Conventional type 1 dendritic cells (cDC1s 1 ) are thought to perform antigen cross-presentation required to prime CD8 T cells 2 , 3 , while cDC2 are considered specialized for priming CD4 T cells 4 , 5 . CD4 T cells are also thought to help CD8 T cell responses through a variety of mechanisms 6 – 11 , including a model in which CD4 T cells ‘license’ cDC1 for CD8 T cell priming 12 . However, this model has not been directly tested in vivo or in the setting of a help-dependent tumour rejection. Here, we generated an Xcr1 -Cre mouse strain to evaluate the cellular interactions that mediate tumour rejection in a model requiring CD4 and CD8 T cells. As expected, tumour rejection required cDC1, and expression of MHC-I by cDC1. Unexpectedly, early priming of CD4 T cell against tumour-derived antigens also required cDC1, which was not simply due to a role in antigen transport to lymph nodes for processing by cDC2, since selective deletion of MHC-II in cDC1 also prevented early CD4 T cell priming. Further, deletion of either MHC-II or CD40 in cDC1 impaired tumour rejection, consistent with a role for cognate CD4 T cell interactions and CD40 signaling in cDC1 licensing. Finally, CD40 signaling in cDC1 was critical not only for CD8 T cell priming, but also for initial CD4 T cell activation. Thus, in the setting of tumour-derived antigens, cDC1 function as an autonomous platform capable of antigen processing and priming for both CD4 and CD8 T cells and directly orchestrating their cross-talk required for optimal anti-tumour immunity.
During the process of cross presentation, viral or tumor-derived antigens are presented to CD8+ T cells by the Batf3-dependent CD8α+/XCR1+ classical dendritic cell (cDC1). We designed a functional CRISPR screen for novel regulators of cross presentation, and identified the BEACH-domain containing protein WDFY4 as essential for cross-presentation of cell-associated antigens by cDC1. WDFY4 was not, however, required for MHC class II presentation or for cross-presentation by monocyte-derived DCs. In contrast to Batf3−/− mice, Wdfy4−/− mice have normal lymphoid and non-lymphoid cDC1 populations that produce IL-12 and protect against Toxoplasma gondii infection. However similar to Batf3−/− mice, Wdfy4−/− mice fail to prime virus- specific CD8+ T cells in vivo or induce tumor rejection, revealing a critical role for cross-presentation in anti-viral and anti-tumor immunity.
NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43−CD73−DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.
Summary Both classical DCs (cDCs) and monocyte-derived DCs (Mo-DCs) are capable of cross-priming CD8+ T cells in response to cell-associated antigens. We found that Ly-6ChiTREML4− monocytes can differentiate into Zbtb46+ Mo-DCs in response to GM-CSF and IL-4, but that Ly-6ChiTREML4+ monocytes were committed to differentiate into Ly-6CloTREML4+ monocytes. Differentiation of Zbtb46+ Mo-DCs capable of efficient cross-priming required both GM-CSF and IL-4, and was accompanied by induction of Batf3 and Irf4. However, monocytes require IRF4, but not BATF3, to differentiate into Zbtb46+ Mo-DCs capable of cross-priming CD8+ T cells. Instead, Irf4−/− monocytes differentiate into macrophages in response to GM-CSF and IL-4. Thus, cDCs and Mo-DCs require distinct transcriptional programs of differentiation in acquiring the capacity to prime CD8+ T cells. These differences may be of consideration in the use of therapeutic DC vaccines based on Mo-DCs.
SummaryThe recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin+CD73−CD235a/CD43− HE and hematopoietic progenitors with myeloid and T lymphoid potential.
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