T-cell Mediated Rejection Research Articles

Spatial Transcriptomic Signatures of Early Acute T Cell-mediated Rejection in Kidney Transplants.

Kidney transplantation significantly improves the quality of life for those with end-stage renal failure, yet allograft rejection resulting from immune cell interactions remains a persistent challenge. Although T cell-directed immunosuppressive drugs effectively contain graft rejection in most patients, a notable proportion still experiences acute T cell-mediated rejection (TCMR). Despite an emphasis on suppressing T cell-mediated immune responses, successful control over TCMR is not always achieved, suggesting the potential involvement of factors beyond T cells. Biopsy samples from suspicious (borderline) for acute TCMR (borderline TCMR) and non-TCMR patients were obtained 9 d postsurgery, and spatial transcriptomics profiling was conducted using the GeoMx Digital Spatial Profiler platform. Regions of interest in the glomerulus and interstitium were selected on the basis of immunohistochemistry staining anti-CD3 to identify areas with T-lymphocyte infiltration. Differential gene expression analysis was performed using unpaired t tests. Unbiased clustering of transcriptional profiles across all regions of interest showed distinct transcriptional profiles between glomeruli and interstitium in non-TCMR samples, whereas borderline TCMR samples displayed no distinct transcriptional profiles between these regions. Contrary to the prevailing T cell-centric view, we observed pathways and genes associated with innate immunity-related inflammatory conditions expressed in glomerular regions of borderline TCMR biopsies. Immunofluorescence staining for CD68 confirmed the presence of macrophages in the glomeruli of the post-TCMR sample in a validation cohort, indicating macrophage involvement in the glomerular response after TCMR. Activation of the innate immune response in borderline TCMR appears to impact not only the interstitium but also the glomerulus. Glomerulus-specific immune signatures suggest the role of the innate immune system in rejection. This nuanced understanding proposes the necessity for tailored therapeutic interventions targeting both innate and adaptive immune pathways to enhance transplant outcomes.

Role of pericoronary adipose tissue inflammation at CCTA after heart transplantation as non-invasive diagnostic tool of cardiac rejection and predictor of major adverse cardiovascular events

Abstract Background Long-term survival of heart transplanted (HT) patients is challenged by cardiac rejection and cardiac allograft vasculopathy (CAV). Until now, there are no established biomarkers for graft dysfunction, and the conventional approach for cardiac rejection surveillance is through the endomyocardial biopsy (EMB), an invasive and costly procedure. Purpose This study investigates the prognostic value of the pericoronary Fat Attenuation Index (FAI) and its role as a marker of inflammation and rejection in HT recipients. Methods A double-center observational retrospective study was conducted on a cohort of HT patients who underwent coronary computed tomography angiography (CCTA) between January 2016 and December 2022. FAI was measured with dedicated software and using the cut-off described in literature, we considered each coronary as "inflamed" if FAI was ≥ - 70,1 HU and "not inflamed" if FAI was < - 70,1 HU. Then, based on the number of inflamed coronary arteries for each patient, we divided the population into two comparison groups: high inflammatory (high INF) phenotype (alle three coronary arteries inflamed) and low inflammatory (low INF) phenotype (at least one coronary artery not inflamed). The primary endpoint was a composite of all-cause mortality, acute myocardial infarction, urgent revascularization and heart failure hospitalization. Furthermore, we investigated the correlation between FAI and cardiac rejection by looking for T cell-mediated rejection (TCMR) and antibody-mediated rejection (AMR) through EMB performed no more than six months before or after CCTA. Results A total of 101 HT patients were included; of this group 58 patients underwent EMBs within six months after or before the CCTA. The median duration of follow-up was 28 months. The composite endpoint occurred in 17 patients (16.8%). We documented 26 significative cardiac rejection episodes, 14 TCMR ≥ 2R and 12 AMR. The composite endpoint was achieved in 37.9% of patients with high INF phenotype compared to 8.3% with low INF phenotype (p < 0.001). In the multivariate analysis, the high INF phenotype remained an independent predictor of the composite endpoint in our population (HR: 10.5; 95% CI: 1.88-58.71; p = 0.007). Patients with TCMR and AMR had significantly higher FAI values. High INF phenotype correlated with the presence of ACR ≥ 2R (55.0% vs 7.9%, p < 0.001) and AMR (55.0% vs 2.6%, p < 0.001). Conclusion In HT patients, FAI is an independent predictor of major cardiovascular events. It is also associated with TCMR and AMR within six months from CCTA. FAI, therefore, may offer a non-invasive approach to stratify the prognosis of HT patients and serve as a valuable inflammatory biomarker for early diagnosis of cardiac rejection, supporting the use of CCTA in this setting of patients.Correlation FAI and MACECorrelation FAI and cardiac rejection

