Marker Of Antibody-mediated Rejection Research Articles

Utility of C4d Immunohistochemistry as an Adjunct Stain in Diagnostic Renal Pathology of Glomerular Diseases.

C4d is a byproduct of the activation of the classic and lectin complement pathways. Being routinely used as a marker for antibody-mediated rejection, the significance of C4d in native kidney disease is currently being widely studied. We evaluated glomerular and extraglomerular C4d staining in 82 biopsies of proliferative and nonproliferative glomerulonephritis diagnosed in our institution. The staining pattern of C4d was tabulated in various glomerular diseases. All biopsies of membranous nephropathy including membranous lupus nephritis (Class V) and immune complex-mediated membranoproliferative glomerulonephritis (MPGN) consistently showed C4d deposits along glomerular basement membrane mirroring the location of immunoglobulin and complement in these conditions. Conversely, other glomerular diseases like IgA nephropathy, postinfectious glomerulonephritis, focal segmental glomerulosclerosis, minimal change disease, and diabetic nephropathy showed variable mesangial and capillary wall C4d deposits. To summarize, the consistent pattern of C4d staining in membranous nephropathy (primary and secondary)and immune complex-mediated MPGN can be used as a valuable adjunct tool in establishing the diagnosis, especially when immunofluorescence findings are limited by inadequate sampling.C4d reactivity in other glomerular diseases are variable and may not aid as a diagnostic tool in renal biopsy evaluation.

Anti-C4d chimeric antigen receptor regulatory T cells suppressed allograft rejection in ABO-incompatible heart transplantation

Abstract Antibody-mediated rejection (ABMR) is one of the main obstacles to successful transplantation, including ABO blood group-incompatible (ABOi) transplantation. Recently, chimeric antigen receptor regulatory T cells (CAR Tregs) were developed to improve the antigen specificity, viability, and suppressive activity of Tregs. C4d deposition is a marker of ABMR and is also found in most ABOi allograft tissues. Based on these findings, we developed anti-C4d CAR Tregs to suppress ABMR in ABOi allografts. Anti-C4d CAR Tregs prepared by retroviral transduction of CAR into CD62L + CD4 + CD25 + Tregs, expressed Foxp3, CD25, CTLA-4, LAP, and GITR to similar extents as non-transduced Tregs. Anti-C4d CAR Tregs were activated by specific binding to C4d and suppressed in vitro T cell proliferation as well as nontransduced Tregs. Furthermore, adoptive transfer of anti-C4d CAR Tregs significantly prolonged mouse ABOi heart allograft survival (P

Circulating Donor Heart Exosome Profiling Enables Noninvasive Detection of Antibody-mediated Rejection.

Endomyocardial biopsy remains the gold standard for distinguishing types of immunologic injury-acute versus antibody-mediated rejection (AMR). Exosomes are tissue-specific extracellular microvesicles released by many cell types, including transplanted heart. Circulating transplant heart exosomes express donor-specific human leukocyte antigen (HLA) I molecules. As AMR is mediated by antibodies to donor HLAs, we proposed that complement deposition that occurs with AMR at tissue level would also occur on circulating donor heart exosomes. Plasma exosomes in 4 patients were isolated by column chromatography and ultracentrifugation. Donor heart exosomes were purified using anti-donor HLA I antibody beads and complement C4d protein expression was assessed in this subset as marker for AMR. Three patients had no rejection episodes. Circulating donor heart exosomes showed troponin protein and mRNA expression at all follow-up time points. One patient developed AMR on day 14 endomyocardial biopsy that was treated with rituximab, IVIG/plasmapheresis. Time-specific detection of C4d protein was seen in donor heart exosome subset in this patient, which resolved with treatment. C4d was not seen in other 3 patients' donor exosomes. Anti-donor HLA I specificity enables characterization of circulating donor heart exosomes in the clinical setting. Further characterization may open the window to noninvasively diagnose rejection type, such as AMR.

Donor-derived cell-free DNA as a biomarker for rejection after kidney transplantation: a systematic review and meta-analysis.

