Project description:Checkpoint inhibitor colitis (CPI colitis) is a frequent immune-related adverse event resulting from cancer immunotherapy that is currently treated with empiric immunosuppression such as corticosteroids. A greater understanding of the pathogenesis of CPI colitis in the steroid-refractory setting is required to develop better colitis interventions targeted to each patient. Using paired colon biopsies and blood from steroid-experienced patients with endoscopically confirmed CPI colitis, combined with control patients, we performed multiplexed single-cell transcriptomics (scRNA/TCRseq) and proteomics (CITEseq, CyTOF) to identify contributing immune and non-immune populations. In CPI colitis patient biopsies compared to healthy controls, we found enrichment of resident memory (RM) T cells in both the CD8+ and unexpectedly the CD4+ compartments, as well as cytotoxic CD8+ T cells. These 3 populations expressed exact matching TCR, suggesting that CD4+ RM and CD8+ RM T cells are clonal progenitors that give rise to tissue-destructive cytotoxic effectors. Unbiased CyTOF and confirmatory scRNAseq and CITEseq analysis identifies specific mobilized subpopulations of both cytotoxic CD8+ and CD4+ RM cells that express both activation markers CD38 and HLA-DR, underscoring their pathogenic potential. CPI colitis induces global upregulation of interferon signaling and antigen presentation, as well as epithelial-specific dysregulation of homeostatic pathways (aquaporin/solute transporters) which is shared with ulcerative colitis. However, distinct from ulcerative colitis, CPI colitis involves specific alterations in stromal cell metabolic (NAD+, tryptophan) or extracellular signaling (BMP) pathways that directly impact epithelial cell survival, arguing for distinct therapeutic targeting. Finally, endothelial cells in CPI colitis demonstrate enrichment of the integrin 47 ligand MADCAM-1 (which is targeted by anti-47 antibody vedolizumab to treat colitis). This ligand is enriched in CPI colitis patients who received TNF blockade and anti-CTLA-4, identifying subsets of patients who may be more responsive to vedolizumab treatment. This work identifies putative pathogenic resident memory and cytotoxic T cells, and non-immune stromal and endothelial populations, whose properties may nominate subsets of CPI colitis patients amenable to distinct targeting strategies.

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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Reverse translational discovery reveals dysregulation of CD4+ and CD8+ resident memory T, myeloid, and stromal cells in steroid-refractory, checkpoint inhibitor colitis

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