Date of Award

Spring 5-15-2015

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Dendritic cells (DCs) orchestrate immune responses to foreign and self proteins by capturing, processing and presenting antigens to naïve CD4+ and CD8+ T cells in specialized regions of lymphoid organs. Consequently, DCs function in many disparate infectious settings, during which their activation can result in both pathogenic and protective responses. The diverse properties of DCs manifest through the actions of a limited set of lineage-specifying DNA-binding proteins called transcription factors (TFs). The precise molecular program controlled by these developmentally important TFs (such as Irf8 and Batf3) is largely unknown because most studies to date have been largely descriptive. We have identified bona fide targets of BATF3 and IRF8 by comparative microarray analysis and chromatin immunoprecipitation followed by sequencing (ChIP-Seq). These targets provided insights into the specialized processes carried out by DCs and may also represent future pathways for manipulation during the development of vaccines.

We characterized the stage-specific actions of BATF3 in the development of CD8+ DCs, a critical initiator of cellular immunity to intracellular pathogens and tumors. Batf3 induction occurs after the expression of Irf8 in the precursor to classical DCs (pre-cDC), in which BATF3 acts subsequent to an Irf8-dependent lineage commitment step to induce the terminal maturation and survival of CD8+ DCs. Genomic regions bound by IRF8 and BATF3 were identified by ChIP-Seq, and notably, the proximal promoters of CD8+ DC-specific genes showed IRF8 and BATF3 co-occupancy. Furthermore, we evaluated IRF8 binding in BATF3-deficient cells to identify BATF3-independent targets. One such candidate was the Myc homolog Mycl1 (L-Myc). Analysis by quantitative real-time PCR (qPCR) of the hematopoietic compartment revealed that Mycl1 expression is restricted to pDCs and cDCs.

To evaluate the significance of L-Myc activity in dendritic cells, we generated a new knock-in mouse model of the Mycl1 locus by replacing the first coding exon with an in-frame GFP cassette. Analysis of heterozygous mice (Mycl1+/gfp) revealed that Mycl1 expression is restricted to dendritic cells. Interestingly, induction of Mycl1 occurs at the CDP to pre-cDC transition concurrent with the loss of Myc (c-Myc) from DCs. Although dispensable for the development of DCs, L-Myc supports the growth and survival of dendritic cells. Moreover, following activation of DCs with pathogen associated molecular patterns (PAMPs) or activating cytokines, L-Myc protein levels either remained constant or increased, suggesting that growth-promoting circumstances are intricately linked to levels of L-Myc. Lastly, L-Myc deficient DCs primed antigen-specific responses poorly and were incapable of supporting the intracellular growth of Listeria monocytogenes.

Collectively, these findings suggest that the switch from Myc to L-Myc expression represents a strategy of growth in the face of disparate inflammatory signals experienced during infections. L-Myc may therefore represent a therapeutic target for selective inhibition or augmentation of immune responses driven by dendritic cell

Language

English (en)

Chair and Committee

Kenneth M Murphy

Committee Members

Deepta Bhattacharya, Marco Colonna, Brian T Edelson, Robert Schreiber, Thaddeus S Stappenbeck

Comments

Permanent URL: https://doi.org/10.7936/K7959FQZ

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