A systematic review and meta-analysis were performed to investigate the value of donor-derived cell-free DNA (dd-cfDNA) as a noninvasive biomarker in diagnosing kidney allograft rejection. We searched PubMed, Web of Science and the Cochrane Library for original research papers published between January 1994 and May 2020 on dd-cfDNA fractions in blood of kidney allograft recipients. A single-group meta-analysis was performed by computing pooled estimates for dd-cfDNA fractions using the weighted median of medians or quantile estimation (QE) approach. Weighted median differences in medians (WMDMs) and median differences based on the QE method were used for pairwise comparisons. Despite heterogeneity among the selected studies, the meta-analysis revealed significantly higher median dd-cfDNA fractions in patients with antibody-mediated rejection (ABMR) than patients without rejection or patients with stable graft function. When comparing patients with T cell-mediated rejection (TCMR) and patients with ABMR, our two statistical approaches revealed conflicting results. Patients with TCMR did not have different median dd-cfDNA fractions than patients without rejection or patients with stable graft function. dd-cfDNA may be a useful marker for ABMR, but probably not for TCMR.

Upregulation of Class II HLA as a Marker of Antibody-Mediated Rejection in Heart Allografts

In 2013, the ISHLT introduced the working classification for pathological changes associated with antibody-mediated rejection of the heart allograft, known as p-AMR. With two components H, for histologic changes, and I for immunolabeled markers associated with AMR, the proposed classification also suggests to use Class II HLA as a marker of endothelial integrity. We hypothesized that Class II HLA rather than a marker of mere endothelial presence, is a marker of endothelial activation, and therefore could be associated with the diagnosis of AMR. 838 heart allograft biopsies, collected from January 2016 to September 2018 at a single institution were evaluated for both morphologic (H/E) and immunolabeled changes of AMR. Biopsies were immunolabeled with immunofluorescence with antibodies against C4d, and for immunohistochemistry with antibodies against C3d, CD68 and Class II HLA. ISHLT criteria was used to classify the biopsies, and for Class II HLA, both the percentage and the stain intensity were recorded. 74.8% of biopsies showed either a H, I or a combined evidence of AMR. The percentage of endothelial HLA Class II staining was significantly correlated with the diagnosis of AMR (r=0.39). HLA Class II showed a spectrum of intensity, quantified as negative (0) to strongly positive (+3). The diagnosis of AMR significantly correlated with moderate (+2) and strong (+3) staining intensity for Class II HLA, as follow: H+I (OR 28.3, p 0.0001); H alone (OR 22.73, p 0.0001) and I alone (32.63, p 0.0001). Our study also confirmed the value of the other AMR markers proposed by the ISHLT, such as C4d, C3d and CD68 (all p 0.0001), but importantly, and in contrast to prior observations, we showed that the C4d focally positive category (10-50%) is also significantly correlated to the diagnosis of AMR (OR 12.25, p 0.0005). We confirmed our hypothesis that in heart allograft biopsies there is a spectrum of both percentage and intensity of HLA Class II in endothelial cells, and that its staining by IHC is a marker significantly correlated with the diagnosis of AMR. In addition, the group of focally positive C4d biopsies should be considered positive for the I component of the 2013 ISHLT classification, as this group of biopsies also correlated with the diagnosis of AMR.

RNA Expression Analysis of Antibody Mediated Rejection after Heart Transplant with or without Donor Specific Antibodies

Antibody mediated rejection (AMR) is an increasingly studied cause of graft failure after heart transplantation. AMR diagnostic criteria previously required serum donor specific antibodies (DSA). However, the most recent criteria only requires pathological findings independent of DSA. This classification defined a subset of patients with AMR without known antibodies. To investigate this complication, we performed RNA sequencing using endomyocardial biopsy samples taken at the time of pathology confirmed AMR in the presence and absence of DSA. We evaluated the transcriptome profile of the paraffin-embedded endomyocardial biopsy specimens of 30 representative heart transplant recipients. Patients were categorized as having no evidence of AMR (controls), immunohistochemical findings of AMR (pAMR1i) in the presence of DSA (1i+D), pAMR1i in the absence of DSA (1i-D), histopathological findings of AMR (pAMR1h) in the presence of DSA (1h+D), pAMR1h in the absence of DSA (1h-D) or presence of both immunohistochemical and histopathological findings of AMR (pAMR2) in the presence of DSA. A total of 5 patients were analyzed per group. All were matched for previous mechanical circulatory support and time from transplant to biopsy. There was no differential RNA expression between patients with no AMR and those in the 1i-D group. There was also no differential expression between patients in the 1h+D and pAMR2 groups. The largest difference was seen between the no AMR and 1i+D groups and the no AMR and pAMR2 groups. In addition, there was RNA expression differences between patients with immunohistochemical evidence of AMR compared to those with histopathological findings, suggesting distinct mechanisms. These studies demonstrate that RNA expression profiling can be performed on endomyocardial biopsy tissue preserved in paraffin from the time of a rejection episode and that different expression patterns are observed dependent on the criteria used to diagnose AMR. In particular, AMR in the presence of DSA showed a different RNA expression profile than AMR without DSAs. The RNA expression associated with histological markers of AMR also appeared different than that associated with immunological markers. Overall, our findings suggest the existence of different subtypes of AMR that may rely on distinct mechanisms of injury.

