Multiomic profiling of transplant glomerulopathy reveals a novel T-cell dominant subclass
Authors: Iacopo Cristoferi, Hilal Varol, Myrthe van Baardwijk, Layla Rahiem, Karishma A. Lila, Thierry P.P. van den Bosch, Carla C. Baan, Dennis A. Hesselink, Rafael Kramann, Robert C. Minnee, Dana A.M. Mustafa, Marlies E.J. Reinders, Dave L. Roelen, Shazia P. Shahzad-Arshad, Rex N. Smith, Andrew P. Stubbs, Robert B. Colvin, Ivy A. Rosales, Marian C. Clahsen-van Groningen
Journal: Kidney International (2023)

Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody–mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype. Kidney transplant (KTx) biopsies showing transplant glomerulopathy (TG) (glomerular basement membrane double contours (cg) > 0) and microvascular inflammation (MVI) in the absence of C4d staining and donor-specific antibodies (DSAs) do not fulfill the criteria for chronic active antibody–mediated rejection (CA-AMR) diagnosis and do not fit into any other Banff category. To investigate this, we initiated a multicenter intercontinental study encompassing 36 cases, comparing the immunomic and transcriptomic profiles of 14 KTx biopsies classified as cg+MVI DSA-/C4d- with 22 classified as CA-AMR DSA+/C4d+ through novel transcriptomic analysis using the NanoString Banff-Human Organ Transplant (B-HOT) panel and subsequent orthogonal subset analysis using two innovative 5-marker multiplex immunofluorescent panels. Nineteen genes were differentially expressed between the two study groups. Samples diagnosed with CA-AMR DSA+/C4d+ showed a higher glomerular abundance of natural killer cells and higher transcriptomic cell type scores for macrophages in an environment characterized by increased expression of complement-related genes (i.e., C5AR1) and higher activity of angiogenesis, interstitial fibrosis tubular atrophy, CA-AMR, and DSA-related pathways when compared to samples diagnosed with cg+MVI DSA-/C4d-. Samples diagnosed with cg+MVI DSA-/C4d- displayed a higher glomerular abundance and activity of T cells (CD3+, CD3+CD8+, and CD3+CD8-). Thus, we show that using novel multiomic techniques, KTx biopsies with cg+MVI DSA-/C4d- have a prominent T-cell presence and activity, putting forward the possibility that these represent a more T-cell dominant phenotype. Lay SummaryIn this study, we compared 2 kidney transplant biopsy rejection diagnoses: one with a rejection type called "chronic-active antibody-mediated rejection" and another that under the microscope looks the same but does not have all the criteria for that diagnosis. Using new and state-of-the-art techniques, we found distinct differences in the biological characteristics between the 2 groups, suggesting that the second group is a different type of rejection with a high presence of T cells. The study will help improve how to diagnose and treat kidney transplant patients with this type of chronic rejection. In this study, we compared 2 kidney transplant biopsy rejection diagnoses: one with a rejection type called "chronic-active antibody-mediated rejection" and another that under the microscope looks the same but does not have all the criteria for that diagnosis. Using new and state-of-the-art techniques, we found distinct differences in the biological characteristics between the 2 groups, suggesting that the second group is a different type of rejection with a high presence of T cells. The study will help improve how to diagnose and treat kidney transplant patients with this type of chronic rejection. The development of chronic-active antibody-mediated rejection (CA-AMR) in kidney transplants (KTxs) represents an intermediate stage in the progression of morphologic lesions from active to chronic AMR and has a large impact on long-term transplant outcome.1Sellarés J. de Freitas D.G. Mengel M. et al.Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence.Am J Transplant. 2012; 12: 388-399Abstract Full Text Full Text PDF PubMed Scopus (1203) Google Scholar, 2Naesens M. Kuypers D.R.J. De Vusser K. et al.The histology of kidney transplant failure: a long-term follow-up study.Transplantation. 