Key genes and immune pathways in T-cell mediated rejection post-liver transplantation identified via integrated RNA-seq and machine learning

Liver transplantation is the definitive treatment for end-stage liver disease, yet T-cell mediated rejection (TCMR) remains a major challenge. This study aims to identify key genes associated with TCMR and their potential biological processes and mechanisms. The GSE145780 dataset was subjected to differential expression analysis, weighted gene co-expression network analysis (WGCNA), and machine learning algorithms to pinpoint key genes associated with TCMR. Gene Set Enrichment Analysis (GSEA), immune infiltration analysis, and regulatory networks were constructed to ascertain the biological relevance of these genes. Expression validation was performed using single-cell RNA-seq (scRNA-seq) data and liver biopsy tissues from patients. We identified 5 key genes (ITGB2, FCER1G, IL-18, GBP1, and CD53) that are associated with immunological functions, such as chemotactic activity, antigen processing, and T cell differentiation. GSEA highlighted enrichment in chemokine signaling and antigen presentation pathways. A lncRNA-miRNA-mRNA network was delineated, and drug target prediction yielded 26 potential drugs. Evaluation of expression levels in non-rejection (NR) and TCMR groups exhibited significant disparities in T cells and myeloid cells. Tissue analyses from patients corroborated the upregulation of GBP1, IL-18, CD53, and FCER1G in TCMR cases. Through comprehensive analysis, this research has identified 4 genes intimately connected with TCMR following liver transplantation, shedding light on the underlying immune activation pathways and suggesting putative targets for therapeutic intervention.

Risk of cellular or antibody-mediated rejection in pediatric kidney transplant recipients with BK polyomavirus replication-an international CERTAIN registry study.

In kidney transplant recipients (KTR), BK polyomavirus-associated nephropathy (BKPyVAN) is a major cause of graft loss. To facilitate the clearance of BKPyV-DNAemia, reduction of immunosuppression is currently the treatment of choice but may increase the risk of graft rejection. This international CERTAIN study was designed to determine the risk of alloimmune response and graft dysfunction associated with immunosuppression reduction for BKPyV treatment in 195 pediatric KTR. BKPyV-DNAemia was associated with a more than twofold increased risk of late T cell-mediated rejection (TCMR) (HR 2.22, p = 0.024), of de novo donor-specific HLA antibodies (dnDSA) and/or antibody-mediated rejection (ABMR) (HR 2.64, p = 0.002), and of graft function deterioration (HR 2.73, p = 0.001). Additional independent risk factors for dnDSA/ABMR development were a higher HLA mismatch (HR 2.72, p = 0.006) and re-transplantation (HR 6.40, p = 0.000). Other independent predictors of graft function deterioration were TCMR (HR 3.98, p = 0.003), higher donor age (HR 1.03, p = 0.020), and re-transplantation (HR 3.56, p = 0.013). These data indicate that reduction of immunosuppression for BKPyV-DNAemia management is associated with increased alloimmune response in pediatric KTR. Therefore, regular dnDSA screening and close monitoring of graft function in case of BKPyV-DNAemia followed by subsequent reduction of immunosuppressive therapy are recommended.

Peptide Sharing Between CMV and Mismatched HLA Class I Peptides Promotes Early T-Cell-Mediated Rejection After Kidney Transplantation.