Upregulation of Endothelial HLA Class II is a Marker of Antibody-Mediated Rejection in Heart Allograft Biopsies

In 2013, the International Society of Heart and Lung Transplant (ISHLT) introduced the working classification for pathologic changes associated with antibody-mediated rejection (AMR) of the heart allograft, known as pathologic AMR (pAMR). With 2 components associated with AMR, histopathologic changes) and immunopathologic markers, the proposed classification also suggests the use of class II HLA as a marker of endothelial integrity. It is known that during allograft rejection, endothelial cells are activated, therefore, we hypothesized that endothelial class II HLA rather than a marker of mere endothelial presence, is a marker of endothelial activation and becomes upregulated in AMR.Eight hundred thirty-eight heart allograft biopsies, collected from January 2016 to September 2018 at a single institution from patients with a current or recent diagnosis of AMR, were evaluated for both histopathologic and immunopathologic changes of AMR. Biopsies were labeled with immunofluorescence with antibodies against C4d and for immunohistochemistry with antibodies against C3d, CD68, and class II HLA. ISHLT criteria were used to classify the biopsies, and for class II HLA, both the percentage and the stain intensity were evaluated.Biopsies (74.8%) from our cohort showed either histopathologic pAMR-1, immunopathologic pAMR-1, or combined histopathologic and immunopathologic pAMR-2 evidence of AMR. Expression of endothelial HLA class II was significantly correlated with the diagnosis of AMR by percentage area (P < .0001) and intensity of staining (P < .0001). The diagnosis of AMR significantly correlated with moderate (+2) and strong (+3) staining intensity for class II HLA as follows: histopathologic and immunopathologic pAMR-2 with odds ratio (OR) = 28.3 (P < .0001);histopathologic pAMR-1 alone with OR = 22.7 (P < .0001); and immunopathologic pAMR-1 alone with OR = 32.6 (P < .0001). Interestingly, our study also suggested that the inclusion of C4d focally positive cases also significantly correlates with the diagnosis of AMR (P < .003).We confirmed our hypothesis that in heart allograft biopsies, there is a spectrum of both percentage and intensity of HLA class II expression due to endothelial activation, and that class II HLA by immunohistochemistry is a marker significantly correlated with the diagnosis of AMR. In addition, the group of focally positive C4d biopsies (10%-50%) should be considered positive for the immunopathologic component of the 2013 ISHLT classification, as this group of biopsies also correlated with the diagnosis of AMR.