2014; 98: 427-435Crossref PubMed Scopus (117) Google Scholar, 3Pouliquen E. Koenig A. Chen C.C. et al.Recent advances in renal transplantation: antibody-mediated rejection takes center stage.F1000Prime Rep. 2015; 7: 51Crossref PubMed Scopus (42) Google Scholar, 4Valenzuela N.M. Reed E.F. Antibody-mediated rejection across solid organ transplants: manifestations, mechanisms, and therapies.J Clin Invest. 2017; 127: 2492-2504Crossref PubMed Scopus (150) Google Scholar According to the Banff Classification of Allograft Pathology 2019 update,5Loupy A. Haas M. Roufosse C. et al.The Banff 2019 Kidney Meeting Report (I): updates on and clarification of criteria for T cell- and antibody-mediated rejection.Am J Transplant. 2020; 20: 2318-2331Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar CA-AMR can be diagnosed when 3 diagnostic criteria are met: (i) the presence of morphologic evidence of chronic tissue injury, for example, transplant glomerulopathy (TG, cg >0); (ii) evidence of current/recent antibody interaction with vascular endothelium, for example, microvascular inflammation (MVI); and (iii) serologic evidence of circulating donor-specific antibodies (DSAs to human leukocyte antigen [HLA] or other antigens) that can be substituted by C4d staining of peritubular capillaries (ptcs) (also diagnostic criterion 2) or the presence of validated gene transcripts/classifiers. Diagnosing CA-AMR remains difficult for several reasons. First, the absence of DSAs at the time of for-cause KTx biopsies does not exclude the presence of CA-AMR. Current antibody testing methods focus on circulating anti-HLA antibodies, thereby missing non-HLA antibodies and possibly leading to false negatives.6Haas M. The revised (2013) Banff classification for antibody-mediated rejection of renal allografts: update, difficulties, and future considerations.Am J Transplant. 2016; 16: 1352-1357Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 7Lucia M. Luque S. Crespo E. et al.Preformed circulating HLA-specific memory B cells predict high risk of humoral rejection in kidney transplantation.Kidney Int. 2015; 88: 874-887Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 8Snanoudj R. Claas F.H. Heidt S. et al.Restricted specificity of peripheral alloreactive memory B cells in HLA-sensitized patients awaiting a kidney transplant.Kidney Int. 2015; 87: 1230-1240Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 9Michielsen L.A. van Zuilen A.D. Krebber M.M. et al.Clinical value of non-HLA antibodies in kidney transplantation: still an enigma?.Transplant Rev (Orlando). 2016; 30: 195-202Crossref PubMed Scopus (36) Google Scholar, 10Dragun D. Catar R. Philippe A. Non-HLA antibodies against endothelial targets bridging allo- and autoimmunity.Kidney Int. 2016; 90: 280-288Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 11Sigdel T.K. Li L. Tran T.Q. et al.Non-HLA antibodies to immunogenic epitopes predict the evolution of chronic renal allograft injury.J Am Soc Nephrol. 2012; 23: 750-763Crossref PubMed Scopus (75) Google Scholar, 12Pineda S. Sigdel T.K. Chen J. et al.Corrigendum: novel non-histocompatibility antigen mismatched variants improve the ability to predict antibody-mediated rejection risk in kidney transplant.Front Immunol. 2018; 9: 107Crossref PubMed Scopus (3) Google Scholar Even when using highly sensitive antibody-detection techniques, in approximately 40% of patients with AMR, no DSAs are detected.13Akalin E. Dinavahi R. Dikman S. et al.Transplant glomerulopathy may occur in the absence of donor-specific antibody and C4d staining.Clin J Am Soc Nephrol. 2007; 2: 1261-1267Crossref PubMed Scopus (56) Google Scholar Second, C4d scoring is affected by subjectivity,14Cohen D. Colvin R.B. Daha M.R. et al.Pros and cons for C4d as a biomarker.Kidney Int. 2012; 81: 628-639Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar and lack of C4d staining of ptcs is also relatively common in CA-AMR,15Gloor J.M. Sethi S. Stegall M.D. et al.Transplant glomerulopathy: subclinical incidence and association with alloantibody.Am J Transplant. 