The causes, significance and consequences of inflammatory fibrosis in kidney transplantation: The Banff i- IFTA lesion

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1 Received: 31 May 2017 Revised: 25 October 2017 Accepted: 28 October 2017 DOI: /ajt ORIGINAL ARTICLE The causes, significance and consequences of inflammatory fibrosis in kidney transplantation: The Banff i- IFTA lesion Brian J. Nankivell 1 Meena Shingde 2 Karen L. Keung 1 Caroline L-S. Fung 2 Richard J. Borrows 1 Philip J. O Connell 1 Jeremy R. Chapman 1 1 Department of Renal Medicine, Westmead Hospital, Sydney, Australia 2 Tissue Pathology and Diagnostic Oncology, Westmead Hospital, Sydney, Australia Correspondence Brian J. Nankivell Brian.Nankivell@health.nsw.gov.au Present addresses Caroline L-S. Fung, Anatomical Pathology, Concord Repatriation General Hospital, Concord, NSW, Australia Richard J. Borrows, Nephrology, University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital, Birmingham, UK Inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) is associated with adverse outcomes in kidney transplantation. We evaluated i- IFTA in 429 indicationand 2052 protocol- driven biopsy samples from a longitudinal cohort of 362 kidney pancreas recipients to determine its prevalence, time course, and relationships with T cell mediated rejection (TCMR), immunosuppression, and outcome. Sequential histology demonstrated that i- IFTA was preceded by cellular interstitial inflammation and followed by IF/TA. The prevalence and intensity of i- IFTA increased with developing chronic fibrosis and correlated with inflammation, tubulitis, and immunosuppression era (P <.001). Tacrolimus era based immunosuppression was associated with reduced histologic inflammation in unscarred and scarred i- IFTA compartments, ameliorated progression of IF, and increased conversion to inactive IF/TA (compared with cyclosporine era, P <.001). Prior acute (including borderline) TCMR and subclinical TCMR were followed by greater 1- year i- IFTA, remaining predictive by multivariate analysis and independent of humoral markers. One- year i- IFTA was associated with accelerated IF/TA, arterial fibrointimal hyperplasia, and chronic glomerulopathy and with reduced renal function (P <.001 versus no i- IFTA). In summary, i- IFTA is the histologic consequence of active T cell mediated alloimmunity, representing the interface between inflammation and tubular injury with fibrotic healing. Uncontrolled i- IFTA is associated with adverse structural and functional outcomes. KEYWORDS classification systems: Banff classification, clinical research/practice, immunosuppressive regimens, kidney transplantation/nephrology, pathology/histopathology, rejection: T cell mediated (TCMR) 1 INTRODUCTION T cell mediated rejection (TCMR) is characterized by interstitial lymphocytic infiltration and tubulitis of the allograft, semiquantitatively Abbreviations: AHR, acute humoral rejection; CI, confidence interval; CSA, cyclosporine; DSA, donor-specific antibody; GFR, glomerular filtration rate; HR, hazard ratio; IF/TA, interstitial fibrosis/tubular atrophy; i-ifta, interstitial inflammation within areas of interstitial fibrosis and tubular atrophy; MFI, median fluorescence intensity; OR, odds ratio; SCR, subclinical rejection; TAC, tacrolimus; TCMR, T cell mediated rejection. coded by i and t Banff scores, respectively, and formulated into diagnostic categories for acute TCMR. 1 Originally optimized for early acute rejection appearing within unscarred kidneys by the Banff consensus in 1991, it still underpins the current diagnostic schema of TCMR. 2 However, modern immunosuppression has altered the presentation of acute rejection: early episodes are fewer and shifted to later presentation of mixed rejection associated with interstitial fibrosis and tubular atrophy (IF/TA). Because only interstitial inflammation within unscarred parenchyma (Banff i) is counted toward the diagnosis of The American Society of Transplantation amjtransplant.com Am J Transplant. 2018;18: and the American Society of Transplant Surgeons

2 365 TCMR, mononuclear cells that are actively destroying tubules are conventionally excluded when IF/TA develops, potentially leading to underdiagnosis. This artificial compartmentalization of inflammation into separate areas according to the presence of allograft scarring makes little biological sense: cells can influence distant microenvironments by secretory signals. 3,4 One solution to account for these uncounted alloreactive cells within IF/TA areas is to score i- IFTA, defined as inflammation within areas of IF/TA and included within the 2015 Banff schema. 1,4-7 i- IFTA mirrors Banff i, with identical thresholds and semiquantitative scores, but is applied only to scarred cortical parenchyma. Its impact has been sporadically reported: by us from 2004 and designated as cdi score using current definitional thresholds 6 ; by Mengel and Sellares (2007, 2009, and 2011, designated as i- IFTA and also quantified by the absolute proportion of total cortex affected) 7-9 ; and by Mannon and DEKAF investigators (in 2010, designated iatr as inflammation in areas of tubular atrophy, using the current definition). 4 i- IFTA in indication biopsies strongly predicted death- censored graft failure (independent of renal function, IF/TA, and inflammation scores) in the DEKAF study, 4 confirming findings from Edmonton. 7 The i- IFTA pattern is not synonymous with the total inflammation (ti) score (counting total cortical inflammation) 1,9 or the Boolean combination of Banff i inflammation occurring with IF/TA ( i+if/ta ), although both demonstrate adverse outcomes, including renal dysfunction, 10,11 nephron attrition by serial pathology, de novo donor- specific antibodies (DSAs), 12,14 and allograft failure. 13,15,16 The mechanisms and causal relationships between i- IFTA and TCMR, their biological, functional, and histologic outcomes, require further investigation. 