Priming of CD8+ T cells against tumors or viral pathogens in vivo primarily depends on the cross-presentation of exogenous antigens by type 1 conventional dendritic cells (cDC1s). While cDC2 or monocyte-derived DCs have been shown to cross-present in vitro, the physiological relevance of these activities in vivo remains unclear. Inthis study, we utilized various genetic models to assess the role of different cDC subsets in presenting cell-associated and immune complexed antigens to CD4+ and CD8+ T cells in vivo. For cell-associated antigens, cDC1 were necessary and sufficient for in vivo priming of both CD4+ and CD8+ T cells. In contrast, for immune complex, either cDC1 or cDC2, but not monocytes or monocyte-derived cells, could carry out cross-presentation to CD8+ T cells in vivo, with cDC2 being superior to cDC1 for priming CD4+ T cells. Mice lacking cDC1s and vaccinated with antibody-tumor antigen immune complexes still could carry out cross-presentation to CD8+ T cells that was sufficient to mediate anti-tumor response. Notably, this cross-presentation was mediated by cDC2 and WDFY4-dependent, like cross-presentation of cell-associated antigens by cDC1, but unlike cross-presentation of soluble antigens. These results demonstrate previously unrecognized activity of WDFY4 in cDC2s and suggest a common pathway for receptor-mediated cross-presentation shared by cDC subsets. NIH R01AI150297, R01CA248919, R01AI162643 and R21AI163421 Antigen and Dendritic Cell Processing, Presentation, and Biology (AGDC)

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.

Interferon regulatory factor-8 (IRF8) is the lineage determining transcription factor for the type one classical dendritic cell (cDC1) subset, a terminal selector for plasmacytoid dendritic cells and important for the function of monocytes. Studies of Irf8 gene regulation have identified several enhancers controlling its activity during development of progenitors in the bone marrow that precisely regulate expression at distinct developmental stages. Each enhancer responds to distinct transcription factors that are expressed at each stage. IRF8 is first expressed in early progenitors that form the monocyte dendritic cell progenitor (MDP) in response to induction of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) acting at the Irf8 +56 kb enhancer. IRF8 levels increase further as the MDP transits into the common dendritic cell progenitor (CDP) in response to E protein activity at the Irf8 +41 kb enhancer. Upon Nfil3-induction in CDPs leading to specification of the cDC1 progenitor, abrupt induction of BATF3 forms the JUN/BATF3/IRF8 heterotrimer that activates the Irf8 +32 kb enhancer that sustains Irf8 autoactivation throughout the cDC1 lifetime. Deletions of each of these enhancers has revealed their stage dependent activation. Surprisingly, studies of compound heterozygotes for each combination of enhancer deletions revealed that activation of each subsequent enhancer requires the successful activation of the previous enhancer in strictly cis-dependent mechanism. Successful progression of enhancer activation is finely tuned to alter the functional accessibility of subsequent enhancers to factors active in the next stage of development. The molecular basis for these phenomenon is still obscure but could have implications for genomic regulation in a broader developmental context.

Monocytes comprise two major subsets, Ly6Chi classical monocytes and Ly6Clo nonclassical monocytes. Notch2 signaling in Ly6Chi monocytes triggers transition to Ly6Clo monocytes, which require Nr4a1, Bcl6, Irf2, and Cebpb. By comparison, less is known about transcriptional requirements for Ly6Chi monocytes. We find transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) is highly expressed in Ly6Chi monocytes, but down-regulated in Ly6Clo monocytes. A few previous studies described the requirement of C/EBPα in the development of neutrophils and eosinophils. However, the role of C/EBPα for in vivo monocyte development has not been understood. We deleted the Cebpa +37 kb enhancer in mice, eliminating hematopoietic expression of C/EBPα, reproducing the expected neutrophil defect. Surprisingly, we also found a severe and selective loss of Ly6Chi monocytes, while preserving Ly6Clo monocytes. We find that BM progenitors from Cebpa +37-/- mice rapidly progress through the monocyte progenitor stage to develop directly into Ly6Clo monocytes even in the absence of Notch2 signaling. These results identify a previously unrecognized role for C/EBPα in maintaining Ly6Chi monocyte identity.

Monocytes comprise two major subsets, Ly6C hi classical monocytes and Ly6C lo nonclassical monocytes. Notch2 signaling in Ly6C hi monocytes triggers transition to Ly6C lo monocytes, which require Nr4a1 , Bcl6 , Irf2 , and Cebpb . By comparison, less is known about transcriptional requirements for Ly6C hi monocytes. We find transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) is highly expressed in Ly6C hi monocytes, but down-regulated in Ly6C lo monocytes. A few previous studies described the requirement of C/EBPα in the development of neutrophils and eosinophils. However, the role of C/EBPα for in vivo monocyte development has not been understood. We deleted the Cebpa +37 kb enhancer in mice, eliminating hematopoietic expression of C/EBPα, reproducing the expected neutrophil defect. Surprisingly, we also found a severe and selective loss of Ly6C hi monocytes, while preserving Ly6C lo monocytes. We find that BM progenitors from Cebpa +37 −/− mice rapidly progress through the monocyte progenitor stage to develop directly into Ly6C lo monocytes even in the absence of Notch2 signaling. These results identify a previously unrecognized role for C/EBPα in maintaining Ly6C hi monocyte identity.

