Molecular Assessment of Microcirculation Injury in Formalin-Fixed Human Cardiac Allograft Biopsies With Antibody-Mediated Rejection.

Precise diagnosis of antibody-mediated rejection (AMR) in cardiac allograft endomyocardial biopsies (EMBs) remains challenging. This study assessed molecular diagnostics in human EMBs with AMR. A set of 34 endothelial, natural killer cell and inflammatory genes was quantified in 106 formalin-fixed, paraffin-embedded EMBs classified according to 2013 International Society for Heart and Lung Transplantation (ISHLT) criteria. The gene set expression was compared between ISHLT diagnoses and correlated with donor-specific antibody (DSA), endothelial injury by electron microscopy (EM) and prognosis. Findings were validated in an independent set of 57 EMBs. In the training set (n = 106), AMR cases (n = 70) showed higher gene set expression than acute cellular rejection (ACR; n = 21, p < 0.001) and controls (n = 15, p < 0.0001). Anti-HLA DSA positivity was associated with higher gene set expression (p = 0.01). Endothelial injury by electron microscopy strongly correlated with gene set expression, specifically in AMR cases (r = 0.62, p = 0.002). Receiver operating characteristic curve analysis for diagnosing AMR showed greater accuracy with gene set expression (area under the curve [AUC] = 79.88) than with DSA (AUC = 70.47) and C4d (AUC = 70.71). In AMR patients (n = 17) with sequential biopsies, increasing gene set expression was associated with inferior prognosis (p = 0.034). These findings were confirmed in the validation set. In conclusion, biopsy-based molecular assessment of antibody-mediated microcirculation injury has the potential to improve diagnosis of AMR in human cardiac transplants.

Banff 2013 meeting report: inclusion of c4d-negative antibody-mediated rejection and antibody-associated arterial lesions.

The 12th Banff Conference on Allograft Pathology was held in Comandatuba, Brazil, from August 19-23, 2013, and was preceded by a 2-day Latin American Symposium on Transplant Immunobiology and Immunopathology. The meeting was highlighted by the presentation of the findings of several working groups formed at the 2009 and 2011 Banff meetings to: (1) establish consensus criteria for diagnosing antibody-mediated rejection (ABMR) in the presence and absence of detectable C4d deposition; (2) develop consensus definitions and thresholds for glomerulitis (g score) and chronic glomerulopathy (cg score), associated with improved inter-observer agreement and correlation with clinical, molecular and serological data; (3) determine whether isolated lesions of intimal arteritis ("isolated v") represent acute rejection similar to intimal arteritis in the presence of tubulointerstitial inflammation; (4) compare different methodologies for evaluating interstitial fibrosis and for performing/evaluating implantation biopsies of renal allografts with regard to reproducibility and prediction of subsequent graft function; and (5) define clinically and prognostically significant morphologic criteria for subclassifying polyoma virus nephropathy. The key outcome of the 2013 conference is defining criteria for diagnosis of C4d-negative ABMR and respective modification of the Banff classification. In addition, three new Banff Working Groups were initiated.

Molecular diagnosis of antibody-mediated rejection in human kidney transplants.

Antibody-mediated rejection is the major cause of kidney transplant failure, but the histology-based diagnostic system misses most cases due to its requirement for C4d positivity. We hypothesized that gene expression data could be used to test biopsies for the presence of antibody-mediated rejection. To develop a molecular test, we prospectively assigned diagnoses, including C4d-negative antibody-mediated rejection, to 403 indication biopsies from 315 patients, based on histology (microcirculation lesions) and donor-specific HLA antibody. We then used microarray data to develop classifiers that assigned antibody-mediated rejection scores to each biopsy. The transcripts distinguishing antibody-mediated rejection from other conditions were mostly expressed in endothelial cells or NK cells, or were IFNG-inducible. The scores correlated with the presence of microcirculation lesions and donor-specific antibody. Of 45 biopsies with scores>0.5, 39 had been diagnosed as antibody-mediated rejection on the basis of histology and donor-specific antibody. High scores were also associated with unanimity among pathologists that antibody-mediated rejection was present. The molecular score also strongly predicted future graft loss in Cox regression analysis. We conclude that microarray assessment of gene expression can assign a probability of ABMR to transplant biopsies without knowledge of HLA antibody status, histology, or C4d staining, and predicts future failure.

Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.

We prospectively studied kidney transplants that progressed to failure after a biopsy for clinical indications, aiming to assign a cause to every failure. We followed 315 allograft recipients who underwent indication biopsies at 6 days to 32 years posttransplant. Sixty kidneys progressed to failure in the follow-up period (median 31.4 months). Failure was rare after T-cell-mediated rejection and acute kidney injury and common after antibody-mediated rejection or glomerulonephritis. We developed rules for using biopsy diagnoses, HLA antibody and clinical data to explain each failure. Excluding four with missing information, 56 failures were attributed to four causes: rejection 36 (64%), glomerulonephritis 10 (18%), polyoma virus nephropathy 4 (7%) and intercurrent events 6 (11%). Every rejection loss had evidence of antibody-mediated rejection by the time of failure. Among rejection losses, 17 of 36 (47%) had been independently identified as nonadherent by attending clinicians. Nonadherence was more frequent in patients who progressed to failure (32%) versus those who survived (3%). Pure T-cell-mediated rejection, acute kidney injury, drug toxicity and unexplained progressive fibrosis were not causes of loss. This prospective cohort indicates that many actual failures after indication biopsies manifest phenotypic features of antibody-mediated or mixed rejection and also underscores the major role of nonadherence.

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.

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.

The molecular phenotype of 6-week protocol biopsies from human renal allografts: reflections of prior injury but not future course.

We assessed the molecular phenotype of 107 6-week protocol biopsies from human renal allografts, using Affymetrix microarrays. Transcript changes were summarized as nonoverlapping pathogenesis-based transcript sets (PBTs) reflecting inflammation (T cells, macrophages, IFNG effects) and the injury-repair response of the parenchyma, stroma and microcirculation-increased ('injury-up') and decreased ('injury-down') transcripts. The molecular changes were highly correlated with each other, even when all rejection and borderline cases were excluded. Inflammation and injury-down PBTs correlated with histologic inflammation and tubulitis, and the inflammation transcripts were greater in kidneys diagnosed as T cell-mediated or borderline rejection. Injury-up PBTs did not correlate with histopathology but did correlate with kidney function: thus functional disturbances are represented in transcript changes but not in histopathology. PBT changes correlated with prior delayed graft function. However, there was little difference between live donor kidneys and deceased donor kidneys that had not shown delayed graft function. Molecular changes did not predict future biopsies for clinical indications, rejection episodes, functional deterioration or allograft loss. Thus while detecting T cell-mediated inflammation, the molecular phenotype of early protocol biopsies mostly reflects the injury-repair response to implantation stresses, and has little relationship to future events and outcomes.

The molecular phenotype of heart transplant biopsies: relationship to histopathological and clinical variables.

Histopathology of endomyocardial biopsies (EMB) is the standard rejection surveillance for heart transplants. However, ISHLT consensus criteria for interpreting biopsies are arbitrarily defined. Gene expression offers an independent re-evaluation of existing diagnostic systems. We performed histologic and microarray analysis on 105 EMB from 45 heart allograft recipients. Histologic lesions, diagnosis and transcripts were compared to one another, time posttransplantation, indication for biopsy and left ventricular ejection fraction (LVEF). Histologic lesions presented in two groups: myocyte-interstitial and microcirculation lesions. Expression of transcript sets reflecting T cell and macrophage infiltration, and γ-interferon effects correlated strongly with each other and with transcripts indicating tissue/myocardium injury. This molecular phenotype correlated with Quilty (p < 0.005), microcirculation lesions (p < 0.05) and decreased LVEF (p < 0.007), but not with the histologic diagnosis of rejection. In multivariate analysis, LVEF was associated (p < 0.03) with γ-interferon inducible transcripts, time posttransplantation, ischemic injury and clinically indicated biopsies, but not the diagnosis of rejection. The results indicate that (a) the current ISHLT system for diagnosing rejection does not reflect the molecular phenotype in EMB and lacks clinical relevance; (b) the interpretation of Quilty lesions has to be revisited; (c) the assessment of molecules in heart biopsy can guide improvements of current diagnostics.

