Treatment of borderline infiltrates with minimal inflammation in kidney transplant recipients has no effect on allograft or patient outcomes

In 2005, the Banff committee expanded the “borderline changes” category to include lesions with minimal (<10%) inflammation: “i0” borderline infiltrates. Clinical significance and optimal treatment of i0 borderline infiltrates are not known. Data suggest that i0 borderline infiltrates may have a more favorable prognosis than borderline infiltrates with higher grades of interstitial inflammation. In this single-center, retrospective, observational study, we assessed 90 renal transplant recipients with i0 borderline infiltrates on biopsies indicated for graft dysfunction. We studied the impact of treatment with corticosteroids on allograft function, allograft survival, and patient survival. We found no differences between treated and untreated groups with respect to eGFR at 4 weeks and 6 months after biopsy. Follow-up biopsies, available in 67% of patients, were negative for rejection in almost half of all cases, regardless of treatment status. The frequencies of persistent borderline infiltrates (38%) and higher-grade T cell–mediated rejection (1A or greater, 14%) on follow-up biopsies were similar between the two groups. There were no differences in rejection-free allograft survival, death-censored graft failure, or patient mortality among treated vs non-treated i0 borderline patients. Our findings suggest that the natural history of i0 borderline infiltrates, in relatively low immunologic risk patients, is not affected by corticosteroid treatment.     

The nature of biopsies with "borderline rejection" and prospects for eliminating this category

In kidney transplantation, many inflamed biopsies with changes insufficient to be called T-cell-mediated rejection (TCMR) are labeled "borderline", leaving management uncertain. This study examined the nature of borderline biopsies as a step toward eventual elimination of this category. We compared 40 borderline, 35 TCMR and 116 nonrejection biopsies. TCMR biopsies had more inflammation than borderline but similar degrees of tubulitis and scarring. Surprisingly, recovery of function after biopsy was similar in all categories, indicating that response to treatment is unreliable for defining TCMR. We studied the molecular changes in TCMR, borderline and nonrejection using microarrays, measuring four published features: T-cell burden; a rejection classifier; a canonical TCMR classifier; and risk score. These reassigned borderline biopsies as TCMR-like 13/40 (33%) or nonrejection-like 27/40 (67%). A major reason that histology diagnosed molecularly defined TCMR as borderline was atrophy-scarring, which interfered with assessment of inflammation and tubulitis. Decision tree analysis showed that i-total >27% and tubulitis extent >3% match the molecular diagnosis of TCMR in 85% of cases. In summary, most cases designated borderline by histopathology are found to be nonrejection by molecular phenotyping. Both molecular measurements and histopathology offer opportunities for more precise assignment of these cases after clinical validation.     

HLA‐G on peripheral blood CD4+ T lymphocytes: a potential predictor for acute renal rejection

HLA‐G Expression in grafts and serum has been shown to improve graft acceptance. However, its expression on peripheral blood lymphocytes (PBLs) during acute rejection (AR) remains unknown. In this study, we serially monitored HLA‐G expression on CD4⁺ and CD8⁺ PBLs of 66 recipients undergoing renal transplantation using flow cytometry at different time points before and after transplantation, as well as during AR episode. In stable recipients, HLA‐G expression on CD4⁺ PBLs declined during the first week after transplantation and increased continuously with immunosuppressive therapy. Then, expression declined gradually after 1 month and remained at a higher level compared with pretransplantation. When AR occurred, HLA‐G expression decreased significantly compared with the stable level. In three recipients suffering from recurrent rejection, it remained at a low level despite impact immunosuppressive treatment. With mix lymphocyte assay, HLA‐G⁺ CD4⁺ T cells showed inhibitory role on proliferation of peripheral blood mononuclear cell. HLA‐G expression on CD8⁺ PBLs was almost undetectable at different time points in the recipients and healthy controls. Our results suggest that HLA‐G on CD4⁺ PBLs might provide a potential marker for the early diagnosis of renal AR and for the immunosuppressive status of recipients.     

