No abstract

The intestinal stem cell fuels the highest rate of tissue turnover in the body and has been implicated in intestinal disease and cancer; understanding the regulatory mechanisms controlling intestinal stem cell physiology is of great importance. Here, we provide evidence that the transcription factor YY1 is essential for intestinal stem cell renewal. We observe that YY1 loss skews normal homeostatic cell turnover, with an increase in proliferating crypt cells and a decrease in their differentiated villous progeny. Increased crypt cell numbers come at the expense of Lgr5 + stem cells. On YY1 deletion, Lgr5 + cells accelerate their commitment to the differentiated population, exhibit increased levels of apoptosis, and fail to maintain stem cell renewal. Loss of Yy1 in the intestine is ultimately fatal. Mechanistically, YY1 seems to play a role in stem cell energy metabolism, with mitochondrial complex I genes bound directly by YY1 and their transcript levels decreasing on YY1 loss. These unappreciated YY1 functions broaden our understanding of metabolic regulation in intestinal stem cell homeostasis. (9), which cooccupy the bottom of crypts with differentiated Paneth cells. Intestinal stem cells marked by leucine rich repeat containing G protein coupled receptor 5 (Lgr5) expression have been the most extensively characterized; these cells maintain their own population through symmetric divisions (10), and when they leave the niche, they give rise to differentiated crypt cells, including a transit amplifying population that ultimately supplies differentiated cells onto luminal projections called villi.Intestinal stem cells are of great importance to human health and regenerative medicine. Mouse models of human colorectal cancer show that intestinal stem cells can function as cells of origin for cancer (11,12). There is a clear imperative to understand the regulatory mechanisms governing intestinal stem cell function. Recent work has shown that intestinal stem cells from both flies and humans are sensitive to the metabolic state of the organism and has implicated cellular metabolism as a critical regulatory input of stem cell homeostasis (13-16). Intestinal stem cells were observed to exhibit higher levels of glycolysis than oxidative phosphorylation compared with their differentiated progeny (17), and the oxidative state of intestinal stem cells impacts the ability of the cells to undergo transformation (18). Caloric restriction was recently shown to increase intestinal stem cell numbers (16). In Drosophila, intestinal stem cell expression of the fly homolog to PGC-1α, a metabolic coregulator, is even coupled to the organism's lifespan (19). These exciting advances highlight a great need to identify additional regulators of intestinal stem cell metabolism.YY1 is a zinc finger transcription factor first discovered for its function in viral gene expression (20, 21) and cloned during investigations of viral (22), immunoglobulin (23), and ribosomal (24) gene expression. YY1 has since been implicated in a number of proc...

Mass cytometry is a powerful tool for high-dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigm's third-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing, we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median and higher coefficients of variation in many antibodyassociated signal intensities. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multisite study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces interinstrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multicenter studies.

Immunotherapy is becoming a mainstay in the treatment of NSCLC. While tumor mutational burden (TMB) has been shown to correlate with response to immunotherapy, little is known about the relation of the baseline immune response with the tumor genotype. Here, we profiled 35 early stage NSCLC lesions using multiscale single cell sequencing. Unsupervised clustering identified in a subset of patients a key cellular module consisting of PDCD1+ CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). Transcriptional data from two NSCLC cohorts confirmed a subset of patients with LCAMhi enrichment, which was independent of overall immune cell content. The LCAMhi module strongly correlated with TMB, expression of cancer testis antigens, and with TP53 mutations in smokers and non-smokers. These data establish LCAM as a key mode of immune cell activation associated with high tumor antigen load and driver mutations.

Conflict of interest statement: AHR is a named inventor of a technology titled "Metal labeled Vericells for mass cytometry", and both he and Mount Sinai have received payments associated with licensing of this technology. CW holds equity in BioNTech, Inc. CW is a co-founder and scientific advisory board member of