1,17 We hypothesized that (1) i- IFTA represents the histologic consequence of prior alloimmune T cell mediated tubular injury, expressed as previous clinical or subclinical TCMR, and (2) inflammatory tubular destruction of i- IFTA is the dominant ( necessary but not sufficient ) pathologic expression of chronic TCMR, contributing to progressive allograft deterioration. The study aims were to understand the pathophysiologic relationships (s) of i- IFTA; correlate i- IFTA with histological inflammation, renal function, immunosuppression, evolution of chronic histologic changes, and graft survival; establish temporal interrelationships between inflammation, tubular destruction, and i- IFTA; and formulate potential mechanistic explanations for tubular destruction. We evaluated a longitudinal data set of 2481 prospective surveillance and indication biopsies obtained up to 10 years posttransplantation, to characterize the time course of i- IFTA relative to influx of inflammatory cells and development of IF/TA, its clinical and subclinical antecedents, and histologic and functional consequences. 2 METHODS AND MATERIALS 2.1 Study population and immunosuppression The study group consisted of kidney pancreas recipients transplanted from 1987 to 2012, for whom a minimum of 1- year histologic follow- up was available. Implanted kidneys were pristine and unscarred at transplantation. Recipients underwent regular, prospective protocol kidney transplant biopsies from implantation until 10 years after transplantation, with additional indication biopsies as clinically indicated. 18 Triple therapy immunosuppression consisted of cyclosporine until its replacement by tacrolimus in 1999 (target levels adjusted clinical events, rejection, nephrotoxicity, and side effects), azathioprine replaced by mycophenolate mofetil in 1996, and prednisolone throughout. Induction by antilymphocyte preparations was reserved for highly sensitized recipients. Patients received optimal kidneys from younger deceased donors, eliminating recurrent disease and donor quality as confounding factors, and leaving alloimmune mechanisms as the dominant driver of injury in this cohort. 2.2 Histologic assessment of i- IFTA Needle- core protocol biopsy samples were taken at implantation and 1 week (early era only); at 1, 3, 6, and 12 months; and then annually or second/third yearly for 10 years, as described. 19 Specialist nephropathologists scored for individual parameters and classified by rejection phenotypes using the Banff schema. 1 Blinded duplicate scoring data were used until Subsequent scores were unblinded to clinical information. The i- IFTA score was defined by Banff 2015 and semiquantitatively graded based on extent of mononuclear infiltration within the areas of tubular atrophy and interstitial fibrosis as i- IFTA0, with mononuclear cells less than 10%; i- IFTA1 = 10% to 25%; i- IFTA2 = 26% to 50%; and i- IFTA3 = greater than 50% of scarred cortical parenchyma, as originally described and used consistently to date. 1,6 2.3 Specific study aims The specific study aims were to: 1. Understand the pathophysiologic cause(s) of i-ifta by evaluation of risk factor, time course, and relationships to clinical events including prior acute rejection number and phenotype 2. Clarify temporal relationships of the relative onset of inflammation, i-ifta, and tubular destruction, against overall IF/TA (and uninflamed fibrosis), to support causal inferences 3. Correlate i-ifta with histologic inflammation (including Banff i, t, g, and ptc scores) and other histologic abnormalities (including the presence of scarring, tubular necrosis, and edema) 4. Evaluate the effect of immunosuppression era on the incidence and prevalence of i-ifta, IF/TA, and inflammation 5. Correlate i-ifta with long-term renal dysfunction 6. Evaluate long-term outcomes of i-ifta on longitudinal isotopic glomerular filtration rate (GFR) and death-censored allograft survival 7. Describe the incidence of chronic pathologic abnormalities: IF/TA, arterial fibrointimal hyperplasia, and transplant glomerulopathy following i-ifta, using actuarial analysis of sequential protocol histology 8. Establish a mechanistic theory linking allograft inflammation with tubular destruction in the context of i-ifta

3 Consider the relevance of the i-ifta lesion to define chronic TCMR 2.4 Data analysis The research design was a single- center, observational cohort study, with retrospective analysis of prospectively collected protocol pathology. STROBE reporting guidelines cohort studies were used. 20 The institutional ethics approvals were HREC LNR/12/WMEAD/114 and LNRSSA/12/WMEAD/117 (3503). All biopsy samples were scored and included. Missing data were omitted, and values were not imputed. Pearson s (r) and Spearman s (ρ) rank coefficients were used for correlations, for parametric and nonparametric data, respectively. The first occurrence of a lesion from serial pathology samples was counted for actuarial analysis. Cox regression was used for survival analysis, preceded by backward elimination. Note that for some analyses, the Kaplan- Meier curves (nonconventionally) begin at transplantation, with stratification by 1- year i- IFTA occurrence, to illustrate the early development of histologic abnormalities. Multiple logistic regression (and ordinal regression) evaluated i- IFTA from 1- year protocol histology. Potential risk factors screened by univariate analysis before multivariable models are listed with detailed descriptions for model construction (Table S1, and detailed methodology). A conditional- binomial- exact test was used for 2 2 categorical data. A least- significantdifferences test was used for multiple group comparisons, after 1- way ANOVA. Linear or binomial generalized estimating equations were used for repeated measurements, as appropriate. Graft loss was censored for patient death (although combined loss data are also presented). Data are expressed as mean ± SD unless stated. All tests were 2- tailed, and a probability <.05 was considered significant. 3 RESULTS 3.1 Demographic and study group data From 395 consecutive recipients of a combined kidney pancreas transplant who have type 1 diabetes (detailed in Table S2), 33 were excluded because of inadequate serial pathology, leaving a study group of 362 patients with longitudinal histology (Table S3). Recipients (n = 362) were 54.1% male and 38.4 ± 6.9 years old and had sustained normoglycemia posttransplantation with a mean glycated hemoglobin (HbA 1c ) of 5.6% ± 0.9%). Donors were 26.9 ± 9.3 years old and 61.9% male, HLA mismatch score was 4.5 ± 1.3, and pretransplantation DSAs (anti- HLA median fluorescence intensity [MFI] >500 by solidphase assay; Luminex, One Lambda, Canoga Park, CA) were detected in 80 (27.3%) of 293 tested patients. Only 5 recipients received repeat grafts. Posttransplantation hemodialysis was required in 20 patients. Early acute cellular rejection (before 3 months posttransplantation) occurred in 123 (34.0%), vascular rejection occurred in 42 (11.6%) and humoral rejection occurred in 46 (12.7%, episodes not mutually exclusive). Antilymphocyte treatment was used in 84 (23.2%) of rejection episodes. 