Ly6C(lo) monocytes are a myeloid subset that specializes in the surveillance of vascular endothelium. Ly6C(lo) monocytes have been shown to derive from Ly6C(hi) monocytes. NOTCH2 signaling has been implicated as a trigger for Ly6C(lo) monocyte development, but the basis for this effect is unclear. Here, we examined the impact of NOTCH2 signaling of myeloid progenitors on the development of Ly6C(lo) monocytes in vitro. NOTCH2 signaling induced by delta-like ligand 1 (DLL1) efficiently induced the transition of Ly6C(hi) TREML4(-) monocytes into Ly6C(lo) TREML4(+) monocytes. We further identified two additional transcriptional requirements for development of Ly6C(lo) monocytes. Deletion of BCL6 from myeloid progenitors abrogated development of Ly6C(lo) monocytes. IRF2 was also required for Ly6C(lo) monocyte development in a cell-intrinsic manner. DLL1-induced in vitro transition into Ly6C(lo) TREML4(+) monocytes required IRF2 but unexpectedly could occur in the absence of NUR77 or BCL6. These results imply a transcriptional hierarchy for these factors in controlling Ly6C(lo) monocyte development.

Supplementary Figure from cDC1 Vaccines Drive Tumor Rejection by Direct Presentation Independently of Host cDC1

The events that initiate autoimmune diabetes in nonobese diabetic (NOD) mice remain poorly understood. CD4 + and CD8 + T cells are both required to develop disease, but their relative roles in initiating disease are unclear. To test whether CD4 + T cell infiltration into islets requires damage to β cells induced by autoreactive CD8 + T cells, we inactivated Wdfy4 in nonobese diabetic (NOD) mice (NOD. Wdfy4 −/−- ) using CRISPR/Cas9 targeting to eliminate cross-presentation by type 1 conventional dendritic cells (cDC1s). Similar to C57BL/6 Wdfy4 −/− mice, cDC1 in NOD. Wdfy4 −/− mice are unable to cross-present cell-associated antigens to prime CD8 + T cells, while cDC1 from heterozygous NOD. Wdfy4 +/− mice cross-present normally. Further, NOD. Wdfy4 −/− mice fail to develop diabetes while heterozygous NOD. Wdfy4 +/− mice develop diabetes similarly to wild-type NOD mice. NOD. Wdfy4 −/− mice remain capable of processing and presenting major histocompatibility complex class II (MHC-II)-restricted autoantigens and can activate β cell-specific CD4 + T cells in lymph nodes. However, disease in these mice does not progress beyond peri-islet inflammation. These results indicate that the priming of autoreactive CD8 + T cells in NOD mice requires cross-presentation by cDC1. Further, autoreactive CD8 + T cells appear to be required not only to develop diabetes, but to recruit autoreactive CD4 + T cells into islets of NOD mice, perhaps in response to progressive β cell damage.

Abstract Skin barrier dysfunction in the setting of atopic dermatitis (AD) leads to cutaneous allergen sensitization (CAS).1 The atopic march hypothesis proposes that CAS leads to the subsequent development of other allergic disorders such as asthma and food allergy.2-5 The latter is associated with IgE-mediated anaphylaxis6. However, the precise mechanisms by which CAS promotes IgE-mediated allergic responses remain unclear. We thus established a CAS model of anaphylaxis and identified a unique role of the Interleukin (IL)-13 signal in DCs to generate anaphylactic IgE responses. Single-cell RNA-seq revealed the emergence of IL-13 receptor alpha 1 chain (IL-13Rα1)-expressing type 2 conventional dendritic cells (cDC2s) in CAS. Similar DC populations were identified in allergic rhinitis humans with cedar pollan-specific IgE antibodies. Lineage-specific disruption of IL-13 signaling resulted in a marked reduction in cDC2s that highly expressed MHC class II: these cells controlled the germinal center entry of TFH cells required for IgE responses. These data reveal a unique role for IL-13 in controlling the expansion of MHCIIhi cDC2s and associated TFH cell responses in the setting of allergy.

