d-positive biopsies without evidence of rejection (C4d+ WER) is unknown. We investigated the transcript levels of genes associated with AbMR in C4d+ WER biopsies from both ABO-compatible and incompatible renal transplant patients.MethodsRNA was extracted from formalin-fixed paraffin-embedded renal transplant biopsies (n = 125) and gene expression analysis of 35 AbMR-associated transcripts carried out using the NanoString nCounter system.ResultsAbMR-associated transcripts were significantly increased in samples with AbMR or suspicious AbMR. A subgroup of 17 of 35 transcripts that best distinguished AbMR from C4d-negative biopsies without evidence of rejection was used to study C4d+ WER samples. There was no differential expression between C4d-negative and C4d+ WER from both ABO-incompatible and -compatible transplants. The geometric mean of 17 differentially expressed genes was used to assign the C4d+ WER biopsies a high- or low-AbMR transcript score. Follow-up biopsies showed AbMR within 1 year of initial biopsy in 5 of 7 high-AbMR transcript patients but only 2 of 46 low-AbMR transcript patients. In multivariate logistic regression analysis, elevated transcript levels in a C4d+ WER biopsy were associated with increased odds for biopsy-proven AbMR on follow-up (P = 0.032, odds ratio 16.318), whereas factors including donor-specific antibody (DSA) status and time since transplantation were not.ConclusionGene expression analysis in C4d+ WER samples has the potential to identify patients at higher risk of developing AbMR.

Clinical Relevance of C4d Deposition in Pediatric Immunoglobulin A Nephropathy

Introduction: Few studies have reported the association between the complement cascade and pediatric immunoglobulin A nephropathy (IgAN). This study aimed to investigate the association between C4d staining positivity and the clinical/histopathological characteristics of pediatric patients with IgAN. Methods: Children diagnosed with IgAN through renal biopsy were retrospectively reviewed. Renal biopsy specimens were stained using C4d immunohistochemistry. Results: Among the 56 patients, 31 (55.4%) showed positive mesangial or peripheral capillary C4d staining in glomeruli. Urine protein-to-creatinine ratio was significantly higher in C4d-positive patients (p = 0.001). The severity of mesangial proliferation according to the Haas and Oxford classification was positively associated with positive C4d staining (p < 0.001). Conclusion: Positive C4d staining was found to be significantly associated with the clinical/histopathological severity of IgAN.

Complement activation associated with ADAMTS13 deficiency may contribute to the characteristic glomerular manifestations in Upshaw-Schulman syndrome

IntroductionUpshaw-Schulman syndrome (USS) is a congenital form of thrombotic thrombocytopenic purpura (TTP) associated with loss-of-function mutations in the ADAMTS13 gene, possibly leading to aberrant complement activation and vascular injury. However, USS is extremely rare, and there have been no systematic studies correlating histopathological severity with local ADAMTS13 expression and complement activation. Materials and methodsHere, we compared histopathological features, ADAMTS13 immunoreactivity, and immunoreactivity of complement proteins C4d and C5b-9 among renal biopsy tissues from five USS cases, ten acquired TTP cases, and eleven controls. ResultsPathological analysis revealed chronic glomerular sclerotic changes in the majority of USS cases (4 of 5), with minor glomerular pathology in the remaining case. In two of these four severe cases, more than half of the glomerular segmental sclerosis area was localized in the perihilar region. The average number of ADAMTS13-positive cells per glomerulus was significantly lower in USS cases than controls (p < 0.05). Conversely, C4d staining was significantly more prevalent in the glomerular capillary walls of USS cases than controls (p < 0.05), while C5b-9 staining did not differ significantly among groups. ConclusionsThese findings suggest that the severity of glomerular injury in USS is associated with deficient ADAMTS13 expression and local complement activation, particularly in vascular regions with higher endothelial shear stress. We suggest that C4d immunostaining provides evidence for complement-mediated glomerular damage in USS.

