Abstract Myeloid dendritic cells (DC) are a critical lineage of innate immunity that serve as a principal point of contact and crosstalk between the innate and adaptive arms of the immune system. CTLA-4 is one of the best characterized of the immune checkpoint proteins, molecules that serve to balance, regulate, and fine-tune immune activation with homeostatic inhibition. CTLA-4 is expressed by all major lymphoid lineage effectors; however, its function- ality has been best characterized in T-cells where it exhibits both cell-extrinsic and cell-intrinsic regulatory func- tions. Until recently, very little was known about CTLA-4 expression or function in non-lymphoid cell types, par- ticularly the myeloid lineage dendritic cell subsets. In the original iteration of this renewal application, we provided preliminary data demonstrating that DC-secreted CTLA-4+ exosomes could bind B7 in paracrine fashion, leading to vesicle internalization and subsequent downregulation of B7 expression among bystander DC that internalized the CTLA-4+ exosomes. Conversely, knockdown of DC-expressed CTLA-4 resulted in a dramatic upregulation of co-cultured CD8+ cell proliferation in vitro as well as enhanced antitumor and antiviral immunity in vivo. These discoveries and concomitant characterization of myeloid CTLA-4 expression signified a paradigm shift in the understanding of CTLA-4’s role in immune governance as well as the mechanisms through which innate and adaptive crosstalk occur. Subsequent data indicated that the expression of DC CTLA-4 is modulated in response to TH polarizing cues and that regulation in DC appears to be governed in part by the transcription factors GATA3 and C/EBP-b. Further, conditional ablation of CTLA-4 in the C57BL/6 background revealed potential new roles for DC expressed CTLA-4 in regulatory processes in the thymus and in other lymphoid tissues including Peyer’s patches. These novel and exciting data have allowed formulation of a refined overarching hypothesis that DC- secreted CTLA-4+ exosomes act as effector vehicles that shape downstream TH polarization of adaptive re- sponses as dictated by DC detection of innate signaling cues. By means of three independent aims we will test this overarching hypothesis by i) defining the role of TH polarizing cues in the governance of DC CTLA-4 expres- sion and the governance of the CEBP/b and GATA3 transcriptional regulators, ii) defining the mechanisms through which DC-secreted CTLA-4+ and CTLA-4neg exosomes regulate downstream adaptive TH polarization, and iii) defining the manner by which DC CTLA-4 expression modulates central tolerance by interrogating the regulatory T-cell deficits observed in the CD11c-Cre CTLA-4flox/flox C57BL/6 mouse. Completion of these inde- pendent aims will further elucidate the novel regulatory role of myeloid CTLA-4, furthering the ability to synthesize effective and powerful vaccination strategies while characterizing critical druggable target interactions and en- h...