IL‐6 production by monocytes is associated with graft function decline in patients with borderline changes suspicious for acute T‐cell‐mediated rejection: a pilot study

Although borderline changes (BL ) suspicious for acute T‐cell‐mediated rejection represent a diagnostic category, its clinical relevance is questioned leading to heterogeneous therapeutic management. We hypothesized that measuring IL ‐6 secretion by peripheral blood mononuclear cells identifies patients with ongoing graft damage. We examined the association between secreted IL ‐6 and the change in estimated glomerular filtration rate at 6 months after the biopsy (ΔeGFR ). We then conducted phenotypic and functional studies on patient and mouse innate immune cells in the blood and the kidney. In a training set, ΔeGFR was strongly associated with IL ‐6 levels, showing a clinically meaningful decline of 4.6 ± 1.5 ml /min per increase in log10 IL ‐6 (P = 0.001). These results were consistent after adjustment and were reproduced in a validation cohort. Phenotyping of peripheral blood cells revealed that the main source of IL ‐6 was CD 14⁺CD 16^?CCR 2⁺HLA‐DR ⁺CD 86⁺CD 11c⁺ inflammatory monocytes. There was a significant correlation between IL ‐6 secretion and interstitial dendritic cell density in the biopsy. Finally, characterization of mouse kidney dendritic cells revealed that they share features with macrophages and function as effector cells secreting IL ‐6. In conclusion, measuring IL ‐6 secreted by peripheral blood cells can be useful in the management of patients with BL in the absence of a concurrent inflammatory condition.     

Non‐organ‐specific and anti‐endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients

Costa C, Touscoz GA, Bergallo M, Terlizzi ME, Astegiano S, Sidoti F, Sinesi F, Segoloni GP, Cavallo R. Non‐organ‐specific and anti‐endothelial antibodies in relation to CMV infection and acute rejection in renal transplant recipients.
Clin Transplant 2010: 24: 488–492.
© 2009 John Wiley & Sons A/S. Abstract: The presence of non‐organ‐specific (NOSA) and anti‐endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65‐antigenemia‐negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluoresce. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post‐transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p < 0.05); NOSAs were detected in 9/48 (18.8%) and 9/48 patients from group 1 and 2, respectively. An acute rejection was diagnosed in ten cases: six of interstitial type (antigenemia‐, and AECA‐negative; two NOSA‐positive); four of vascular type (all of them NOSA‐negative, 3/4 antigenemia‐, and AECA‐positive). CMV infection did not seem to be significantly associated with the appearance of NOSAs, while there was a significant correlation with the occurrence of AECAs. No significant correlation was found between acute rejection and the occurrence of NOSAs, while 75% of the cases of vascular rejection was associated to CMV infection and AECA‐positivity, suggesting the pathogenic role of CMV‐mediated endothelial damage.     

Subclass analysis of donor HLA‐specific IgG in antibody‐incompatible renal transplantation reveals a significant association of IgG4 with rejection and graft failure

Donor HLA‐specific antibodies (DSAs) can cause rejection and graft loss after renal transplantation, but their levels measured by the current assays are not fully predictive of outcomes. We investigated whether IgG subclasses of DSA were associated with early rejection and graft failure. DSA levels were determined pretreatment, at the day of peak pan‐IgG level and at 30 days post‐transplantation in eighty HLA antibody‐incompatible kidney transplant recipients using a modified microbead assay. Pretreatment IgG₄ levels were predictive of acute antibody‐mediated rejection (P = 0.003) in the first 30 days post‐transplant. Pre‐treatment presence of IgG₄ DSA (P = 0.008) and day 30 IgG₃ DSA (P = 0.03) was associated with poor graft survival. Multivariate regression analysis showed that in addition to pan‐IgG levels, total IgG₄ levels were an independent risk factor for early rejection when measured pretreatment, and the presence of pretreatment IgG₄ DSA was also an independent risk factor for graft failure. Pretreatment IgG₄ DSA levels correlated independently with higher risk of early rejection episodes and medium‐term death‐censored graft survival. Thus, pretreatment IgG₄ DSA may be used as a biomarker to predict and risk stratify cases with higher levels of pan‐IgG DSA in HLA antibody‐incompatible transplantation. Further investigations are needed to confirm our results.     

The therapeutic challenge of late antibody‐mediated kidney allograft rejection

Late antibody‐mediated rejection (ABMR) is a cardinal cause of kidney allograft failure, manifesting as a continuous and, in contrast with early rejection, often clinically silent alloimmune process. While significant progress has been made towards an improved understanding of its molecular mechanisms and the definition of diagnostic criteria, there is still no approved effective treatment. In recent small randomized controlled trials, therapeutic strategies with promising results in observational studies, such as proteasome inhibitor bortezomib, anti‐C5 antibody eculizumab, or high dose intravenous immunoglobulin plus rituximab, had no significant impact in late and/or chronic ABMR. Such disappointing results reinforce a need of new innovative treatment strategies. Potential candidates may be the interference with interleukin‐6 to modulate B cell alloimmunity, or innovative compounds that specifically target antibody‐producing plasma cells, such as antibodies against CD38. Given the phenotypic heterogeneity of ABMR, the design of adequate systematic trials to assess the safety and efficiency of such therapies, however, is challenging. Several trials are currently being conducted, and new developments will hopefully provide us with effective ways to counteract the deleterious impact of antibody‐mediated graft injury. Meanwhile, the weight of evidence would suggest that, when approaching using existing treatments for established antibody‐mediated rejection, “less may be more”.     