3.2 Renal transplant pathology Each patient underwent 6.9 ± 3.4 biopsies (range 3-24) until a mean of 5.3 ± 4.7 years posttransplantation, producing a total of 2481 biopsies. Suboptimal samples (<7 glomeruli or no artery) characterized 606 (23.2%) of 2616 biopsies, but all were included to formulate time series. Evaluated tissue contained 12.5 ± 8.6 glomeruli and 2.0 ± 1.1 arteries (full data given in Table S4). Implantation biopsies displayed Banff ci and ct scores of 0.03 ± 0.15 and 0.03 ± 0.16, respectively, and 1.4 ± 3.5% glomeruli were sclerosed. Biopsies were per protocol in 2051 (82.7%, taken 2.27 ± 1.31 years posttransplantation) and by indication in 430 (15.1%, at 1.45 ± 3.45 years). Of 2481 available biopsies, 1261 samples were scored as Banff ci0, leaving 1220 scarred kidneys (49.2%, as Banff ci1 = 844, ci2 = 299, ci3 = 77) where 1105 fibrotic kidneys were scored for i- IFTA. From 1175 total biopsies assigned an i- IFTA score, 1138 final samples were used for primary descriptive categorization of i- IFTA associated with scarring and TA (see Figure S1). These included 33 (3.3% of kidneys) of 2481 with localized inflamed tubular atrophy with IF less than 10% cortex (Banff ci0) and 82 ci1/ct0 cases (where TA is less than 10% of cortex) but excluded 37 samples because of minimal/absent IF/TA. Data for isolated inflamed TA are presented separately (Figure 1, minimal ), leaving the final i- IFTA group being subsequently defined as having a minimal fibrotic area of 10% or greater (Banff ci 1). 3.3 Correlations of i- IFTA with contemporaneous histology From those evaluable posttransplantation biopsy samples (n = 1138 scored), the majority of scarred kidneys (61.7%, 702/1138) displayed i- IFTA, although 81.8% were mild (574/702, i- IFTA1; Table 1). Beyond the early posttransplantation period when inflammation from early acute rejection had subsided (conventionally defined 3 months, n = 1641 late biopsies), i- IFTA associated with edema (30/612, 4.9% versus 23/867, 2.7%, P =.022) but not with histologic acute tubular necrosis (31/613, 5.1% versus 35/868, 4.9%, P =.347). Markers of T cell activity positively correlated with i- IFTA (presence versus i- IFTA0 and unscarred, P <.001) or the i- IFTA score (reflecting extent of inflammation) from the subset of IF/TA kidneys (illustrated Figure 1) but not with humoral markers. Banff i- IFTA scores best correlated with Banff i (Spearman s ρ = 0.275, P <.001, IF/TA samples, n = 1138) and t scores (ρ = 0.188, P <.001), chronic fibrosis (ρ = 0.401, P <.001) and tubular atrophy scores (ρ = 0.387, P <.001), and peritubular capillaritis (ρ = 0.110, P <.001), whereas the humoral markers, C4d scores (ρ = 0.072, P =.10), and glomerulitis (ρ = 0.031, P =.30) were unrelated (Figure 1). 3.4 Time course of i- IFTA by sequential histology The natural history of i- IFTA and its interrelationships with individual risk factors were evaluated from protocol (n = 429) and indication (n = 2052) histology. Sequential histology demonstrated that

4 367 FIGURE 1 The relationships of interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) with individual histologic features, including inflammation, fibrosis and tubular, and glomerular pathology. Stack column graphs demonstrate acute and chronic Banff histologic categories against the intensity Banff i- IFTA scores (within areas of scarred cortex, n = 1138 i- IFTA samples), unscarred cortex (no interstitial fibrosis/tubular atrophy [IF/TA], n = 1261 samples), and minimal inflammation of tubular atrophy without fibrosis (n = 37) from the overall cohort. Only T cell histologic markers (inflammation and tubulitis, top panels) correlated with i- IFTA, whereas antibody markers were not related (bottom panels, see text for correlation coefficients) interstitial inflammation (Banff i) occurred before the appearances of i- IFTA and chronic fibrosis, by using actuarial analysis and averaged scores from sequential pathology (Figure 2). The prevalence of i- IFTA progressively increased with time, commensurate with increasing area of chronic fibrosis (IF/TA scores), although i- IFTA scores (relative intensity of inflammation) only marginally increased during that period (linear regression β coefficient = ± SE0.01 for slope, P <.001 vs 0; Figure 2). Allograft fibrosis steadily accumulated, with most kidneys displaying IF/TA and inflammation by 10 years. Some i- IFTA allografts resolved their inflammation by analysis of the pointprevalence of active i- IFTA (>0) of yearly sequential histology samples (sometimes transiently, with later reoccurrence; Figure 2). Resolution of active i- IFTA reduced the incidence of fibrosis (Banff ci2, hazard ratio [HR] = 0.555, 95% confidence interval [CI] = , P =.002) and arterial hyperplasia (Banff cv, HR = 0.612, 95% CI = , P =.010). By multivariable binomial generalized estimating equation (2161 evaluable biopsy samples from 283 patients), the presence of i- IFTA was predicted by coexistent interstitial infiltrate (P =.002), fibrosis (P <.001), and glomerulopathy scores (P =.047; Table 2). The percentage of scarred cortex with active inflammation was 38.7% at 3 months and 70.8% for late biopsies ( 3 months, n = 430 beyond the early acute rejection period); with 61.5% to 68.9% for 5 to 10 years posttransplantation (n = 370 biopsies, mean = 77.8% overall). 3.5 One- year i- IFTA and antecedent events One- year protocol histology found i- IFTA in 31.9% (91/285 available; Table 3, full data given in Table S5), which correlated with prior events, immunosuppression era, and subsequent histology. The 1- year i- IFTA intensity score correlated with early TCMR (χ 2 = , P =.002, only IF/TA biopsies, n = 165), the number of prior TCMR episodes (ρ = 0.272, P <.001; Figure 3), and use of antithymocyte therapy (χ 2 = , P <.001). The 1- year i- IFTA score (i- IFTA0 plus non- IF/ TA biopsies counted as 0, n = 288) correlated with early TCMR (ρ = 0.344, P <.001), numbers of prior TCMR (ρ = 0.346, P <.001), vascular (ρ = 0.130, P =.029) and humoral rejection episodes (ρ = 0.272, P <.001; Figure 3), and antithymocyte therapy use (χ 2 = , P <.001). T cell mediated events predated the appearance of i- IFTA. Oneyear i- IFTA was preceded by greater inflammation (Banff i scores, P <.001) and tubulitis (P <.001, compared with allografts without

