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Single-Cell Transcriptomics Unravels the Early Immune Landscape of Renal Allograft Rejection and Nominates Ccl3-Ccr5 as a Therapeutic Target

ABSTRACT: Background Acute rejection is a primary determinant of long-term allograft survival, and the early-stage immune cell dynamics with local intercellular communication during this process require further elucidation. Methods We performed single-cell RNA sequencing (scRNA-seq) on CD45+ immune cells isolated from rat renal allografts during the early phase of acute rejection (days 0, 1, 3, and 7). Using unsupervised clustering, functional enrichment analysis, cellular trajectory inference, and intercellular communication network mapping, we delineated the immune cell dynamics and local communication networks at single-cell resolution. Our findings were subsequently validated through multiplex immunofluorescence and therapeutic intervention experiments. Results Macrophages constituted the dominant immune population during acute rejection. Sub-clustering analysis revealed a rapid expansion of the Isg15+Mac subset by post-transplant day 1, which persisted at elevated levels thereafter. Functional enrichment and trajectory inference demonstrated the pro-inflammatory properties of Isg15+Mac, implicating this subset in acute rejection. Cell-cell communication analysis identified Ccl3-Ccr5 ligand-receptor interactions between Isg15+Mac and T cells. Multiplex immunofluorescence confirmed abundance of Isg15+Mac within the allografts. Moreover, We alleviated the acute rejection after kidney transplantation using the FDA-approved Ccr5 blocker Maraviroc. Conclusions Our study establishes an in-depth, early-stage immune landscape of renal transplantation, uncovering the Isg15+Mac subset as a critical driver of acute rejection via the Ccl3-Ccr5 axis. And also indicating that Maraviroc may potentially be a therapeutic candidate for transplant rejection.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE303904 | GEO | 2026/03/01

REPOSITORIES: GEO

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Project description:CONTEXT Slowly progressive chronic tubulo-interstitial damage jeopardizes long-term renal allograft survival. Both immune and non-immune mechanisms are thought to contribute, but the most promising targets for timely intervention have not been identified. OBJECTIVE In the current study we seek to determine the driving force behind progressive histological damage of renal allografts, without the interference of donor pathology, delayed graft function and acute graft rejection. DESIGN We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. PATIENTS Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection. RESULTS In this highly cross-validated study, we demonstrate the significant association of established, ongoing and future chronic histological damage with regulation of adaptive immune gene expression (T-cell and B-cell transcript sets) and innate immune response gene expression (dendritic cell, NK-cell, mast cell and granulocyte transcripts). We demonstrate the ability of gene expression analysis to perform as a quantitative marker for ongoing inflammation with a wide dynamic range: from subtle subhistological inflammation prior to development of chronic damage, over moderate subclinical inflammation associated with chronic histological damage, to marked inflammation of Banff-grade acute T-cell mediated rejection. CONCLUSION Progressive chronic histological damage after kidney transplantation is associated with significant regulation of both innate and adaptive immune responses, months before the histological lesions appear. This study therefore corroborates the hypothesis that quantitative inflammation below the diagnostic threshold of classic T-cell or antibody-mediated rejection is associated with early subclinical stages of progressive renal allograft damage. We used microarrays to examine whole genome expression profiles in renal allograft protocol biopsies, and analyzed the correlation between gene expression and the histological appearance over time. The gene expression profiles in these protocol biopsies were then compared with gene expression of biopsies with acute T-cell mediated rejection. Human renal allograft biopsies (N=120) were included: 96 rejection-free protocol biopsies and 24 biopsies with T-cell mediated acute rejection.

Project description:The data set contains 67 array (lymphochip cDNA array), published in N Engl J Med 2003 Jul 10;349(2):125-38. PMID: 12853585. TITLE: Molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling. BACKGROUND: The causes and clinical course of acute rejection vary, and it is not possible to predict graft outcome reliably on the basis of available clinical, pathological, and genetic markers. We hypothesized that previously unrecognized molecular heterogeneity might underlie some of the variability in the clinical course of acute renal allograft rejection and in its response to treatment. METHODS: We used DNA microarrays in a systematic study of gene-expression patterns in biopsy samples from normal and dysfunctional renal allografts. A combination of exploratory and supervised bioinformatic methods was used to analyze these profiles. : We found consistent differences among the gene-expression patterns associated with acute rejection, nephrotoxic effects of drugs, chronic allograft nephropathy, and normal kidneys. The gene-expression patterns associated with acute rejection suggested at least three possible distinct subtypes of acute rejection that, although indistinguishable by light microscopy, were marked by differences in immune activation and cellular proliferation. Since the gene-expression patterns pointed to substantial variation in the composition of immune infiltrates, we used immunohistochemical staining to define these subtypes further. This analysis revealed a striking association between dense CD20+ B-cell infiltrates and both clinical glucocorticoid resistance (P=0.01) and graft loss (P<0.001). CONCLUSIONS: Systematic analysis of gene-expression patterns provides a window on the biology and pathogenesis of renal allograft rejection. Biopsy samples from patients with acute rejection that are indistinguishable on conventional histologic analysis reveal extensive differences in gene expression, which are associated with differences in immunologic and cellular features and clinical course. The presence of dense clusters of B cells in a biopsy sample was strongly associated with severe graft rejection, suggesting a pivotal role of infiltrating B cells in acute rejection. A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied. Disease State: normal vs disease Keywords: disease_state_design Using regression correlation

Project description:Steroid-refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection. Eighty-three kidney transplant recipients (1995-2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (P<0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT-1 as an independent covariate (AUC=0.88, P<0.0000001). In the validation set, MT-1 expression was also significantly associated with steroid resistance (P=0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that non-response to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc-requiring anti-inflammatory effect of the glucocorticoid receptor. Transcriptional profiles of 40 renal allograft biopsy samples were analyzed using Illumina HumanRef-8 v3.0 BeadChips (Illumina, San Diego, CA). Expression profiles were investigated in total RNA isolated from biopsy samples of 18 patients with steroid responsive acute rejection and 18 patients with steroid resistant acute rejection. Four biopsies, taken during acute decrease of graft function but with no histomorphologic indication of rejection, were included as controls.

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Single-Cell Transcriptomics Unravels the Early Immune Landscape of Renal Allograft Rejection and Nominates Ccl3-Ccr5 as a Therapeutic Target

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