CaSSiDI: novel single-cell "Cluster Similarity Scoring and Distinction Index" reveals critical functions for PirB and context-dependent Cebpb repression.

PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called "Cluster Similarity Scoring and Distinction Index" (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB's effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ "neuronal-primed" subset and an CD68hiFtl1+ "iron-loaded" subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.

Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

Cholinergic control of Th17 cell pathogenicity in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFß, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.

Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs.

Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.

Choline acetyltransferase-expressing T cells are required to control chronic viral infection.

Although widely studied as a neurotransmitter, T cell-derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of Chat within the T cell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral T cells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in T cells during viral infection and identify a pathway of T cell migration that sustains antiviral immunity.

Smg1 is required for embryogenesis and regulates diverse genes via alternative splicing coupled to nonsense-mediated mRNA decay.

Smg1 is a PI3K-related kinase (PIKK) associated with multiple cellular functions, including DNA damage responses, telomere maintenance, and nonsense-mediated mRNA decay (NMD). NMD degrades transcripts that harbor premature termination codons (PTCs) as a result of events such as mutation or alternative splicing (AS). Recognition of PTCs during NMD requires the action of the Upstream frameshift protein Upf1, which must first be phosphorylated by Smg1. However, the physiological function of mammalian Smg1 is not known. By using a gene-trap model of Smg1 deficiency, we show that this kinase is essential for mouse embryogenesis such that Smg1 loss is lethal at embryonic day 8.5. High-throughput RNA sequencing (RNA-Seq) of RNA from cells of Smg1-deficient embryos revealed that Smg1 depletion led to pronounced accumulation of PTC-containing splice variant transcripts from approximately 9% of genes predicted to contain AS events capable of eliciting NMD. Among these genes are those involved in splicing itself, as well as genes not previously known to be subject to AS-coupled NMD, including several involved in transcription, intracellular signaling, membrane dynamics, cell death, and metabolism. Our results demonstrate a critical role for Smg1 in early mouse development and link the loss of this NMD factor to major and widespread changes in the mammalian transcriptome.

The apoptotic protease-activating factor 1-mediated pathway of apoptosis is dispensable for negative selection of thymocytes.

Negative selection is a process to delete potentially autoreactive clones in developing thymocytes. Programmed cell death or apoptosis is thought to play an important role in this selection process. In this study, we investigated the role of apoptotic protease-activating factor 1 (Apaf1), a mammalian homologue of CED-4, in programmed cell death during the negative selection in thymus. There was no developmental abnormality in thymocytes from newborn Apaf1(-/-) mice in terms of CD4 and CD8 expression pattern and thymocyte number. Clonal deletion by endogenous male H-Y Ag of Apaf1-deficient thymocytes with transgenic expression of H-Y Ag-specific TCRs (H-Y Tg/Apaf1(-/-) thymocytes) was normally observed in lethally irradiated wild-type mice reconstituted with fetal liver-derived hemopoietic stem cells. Clonal deletion induced in vitro by a bacterial superantigen was also normal in fetal thymic organ culture. Thus, Apaf1-mediated pathway of apoptosis is dispensable for the negative selection of thymocytes. However, H-Y Tg/Apaf1(-/-) thymocytes showed partial resistance to H-Y peptide-induced deletion in vitro as compared with H-Y Tg/Apaf1(+/-) thymocytes, implicating the Apaf1-mediated apoptotic pathway in the negative selection in a certain situation. In addition, the peptide-induced deletion was still observed in H-Y Tg/Apaf1(-/-) thymocytes in the presence of a broad spectrum caspase inhibitor, z-VAD-fmk, suggesting the presence of caspase-independent cell death pathway playing roles during the negative selection. We assume that mechanisms for the negative selection are composed of several cell death pathways to avoid failure of elimination of autoreactive clones.