5 368 TABLE 1 Summary data of all scored interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) categories (n = 1138) and their relationships with other histologic parameters from scarred IF/TA kidneys only Factor i-ifta0 i-ifta1 i-ifta2/3 Number of biopsies Months 2.89 ± ± 3.90 c 4.57 ± 4.25 c posttransplantation Indication biopsy (%) 11.9% 11.5% 14.1% Isotopic GFR (ml/min) 62.3 ± ± ± 24.1 Glomeruli (n) 11.9 ± ± 8.7 c 17.4 ± 14.2 c Arteries (n) 2.0 ± ± 1.2 c 2.5 ± 1.4 c Banff g score 0.05 ± ± ± 0.38 Banff i score 0.48 ± ± 0.72 c 1.23 ± 0.87 c Banff t score 0.52 ± 0.67 c 0.67 ± 0.70 c 0.99 ± 0.87 c Banff v score 0.04 ± ± ± 0.44 a Banff ah score 0.61 ± ± 0.82 c 0.99 ± 1.04 c Banff ptc score 0.34 ± ± 0.69 c 0.61 ± 0.77 c Banff ci score 1.07 ± ± 0.62 c 1.98 ± 0.74 c Banff ct score 0.98 ± ± 0.64 c 1.93 ± 0.81 c Banff cv score 0.22 ± ± 0.54 c 0.51 ± 0.75 c Banff mm score 0.26 ± ± 0.50 c 0.49 ± 0.63 c Banff cg score 0.05 ± ± 0.35 c 0.23 ± 0.52 c SV40T positive (n, %) 2/115 (1.7) 6/102 (5.9) 3/33 (9.1) C4d scores 0.50 ± ± ± 1.03 Edema (%) 11.2% 10.0% 16.0 Acute tubular c necrosis (%) Sclerosed glomeruli (%) c 28.8 c Unless otherwise indicated, values given as mean ± SD. SV40T, simian virus 40T antigen; GFR, glomerular filtration rate. P values: a P <.05; b P <.01; c P <.001 versus i- IFTA0 by multiple comparison. i- IFTA; Figure 3) and prior subclinical rejection by 1- and 3- month protocol histology (P <.001 for both borderline and acute subclinical TCMR, versus 0 Banff i score; Figure 3). While all phenotypes of prior acute rejection were associated with increased 1- year i- IFTA scores, only early TCMR remained the dominant driver by multivariable analysis. Abnormalities of chronic histology generally developed before the 1- year protocol biopsy time-point. Using logistic regression, 1- year i- IFTA was predicted by all prior rejection phenotypes; requirement of antilymphocyte therapy to reverse severe rejection; markers of sensitization including prior transfusions, PRA, and DSA; and nonimmune factors such as anastomosis time (Table 4). Only early T cell rejection and peak PRA remained significant by multivariable regression (Table 4). Most early humoral rejection episodes were of mixed phenotype, with accompanying interstitial inflammation (data not presented). Cox regression of all 2361 sequential posttransplantation kidney biopsy samples (to detect the new onset of a histologic change from the time of transplantation) identified the same immune (and some nonimmune/ischemic) risk factors for development of i- IFTA (Table 5, full data given in Table S6). Multivariable Cox regression identified early T cell and vascular rejection as predictive risk factors for 1- year i- IFTA, when statistically controlled by immunosuppression era, where tacrolimus exerted a powerful protective effect (HR= 0.219, 95% CI = vs cyclosporine, P <.001). Subanalysis of individual on- treatment therapies at 3 months found greater protection with the antiproliferative agent (mycophenolate HR = 0.223, 95% CI = , P <.001 vs azathioprine) relative to the calcineurin inhibitor used (tacrolimus HR = 0.582, 95% CI = , P <.027 vs cyclosporine; Table S7). Additional sensitivity analyses of the forced addition of antibody- mediated rejection into the multivariable Cox regression model demonstrated no effect on i- IFTA development (Table S7). 3.6 Effect of immunosuppression era on i- IFTA The incidence, point- prevalence, and intensity of i- IFTA were evaluated by baseline immunosuppression era to assess potential treatment effects and to distinguish an alloimmune etiology from nonspecific injury responses. Of 2481 tissue samples, 100 patients received cyclosporine (n = 1039 biopsies, 36.6%) or tacrolimus (1442 samples, 58.1% from 262 patients) as their calcineurin inhibitor at biopsy. Tacrolimus- era therapy was associated with reduced early TCMR rates to 19.7% (51/262) from 72.0% (72/100) with cyclosporine (χ 2 = 88.8, P <.001), accompanied by fewer early vascular (8.0% vs 21.0%; χ 2 = 11.9, P <.001) and humoral (6.9% vs 28.0%; χ 2 = 29.0, P <.001) rejection episodes. Tacrolimus era was associated with ameliorated 1- year interstitial inflammation (Banff i ± vs ± for cyclosporine, P <.001), fibrosis (Banff ci score ± vs ± for cyclosporine, P <.001), i- IFTA occurrence (16.1% vs 74.0% 2 ; χ 2 = 87.2, P <.001), and intensity of inflammation (i- IFTA score ± vs ± for cyclosporine, P <.001; Figure 4). Tacrolimus was also associated with less prior subclinical rejection (on protocol histology), inflammation, and tubulitis (by Banff i and t scores of all samples, including indication biopsies, P <.001 vs cyclosporine; Figure 4, see also Tables 3, 4, 5, S5, S6, and S7). Tacrolimus- era therapy was associated with altered cellular kinetics into the i- IFTA compartment involving: (i) reduced intensity and duration of early inflammation within the interstitial compartment, (ii) lower de novo i- IFTA formation, prevalence, and intensity of inflammation, and (iii) increased conversion rates of active i- IFTA to inactive IF/TA (all P <.001; Figure 4, methodology detailed in Table S1) compared with cyclosporine. By Cox regression, tacrolimus increased conversion of active to inactive fibrosis (median 1 year, 95% CI years) compared with cyclosporine (6 years, 95% CI 3-9 years, P <.001). 3.7 Histologic and functional associations with i-ifta The histologic associations of i- IFTA from 1- year pathology were determined by sequential histologic analysis of repeated protocol samples, from the time of transplantation. One- year i- IFTA was associated with accelerated IF/TA by longitudinal actuarial analysis

