Donor Fas Is Not Necessary for T‐Cell‐Mediated Rejection of Mouse Kidney Allografts

It is important to resolve whether T‐cell‐mediated rejection (TCMR) is mediated by contact‐dependent cytotoxicity or by contact‐independent inflammatory mechanisms. We recently showed that the cytotoxic molecules perforin and granzymes A and B are not required for TCMR of mouse kidney transplants. Nevertheless, TCMR could still be mediated by cytotoxicity via Fas on donor cells engaging Fas ligand on host T cells. We examined whether the diagnostic TCMR lesions would be abrogated if donor Fas was absent, particularly in hosts deficient in perforin or granzymes A and B. Kidneys from Fas‐deficient donors transplanted into major histocompatibility complex (MHC)‐ mismatched hosts developed tubulitis and diffuse interstitial infiltration indistinguishable from wild‐type (WT) allografts, even in hosts deficient in perforin and granzymes A and B. Gene expression analysis revealed similar molecular disturbances in Fas‐deficient and WT allografts at day 21 transplanted into WT, perforin and granzyme A/B‐deficient hosts, indicating epithelial injury and dedifferentiation. Thus, donor Fas is not necessary for TCMR diagnostic lesions or molecular changes, even in the absence of perforin–granzyme mechanisms. We propose that in TCMR, interstitial effector T cells mediate parenchymal injury by inflammatory mechanisms that require neither the perforin–granzyme nor the Fas–Fas ligand cytotoxic mechanisms.     

Tubulitis and Epithelial Cell Alterations in Mouse Kidney Transplant Rejection Are Independent of CD103, Perforin or Granzymes A/B

One of the defining lesions of kidney allograft rejection is epithelial deterioration and invasion by inflammatory cells (tubulitis). We examined epithelial changes and their relationship to effector T cells and to CD103/E-cadherin interactions in mouse kidney allografts. Rejecting allografts showed interstitial mononuclear infiltration from day 5. Loss of epithelial mass, estimated by tubular surface area, and tubulitis were minimal through day 7 and severe by day 21. Tubules in day 21 allografts manifested severe reduction of E-cadherin and Ksp-cadherin by immunostaining with redistribution to the apical membrane, indicating loss of polarity. By flow cytometry T cells isolated from allografts were 25% CD103+. Laser capture microdissection and RT-PCR showed increased CD103 mRNA in the interstitium and tubules. However, allografts in hosts lacking CD103 developed tubulitis, cadherin loss, and epithelial deterioration similar to wild-type hosts. The loss of cadherins and epithelial mass was also independent of perforin and granzymes A and B. Thus rejection is characterized by severe tubular deterioration associated with CD103+ T cells but not mediated by CD103/cadherin interactions or granzyme-perforin cytotoxic mechanisms. We suggest that alloimmune effector T cells mediate epithelial injury by contact-independent mechanisms related to delayed type hypersensitivity, followed by invasion of the altered epithelium to produce tubulitis.     

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.     

The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell–mediated rejection,antibody‐mediated rejection,and prospects for integrative endpoints for next‐generation clinical trials

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i‐IFTA) and its relationship to T cell–mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody‐mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i‐IFTA is associated with reduced graft survival. Furthermore, these groups presented that i‐IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i‐IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor‐specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next‐generation clinical trials.     

Does tubulitis without interstitial inflammation represent borderline acute T cell mediated rejection?

