Project description:Conventional dendritic cells (cDC) consist of two functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in mature cDC1 remains unclear. Here we used XCR1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identify of cDC1 but not their survival. In the absence of Irf8, committed cDC1 (“ex-cDC1”) acquired the transcriptional, functional and chromatin accessibility properties of cDC2. This conversion was independent on Irf4 and was associated with decreased accessibility in putative IRF8, Batf3 and composite AP-1-IRF (AICE) binding elements, together with increased accessibility of cDC2 associated transcription factor binding elements. Thus, IRF8 expression by committed cDC1 is required for preventing their conversion into cDC2.
Project description:ChIP-seq analysis was performed in an adult T-cell leukemia/lymphoma cell line (TL-Om1) to analyze DNA bindings of IRF4 in TL-Om1 cells.
Project description:Flt3 ligand (Flt3L) promotes an increased generation of type 1 conventional dendritic cells (cDC1s), resulting in enhanced immunity against infections and cancer. Here, we employ cellular barcoding to understand how Flt3L regulates single haematopoietic stem and progenitor cell (HSPC) fate. Our results demonstrate that although Flt3L stimulation can recruit some additional cDC1-generating HSPCs, the major contributing factor to higher cDC1 numbers is through enhanced clonal expansion. This selective cDC1 expansion occurs primarily via multi-/oligo-potent clones, without compromising their clonal output to other lineages. We then develop Divi-Seq to simultaneously profile division history, surface phenotype and the transcriptional state of single HSPCs during the early phase of the response. We discover that Flt3L-responsive HSPCs maintain a proliferative ‘early progenitor’-like state, which leads to a selective emergence of CD11c+cKit+ transitional precursors with high cellular output to cDC1s. These findings inform the mechanistic action of Flt3L in natural immunity and immunotherapy.
Project description:Conventional dendritic cells (cDCs) are at the forefront of activating the immune system to mount an anti‐tumor immune response. Flt3L is a cytokine required for DC development that can increase DC abundance in the tumor when administered therapeutically. However, the impact of Flt3L on the phenotype of distinct cDC subsets in the tumor microenvironment is still largely undetermined. Here, using multi‐omic single‐cell analysis, we show that Flt3L therapy increases all cDC subsets in orthotopic E0771 triple‐negative breast cancer, but this did not result in a reduction of tumor growth. Interestingly, a CD81+migcDC1 population, likely developing from cDC1, was induced upon Flt3L treatment. This subset is characterized by the expression of both canonical cDC1 markers as well as migratory cDC activation and regulatory markers and displayed a higher Treg‐inducing potential compared to other migcDCs. To shift the cDC phenotype towards a T‐cell stimulatory phenotype, CD40 agonist therapy was administered in combination with Flt3L. However, while αCD40 reduced tumor growth, Flt3L failed to improve the therapeutic response to αCD40 therapy. Interestingly, Flt3L+αCD40 combination therapy increased the abundance of Treg promoting CD81+migcDC1. Nonetheless, while Treg‐depletion and αCD40 therapy were synergistic, the addition of Flt3L to this combination did not result in any added benefit. Overall, these results indicate that merely increasing cDCs in the tumor by Flt3L treatment cannot improve anti‐tumor responses and therefore might not be beneficial for the treatment of triple‐negative breast cancer, though could still be of use to increase cDC numbers for autologous DC‐therapy.
Project description:Conventional Dendritic Cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2 subsets, responsible for priming naïve CD8+ and CD4+ T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300ciCre-hCD2R26tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompass myeloid-derived pre-cDC2 and lymphoid-derived pDC-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared to Ms4a3CreR26tdTomato lineage-traced DC3, a monocyte-DC progenitor-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to a blood-borne antigen. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 cells across tissues. Thus, ontogenesis may impact tissue immune responses.
Project description:Conventional Dendritic Cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2 subsets, responsible for priming naïve CD8+ and CD4+ T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300ciCre-hCD2R26tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompass myeloid-derived pre-cDC2 and lymphoid-derived pDC-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared to Ms4a3CreR26tdTomato lineage-traced DC3, a monocyte-DC progenitor-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to a blood-borne antigen. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 cells across tissues. Thus, ontogenesis may impact tissue immune responses.