Background

Dendritic cells (DCs) are professional antigen presenting cells specialized in the recognition, processing and presentation of antigens to T cells, playing a pivotal role in the induction of adaptive immune responses and immunological memory (Merad et al., 2013). DCs can be divided into 4 main subsets: plasmacytoid DCs (pDC), producers of large amounts of type 1 interferons, monocyte-derived DCs, derived from circulating monocytes, and conventional DCs (cDC), a functionally heterogeneous subset further subdivided in conventional DCs type 1 (cDC1) and type 2 (cDC2). While cDC1s excel on priming cytotoxic CD8⁺ T cell responses by cross-presenting exogenous antigens on major histocompatibility complex (MHC) class I, cDC2s are specialized in the presentation of extracellular antigens to CD4⁺ T cells via MHC class II. This functional specialization results from stage-specific interplay between different transcription factors (TFs) that specify DC subsets. For instance, it has been shown that conditional deletion of E26 transformation-specific (ETS) family TF PU.1 in hematopoietic progenitors impacts the specification of the whole DC lineage (Carotta et al., 2010). Interferon regulatory factor 8 (IRF8) knock-out mice lack pDC and cDC1 subsets, and basic leucine zipper ATF-Like transcription factor 3 (BATF3) deficient mice have impaired cDC1 specification (Schiavoni et al., 2002; Hildner et al., 2008; Carotta et al., 2010).

Growing evidence supports the crucial role of cDC1s in initiating immune responses in the context of cancer (Bottcher and Reis, 2018), and their in vivo rarity has propelled attempts to generate cDC1 in vitro (Perez and De Palma, 2019; Wculek et al., 2019). Several strategies have been explored to generate bona fide DCs from blood monocytes or bone marrow (BM) precursors in the presence of different hematopoietic cytokines (Inaba et al., 1992; Lu et al., 1995; Lutz et al., 1999; Son et al., 2002; Naik et al., 2005; Poulin et al., 2010; Proietto et al., 2012; Balan et al., 2014; Mayer et al., 2014; Lee et al., 2015; Balan et al., 2018; Kirkling et al., 2018). However, limiting numbers of BM progenitors, together with the generation of mixed populations of DC subsets with conflicting functions, strongly limits the use of these approaches to elucidate DC development and harness cDC1s for therapy (Yona and Mildner, 2018).

Cell reprogramming offers an exciting opportunity to overcome these challenges. Through enforced expression of TFs, it is possible to reprogram somatic cells into induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Protocols to differentiate DCs from iPSCs have been described (Choi et al., 2009; Senju et al., 2009 and 2011; Horton et al., 2019). However, these rely on complex and long culture conditions that give rise to mixed populations of distinct DC subsets. Alternatively, somatic cells can be reprogrammed directly into other specialized cell types without transiting through pluripotency or multipotency. Mouse and human fibroblasts were directly reprogrammed into several cell types, such as neurons, cardiomyocytes and hematopoietic stem and progenitor cells, using TFs specifying target-cell identity (Pereira et al., 2012 and 2013, Xu et al., 2015).

Here, we describe a direct cell reprogramming approach to generate cDC1-like cells (induced DCs, or iDCs) by enforced expression of the TFs PU.1, IRF8 and BATF3 in mouse embryonic fibroblasts (MEFs) (Rosa et al., 2018). MEF-derived iDCs acquire DC morphology, cDC1 phenotype and transcriptional program characteristic of natural cDC1s. Functionally, iDCs acquire the ability to respond to toll-like receptor 3 (TLR3) and TLR4 stimuli, engulf protein and dead cells and, importantly, cross-present antigens to CD8⁺ T cells. Thus, the protocol described here enables the fast generation (within 9 days) of cDC1-like cells in vitro using a controlled and adaptable direct reprogramming approach suitable for screening studies aiming to better understand cDC1 developmental specification and functional maturation. This approach, coupled with pharmacological inhibition, gene knock-out or knock-down, will enable the discovery of regulators of DC specification acting individually or in combination (Pires et al., 2019). Employing high-content screening methodologies based on recent CRISPR-Cas9 technologies in MEFs paves the way for defining novel critical regulators of cDC1 fate and how cross-presentation ability is established de novo in multiple cell types. In the future, DC reprogramming will serve as a platform to develop immunotherapies for cancer and infectious disease.