SummaryThe IRE1a-XBP1 pathway, a conserved adaptive mediator of the unfolded protein response, is indispensable for the development of secretory cells. It maintains endoplasmic reticulum homeostasis by facilitating protein folding and enhancing secretory capacity of the cells. Its role in immune cells is emerging. It is involved in dendritic cell, plasma cell and eosinophil development and differentiation. Using genome-wide approaches, integrating ChIPmentation and mRNA-sequencing data, we have elucidated the regulatory circuitry governed by the IRE1a-XBP1 pathway in type-2 T helper cells (Th2). We show that the XBP1 transcription factor is activated by splicing in vivo in T helper cell lineages. We report a comprehensive repertoire of XBP1 target genes in Th2 lymphocytes. We found that the pathway is conserved across cell types in terms of resolving secretory stress, and has T helper cell-specific functions in controlling activation-dependent Th2 cell proliferation and regulating cytokine expression in addition to secretion. These results provide a detailed picture of the regulatory map governed by the XBP1 transcription factor during Th2 lymphocyte activation.

Non-cancerous stromal cells represent a highly diverse compartment of the tumour, yet their role across tumour evolution remains unclear. We employed single-cell RNA sequencing to determine stromal adaptations in murine melanoma at different points of tumour development. Naive lymphocytes recruited from lymph nodes underwent activation and clonal expansion within the tumour, prior to PD1 and Lag3 expression, while tumourassociated myeloid cells promoted the formation of a suppressive niche through cytokine secretion and inhibitory T cell interactions. We identified three temporally distinct cancerassociated fibroblast (CAF) populations displaying unique signatures, and verified these in human datasets. In early tumours, immune CXCL12/CSF1 and complement -expressing CAFs supported recruitment of macrophages, whereas contractile CAFs became more prevalent in later tumours. This study highlights the complex interplay and increasing diversity among cells that co-evolve with the tumour, indicating that from early stages of development, stromal cells acquire the capacity to modulate the immune landscape towards suppression.In particular, extensive heterogeneity has been reported within tumour fibroblast populations.Cancer associated fibroblasts (CAFs) are the most abundant non-immune stromal component, secreting growth factors, promoting angiogenesis, facilitating metastasis and regulating immune infiltrates 9-15 . Although they express typical fibroblast markers, such as fibroblast activation protein (FAP), platelet derived growth factor receptors α (PDGFRα) and β (PDGFRβ), podoplanin (PDPN), Thy-1 and α-smooth muscle actin (αSMA), no single marker universally identifies all CAFs within the tumour stroma 16-18 . To date, many studies rely on positive selection approaches, in which one or two markers are used to isolate CAFs for functional characterisation. Consequently, these findings likely reflect a sub-population of cells and may bias our perceptions of CAF function. It remains unclear whether fibroblast subpopulations with distinct roles are present in the tumour microenvironment.Current approaches lack the resolution to visualise the true extent of stromal heterogeneity and may mask rare populations, or cellular phenotypes, that could be critical for tumour survival. Therefore, we have employed single-cell RNA sequencing (scRNAseq) to profile both immune and non-immune stromal populations from the B16-F10 murine model of melanoma. Furthermore, cells were isolated from both primary tumours and draining lymph nodes, at different stages of tumour development, enabling a systems level interrogation of the melanoma microenvironment in real-time. Here, we identified the presence of a diverse immune landscape, in which the composition and phenotype of leukocytes change as the tumour evolves. In particular, effector T cells displaying signs of dysfunction, were detected predominantly in late stage tumours. This work also highlighted significant heterogeneity within the CAF compartment of the primary tumour. Three distinc...

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

The relationship between single cell RNA sequencing Clustered-Populations (scCPops) and cell surface marker-defined classic T cell subsets remain unclear. Here, we interrogated 6 bead-enriched T cell subsets with 62,235 single cell transcriptomes and re-grouped them into 9 scCPops. Bead-enriched CD4 Naïve and CD8 Naïve were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops. Interestingly, we discovered and named IFNhi T, a new T cell subpopulation that highly expressed Interferon Signaling Associated Genes (ISAGs). We further enriched IFNhi T by FACS sorting. IFNhi T cluster disappeared on tSNE plot after removing ISAGs, while IFNhi T cluster showed up by tSNE analyses of ISAGs alone, indicating ISAGs are the major contributor of IFNhi T cluster. BST2+ T cells and BST2- T cells showing different efficiencies of T cell activation indicates high level of ISAGs may contribute to quick immune responses.

Single-cell genomic analysis has grown rapidly in recent years and will find widespread applications in various fields of biology, including cancer biology, development, immunology, pre-implantation genetic diagnosis, and neurobiology. In this study, we amplified genomic DNA from individual hippocampal neurons using one of three single-cell DNA amplification methods (multiple annealing and looping-based amplification cycles (MALBAC), multiple displacement amplification (MDA), and GenomePlex whole genome amplification (WGA4)). We then systematically evaluated the genome coverage, GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. Chromosome-level and subchromosomal-level copy number variations among individual neurons can be detected.