Cytomegalovirus (CMV) infection is related to acute rejection and graft loss after kidney transplantation, though the underlying mechanism remains largely unknown. Some CMV strains produce a peptide that is identical to a peptide sequence found in the leader peptide of specific HLA-A and -C alleles. In this retrospective study of 351 kidney transplantations, we explored whether CMV-seropositive recipients without the VMAPRTLIL, VMAPRTLLL or VMAPRTLVL HLA class I leader peptide receiving a transplant from a donor with this peptide, faced an increased risk of T-cell-mediated rejection (TCMR) in the first 90 days after transplantation. An independent case-control cohort was used for validation (n = 122). The combination of recipient CMV seropositivity with the VMAPRTLIL peptide mismatch was associated with TCMR with a hazard ratio (HR) of 3.06 (p = 0.001) in a multivariable analysis. Similarly, the VMAPRTLLL peptide mismatch was associated with TCMR revealing a HR of 2.61 (p = 0.008). Transplantations featuring either a VMAPRTLIL or a VMAPRTLLL peptide mismatch had a significantly higher cumulative TCMR incidence (p < 0.0001), with the primary impact observed in the first 2 weeks post-transplantation. The findings could be validated in an independent cohort. Together, our data strongly suggest that CMV-positive recipients without an HLA peptide identical to a CMV peptide yet transplanted with a donor who does possess this peptide, have a significantly increased risk of early TCMR. Considering the prevention of such an leader peptide mismatch in these patients or adjusting immunosuppression protocols accordingly may hold promise in reducing the incidence of early TCMR.

S1871 To Biopsy or Not to Biopsy: A Cochrane Diagnostic Meta-Analysis of Non-Invasive Biomarkers for Acute T-Cell Mediated Liver Transplant Rejection

S1871 To Biopsy or Not to Biopsy: A Cochrane Diagnostic Meta-Analysis of Non-Invasive Biomarkers for Acute T-Cell Mediated Liver Transplant Rejection

Pig-to-human kidney xenotransplants using genetically modified minipigs

This study develops an observational model to assess kidney function recovery and xenogeneic immune responses in kidney xenotransplants, focusing on gene editing and immunosuppression. Two brain-dead patients undergo single kidney xenotransplantation, with kidneys donated by minipigs genetically modified to include triple-gene knockouts (GGTA1, β4GalNT2, CMAH) and human gene transfers (hCD55 or hCD55/hTBM). Renal xenograft functions are fully restored; however, immunosuppression without CD40-CD154 pathway blockade is ineffective in preventing acute rejection by day 12. This rejection manifests as both Tcell-mediated rejection and antibody-mediated rejection (AMR), confirmed by natural killer (NK) cell and macrophage infiltration in sequential xenograft biopsies. Despite donor pigs being pathogen free before transplantation, xenografts and recipient organs test positive for porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) by the end of the observation period, indicating reactivation and contributing to significant immunopathological changes. This study underscores the critical need for extended clinical observation and comprehensive evaluation using deceased human models to advance xenograft success.

Donor-derived cell-free DNA-based liquid biopsies to determine future kidney transplant rejection.

Donor-derived cell-free DNA (dd-cfDNA) based liquid kidney biopsies have the potential to detect the chances of kidney transplant rejection. Several studies have found that dd-cfDNA can be used to determine the risk of kidney transplant rejection and may correlate with antibody-mediated rejection (ABMR), T cell-mediated rejection (TCMR), and estimated glomerular filtration rate (eGFR). A high concentration of dd-cfDNA in the body fluids may indicate possible transplant rejection since dd-cfDNA is released as a result of apoptotic and necrotic processes initiated by the recipient's immune system. dd-cfDNA assays have advantages over conventional biopsies since they are noninvasive, and therefore, have the potential to provide a safe and reliable biomarker. Different dd-cfDNA levels have been reported above a number of cutoff thresholds: ABMR at 2.45% and TCMR at 1.3%, compared with 0.44% in healthy patients; and eGFR at 2.5%, a decrease of 25% compared with healthy patients. These results indicate the levels of dd-cfDNA that may be used to signal possible kidney rejection. dd-cfDNA assay is a rapid technique, making it particularly useful in emergencies, and further research into its use in the study of kidney rejection should prove beneficial.