P042 Accelerated antibody mediated rejection in a heart patient with weak donor specific antibodies present pre-transplant

AbstractWe present a case on a 46 year old Caucasian female patient with hypertrophic obstructive cardiomyopathy. The patient’s sensitization history included 3 pregnancies and a flu vaccination 2 months prior to transplant. The pre-transplant single antigen bead assay revealed weak reactivity to the Bw6 epitope including B7 (1,149 MFI) and B8 (775 MFI). The patient received a heart transplant in which B7 and B8 were crossed. As expected, the retrospective flow crossmatch was weakly positive. The patient was treated with Simulect on the day of transplant (Day 0) and with anti-thymoglobulin on Day 3 (Figure 1A). On Day 7, the patient went into cardiac arrest and was placed on extracorporeal membrane oxygenation (ECMO). The antibody screen on Day 7 revealed a large increase in donor specific antibodies (DSA) to B7 (21,694 MFI) and B8 (21,460 MFI). Positive ERG, CD68, and C4d staining on the Day 7 biopsy corroborated antibody mediated rejection (AMR). The patient was treated with plasma exchange, IVIG, Rituxan, and Cytoxan. At Day 14, ECMO was discontinued. DSAs declined to levels observed at pre-transplant (Figure 1B). Although antibody and biopsy results demonstrated a reduction in AMR, the patient became neutropenic and developed fever, bacteremia, a necrotic leg wound, and a fungal infection in the brain. She progressed to brain death. This case describes an amnestic response to weak DSA which contributed to cardiac arrest. Augmented immunosuppression used to treat an anamnestic AMR is associated with a significant risk of fatal infectious complications.▪

Intragraft vasculitis and gene expression analysis: Association with acute rejection and prediction of mortality in long-term heart transplantation.

Vasculitis entails heterogeneous origins; it starts with an inflammatory process that leads to small vessels' necrosis, hemorrhage, and ischemic lesion, and may further result in occlusion of the vascular lumen. Vasculitis' contribution to allograft rejection is still unclear. This study aims to investigate the incidence of vasculitis in the early stages of heart transplantation as well as to assess the intragraft genes' expression associated with vascular function and subsequently to verify the way in which it affects the outcome of the allograft. In this retrospective study, 300 archive paraffin-embedded endomyocardial biopsies from 63 heart allograft recipients were assessed. Cellular rejection and vasculitis were diagnosed through histological analysis, and antibody-mediated rejection was performed with immunohistochemical C4d staining. The transcripts of ICAM, VCAM, VEGF, CCL2, IFNG, TGFB, TNF, ADIPOR1, and ADIPOR2 genes were examined through quantitative polymerase chain reaction using B2M for normalization. We observed a higher prevalence of severe vasculitis in the early period of post-transplant, and recovery was observed to take place around 1year post-transplant. Additionally, vasculitis was found to be directly associated with acute cellular rejection and antibody-mediated rejection. The intense C4d capillary positivity predicts higher long-term cardiovascular disease mortality. In comparison with the vasculitis-free group, the group with severe vasculitis displayed reduced left ventricular ejection fraction and an upregulation of VCAM and IFNG associated with the downregulation of VEGF, ADIPOR1, and ADIPOR2. The vasculitis associated with the presence of C4d and the change in intragraft gene expression profile may contribute to poor allograft outcomes.

P054 Unexpected quiescence of expected memory response against prior kidney allografts – Novel lessons learned from an 11 year old recipient of 3 kidney transplants