SUN-371 C4D STAINING IN INFECTION RELATED GLOMERULONEPHRITIS: A RETROSPECTIVE STUDY

C4d is a byproduct of activation of the classic and lectin pathways. It is widely used as marker of antibody-mediated rejection. However, recent studies shown that C4d can be positive in immune complex glomerulonephritis. There is paucity in literature regarding utility C4d staining in suspected cases of infection related glomerulonephritis (IRGN) to demonstrate lack of activation of alternate complement pathway. In this retrospective observational study we included all biopsy proven IRGN from December 2016 to December 2017. Demographic data, initial lab values biopsy findings and treatment details were documented. C4d was done with IHC. Patients were followed up through a period of one year. Outcome variables were defined as persistence of proteinuria/ hematuria at one year, dialysis dependency and death. Of 90 patients with biopsy proven IRGN, C4d staining was done in 53 patients out of which 29 (55%) were C4d positive and 24 patients (45%) were negative. Out of the patients negative for C4d, 62% were C3 dominant and co-dominance was seen in 38%. The mean age of presentation was 41.89±14.58 years, with 51% Males. We observed that C4d IHC showed intense, diffuse >2+ glomerular capillary wall staining. In C4d +ve group mean creatinine at presentation was 2.9±1.8 g/dl and mean 24 hour urine protein was 2.84±1.5. Nephrotic range proteinuria was seen in 5 patients. Immunosuppression (IS) was started in 7 patients, HD was given to 6 patients and PLEX done in 2. At 1 year, 4 patients progressed to CKD, 4 patients were dialysis Dependent and 3 died. In C4d neg, C3 dominant group the mean creatinine at presentation was 4.1±2.6 g/dl and mean 24 hour urine protein was 3.1±1.3. Nephrotic range proteinuria was seen in 5 patients. IS & HD was started in 2 & 5 patients each. At 1 year 5 patients progressed to CKD, 1 patient became dialysis dependent and one died. In C4d neg, C3 co - dominant group the mean serum creatinine at presentation was 3.3±2.5g/dl and mean protenuria was 2.3±1.4gm and nephrotic range proteinuria seen in 1 patient. At 1 year 2 patients progressed to CKD, 1 patient became dialysis dependent. Our study demonstrates that C4d staining helps to differentiate IC GN from complement mediated GN. There was significant difference between the clinical presentation, and follow up between C4d positive, c4d negative - c3 dominant and Codominant IRGN. But the true differentiation of glomerulonephritis with c3 dominance (infection related) and C3 glomerulonephritis is not well established.

C4d detection and histological patterns in the diagnosis ofantibody-mediated rejection after lung transplantation: a single-centre study.

Antibody-mediated rejection (AMR) is an emerging and challenging issue in transplantation. Endothelial deposition of C4d and microvascular inflammation (MI) are reliable markers of AMR in renal and cardiac transplantation, but remain controversial in the lung. Our aim was to assess C4d immunohistochemistry and histological patterns for the diagnosis of lung AMR. We reviewed 158 transbronchial biopsies (TBBs) (n=85 clinically indicated, and n=73 surveillance TBBs) from 48 recipients, blinded to clinical and serological data. C4d was scored as 0, 1+ (<10%), 2+ (10-50%) or 3+ (>50%). TBBs were reassessed for MI and acute lung injury (ALI). Donor-specific antibodies (DSAs), acute clinical graft dysfunction and chronic lung allograft graft dysfunction (CLAD) were recorded. C4d3+, C4d2+, C4d1+ and C4d0 occurred respectively in four (2.5%), six (3.8%), 28 (17.7%) and 120 (75.9%) TBBs. MI and ALI were rare but more frequent in C4d1-3+ TBBs than in the absence of C4d. C4d2+ was frequently observed with concomitant infection. Among the surveillance TBBs, only two (2.7%) showed MI. Neither ALI nor C4d3+ was diagnosed on surveillance TBBs. No significant association was found between histopathological findings and DSAs. All four patients with C4d3+ could retrospectively be diagnosed with AMR and developed CLAD. Although rare, diffuse C4d deposition appears to be a strong indication of acute clinical AMR in lung transplant patients, whereas intermediate C4d2+ requires more investigations. In stable patients, histopathology and C4d may lack the sensitivity to diagnose subclinical AMR. This emphasises the need for a multidisciplinary evaluation of each suspected AMR case, and the need for complementary diagnostic tools.

Detection of T and B cells specific complement-fixing alloantibodies using flow cytometry: A diagnostic approach for a resource limited laboratory.

BACKGROUND AND OBJECTIVES:Various methods have been reported for the detection of antibodies in recipient sera, which can be human leukocyte antigens (HLAs) or non-HLA specific, complement- or noncomplement fixing, as well as donor T (HLA-Class-I) and/or B cell (HLA-Class-I and II) specific. These alloantibodies play a pivotal role in antibody-mediated renal transplantation rejection. Deposition of C4d in peritubular capillaries of a kidney biopsy is a marker of antibody-mediated rejection. The C4d flow-panel reactive antibodies (PRAs) are a screening method for HLA-specific and complement fixing antibodies. However, the method is limited by the lack of donor specificity.DESIGN AND SETTINGS:Here, we present a new and simple flow cytometric method referred to as C4d-flow cytometry crossmatch (C4d-FCXM) for the detection of donor-specific (T and/or B cell) and C4d-fixing alloantibodies.RESULTS:The method was applied in a series of clinical cases and judged to be useful. The method may limit unwanted deferral of the donor due to positivity in C4d Flow-PRA and/or FCXM and may be helpful in prediction of antibody mediated rejections. Furthermore, this method can provide information pretransplant in contrast to kidney biopsy and C4d evaluation done posttransplant.CONCLUSIONS:We postulate that this method incorporates most of the features of all the available modalities (i.e., National Institute of Health-complement dependent lymphocytotoxicity, FCXM, cytotoxic FCXM and C4d-flowPRA) yet cost-effective and best suited for resource-limited laboratory/ies which is a common scenario in developing countries.

