Bioinformatic analysis of human dendritic cell development

Ph. D. ThesisDendritic 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 DC

Ikaros family zinc finger 1 regulates dendritic cell development and function in humans

Ikaros family zinc finger 1 (IKZF1) is a haematopoietic transcription factor required for mammalian B-cell development. IKZF1 deficiency also reduces plasmacytoid dendritic cell (pDC) numbers in mice, but its effects on human DC development are unknown. Here we show that heterozygous mutation of IKZF1 in human decreases pDC numbers and expands conventional DC1 (cDC1). Lenalidomide, a drug that induces proteosomal degradation of IKZF1, also decreases pDC numbers in vivo, and reduces the ratio of pDC/cDC1 differentiated from progenitor cells in vitro in a dose-dependent manner. In addition, non-classical monocytes are reduced by IKZF1 deficiency in vivo. DC and monocytes from patients with IKZF1 deficiency or lenalidomide-treated cultures secrete less IFN-alpha, TNF and IL-12. These results indicate that human DC development and function are regulated by IKZF1, providing further insights into the consequences of IKZF1 mutation on immune function and the mechanism of immunomodulation by lenalidomide

Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+ SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using highdimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+ DC) heterogeneity originates from two distinct pathways of development. The lymphoidprimed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+ SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency

MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology

Abstract Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material

MicroRNA profiling of low concentration extracellular vesicle RNA utilizing NanoString nCounter technology

Extracellular vesicles (EV) and the microRNAs that they contain are increasingly recognised as a rich source of informative biomarkers, reflecting pathological processes and fundamental biological pathways and responses. Their presence in biofluids makes them particularly attractive for biomarker identification. However, a frequent caveat in relation to clinical studies is low abundance of EV RNA content. In this study, we used NanoString nCounter technology to assess the microRNA profiles of n = 64 EV low concentration RNA samples (180–49125 pg), isolated from serum and cell culture media using precipitation reagent or sequential ultracentrifugation. Data was subjected to robust quality control parameters based on three levels of limit of detection stringency, and differential microRNA expression analysis was performed between biological subgroups. We report that RNA concentrations > 100 times lower than the current NanoString recommendations can be successfully profiled using nCounter microRNA assays, demonstrating acceptable output ranges for imaging parameters, binding density, positive/negative controls, ligation controls and normalisation quality control. Furthermore, despite low levels of input RNA, high‐level differential expression analysis between biological subgroups identified microRNAs of biological relevance. Our results demonstrate that NanoString nCounter technology offers a sensitive approach for the detection and profiling of low abundance EV‐derived microRNA, and may provide a solution for research studies that focus on limited sample material