6 369 FIGURE 2 The natural history and kinetics of inflammatory fibrosis using sequential histology of overall cohort (2481 biopsies from 362 recipients). Left panel shows sequential kinetics of initial inflammation (Banff i), subsequent interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA), and then chronic fibrosis (Banff ci) according to time after transplantation, using Kaplan- Meier actuarial onset of i- IFTA relative to preceding interstitial inflammation and subsequent chronic interstitial fibrosis (Banff i and ci scores, respectively, top left) and the averaged Banff scores (bottom left panel, see text for interpretation). The long- term time course of graded i- IFTA categories and mean Banff scores (top middle and right panels) illustrate the increasing intensity and prevalence of i- IFTA to 10 years posttransplantation. The time course of relative intensity of inflammation within the scarred IF/TA areas (middle bottom) and their Kaplan- Meier actuarial outcomes, including resolution of inflammatory markers, by time after transplantation (right bottom panel, numbers at risk; see Figure S2). Values given in mean ± SEM TABLE 2 Histologic predictors of the presence of interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) by multivariable binomial generalized estimating equation from 1138 evaluable biopsies (from 292 patients with full repeated biopsy data) Occurrence of i-ifta Odds ratio 95% CI P value Interstitial infiltrate (i score) Interstitial fibrosis (ci score) Glomerulopathy (cg score) Interstitial edema (present) < This describes the risk of any i- IFTA occurring against other contemporaneous pathologic parameters on biopsy. (P <.001; Figure 5), excess fibrointimal hyperplasia, and a mild increase in chronic transplant glomerulopathy (coded Banff cg, all cases confirmed by electron microscopy; Figure 5). Abnormalities began soon after transplantation and continued for the study duration. Measured 99m Tc DTPA glomerular filtration rate (mgfr) was proportionally reduced when inflammation, IF/TA, and i- IFTA were concomitantly present (Figure 6). Measured GFR inversely correlated with i- IFTA scores (ρ = v0.19, P <.001, n = 823 measurements), Banff ci (ρ = 0.43, P <.001, n = 1363), and Banff i scores (ρ = 0.23, P <.001, n = 1363 samples with mgfr). Isotopic GFR was independently predicted by i- IFTA (P =.009) and interstitial fibrosis (P <.001), adjusted for acute tubular necrosis and interstitial edema (both P <.05; Table 6). In kidneys developing i- IFTA at 1 year, early serum creatinine concentrations were elevated from hospital discharge (P <.05 to.001 to 3 months), remaining abnormal from 3- month protocol isotopic mgfr (P <.001) until 10 years (Figure 6). The 10- year death- censored graft survival for 1- year i- IFTA was 87.6% (versus 93.0% for no i- IFTA, log- rank 0.100, P =.752); however, i- IFTA modestly increased late graft loss with longer follow- up (log- rank 3.027, P =.082; Figure 7). The overall 10- year graft survival (including death with function) was 71.6% (and unrelated to i- IFTA, log- rank 1.351, P =.245). One- year i- IFTA had no effect on patient mortality (median survival and years, respectively, logrank 0.100, P =.752; Figure 7). Deaths (53/362) were primarily from cardiovascular causes (35/52, 67.3%). 4 DISCUSSION This large, longitudinal cohort study illustrates i- IFTA is an important time- dependent histologic lesion: denoting the interface between T

7 370 Factor Nil i-ifta 1-year i-ifta P value Number (patients/biopsies) HLA mismatch (of 6) 4.5 ± ± Delayed graft function (n, %) 9 (4.6) 4 (4.4).93 Pretransplantation DSA 39/172 (22.7) 28/72 (38.9).01 MFI immunodominant DSA 3781 ± ± 14, Retransplantation (yes) 2/194 (1.0) 2/96 (2.2).61 Initial immunosuppression (n, %) Cyclosporine/azathioprine 11 (5.7) 48 (52.7) Cyclosporine/mycophenolate 9 (4.6) 9 (9.8) Tacrolimus/mycophenolate 174 (89.7) 34 (37.4).001 Early cellular rejection (n, %) 45 (23.2) 31 (58.2) <.001 Number of TCMR episodes 0.30 ± ± 1.04 <.001 Early antibody rejection (n, %) 18 (9.1) 24 (26.4).02 Number of AHR episodes 0.11 ± ± 0.89 <.001 Early vascular rejection (n, %) 20 (10.2) 18 (19.8).03 Number of rejection episodes 0.12 ± ± Antithymocyte therapy (n, %) 31 (15.8) 41 (45.1) <.001 TABLE 3 Comparison of selected risk factors for 1- year interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) using univariable and multivariable analysis (285 biopsies undertaken and scored) Unless otherwise indicated, values are given as mean ± SD. AHR, acute humoral rejection; DSA, donor- specific antibodies; MFI, median fluorescence intensity; TCMR, T cell mediated rejection. FIGURE 3 Early T cell mediated inflammation was followed by interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) on 1- year protocol histology (285 scored biopsies). Early prior clinical acute T cell mediated rejection increased i- IFTA scores and categories by 1 year (left and middle top panels). Prior subclinical rejection (Banff i score) on 1- and 3- month protocol histology also increased i- IFTA scores (right top panel). The time course and relationships of 1- year i- IFTA, with preceding T cellular inflammation (Banff i score), and tubulitis (Banff t score, left and middle bottom panels). Greater numbers of acute rejection episodes of all phenotypes increased unadjusted 1- year i- IFTA scores (right bottom panel). Values given as mean ± SEM. *P <.05, **P <.01, ***P <.001

8 371 TABLE 4 Risk factors for the occurrence of any 1- year interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA), using logistic regression (n = 285 biopsies and patients, selected results are presented; for other data, see Tables 3 and S5) Univariable risk factors OR 95% CI P value Peak PRA (%) <.001 Current PRA (%) Solid state DSAs present <.01 DSA strength (MFI) 1.00 incalculable.590 Late de novo DSAs (any) HLA mismatch (of 6) Anastomosis time (min) <.001 Total ischemic time (min) Transfusions (n) <.001 Retransplantation (yes/no) Recipient hypertension Tacrolimus era (vs cyclosporine) Early T cell rejection <0.001 Early vascular rejection Early antibody rejection <0.001 Antilymphocyte required <0.001 Multivariable model HR 95%CI P value Early T cell rejection <.001 Peak PRA (%) DSA, donor- specific antibody; MFI, median fluorescence intensity; OR, odds ratio; CI, confidence interval. cell mediated inflammation and established IF/TA and independent of antibody markers. While i- IFTA was associated with multiple adverse histologic outcomes reflecting persistent injury to diverse anatomical compartments, the pattern of sequential histology was dominated by progressive IF/TA. IF/TA has been previously correlated with alloimmune molecular signatures from tissue and blood 5,21-23 and prior clinical or subclinical rejection. 6,13-16,19,24,25 Experimental models of T cell rejection demonstrate a stereotypical sequence of initial interstitial cellular infiltration, tubulitis, and subsequent parenchymal injury response with wound healing. 