Soluble BAFF Cytokine Levels and Antibody-Mediated Rejection of the Kidney Allograft.

The B-cell activating factor (BAFF) cytokine has important functions for the survival and maturation of B lymphocytes, which implies that this cytokine might play a role in the development of antibody-mediated rejection (AMR) after kidney transplantation. In our study, we compared the concentrations of the soluble BAFF cytokine in kidney graft recipients with AMR and patients without rejection with the goal of testing the hypothesis whether BAFF level measurement might be useful as a diagnostic marker of AMR. The study included a cohort of 19 high-risk patients with diagnosed AMR and 17 control patients free of rejection. BAFF was measured in all patients before transplantation, during the rejection episodes, and three months after transplantation in patients free of rejection using the Luminex technique. Before transplantation, the serum concentrations of BAFF in patients with AMR and kidney recipients without rejection did not significantly differ. After transplantation, however, BAFF levels were significantly lower in patients with AMR and also in patients with concurrent humoral and cellular rejection compared with patients without rejection (p<0.05 and p<0.01, respectively). No correlation was found between BAFF and the production of donor-specific antibodies (DSA) before and after transplantation. Patients experiencing AMR and simultaneous cellular and AMR had significantly lower concentrations of BAFF in comparison with patients free of rejection.

Evaluation of Renal Allograft Biopsies for Graft Dysfunction and Relevance of C4d Staining in Antibody Mediated Rejection.

Biopsy remains gold standard for diagnosis of Graft Dysfunction (GD). It guides clinical management, provides valuable insights into pathogenesis of early and late allograft injury and is indispensable for distinguishing rejection from non- rejection causes of GD. The primary aim of the study was to evaluate the diverse histomorphological lesions in renal allograft biopsy (RAB). Further, we determined the frequency of peritubular capillary (PTC) C4d positivity and its correlation with microvascular inflammation in Antibody Mediated Rejection (AMR). This was a prospective study on RAB over a period of 2 months. Histopathological evaluation was undertaken as per revised Banff'13 schema. Immunohistochemistry was performed to detect PTC C4d deposition. Sixty five diagnostic biopsies were evaluated. Mean patient age was 34 years and males were predominant. The time interval between graft biopsy and transplantation ranged from 5 days to 8 years, with 52.3% biopsies belonging to period of ≤ 6 months post-transplant. Immune injuries were observed in 40 biopsies out of which AMR was observed in 35 biopsies. Calcineurin inhibitor toxicity (CNI Toxicity) was the second commonest cause observed in 12 biopsies and other lesions including de novo glomerulopathies were observed in the remaining biopsies. The sensitivity of C4d in detecting acute AMR was 55% and chronic AMR was 23.5. AMR and CNI Toxicity account for majority of graft dysfunction. C4d is not as sensitive a marker of AMR, as was initially thought. Higher proportion of moderate microvascular inflammation is found in diffuse C4d positive cases compared to focal C4d positive cases.

OR21 Is circulating angiotensin II receptor a potential marker of renal allograft injury?