2007; 7: 2124-2132Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar,16Becker L.E. Morath C. Suesal C. Immune mechanisms of acute and chronic rejection.Clin Biochem. 2016; 49: 320-323Crossref PubMed Scopus (33) Google Scholar suggesting that TG could occur in the absence of complement activation. Lastly, although promising developments are made in transcriptomic analysis of for-cause kidney biopsies with DSA-selective transcripts,17Hidalgo L.G. Sis B. Sellares J. et al.NK cell transcripts and NK cells in kidney biopsies from patients with donor-specific antibodies: evidence for NK cell involvement in antibody-mediated rejection.Am J Transplant. 2010; 10: 1812-1822Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar it is not commonly used in clinical practice. Therefore, clinicians identify patients fulfilling only the first 2 diagnostic criteria for the CA-AMR diagnosis showing TG and MVI, testing negative for circulating DSAs with no C4d staining of ptcs, but lacking the opportunity of performing transcript analysis. Hence, there is an unmet need to correctly classify those cases with cg+MVI that do not have C4d positivity and DSAs. To improve classification and therapeutic management, comparison of cg+MVI DSA−/C4d− samples with CA-AMR DSA+/C4d+ samples is needed. Clinical centers encountering cg+MVI DSA−/C4d− patients are usually not able to perform molecular assessments and therefore cannot properly classify their patients within one of the current Banff categories. These cases are often deemed as "histologically comparable to CA-AMR," initiating the same therapeutic strategy as cases fulfilling Banff category 2, CA-AMR. Discussions during the most recent Banff meeting (September 2022 in Banff, Canada) have additionally emphasized the need to not classify those cases as AMR if they do not meet all criteria.18Naesens M. Roufosse C. Colvin R.B. et al.The Banff 2022 Kidney Meeting Report: re-appraisal of microvascular inflammation and the role of biopsy-based transcript diagnostics.Am J Transplant. 2023; (Published online October 28)https://doi.org/10.1016/j.ajt.2023.10.016Abstract Full Text Full Text PDF Scopus (1) Google Scholar cg+MVI DSA−/C4d− samples should be diagnosed as "HLA-DSA-neg MVI cause unclear." It has previously been shown that DSA+ active AMR (A-AMR) cases displayed similar gene expression profiles to cases meeting only the first 2 diagnostic criteria for the AMR diagnosis (MVI) in the absence of DSAs.19Callemeyn J. Lerut E. de Loor H. et al.Transcriptional changes in kidney allografts with histology of antibody-mediated rejection without anti-HLA donor-specific antibodies.J Am Soc Nephrol. 2020; 31: 2168-2183Crossref PubMed Scopus (45) Google Scholar Multiomic profiling comparing cg+MVI DSA−/C4d− with CA-AMR DSA+/C4d+ cases could render a better understanding of these chronic conditions to improve the current classification and subsequently tailor the therapeutic management. In this multicenter intercontinental study, we compared the molecular profiles of for-cause KTx biopsies classified as CA-AMR DSA+/C4d+ with those classified as cg+MVI DSA−/C4d− using the comprehensive Banff Human Organ Transplant panel with the innovative NanoString nCounter technology. Afterward, we cross-checked our results on a subset of the cohort through orthogonal analyses including a state-of-the-art multiplex immunofluorescence (mIF) protein analysis using 2 panels focused on the cell types highlighted by our gene expression analysis. An extensive description of Materials and Methods is provided in the Supplementary Methods. This retrospective multicenter intercontinental study included a highly selected population of KTx recipients. KTx biopsy samples were retrieved from the archives of the Department of Pathology, Erasmus MC, Rotterdam, the Netherlands (Erasmus MC cases, n = 19) and the Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA (Massachusetts General Hospital cases, n = 17). Coherently with the purpose of