We present an 11 yr old girl who received 3 kidney transplants (KTx). She received 1st-KTx from a living donor at age 4 due to the end-stage renal disease. She had no HLA antibodies (Ab) at pre-1st-KTx, and thus pre-tx crossmatch (XM) and donor-specific HLA antibodies (DSA) were −ve. Six weeks later, the allograft was removed due to torsion (twisting of blood vessels) and thrombosis (formation of a blood clot). The C4d staining of Tx-biopsy was −ve indicating no antibody-mediated rejection (ABMR). She was relisted for the 2nd-KTx. Two-mo post tx-nephrectomy sample revealed Abs to all 10 mismatched (MM) donor’s HLA types (and associated CREG) resulting in 97% CPRA. After a 3 yr wait, she received 2nd-KTx from a deceased donor (DD) with −ve XM and DQ8 DSA (MFI = 1961). Shortly, she developed complications by BK viremia/nephropathy and immunosuppression was reduced, which potentially increased alloimmune activation. At 2-mo post-2nd-KTx, she developed de novo DSAs directed against 4 mismatched HLAs of 2nd-KTx (1600–14600 MFI). She was treated with multiple rounds of eculizumab, plasmapheresis and IVIG until Dec. 2015, when she presented with an acute increase in serum creatinine to 8.7 mg/dL. KTx biopsy showed acute ABMR. She was relisted for 3rd-KTx on Feb. 2016 with a cPRA 100%. After 2 yr wait, she received 3rd-KTx on Feb. 2018 from a DD with −ve XM and no DSA. The 3rd donor was mismatched by 8 HLA antigens - 1 of them was a repeat-MM with 1st donor and another was repeat-MM with both 1st and 2nd donors. However, Abs to 5 mismatched HLAs of the 3rd-KTx (B8, B37, Cw6, DP2, DP4) found in the historical sera (MFIs 1000–16,000) were completely −ve in multiple sera tested during 2 mo post-3rd-KTx (Fig.). No rejection was noted. Lessons learned from this case were: A 4-yr old human immune system can recognize and respond to alloantigens, and can trigger HLA-Abs production efficiently. However, acquired memory response, an integral component of adaptive immunity is not fully developed until 11 yr of age. These findings have vital implications in pediatric KTxs. Download : Download high-res image (508KB) Download : Download full-size image

Graft dysfunction in simultaneous pancreas kidney transplantation (SPK): Results of concurrent kidney and pancreas allograft biopsies.

Simultaneous pancreas and kidney transplants offer significant therapeutic advantages but present a diagnostic approach dilemma in the diagnosis of rejection. Because both organs are from the same donor, the kidney has been treated traditionally as the "sentinel" organ to biopsy, presumably representing the status of both allografts. Truly concurrent biopsy studies, however, are needed to confirm this hypothesis. We examined 101 concurrent biopsies from 70 patients with dysfunction in either or both organs. Results showed concurrent rejection in 23 of 57 (40%) of cases with rejection; 19 of 57 (33.5%) and 15 of 57 (26.5%) showed kidney or pancreas only rejection, respectively. The degree and type of rejection differed in the majority (13 of 23, 56.5%) of cases with concurrent rejection, with the pancreas more often showing higher rejection grade. Taking into account pancreas dysfunction, a positive kidney biopsy should correctly predict pancreas rejection in 86% of the instances. However, the lack of complete concordance between the 2 organs, the discrepancies in grade and type of rejection, and the tendency for higher rejection grades in concurrent or pancreas only rejections, all support the rationale for pancreas biopsies. The latter provide additional data on the overall status of the organ, as well as information on nonrejection-related pathologies.

Banff survey on antibody-mediated rejection clinical practices in kidney transplantation: Diagnostic misinterpretation has potential therapeutic implications.

The aim of this study was to determine how the Banff antibody-mediated rejection (ABMR) classification for kidney transplantation is interpreted in practice and affects therapy. The Banff Antibody-Mediated Injury Workgroup electronically surveyed clinicians and pathologists worldwide regarding diagnosis and treatment for 6 case-based scenarios. The participants' (95 clinicians and 72 renal pathologists) assigned diagnoses were compared to the Banff intended diagnoses (reference standard). The assigned diagnoses and reference standard differed by 26.1% (SD 28.1%) for pathologists and 34.5% (SD 23.3%) for clinicians. The greatest discordance between the reference standard and clinicians' diagnosis was when histologic features of ABMR were present but donor-specific antibody was undetected (49.4% [43/87]). For pathologists, the greatest discordance was in the case of acute/active ABMR C4d staining negative in a positive crossmatch transplant recipient (33.8% [23/68]). Treatment approaches were heterogeneous but linked to the assigned diagnosis. When acute/active ABMR was diagnosed by the clinician, treatment was recommended 95.3% (SD 18.4%) of the time vs only 77.7% (SD 39.2%) of the time when chronic active ABMR was diagnosed (P<.0001). In conclusion, the Banff ABMR classification is vulnerable to misinterpretation, which potentially has patient management implications. Continued efforts are needed to improve the understanding and standardized application of ABMR classification in the transplant community.