The molecular phenotype of kidney transplants.

Microarray studies of kidney transplant biopsies provide an opportunity to define the molecular phenotype. To facilitate this process, we used experimental systems to annotate transcripts as members of pathogenesis-based transcript sets (PBTs) representing biological processes in injured or diseased tissue. Applying this annotation to microarray results revealed that changes in single molecules and PBTs reflected a large-scale coordinate disturbance, stereotyped across various diseases and injuries, without absolute specificity of individual molecules or PBTs for rejection. Nevertheless, expression of molecules and PBTs was quantitatively specific: IFNG effects for rejection; T cell and macrophage transcripts for T cell-mediated rejection; endothelial and NK transcripts for antibody-mediated rejection. Various diseases and injuries induced the same injury-repair response, undetectable by histopathology, involving epithelium, stroma and endothelium, with increased expression of developmental, cell cycle and apoptosis genes and decreased expression of differentiated epithelial features. Transcripts reflecting this injury-repair response were the best correlates of functional disturbance and risk of future graft loss. Late biopsies with atrophy-fibrosis, reflecting their cumulative burden of injury, displayed more transcripts for B cells, plasma cells and mast cells. Thus the molecular phenotype is best described in terms of three elements: specific diseases, including rejection; the injury-repair response and the cumulative burden of injury.

An integrated view of molecular changes, histopathology and outcomes in kidney transplants.

Data-driven approaches to deteriorating kidney transplants, incorporating histologic, molecular and HLA antibody findings, have created a new understanding of transplant pathology and why transplants fail. Transplant dysfunction is best understood in terms of three elements: diseases, the active injury-repair response and the cumulative burden of injury. Progression to failure is mainly attributable to antibody-mediated rejection, nonadherence and glomerular disease. Antibody-mediated rejection usually develops late due to de novo HLA antibodies, particularly anti-class II, and is often C4d negative. Pure treated T cell-mediated rejection does not predispose to graft loss because it responds well, even with endothelialitis, but it may indicate nonadherence. The cumulative burden of injury results in atrophy-fibrosis (nephron loss), arterial fibrous intimal thickening and arteriolar hyalinosis, but these are not progressive without ongoing disease/injury, and do not explain progression. Calcineurin inhibitor toxicity has been overestimated because burden-of-injury lesions invite this default diagnosis when diseases such as antibody-mediated rejection are missed. Disease/injury triggers a stereotyped active injury-repair response, including de-differentiation, cell cycling and apoptosis. The active injury-repair response is the strongest correlate of organ function and future progression to failure, but should always prompt a search for the initiating injury or disease.

NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection.

To explore the mechanisms of antibody-mediated rejection (ABMR) in kidney transplants, we studied the transcripts expressed in clinically indicated biopsies from patients with donor-specific antibody (DSA). Comparison of biopsies from DSA-positive versus DSA-negative patients revealed 132 differentially expressed transcripts: all were associated with class II DSA but none with class I DSA. Many transcripts were expressed in DSA-positive ABMR but were also expressed in T-cell-mediated rejection (TCMR), reflecting shared molecular features. Removal of shared transcripts created 23 DSA selective transcripts (DSASTs). Some DSASTs (6/23) showed selective high expression in NK cells, whereas others (8/23) were expressed in endothelium or in endothelium plus other cell types (7/23). Of 145 biopsies ranked by DSAST expression, the 25 with highest DSAST expression primarily consisted of ABMR (22/25, 88%), either C4d-positive or C4d-negative. By immunostaining, CD56+ and CD68+ cells in peritubular capillaries, but not CD3+ cells, were increased in ABMR compared to TCMR, compatible with a role for NK cells, as well as macrophages, as effectors in endothelial injury during ABMR. Thus, the strategy of using DSASTs in the biopsy to identify mechanism-related transcripts in biopsies from patients with clinical phenotypes indicates the selective involvement of NK cells in ABMR.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Alternative macrophage activation-associated transcripts in T-cell-mediated rejection of mouse kidney allografts.