Aim Angiotensin II receptor-1 (AngII) is a membrane-bound protein in vascular endothelium that is responsible for vasoconstriction and blood pressure regulation. Current data suggests AngII may be a target of antibody mediated rejection (AMR) following renal transplantation. We speculated that detection of AngII in the peripheral circulation might reflect allograft injury and be a marker for AMR. Methods This retrospective study selected 20 patients without evidence of anti-HLA antibody and graft survival ⩾ 1 year: Ten patients were AMR-free and blood tested at 0, 6 and 12 months post-transplant. Ten patients had AMR and blood was tested pretransplant, during and after AMR). Blood was also collected from 63 healthy volunteers. AngII was measured by commercial elisa (Cusabio). Results AngII was absent in 63/63 volunteers (100%). In contrast, circulating AngII was detected in half the AMR (4/10) and AMR-free (6/10) patients pretransplant ( p p = ns). Posttransplant, circulating AngII declined from 6/10 to 4/10 non-AMR patients by 6 months. In addition, AngII concentration declined to 84 ± 135 and 22 ± 50 pg/ml by 6 and 12 months posttransplant. In contrast, AngII was present in 7/10 patients during AMR ( p p Conclusions This pilot study showed that circulating AngII is lacking among healthy volunteers but present in half of patients awaiting renal transplantation. Neither the presence or the concentration of AngII pretransplant predicted risk of AMR. However, the demonstration that circulating AngII was common during AMR and resolved upon clinical stability and the finding that de novo AngII expression was only seen among patients with AMR suggests that circulating AngII might be a useful marker in detection and treatment of AMR, particularly when anti-HLA DSA are not present.

C4d as a Diagnostic Tool in Proliferative GN.

Proliferative GN is classified as immune complex-mediated or complement-mediated (C3 glomerulopathy). Immune complex-mediated GN results from glomerular deposition of immune-complexes/Ig and C3; the C3 is derived from activation of the classical and/or lectin pathways of complement. C3 glomerulopathy results from deposition of C3 and other complement fragments with minimal or no deposition of immune complexes/Ig; the C3 is derived from activation of the alternative pathway of complement. C4d is a byproduct of activation of the classic and lectin pathways. Although widely used as a marker for antibody-mediated rejection, the significance of C4d in C3 glomerulopathy is undetermined. We studied glomerular C4d staining in 18 biopsy specimens of immune-complex GN, 30 biopsy specimens of C3 GN, and 13 biopsy specimens of postinfectious GN. All specimens of immune complex-mediated GN, except two specimens of IgA nephropathy and one specimen of sclerosing membranoproliferative GN, showed bright (2-3+) C4d staining. The staining pattern of C4d mirrored the staining patterns of Ig and C3. Conversely, C4d staining was completely negative in 24 (80%) of 30 specimens of C3 glomerulopathy, and only trace/1+ C4d staining was detected in six (20%) specimens. With regard to postinfectious GN, C4d staining was negative in six (46%) of 13 specimens, suggesting an abnormality in the alternative pathway, and it was positive in seven (54%) specimens. To summarize, C4d serves as a positive marker for immune complex-mediated GN but is absent or minimally detected in C3 glomerulopathy.

Urinary C-X-C Motif Chemokine 10 Independently Improves the Noninvasive Diagnosis of Antibody-Mediated Kidney Allograft Rejection.

Urinary levels of C-X-C motif chemokine 9 (CXCL9) and CXCL10 can noninvasively diagnose T cell-mediated rejection (TCMR) of renal allografts. However, performance of these molecules as diagnostic/prognostic markers of antibody-mediated rejection (ABMR) is unknown. We investigated urinary CXCL9 and CXCL10 levels in a highly sensitized cohort of 244 renal allograft recipients (67 with preformed donor-specific antibodies [DSAs]) with 281 indication biopsy samples. We assessed the benefit of adding these biomarkers to conventional models for diagnosing/prognosing ABMR. Urinary CXCL9 and CXCL10 levels, normalized to urine creatinine (Cr) levels (CXCL9:Cr and CXCL10:Cr) or not, correlated with the extent of tubulointerstitial (i+t score; all P<0.001) and microvascular (g+ptc score; all P<0.001) inflammation. CXCL10:Cr diagnosed TCMR (area under the curve [AUC]=0.80; 95% confidence interval [95% CI], 0.68 to 0.92; P<0.001) and ABMR (AUC=0.76; 95% CI, 0.69 to 0.82; P<0.001) with high accuracy, even in the absence of tubulointerstitial inflammation (AUC=0.70; 95% CI, 0.61 to 0.79; P<0.001). Although mean fluorescence intensity of the immunodominant DSA diagnosed ABMR (AUC=0.75; 95% CI, 0.68 to 0.82; P<0.001), combining urinary CXCL10:Cr with immunodominant DSA levels improved the diagnosis of ABMR (AUC=0.83; 95% CI, 0.77 to 0.89; P<0.001). At the time of ABMR, urinary CXCL10:Cr ratio was independently associated with an increased risk of graft loss. In conclusion, urinary CXCL10:Cr ratio associates with tubulointerstitial and microvascular inflammation of the renal allograft. Combining the urinary CXCL10:Cr ratio with DSA monitoring significantly improves the noninvasive diagnosis of ABMR and the stratification of patients at high risk for graft loss.

