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Many lung transplants fail due to chronic lung allograft dysfunction (CLAD). We recently showed that transbronchial biopsies (TBBs) from CLAD patients manifest severe parenchymal injury and dedifferentiation, distinct from time-dependent changes. The present study explored time-selective and CLAD-selective transcripts in mucosal biopsies from the third bronchial bifurcation (3BMBs), compared to those in TBBs.We used genome-wide microarray measurements in 324 3BMBs to identify CLAD-selective changes as well as time-dependent changes and develop a CLAD classifier. CLAD-selective transcripts were identified with linear models for microarray data (limma) and were used to build an ensemble of 12 classifiers to predict CLAD. Hazard models and random forests were then used to predict the risk of graft loss using the CLAD classifier, transcript sets associated with rejection, injury, and time.T cell-mediated rejection and donor-specific antibody were increased in CLAD 3BMBs but most had no rejection. Like TBBs, 3BMBs showed a time-dependent increase in transcripts expressed in inflammatory cells that was not associated with CLAD or survival. Also like TBBs, the CLAD-selective transcripts in 3BMBs reflected severe parenchymal injury and dedifferentiation, not inflammation or rejection. While 3BMBs and TBBs did not overlap in their top 20 CLAD-selective transcripts, many CLAD-selective transcripts were significantly increased in both for example LOXL1, an enzyme controlling matrix remodeling. In Cox models for one-year survival, the 3BMB CLAD-selective transcripts and CLAD classifier predicted graft loss and correlated with CLAD stage. Many 3BMB CLAD-selective transcripts were also increased by injury in kidney transplants and correlated with decreased kidney survival, including LOXL1.Mucosal and transbronchial biopsies from CLAD patients reveal a diffuse molecular injury and dedifferentiation state that impacts prognosis and correlates with the physiologic disturbances. CLAD state in lung transplants shares features with failing kidney transplants, indicating elements shared by the injury responses of distressed organs.

Transplanted lungs suffer worse outcomes than other organ transplants with many developing chronic lung allograft dysfunction (CLAD), diagnosed by physiologic changes. Histology of transbronchial biopsies (TBB) yields little insight, and the molecular basis of CLAD is not defined. We hypothesized that gene expression in TBBs would reveal the nature of CLAD and distinguish CLAD from changes due simply to time posttransplant. Whole-genome mRNA profiling was performed with microarrays in 498 prospectively collected TBBs from the INTERLUNG study, 90 diagnosed as CLAD. Time was associated with increased expression of inflammation genes e.g. CD1E and immunoglobulins. After correcting for time, CLAD manifested not as inflammation but as parenchymal response-to-wounding, with increased expression of genes such as HIF1A, SERPINE2, and IGF1 that are increased in many injury and disease states and cancers, associated with development, angiogenesis, and epithelial response-to-wounding in pathway analysis. Fibrillar collagen genes were increased in CLAD, indicating matrix changes, and normal transcripts were decreased - dedifferentiation. Gene-based classifiers predicted CLAD with AUC 0.70 (no time-correction) and 0.87 (time-corrected). CLAD related gene sets and classifiers were strongly prognostic for graft failure and correlated with CLAD stage. Thus, in TBBs molecular changes indicate that CLAD primarily reflects severe parenchymal injury-induced changes and dedifferentiation.

BACKGROUND We previously developed molecular assessment systems for lung transplant transbronchial biopsies (TBBs) with high surfactant and bronchial mucosal biopsies, identifying T-cell‒mediated rejection (TCMR) on the basis of the expression of rejection-associated transcripts, but the relationship of rejection to graft loss is unknown. This study aimed to develop molecular assessments for TBBs and mucosal biopsies and to establish the impact of molecular TCMR on graft survival. METHODS We used microarrays and machine learning to assign TCMR scores to an expanded cohort of 457 TBBs (367 high surfactant plus 90 low surfactant) and 314 mucosal biopsies. We tested the score agreement between TBB–TBB, mucosal–mucosal, and TBB–mucosal biopsy pairs in the same patient. We also assessed the association of molecular TCMR scores with graft loss (death or retransplantation) and compared it with the prognostic associations for histology and donor-specific antibodies. RESULTS The molecular TCMR scores assigned in all the TBBs performed similarly to those in high-surfactant TBBs, indicating that variation in alveolation in TBBs does not prevent the detection of TCMR. Mucosal biopsy pieces showed less piece-to-piece variation than TBBs. TCMR scores in TBBs agreed with those in mucosal biopsies. In both TBBs and mucosal biopsies, molecular TCMR was associated with graft loss, whereas histologic rejection and donor-specific antibodies were not. CONCLUSIONS Molecular TCMR can be detected in TBBs regardless of surfactant and in mucosal biopsies, which show less variability in the sampled tissue than TBBs. On the basis of these findings, molecular TCMR appears to be an important predictor of the risk of future graft failure. Trial registration ClinicalTrials.gov NCT02812290.