Macrophages display two activation states that are considered mutually exclusive: classical macrophage activation (CMA), inducible by IFNG, and alternative macrophage activation (AMA), inducible by IL4 and IL13. CMA is prominent in allograft rejection and AMA is associated with tissue remodeling after injury. We studied expression of AMA markers in mouse kidney allografts and in kidneys with acute tubular necrosis (ATN). In rejecting allografts, unlike interferon gamma (IFNG) effects and T-cell infiltration that developed rapidly and plateaued by day 7, AMA transcripts (Arg1, Mrc1, Mmp12 and Ear1) rose progressively as tubulitis and parenchymal deterioration developed at days 21 and 42, despite persistent IFNG effects. AMA in allografts was associated with transcripts for AMA inducers IL4, IL13 and inhibin A, but also occurred when hosts lacked IL4/IL13 receptors, suggesting a role for inhibin A. Kidneys with ATN injured by ischemia/reperfusion also had increased expression of AMA markers and inhibin A. Thus kidneys undergoing T-cell-mediated rejection progressively acquire macrophages with alternative activation phenotype despite strong local IFNG effects, independent of IL4 and IL13. Although the mechanisms and causal relationships remain to be determined, high AMA transcript levels in rejecting allografts are strongly associated with and may be a consequence of parenchymal deterioration similar to ATN.

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.

Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.

We studied the phenotype of late kidney graft failure in a prospective study of unselected kidney transplant biopsies taken for clinical indications. We analyzed histopathology, HLA antibodies and death-censored graft survival in 234 consecutive biopsies from 173 patients, taken 6 days to 31 years posttransplant. Patients with late biopsies (>1 year) frequently displayed donor-specific HLA antibody (particularly class II) and microcirculation changes, including glomerulitis, glomerulopathy, capillaritis, capillary multilayering and C4d staining. Grafts biopsied early rarely failed (1/68), whereas grafts biopsied late often progressed to failure (27/105) within 3 years. T-cell-mediated rejection and its lesions were not associated with an increased risk of failure after biopsy. In multivariable analysis, graft failure correlated with microcirculation inflammation and scarring, but C4d staining was not significant. When microcirculation changes and HLA antibody were used to define antibody-mediated rejection, 17/27 (63%) of late kidney failures after biopsy were attributable to antibody-mediated rejection, but many were C4d negative and missed by current diagnostic criteria. Glomerulonephritis accounted for 6/27 late losses, whereas T-cell-mediated rejection, drug toxicity and unexplained scarring were uncommon. The major cause of late kidney transplant failure is antibody-mediated microcirculation injury, but detection of this phenotype requires new diagnostic criteria.

De novo donor-specific antibody at the time of kidney transplant biopsy associates with microvascular pathology and late graft failure.

We studied whether de novo donor-specific antibodies (DSA) in sera from patients undergoing kidney transplant biopsies associate with specific histologic lesions in the biopsy and prognosis. DSA were assessed in 145 patients at the time of biopsy between 7 days to 31 years posttransplant. DSA was detected in 54 patients (37%), of which 32 represented de novo DSA. De novo DSA was more frequent in patients having late biopsies (34%) versus early biopsies (4%), and was usually either against class II alone or class I and II but rarely against class I alone. Microcirculation inflammation (glomerulitis, capillaritis) and damage (glomuerulopathy, capillary basement membrane multilayering), and C4d staining were associated with de novo DSA. However, the degree of scarring, arterial fibrosis and tubulo-interstitial inflammation did not correlate with the presence of de novo DSA. De novo DSA correlated with reduced graft survival after the biopsy. Thus, de novo DSA at the time of a late biopsy for clinical indication is primarily against class II, and associates with microcirculation changes in the biopsy and subsequent graft failure. We propose careful assessment of de novo DSA, particularly against class II, be performed in all late kidney transplant biopsies.

