Similarly, the CTCAE grade was significantly higher in patients carrying the CC genotype compared with patients with a GC or GG genotype (Figure 4B). the effect of a SNP in the gene on irAE severity in 167 patients treated with ICIs. We found that the SNP rs2910164 leading to reduced miR-146a expression was associated with an increased risk of developing severe irAEs, reduced progression-free survival, and increased neutrophil counts both at baseline and during ICI therapy. In conclusion, we characterized miR-146a as a molecular target for preventing ICI-mediated autoimmune dysregulation. Furthermore, we identified the SNP rs2910164 as a biomarker to predict severe irAE development in ICI-treated patients. mice with antiCPD-1 led to significantly more severe irAEs compared with WT mice treated with antiCPD-1, indicated by increased neutrophil and lymphocyte infiltration in the major irAE target organs, comprising the lungs (Figure 1, ACC), liver (Figure 1, DCF), colon (Figure 1, G and H), and skin (Figure 1, I and J). To control for nonspecific effects of the antibody or low-dose LPS treatment, WT or mice treated with LPS and isotype control antibody were analyzed and did not develop signs of significant immune infiltration (Figure 1, ACJ). In addition to the increased irAE severity, as assessed by histopathology, the inflammatory side effects caused reduced survival of the + antiCPD-1 group, compared with all other groups (Supplemental Figure 1B). Open in a separate window Figure 1 deficiency increases TC-E 5002 irAE severity in antiCPD-1Ctreated mice.WT or mice (= 9C10 per group) were treated with LPS and antiCPD-1/isotype control antibody for 3 weeks as described. The lungs, liver, colon, and skin were isolated on day 22 after the first treatment for histopathological assessment. irAE grading was performed by an experienced pathologist blinded to the treatment groups. 0 = absent, 1 = mild, 2 = massive neutrophil/lymphocyte infiltration. Data were pooled from 2 independent experiments. Statistical significance was analyzed by Kruskal-Wallis test followed by 2-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Adjusted value is depicted: **< 0.01, ***< 0.001. (A) Representative H&E staining of lung sections at original magnification 200. Black arrows point towards inflammatory infiltrates. (B and C) Histopathology scores for neutrophil infiltration and lymphocyte infiltration into the lung. (D) Representative H&E staining of liver sections at original magnification 200. Black arrows point towards inflammatory infiltrates. (E and F) Histopathology scores for neutrophil infiltration and lymphocyte infiltration into the liver. (G and H) Histopathology scores for neutrophil infiltration and lymphocyte infiltration into the colon. (I and J) Histopathology scores for neutrophil infiltration and lymphocyte infiltration into the skin. deficiency in the hematopoietic system also increased checkpoint inhibitorCmediated autoimmunity in the major irAE target organs in mice treated with antiCPD-1 and antiCCTLA-4 combination therapy compared with WT mice (Supplemental Figure 2). Taken together, our data indicate that miR-146a negatively regulates inflammatory side effects of ICI therapy while mice lacking miR-146a exhibit increased ICI-induced organ Rabbit Polyclonal to ABCC2 toxicity. miR-146aCdeficient mice treated with ICIs show increased TC-E 5002 CD4 T cell activation. Since antiCPD-1 treatment is therapeutically used to enhance T cell activation, we next used an unbiased approach to assess whether miR-146a regulates the T cell response during ICI therapy. To analyze the T cell phenotype at the single cell level, we performed 10 Genomics single-cell RNA sequencing (scRNA-seq) from murine splenic T cells of WT or mice were used for further analysis. Unsupervised clustering showed that both WT and cells separated into clearly defined clusters expressing genes characteristic of distinct T cell subsets (Figure 2A and Supplemental Figure 3). Differential expression analysis followed by gene set enrichment TC-E 5002 analysis (GSEA) of the main T cell clusters using Hallmark immune gene sets revealed an enrichment of pathways involved in inflammation and immune activation in mice (= 2 per group) were treated with low-dose LPS and antiCPD-1/isotype control antibody for 3 weeks before capturing of MACS purified splenic T cells for scRNA-seq using 10 v3.1 Next GEM chemistry. Data were processed, visualized, and analyzed using the Seurat pipeline v3.0 (45, 46). (A) Uniform Manifold Approximation and Projection (UMAP) TC-E 5002 plots showing distinct T cell clusters in both and WT mice. (B) Gene set enrichment analysis of major T cell clusters. Bivariate heatmap depicts normalized enrichment score as color TC-E 5002 code and Clog10 of the adjusted value as dot size. Hallmark gene.