this study, samples were selected for inclusion if diagnosed with the conditions of interest (including DSA assessment) and if sufficient sample material was available for analysis. Thirty-six transplant recipients who fulfilled the first 2 diagnostic criteria for CA-AMR were retrospectively included in this study (n = 36). Twenty-two patients were classified as CA-AMR DSA+/C4d+, as they had positive C4d staining of ptcs (Banff C4d lesion score ≥1) and showed circulating DSAs. The remaining 14 patients were classified as cg+MVI DSA−/C4d− as they did not show C4d-positive staining of ptcs (Banff C4d lesion score = 0) and had no circulating DSAs. Alternative diagnoses compatible with the histomorphologic findings characterizing TG and MVI (and therefore CA-AMR) were excluded20Haas M. Transplant glomerulopathy: it's not always about chronic rejection.Kidney Int. 2011; 80: 801-803Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar,21Baid-Agrawal S. Farris A.B. Pascual M. et al.Overlapping pathways to transplant glomerulopathy: chronic humoral rejection, hepatitis C infection, and thrombotic microangiopathy.Kidney Int. 2011; 80: 879-885Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar through the use of clinical information, immunofluorescence, and electron microscopy. The details of the Massachusetts General Hospital cases are presented in a previous report.22Rosales I.A. Mahowald G.K. Tomaszewski K. et al.Banff human organ transplant transcripts correlate with renal allograft pathology and outcome: importance of capillaritis and subpathologic rejection.J Am Soc Nephrol. 2022; 33: 2306-2319Crossref PubMed Scopus (14) Google Scholar Formalin-fixed paraffin-embedded specimens were re-evaluated using the 2019 Banff Classification of Allograft Pathology by 2 experienced nephropathologists (IAR and MCC-vG) blinded to the study group the cases belonged to. Circulating donor-specific anti-HLA DSA assessment was performed with the LIFECODES Donor Specific Antibody Assay (Immucor) using bead-specific cutoffs in combination with a raw mean fluorescence intensity of more than 750 arbitrary units. Data were successively analyzed using MATCH IT! Antibody Analysis Software version 1.3.1 (Immucor). The relationship between the diagnosis group and the occurrence of graft failure was investigated through Cox proportional hazards regression adjusted for donor type. Graft survival data were available for the Erasmus MC cases. Details about antirejection therapeutic management of these cases were included in the demographic analysis. Proportional hazards and log-linearity assumptions were verified through the use of Schoenfeld and Martingale residuals, respectively. An additional Kaplan-Meier analysis was performed for visualization purposes. Gene expression profiles of 35 KTx biopsies (n = 21 CA-AMR DSA+/C4d+ and n = 14 cg+MVI DSA−/C4d−) were compared after one initially included sample did not render sufficient amount of RNA for further analysis. The Banff Human Organ Transplant panel using NanoString nCounter technology was used to target the most relevant genes associated with transplant damage.23Mengel M. Loupy A. Haas M. et al.Banff 2019 Meeting Report: molecular diagnostics in solid organ transplantation-consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation.Am J Transplant. 2020; 20: 2305-2317Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar The details of the sample preparation and gene expression analysis of the Massachusetts General Hospital cases were presented in a previous report.22Rosales I.A. Mahowald G.K. Tomaszewski K. et al.Banff human organ transplant transcripts correlate with renal allograft pathology and outcome: importance of capillaritis and subpathologic rejection.J Am Soc Nephrol. 