Diagnosing lung transplant rejection currently depends on histologic assessment of transbronchial biopsies (TBB) with limited reproducibility and considerable risk of complications. Mucosal biopsies are safer but not histologically interpretable. Microarray-based diagnostic systems for TBBs and other transplants suggest such systems could assess mucosal biopsies as well. We studied 243 mucosal biopsies from the third bronchial bifurcation (3BMBs) collected from seven centers and classified them using unsupervised machine learning algorithms. Using the expression of a set of rejection-associated transcripts annotated in kidneys and validated in hearts and lung transplant TBBs, the algorithms identified and scored major rejection and injury-related phenotypes in 3BMBs without need for labeled training data. No rejection or injury, rejection, late inflammation, and recent injury phenotypes were thus scored in new 3BMBs. The rejection phenotype correlated with IFNG-inducible transcripts, the hallmarks of rejection. Progressive atrophy-related changes reflected by the late inflammation phenotype in 3BMBs suggest widespread time-dependent airway deterioration, which was especially pronounced after two years posttransplant. Thus molecular assessment of 3BMBs can detect rejection in a previously unusable biopsy format with potential utility in patients with severe lung dysfunction where TBB is not possible and provide unique insights into airway deterioration. ClinicalTrials.gov NCT02812290.

BACKGROUND Improved understanding of lung transplant disease states is essential because failure rates are high, often due to chronic lung allograft dysfunction. However, histologic assessment of lung transplant transbronchial biopsies (TBBs) is difficult and often uninterpretable even with 10 pieces. METHODS We prospectively studied whether microarray assessment of single TBB pieces could identify disease states and reduce the amount of tissue required for diagnosis. By following strategies successful for heart transplants , we used expression of rejection-associated transcripts (annotated in kidney transplant biopsies) in unsupervised machine learning to identify disease states. RESULTS All 242 single-piece TBBs produced reliable transcript measurements. Paired TBB pieces available from 12 patients showed significant similarity but also showed some sampling variance. Alveolar content, as estimated by surfactant transcript expression, was a source of sampling variance. To offset sampling variation, for analysis, we selected 152 single-piece TBBs with high surfactant transcripts. Unsupervised archetypal analysis identified 4 idealized phenotypes (archetypes) and scored biopsies for their similarity to each: normal; T-cell‒mediated rejection (TCMR; T-cell transcripts); antibody-mediated rejection (ABMR)-like (endothelial transcripts); and injury (macrophage transcripts). Molecular TCMR correlated with histologic TCMR. The relationship of molecular scores to histologic ABMR could not be assessed because of the paucity of ABMR in this population. CONCLUSIONS Molecular assessment of single-piece TBBs can be used to classify lung transplant biopsies and correlated with rejection histology. Two or 3 pieces for each TBB will probably be needed to offset sampling variance.

