Comprehensive Analysis of Transcript Changes Associated With Allograft Rejection: Combining Universal and Selective Features.

We annotated the top transcripts associated with kidney transplant rejection by p-value, either universal for all rejection or selective for T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR; ClinicalTrials.gov NCT01299168). We used eight class-comparison algorithms to interrogate microarray results from 703 biopsies, 205 with rejection. The positive comparators were all rejection, TCMR, or ABMR; the negative comparators varied from normal biopsies to all nonrejecting biopsies, including other diseases. The universal algorithm, rejection versus all nonrejection, identified transcripts mainly inducible by interferon γ. Selectivity for ABMR or TCMR required the other rejection class as well as nonrejection biopsies in the comparator to avoid selecting universal transcripts. Direct comparison of ABMR versus TCMR yielded only transcripts related to TCMR, the stronger signal. Transcripts highly associated with rejection were never completely specific for rejection: Many were increased in biopsies without rejection, reflecting sharing between rejection and injury-induced innate immunity. Union of the top 200 transcripts from universal and selective algorithms yielded 454 transcripts that permitted unsupervised analysis of biopsies in principal component analysis: PC1 was rejection, and PC2 was separation of TCMR from ABMR. Appreciating rejection-associated molecular changes requires a diverse case mix, accurate histologic classification (including C4d-negative ABMR), and both selective and universal algorithms.

A Probabilistic Approach to Histologic Diagnosis of Antibody-Mediated Rejection in Kidney Transplant Biopsies.

Histologic diagnosis of antibody-mediated rejection (ABMR) in kidney transplant biopsies uses lesion score cutoffs such as 0 versus >0 rather than actual scores and requires donor-specific antibody (DSA); however, cutoffs lose information, and DSA is not always reliable. Using microarray-derived molecular ABMR scores as a histology-independent estimate of ABMR in 703 biopsies, we reassessed criteria for ABMR to determine relative importance of various lesions, the utility of equations using actual scores rather than cutoffs, and the potential for diagnosing ABMR when DSA is unknown or negative. We confirmed that the important features for ABMR diagnosis were peritubular capillaritis (ptc), glomerulitis (g), glomerular double contours, DSA and C4d staining, but we questioned some features: arterial fibrosis, vasculitis, acute tubular injury, and sum of ptc+g scores. Regression equations using lesion scores predicted molecular ABMR more accurately than score cutoffs (area under the curve 0.85-0.86 vs. 0.75). DSA positivity improved accuracy, but regression equations predicted ABMR with moderate accuracy when DSA was unknown. Some biopsies without detectable DSA had high probability of ABMR by regression, although most had HLA antibody. We concluded that regression equations using lesion scores plus DSA maximized diagnostic accuracy and can estimate probable ABMR when DSA is unknown or undetectable.

The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors.

The recent recognition that antibody-mediated rejection (ABMR) is the major cause of kidney transplant loss creates strong interest in its pathogenesis. We used microarray analysis of kidney transplant biopsies to identify the changes in pure ABMR. We found that the ABMR transcript changes in the initial Discovery Set were strongly conserved in a subsequent Validation Set. In the Combined Set of 703 biopsies, 2603 transcripts were significantly changed (FDR < 0.05) in ABMR versus all other biopsies. In cultured cells, the transcripts strongly associated with ABMR were expressed in endothelial cells, e.g. cadherins CDH5 and CDH13; IFNG-treated endothelial cells, e.g. phospholipase PLA1A and chemokine CXCL11; or NK cells, e.g. cytotoxicity molecules granulysin (GNLY) and FGFBP2. Other ABMR transcripts were expressed in normal kidney but not cell lines, either increased e.g. Duffy chemokine receptor (DARC) or decreased e.g. sclerostin (SOST). Pathway analysis of ABMR transcripts identified angiogenesis, with roles for angiopoietin and vascular endothelial growth factors; leukocyte-endothelial interactions; and NK signaling, including evidence for CD16a Fc receptor signaling elements shared with T cells. These data support a model of ABMR involving injury-repair in the microcirculation induced by cognate recognition involving antibody and CD16a, triggering IFNG release and antibody-dependent NK cell-mediated cytotoxicity.