2022; 33: 2306-2319Crossref PubMed Scopus (14) Google Scholar Details concerning the Erasmus MC cases RNA extraction are provided in the Supplementary Methods. Gene expression profiles of the Erasmus MC cases were generated by using the Banff Human Organ Transplant panel within the nCounter FLEX Analysis System (NanoString Technologies, Inc.) following manufacturer's instructions. A total of 490 fields of view were scanned to count the number of copies of each gene in each sample. mIF protein orthogonal analysis was performed on the Erasmus MC cases (n = 9 CA-AMR DSA+/C4d+ and n = 10 cg+MVI DSA−/C4d−) to cross-check results from gene expression analysis. Two 5-plex mIF panels feasible on formalin-fixed paraffin-embedded material were custom-developed to investigate the immune cell repertoire in KTx biopsies. Panel 1 is a broad transplantation inflammatory panel using the markers CD3 (pan T cells), CD8 (cytotoxic T cells), CD68 (macrophages), CD31 (endothelial cells), and C4d. Panel 2 focuses on macrophages and monocytes using CD68 (macrophages), CD163 (M2 macrophages), CD14 (M2 macrophages), CD16 (monocytes), and CD56 (natural killer cells). These 2 panels were performed using automated mIF on the BenchMark ULTRA System (Ventana Medical Systems–F. Hoffmann-La Roche AG). An overview of the products and antibodies used for the mIF panels is presented in Supplementary Table S1. A more detailed description of the methodology used for the mIF analysis is provided in the Supplementary Methods. All mIF slides were scanned at ×20 magnification using an Axio lmager 2 Fluorescence color microscope (ZEISS). Biopsy samples were analyzed using QuPath Quantitative Pathology & Bioimage Analysis software (version 0.3.0), an open software platform for bioimage analysis.24Bankhead P. Loughrey M.B. Fernández J.A. et al.QuPath: open source software for digital pathology image analysis.Sci Rep. 2017; 716878Crossref PubMed Scopus (2956) Google Scholar Biopsies were annotated manually to include only cortical tissue and perform compartmental analysis. Using the simple thresholding method, an individual classifier was made for each marker so that the absolute number of cells could be assessed. The data measurements were then exported for further statistical analysis. To assess the amount of interstitial fibrosis, Sirius Red staining was performed on the same slides used for mIF panel 2. Slides were scanned using a NanoZoomer 2.0-HT digital slide scanner (Hamamatsu Photonics K.K.) with high resolution (×40). Using QuPath, a training image was created as previously described, and the simple thresholding method was then applied to quantify fibrosis. Medulla, glomeruli, and large blood vessels were manually excluded. Quantitative outcomes and variables (i.e., estimated glomerular filtration rate and Banff scores) were handled as continuous variables. Missing data were excluded from analyses. Cell counts differing from the specific per cell type median value of more than 3 times the per cell type median absolute deviation were labeled as outliers and removed from the analysis. To compare 2 unmatched groups, the Student t test was performed after verifying normal distribution. When data were not normally distributed, the Mann-Whitney U test was performed. For this study, we considered P values and Benjiamini-Hochberg–adjusted P values (q values) lower than 0.05 to be statistically significant. Unless noted otherwise, results are expressed as mean ± SEM. Gene expression data of all the included cases were imported within the R environment for further analysis. Quality control and normalization were performed according to Bhattacharya et al.25Bhattacharya A. Hamilton A.M. Furberg H. et al.An approach for normalization and quality control for NanoString RNA expression data.Brief Bioinform. 2021; 22: bbaa163Crossref PubMed Scopus (48) Google Scholar Hierarchical clustering was used to generate heatmaps for additional quality control. Differential expression analysis was performed using DESeq2.26Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15: 550Crossref PubMed Scopus (42425) Google Scholar The Benjamini-Hochberg method was used to adjust the P values for multiple testing.27Haynes W. Benjamini–Hochberg method.in: Dubitzky W. Wolkenhauer O. Cho K.