26 In our study, the ordered appearance of T cell characteristics from early sequential histology was (i) initial interstitial inflammation and tubulitis, (ii) appearance of i- IFTA, and then (iii) either transition to inactive IF/TA or continued i- IFTA activity that failed to resolve (see Figure 2). Conceptually, this positions i- IFTA as a transitional lesion, bridging its 2 nominated histologic components: cellular inflammation and IF/TA. The prevalence of i- IFTA gradually increased with expanding areas of chronic fibrosis, and correlated with contemporaneous inflammation, tubulitis, and IF/TA (Figures 1 and 2). While the absolute area occupied by i- IFTA increased along with progressive fibrosis, Banff i- IFTA scores representing relative intensity remained more stable, governed primarily by immunosuppression era. Active inflammation occurred in 70.8% of biopsy samples from 3 months on posttransplantation TABLE 5 Univarible and multivariable Cox regression for interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) development within all sequential histology (n = 2361 postimplantation samples) of selected risk factors Risk factor HR 95% CI P value Peak PRA (%) <.001 Current PRA (%) Solid state DSAs present DSA strength (MFI) Incalculable.717 Late de novo DSAs (any) HLA mismatch (of 6) Anastomosis time (min) Total ischemic time (min) Transfusions (number) <.001 Retransplantation Recipient hypertension Tacrolimus- era therapy (vs cyclosporine) At transplantation < months posttransplantation <.001 Early T cell rejection <.001 Early vascular rejection <.001 Early antibody rejection <.001 Antilymphocyte required <.001 Multivariable model 1 HR 95% CI P value Early T cell rejection Early vascular rejection Tacrolimus era (vs cyclosporine) <.001 For full data, event rates, and numbers see Table S6; for additional models, see Table S7. DSA, donor- specific antibody; MFI, median fluorescence intensity. (predominantly protocol samples), with proportions varying by time of biopsy, indication- versus- protocol, and coexistent acute rejection (see Figure 7, right panel). These percentages of active i- IFTA were comparable to those of other studies from Hannover (stable protocol histology, 44.9%; indication, 62.3%), 8 North America (68.8% from late indication biopsies), 4 and Edmonton ( %, where it was unrelated to IF/TA extent). 7 T cell mediated alloimmunity appeared to facilitate i- IFTA. Early acute (including borderline) and subclinical TCMR correlated with 1- year i- IFTA, which remained significant by multivariate analysis and independent of humoral mechanisms and multiple other risk factors (Figure 1; Tables 2, 3, 4, S5, S6, and S7). Its incidence and prevalence rates were strongly modified by immunosuppression era: tacrolimusera therapy (with mycophenolate) reduced inflammation within unscarred and scarred i- IFTA compartments, limited progression of chronic fibrosis, and increased conversion to inactive IF/TA states, compared with cyclosporine (with azathioprine) therapy. Subanalysis suggested the antiproliferative agent was more influential than the

9 372 FIGURE 4 Parallel reductions of T cell inflammation and interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) associated with potent tacrolimus (TAC)- era immunosuppression (TAC usually with mycophenolate; n = 262 patients and 1442 biopsy samples), compared with weaker cyclosporine (CSA)- era group, with azathioprine therapy, 100 patients and 1039 samples. Greater pointprevalence of i- IFTA categories (top panels, by time posttransplantation), intensity of inflammation (Banff i score time course), inflammatory fibrosis (i- IFTA), and chronic fibrosis (Banff ci as part of interstitial fibrosis/tubular atrophy [IF/TA]) occurred in the CSA- era group, compared with the TAC era (left, middle, and right middle panels, respectively). The kinetics of inflammatory fibrosis, encompassing the onset and resolution of i- IFTA according to immunosuppression era, are illustrated in the bottom panels using actuarial methodology. CSA era was associated with greater de novo development of i- IFTA (left bottom), whereas TAC era produced a greater likelihood of resolution of active i- IFTA to inactive IF/TA (i- IFTA0 state), illustrated by Kaplan- Meier conversion rates (bottom middle) and the instantaneous hazard (as the conversion to inactive fibrosis, bottom right panel). Overall, TAC- era therapy was associated with a comprehensive reduction in the intensity of early inflammation, i- IFTA and IF/TA development, ongoing mean i- IFTA intensity scores, and enhanced abatement to inactive i- IFTA states, compared with CSA era (numbers at risk, see Figure S2). Values given as mean ± SEM. ***P <.001 calcineurin inhibitor. Alterations of kinetics by immunosuppression era suggest i- IFTA intensity scores reflect the summated effects of cellular influx, available IF/TA area, and (a variable) resolution phase where i- IFTA could transition to inactive fibrosis (although potential reactivation from underimmunosuppression could still recur; Figure 4). To evaluate whether i- IFTA was initiated and driven by T cell mediated alloimmunity, we applied published epistemological criteria 27 to these data to test the causal hypothesis (detailed in Table S8). The temporal sequence consisted of initial cellular inflammation followed by i- IFTA, demonstrated by Kaplan- Meier sequential histology. Prior inflammation demonstrated very high specificity ( necessary ) for later i- IFTA at (76.8% specificity of early TCMR for 1- year i- IFTA). A direct and independent relationship was confirmed by extensive multivariable analysis, where confounding humoral mechanisms were controlled and excluded. The consistently strong correlations between i- IFTA and T cell mediated processes, including contemporaneous inflammation and tubulitis scores (but not antibody- specific markers); prior TCMR and cellular infiltration; and its amelioration by potent tacrolimus- era immunosuppression (paralleling reductions in acute and subclinical TCMR) all suggest a cellular alloimmunity was responsible for i- IFTA, rather than nonspecific response to injury. The colocalization of interstitial inflammation with i- IFTA also supports a specific mechanistic effect and is implicit by definition. 3 Other studies using alternative experimental designs (eg, cross- sectional indication pathology) reached the same conclusions. 4-8 The hypothesis that T cell mediated injury causes i- IFTA is biologically plausible, logically coherent, and consistent with known relationships of subclinical inflammation and IF/TA 14,28,29 and constitutes the best explanation of observed pathology.