Pattern of Glomerular Staining for C4d Immunohistochemistry is Useful for the Diagnosis of Immune Complex Mediated Glomerulonephritis in Renal Allograft Biopsies

Introduction Performance of the C4d stain is required for the adequate evaluation of kidney transplant biopsies (KTBx). Positive C4d staining in the renal peritubular capillaries is very useful for the diagnosis of antibody mediated allograft rejection (AMR) and correlates with poorer graft outcomes. Glomerular C4d staining is common in the chronic phases of AMR but this is not diagnostically useful due to inconsistent distribution and intensity of staining. Deposition of the C4d complement component in immune complex mediated (ICM) glomerulonephritis (GN) is expected but this has not been systematically studied in KTBx. Materials and Methods C4d stain (immunohistochemical method) was applied to 2432 consecutive, paraffin embedded KTBx, over a period of 5 years. In KTBx due to abrupt increase in proteinuria or history of GN in the native kidney, immunofluorescence and electron microscopy studies for standard evaluation of GN were also used for diagnosis. Results and Discussion In this unselected biopsy cohort, 33 KTBx from 27 patients showed recurrent or primary GN: 12 membranous glomerulopathy (MGN), 4 lupus nephritis (SLE), 9 IgA nephropathy and 8 focal segmental glomerulosclerosis (FSGS), Distinctive and consistent glomerular C4d stainining was noted in patients with MGN with a pattern paralleling the IF and EM findings. SImilarly, in recurrnet SLE, glomerular C4d staining corresponded to the histological lupus Class determined by light microscopy, IF and EM. The C4d staining pattern in these GNs was clearly different from that seen in transplant glomerulopathy. C4d staining in IgA nephropathy was inconsistent, mostly mesangial and did not correlate clearly with the strength of IF IgA staining. Only the segmental sclerotic lesions in FSGS were highlighted by the C4d staining in a manner that appeared nonspecific. Conclusions C4d staining is routinely evaluated in transplant biopsies and this stain consistently highlights immune complex deposits in MGN and SLE. Recognition of this distinctive staining pattern is helpful in the diagnosis of these processes and should prompt complete evaluation with IF and EM if these studies were not initially available. The findings in this study are based on C4d stain evaluation of paraffin embedded tissue sections. Since C4d staining corresponds to the classical pathway of complement activation, similar findings are expected with the IF staining method in fresh frozen tissue sections.

Humoral Rejection in Lung Transplantation

Introduction The diagnosis of humoral rejection in lung transplant recipients is sometimes elusive, involving histopathologic (capillaritis), immunopathologic (C4d staining), and serologic (donor-specific anti-HLA antibodies, DSA) parameters. These criteria are not always present in cases and, moreover, the involvement of non-HLA antibodies in lung-allograft humoral rejection remains largely unclear. The aim of the study was to assess the involvement of HLA and non-HLA antibodies in humoral rejection diagnosis after lung transplantation. Materials and Methods A total of 60 consecutive lung transplant recipients were followed in our Institution from 2015. Protocol biopsy at day 21 post transplantation and screening for serum HLA and non-HLA antibodies by Luminex, LABScreen Autoantibody (One Lambda), before lung transplant and at the moment of the biopsy, were performed. Results and Discussion A total of 5 lung transplant recipients (8.33%) were C4d+ on biopsy but only one of them developed de novo DSA without evidence of non-HLA antibodies. The remaining 4 patients C4d+, were serum positive for non-HLA antibodies (2 against LG3 and other 2 against myosin) prior lung transplantation (6.67%) but negative at the moment of the biopsy. The incidence observed of preformed non-HLA antibodies in patients awaiting for lung transplantation was significant and potentially could drive C4d deposition on lung tissue with subsequent humoral rejection. Conclusion The screening of non-HLA and HLA antibodies could help to identify patient at risk of lung-allograft rejection.