-H. Yokota H. Encyclopedia of Systems Biology. Springer, 2013: 78Crossref Google Scholar The 15 differentially expressed genes with the lowest q values were selected to perform a principal component analysis. Successively, gene-set analyses for cell types and pathway scores were performed using renal allograft pathology–specific custom pathway definitions as previously described22Rosales I.A. Mahowald G.K. Tomaszewski K. et al.Banff human organ transplant transcripts correlate with renal allograft pathology and outcome: importance of capillaritis and subpathologic rejection.J Am Soc Nephrol. 2022; 33: 2306-2319Crossref PubMed Scopus (14) Google Scholar and generally applicable gene-set enrichment for pathway analysis (GAGE).28Luo W. Friedman M.S. Shedden K. et al.GAGE: generally applicable gene set enrichment for pathway analysis.BMC Bioinformatics. 2009; 10: 161Crossref PubMed Scopus (891) Google Scholar Thirty-six patients were included in this study, with 22 diagnosed with CA-AMR DSA+/C4d+ and 14 with cg+MVI DSA−/C4d−. No significant difference was found in demographics and antirejection management between the 2 study groups as presented in Table 1.Table 1Demographics and clinical characteristics of the included study populationFull datasetCA-AMR DSA+/C4d+ (n = 22)cg+MVI DSA−/C4d− (n = 14)P valueRecipient Sex (female/male)8/144/100.90 Age, yr43.1 ± 18.6 (2.7; 79.5)49.7 ± 16.8 (15.5; 80.5)0.23 Time after transplantation, yr6.4 ± 7.9 (0.2; 31.0)4.6 ± 3.4 (1.0; 11.2)0.96 Creatinine at biopsy, mg/dl2.32 ± 1.03 (0.81; 5.29)1.85 ± 0.82 (1.01; 4.00)0.12 Creatinine at 3 yr, mg/dl4.48 ± 3.35 (0.89; 11.5)2.10 ± 1.01 (1.29; 4.31)0.14Erasmus MC subsetCA-AMR DSA+/C4d+ (n = 9)cg+MVI DSA−/C4d− (n = 10)P valueTreatment i.v. Ig9/010/01.00 Methylprednisolone9/010/01.00 Alemtuzumab8/16/40.36 Tocilizumab5/41/90.10CA-AMR, chronic-active antibody-mediated rejection; cg, Banff glomerular basement membrane double contours lesion score; DSA, circulating donor-specific antibodies; i.v. Ig, intravenous immunoglobulin; MVI, microvascular inflammation.Values are expressed as mean ± SD (minimum value; maximum value). Open table in a new tab CA-AMR, chronic-active antibody-mediated rejection; cg, Banff glomerular basement membrane double contours lesion score; DSA, circulating donor-specific antibodies; i.v. Ig, intravenous immunoglobulin; MVI, microvascular inflammation. Values are expressed as mean ± SD (minimum value; maximum value). After re-evaluation using the Banff 2019 classification, 5 samples in the CA-AMR DSA+/C4d+ group also fulfilled the criteria for borderline (suspicious) acute T cell–mediated rejection. However, the 2 study groups were comparable concerning all the individual Banff lesion scores, including interstitial inflammation (i), tubulitis (t), total inflammation (ti), glomerulitis (g), and peritubular capillaritis (ptc) (Supplementary Table S2). Data concerning graft failure, donor type, and antirejection therapy were available only for the Erasmus MC cases. Graft failure occurred in 7 and 3 patients within the CA-AMR DSA+/C4d+ and cg+MVI DSA−/C4d− groups, respectively. When performing Cox proportional hazards regression, patients diagnosed with CA-AMR DSA+/C4d+ showed a higher risk of graft failure occurrence when compared with patients diagnosed with cg+MVI DSA−/C4d− (hazard ratio = 8.7, confidence interval: 1.1–68.7; P = 0.040) after adjusting by donor type (Figure 1). Sufficient RNA was successfully isolated from 35 samples (n = 21 CA-AMR DSA+/C4d+ and n = 14 cg+MVI DSA−/C4d−), while 1 sample was excluded because of insufficient amount of material. The significant differentially expressed genes are reported in Table 2. The full list of differential gene expression analysis is presented in Supplementary Table S3. A volcano plot representing the retrieved differential expression analysis data is presented in Figure 2. Nineteen genes were differentially expressed when comparing the 2 study groups. Ten genes displayed higher expression in samples diagnosed with CA-AMR DSA+/C4d+ compared with samples diagnosed with cg+MVI DSA−/C4d−, including the complement