Bioinformatic analysis of human dendritic cell development

Abstract

Dendritic cells (DCs) are professional antigen presenting cells of the mammalian immune system, and constitute a vital link between the adaptive and the innate immune systems. These cells are phenotypically and functionally highly heterogeneous, comprising at least 3 subsets in human: classical/conventional DC types 1 and 2 (cDC1, cDC2), and plasmacytoid DC (pDC). Additional heterogeneity has been described within the cDC2 compartment which can be divided into two populations, termed DC2 and DC3, more closely related to cDC1s or monocytes, respectively. DCs develop in the bone marrow (BM) under the control of lineagespecific transcription factors (TFs). However, the cellular pathways and genetic factors that govern the development of human DC subpopulations from haematopoietic stem cells are not well known, in part due to their rarity in vivo. First, this work addressed the rarity of DCs via a novel in vitro culture system that favoured the production of large numbers of DCs from primary human CD34+ stem/progenitor cells. Two transcriptomic approaches were employed to verify the culture output: the NanoString assay and bulk RNA-Seq. The transcriptomic analyses confirmed that all DC subsets produced in culture exhibited appropriate transcription profiles and bore close resemblance their ex vivo-derived counterparts. Furthermore, these methods attested that Notch stimulation predisposed the culture output toward the production of cDC1, the rarest of the DC subsets. The culture system, confirmed to produce bona fide DC subsets, facilitated the interrogation of DC haematopoiesis to establish the phenotypic identities of putative progenitor and precursor populations. These early populations, derived from human BM, along with mature DCs, were subjected to single cell transcriptomics. Pseudotemporal ordering and lineage branching reconstruction analyses revealed two pathways of DC development, marked by differential expression of the TF IRF8 and explaining the origin of cDC2 heterogeneity. The IRF8high pathway generated pDC, cDC1 and DC2, while DC3 and monocytes developed along an IRF8low trajectory. Mass cytometry analysis validated the link between the two pathways in BM and DC populations found in peripheral blood. III Finally, the project focused on determining the role of IRF8 in the homeostasis of human cDC1s and pDCs, both of which develop through the IRF8high pathway and retain IRF8 expression as they mature. The previously established in vitro culture techniques were employed to generate sufficient DCs for low-input IRF8 chromatin immunoprecipitation, followed by high-throughput DNA sequencing (ChIP-Seq). The analysis of the ChIP-Seq data revealed that IRF8 maintains both the function and surface phenotype of cDC1s, while in pDCs it controls important functional modules. During this work, a wide variety of transcriptomic and genomic bioinformatic techniques and analyses enabled the verification of a novel human DC culture system (Kirkling and Cytlak et al., 2018), the identification of two pathways of human DC development (Cytlak and Resteu et al., 2020), and have generated new insights into the role of IRF8 in human DCs