Significance of Antibody-Mediated Vascular Rejection (AMVR) on Graft Survival

Introduction Traditionally, endarteritis accepted to associate with cellular rejection. Nevertheless, some of the patients with AMR also had endarteritis at the same time. The risk of graft loss was nine times higher in AMVR than T cell-mediated rejection without endarteritis. However, the importance of the endarteritis on the graft survival in recipients with AMR is controversial. Thus we aimed to understand the prognostic value of the presence of endarteritis in recipients with AMR. Materials and Methods Among 155 recipients 72 (46,5%)had pure AMR (Group 1) while 83 (53,5%) had both AMR and endarteritis (Group 2). Endarteritis graded according to Banff. First indication biopsies of all cases reevaluated and the intensity of interstitial, glomerular and peritubular capillary (PTC) inflammation, neutrophil, macrophage and lymphocyte infiltration graded. HLA-DR expression in the arteries, PTCs, and tubules examined. The loss of DR expression on PTCs accepted as the destruction of PTCs. Follow-up biopsies analyzed for the development of interstitial fibrosis (IF), transplant glomerulopathy (TG) and transplant arteriopathy (TA). Results The extensity of the PTC and arterial C4d expression and the degree of PTC destruction found higher in Group 2 compared to Group 1 (P<.001).The degree of the interstitial eosinophil, plasma, and macrophage infiltration found higher in Group 2 patients than Group 1 (p<.001). Also, the intensity of inflammatory cells and neutrophils in both glomeruli and PTCs found higher in Group 2 compared to Group 1 (p<.001). Compared to Group 1 patients, Group 2 patients showed a higher incidence of IF, TG and TA in follow-up biopsies (p<.01).The development of IF and TG increases with the increasing degree of glomerulitis, PTC-itis, C4d expression and PTC destruction (p<.01). Also, the time of the development of IF and TG decreased with increasing intensity of PTC and interstitial infiltration, glomerulitis, PTC destruction and C4d expression (p<.01). The presence of the eosinophil and macrophage infiltration on the wall of arteries of recipients with AMVR rises the risk of the development of TG and TA (p<.01). Also, patients who had arterial C4d staining had a higher risk of TG, TA and graft loss (p<.001). The response to rejection therapy was lower in Group 2 recipients compared to Group 1 patients (p<.001). Overall the 3- and 5-year graft survival was 98%, and 85% respectively for Group 1 patients while it was 57%, and 27% respectively for Group 2 (p<.001). Conclusion Our results have underlined the importance of the presence of endarteritis in AMR. We showed that the course of AMVR are noticeably different from pure AMR, with AMVR having the worst outcome through leading the early development of IF, TG, and TA via augmenting inflammatory and fibrotic pathways. Thus the development of new treatment strategies for AMVR could salvage many kidney allografts.

Do C1q-Positive Donor Specific Antibodies have Prognostic Effect in Patients with Resistant Antibody-Mediated Rejection Treated with Bortezomib? Single-Centre Experience

