Highlights d Human dendritic cell and monocyte subsets show one-toone equivalence in mouse d Neutrophils exhibit tumor-associated phenotypes that are conserved across species d Myeloid subsets in patient blood only partially overlap with those in their tumors d Unique markers define myeloid cell subsets and associate with clinical prognosis

T regulatory cells that express the transcription factor Foxp3 (Foxp3+ Treg) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction, which we find to map to a broad, but specific, array of individual bacterial species, requires the transcription factor Rorγ, paradoxically in that Rorγ is thought to antagonize FoxP3 and promote T helper 17 (Th17) cell differentiation. Rorγ's transcriptional footprint differs in colonic Tregs and Th17 cells, controlling important effector molecules. Rorγ, and the Tregs that express it, contribute substantially to regulating colonic Th1/Th17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell-types.

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

CD4+ T regulatory (Treg) cells are central to immune homeostasis, their phenotypic heterogeneity reflecting the diverse environments and target cells they regulate. To understand this heterogeneity, we combined single-cell RNAseq, activation reporter and TCR analysis to profile thousands of Tregs or Tconvs from mouse lymphoid organs or human blood. Treg and Tconv pools showed areas of overlap, as resting “furtive” Tregs with overall similarity to Tconv, or as a convergence of activated states. All Tregs express a small core of FoxP3-dependent transcripts, onto which additional programs are added less uniformly. Among suppressive functions, Il2ra and Ctla4 were quasi-constant, inhibitory cytokines being more sparsely distributed. TCR signal intensity didn’t affect resting/activated Treg proportions, but molded activated Treg programs. The main lines of Treg heterogeneity in mice were strikingly conserved in human blood. These results reveal unexpected TCR-shaped states of activation, providing a framework to synthesize previous observations about Treg heterogeneity.