Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells
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PROVIDER: PRJNA682551 | ENA |
REPOSITORIES: ENA
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Project description:Type 1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T cells, although the genetic program driving human cDC1 specification remains largely unexplored. We previously identified PU.1, IRF8, and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts, but reprogramming of human somatic cells was limited by low efficiency. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDC1s) generated from embryonic fibroblasts gradually acquired a global cDC1 transcriptional profile and expressed antigen presentation signatures, whereas other DC subsets were not induced at the single-cell level during the reprogramming process. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β, and TNF-α with constitutive expression of cDC1-inducing transcription factors led to improvement of reprogramming efficiency by 190-fold. hiDC1s engulfed dead cells, secreted inflammatory cytokines, and performed antigen cross-presentation, key cDC1 functions. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 showed dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters led to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.
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Project description:Type-1 conventional dendritic cells (cDC1s) are rare immune cells critical for the induction of antigen-specific cytotoxic CD8+ T-cells. The genetic program driving human cDC1 specification remains largely unexplored. We have previously identified PU.1, IRF8 and BATF3 transcription factors as sufficient to induce cDC1 fate in mouse fibroblasts but reprogramming of human somatic cells was limited by low efficiencies. Here, we investigated single-cell transcriptional dynamics during human cDC1 reprogramming. Human induced cDC1s (hiDCs) generated from embryonic fibroblasts gradually acquire a global cDC1 transcriptional profile and activate antigen presentation signatures. Importantly, other DC subsets are not induced at the single-cell level. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the cDC1-inducing transcription factor network as key drivers of the process. Combining IFN-γ, IFN-β and TNF-α with constitutive expression of cDC1-inducing transcription factors lead to improvement of reprogramming efficiency by 190-fold. hiDCs uptake dead cells, secrete inflammatory cytokines and perform antigen cross-presentation, a key cDC1 function. This approach allowed efficient hiDC1 generation from adult fibroblasts and mesenchymal stromal cells. Mechanistically, PU.1 shows dominant and independent chromatin targeting at early phases of reprogramming, recruiting IRF8 and BATF3 to shared binding sites. The cooperative binding at open enhancers and promoters leads to silencing of fibroblast genes and activation of a cDC1 program. These findings provide mechanistic insights into human cDC1 specification and reprogramming and represent a platform for generating patient-tailored cDC1s, a long-sought DC subset for vaccination strategies in cancer immunotherapy.
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Project description:Type-1 dendritic cells (DC1s) are critical cellular mediators of anti-tumor immunity, as their ability to prime CD8+ T-cells is crucial for response to checkpoint blockade and adoptive T-cell transfer. Vaccination strategies to harness this unique DC subset for immunotherapy have been limited by their rarity in peripheral blood and lack of homogeneous alternative cell sources. We have previously identified PU.1, IRF8 and BATF3 transcription factors (TF) as sufficient to induce DC1 program in mouse fibroblasts but reprogramming efficiency in human cells was low. Here, we investigated single-cell transcriptional dynamics during human DC1 reprogramming. Human induced DC1s (hiDC1s) generated from embryonic fibroblasts acquire global DC1 transcriptional profile and activate antigen presentation signatures. Interestingly, other DC subsets are not induced at the single cell level. We extracted gene modules associated with successful reprogramming and identified inflammatory signaling and the DC1 reprogramming TF network as key drivers of the process. Combining inflammatory cytokine signaling with TF constitutive overexpression lead to improved reprogramming efficiency by 190-fold. HiDC1s acquire the ability to uptake dead cells, respond to stimuli, secrete inflammatory cytokines and perform antigen presentation. Remarkably, intra-tumoral vaccination in mouse models increased infiltration of antigen-specific CD8+ T-cells, promoted a T-cell cytotoxic profile and conferred protection against tumor growth. Finally, we demonstrated efficient hiDC1 generation from human adult somatic cells including dermal fibroblasts and mesenchymal stem cells with a xeno-free protocol. These findings provide insights into human DC1 specification and reprogramming and represent a platform for generating patient-specific DC1s, a long-sought DC subset for cancer immunotherapy.
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