Differentiation of multipotent cells is a complex process governed by interactions of thousands of genes subject to substantial expression fluctuations. Resolving cell state heterogeneity arising during this process requires quantification of gene expression within individual cells. However, computational methods linking this heterogeneity to biases towards distinct cell fates are not well established. Here, we perform deep single-cell transcriptome sequencing of ~2,000 bone-marrow derived mouse hematopoietic progenitors enriched for lymphoid lineages. To resolve subtle transcriptome priming indicative of distinct lineage biases, we developed FateID, an iterative supervised learning algorithm for the probabilistic quantification of cell fate bias. FateID delineates domains of fate bias within progenitor populations and permits the derivation of high-resolution differentiation trajectories, revealing a common progenitor population of B cells and plasmacytoid dendritic cells, which we validated by in vitro differentiation assays. We expect that FateID will enhance our understanding of the process of cell fate choice in complex multi-lineage differentiation systems. INTRODUCTIONRecent studies utilizing scRNA-seq 1-5 and single-cell lineage tracing techniques 6-8 , call into question the traditional view of hematopoietic differentiation as a sequence of binary fate choices giving rise to a succession of increasingly fate restricted progenitor types 9 . Evidence from these studies rather suggests early cell fate priming starting at the level of multipotent progenitors (MPP) or even within the HSC pool. Pronounced heterogeneity of common myeloid progenitors (CMP) was elucidated with high resolution 1 , and an early fate bias emerging in human short term HSCs was suggested in a more recent study 2 . However, heterogeneity of lymphoid progenitors has not been well investigated with single-cell resolution. Since lymphoid progenitor heterogeneity was previously found by flow cytometry 10,11 , utilizing distinct sets of cell surface markers in combination with differentiation assays, we here perform an scRNA-seq analysis to comprehensively elucidate heterogeneity across lymphoid progenitors purified from the bone-marrow of adult mice. Although a number of methods for lineage reconstruction have been developed [12][13][14][15] , these algorithms are not specifically designed to uncover subtle transcriptome changes and uniquely assign cells to individual branches without accounting for multi-lineage bias. Weak transcriptome modulations also remain undiscovered by state-of-the-art clustering methods, which partition cells into groups without accounting for the co-existence of fate bias towards multiple lineages within individual cells. To elucidate the process of cell fate emergence, we introduce FateID, a computational method for the quantification of fate bias, manifested by subtle lineage specific transcriptome modulations within a multipotent progenitor . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the aut...

Keywords 20Gastrulation, mouse embryo, Eomes, definitive endoderm, mesoderm, lineage 21 specification 22 Summary statement 24 Cells lineages are specified in the mouse embryo already within the primitive streak 25 where Mesp1+ mesoderm and Foxa2+ endoderm are generated in a spatial and 26 temporal sequence from unbiased progenitors. Abstract 29 Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the 30 earliest embryonic cell types that are specified during germ layer formation at the 31 primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both 32 lineages segregate from Eomes expressing progenitors in response to different 33 NODAL signaling levels. However, the precise spatiotemporal pattern of the 34 emergence of these cell types and molecular details of lineage segregation remain 35 unexplored. We combined genetic fate labeling and imaging approaches with scRNA-36 seq to follow the transcriptional identities and define lineage trajectories of Eomes 37 dependent cell types. All cells moving through the PS during the first day of 38 gastrulation express Eomes. AM and DE specification occurs before cells leave the 39 PS from discrete progenitor populations that are generated in distinct spatiotemporal 40 patterns. Importantly, we don't find evidence for the existence of progenitors that co-41 express markers of both cell lineages suggesting an immediate and complete 42 separation of AM and DE lineages.43 44 48 embryo under the influence of elevated levels of the instructive signals of 49 TGFß/NODAL, WNT and FGF. These signals induce an epithelial-to-mesenchymal 50 transition (EMT) of epiblast cells at the primitive streak (PS) leading to their 51 delamination and the formation of the mesoderm and DE cell layer. The nascent 52 mesoderm layer rapidly extends towards the anterior embryonic pole by cell migration 53 between the epiblast and the visceral endoderm (VE) (reviewed by (Arnold and 54 Robertson 2009; Rivera-Pérez et al. 2003)). DE progenitors migrate from the epiblast 55 together with mesoderm cells, before they eventually egress into the VE layer to 56 constitute the DE (reviewed by (Rivera-Pérez and Hadjantonakis 2014; Viotti, Foley, 57 et al. 2014)). 58 Current concepts suggest that different cell fates are specified according to the time 59 and position of cell ingression through the PS reflecting different instructive signaling 60 environments (Rivera-Pérez and Hadjantonakis 2014). However, the precise 61 morphogenetic mechanisms guiding the emergence of various cell types along the 62 PS still remain uncertain. This is at least in parts due to the lack of detailed knowledge 63 about the precise timing and location of individual cells becoming lineage specified, 64 and the challenge to exactly determine the signaling pathway activities during fate 65 commitment. 66 Clonal cell labeling and transplantation experiments have proposed the gross patterns 67 and dynamics of cell specification during gastrulation, which have been represented 68 in fate maps...

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SARS-CoV-2 spike mRNA vaccines mediate protection from severe disease as early as 10 days post prime vaccination, when specific antibodies are hardly detectable and still lack neutralizing activity. Vaccine-induced T cells, especially CD8+ T cells, may thus be the main mediators of protection at this early stage. The details of antigen-specific CD8+ T cell induction after prime/boost vaccination, their comparison to naturally induced CD8+ T cell responses and their association with other arms of vaccine-induced adaptive immunity remain, however, incompletely understood. Here, we show on a single epitope level that both, a stable memory precursor pool of spike-specific CD8+ T cells and fully functional spike-specific effector CD8+ T cell populations, are vigorously mobilized as early as one week after prime vaccination when CD4+ T cell and spike-specific antibody responses are still weak and neutralizing antibodies are lacking. Boost vaccination after 3 weeks induced a full-fledged recall expansion generating highly differentiated CD8+ effector T cells, however, neither the functional capacity nor the memory precursor T cell pool was affected. Compared to natural infection, vaccine-induced early memory T cells exhibited similar frequencies and functional capacities but a different subset distribution dominated by effector memory T cells at the expense of self-renewing and multipotent central memory T cells. Our results indicate that spike-specific CD8+ T cells may represent the major correlate of early protection after SARS-CoV-2 mRNA/bnt162b2 prime vaccination that precede other effector arms of vaccine-induced adaptive immunity and are stably maintained after boost vaccination.

Recent studies have established gd T cells as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases and tissue homeostasis. However, differentiation of gd T cells in the adult thymus remains poorly understood, due to the rare frequency of this lineage. Here, we infer high-resolution developmental trajectories of this rare population by single-cell RNA-sequencing. We reveal previously unknown subtypes and identify the transcription factor c-MAF as a novel key regulator of IL-17-producing gd T cell (gdT17) differentiation. c-MAF knockout mice exhibit a complete block in gdT17 differentiation, absence of these cells from peripheral organs, and protection from an autoimmune phenotype in a psoriasis model. Single-cell RNA-sequencing of Sox13 and Rorc knockout mice pinpoints c-MAF as an essential missing link between these lineage-specifying factors. These findings significantly enhance our understanding of gd T cell ontogeny. Our experimental strategy provides a blueprint for deciphering differentiation of rare cell types.