IVIG and rituximab for treatment of chronic antibody‐mediated rejection: a prospective study in paediatric renal transplantation with a 2‐year follow‐up

Chronic antibody‐mediated rejection (AMR) is the major cause of late renal allograft loss. There is, however, no established treatment for this condition. We report the results of a prospective pilot study on an antihumoral therapy (AHT) consisting of high‐dose intravenous immunoglobulin G (IVIG) and rituximab in 20 paediatric renal transplant recipients. Donor‐specific HLA antibodies (HLA DSA) were quantified by Luminex‐based bead array technology. Loss of eGFR decreased significantly from 7.6 ml/min/1.73 m² during 6 months prior to AHT to 2.1 ml/min/1.73 m² (P = 0.0013) during 6 months after AHT. Fourteen patients (70%) responded: nine of nine patients (100%) without and five of 11 (45%) with transplant glomerulopathy (P = 0.014). C4d positivity in PTC decreased from 40 ± 18.5% in the index biopsy to 11.6 ± 12.2% (P = 0.002) in the follow‐up biopsy. In four of nine biopsies (44%) C4d staining turned negative. During 2 years of follow‐up, the median loss of eGFR in each of the four 6‐month periods remained significantly lower compared with prior to AHT. Class I DSA declined in response to AHT by 61% (p = 0.044), class II DSA by 63% (p = 0.033) 12 months after intervention. AHT with IVIG and rituximab significantly reduces or stabilizes the progressive loss of transplant function in paediatric patients with chronic AMR over an observation period of 2 years, apparently by lowering circulating DSA and reducing intrarenal complement activation.     

High levels of dd‐cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury

The clinical importance of subclinical, early T cell–mediated rejection (Banff TCMR 1A and borderline lesions) remains unclear, due, in part to the fact that histologic lesions used to characterize early TCMR can be nonspecific. Donor‐derived cell‐free DNA (dd‐cfDNA) is an important molecular marker of active graft injury. Over a study period from June 2017 to May 2019, we assessed clinical outcomes in 79 patients diagnosed with TCMR 1A/borderline rejection across 11 US centers with a simultaneous measurement of dd‐cfDNA. Forty‐two patients had elevated dd‐cfDNA (≥0.5%) and 37 patients had low levels (<0.5%). Elevated levels of dd‐cfDNA predicted adverse clinical outcomes: among patients with elevated cfDNA, estimated glomerular filtration rate declined by 8.5% (interquartile rate [IQR] ?16.22% to ?1.39%) (?3.50 mL/min/1.73 m² IQR ?8.00 to ?1.00) vs 0% (?4.92%, 4.76%) in low dd‐cfDNA patients (P = .004), de novo donor‐specific antibody formation was seen in 40% (17/42) vs 2.7% (P < .0001), and future or persistent rejection occurred in 9 of 42 patients (21.4%) vs 0% (P = .003). The use of dd‐cfDNA may complement the Banff classification and to risk stratify patients with borderline/TCMR 1A identified on biopsy.     

The influence of non‐HLA antibodies directed against angiotensin II type 1 receptor (AT1R) on early renal transplant outcomes

Non‐HLA antibodies (Abs) targeting vascular receptors are thought to have an impact on renal transplant injury. Anti‐angiotensin II type 1‐receptor‐activating antibodies (anti‐AT1R) have been mentioned to stimulate a severe vascular rejection, but the pretransplant screening has not been introduced yet. The aim of our study was to assess the incidence and importance of anti‐AT1R antibodies and their influence on renal transplant in the 1st year of observation. We prospectively evaluated the presence of anti‐AT1R antibodies in 117 consecutive renal transplant recipients in pre‐ and post‐transplant screening. Anti‐AT1R antibodies were observed in 27/117 (23%) of the analyzed recipients already before transplantation. The function of renal transplant was considerably worse in anti‐AT1R(+) group. The patients with anti‐AT1R Abs >9 U/ml lost their graft more often. Biopsy‐proven AR was described in 4/27 (15%) pts in the anti‐AT1R(+) group and 13/90 (14.4%) in the anti‐AT1R(?) group, but more severe cases of Banff IIB or antibody‐mediated rejection (AMR) were more often observed in anti‐AT1R (+) 4/27 (15%) vs. 1/90 (1.1%) in anti‐AT1R(+) (P = 0.009). Patients with anti‐AT1R Abs level >9 U/ml run a higher risk of graft failure independently of classical immunological risk factors. The recipients with anti‐AT1R Abs developed more severe acute rejections described as IIB or AMR in Banff classification. More recipients among the anti‐AT1R‐positive ones lost the graft. Our study suggests monitoring of anti‐AT1R Abs before renal transplantation for assessment of immunologic risk profiles and the identification of patients highly susceptible to immunologic events, graft failure, and graft loss.     