Introduction and Aims Donor-specific antibodies (DSAs) examination is a crucial part of antibody-mediated rejection (AMR) diagnosis. C1q-positive (+) DSAs are associated with poor graft survival. We analyzed results of patients (pts) treated for acute resistant AMR by a bortezomib-based regimen with retrospectively performed C1q assay. The aim of this work was to analyze the treatment effect on DSA levels and potential effect of C1q+ DSAs on graft survival. Methods We retrospectively analyzed documentation of 772 pts who underwent renal transplantation (Tx) between 1/2012-6/2015. Novel therapeutic approach to resistant acute AMR in kidney transplant recipients was applied in 23 pts (3%) based on administration of bortezomib (B) [1 cycle of 4 doses of B (1.3 mg/m2)], small doses of i.v. steroids, plasmapheresis (PP) and a dose of rituximab (375mg/m2). This protocol was administered after conventional treatment had failed. Resistant AMR was defined as persisting deterioration or non-function of renal allograft in pts with histological verification of AMR, positive C4d staining and detection of DSA receiving standard antirejection treatment with PP + IVIG. C1q assay was performed 3 times – before Tx, at the time of diagnosis and after AMR treatment. Pts were followed for minimum of 24 months. Results Therapy of resistant acute AMR was administered to 23 pts after kidney Tx with median peak PRA 52%, actual PRA 36%, mean HLA mismatch in HLA-A 1.2 ± 0.4, HLA-B 1.7 ± 0.5, HLA-DR 1.3 ± 0, with median of 5.8 years on dialysis. 3 pts underwent 1st kidney Tx, while 20 pts reTx. All pts received induction therapy with antithymocyte globulin (n=22) or basiliximab (n=1), and maintenance immunosuppression with tacrolimus, mycophenolate mofetil/enteric-coated mycophenolate sodium and corticosteroids. Diagnosis of resistant acute AMR was made on 14th POD (7- 60 days). Using bortezomib regimen in treating resistant acute AMR led to decrease in DSA quantity in HLA especially in class I (p=0.005), class II (p = 0.015), but not in DQ (p= 0.2). No significant improvement of renal function was observed during the follow-up. The pts whose levels of serum creatinine increased more than 25% of baseline level in 6 months after administration of protocol with B, are progressors (n=11). The progressors graft survival was 57% in 2 years. The treatment protocol was not effective in decreasing C1q+ DSAs class I (p=0.25), class II (p=0.69), DQ (p=0.58). We detected C1q+ in 11 out of 11 progressors, compared with 3 out of 12 non-progressors (p<0.001). C1q+ DSAs correlated with C4d+ in graft biopsies at the time of AMR diagnosis (p=0.0012). Conclusions Bortezomib-based regimen was effective against class I and II DSAs, but it did not affect DQ DSAs. Pts with any C1q+ DSAs were more likely to have progressive graft dysfunction compared to those with C1q-negative DSAs. C1q positivity is a promising prognostic marker in pts with resistant AMR.

Ultrastructural Changes in Lung Allografts

Introduction Antibody mediated rejection (AMR) is a well-recognised cause of allograft injury; however only recently there is a growing recognition in lung transplantation (LTx). Allograft dysfunction can be multifactorial and challenges with C4d stain interpretation and the lack of specificity of histological criteria account for some of the difficulties in diagnosing pulmonary AMR. Donor Specific Antibodies (DSA) target the endothelium. In fact, in renal transplantation, endothelial activation markers and early ultrastructural changes of the endothelium correlate with AMR. The aim of this study was to identify ultrastructural changes in the endothelium of lung allografts in the presence of DSA, with the intent of determining if prospective use of electron microscopy might be helpful in establishing a diagnosis of AMR in LTx recipients. Materials & Methods The LTx database was queried for cases of pulmonary AMR between 07/2009 & 09/2015. The ISHLT guidelines were used to categorise these cases into definite, probable & possible AMR. Retrospective ultrastructural studies were performed on all these biopsies- either fresh or paraffin embedded. Cases of acute cellular rejection (4) and normal lung (3) were evaluated as controls. The findings on ultrastructural examination were correlated with the presence of DSA & diagnosis of AMR using the Pearson’s chi-square test of statistical significance. Results & Discussion Eleven patients (male, 54.5%) were included in this study.Mean age was 53yrs. Three cases were combined lung & heart Tx. We identified 7 cases of AMR (1 definite, 1 probable & 5 possible). The median time from LTx to biopsy (bx) was 210 days, with median follow up of 1063 days. Bronchiolitis Obliterans Syndrome was diagnosed in 5 patients. Ultrastructural studies were performed on 10 AMR bxs (pt 6:2 bxs; pt 7:3 bxs; all other pts: 1bx each), 4 ACR bxs & 4 controls. The results are shown.The presence of DSA as well as the ISHLT diagnosis of AMR significantly correlated with endothelial swelling, cytoplasmic vacuolisation & septal edema (p<0.01; each). Septal infiltration by neutrophils was only identified in AMR cases, although it did not achieve statistical significance. Importantly, these differences were still significant when using resin or paraffin based samples for EM, allowing larger retrospective studies to be performed; although endothelial swelling & vacuolisation occurred in non-DSA paraffin samples with EM.Conclusion Donor specific anti-HLA antibodies are associated with endothelial injury of the allograft. Ultrastructural findings such as endothelial swelling & vacuolization help in diagnosing antibody mediated allograft injury. Although paraffin embedded biopsy material can be used for this purpose, it is not without limitations and larger prospective studies are needed to validate these findings. The effect of DSA on the endothelial pathways will be important in all solid organ transplantation.