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to posttransplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we have previously reported that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia-reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 h of reoxygenation and incubating NRK-52E cells in a chemical anoxia-recovery environment. We found that during renal IRI MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein, programmed death ligand 1 (PD-L1), a coinhibitory second signal for T cells in TECs. Using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Finally, we set up a coculture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T-cell activation and TCMR.NEW & NOTEWORTHY Ischemic acute kidney injury (AKI) donors can effectively shorten the waiting time for kidney transplantation but increase immune rejection, especially T cell-mediated rejection (TCMR), the mechanism of which remains to be elucidated. Our study demonstrates that during ischemia-reperfusion injury (IRI), the translocation of tubular murine double minute 2 leads to basolateral programmed death ligand 1 degradation, which ultimately results in the occurrence of TCMR, which may provide a new therapeutic strategy for preventing AKI donor-associated TCMR.

Unique Lessons From the Natural Progression of Rejection in Human Uterine Allografts.

Uterus transplantation (UTx) is a novel treatment for absolute uterine infertility. Acute T cell-mediated rejection (TCMR) can be monitored only through serial cervical biopsies. This study, the first of its kind in human transplantation, evaluated clinical, serological, and pathophysiological manifestations of allograft rejection from immunosuppression withdrawal (ISW) to graft hysterectomy (Hx). Following live birth, immunosuppression was abruptly withdrawn from six living-donor UTx recipients. ISW occurred at a median of 7.4 weeks before graft Hx. Post-ISW signs of rejection included: (1) discoloration of the cervix; (2) increased uterine size compared to day of ISW; (3) serological evidence of eosinophilia and progressive development of donor-specific antibodies (DSA) or child-specific antibodies (CSA); (4) histopathological evidence of TCMR in cervical biopsies preceding the development of antibodies in serum; and (5) C4d deposition in tissue before formation of DSA or CSA in all but two recipients. At graft Hx, endometrial glands were preferentially targeted for destruction over stroma while parametrial arteries displayed variable arteritis and fibrointimal hyperplasia. Recognition of the progression of uterine allograft rejection may be important for other human organ recipients and drive research on modulation of immunosuppression and the paradoxical relationship between adaptive cellular and humoral immunity in natural pregnancies. ClinicalTrials.gov identifier: NCT02656550.

Antibody Mediated Rejection and T-cell Mediated Rejection Molecular Signatures Using Next-Generation Sequencing in Kidney Transplant Biopsies.

Recently, interest in transcriptomic assessment of kidney biopsies has been growing. This study investigates the use of NGS to identify gene expression changes and analyse the pathways involved in rejection. An Illumina bulk RNA sequencing on the polyadenylated RNA of 770 kidney biopsies was conducted. Differentially-expressed genes (DEGs) were determined for AMR and TCMR using DESeq2. Genes were segregated according to their previous descriptions in known panels (microarray or the Banff Human Organ Transplant (B-HOT) panel) to obtain NGS-specific genes. Pathway enrichment analysis was performed using the Reactome and Kyoto Encyclopaedia of Genes and Genomes (KEGG) public repositories. The differential gene expression using NGS analysis identified 6,141 and 8,478 transcripts associated with AMR and TCMR. While most of the genes identified were included in the microarray and the B-HOT panels, NGS analysis identified 603 (9.8%) and 1,186 (14%) new specific genes. Pathways analysis showed that the B-HOT panel was associated with the main immunological processes involved during AMR and TCMR. The microarrays specifically integrated metabolic functions and cell cycle progression processes. Novel NGS-specific based transcripts associated with AMR and TCMR were discovered, which might represent a novel source of targets for drug designing and repurposing.

Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade Tcell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) Tcells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells invitro and invivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

Inter- and intra-individual differences regarding SARS-CoV-2 and influenza vaccination in pediatric kidney transplant recipients: An observational study.