Clinicopathological relevance of granular C4d deposition in peritubular capillaries of kidney allografts

While linear C4d staining in peritubular capillaries (PTC) is established as a marker of antibody-mediated rejection, the significance of a distinct granular C4d deposition pattern has not yet been clarified. In this study, 329 renal allograft recipients who underwent indication biopsies were analysed for immunohistochemical C4d staining characteristics. Fifty-six (17%) recipients showed granular C4d in PTC, without any relationship to conventional risk factors and morphological features of rejection. We found a strong association with long-term overall graft survival (7-year survival: 41% vs. 66% in granular C4d-negative subjects, P=0.001), which was mainly driven by a greater risk of mortality [hazard ratio: 3.12 (95% confidence interval: 1.23-7.94); P=0.02]. Granular C4d was associated with delayed graft function [39% vs. 22% (C4d-negative subjects), P=0.007], higher 1-year serum creatinine [median 2.1 (interquartile range: 1.7-2.6) mg/dl vs. 1.6 (1.3-2.0) mg/dl, P=0.001] and a trend towards worse death-censored graft survival (P=0.07). In support of a role of capillary immune complex formation, granular C4d was associated with electron-dense deposits in PTC basement membranes, which were occasionally accompanied by focally distributed capillary IgG deposits. In conclusion, our study suggests clinical relevance of detecting capillary granular C4d deposition. Our results point to a pathogenetic role of alloimmune-independent immune complex deposition.

Pathologic classification of antibody-mediated rejection correlates with donor-specific antibodies and endothelial cell activation

Humoral immune responses during heart transplantation may result in antibody-mediated rejection (AMR), which is now taken into account on endomyocardial biopsy (EMB) specimens and ranked according to the pathologic AMR (pAMR) grades of the International Society for Heart and Lung Transplantation classification. This classification might benefit from new immunohistological markers and validation by others biomarkers, namely donor-specific antibodies (DSA). From the 293 protocol EMBs performed in 113 patients in our institution during a 1-year period for this prospective study, 280 EMB specimens were available with both histology and immunohistochemistry. C4d and labeling of intravascular cells by cluster of differentiation (CD) 68 were performed on paraffin sections. Available sera (n = 150) concomitant of EMB specimens were tested for the presence of DSA. All of the pAMR+ EMB specimens, along with a set of randomized pAMR0 EMB specimens, were immunolabeled for mammalian target of rapamycin (mTOR) effectors, phosphorylated 70 S6-kinase (p70S6K) and phosphorylated S6 ribosomal protein (pS6RP). AMR was diagnosed in 37 EMB specimens (13.2%): 1 pAMR1(I+), 27 pAMR1(H+), and 9 pAMR2. The proportion of DSA-positive EMB varied according to the pAMR grade, with pAMR0, pAMR1(H+), and pAMR2 EMB presenting 17.6%, 77.3%, and 100% of DSA-positivity, respectively. Among the 30 pAMR+ specimens with available DSA testing and the 30 pAMR0 randomized specimens, mTOR pathway immunohistochemistry showed endothelial cell positivity for p70S6K in 17 pAMR+ EMB specimens (56.7%) and in 1 pAMR0 EMB specimen (3.3%). pS6RP was detected in 8 pAMR+ EMB specimens (26.7%) and in 1 pAMR0 EMB specimen (3.3%). p70S6K and pS6RP immunohistochemistry afford new markers of AMR on EMB specimens because their expression is correlated with microcirculation inflammation and DSA. The correlation of DSA with pAMR grade suggests that this grading system is valid.

