Conserved transcriptional connectivity of regulatory T cells in the tumor microenvironment informs new combination cancer therapy strategies.

While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell 'connectivity' to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity.

Dendritic cells (DCs) play a critical role in orchestrating adaptive immune responses due to their unique ability to initiate T cell responses and direct their differentiation into effector lineages. Classical DCs have been divided into two subsets, cDC1 and cDC2, based on phenotypic markers and their distinct abilities to prime CD8 and CD4 T cells. While the transcriptional regulation of the cDC1 subset has been well characterized, cDC2 development and function remain poorly understood. By combining transcriptional and chromatin analyses with genetic reporter expression, we identified two principal cDC2 lineages defined by distinct developmental pathways and transcriptional regulators, including T-bet and RORγt, two key transcription factors known to define innate and adaptive lymphocyte subsets. These novel cDC2 lineages were characterized by distinct metabolic and functional programs. Extending our findings to humans revealed conserved DC heterogeneity and the presence of the newly defined cDC2 subsets in human cancer.

The Innate Lymphoid Cell Precursor.

The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming. Our findings provide insight into the divergent mechanisms of the differentiation of the ILC lineage and LTi cell lineage and establish a high-resolution 'blueprint' of their development.

PLZF expression maps the early stages of ILC1 lineage development.

Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre-pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny. Although a limited set of ILC1 genes depended on PLZF for expression, characteristically including Il7r, most of these genes were also differentially expressed between ILC1s and cNKs, indicating that PLZF together with other, yet to be defined, factors contribute to the divergence between these lineages.