10 373 FIGURE 5 Increased development of chronic histologic abnormalities associated with the occurrence of interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) at the 1- year protocol biopsy (arrow), using longitudinal histologic actuarial analysis from the time of transplantation. i- IFTA occurred in 91/285 (31.9% of available samples) at 1 year. Substantial increases in moderate- severity interstitial fibrosis/tubular atrophy (IF/TA) (Banff ci score 2), fibromuscular hyperplasia of arteries (a marker of chronic T cell mediated rejection), and minor increases in chronic transplant glomerulopathy (Banff cg, confirmed by electron microscopy) were associated with the development of 1- year i- IFTA (methodology and numbers at risk; Figures S1 and S2). Early separation of curves had already begun before 1 year, indicating the existence of antecedent subclinical pathologic processes. ***P <.001 FIGURE 6 Allograft function was impaired by interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA), cellular inflammation, and scarring, representing the summated loss and/or inflammatory dysfunction of individual nephrons. Transplant dysfunction began early after transplantation in kidneys destined for i- IFTA (91/285, 31.9% by 1 year), with increased serum creatinine concentrations (left panel, conversion: l µmol/l to mg/dl, divide by 88.42). Dysfunction persisted over the 10- year observation period measured by isotopic glomerular filtration rate (GFR, middle panel), when stratified by 1- year i- IFTA. Isotopic GFR (n = 1363 measurements in stable patients) was proportionally reduced by all patterns of histologic inflammation and scarring, including increased inflammation (Banff i score), i- IFTA, and chronic interstitial fibrosis (Banff ci scores, right panel). Values are given as mean ± SEM. *P < 0.05, **P <.01, ***P <.001 versus nil 1- year i- IFTA or Banff score of 0 TABLE 6 Relationship of renal transplant function and structure 99m Tc DTPA GFR (ml/min) Coefficient SE P value Any i- IFTA (vs none) Interstitial fibrosis (ci >1) Acute tubular necrosis present < Edema present Determinants of measured isotopic GFR from contemporaneous transplant histology features (n = 1363 GFR measurements with corresponding protocol biopsy pathology in 347 recipients), using a linear generalized estimating equation to account for repeated measurements, following backward elimination. The coefficient estimates the relative (negative) impacts of individual pathological features on function. i- IFTA, interstitial inflammation within areas of interstitial fibrosis and tubular atrophy. i- IFTA was associated with, and reflected, unfavorable histologic and functional allograft outcomes. One- year i- IFTA was associated with accelerated progression of IF/TA from sequential protocol histology, with abnormalities developing early posttransplantation (Figures 2 and 5). It was also associated with increased expression of chronic fibrointimal hyperplasia (currently, the only accepted marker of chronic TCMR 28,30 ) and modestly affected transplant glomerulopathy (confirmed by electron microscopy as chronic humoral rejection). One- year i- IFTA correlated with early renal dysfunction (from early alloimmune injury and predating histology) and impaired GFR extending over the allograft s lifetime (Figure 6). The numerically inferior allograft survival following 1- year protocol i- IFTA from these young deceaseddonor kidneys echoed findings of late indication biopsy studies with allograft dysfunction. 4,5,7,31 The multicenter DEKAF study of 337 indication biopsies found i- IFTA strongly predicted death- censored graft failure compared with no inflammation, independent of renal

11 374 FIGURE 7 Death- censored allograft survival was only modestly worse in kidneys with 1- year interstitial inflammation within areas of interstitial fibrosis and tubular atrophy (i- IFTA) on protocol histology, occurring relatively late posttransplantation (left panel, P =.082) and without any effect on recipient survival (middle panel, numbers at risk, see Figure S2). Right panel, the prevalence of i- IFTA according to clinical scenario (see discussion for comparative data from other studies). Stack bar illustrates i- IFTA categories from protocol biopsy samples (no rejection or subclinical rejection [SCR]), and indication biopsy by acute T cell rejection phenotype (BL borderline, Banff IA or IB interstitial rejection, and Banff type II vascular rejection) for comparisons with other studies (see discussion) function, IF/TA, and Banff i scores and unrelated to forced addition of C4d and DSA into the multivariable model. 4 Similarly, marked inflammatory scarring within late indication biopsies from Edmonton ( 50% Banff relative i- IFTA3, n = 46) reduced 40- month allograft survival to 69.6%, compared with 93.5% for lesser inflammation (<50% scarred area, Banff i- IFTA2, n = 31, P =.02). 5 Further analysis confirmed an inferior 51.3% allograft survival with i- IFTA (assessed proportional to whole cortex) compared with 82% with i- IFTA0 (ie, uninflamed IF/TA, P <.0001), and by stratification within absolute i- IFTA categories (74% i- IFTA1, 39% i- IFTA 2). 7 All representations of inflammation from late biopsies, regardless of scoring methodology (i- Banff, i- IFTA, and Banff ti), predicted future graft loss in that study. 7 Molecular studies showed tissue injury and repair- associated transcripts were common in i- IFTA (exceeding expression from bland IF/TA) and independently predicted adverse outcomes. 32 Hence, the deleterious graft survival effect of i- IFTA in a suboptimal deceased- donor kidney with acute rejection was greater than our protocol biopsy patients from optimal allografts and stable function. The concordance of multiple association data denotes i- IFTA as an adverse pathologic lesion with unfavorable histologic and functional consequences. The Banff consensus definition of chronic TCMR is acknowledged to be incomplete: its only diagnostic occupant is arterial fibrointimal hyperplasia with mononuclear cell infiltration. 