Tubulitis without interstitial inflammation (Banff i0), termed “isolated tubulitis” (ISO‐T), has been controversially included within the Banff “borderline” category of acute T cell mediated rejection (TCMR). This single‐center, retrospective, observational study of 2055 consecutive biopsies from 775 recipients, determined the clinical significance of ISO‐T. ISO‐T prevalence was 19.1%, comprising mild tubulitis (i0t1) in 97.2%. Independent clinical predictors of tubulitis were HLA mismatch, prior TCMR and antibody‐mediated rejection, pulse corticosteroids, and BKVAN (P = .006 to P < .001 by multivariable analysis). Histological associations of tubulitis included interstitial inflammation, peritubular capillaritis, tubular atrophy, and SV40T (P = .005 to <.001). The dominant pathological diagnoses in ISO‐T (n = 393) were interstitial fibrosis/tubular atrophy (IF/TA, 44.5%) or normal/minimal (31.8%). Subanalysis of ISO‐T from indication biopsies (n = 107) found acute tubular injury (37.4%), IF/TA (28.0%), normal/minimal (12.1%), acute rejection (9.3%, vascular or antibody), chronic‐active TCMR (2.8%), and BKVAN (5.6%). Allograft function of ISO‐T frequently improved, affected by early biopsy timing and underlying disease diagnosis. Subsequent histology of 1197 ISO‐T biopsy‐pairs was generally benign. The 1‐ and 5‐year death‐censored graft survivals of ISO‐T were 98.8% and 92.7%. In summary, tubulitis without inflammation does not represent borderline TCMR. We suggest its removal from the borderline category, and reinstatement of i1 as the diagnostic threshold.     

Inflammation Lesions in Kidney Transplant Biopsies: Association with Survival Is Due to the Underlying Diseases

Assessment of kidney transplant biopsies relies on nonspecific inflammatory lesions: Interstitial infiltrates (i), tubulitis (t) and intimal arteritis (v). We studied the relationship between inflammation and prognosis in biopsies for clinical indications from 314 patients (median follow‐up 25 months). We used a modified Banff classification, separately assessing inflammation (i‐) in nonscarred (i‐Banff), scarred (i‐IFTA) and whole cortex (i‐total), plus tubulitis and intimal arteritis. In early biopsies (<1 year), i‐ and t‐lesions had no association with graft survival. In late (>1 year) biopsies, all i‐scores correlated with progression to failure, due to the association of these infiltrates with progressive diseases: antibody‐mediated rejection (ABMR) and glomerulonephritis. Tubulitis in nonscarred areas had no impact on survival. Severe tubulitis including scarred areas (tis3) was associated with worse survival, but reflected polyoma virus nephropathy or ABMR, not T‐cell‐mediated rejection. Intimal arteritis (v‐lesions) had no association with allograft loss in early or late biopsies. In multivariate analysis, outcome was better predicted by the presence of progressive disease than by inflammation. Thus inflammation in late kidney transplants has no inherent prognostic impact, but predicts reduced survival because inflammation indicates actively progressing diseases. The most important predictor of outcome is the diagnosis of a progressive disease.     

Potential Impact of Microarray Diagnosis of T Cell–Mediated Rejection in Kidney Transplants: The INTERCOM Study

We previously developed a microarray‐based test for T cell‐mediated rejection (TCMR) in a reference set of 403 biopsies. To determine the potential impact of this test in clinical practice, we undertook INTERCOM, a prospective international study of 300 indication biopsies from 264 patients ( ClinicalTrials.gov NCT01299168). Biopsies from six centers—Baltimore, Barcelona, Edmonton, Hannover, Manchester and Minneapolis—were analyzed by microarrays, assigning TCMR scores by an algorithm developed in the reference set and comparing TCMR scores to local histology assessment. The TCMR score correlated with histologic TCMR lesions—tubulitis and interstitial infiltration. The accuracy for primary histologic diagnoses (0.87) was similar to the reference set (0.89). The TCMR scores reclassified 77/300 biopsies (26%): 16 histologic TCMR were molecularly non‐TCMR; 15 histologic non‐TCMR were molecularly TCMR, including 6 with polyoma virus nephropathy; and all 46 “borderline” biopsies were reclassified as TCMR (8) or non‐TCMR (38). Like the reference set, discrepancies were primarily in situations where histology has known limitations, for example, in biopsies with scarring and inflammation/tubulitis potentially from other diseases. Neither the TCMR score nor histologic TCMR was associated with graft loss. Thus the molecular TCMR score has potential to add new insight, particularly in situations where histology is ambiguous or potentially misleading.     

