Differential IRF8 Transcription Factor Requirement Defines Two Pathways of Dendritic Cell Development in Humans

The formation of mammalian dendritic cells (DCs) is controlled by multiple hematopoietic transcription factors, including IRF8. Loss of IRF8 exerts a differential effect on DC subsets, including plasmacytoid DCs (pDCs) and the classical DC lineages cDC1 and cDC2. In humans, cDC2-related subsets have been described including AXL+ SIGLEC6+ pre-DC, DC2 and DC3. The origin of this heterogeneity is unknown. Using highdimensional analysis, in vitro differentiation, and an allelic series of human IRF8 deficiency, we demonstrated that cDC2 (CD1c+ DC) heterogeneity originates from two distinct pathways of development. The lymphoidprimed IRF8hi pathway, marked by CD123 and BTLA, carried pDC, cDC1, and DC2 trajectories, while the common myeloid IRF8lo pathway, expressing SIRPA, formed DC3s and monocytes. We traced distinct trajectories through the granulocyte-macrophage progenitor (GMP) compartment showing that AXL+ SIGLEC6+ pre-DCs mapped exclusively to the DC2 pathway. In keeping with their lower requirement for IRF8, DC3s expand to replace DC2s in human partial IRF8 deficiency

Dendritic cells in cancer immunology and immunotherapy

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.S.K.W. is supported by a European Molecular Biology Organization Long- Term Fellowship (grant ALTF 438– 2016) and a CNIC–International Postdoctoral Program Fellowship (grant 17230–2016). F.J.C. is the recipient of a PhD ‘La Caixa’ fellowship. Work in the D.S. laboratory is funded by the CNIC, by the European Research Council (ERC Consolidator Grant 2016 725091), by the European Commission (635122-PROCROP H2020), by the Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R), by the Comunidad de Madrid (B2017/BMD-3733 Immunothercan- CM), by FIS- Instituto de Salud Carlos III, MCNU and FEDER (RD16/0015/0018-REEM), by Acteria Foundation, by Atresmedia (Constantes y Vitales prize) and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III, the MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015-0505).S

Notch signaling facilitates in vitro generation of cross-presenting classical dendritic cells

Dendritic cells (DCs) comprise a heterogeneous population of mononuclear phagocytes that play a critical role in both innate and adaptive immunity. DCs in mice can be divided into two main types. Plasmacytoid DCs (pDCs) secrete type I interferons (IFN-α/β) in response to viruses. Classical or conventional dendritic cells (cDCs) are highly adept at Ag presentation. There are two main subsets of cDCs; the CD11b+ cDC subset presents exogenous Ag to CD4+ T cells on major histocompatibility complex class II (MHCII) and the CD8α+/CD103+ cDCs uniquely capable of cross-presenting exogenous Ag to CD8+ T cells on MHCI. Functional equivalents of both subsets have been identified in humans and have been designated cDC2 and cDC1, respectively. All DCs develop from progenitors found in the bone marrow (BM) by a process directed primarily by the cytokine Fms-like tyrosine kinase 3 ligand (FL). The specification of DC types is driven by several transcription factors such as IRF8, while terminal cDC differentiation is guided by tissue-specific signals mediated through signaling pathways such as Notch and lymphotoxin-β. Notch is an evolutionarily conserved pathway of cell-cell communication that plays an essential role in the development of immune cell types, including T and B lymphocytes. DC-specific gene targeting, has been used to establish the role of Notch2 receptor signaling in the differentiation of cDC2 subset in the spleen and intestine and splenic cDC1. Because primary cDCs, particularly cDC1, are rare in vivo their study and use in translational applications require methods to generate functional cDC subsets in vitro. Commonly used cultures of primary BM with the cytokines FL or granulocyte-monocyte colony stimulating factor (GM-CSF) produce a mixture of pDC, cDC2 and cDC1-like cells, or cDC2-like cells and macrophages, respectively. Thus, new approaches are needed to yield high numbers of fully differentiated cDCs, particularly of mature cDC1. Given the critical role of Notch signaling in cDC differentiation in vivo, I hypothesized that it would facilitate cDC differentiation in vitro. Indeed, coculture of murine primary BM cells with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) facilitated the generation of bona fide, IRF8-dependent CD8α+ CD103+ Dec205+ cDC1 with an expression profile resembling ex vivo cDC1. Critically, the resulting cDC1 showed improved Ccr7-dependent migration, superior T cell cross-priming capacity and antitumor vaccination in vivo. Further, OP9-DL1 cocultures of immortalized progenitors allowed for the de novo generation CD8α+ cDC1. This discovery can help further our understanding of the mechanisms of DC differentiation while providing a tool to allow for the generation of unlimited numbers of cDCs for functional studies. Further, as cDC1 are essential for the cross-priming of cytotoxic T cells against tumors, they hold great promise as cellular vaccines. However, the use of DCs in clinical applications has been hampered by inadequate methods to generate large quantities of functionally mature cDC1 in vitro. As such, these findings should help to advance the use of cDCs in translational and therapeutic applications, such as antitumor vaccination and immunotherapy

TAO-kinase 3 governs the terminal differentiation of conventional dendritic cells

Antigen-presenting conventional dendritic cells (cDCs) are broadly divided into type 1 and type 2 subsets that further adapt their phenotype and function to perform specialized tasks in the immune system. The precise signals controlling tissue-specific adaptation and differentiation of cDCs are currently poorly understood. We found that mice deficient in the Ste20 kinase Thousand and One Kinase 3 (TAOK3) lacked terminally differentiated ESAM(+) CD4(+) cDC2s in the spleen and failed to prime CD4(+) T cells in response to allogeneic red-blood-cell transfusion. These NOTCH2and ADAM10dependent cDC2s were absent selectively in the spleen, but not in the intestine of Taok3(-/-) and CD11c-cre Taok3(fl/fl) mice. The loss of splenic ESAM(+) cDC2s was cell-intrinsic and could be rescued by conditional overexpression of the constitutively active NOTCH intracellular domain in CD11c-expressing cells. Therefore, TAOK3 controls the terminal differentiation of NOTCH2-dependent splenic cDC2s