and macrophage gene C5AR1 (complement C5a receptor 1, P = 3.4E−07, q = 2.6E−04, logarithm of fold change [Log2FC] = 0.618), the macrophage surface marker CD163 (P = 0.0012, q = 0.046, Log2FC = 0.884), and genes related to endothelial processes such as ACKR1 (P = 5.6E−05, q = 0.011, Log2FC = 0.902), CDH13 (cadherin 13, P = 4.6E−04, q = 0.025, Log2FC = 0.748), and CAV1 (caveolin 1, P = 9.2E−04, q = 0.042, Log2FC = 0.757). Nine genes displayed higher expression in samples diagnosed with cg+MVI DSA−/C4d− compared with samples diagnosed with CA-AMR DSA+/C4d+, including the cytotoxic T cell–specific gene CD8B (P = 1.9E−05, q = 0.0048, Log2FC = −0.882).Table 2Differential gene expression analysisNo.TargetLog2 fold changeP valueq value1C5AR10.6193.35E−070.0002582FCER1A−1.461.66E−050.004783CD8B−0.8821.86E−050.004784ACKR10.9035.62E−050.01085CD207−0.8457.77E−050.01206AGR2−1.340.0001230.01587SLC11A10.9200.0001680.01628CTNNB1−0.2410.0001890.01629LHX60.5080.0001530.016210ABCE1−0.2670.0002130.016411LILRB20.5170.0003190.022312PPIA−0.2300.0003580.023013CMKLR10.4130.0004030.023914CDH130.7480.0004570.025115C5−0.4600.0007240.037216IFI60.8840.0007870.037917CAV10.7570.0009160.041518CD40LG−0.7040.0009810.042019CD1630.8840.001150.0465CA-AMR, chronic-active antibody-mediated rejection; cg, Banff glomerular basement membrane double contours lesion score; DSA, circulating donor-specific antibodies.Gene expression level comparison of the 15 most differentially expressed genes between cases diagnosed with CA-AMR DSA+/C4d+ and cg+MVI DSA−/C4d− cases using the Banff-Human Organ Transplant Panel with NanoString nCounter technology sorted by the Benjamini-Hochberg–adjusted P value (q value). Genes with positive Log2 fold change have higher expression in CA-AMR DSA+/C4d+ cases. Open table in a new tab CA-AMR, chronic-active antibody-mediated rejection; cg, Banff glomerular basement membrane double contours lesion score; DSA, circulating donor-specific antibodies. Gene expression level comparison of the 15 most differentially expressed genes between cases diagnosed with CA-AMR DSA+/C4d+ and cg+MVI DSA−/C4d− cases using the Banff-Human Organ Transplant Panel with NanoString nCounter technology sorted by the Benjamini-Hochberg–adjusted P value (q value). Genes with positive Log2 fold change have higher expression in CA-AMR DSA+/C4d+ cases. Using GAGE and custom-made Banff Human Organ Transplant panel–specific pathways,22Rosales I.A. Mahowald G.K. Tomaszewski K. et al.Banff human organ transplant transcripts correlate with renal allograft pathology and outcome: importance of capillaritis and subpathologic rejection.J Am Soc Nephrol. 2022; 33: 2306-2319Crossref PubMed Scopus (14) Google Scholar DSA-, CA-AMR-, AMR-, endothelium-, angiogenesis-, interstitial fibrosis and tubular atrophy-, and extracellular matrix–related pathways were enriched in samples diagnosed with CA-AMR DSA+/C4d+ when compared with samples diagnosed with cg+MVI DSA−/C4d−. In the same analysis, the T-cell receptor signaling and B cell–associated transcripts (BAT) pathways were enriched in samples diagnosed with cg+MVI DSA−/C4d− when compared with samples diagnosed with CA-AMR DSA+/C4d+ (P < 0.05; q < 0.05). The 20 most significant pathways are presented in Supplementary Table S4. Cell type profiling was performed to compare the gene expression analysis findings with those of the mIF analysis. Samples diagnosed with CA-AMR DSA+/C4d+ showed an increase in pan-endothelial cells (P = 9.2E–07, q = 1.7–05) and macrophages (P = 1.9E–04, q = 0.0017) in comparison with samples diagnosed with cg+MVI DSA−/C4d−. In the same analysis, samples diagnosed with cg+MVI DSA−/C4d− showed an increase in B cells (P = 3.2E–04, q = 0.0031), T cells (P = 3.4E–04, q = 0.0031), and CD45+ cells (P = 9.8E–04, q = 0.0059) in comparison with samples diagnosed with CA-AMR DSA+/C4d+. The complete results of cell type profiling are presented in Supplementary Table S5. The 15 genes with the lowest q values in the differential expression analysis were used to generate a principal component a