Purpose The INTERHEART study previously used microarray assessment of 889 heart transplant biopsies to develop the Molecular Microscope Diagnostic System (MMDx) based on expression of rejection-associated transcripts (RATs). The present study reclassified the rejection-related states in an expanded set of 1320 prospectively collected biopsies from 645 patients from 13 centers. Methods Biopsies were classified by ensembles of classifiers and analyzed for left ventricular ejection fraction (LVEF) and survival. Results New algorithms identified 853 No rejection (NR), 179 ABMR, 76 TCMR, 13 Mixed, 161 possible ABMR (pABMR), and 38 possible TCMR (pTCMR). No rejection was subclassified as NR-Normal 462, NR-Minor 359, and NR-Early-injury 32 (Figure 1A). Compared to NR-Normal, NR-Minor biopsies had mild elevation of many inflammation transcripts (e.g. IFNG-inducible genes) and to a lesser extent parenchymal injury transcripts. NR-minor biopsies were often designated as TCMR1R by histology. In all NR biopsies, NR-Minor scores and histologic TCMR1R increased through the first year, peaking about one year, suggesting that Minor inflammation is a late response to injury, unrelated to rejection. LVEF was similar in NR-Normal and NR-Minor but depressed in TCMR and Early injury. In a 3-year post-biopsy survival analysis, NR-Minor and NR-Normal had similar survival. TCMR and Early-injury were associated with increased graft loss, but many losses were not related to rejection. Surprisingly, 76 hearts with ABMR (149 biopsies) and follow-up data had only 3 losses within 3 years post-biopsy (Figure 1B). Conclusion Many biopsies with molecular no rejection develop minor increases in rejection-related transcripts in the first year, often called TCMR1R by histology, with no apparent effects on function or survival. An unexpected finding was that molecular ABMR was associated with very few graft losses over three years. (ClinicalTrials.gov #NCT02670408).

Objective COPA syndrome is a genetic disorder of retrograde cis‐Golgi vesicle transport that leads to upregulation of pro‐inflammatory cytokines (mainly IL‐1β and IL‐6) and the development of interstitial lung disease (ILD). The impact of COPA syndrome on post‐lung transplant (LTx) outcome is unknown but potentially detrimental. In this case report, we describe progressive allograft dysfunction following LTx for COPA‐ILD. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy. Methods Multiplexed cytokine analysis was performed on serum and bronchoalveolar lavage (BAL) fluid obtained pre‐ and post‐LTx. Peripheral blood mononuclear cells (PMBCs) obtained pre‐ and post‐LTx were stimulated with PMA, LPS and anti‐CD3/CD28 antibodies. Post‐LTx endobronchial biopsies underwent microarray‐based gene expression analysis. Results were compared to non‐COPA LTx recipients and non‐LTx healthy controls. Results Multiplexed cytokine analysis showed rising type I/II IFNs, and IL‐6 in BAL post‐LTx that decreased following treatment of acute rejection but rebounded with further clinical deterioration. In vitro stimulation of PMBCs suggested that myeloid cells were driving deterioration, through IL‐6 signalling pathways. Tocilizumab (IL‐6 receptor antibody) administration for 3 months (4 mg kg−1, monthly) effectively suppressed IL‐6 levels in BAL. Mucosal gene expression profile following tocilizumab suggested greater similarity to normal. Conclusion Clinical effectiveness of IL‐6 receptor blockade was not observed. However, we identified IL‐6 upregulation associated with graft injury, effective IL‐6 suppression with tocilizumab and evidence of beneficial effect on molecular transcripts. This mechanistic analysis suggests a role for IL‐6 blockade in post‐LTx care that should be investigated further., In this case report, we describe progressive allograft dysfunction following lung transplant for interstitial lung disease due to COPA syndrome. Following the failure of standard immunosuppressive approaches, detailed cytokine analysis was performed with the intention of personalising therapy. We identified IL‐6 upregulation associated with graft injury, effective IL‐6 suppression with tocilizumab and evidence of beneficial effect of tocilizumab on molecular transcripts of rejection/injury. This mechanistic analysis suggests a role for IL‐6 blockade in post‐LTx care that should be investigated further.

The molecular mechanisms operating in human organ transplant rejection are best inferred from the mRNAs expressed in biopsies because the corresponding proteins often have low expression and short half-lives, while small non-coding RNAs lack specificity. Associations should be characterized in a population that rigorously identifies T cell-mediated (TCMR) and antibody-mediated rejection (ABMR). This is best achieved in kidney transplant biopsies, but the results are generalizable to heart, lung, or liver transplants. Associations can be universal (all rejection), TCMR-selective, or ABMR-selective, with universal being strongest and ABMR-selective weakest. Top universal transcripts are IFNG-inducible (eg, CXCL11 IDO1, WARS) or shared by effector T cells (ETCs) and NK cells (eg, KLRD1, CCL4). TCMR-selective transcripts are expressed in activated ETCs (eg, CTLA4, IFNG), activated (eg, ADAMDEC1), or IFNG-induced macrophages (eg, ANKRD22). ABMR-selective transcripts are expressed in NK cells (eg, FGFBP2, GNLY) and endothelial cells (eg, ROBO4, DARC). Transcript associations are highly reproducible between biopsy sets when the same rejection definitions, case mix, algorithm, and technology are applied, but exact ranks will vary. Previously published rejection-associated transcripts resemble universal and TCMR-selective transcripts due to incomplete representation of ABMR. Rejection-associated transcripts are never completely rejection-specific because they are shared with the stereotyped response-to-injury and innate immunity.