Concurrent Antibody-Mediated Rejection in Post-Transplant IgA Nephropathy – Diagnosis Using a Stepwise Approach of Graft Morphology

Background Diagnosis of concurrent antibody-mediated rejection (AMR) is difficult in cases of post-transplant IgA nephropathy (TxIgAN) with glomerular inflammation. Glomerular inflammatory cell infiltration may be seen either in the contexts of AMR or immune complex-mediated injury and described as ‘glomerulitis’ (‘G’) or ‘endocapillary hypercellularity’ (‘E’), respectively. Methods We analyzed 40 renal allograft biopsy samples of 37 TxIgAN patients having glomerular inflammatory cells (‘E’ and/or ‘G’) between January 1992 and December 2016, in search for ancillary morphologic features in the diagnosis of concurrent AMR. Results Glomerular inflammation was described as ‘G’, ‘E’, or both in 11, 14, and 15 biopsies, respectively. Morphologic features supportive of acute and chronic AMR (AAMR and CAMR, respectively), comprising peritubular capillaritis, glomerular basement membrane doubling, peritubular capillary basement membrane multilayering, and peritubular capillary C4d staining, were sequentially examined, which revealed nine AAMR and 14 CAMR. C4d was positive in 77.8% of AAMR and 42.9% of CAMR. Of the remaining 17 biopsy samples without AMR, mesangial hypercellularity and/or crescents were present in 14 samples. In four samples, acute T cell-mediated rejection coexisted. Conclusions Diagnosis of concurrent AMR can be aided by using a stepwise approach of morphologic features of AMR in cases of TxIgAN with glomerular inflammation.

Donor Specific Antibodies are Associated with more Inflammation, More Fibrosis and Higher Expression of Rejection Associated Transcripts in Subclinically Rejected Liver Allografts

Background and Aims Subclinical rejection (SCR) is a common event in protocol biopsies after liver transplantation and has a good medium-term prognosis even if left untreated. We recently found hints for an intrahepatic regulation of cytotoxic T cells by regulatory T cells (Treg) in SCR. However, the role of donor-specific anti-HLA antibodies (DSA) has not been investigated in this setting. Material and Methods We included all liver biopsies with SCR (rejection activity index (RAI)≥1+1+1; liver enzymes (ALT, AST, AP)≤2x upper limit of normal (ULN)) from virus negative patients in our program. Comparator cohorts consisted of biopsies with acute cellular rejection (ACR: RAI≥3; liver enzymes >2x ULN), and no histological rejection (NHR: RAI<2, normal liver enzymes). RT-PCR was performed from 77 liver biopsies with gene panels using 90 genes for rejection, endothelial cell activation, operational tolerance, T cell exhaustion and immune regulation. DSA were detected with bead assays. DSA positivity was analyzed using an MFI threshold of 1000 or more. Results and Discussion Graft gene expression in SCR biopsies broadly overlapped with the distinct expression pattern from ACR and NHR in a cluster analysis of RT-PCR results (p<0.05 and q<0.08). Thereby, SCR was characterized rather by an intermediate expression level of those genes and molecular pathways that were upregulated in ACR than by a unique set of genes or pathways. However, expression levels of GARP, a marker for activated Treg, but not of FOXP3, the Treg lineage marker, were higher in grafts with SCR compared ACR. Additionally, granzyme B, an effector molecule of cytotoxic lymphocytes, was downregulated in SCR compared to ACR, while CD8 was not significantly different in SCR and ACR. Next, the influence of DSA on the graft gene expression was evaluated, because the graft gene expression in SCR was inhomogeneous. DSAs were found in paired blood samples in 30% of SCR, 8% of NHR and 47% of ACR biopsies, while the overall DSAs frequency was 27% in our biopsy program. DSA+ SCR biopsies had significantly higher scores for lobular and portal inflammation, central perivenulitis and perisinusoidal fibrosis compared to DSA- SCR biopsies. Thereby, rejection associated transcripts were significantly overexpressed in DSA+ compared to DSA- SCR biopsies. No DSA+ liver biopsy showed a relevant C4d staining with our protocols. Nonetheless, eight SCR biopsies (10% of all SCR biopsies, 43% of DSA+ SCR biopsies) additionally fulfilled the 2016 Banff criteria for possible chronic antibody mediated rejection (cAMR). However, the biopsies numbers are still too low for prognostic evaluations. Conclusions SCR overlaps transcriptionally with NHR and ACR. Thereby, DSA seems to mark SCR biopsies with more inflammation and fibrosis. The combination of SCR in biopsies with DSA positivity might therefore identify patients with more pronounced graft inflammation, which might require a change in immunosuppression.