Cluster Analysis of Lesions in Nonselected Kidney Transplant Biopsies: Microcirculation Changes,Tubulointerstitial Inflammation and Scarring

Banff classification empirically established scoring of histologic lesions, but the relationships of lesions to each other and to underlying biologic processes remain unclear. We hypothesized that class discovery tools would reveal new relationships between individual lesions, and relate lesions to C4d staining, anti‐HLA donor‐specific antibody (DSA) and time posttransplant. We studied 234 nonselected renal allograft biopsies for clinical indications from 173 patients. Silhouette plotting and principal component analysis revealed three groups of lesions: microcirculation changes, including inflammation (glomerulitis, capillaritis) and deterioration (double contours, mesangial expansion); scarring/hyalinosis; and tubulointerstitial inflammation. DSA and C4d grouped with microcirculation inflammation, whereas time posttransplant grouped with scarring/hyalinosis lesions. Intimal arteritis clustered with DSA, C4d and microcirculation inflammation, but also showed correlations with tubulitis. Fibrous intimal thickening in arteries clustered with scarring/hyalinosis. Capillary basement membrane multilayering showed intermediary relationships between microcirculation deterioration and time‐dependent scarring. Correlation analysis and hierarchical clustering confirmed the lesion relationships. Thus, we propose that the pathologic lesions in biopsies are not independent but are members of groups that represent distinct pathogenic forces: microcirculation changes, reflecting the stress of DSA; scarring, hyalinosis and arterial fibrosis, reflecting the cumulative burden of injury over time; and tubulointerstitial inflammation. Interpretation of lesions should reflect these associations.     

Using Molecular Phenotyping to Guide Improvements in the Histologic Diagnosis of T Cell–Mediated Rejection

Recognition that some lesions typical of T cell–mediated rejection (TCMR) also occur in antibody‐mediated rejection requires revision of the histologic TCMR definition. To guide this process, we assessed the relative importance of various lesions and the performance of new histology diagnostic algorithms, using molecular TCMR scores as histology‐independent estimates of true TCMR. In 703 indication biopsies, random forest analysis and logistic regression indicated that interstitial infiltrate (i‐lesions) and tubulitis (t‐lesions) were the key histologic predictors of molecular TCMR, with arteritis (v‐lesions) having less importance. Histology predicted molecular TCMR more accurately when diagnoses were assigned by strictly applying the Banff rules to the lesion scores and redefining isolated v‐lesion TCMR. This improved prediction from area under the curve (AUC) 0.70 with existing rules to AUC 0.80. Further improvements were achieved by introducing more categories to reflect inflammation (AUC 0.84), by summing the lesion scores (AUC 0.85) and by logistic regression (AUC 0.90). We concluded that histologic assessment of TCMR can be improved by placing more emphasis on i‐ and t‐lesions and incorporating new algorithms for diagnosis. Nevertheless, some discrepancies between histologic and molecular diagnoses persist, partially due to the inherent nonspecificity of i‐ and t‐lesions, and molecular methods will be required to help resolve these cases.     

Subclinical Rejection Phenotypes at 1 Year Post-Transplant and Outcome of Kidney Allografts