In kidney transplant recipients (KTRs), viral infection can lead to antibody and/or T-cell mediated rejection, resulting in kidney transplant dysfunction. Therefore, it is critical to prevent infections. However, KTRs exhibit suboptimal responses to SARS-CoV-2 and/or influenza vaccines, partly due to immunosuppressant therapy. Inter- and intra-individual differences in the biological responses to vaccines may also affect patients' antibody production ability. This study included KTRs who received an messenger RNA SARS-CoV-2 vaccine (3 doses), and an inactivated quadrivalent influenza vaccine (1 or 2 doses). We measured the patients' total antibody titers against SARS-CoV-2 spike antigen, and hemagglutination inhibition (HI) titers against influenza A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. Five patients were eligible for this study. Of these 5 KTRs, two produced anti-SARS-CoV-2 spike antibody titers to a seroprotective level, and also produced HI titers against A/H1N1 to a seroprotective level. Another 2 KTRs did not produce seroprotective anti-SARS-CoV-2 antibody titers, but produced seroprotective HI titers against A/H1N1. The remaining KTR produced a seroprotective anti-SARS-CoV-2 antibody titer, but did not produce a seroprotective HI titer against A/H1N1. The 2 KTRs who did not produce seroprotective anti-SARS-CoV-2 antibody titers following vaccination, later developed COVID-19, and this infection increased their titers over the seroprotective level. This study demonstrated that inter- and intra-individual differences in biological responses to vaccines should be considered in pediatric KTRs, in addition to immunosuppressant effects. Personalized regimens, such as augmented or booster doses of vaccines, could potentially improve the vaccination efficacy against SARS-CoV-2 and influenza.

T-cell Mediated Rejection Associated Microvascular Inflammation in the Allograft Kidney: RNAseq Analysis Using the Banff Human Organ Transplant Gene Panel.

Microvascular inflammation (MVI) can occur in biopsies showing T-cell mediated rejection (TCMR), but it is not well established that T-cells can directly mediate microvascular injury (TCMR-MVI). This was a cross sectional RNAseq based Banff Human Organ Transplant (BHOT) gene expression (GE) analysis. The objective of this study was to probe the molecular signature of TCMR-MVI in comparison with C4d+, DSA+ antibody mediated rejection (ABMR), stable renal function (STA), and TCMR without MVI. Transcriptome analysis utilized CLC genomic workbench and R-studio software. No gene set was specific for any diagnostic category, and all were expressed at low levels in STA biopsies. BHOT gene set scores could differentiate ABMR from TCMR and TCMR-MVI, but not TCMR from TCMR-MVI. TCMR-MVI underexpressed several genes associated with ABMR including DSATs, ENDAT, immunoglobulin genes, ADAMDEC1, PECAM1 and NK cell transcripts (MYBL1, GNLY), but overexpressed C3, NKBBIZ, and LTF. On the other hand, there was no significant difference in the expression of these genes in TCMR-MVI versus TCMR. This indicates that the GE profile of TCMR MVI aligns more closely with TCMR than ABMR. The limitations of classifying biopsies using the binary ABMR-TCMR algorithm, and the occurrence of common pathogenesis mechanisms amongst different rejection phenotype was highlighted by the frequent presence of molecular mixed rejection. T-cell mediated mechanisms play a significant role in the pathogenesis of MVI. GE was broadly different between rejection phenotypes, but molecular scores varied substantially between biopsies with the same Banff grade. It was not always possible to achieve precise molecular score-based diagnostic categorization of individual patients.

A universal urinary cell gene signature of acute rejection in kidney allografts

IntroductionAcute rejection (AR) undermines the life-extending benefits of kidney transplantation and is diagnosed using the invasive biopsy procedure. T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (Mixed Rejection [MR]) are the three major types of AR. Development of noninvasive biomarkers diagnostic of AR due to any of the three types is a useful addition to the diagnostic armamentarium. MethodsWe developed customized RT-qPCR assays and measured urinary cell mRNA copy numbers in 145 biopsy-matched urine samples from 126 kidney allograft recipients. We determined whether the urinary cell three-gene signature diagnostic of TCMR (Suthanthiran et al., 2013) discriminates patients with no rejection biopsies (NR, n = 50) from those with ABMR (n = 28) or MR (n = 20) biopsies. ResultsThe urinary cell three-gene signature discriminated all three types of rejection biopsies from NR biopsies (P < 0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded P < 0.0001 for NR vs. TCMR; P < 0.001 for NR vs. ABMR; and P < 0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (P < 0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (P < 0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (P < 0.0001). All three rejection categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons P > 0.05). ConclusionThe urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients.

