ABSTRACT
Microglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease.
Subject(s)
Germ-Free Life , Microbiota , Microglia/cytology , Prenatal Exposure Delayed Effects/microbiology , Transcriptome , Animals , Brain/cytology , Brain/embryology , Brain/metabolism , Cell Differentiation , Cells, Cultured , Chromatin Assembly and Disassembly , Female , Humans , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Pregnancy , Sex FactorsABSTRACT
BACKGROUND: Casitas B lymphoma-b (Cbl-b) is a central negative regulator of cytotoxic T and natural killer (NK) cells and functions as an intracellular checkpoint in cancer. In particular, Th9 cells support mast cell activation, promote dendritic cell recruitment, enhance the cytolytic function of cytotoxic T lymphocytes and NK cells, and directly kill tumor cells, thereby contributing to tumor immunity. However, the role of Cbl-b in the differentiation and antitumor function of Th9 cells is not sufficiently resolved. METHODS: Using Cblb-/- mice, we investigated the effect of knocking out Cblb on the differentiation process and function of different T helper cell subsets, focusing on regulatory T cell (Treg) and Th9 cells. We applied single-cell RNA (scRNA) sequencing of in vitro differentiated Th9 cells to understand how Cbl-b shapes the transcriptome and regulates the differentiation and function of Th9 cells. We transferred tumor-model antigen-specific Cblb-/- Th9 cells into melanoma-bearing mice and assessed tumor control in vivo. In addition, we blocked interleukin (IL)-9 in melanoma cell-exposed Cblb-/- mice to investigate the role of IL-9 in tumor immunity. RESULTS: Here, we provide experimental evidence that Cbl-b acts as a rheostat favoring Tregs at the expense of Th9 cell differentiation. Cblb-/- Th9 cells exert superior antitumor activity leading to improved melanoma control in vivo. Accordingly, blocking IL-9 in melanoma cell-exposed Cblb-/- mice reversed their tumor rejection phenotype. Furthermore, scRNA sequencing of in vitro differentiated Th9 cells from naïve T cells isolated from wildtype and Cblb-/- animals revealed a transcriptomic basis for increased Th9 cell differentiation. CONCLUSION: We established IL-9 and Th9 cells as key antitumor executers in Cblb-/- animals. This knowledge may be helpful for the future improvement of adoptive T cell therapies in cancer.