Molecular landscape of T cell-mediated rejection in human kidney transplants: prominence of CTLA4 and PD ligands.

We used expression microarrays to characterize the changes most specific for pure T cell-mediated rejection (TCMR) compared to other diseases including antibody-mediated rejection in 703 human kidney transplant biopsies, using a Discovery Set-Validation Set approach. The expression of thousands of transcripts--fold change and association strength--changed in a pattern that was highly conserved between the Discovery and Validation sets, reflecting a hierarchy of T cell signaling, costimulation, antigen-presenting cell (APC) activation and interferon-gamma (IFNG) expression and effects, with weaker associations for inflammasome activation, innate immunity, cytotoxic molecules and parenchymal injury. In cell lines, the transcripts most specific for TCMR were expressed most strongly in effector T cells (e.g. CTLA4, CD28, IFNG), macrophages (e.g. PDL1, CD86, SLAMF8, ADAMDEC1), B cells (e.g. CD72, BTLA) and IFNG-treated macrophages (e.g. ANKRD22, AIM2). In pathway analysis, the top pathways included T cell receptor signaling and CTLA4 costimulation. These results suggest a model in which TCMR creates an inflammatory compartment with a rigorous hierarchy dominated by the proximal aspects of cognate engagement of effector T cell receptor and costimulator triggering by APCs. The prominence of inhibitors like CTLA4 and PDL1 raises the possibility of active negative controls within the rejecting tissue.

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.

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.

Kidney transplants with progressing chronic diseases express high levels of acute kidney injury transcripts.

We previously reported that kidney transplants with early acute injury express transcripts indicating injury repair--the acute kidney injury signal. This study investigated the significance of this signal in transplants with other conditions, including rejection and recurrent disease. The injury signal was elevated in biopsies in many different conditions, including T cell-mediated rejection and potentially progressive diseases such as antibody-mediated rejection and glomerulonephritis. A high injury signal correlated with poor function and with inflammation in areas of fibrosis, but not with fibrosis without inflammation. In multivariate survival analysis, the injury signal in late kidney transplant biopsies strongly predicted future graft loss, similar to a published molecular risk score derived in late kidneys. Indeed, the injury signal shared many individual transcripts with the risk score, e.g. ITGB6, VCAN, NNMT. The injury signal was a better predictor of future graft loss than fibrosis, inflammation or expression of collagen genes. Thus the acute injury signal, first defined in early reversible injury, is present in many diseases as a reflection of parenchymal distress, where its significance is dictated by the inducing insult, i.e. treatable/self-limited versus untreatable and sustained. Progression in troubled transplants is primarily a function of ongoing parenchymal injury by disease, not fibrogenesis.

Comparing molecular assessment of implantation biopsies with histologic and demographic risk assessment.

We hypothesized that measurement of previously defined acute kidney injury-induced transcripts at the time of implantation would add a new dimension to existing methods based on donor factors, histology and recipient factors. We analyzed microarray results from implantation biopsies taken after reperfusion from 70 kidneys from 53 deceased donors. We used two definitions of early dysfunction: serum creatinine > 265 umol/L at day 7 posttransplant; and dialysis in the first week. The strongest correlate with early dysfunction was the mean expression of 30 injury transcripts. Older donor and recipient age were associated with early dysfunction, but histologic lesions were not. Prediction was best when the injury transcript expression was combined with donor or recipient age, particularly in standard criteria donors. In contrast, although extended criteria donor kidneys had a high risk of early dysfunction, no variables tested, including injury transcripts, predicted risk significantly, probably because these kidneys were allocated preferentially to old, high risk recipients. The injury transcripts did not predict late function, which was mainly associated with donor age. Thus, measurement of injury-induced transcripts at the time of implantation improves the prediction of early kidney dysfunction, but risk prediction may fail when old kidneys are transplanted into old recipients.

