Single-cell transcriptional profiling informs efficient reprogramming of human somatic cells to cross-presenting dendritic cells

Abstract

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.

Single-cell analysis of myeloid cells in HPV+tonsillar cancer

“…Here we identified 7 DC populations corresponding to 4 different lineages and diverse cell states. We observed that the cDC1 cluster was consistently homogeneous through tissue types, and its identity was dictated by the IRF8 and STAT2 regulons, as described previously (48, 49). Interestingly, cDC1s also featured high regulon activity and expression of SMARCA5 .…”

Epigenetics, Enhancer Function and 3D Chromatin Organization in Reprogramming to Pluripotency

“…For instance, direct reprogramming of fibroblasts to neurons requires the transcription factors ASCL1, BRN2 and Myt1l [144], while hemogenic reprogramming depends on GATA2, GFI1B and FOS expression [145]. Efficient reprogramming of somatic cells to hepatocytes [146] and cross-presenting dendritic cells (cDC1s) [147] has also been described. Further studies will elucidate which specific reprogramming machineries are required to induce additional cell identities.…”

The Human Dendritic Cell Atlas: An integrated transcriptional tool to study human dendritic cell biology

“…Given the importance of DCs in immunity, and their distinctive phenotype and functions compared to other myeloid components including macrophages and monocytes, the lack of laboratory models of these cells is becoming a critical bottleneck for the field. The current in vitro models of human DC biology rely on the differentiation of CD34+ hematopoietic stem cells (HSC) from blood or bone marrow (Balan et al, 2014), are differentiated in vitro from peripheral blood monocytes (Pacis et al, 2015), or use induced pluripotent stem cells (iPSC)(Monkley et al, 2020) or directed differentiation from fibroblasts (Rosa et al, 2018, 2022). Concordance between cord-blood derived cDC1 with their in vivo blood counterparts was previously reported (Balan et al, 2018).…”