Scoring total inflammation is superior to the current Banff inflammation score in predicting outcome and the degree of molecular disturbance in renal allografts.

Emerging molecular analysis can be used as an objective and independent assessment of histopathological scoring systems. We compared the existing Banff i-score to the total inflammation (total i-) score for assessing the molecular phenotype in 129 renal allograft biopsies for cause. The total i-score showed stronger correlations with microarray-based gene sets representing major biological processes during allograft rejection. Receiver operating characteristic curves showed that total-i was superior (areas under the curves 0.85 vs. 0.73 for Banff i-score, p = 0.012) at assessing an abnormal cytotoxic T-cell burden, because it identified molecular disturbances in biopsies with advanced scarring. The total-i score was also a better predictor of graft survival than the Banff i-score and essentially all current diagnostic Banff categories. The exception was antibody-mediated rejection which is able to predict graft loss with greater specificity (96%) but at low sensitivity (38%) due to the fact that it only applies to cases with this diagnosis. The total i-score is able to achieve moderate sensitivities (60-80%) with losses in specificity (60-80%) across the whole population. Thus, the total i-score is superior to the current Banff i-score and most diagnostic Banff categories in predicting outcome and assessing the molecular phenotype of renal allografts.

Diagnosing rejection in renal transplants: a comparison of molecular- and histopathology-based approaches.

The transcriptome has considerable potential for improving biopsy diagnoses. However, to realize this potential the relationship between the molecular phenotype of disease and histopathology must be established. We assessed 186 consecutive clinically indicated kidney transplant biopsies using microarrays, and built a classifier to distinguish rejection from nonrejection using predictive analysis of microarrays (PAM). Most genes selected by PAM were interferon-gamma-inducible or cytotoxic T-cell associated, for example, CXCL9, CXCL11, GBP1 and INDO. We then compared the PAM diagnoses to those from histopathology, which are based on the Banff diagnostic criteria. Disagreement occurred in approximately 20% of diagnoses, principally because of idiosyncratic limitations in the histopathology scoring system. The problematic diagnosis of 'borderline rejection' was resolved by PAM into two distinct classes, rejection and nonrejection. The diagnostic discrepancies between Banff and PAM in these cases were largely due to the Banff system's requirement for a tubulitis threshold in defining rejection. By examining the discrepancies between gene expression and histopathology, we provide external validation of the main features of the histopathology diagnostic criteria (the Banff consensus system), recommend improvements and outline a pathway for introducing molecular measurements.

Molecular correlates of scarring in kidney transplants: the emergence of mast cell transcripts.

In the Banff consensus, infiltrates in areas of scarring are ignored. This study aimed to characterize the molecular correlates and clinical significance of scarring and inflammation in scarred areas. We assessed the extent of interstitial infiltrates, tubulitis and scarring in 129 clinically indicated renal allograft biopsies, and correlated the results with microarray expression data and allograft survival. Findings were validated in 50 additional biopsies. Transplants with scarring had a worse prognosis if the scarred area showed infiltrates. Infiltration in unscarred and scarred areas was associated with reduced death censored graft survival. In microarray analysis, infiltration in unscarred areas strongly (>r +/- 0.4) correlated with 484 transcripts associated with cytotoxic T cells, interferon-gamma, macrophages and injury. Scarring correlated with a distinct set of 172 transcripts associated with B cells, plasma cells, and others of unknown significance. The strongest correlation was with four mast cell transcripts. In biopsies with scarring, high expression of mast cell transcripts was associated with reduced graft survival and poor functional recovery. In renal allograft biopsies, infiltrates in scarred areas have implications for poor outcomes. Scarring is associated with a distinct pattern of inflammatory molecules, including B cell/immunoglobulin but particularly mast cell-associated transcripts, which correlated with poor outcomes.

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.