A prospective observational study of immune reconstitution following transplantation with post‐transplant reduced‐dose cyclophosphamide from HLA‐haploidentical donors

Allogeneic hematopoietic cell transplantation (HCT) from HLA‐haploidentical donors with post‐transplantation high‐dose cyclophosphamide (PT/Cy‐haplo) now predominates worldwide. However, to our knowledge, no prospective study has compared immune reconstitution after PT/Cy‐haplo with that after conventional HCT. The mechanism by which chronic graft‐versus‐host disease (GVHD) is inhibited by PT/Cy‐haplo also remains unknown. We prospectively compared immune recovery patterns of lymphocyte subsets among four groups of adult patients with hematological disease who received HCT from either HLA‐matched related or HLA‐matched unrelated donors, cord blood transplantation, or reduced‐dose PT/Cy‐haplo. Counts of CD4+ T‐cell subsets, CD8+ T‐cell subsets, and NK cells on days 30 and 60 were often lower in PT/Cy‐haplo than those in HLA‐matched related HCT. The immune recovery pace in PT/Cy‐haplo subsequently caught up with that of the other grafts. The regulatory T cells (Tregs) to conventional CD4+ T‐cell (Tcon) ratio was significantly higher until day 90 in PT/Cy‐haplo. In multivariate analysis, a higher Tregs‐to‐Tcon ratio on day 60 was significantly associated with a lower incidence of chronic GVHD (P < 0.01). The preservation of Tregs by PT/Cy in the early phase might have resulted in a lower incidence of chronic GVHD.     

Postoperative rebound of antiblood type antibodies and antibody‐mediated rejection after ABO‐incompatible living‐related kidney transplantation

The purpose of this study is to examine whether postoperative antiblood type antibody rebound is attributed to kidney allograft rejection in ABO blood type‐incompatible (ABO‐I) living‐related kidney transplantation (KTx). A total of 191 ABO‐I recipients who received ABO‐I living‐related KTx between 2001 and 2013 were divided into two groups: Group 1 consisted of low rebound [(≦1:32), N = 170] and Group 2 consisted of high rebound [(≧1:64), N = 21], according to the levels of the rebounded antiblood type antibodies within 1 year after transplantation. No prophylactic treatment for rejection was administered for elevated antiblood type antibodies, regardless of the levels of the rebounded antibodies. Within 1 year after transplantation, T‐cell‐mediated rejection was observed in 13 of 170 recipients (13/170, 8%) in Group 1 and in 2 of 21 recipients (2/21, 10%) in Group 2 (Groups 1 vs. 2, P = 0.432). Antibody‐mediated rejection was observed in 15 of 170 recipients (15/170, 9%) and 2 of 21 recipients (2/21, 10%) in Groups 1 and 2, respectively (P = 0.898). In this study, we found no correlation between the postoperative antiblood type antibody rebound and the incidence of acute rejection. We concluded that no treatment is necessary for rebounded antiblood type antibodies.     

Compartment‐specific expression of natural killer cell markers in renal transplantation: immune profile in acute rejection

Natural killer (NK) cells have been implicated in graft dysfunction. Here, we formulated hypothesis that distinct patterns of expression NK cells markers correlated with acute rejection in kidney transplantation. Therefore, we studied the pattern of NK cell markers CD56, CD57, and CD16 in different compartments of biopsies obtained from recipients diagnosed with acute graft rejection, with or without donor‐specific antibodies (DSA). DSA‐negative biopsies‐from patients with acute T‐cell mediated rejection (aTCMR) had an increased expression of CD56+ and CD57+ cells (P = 0.004 and P = 0.001) in the interstitial compartment in comparison with DSA‐positive biopsies from patients acute antibody‐mediated rejection (aABMR) with (aABMR C4d+) and without C4d deposition (aABMR C4d‐). CD16+ cells was increased (P = 0.03) in the glomerular compartment in DSA‐positive biopsies. We assume that CD16+ expression and antibody‐dependent cellular cytotoxicity (ADCC) in microvascular injury can be associated with aABMR. IFN‐γ release from cytoplasmic granules of NK cell could be associated with aTCMR. Our findings suggest that NK cells need to be carefully evaluated because variations in NK cell marker expression might imply the involvement of different immune system pathways in graft rejection.     