Diagnosis and classification of kidney transplant rejection using machine learning-assisted surface-enhanced Raman spectroscopy using a single drop of serum

The quest to reduce kidney transplant rejection has emphasized the urgent requirement for the development of non-invasive, precise diagnostic technologies. These technologies aim to detect antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR), which are asymptomatic and pose a risk of potential kidney damage. The protocols for managing rejection caused by ABMR and TCMR differ, and diagnosis has traditionally relied on invasive biopsy procedures. Therefore, a convergence system using a nano-sensing chip, Raman spectroscopy, and AI technology was introduced to facilitate diagnosis using serum samples obtained from patients with no major abnormality, ABMR, and TCMR after kidney transplantation. Tissue biopsy and Banff score analysis were performed across the groups for validation, and 5 μL of serum obtained at the same time was added onto the Au–ZnO nanorod-based Surface-Enhanced Raman Scattering sensing chip to obtain Raman spectroscopy signals. The accuracy of machine learning algorithms for principal component-linear discriminant analysis and principal component-partial least squares discriminant analysis was 93.53% and 98.82%, respectively. The collagen (an indicative of kidney injury), creatinine, and amino acid-derived signals (markers of kidney function) contributed to this accuracy; however, the high accuracy was primarily due to the ability of the system to analyze a broad spectrum of various biomarkers.

Unveiling Spatial Immune Cell Profile in Kidney Allograft Rejections Using 36-plex Immunofluorescence Imaging.

Kidney allograft rejections are orchestrated by a variety of immune cells. Because of the complex histopathologic features, accurate pathological diagnosis poses challenges even for expert pathologists. The objective of this study was to unveil novel spatial indices associated with transplant rejection by using a spatial bioinformatic approach using 36-plex immunofluorescence image data. The image obtained from 11 T cell-mediated rejection (TCMR) and 12 antibody-mediated rejection (AMR) samples were segmented into 753 737 single cells using DeepCell's Mesmer algorithm. These cells were categorized into 13 distinct cell types through unsupervised clustering based on their biomarker expression profiles. Cell neighborhood analysis allowed us to stratify kidney tissue into 8 distinct neighborhood components consisting of unique cell type enrichment profiles. In contrast to TCMR samples, AMR samples exhibited a higher frequency of neighborhood components that were characterized by an enrichment of CD31+ endothelial cells. Although the overall frequency of CD68+ macrophages in AMR samples was not significantly high, CD68+ macrophages within endothelial cell-rich lesions exhibited a significantly higher frequency in AMR samples than TCMR samples. Furthermore, the frequency of interactions between CD31+ cells and CD68+ cells was significantly increased in AMR samples, implying the pivotal role of macrophages in AMR pathogenesis. Importantly, patients demonstrating a high frequency of CD31:CD68 interactions experienced significantly poorer outcomes in terms of chronic AMR progression. Collectively, these data indicate the potential of spatial bioinformatic as a valuable tool for aiding in pathological diagnosis and for uncovering new insights into the mechanisms underlying transplant rejection.

Alloreactive T cells temporarily increased in the peripheral blood of patients before liver allograft rejection.

