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Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (T reg ) cell development 1 – 4 . Within weeks of birth, a separate wave of T reg cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota 5 – 8 , yet the cell types responsible for the generation of peripheral T reg (pT reg ) cells have not been identified. Here we describe the identification of a class of RORγt + antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I–TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pT reg cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pT reg generation, including the TGF-β-activating integrin αvβ8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pT reg differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt + group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pT reg generation, further implicating TC IV as the tolerogenic RORγt + antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes. Single-cell transcriptomic and epigenetic analysis has enabled the identification of Thetis cells, a class of RORγt + antigen-presenting cells with a key role in the differentiation of commensal microbiota-induced peripheral regulatory T cells.

Despite infiltrating immune cells having an essential function in human disease and patients’ responses to treatments, mechanisms influencing variability in infiltration patterns remain unclear. Here, using bulk RNA-seq data from 46 tissues in the Genotype-Tissue Expression project, we apply cell-type deconvolution algorithms to evaluate the immune landscape across the healthy human body. We discover that 49 of 189 infiltration-related phenotypes are associated with either age or sex ( FDR  < 0.1). Genetic analyses further show that 31 infiltration-related phenotypes have genome-wide significant associations (iQTLs) ( P  < 5.0 × 10 −8 ), with a significant enrichment of same-tissue expression quantitative trait loci in suggested iQTLs ( P  < 10 −5 ). Furthermore, we find an association between helper T cell content in thyroid tissue and a COMMD3 / DNAJC1 regulatory variant ( P  = 7.5 × 10 −10 ), which is associated with thyroiditis in other cohorts. Together, our results identify key factors influencing inter-individual variability of immune infiltration, to provide insights on potential therapeutic targets. Immune infiltration provides critical information for health and disease, yet it is unclear what factors influence infiltration levels. Here, the authors analyze human tissue transcriptomes from the Genotype-Tissue Expression project to find infiltration patterns regulated by age, sex and host genetic information.

Standard scATAC sequencing (scATAC-seq) analysis pipelines represent cells as sparse numeric vectors relative to an atlas of peaks or genomic tiles and consequently ignore genomic sequence information at accessible loci. Here we present CellSpace, an efficient and scalable sequence-informed embedding algorithm for scATAC-seq that learns a mapping of DNA k -mers and cells to the same space, to address this limitation. We show that CellSpace captures meaningful latent structure in scATAC-seq datasets, including cell subpopulations and developmental hierarchies, and can score transcription factor activities in single cells based on proximity to binding motifs embedded in the same space. Importantly, CellSpace implicitly mitigates batch effects arising from multiple samples, donors or assays, even when individual datasets are processed relative to different peak atlases. Thus, CellSpace provides a powerful tool for integrating and interpreting large-scale scATAC-seq compendia. By learning to embed DNA k -mers and cells into a joint space, CellSpace improves single-cell ATAC-seq analysis in multiple tasks such as latent structure discovery, transcription factor activity inference and batch effect mitigation.

Single-cell next-generation sequencing has revolutionized our understanding of cellular heterogeneity and functional genomics. Single-cell RNA sequencing (scRNA-seq), which quantifies gene expression levels, and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq), which profiles genome-wide chromatin accessibility, are the most popular assays of transcriptional and epigenetic variability within individual cells, respectively.Here, I present recent technological advancements, as well as best practices and state-of-the-art methods of data analysis for scRNA-seq and scATAC-seq; particularly, methods for reconstructing cell differentiation trajectories mapping the continuous epigenetic landscape during development. The work in my Ph.D. thesis showcases the applications of single-cell analysis in studying cellular heterogeneity and developmental processes.First, I present CellSpace, a sequence-informed algorithm for scATAC-seq data analysis that learns a shared latent space embedding for cells and DNA k-mers, and I evaluate CellSpace in recovering meaningful biological structure and developmental trajectories, computing accurate transcription factor motif scores, scaling to large datasets, mitigating batch effects while conserving biological heterogeneity, and learning a shared embedding for datasets with different peak atlases.

The pancreas and liver arise from a common pool of progenitors. However, the underlying mechanisms that drive their lineage diversification from the foregut endoderm are not fully understood. To tackle this question, we undertook a multifactorial approach that integrated human pluripotent-stem-cell-guided differentiation, genome-scale CRISPR–Cas9 screening, single-cell analysis, genomics and proteomics. We discovered that HHEX, a transcription factor (TF) widely recognized as a key regulator of liver development, acts as a gatekeeper of pancreatic lineage specification. HHEX deletion impaired pancreatic commitment and unleashed an unexpected degree of cellular plasticity towards the liver and duodenum fates. Mechanistically, HHEX cooperates with the pioneer TFs FOXA1, FOXA2 and GATA4, shared by both pancreas and liver differentiation programmes, to promote pancreas commitment, and this cooperation restrains the shared TFs from activating alternative lineages. These findings provide a generalizable model for how gatekeeper TFs like HHEX orchestrate lineage commitment and plasticity restriction in broad developmental contexts. Yang et al. report that HHEX acts as a gatekeeper of pancreatic lineage specification against the liver and duodenum fates via guiding FOXA1, FOXA2 and GATA4 to activate pancreatic genes, while restraining them from activating alternative lineages.

Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (T ) cell development . Within weeks of birth, a separate wave of T cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota , yet the cell types responsible for the generation of peripheral T (pT ) cells have not been identified. Here we describe the identification of a class of RORγt antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I-TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pT cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pT generation, including the TGF-β-activating integrin αvβ8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pT differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pT generation, further implicating TC IV as the tolerogenic RORγt antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.

The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This \"push and pull\" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease.The mechanisms underlying the ability of embryonic stem cells (ESCs) to rapidly activate lineage-specific genes during differentiation remain largely unknown. Through multiple CRISPR-activation screens, we discovered human ESCs have pre-established transcriptionally competent chromatin regions (CCRs) that support lineage-specific gene expression at levels comparable to differentiated cells. CCRs reside in the same topological domains as their target genes. They lack typical enhancer-associated histone modifications but show enriched occupancy of pluripotent transcription factors, DNA demethylation factors, and histone deacetylases. TET1 and QSER1 protect CCRs from excessive DNA methylation, while HDAC1 family members prevent premature activation. This \"push and pull\" feature resembles bivalent domains at developmental gene promoters but involves distinct molecular mechanisms. Our study provides new insights into pluripotency regulation and cellular plasticity in development and disease.We report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.One sentence summaryWe report a class of distal regulatory regions distinct from enhancers that confer human embryonic stem cells with the competence to rapidly activate the expression of lineage-specific genes.

Standard scATAC-seq analysis pipelines represent cells as sparse numeric vectors relative to an atlas of peaks or genomic tiles and consequently ignore genomic sequence information at accessible loci. We present CellSpace, an efficient and scalable sequence-informed embedding algorithm for scATAC-seq that learns a mapping of DNA k-mers and cells to the same space. CellSpace captures meaningful latent structure in scATAC-seq datasets, including cell subpopulations and developmental hierarchies, and scores the activity of transcription factors in single cells based on proximity to binding motifs embedded in the same space. Importantly, CellSpace implicitly mitigates batch effects arising from multiple samples, donors, or assays, even when individual datasets are processed relative to different peak atlases. Thus, CellSpace provides a powerful tool for integrating and interpreting large-scale scATAC-seq compendia.Competing Interest StatementThe authors have declared no competing interest.Footnotes* - A systematic method comparison with both sequence-aware and -unaware methods for analysis of scATAC-seq data (chromVAR, ArchR itLSI, scBasset, SIMBA, PeakVI) on multiple data sets using quantitative metrics for assessing batch correction and clustering. - Additional comparison with SIMBA, the most closely related alternative embedding method, explaining how CellSpace's representational and algorithmic choices lead to an improvement over SIMBA for batch mitigation, and showing that the SIMBA embedding does not yield meaningful transcription factor (TF) motif activities. - A demonstration that CellSpace can scale to very large data sets via application to a ~720K cell scATAC-seq data set from Domcke et al., Science 2020. - A proof-of-principle demonstration that CellSpace's k-mer embedding can be used for de novo TF motif discovery, showing that the approach could potentially yield novel biological insights.* https://github.com/zakieh-tayyebi/CellSpace

Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. Within weeks of birth, a separate wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new class of tolerogenic RORγt+Aire+ antigen-presenting cells (APC), dubbed Thetis cells (TCs), with a hybrid dendritic cell (DC)-mTEC phenotype, comprising 4 major sub-groups (TC I-IV). We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While TC I and III expressed the signature mTEC nuclear factor Aire, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, TC IV were specifically enriched for critical molecules required for pTreg generation, including the TGF-β activating integrin αvβ8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical DCs was was neither sufficient nor required for pTreg generation, further implicating TCs as the tolerogenic RORγt+ APC with an essential early life function. Our studies reveal parallel pathways for establishment of tolerance within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs. Competing Interest Statement M.vdB. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he has received royalties from Wolters Kluwer; has consulted, received honorarium from or participated in advisory boards for Seres Therapeutics, WindMIL Therapeutics, Rheos Medicines, Merck & Co, Inc., Magenta Therapeutics, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Forty Seven Inc., Priothera, Ceramedix, Lygenesis, Pluto Therapeutics, GlaskoSmithKline, Da Volterra, Vor BioPharma, Novartis (Spouse), Synthekine (Spouse), and Beigene (Spouse); he has IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). A.Y.R. is a member of SAB and has equity in Surface Oncology, RAPT Therapeutics, and holds an IP licensed to Takeda, which is not related to the content of this study. Footnotes * New data with revised figures.