BACKGROUND: Natural killer (NK) cells localize in the microcirculation in antibody-mediated rejection (AMR) and have been postulated to be activated by donor-specific anti-HLA antibodies triggering their CD16a Fc receptors. However, direct evidence for NK cell CD16a triggering in AMR is lacking. We hypothesized that CD16a-inducible NK cell-selective transcripts would be expressed in human AMR biopsies and would offer evidence for CD16a triggering. METHODS: We stimulated human NK cells through CD16a in vitro, characterized CD16a-inducible transcripts, and studied their expression in human kidney transplant biopsies with AMR and in an extended human cell panel to determine their selectivity. RESULTS: In NK cells, CD16a stimulation induced increased expression of 276 transcripts (FC > 2x, false discovery rate < 0.05), including IFNG, TNF, CSF2, chemokines, such as CCL3, CCL4, and XCL1, and modulators of NK cell effector functions (TNFRSF9, CRTAM, CD160). Examination in an extended human cell panel revealed that CD160 and XCL1 were likely to be selective for NK cells in AMR. In biopsies, 8 of the top 30 CD16a-inducible transcripts were highly associated with AMR (P < 5 × 10(−6)): CCL4, CD160, CCL3, XCL1, CRTAM, FCRL3, STARD4, TNFRSF9. Other NK cell transcripts (eg, GNLY) were increased in AMR but not CD16a-inducible, their presence in AMR probably reflecting NK cell localization. CONCLUSIONS: The association of CD16a-inducible NK cell-selective transcripts CD160 and XCL1 with biopsies with AMR provides evidence for NK cell CD16a activation in AMR. This raises the possibility of other CD16a-triggered effects that are not necessarily transcriptional, including NK localization and cytotoxicity.

Purpose The molecular pathogenesis of chronic lung allograft dysfunction (CLAD) is poorly understood. We hypothesized that microarray analysis of TBBs and mucosal biopsies from the third bronchial bifurcation (3BMBs) would provide insight into biological pathways through gene ontology (GO) analysis. Methods We performed gene expression microarray analysis of 498 TBBs and 324 3BMBs from 10 international centers, comparing CLAD+ and CLAD- cases. CLAD was defined per 2019 ISHLT guidelines. Bayes-moderated t-tests were used to identify CLAD-associated genes. We performed GO biological process analysis with CLAD-associated genes with false discovery rate Results In TBBs CLAD-associated GO terms described lung development regulation, injury response, epithelial proliferation, epithelial-mesenchymal transition, and angiogenesis (Fig. 1A). In 3BMBs the GO terms included endocytosis and intracellular trafficking, cell-cell/cell-substrate adhesion, collagen/matrix metabolism, but unlike TBBs included immunity (Fig. 1B). Time correction did not affect transcript associations with CLAD in TBBs but diminished them in 3BMBs. Excluding biopsies with coexistent rejection or infection removed associations in TBBs but strengthened them in 3BMBs even after correcting for time post-transplant. Conclusion In our biopsy set the molecular CLAD phenotype resembles a parenchymal response to wounding that overlaps other pathologies. CLAD-associated molecular changes were independent of time in TBBs but not 3BMBs, suggesting that CLAD is primarily a time dependent airway process involving injury and either adaptive or innate immunity. Unlike TBBs, molecular changes in 3BMBs were identifiable despite coexistent rejection or infection, suggesting this biopsy format may better capture CLAD information.

