Project description:Droplet-based massively parallel single-cell RNA sequencing (scRNAseq) was performed by encapsulating sorted live CD45+ tumour-infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 5′ Reagent Kits v2 according to the manufacturer’s protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using an Illumina HiSeq2500.
Project description:T cell immunoglobulin and mucin-containing molecule 3 (TIM-3), first identified as a molecule expressed on interferon-γ producing T cells1, is emerging as an important immune-checkpoint molecule, with therapeutic blockade of TIM-3 being investigated in multiple human malignancies. Expression of TIM-3 on CD8+ T cells in the tumour microenvironment is considered a cardinal sign of T cell dysfunction; however, TIM-3 is also expressed on several other types of immune cell, confounding interpretation of results following blockade using anti-TIM-3 monoclonal antibodies. Here, using conditional knockouts of TIM-3 together with single-cell RNA sequencing, we demonstrate the singular importance of TIM-3 on dendritic cells (DCs), whereby loss of TIM-3 on DCs-but not on CD4+ or CD8+ T cells-promotes strong anti-tumour immunity. Loss of TIM-3 prevented DCs from expressing a regulatory program and facilitated the maintenance of CD8+ effector and stem-like T cells. Conditional deletion of TIM-3 in DCs led to increased accumulation of reactive oxygen species resulting in NLRP3 inflammasome activation. Inhibition of inflammasome activation, or downstream effector cytokines interleukin-1β (IL-1β) and IL-18, completely abrogated the protective anti-tumour immunity observed with TIM-3 deletion in DCs. Together, our findings reveal an important role for TIM-3 in regulating DC function and underscore the potential of TIM-3 blockade in promoting anti-tumour immunity by regulating inflammasome activation.
Project description:The danger signals that activate the NLRP1 inflammasome have yet to be firmly established. NLRP1 undergoes autoproteolysis to generate N-terminal (NT) and C-terminal (CT) fragment, which importantly, is a necessary step for its check-point regulation by the DPP9 ternary complex and the mechanistic activation of NLRP1 through functional degradation. Here, we report an added layer of regulatory complexity to NLRP1 activity, in the form of a repressive interaction that NLRP1 forms with the oxidized, but not reduced, form of thioredoxin-1 (TRX1). Loss of TRX1 destabilizes the NT fragment of NLRP1 and promotes enhanced inflammasome activation. The TRX1 interaction occurs through the NACHT-LRR of NLRP1 and requires nucleotide binding in its ATPase domain. In addition, we found that several patient-derived and ATPase-inactivating mutations in the NACHT-LRR region hyperactive the inflammasome by destabilize protein folding and are also shown to abrogate TRX1 binding. Thus, NLRP1 appears to detect intracellular reductive stress through a decrease in the fraction of intracellular oxidized TRX1, which enhances protein disorder, leading to inflammasome signaling. These findings link the cellular redox environment to NLRP1-mediated innate immunity.
Project description:The danger signals that activate the NLRP1 inflammasome have yet to be firmly established. NLRP1 undergoes autoproteolysis to generate N-terminal (NT) and C-terminal (CT) fragment, which importantly, is a necessary step for its check-point regulation by the DPP9 ternary complex and the mechanistic activation of NLRP1 through functional degradation. Here, we report an added layer of regulatory complexity to NLRP1 activity, in the form of a repressive interaction that NLRP1 forms with the oxidized, but not reduced, form of thioredoxin-1 (TRX1). Loss of TRX1 destabilizes the NT fragment of NLRP1 and promotes enhanced inflammasome activation. The TRX1 interaction occurs through the NACHT-LRR of NLRP1 and requires nucleotide binding in its ATPase domain. In addition, we found that several patient-derived and ATPase-inactivating mutations in the NACHT-LRR region hyperactive the inflammasome by destabilize protein folding and are also shown to abrogate TRX1 binding. Thus, NLRP1 appears to detect intracellular reductive stress through a decrease in the fraction of intracellular oxidized TRX1, which enhances protein disorder, leading to inflammasome signaling. These findings link the cellular redox environment to NLRP1-mediated innate immunity.
Project description:Here we report RNA sequencing data from sorted Tim-4low vs Tim-4high macrophages in steady-state (no tumor) mice and also MC38-LG-tumor-bearing animals that were treated with isotype or anti-Tim-4 antibody.
Project description:Bladder cancer (BC) is one of the most common cancers in the world. T-cell immunoglobulin and mucin domain 1 (TIM-1) are involved in the progression of multiple tumors. However the role of TIM-1 in BC progression is poorly understood. In this study, we searched the Gene Expression Profiling Interactive Analysis (GEPIA) database and performed immunohistochemistry (IHC) to assess TIM-1 protein expression in bladder cancer (BC) patients. The results demonstrated that BC with high TIM-1 expression was associated with longer overall survival (OS) and disease-specific survival (DSS) than BC with low TIM-1 expression. Overexpression of TIM-1 inhibits BC cell proliferation in both cell culture and animal experiments. RNA sequencing data indicated that interferon-induced protein with tetratricopeptide repeats (IFIT) genes induced by interferon-α (IFN-α) were significantly enriched among the genes upregulated by TIM-1 overexpression. Mechanistically, our data revealed that TIM-1 promotes IFN-α release and activates the IFIT2/p-STAT1 pathway, which is known to be related to tumor cell proliferation. Moreover, knockdown of IFIT2 in TIM-1-overexpressing BC cells hinders the tumor suppressive effect of TIM-1. Our results revealed that TIM-1 is a potential molecular marker for BC prognosis and indicate that high TIM-1 expression suppresses BC cell proliferation in an IFIT2/p-STAT1-dependent manner.