Defining Transcriptional Networks Underlying Dendritic Cell Heterogeneity Using Direct Cell Reprogramming

September 24, 2018
MSc. Thesis - University of Coimbra

Cláudia de Jesus Azenha

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Cellular reprogramming strategies have highlighted the flexibility of cell fates with the possibility to use cell-type-specific transcription factors (TFs) to convert somatic cells into pluripotency. Direct lineage conversions of one differentiated cell-type into another have also been demonstrated and explored for elucidating cell biology mechanisms and for regenerative medicine purposes. Recently, we have demonstrated that antigen presenting Dendritic cells (DCs) can be reprogrammed into unrelated cell-types by a small combination of TFs. Classically, it is thought that a myeloid DC committed progenitor gives rise to the functionally different DC subsets: conventional DCs (cDCs) are professional Antigen Presenting Cells (APC) driving antigen-specific immune responses; plasmacytoid DCs (pDCs) are professional producers of type I interferons during viral infection. However, the timing and exact mechanisms regulating the divergence of the different subsets during DC development is still to be established.
We have recently identified Irf8, Pu.1 and Batf3 as sufficient and necessary to induce a cDC type 1 fate in fibroblasts. Given the important role of TFs in cell-fate decisions of the different DC subsets, the aim of this study is to investigate DC heterogeneity by fine-tuning the minimal TF network necessary to induce DC fate in order to program pDCs from fibroblasts.
By combining literature review and computational analysis, we have identified 23 pDC-inducing candidate TFs with important roles in pDC specification and restricted pDC expression. We have then validated that in Clec9a reporter mouse, pDCs are labelled with tdTomato fluorescent protein making this model suitable for screening pDC-inducing factor. Then we have transduced Clec9a reporter mouse embryonic fibroblasts (MEFs) with a set of the pDC-inducing TFs using a doxycycline-induced lentiviral system. By sequential individual elimination of each TF, we have identified Irf8 and TF2 as the minimal combination required for reporter activation. Major
histocompatibility complex (MHC) class II molecules’ expression important for DC functionality was also observed to be dependent of Irf8 and TF2. Moreover, our study highlighted the role of TF1 for pDC specification. Whilst not being intrinsically required, when combined with Irf8 and TF2, TF1 increases the expression of pDC- typical surface markers with the generation of tdTomato+ B220+ Bst2+ pDC-like cells by direct reprogramming.
In summary, we provide evidence that Irf8 when combined with TF2 and TF1 kicks-start a pDC program in fibroblasts. These findings provide valuable insights into pDC specification. In the future, the generation of pDCs by direct reprogramming opens avenues for inducing anti-viral immune responses with autologous-engineered cells.