Gut CD4+ T cell phenotypes are a continuum molded by microbes, not by TH archetypes.

CD4+ effector lymphocytes (Teff) are traditionally classified by the cytokines they produce. To determine the states that Teff cells actually adopt in frontline tissues in vivo, we applied single-cell transcriptome and chromatin analyses to colonic Teff cells in germ-free or conventional mice or in mice after challenge with a range of phenotypically biasing microbes. Unexpected subsets were marked by the expression of the interferon (IFN) signature or myeloid-specific transcripts, but transcriptome or chromatin structure could not resolve discrete clusters fitting classic helper T cell (TH) subsets. At baseline or at different times of infection, transcripts encoding cytokines or proteins commonly used as TH markers were distributed in a polarized continuum, which was functionally validated. Clones derived from single progenitors gave rise to both IFN-γ- and interleukin (IL)-17-producing cells. Most of the transcriptional variance was tied to the infecting agent, independent of the cytokines produced, and chromatin variance primarily reflected activities of activator protein (AP)-1 and IFN-regulatory factor (IRF) transcription factor (TF) families, not the canonical subset master regulators T-bet, GATA3 or RORγ.

Deconvoluting the diversity of within-host pathogen strains in a multi-locus sequence typing framework.

BACKGROUND: Bacterial pathogens exhibit an impressive amount of genomic diversity. This diversity can be informative of evolutionary adaptations, host-pathogen interactions, and disease transmission patterns. However, capturing this diversity directly from biological samples is challenging. RESULTS: We introduce a framework for understanding the within-host diversity of a pathogen using multi-locus sequence types (MLST) from whole-genome sequencing (WGS) data. Our approach consists of two stages. First we process each sample individually by assigning it, for each locus in the MLST scheme, a set of alleles and a proportion for each allele. Next, we associate to each sample a set of strain types using the alleles and the strain proportions obtained in the first step. We achieve this by using the smallest possible number of previously unobserved strains across all samples, while using those unobserved strains which are as close to the observed ones as possible, at the same time respecting the allele proportions as closely as possible. We solve both problems using mixed integer linear programming (MILP). Our method performs accurately on simulated data and generates results on a real data set of Borrelia burgdorferi genomes suggesting a high level of diversity for this pathogen. CONCLUSIONS: Our approach can apply to any bacterial pathogen with an MLST scheme, even though we developed it with Borrelia burgdorferi, the etiological agent of Lyme disease, in mind. Our work paves the way for robust strain typing in the presence of within-host heterogeneity, overcoming an essential challenge currently not addressed by any existing methodology for pathogen genomics.

The impact of similarity metrics on cell-type clustering in highly multiplexed in situ imaging cytometry data.

Motivation: The advent of highly multiplexed in situ imaging cytometry assays has revolutionized the study of cellular systems, offering unparalleled detail in observing cellular activities and characteristics. These assays provide comprehensive insights by concurrently profiling the spatial distribution and molecular features of numerous cells. In navigating this complex data landscape, unsupervised machine learning techniques, particularly clustering algorithms, have become essential tools. They enable the identification and categorization of cell types and subsets based on their molecular characteristics. Despite their widespread adoption, most clustering algorithms in use were initially developed for cell suspension technologies, leading to a potential mismatch in application. There is a critical gap in the systematic evaluation of these methods, particularly in determining the properties that make them optimal for in situ imaging assays. Addressing this gap is vital for ensuring accurate, reliable analyses and fostering advancements in cellular biology research. Results: In our extensive investigation, we evaluated a range of similarity metrics, which are crucial in determining the relationships between cells during the clustering process. Our findings reveal substantial variations in clustering performance, contingent on the similarity metric employed. These variations underscore the importance of selecting appropriate metrics to ensure accurate cell type and subset identification. In response to these challenges, we introduce FuseSOM, a novel ensemble clustering algorithm that integrates hierarchical multiview learning of similarity metrics with self-organizing maps. Through a rigorous stratified subsampling analysis framework, we demonstrate that FuseSOM outperforms existing best-practice clustering methods specifically tailored for in situ imaging cytometry data. Our work not only provides critical insights into the performance of clustering algorithms in this novel context but also offers a robust solution, paving the way for more accurate and reliable in situ imaging cytometry data analysis. Availability and implementation: The FuseSOM R package is available on Bioconductor and is available under the GPL-3 license. All the codes for the analysis performed can be found at Github.

Atlas-scale single-cell multi-sample multi-condition data integration using scMerge2.

The recent emergence of multi-sample multi-condition single-cell multi-cohort studies allows researchers to investigate different cell states. The effective integration of multiple large-cohort studies promises biological insights into cells under different conditions that individual studies cannot provide. Here, we present scMerge2, a scalable algorithm that allows data integration of atlas-scale multi-sample multi-condition single-cell studies. We have generalized scMerge2 to enable the merging of millions of cells from single-cell studies generated by various single-cell technologies. Using a large COVID-19 data collection with over five million cells from 1000+ individuals, we demonstrate that scMerge2 enables multi-sample multi-condition scRNA-seq data integration from multiple cohorts and reveals signatures derived from cell-type expression that are more accurate in discriminating disease progression. Further, we demonstrate that scMerge2 can remove dataset variability in CyTOF, imaging mass cytometry and CITE-seq experiments, demonstrating its applicability to a broad spectrum of single-cell profiling technologies.