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.

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 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 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.

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.

Loss of solute carriers in T cell-mediated rejection in mouse and human kidneys: an active epithelial injury-repair response.

T cell-mediated rejection of kidney allografts causes epithelial deterioration, manifested by tubulitis, but the mechanism remains unclear. We hypothesized that interstitial inflammation triggers a stereotyped epithelial response similar to that triggered by other types of injury such as ischemia-reperfusion. We identified solute carrier transcripts with decreased expression in mouse allografts, and compared their behavior in T cell-mediated rejection to native kidneys with ischemic acute tubular necrosis (ATN). Average loss of solute carrier expression was similar in ATN (77%) and T cell-mediated rejection (75%) with high correlation of individual transcripts. Immunostaining of SLC6A19 confirmed loss of proteins. Analysis of human kidney transplant biopsies confirmed that T cell-mediated rejection and ATN showed similar loss of solute carrier mRNAs. The loss of solute carrier expression was weakly correlated with interstitial inflammation, but kidneys with ATN showed decreased solute carriers despite minimal inflammation. Loss of renal function correlated better with decreased solute carrier expression than with histologic lesions (r = 0.396, p < 0.001). Thus the loss of epithelial transcripts in rejection is not a unique consequence of T cell-mediated rejection but an active injury-repair response of epithelium, triggered by rejection but also by other injury mechanisms.

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.

Defining the canonical form of T-cell-mediated rejection in human kidney transplants.

Banff defines T-cell-mediated rejection (TCMR) using nonspecific lesions and arbitrary cutoffs, with no external gold standard. We reexamined features of TCMR using exclusively molecular definition independent of histopathology. The definition was derived from mouse kidney transplants with fully developed TCMR, and is based on high expression of transcripts reflecting IFNG effects and alternative macrophage activation. In 234 human kidney transplant biopsies for cause phenotyped by microarrays, we identified 26 biopsies meeting these criteria. After excluding three biopsies with unrelated diseases, all 23 biopsies had typical Banff lesions of TCMR (inflammation, tubulitis), with v lesions in 10/23. Banff histopathology diagnosed 18 as TCMR, 1 as mixed and 4 as borderline. Despite marked changes in transcriptome indicating tissue injury and dedifferentiation, all kidneys with molecularly defined TCMR, even with v lesions or late rejection, demonstrated excellent recovery of function at 6 months with no graft loss (mean follow-up 2.5 years). Thus TCMR defined exclusively by molecules manifests TCMR-related lesions and function impairment, but good recovery and survival, even with late rejection or arteritis. This combination of pathologic, clinical and molecular features constitutes the typical or canonical T-cell-mediated rejection.

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.

The early course of kidney allograft rejection: defining the time when rejection begins.

We studied the early events in mouse kidney allografts and isografts to define when allorecognition begins and when alloimmune tissue injury begins. Allografts but not isografts showed T-cell infiltration in perivascular areas from day 1, but tubulitis and arteritis did not develop until day 7. Flow cytometry confirmed the early allospecific CD3(+)CD8(+) T-cell infiltrate. At day 1, both allografts and isografts showed extensive transcriptome changes, reflecting the response to surgery, but only allografts showed expression of interferon-gamma (IFN-gamma)-inducible transcripts and T-cell-associated transcripts. Although the number of CD68(+) myeloid cell numbers did not increase in day 1 isografts or allografts, mRNA expression for myeloid markers was increased in isografts and allografts, suggesting activation of resident cells of the macrophage-dendritic cell series (MMDCs) in response to injury, followed by increased CD68(+) cell numbers from day 2. By day 3, an interstitial T-cell and MMDC infiltrate was established in allografts, corresponding with the emergence of allospecific tissue injury, as reflected by decreased parenchymal transcripts. Thus, in renal allografts, allorecognition by T cells occurs in perivascular sites by day 1, but alloimmune parenchymal damage begins at day 3, coinciding with the emergence of the interstitial T-cell-MMDC infiltrate.

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.