T cells are key mediators of alloresponse during liver transplantation (LTx). However, the dynamics of donor-reactive T-cell clones in peripheral blood during a clinical T-cell-mediated rejection (TCMR) episode remain unknown. Here, we collected serial peripheral blood mononuclear cell samples spanning from pre-LTx to 1 year after LTx and available biopsies during the TCMR episodes from 26 rejecting patients, and serial peripheral blood mononuclear cell samples were collected from 96 nonrejectors. Immunophenotypic and repertoire analyses were integrated on T cells from rejectors, and they were longitudinally compared to nonrejected patients. Donor-reactive T-cell clone was identified and tracked by cross-matching with the mappable donor-reactive T-cell receptor repertoire of each donor-recipient pair in 9 rejectors and 5 nonrejectors. Before transplantation, the naive T-cell percentage and T-cell receptor repertoire diversity of rejectors was comparable to that of healthy control, but it was reduced in nonrejectors. After transplantation, the naïve T-cell percentages decreased, and T-cell receptor repertoires were skewed in rejectors; the phenomenon was not observed in nonrejectors. Alloreactive clones increased in proportion in the peripheral blood of rejectors before TCMR for weeks. The increase was accompanied by the naïve T-cell decline and memory T-cell increase and acquired an activated phenotype. Intragraft alloreactive clone tracking in pre-LTx and post-LTx peripheral blood mononuclear cell samples revealed that the pretransplant naïve T cells were significant contributors to the donor-reactive clones, and they temporarily increased in proportion and subsequently reduced in blood at the beginning of TCMR. Together, our findings offer an insight into the dynamic and origin of alloreactive T cells in clinical LTx TCMR cases and may facilitate disease prediction and management.

Implementation of a multicomponent medication adherence promotion system is associated with a decrease in late T-cell-mediated rejection.

Poor immunosuppression adherence in pediatric recipients of liver transplant (LT) contributes to late T-cell-mediated rejection (TCMR) in ~90% of cases and increases the risk of mortality. A medication adherence promotion system (MAPS) was found to reduce late rejection in pediatric recipients of kidney transplants. Using quality improvement methodology, we adapted and implemented the MAPS in our LT clinic. Our primary outcome was population-level rates of late TCMR, measured as a monthly incident rate. Three-hundred fourteen patients undergoing LT are currently cared for at our institution. One-hundred sixty-two (52%) are females with a median age of 16 years and a median age at LT of 2 years. Preimplementation, monthly rejection rates were 0.84 rejections per 100 patient-months. After iterative implementation of MAPS over 2.3 years, monthly rejection rates decreased to 0.46 rejections per 100 patient-months, a 45% decrease in late TCMR. Implementation of MAPS was associated with a sustained 45% decrease in TCMR at a single center, suggesting that quality improvement tools may help improve clinical outcomes. MAPS may be an important tool to ensure long-term allograft health. Future studies should rigorously test MAPS across a multicenter sample.

#2566 Clinicopathological and molecular characteristics of plasma cell rich rejection in renal transplant biopsies

Abstract Background and Aims Plasma cell rich rejection (PCRR) is an uncommon, ill-defined type of renal allograft rejection in the literature considered as a subtype of T cell-mediated rejection (TCMR). PCRR has poorer clinical outcome and is often refractory to classic immunosuppressive therapy. Our study analyzed In this study, we analyzed PCRR in a large cohort of patients who underwent renal transplantation at the Amsterdam UMC. Our objective was to study clinical, histological and molecular signatures of PCRR in comparison to other types of rejection, especially late presentations of TCMR and ABMR. We hypothesized PCRR could be a specific subtype of allograft rejection with different molecular and cellular characteristics compared to (late) TCMR and ABMR with differences in clinical outcome. Method We retrospectively scored and reclassified the last known biopsy of 263 renal transplant recipients, morphologically classified as rejection according to the 2019 Banff classification. mRNA expression analysis was performed using the Nanostring B-HOT panel on a subset of cases. PCRR was compared to (late) TCMR, ABMR and mixed rejection for renal function follow-up and graft survival. Results mRNA analysis revealed uniquely expressed genes in PCRR including LOX, CPA3, IL4, IL17F, and MMP12. PCRR is enriched for genes related to mast cells, memory B- and T-cells and transcripts involved in NK cells and allograft fibrosis with heterogeneity in gene expression in biopsies with PCRR. PCRR might be a late event compared to late TCMR and ABMR, with a higher degree of total inflammation and fibrosis. Graft survival and kidney function was similar to late TCMR and ABMR during a 5-year follow-up period after renal biopsy Conclusion PCRR represents a distinct late-onset stage of inflammation displaying diverse gene expression patterns, with presence of mainly mast cells, NK cells and transcripts involved in renal allograft fibrosis. Such insights could enable a better and more tailored treatment for these patients and ultimately enhance long-term graft survival for patients affected by PCRR. Clinical outcomes in patients with PCRR appeared similar to late TCMR and ABMR.