Kidney allograft rejection can occur in clinically stable patients, but long-term significance is unknown. We determined whether early recognition of subclinical rejection has long-term consequences for kidney allograft survival in an observational prospective cohort study of 1307 consecutive nonselected patients who underwent ABO-compatible, complement-dependent cytotoxicity-negative crossmatch kidney transplantation in Paris (2000–2010). Participants underwent prospective screening biopsies at 1 year post-transplant, with concurrent evaluations of graft complement deposition and circulating anti-HLA antibodies. The main analysis included 1001 patients. Three distinct groups of patients were identified at the 1-year screening: 727 (73%) patients without rejection, 132 (13%) patients with subclinical T cell-mediated rejection (TCMR), and 142 (14%) patients with subclinical antibody-mediated rejection (ABMR). Patients with subclinical ABMR had the poorest graft survival at 8 years post-transplant (56%) compared with subclinical TCMR (88%) and nonrejection (90%) groups (P<0.001). In a multivariate Cox model, subclinical ABMR at 1 year was independently associated with a 3.5-fold increase in graft loss (95% confidence interval, 2.1 to 5.7) along with eGFR and proteinuria (P<0.001). Subclinical ABMR was associated with more rapid progression to transplant glomerulopathy. Of patients with subclinical TCMR at 1 year, only those who further developed de novo donor-specific antibodies and transplant glomerulopathy showed higher risk of graft loss compared with patients without rejection. Our findings suggest that subclinical TCMR and subclinical ABMR have distinct effects on long-term graft loss. Subclinical ABMR detected at the 1-year screening biopsy carries a prognostic value independent of initial donor-specific antibody status, previous immunologic events, current eGFR, and proteinuria.     

Molecular Diagnosis of T Cell‐Mediated Rejection in Human Kidney Transplant Biopsies

Histologic diagnosis of T cell‐mediated rejection is flawed by subjective assessments, nonspecific lesions and arbitrary rules. This study developed a molecular test for T cell‐mediated rejection. We used microarray results from 403 kidney transplant biopsies to derive a classifier assigning T cell‐mediated rejection scores to all biopsies, and compared these with histologic assessments. The score correlated with histologic lesions of T cell‐mediated rejection (infiltrate, tubulitis). The accuracy of the classifier for the histology diagnoses was 89%. Very high and low molecular scores corresponded with unanimity among three pathologists on the presence or absence of T cell‐mediated rejection, respectively. The molecular score had low sensitivity (50%) and positive predictive value (62%) for the histology diagnoses. However, histology showed similar disagreement between pathologists—only 45–56% sensitivity of one pathologist with diagnoses of T cell‐mediated rejection by another. Discrepancies between molecular scores and histology were mostly when histology was ambiguous (“borderline”) or unreliable, e.g. in cases with scarring or inflammation induced by tissue injury. Vasculitis (isolated v‐lesion TCMR) was particularly discrepant, with most cases exhibiting low TCMR scores. We propose new rules to integrate molecular tests and histology into a precision diagnostic system that can reduce errors, ambiguity and interpathologist disagreement.     

Absence of donor CD40 protects renal allograft epithelium and preserves renal function

Blocking the CD40‐CD154 pathway prevents allograft rejection and induces donor‐specific tolerance in various experimental models. However, the translation to clinical studies has been hampered by unexpected thromboembolic complications of CD154‐blocking antibodies. Thus, blocking CD40 instead is now considered as an alternative strategy. Here, we evaluated the role of donor CD40 in allospecific T‐cell responses in vitro and in an in vivo model for renal transplantation. Fully MHC‐mismatched allografts from CD40‐deficient donors displayed better renal function than wild type. These functional data correlated with a lower level of apoptosis in renal tubular epithelial cells and higher expression of PD‐L1, which is most probably because of a reduced Th17 response in recipients of a CD40‐deficient donor. This hypothesis was supported in vitro, where donor CD40 expression was important for the induction of direct allospecific T‐cell responses. Especially the induction of Th17 cells was critically dependent on donor CD40. IL‐17A in conjunction with interferon‐γ in turn rendered renal tubular epithelial cells to a more costimulatory state by upregulating CD40 and downregulating PD‐L1 expression. In conclusion, CD40 blockade not only reduces the allospecific T‐cell responses, but might also lead to protection of tubular epithelium from apoptosis and thereby preserve kidney allograft function.