The use of NGAL and IP‐10 in the prediction of early acute rejection in highly sensitized patients following HLA‐incompatible renal transplantation

Acute rejection is a significant problem for patients undergoing HLA‐incompatible renal transplantation, affecting between 12 and 53% of patients. Any mechanism of detecting rejection in advance of current methods would offer significant benefit. This study aimed to evaluate whether serum biomarkers could predict rejection in HLAi transplants recipients. Sera from 94 HLAi transplant recipients from a single centre were analysed for a panel of biomarkers including: NGAL, KIM‐1, IP‐10, cystatin C, cathepsin L and VEGF. Biomarker levels pre‐operatively, day 1 and at day 30 post‐transplant were correlated with the development of early rejection. Significantly higher levels of IP‐10 and NGAL were seen on day 1 following transplant in those patients who developed acute rejection (P < 0.001 and 0.005) and generated AUC of 0.73 and 0.67, respectively. No differences were seen for the other biomarkers or at the other time points. In this study cohort, IP‐10 and NGAL have demonstrated good predictive ability for the development of acute rejection at a very early time point. They may have a role in identifying patients at higher risk for rejection and stratifying immunosuppression or surveillance.     

Isolated v‐lesion represents a benign phenotype of vascular rejection of the kidney allograft – a retrospective study

While the detrimental impact of the humoral acute vascular rejection (AVR) phenotype is recognized, the prognostic significance of isolated v‐lesion (IV) remains unclear. In this retrospective single‐centre study, AVR was found in 98 of 1015 patients (9.7%) who had undergone kidney transplantation in 2010–2014, with donor‐specific antibodies (DSA) evaluated in all of them. The outcome of four AVR phenotypes was evaluated during median follow‐up of 59 months; in 25 patients with IV, 18 with T‐cell‐mediated vascular rejection (TCMVR), 19 with antibody‐mediated vascular rejection (AMVR) and 36 with suspected antibody‐mediated rejection (sAMVR). AVR was diagnosed mainly by for‐cause biopsy (81%) early after transplantation (median 19 POD) and appeared as mild‐grade intimal arteritis. IV occurred in low‐sensitized patients after the first transplantation (96%) in the absence of DSA. IV responded satisfactorily to treatment (88%), showed no persistence of rejection in surveillance biopsy, and had stable graft function, minimal proteinuria and excellent DCGS (96%). Contrary to that, Kaplan–Meier estimate of 3‐year DCGS of AMVR was 66% (log‐rank = 0.0004). Early IV represents a benign phenotype of AVR with a favourable outcome. This study prompts further research to evaluate the nature of IV before considering any change in the classification and management.     

The influence of CTLA‐4 single nucleotide polymorphisms on acute kidney allograft rejection in Turkish patients

Cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) is a cell surface protein, which down‐regulates the immune response at CTLA‐4/CD28/B7 pathway. We aimed to investigate the influence of the ?318C/T, +49A/G, ?1661A/G and CT60A/G, and CTLA‐4 gene polymorphisms on acute rejection of kidney allograft in Turkish patients. The study design was a case–control study that consists of three groups: Group 1 (n = 34) represented the kidney transplant (Ktx) recipients who experienced acute rejection, Group 2 (n = 47) was randomly assigned Ktx recipients without acute rejection, and Group 3 (n = 50) consisting of healthy volunteers to evaluate the normal genomic distribution. The polymerase chain reaction–restriction fragment length polymorphism technique was used to determine the polymorphisms. Genotype and allele frequencies among three groups denoted similar distributions for +49A/G, ?1661A/G, and CT60A/G. Conversely, ?318C/T genotype was three times more frequent in the acute rejection group than in the non‐rejection group (OR = 3.45; 95%CI = 1.18–10.1, p = 0.015) and two times more frequent than the healthy control group (OR = 2.45; 95% CI = 0.98 – 6.11, p = 0.047). Additionally, having a T allele at ?318 position was significantly associated with acute rejection (0.147 vs. 0.043, OR = 3.45; 95% CI = 1.13–10.56, p = 0.02). 318C/T gene polymorphism and T allelic variant were found to be associated with increased acute rejection risk in Turkish kidney allograft recipients.