Lupus nephritis: the roles of C1q and C3 in preventing antibody mediated injury

Abstract Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). While defective clearance of apoptotic cells (AC) and immune complexes (IC) is implicated in LN pathogenesis, the precise way in which different complement components impact kidney protection and injury is unknown. To address this, we created C57BL/6 mice with defective clearance of AC (Mfge8 deficiency) and anti-chromatin antibodies (sle1 interval) that were also deficient in either C1q [C1q Triple mutant (C1qTM)] or C3 (C3TM). Both C1q and C3 TM mice spontaneously developed anti-DNA antibodies and glomerulonephritis associated with glomerular IgG deposits after the age of 6 months. To dissociate the effects of complement on B cells versus effects on the kidney, we examined the effects of antibody mediated kidney injury (nephrotoxic nephritis, NTN) in mice deficient in the clearance of AC with complement deficiency [double knockout (DKO) (Mfge8 C1q or Mfge8 C3) mice]. In NTN, 50% of C1q DKO and 25% of C3 DKO, but no Mfge8 KO, died after NTN induction and the surviving C1q DKO mice developed severe proteinuria. Surprisingly, both spontaneous and NTN-induced nephritis in C1q TM or C1q DKO had strong glomerular C3 and C3d staining, while strong staining for the membrane attack complex (MAC) was observed in C3 TM or C3 DKO mice. These findings indicate that: 1) C1q and C3 independently protect against kidney injury separate from their effects on B cell function 2) In the presence of excess AC and low C1q, antibody mediated kidney injury is associated with C3 activation, presumably by the lectin or alternative pathway and 3) In the presence of excess AC and low C3, antibodies can cause MAC activation by pathways that remain to be determined.

Recurrent Membranous Nephropathy After Kidney Transplantation Associated With Phospholipase A2 Receptor and Successfully Treated With Rituximab: A Case Report

Primary membranous nephropathy (MN) is an organ-specific autoimmune disease mainly caused by autoantibodies acting against the podocyte antigen M-type phospholipase A2 receptor 1 (PLA2R). Herein we present the clinical and histologic findings, including PLA2R staining, of early recurrent MN after kidney transplantation that was successfully treated with rituximab.A 60-year-old Japanese man had end-stage renal failure due to steroid-resistant primary MN and underwent ABO-incompatible living donor kidney transplantation. At 1 month after transplantation, a protocol biopsy revealed positive granular staining of IgG, C4d, and PLA2R on glomerular capillaries (GCs) without any abnormalities on light microscopy (LM). Although the patient had low-level proteinuria, recurrent MN was suspected based on the positive PLA2R staining; he was treated with an angiotensin receptor blocker and a single dose of 200 mg rituximab. However, proteinuria gradually increased to 877 mg/d. At 21 months after transplantation, a graft biopsy revealed spikes along the outer aspects of GC on LM, with stronger staining for PLA2R than that at 1 month after transplantation. A single dose of 500 mg rituximab was added, which effectively reduced proteinuria, and clinical remission continued until 3 years after transplantation. The latest graft biopsy showed reduced staining of PLA2R. The disease activity and therapeutic effect were well-reflected in the intensity of PLA2R staining.An approach intending an early diagnosis by protocol biopsy using PLA2R immunostaining is made and early treatment with rituximab will help reduce the risk of kidney graft loss due to recurrent primary MN.