Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study.

BACKGROUND: Cross-species immunological incompatibilities have hampered pig-to-human xenotransplantation, but porcine genome engineering recently enabled the first successful experiments. However, little is known about the immune response after the transplantation of pig kidneys to human recipients. We aimed to precisely characterise the early immune responses to the xenotransplantation using a multimodal deep phenotyping approach. METHODS: We did a complete phenotyping of two pig kidney xenografts transplanted to decedent humans. We used a multimodal strategy combining morphological evaluation, immunophenotyping (IgM, IgG, C4d, CD68, CD15, NKp46, CD3, CD20, and von Willebrand factor), gene expression profiling, and whole-transcriptome digital spatial profiling and cell deconvolution. Xenografts before implantation, wild-type pig kidney autografts, as well as wild-type, non-transplanted pig kidneys with and without ischaemia-reperfusion were used as controls. FINDINGS: The data collected from xenografts suggested early signs of antibody-mediated rejection, characterised by microvascular inflammation with immune deposits, endothelial cell activation, and positive xenoreactive crossmatches. Capillary inflammation was mainly composed of intravascular CD68+ and CD15+ innate immune cells, as well as NKp46+ cells. Both xenografts showed increased expression of genes biologically related to a humoral response, including monocyte and macrophage activation, natural killer cell burden, endothelial activation, complement activation, and T-cell development. Whole-transcriptome digital spatial profiling showed that antibody-mediated injury was mainly located in the glomeruli of the xenografts, with significant enrichment of transcripts associated with monocytes, macrophages, neutrophils, and natural killer cells. This phenotype was not observed in control pig kidney autografts or in ischaemia-reperfusion models. INTERPRETATION: Despite favourable short-term outcomes and absence of hyperacute injuries, our findings suggest that antibody-mediated rejection in pig-to-human kidney xenografts might be occurring. Our results suggest specific therapeutic targets towards the humoral arm of rejection to improve xenotransplantation results. FUNDING: OrganX and MSD Avenir.

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.

Banff Human Organ Transplant Consensus Gene Panel for the Detection of Antibody Mediated Rejection in Heart Allograft Biopsies.

The molecular refinement of the diagnosis of heart allograft rejection based on whole-transcriptome analyses faces several hurdles that greatly limit its widespread clinical application. The targeted Banff Human Organ Transplant gene panel (B-HOT, including 770 genes of interest) has been developed to facilitate reproducible and cost-effective gene expression analysis of solid organ allografts. We aimed to determine in silico the ability of this targeted panel to capture the antibody-mediated rejection (AMR) molecular profile using whole-transcriptome data from 137 heart allograft biopsies (71 biopsies reflecting the entire landscape of histologic AMR, 66 non-AMR control biopsies including cellular rejection and non-rejection cases). Differential gene expression, pathway and network analyses demonstrated that the B-HOT panel captured biologically and clinically relevant genes (IFNG-inducible, NK-cells, injury, monocytes-macrophage, B-cell-related genes), pathways (interleukin and interferon signaling, neutrophil degranulation, immunoregulatory interactions, endothelial activation) and networks reflecting the pathophysiological mechanisms underlying the AMR process previously identified in whole-transcriptome analysis. Our findings support the potential clinical use of the B-HOT-gene panel as a reliable proxy to whole-transcriptome analysis for the gene expression profiling of cardiac allograft rejection.

Low fluid shear stress stimulates the uptake of noxious endothelial extracellular vesicles via MCAM and PECAM-1 cell adhesion molecules.

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS-EVs have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

Archetypal Analysis of Kidney Allograft Biopsies Using Next-generation Sequencing Technology.

BACKGROUND: In kidney transplantation, molecular diagnostics may be a valuable approach to improve the precision of the diagnosis. Using next-generation sequencing (NGS), we aimed to identify clinically relevant archetypes. METHODS: We conducted an Illumina bulk RNA sequencing on 770 kidney biopsies (540 kidney recipients) collected between 2006 and 2021 from 11 European centers. Differentially expressed genes were determined for 11 Banff lesions. An ElasticNet model was used for feature selection, and 4 machine learning classifiers were trained to predict the probability of presence of the lesions. NGS-based classifiers were used in an unsupervised archetypal analysis to different archetypes. The association of the archetypes with allograft survival was assessed using the iBox risk prediction score. RESULTS: The ElasticNet feature selection reduced the number of the genes from a range of 859-10 830 to a range of 52-867 genes. NGS-based classifiers demonstrated robust performances (precision-recall area under the curves 0.708-0.980) in predicting the Banff lesions. Archetypal analysis revealed 8 distinct phenotypes, each characterized by distinct clinical, immunological, and histological features. Although the archetypes confirmed the well-defined Banff rejection phenotypes for T cell-mediated rejection and antibody-mediated rejection, equivocal histologic antibody-mediated rejection, and borderline diagnoses were reclassified into different archetypes based on their molecular signatures. The 8 NGS-based archetypes displayed distinct allograft survival profiles with incremental graft loss rates between archetypes, ranging from 90% to 56% rates 7 y after evaluation (P < 0.0001). CONCLUSIONS: Using molecular phenotyping, 8 archetypes were identified. These NGS-based archetypes might improve disease characterization, reclassify ambiguous Banff diagnoses, and enable patient-specific risk stratification.

Increased Circulating CD62E+ Endothelial Extracellular Vesicles Predict Severity and in- Hospital Mortality of COVID-19 Patients.

COVID-19 and infectious diseases have been included in strategic development goals (SDG) of United Nations (UN). Severe form of COVID-19 has been described as an endothelial disease. In order to better evaluate Covid-19 endotheliopathy, we characterized several subsets of circulating endothelial extracellular vesicles (EVs) at hospital admission among a cohort of 60 patients whose severity of COVID-19 was classified at the time of inclusion. Degree of COVID-19 severity was determined upon inclusion and categorized as moderate to severe in 40 patients and critical in 20 patients. We measured citrated plasma EVs expressing endothelial membrane markers. Endothelial EVs were defined as harboring VE-cadherin (CD144+), PECAM-1 (CD31 + CD41-) or E-selectin (CD62E+). An increase in CD62E + EV levels on admission to the hospital was significantly associated with critical disease. Moreover, Kaplan-Meier survival curves for CD62E + EV level showed that level ≥ 88,053 EVs/µL at admission was a significant predictor of in hospital mortality (p = 0.004). Moreover, CD62E + EV level ≥ 88,053 EV/µL was significantly associated with higher in-hospital mortality (OR 6.98, 95% CI 2.1-26.4, p = 0.002) in a univariate logistic regression model, while after adjustment to BMI CD62E + EV level ≥ 88,053 EV/µL was always significantly associated with higher in-hospital mortality (OR 5.1, 95% CI 1.4-20.0, p = 0.01). The present findings highlight the potential interest of detecting EVs expressing E-selectin (CD62) to discriminate Covid-19 patients at the time of hospital admission and identify individuals with higher risk of fatal outcome.