1,17,30 Our data suggest that i- IFTA should be included as the quintessential pathologic expression of chronic TCMR (with important caveats, see next). Supportive evidence for this concept includes its anatomical colocalization of inflammatory cells within areas of tubular destruction and chronic fibrotic healing (the definition i- IFTA), active i- IFTA that persists by serial repeated histology ( smoldering ), the multiple associations of i- IFTA with T cell mediated processes (eg, prior clinical and subclinical TCMR, contemporaneous interstitial inflammation, and tubulitis), demonstrable independence from antibody- mediated features, amelioration by potent T- centric immunosuppression, the histologic acceleration of progressive IF/TA and arterial fibrointimal hyperplasia, chronic renal impairment, and excess death- censored allograft loss. 4,5,8,10,31 Use of i- IFTA to diagnose chronic TCMR warrants some cautions. First, because i- IFTA is a common end- pathway phenotypic pattern (comparable to IF/TA or chronic allograft nephropathy ) with several potential etiologic causes, other diagnostic entities should be considered and actively excluded by specific evaluation. 17 The differential diagnoses of i- IFTA include BK viral infection (SV40T immunochemistry is thus recommended to exclude this diagnosis), drug- induced interstitial nephritis (where the presence of eosinophils should alert one), posttransplant lymphoproliferative disease (PTLD, morphology and immunophenotyping), and pyelonephritis (often characterized by neutrophilic infiltration and clinical urosepsis). Second, while the alloimmune specificity of i- IFTA may be improved by phenotype, the cellular infiltrate within the i- IFTA areas could deviate away from T cells and include other cells such as macrophages, B cells, and histiocytes. 8 Microarrays of classic TCMR display T cell and macrophage genes, whereas i- IFTA differentially express B cell, plasma cell, mast cell, and immunoglobulin- associated transcripts. 5,21 The extent of reactive cells expected from nonspecific tubular injury or whether the fibrotic pattern can aid diagnosis (eg, not striped fibrosis from calcineurin inhibitor nephrotoxicity) is unknown. Further research to evaluate the sensitivity, specificity, and clinical utility of i- IFTA for the diagnosis of chronic TCMR is needed. The study strengths include its large size, granular data, comprehensive histologic assessment, and availability of subsequent repeated protocol follow- up pathology spanning the kidney transplant s lifespan to accurately estimate the incidence, point- prevalence, time course, and actuarial outcomes of i- IFTA. Use of a single, standardized observational start- point (the 1- year i- IFTA score) for outcomes avoids the inescapable ascertainment bias from indication biopsy studies where the posttransplantation sample time can vary from days to decades. Comparison by immunosuppression era allows differentiation between immune i- IFTA from reactive inflammation, although historically controlled data require cautious interpretation because of potential residual confounding bias (including the presence of pretransplantation DSAs and unrecognized era effects). However, patient and implanted kidney demographics were exceptionally constant between eras, and

12 375 our longitudinal protocol biopsy regimen has continued. The 2 eras were easily distinguishable by their overall level of alloimmune activity, including acute and subclinical rejection rates. Because this study includes older historical patients, the overall rejection rates (and i- IFTA incidence) were greater than those of the current era. Our study was a nonrandomized, observational, single- center cohort design. Although analyses were retrospectively undertaken, histologic and clinical data were prospectively collected as per protocol and scored according to the standardized Banff schema. This study presents the largest histologic evaluation of i- IFTA with comprehensive clinical data to date. In summary, these data support the concept that i- IFTA is mediated by chronic active T cellular processes and is associated with progressive tubular atrophy and secondary interstitial fibrosis, renal dysfunction, and histologic deterioration. i- IFTA appears susceptible to an environment of effective immunosuppression and, when persistent, represents a pathologic expression of chronic TCMR, albeit as a nonspecific phenotype of active tubular destruction. ACKNOWLEDGMENTS We thank Ms Julie McKelvey for organizational help, our nursing colleagues in Westmead Hospital, and our referring medical practitioners. The Australian National Pancreas Transplant Unit at Westmead is funded by the Commonwealth Government of Australia. DISCLOSURE The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. POC has received honoraria from Novartis, Astellas, and Janssen Cilag. The other authors have no conflicts of interest to disclose. AUTHOR S CONTRIBUTIONS All authors participated in manuscript writing and revision; MS, CLSF, RB, and BJN undertook histologic evaluation; BJN was responsible for research design and data analysis. REFERENCES 1. Loupy A, Haas M, Solez K, et al. The Banff 2015 kidney meeting report: current challenges in rejection classification and prospects for adopting molecular pathology. Am J Transplant. 2017;17(1): Solez K, Axelsen RA, Benediktsson H, et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: the Banff working classification of kidney transplant pathology. Kidney Int. 1993;44(2): Farris AB, Colvin RB. Renal interstitial fibrosis: mechanisms and evaluation. Curr Opin Nephrol Hypertens. 2012;21(3): Mannon RB, Matas AJ, Grande J, et al. Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure. Am J Transplant. 2010;10(9): Mengel M, Reeve J, Bunnag S, et al. 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