C4d by Immunofluorescence and Immunohistochemistry in Routine Lung Allograft Biopsies

Purpose The role of C4d in diagnosing antibody mediated rejection (AMR) in lung allografts is unclear. We compared C4d by immunofluorescence (IF) and immunohistochemistry (IHC) in routine allograft transbronchial bx (TBBx) and correlated the results with clinical findings. Methods and Materials Prospectively, consecutive paired frozen and paraffin-embedded samples from the same TBBx were stained for C4d by IF and IHC and scored as negative (neg), focal+ or diffuse 2+ or 3+. Serum HLA antibodies (ab) (within 6 mo of TBBx) were noted. Poisson regression (SAS version 9) was used to compare HLA ab between neg and focal+. Results 228 TBBx from 76 patients (pts) were reviewed. No pt was clinically suspected to have AMR. Pt 1 with C4d 3+ had pleural effusions; TBBx was unremarkable; apheresis was performed. The pt died 1 mo later (subdural hematoma). Pt 2 with C4d 3+ had pseudomonas bacteremia and progressive multifocal leukoencephalopathy (PML). TBBx showed intimitis. The pt died 4 mo later (PML). A pt (heart-lung transplant) had AMR on heart bx. A TBBx 8 mo later was C4d focal+ (IF); neg. by IHC; HLA ab 8% & 28% (class I & II, respectively) at time of TBBx. HLA ab were not significantly different between C4d (IHC) neg and C4d (IHC) focal+ TTBx. Conclusions IHC and IF are similar in detecting diffuse 3+C4d in routine TBBx. IHC appears superior in identifying focal C4d, likely because IHC provides better architecture that helps to neglect the high background of C4d stain in the lung. Routine C4d might not be a reliable marker of AMR in lung transplant. Results of C4d and HLA abs C4d by IHC (#pts) C4d by IF (#pts) HLA Abs (#pts with + abs), median (range) Neg (196) Neg (188) Screen (3/68), 2 (2-7) Class (Cl) I (50/76), 3 (0-76) Cl II (53/76), 5.5 (0-65) Diffuse 2+(3), focal + (4), equivocal (1) Screen (0/8) Cl I (4/8), 0.5 (0-9) Cl II (4/8), 3.5 (0-28) Focal+ (30) Neg (30) Screen, neg. Cl I (13/19), 3 (0-52) Cl II (13/19), 25 (0-65) Diffuse 3+ (2) Diffuse 3+ (2) Screen (1) neg. Cl I (2), 16.5 (3-30) Cl II (2), 1 (0-2)

Pathologic findings in lung allografts with anti-HLA antibodies

Despite data indicating a positive correlation between donor-specific anti-HLA antibodies (DSAs) and early development of bronchiolitis obliterans syndrome (BOS) in lung allografts, the role of an antibody-mediated process in acute and chronic lung allograft rejection has not been elucidated. In this study we evaluated pathologic features of transplant lung biopsies in patients with and without DSAs. Forty-one lung transplant biopsies from 41 patients at our institution were included in our study. The biopsy H&E slides were reviewed in a blinded fashion, and scored for presence of microvascular inflammation, acute rejection, bronchiolar inflammation and acute lung injury, as well as diffuse alveolar damage (DAD). Microvascular inflammation was graded by the presence of capillary neutrophils on a scale of 0 to 4(+). For immunohistochemical analysis, the pattern and intensity of staining for C4d and C3d deposition were evaluated in airways and alveolar capillaries. Histopathology suspicious for antibody-mediated rejection (AMR)-defined as≥2(+) neutrophilic infiltration and/or DAD-were more common in DSA-positive cases than controls (11 of 16 vs 6 of 25, p<0.01). Evidence of allograft dysfunction was significantly more common among patients with both DSA and suspicious histopathology compared with controls (5 of 10 vs 3 of 25, p = 0.03). The combination of DSAs and histopathology suspicious for AMR was associated with both BOS (p = 0.002) and mortality (p = 0.03). Immunohistochemistry for C3d and C4d showed no correlation with each other, DSAs or histopathology. Grade 2(+) neutrophilic infiltration is the histopathologic finding most closely related to DSAs with graft dysfunction and development of BOS in lung transplant recipients and may be a marker for AMR.

Late and Chronic Antibody-Mediated Rejection: Main Barrier to Long Term Graft Survival

Antibody-mediated rejection (AMR) is an important cause of graft loss after organ transplantation. It is caused by anti-donor-specific antibodies especially anti-HLA antibodies. C4d had been regarded as a diagnosis marker for AMR. Although most early AMR episodes can be successfully controlled or reversed, late and chronic AMR remains the leading cause of late graft loss. The strategies which work in early AMR have limited effect on late/chronic episodes. Here, we reviewed the lines of evidence that late/chronic AMR is the leading cause of late graft loss, characteristics of late AMR, and current strategies in managing late/chronic AMR. More effort should be put on the management of late/chronic AMR to make a better long term graft survival.