BACKGROUND. Because injury is universal in organ transplantation, heart transplant endomyocardial biopsies present an opportunity to explore response to injury in heart parenchyma. Histology has limited ability to assess injury, potentially confusing it with rejection, whereas molecular changes have potential to distinguish injury from rejection. Building on previous studies of transcripts associated with T cell–mediated rejection (TCMR) and antibody-mediated rejection (ABMR), we explored transcripts reflecting injury. METHODS. Microarray data from 889 prospectively collected endomyocardial biopsies from 454 transplant recipients at 14 centers were subjected to unsupervised principal component analysis and archetypal analysis to detect variation not explained by rejection. The resulting principal component and archetype scores were then examined for their transcript, transcript set, and pathway associations and compared to the histology diagnoses and left ventricular function. RESULTS. Rejection was reflected by principal components PC1 and PC2, and by archetype scores S2TCMR, and S3ABMR, with S1normal indicating normalness. PC3 and a new archetype score, S4injury, identified unexplained variation correlating with expression of transcripts inducible in injury models, many expressed in macrophages and associated with inflammation in pathway analysis. S4injury scores were high in recent transplants, reflecting donation-implantation injury, and both S4injury and S2TCMR were associated with reduced left ventricular ejection fraction. CONCLUSION. Assessment of injury is necessary for accurate estimates of rejection and for understanding heart transplant phenotypes. Biopsies with molecular injury but no molecular rejection were often misdiagnosed rejection by histology. TRAIL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02670408","term_id":"NCT02670408"}}NCT02670408 FUNDING. Roche Organ Transplant Research Foundation, the University of Alberta Hospital Foundation, and Alberta Health Services.

Purpose In heart and kidney transplants, rejection is a major cause of graft loss. In kidneys, antibody-mediated rejection (ABMR) is more important than T cell-mediated rejection (TCMR), and molecules predict graft loss better than histology (JASN 26 (7):1711-1720, 2015). We examined the relative importance of ABMR vs TCMR in heart transplant endomyocardial biopsies (EMBs), and the molecules predicting graft survival. Methods The INTERHEART population includes 1219 transplant biopsies from 8 centers in Canada, USA, Australia and Europe. Gene expression was studied using microarrays, selecting the most recent biopsy per patient. Random forest classifiers were used to assess predictive accuracy and determine the importance of molecular predictors, including gene sets and scores from analyses in a reference set of 889 EMBs. Results We studied 3-year survival in 484 patients with follow-up times. Graft failure occurred in 60 patients. Median follow-up was 435 days; biopsies were mainly for indications. Surprisingly, TCMR was a greater short-term hazard than ABMR. The molecular archetype clusters for TCMR and injury (Fig. 1) had the highest risk of graft failure. Fig. 2 combines these clusters since they have similar characteristics. Conclusion Unlike kidneys, graft loss (particularly within one year) after EMB is highly associated with TCMR but not ABMR. TCMR may reflect failure of immunosuppression or non-adherence. This difference between the heart and renal transplant populations raises the possibility that TCMR is relatively more destructive, and ABMR less destructive, in heart than in kidney transplants. ClinicalTrials.gov # NCT02670408

Purpose Gene expression profiling (GEP) of endomyocardial biopsies (EMB) by the MMDx system has been shown to estimate the probability of T-cell mediated (TCMR) and antibody-mediated rejection (AMR), improving the histology-based diagnoses in heart transplant (HT) recipients. Second generation analysis allowed to identify an additional phenotype characterised by molecules related to tissue injury (INJ), such as de-differentiation pathways, macrophages transcripts, and heart transcripts. The clinical correlates of this novel profile and its association with ISHLT pathology grading is under development. In this study we analyse the association of MMDx novel profiles with cardiac function and with ISHLT grades for TCMR and AMR. Methods 254 EMBs from 121 patients were analysed with MMDx platform. EMBs were performed according to standard protocol during the first 5 years after transplant and by clinical indication later on. Right heart catheretization was performed at the time of EMB, which were graded by 1990 and 2006 ISHLT classifications for TCMR and 2014 guidelines for AMR. Results Prevalence of molecular phenotypes varied during time course: INJ was frequent in the first months, absent during the first 5 years, and rose again in late for-cause EMBs; AMR peaked early and late after transplant, in line with DSA time-course; TCMR probability was meaningful in the early months only. Cardiac function also varied across the three profiles: INJ was significantly associated with low cardiac index (CI) and high right atrial pressure, AMR with elevated wedge pressure and low CI, and TCMR with low CI only. Overall, GEP was in agreement with pathology readings in most cases, being normal in 90% of the 0 graded EMB, and abnormal in over 70% of the 2R or pAMR2. While current 1R grade showed abnormal GEP only in 37% of cases, the old 1B grade had abnormal GEP in 60% of cases, mostly associated with AMR profile. INJ profile was found in 54% of the 2R or greater EMBs. Conclusion GEP reveals variable molecular patterns during transplant time course; INJ phenotype appears associated mainly with low CI in the context of post-operative injury and severe mixed rejection, while AMR seems to increase markers of graft stiffness. Despite good overall agreement with pathology readings, current ISHLT grading is inaccurate, with the “old” 1B grade being highly associated with AMR molecular profiling.

