Date of Award
Doctor of Philosophy (PhD)
Dendritic cells (DCs) are a group of immune cells that include both classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). cDCs are further comprised of two distinct subsets, cDC1s and cDC2s, which play critical roles in the initiation of innate and adaptive immune responses. Understanding how these lineages develop and function is therefore paramount. All DCs require the receptor tyrosine kinase Flt3 and its ligand Flt3L for their development, but the loss of Flt3L in mice leads to a more severe DC deficiency than does the loss of Flt3. This has led to speculation that Flt3L can bind to an alternate receptor that also supports DC development. However, we found that Flt3L administration to Flt3-/- mice does not generate DCs, arguing against a second receptor. Instead, Flt3-/- DC progenitors matured in response to macrophage colony-stimulating factor (M-CSF) or stem cell factor, and deletion of Csf1r in Flt3-/- mice further reduced DC development, indicating that these cytokines could compensate for Flt3. Surprisingly, Flt3-/- DC progenitors displayed enhanced M-CSF signaling, suggesting that loss of Flt3 increased responsiveness to other cytokines. In agreement, deletion of Flt3 in Flt3l-/- mice paradoxically rescued their severe DC deficiency. We therefore conclude that cytokines play a supportive role in DC development, and that the discrepancy between Flt3-/- and Flt3l-/- mice results from the increased sensitivity of Flt3-/- progenitors to these supportive cytokines. The specification of immune lineages such as DCs during hematopoiesis is dependent upon the precise induction of lineage-determining transcription factors in multipotent progenitors, such as the induction of Irf8 in the common dendritic cell progenitor (CDP) that drives cDC1 development. To uncover the mechanisms regulating this induction, we identified Irf8 enhancers via chromatin profiling of DCs and used CRISPR/Cas9 genome editing to test their functions in vivo. A +32 kb Irf8 enhancer active in mature cDC1s was required for the development and anti-tumor functions of this lineage, but was not required for its specification. To identify enhancers controlling cDC1 specification, we performed ATAC-seq on DC progenitors. Unexpectedly, a +41 kb Irf8 enhancer, which binds E2A and is active in pDCs, was transiently accessible in cDC1 progenitors but not in mature cDC1s. Deleting this enhancer reduced Irf8 expression in pDCs, as expected, but also surprisingly prevented Irf8 induction in CDPs and completely abolished cDC1 specification. We thus found that cryptic activation of the +41 kb Irf8 enhancer was responsible for specifying cDC1 fate. Collectively, our studies have provided insight into the transcriptional mechanisms regulating the development of DCs. Manipulation of these mechanisms in humans may prove useful in enhancing cytotoxic immune responses against viruses and cancer.
Chair and Committee
Kenneth M. Murphy
Brian T. Edelson, Takeshi Egawa, Daved H. Fremont, Chyi S. Hsieh,
Durai, Vivek, "Dendritic Cell Development and Function" (2020). Arts & Sciences Electronic Theses and Dissertations. 2179.