Inducing regulatory dendritic cells with cellular reprogramming — ASN Events
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  2. Poster Session 2
  3. Inducing regulatory dendritic cells with cellular reprogramming

Inducing regulatory dendritic cells with cellular reprogramming (#210)

Abigail R Altman 1 2 , Ilia Kurochkin 1 2 , Katherine Hampton 1 2 , Carlos-Filipe Pereira 1 2 3 4
  1. Molecular Medicine and Gene Therapy, Science for Life Laboratory, Lund Stem Cell Centre, Lund University, Lund, Sweden
  2. Wallenberg Centre for Molecular Medicine, Lund University, Lund, Sweden
  3. Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
  4. Asgard Therapeutics AB, Medicon Village, Lund, Sweden

Dendritic cells (DCs) constitute a heterogeneous lineage that orchestrates immunogenic and tolerogenic adaptive immune responses. Mature DCs enriched in immunoregulatory molecules (mregDCs) promote anti-tumor responses and also suppress autoreactive responses through regulatory T cell (Treg) activation. However, incomplete understanding of mregDC transcription factor (TF) networks has prevented their stable, programmable generation, limiting therapeutic potential. To uncover the transcriptional circuits underlying mregDC identity, we employed REPROcode, a combinatorial screening platform enabling simultaneous detection of cellular transcriptomes and exogenous TF expression. Human fibroblasts transduced with a DC-enriched 48-TF pool were sorted at day 9 for non-reprogrammed (CD45-HLA-DR-) and reprogrammed (CD45+) cells, and profiled by single-cell RNA sequencing, totaling 210,813 cells across three experiments. We identified a cluster of induced mregDC-like cells co-expressing genes related to DC maturation (HLA-DR, CD40, CD80, CCR7, LAMP3, FSCN1, SOCS2), Treg induction and maintenance (CCL22, IDO1, ITGB8, SCUBE3), and immunoregulatory function (PDL1, EPOR, ETV3). Gene set enrichment analysis revealed enrichment of homeostatic gene signatures and downregulation of interferon gene signatures relative to induced immunogenic DC1s, defined by a PU.1, IRF8, and BATF3 TF network. Flow cytometry confirmed expression of maturation (HLA-DR, CD40, CD80/86, CCR7) and immunoregulatory markers (ITGB8, IDO1, LAMP3, ICOSL, PDL2) on fibroblasts reprogrammed with the mregDC TF circuit in vitro. To assess robustness of the mregDC TF circuit, we reprogrammed murine cancer cell lines of distinct tissue origins, including YUMM1.7 (melanoma), MC38 (colon adenocarcinoma), and PANCO2 (pancreatic adenocarcinoma), and confirmed species conservation and maintenance of a regulatory program. Functionally, in vivo reprogramming of YUMM1.7 and MC38 increased intratumoral Treg infiltration compared to induced DC1 and empty-vector controls. Using the proximity labeling FOXP3-LIPSTIC model, in which Tregs biotinylate physically interacting cells, we confirmed direct induced mregDC-Treg interactions in vivo. Collectively, we uncover TF networks governing mregDC identity and provide a framework for induction of immunoregulatory DCs.