Purpose Chronic lung allograft dysfunction (CLAD) is a clinical/functional diagnosis that cannot be defined by histology in transbronchial biopsies (TBBs). Moreover, understanding the biology of CLAD is crucial for prevention and potential treatment. We studied gene expression associated with CLAD in TBBs, but also studied mucosal biopsies from the third bronchial bifurcation (3BMBs). Methods 223 highly alveolated TBBs (54 CLAD, 169 no CLAD) and 182 3BMBs (43 CLAD, 139 no CLAD) were collected from 7 centers and processed on microarrays. The top 100 non-overlapping genes associated with a diagnosis of CLAD prior to biopsy were identified, annotated, and used for gene set analysis and gene ontology (GO) biological process analysis. Results The top genes associated with CLAD reflected de-differentiation (expression loss), injury (expression gain), and inflammation in both TBBs and 3BMBs. These associations were stronger in 3BMBs. Pathway analysis using the top genes also suggested parenchymal de-differentiation (e.g. loss of transcriptional regulation and mitochondrial function in TBBs) and inflammatory pathway activation, particularly in 3BMBs (Table 1). Gene sets previously annotated in atrophy-scarring, including immunoglobulin transcripts (plasma cells), were elevated in CLAD in TBBs and 3BMBs, but 3BMBs also showed more inflammation-related changes (e.g. interferon gamma effects). Conclusion Molecular changes associated with CLAD were apparent in TBBs and especially in 3BMBs, and indicate that CLAD is a combination of atrophy/scarring, function loss, and, particularly in 3BMBs, inflammation. Molecular assessment of 3BMBs is a particularly promising opportunity to dissect CLAD biology and potentially gain insight into the BOS variant. ClinicalTrials.gov: NCT02812290

BACKGROUND: Ulcerative colitis (UC) is a chronic, idiopathic inflammatory condition affecting the colonic epithelium, with the inflammasome, T cells, complement activation, and microbiome dysbiosis contributing to pathogenesis. AIMS: We applied a previously established method of microarray molecular analysis to a set of 71 UC biopsies (from 61 patients), to elucidate the molecular changes associated with active UC, specifically comparing UC to T-cell mediated rejection (TCMR), as a prototype of a sterile, T-cell mediated disease. METHODS: 71 for-cause UC colonic biopsies were collected at the U of A Hospital in Edmonton, AB and Cedars-Sinai Hospital in LA, California. These biopsies were processed using Affymetrix GeneChip microarrays and the data analyzed in R programming language. Gene expression data was displayed using volcano plots (showing the fold change and association between the genes and endoscopic Mayo score) and heatmaps (showing expression of the top 30 genes in a cell panel). We then analyzed overexpression of the top genes using the DAVID tool(1), and compared the results to similar results for top genes overexpressed in TCMR. RESULTS: The volcano plot (Figure 1) showed strong associations between the endoscopic Mayo score and decay accelerating factor (CD55), and moderate associations with calprotectin genes (S100A8/S100A9), with lesser associations for effector T cell transcripts (i.e. CTLA4, interferon gamma (IFNG), and chemokine ligand (CXCL13)), many IFNG inducible transcripts, inflammasome transcripts (CASP1), and toll-like receptors (TLR5). NLRP3 (incriminated in mouse UC models) did not pass the IQR filter and was not significant. Expression of the top genes in a cell panel showed primary expression in monocytes, macrophages, and dendritic cells, with some expression in epithelial and endothelial cells, while minimal expression was found in CD4/CD8 T cells, or in NK cells. Pathway analysis showed major differences between pathway terms. CONCLUSIONS: Our findings show that while transcripts associated with cognate T cell inflammatory processes (TCMR) are expressed in UC, there are substantial differences between the pathogenesis of a pure T cell-mediated disease and UC. In our cohort, the top genes associated with the endoscopic Mayo score in UC were not those associated with effector T cells: T cell-associated transcripts were only moderately associated with the Mayo score, and pathway analysis showed significant differences between TCMR and UC, which is much more an inflammatory environment with strong associations to CD55. This suggests that cognate T cell recognition is present in UC but supplemented by a second source of inflammation. REFERENCES: 1. Huang et al. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols (2009). FUNDING AGENCIES: None

Aim Antibody-mediated rejection (ABMR) involves a cellular component where NK cell CD16 activation plays a pivotal role. In T-cell mediated rejection (TCMR) CD8+ T cells; the most closely related cell to NK cells, are most prevalent. We used global gene expression analyses to determine the similarities in gene expression changes following CD16 and CD3 stimulation of NK cells and CD8+ T cells, respectively and examined their expression in kidney transplant biopsies. Methods Cells were purified from PBMCs by immunomagnetic separation and stimulated in vitro on plates coated with crosslinking antibodies or uncoated plates. NK (N = 3) and CD8+ T cells (N = 3) were cultured for 4 h in the presence of IL2 prior to harvest. RNA was extracted and analyzed on Affymetrix gene expression arrays and supernatants underwent multiplex analysis for soluble mediators. Results We identified 276 transcripts increased (>2X, FDR Conclusion The cognate events that drive the rejection process in ABMR (NK cell CD16 activation) and TCMR (CD8+ T cell TCR/CD3 activation) are surprisingly similar. Our study helps us understand the confusion regarding the accurate diagnosis of rejection due to similarities and invites exploration of T-cell directed therapies for potential treatment of ABMR. P.F. Halloran: Stock Shareholder; Company/Organization; P F Halloran holds shares in Transcriptome Sciences Inc., a company with an interest in molecular diagnostics..

Background Complement (C′) fixing DSA increases the risk of kidney allograft loss and degree of antibody-mediated damage. However, the immunologic mechanisms responsible for this process remain ill defined. We hypothesized that IFN-g-activated monocytes would play a key role in the rejection process involving C′ fixing DSA given the NK cell Fc receptor CD16 preference for IgG1 and IgG3 subclasses. Methods Global gene expression was assessed in kidney transplant biopsies from 150 patients with DSA detected in the first year post-transplant (48 C1Q+ DSA), and on purified monocytes stimulated with IFN-g over a time course (2, 4, and 8 h). Results Compared to biopsies from C1Q-DSA patients, those from C1Q+ DSA patients showed higher expression of transcripts depicting monocyte activation (p = 0.0014), IFN-g effects (p = 0.02), and endothelial activation (p = 0.0273). With in vivo evidence of increased monocyte activation and IFN-g effects in the presence of C1Q+ DSA, we examined an IFN-g time course treatment of monocytes. IFN-g treatment of monocytes induced 189 transcripts by >5-fold at either 2, 4, or 8 h compared to 2 h unstimulated monocytes. 7 IFN-g responsive transcripts showed a direct contribution to antibody-mediated damage (Fig. 1): Chemokines CCL2 and CCL7 are both potent monocyte chemoattractants while CXCL11 contributes to NK cell recruitment, IL15 and IL15RA maximize monocyte’s ability to express and present this key NK cell growth factor, the high affinity FcR (FCGR1) was also highly inducible by IFN-g as were transcripts for C1Q components which allows for higher local concentrations of C1Q in addition to that present in blood. Conclusion Patients with C1Q+ DSA show a distinct molecular phenotype characterized by higher expression of genes related to monocyte activation, IFN-g effects, and endothelial activation. Multiple transcripts are rapidly upregulated by IFN-g in monocytes which elucidate the significant contribution that these cells make towards injury by C′ fixing DSA.