In vitro culture of bone marrow (BM) with Fms-like tyrosine kinase 3 ligand (Flt3L) is widely used to study development and function of type 1 conventional dendritic cells (cDC1). Hematopoietic stem cells (HSCs) and many progenitor populations that possess cDC1 potential in vivo do not express Flt3 and thus may not contribute to Flt3L-mediated cDC1 production in vitro. Here, we present a KitL/Flt3L protocol that recruits such HSCs and progenitors into the production of cDC1. Kit ligand (KitL) is used to expand HSCs and early progenitors lacking Flt3 expression into later stage where Flt3 is expressed. Following this initial KitL phase, a second Flt3L phase is used to support the final production of DCs. With this two-stage culture, we achieved approximately tenfold increased production of both cDC1 and cDC2 compared to Flt3L culture. cDC1 derived from this culture are similar to in vivo cDC1 in their dependence on IRF8, ability to produce IL-12, and induction of tumor regression in cDC1-deficient tumor-bearing mice. This KitL/Flt3L system for cDC1 production will be useful in further analysis of cDC1 that rely on in vitro generation from BM.

Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified anIrf8superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kbIrf8enhancer is required for pre-cDC1 specification, while the +32-kbIrf8enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggestingcisdependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kbIrf8enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located incis. Thus, the +41-kbIrf8enhancer controls the subsequent activation of the +32-kbIrf8enhancer in a manner that is independent of associated lncRNA transcription.

Cytokines produced in association with tumors can impair antitumor immune responses by reducing the abundance of type 1 conventional dendritic cells (cDC1), but the mechanism remains unclear. Here, we show that tumor-derived IL-6 generally reduces cDC development but selectively impairs cDC1 development in both murine and human systems through the induction of C/EBPβ in the common dendritic cell progenitor (CDP). C/EBPβ and NFIL3 compete for binding to sites in the Zeb2 −165 kb enhancer and support or repress Zeb2 expression, respectively. At homeostasis, pre-cDC1 specification occurs upon Nfil3 induction and consequent Zeb2 suppression. However, IL-6 strongly induces C/EBPβ expression in CDPs. Importantly, the ability of IL-6 to impair cDC development is dependent on the presence of C/EBPβ binding sites in the Zeb2 −165 kb enhancer, as this effect is lost in Δ1+2+3 mutant mice in which these binding sites are mutated. These results explain how tumor-associated IL-6 suppresses cDC1 development and suggest therapeutic approaches preventing abnormal C/EBPβ induction in CDPs may help reestablish cDC1 development to enhance antitumor immunity.

Characterization of the functional effects of cDC2s in vivo requires model systems in which cDC2s are depleted. Previous literature has reported a loss of cDC2s in mice lacking the transcription factor IRF2 1,2 . We sought to further characterize the cDC2 defect in these animals. Here, we find that the requirement for IRF2 in cDC2 development and survival is cell-extrinsic and correlated to the development of dermatitis in the Irf2 -/- model system. We also find that Flt3L-mediated in vitro development of cDC1s and cDC2s, but not pDCs, is abrogated in Irf2 -/- bone marrow, as well as in wild-type bone marrow cultured with IFNα. Loss of interferon α (IFNα) signaling in Irf2 -/- mice restored cDC2 development in vivo and cDC1 and cDC2 development in vitro. We therefore conclude that IRF2 is required for cDC2 development in a cell-extrinsic manner dependent on IFNα signaling.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear At homeostasis, only mature CCR7(+) cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived L-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite L-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

CD40 signaling in classical type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1s, including Cd70, Tnfsf9, Ptgs2 and Bcl2l1, and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased caspase activation in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation by Cd40−/− cDC1s to CD8+ T cells, which were reversed by re-expression of Bcl2l1. Thus, CD40 signaling in cDC1s not only induces costimulatory ligands for CD8+ T cells but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses. CD40 is typically understood as a costimulatory molecule. Here, the authors show CD4+ T cell-induced CD40 signaling in conventional type 1 dendritic cells results in complicated gene expression that can enhance CD8+ T cell priming by various underappreciated and independent mechanisms.

Efforts to determine the unique functions of the two types of classical dendritic cells, cDC1 and cDC2, are hindered by limited understanding of the divergence of the common dendritic cell progenitor (CDP). Some transcription factors act in commitment of already specified progenitors, such as Batf3 which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer, but how other factors control CDP divergence remains unknown. Here, we report the transcriptional mechanism of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis suggested that Nfil3 acts upstream of Id2, Batf3, and Zeb2 in cDC1 development but has not revealed its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and ChIP-seq analysis identified NFIL3 binding in the −165 kb Zeb2 enhancer at sites that also bind CCAAT-enhancer-binding proteins (C/EBPs). Mutation of these NFIL3/C/EBP sites by in vivo CRISPR/Cas9 targeting revealed functional redundancy, with C/EBPs and NFIL3 competing in binding these sites to support or repress Zeb2 expression, respectively. Mutation of these three NFIL3/C/EBP sites eliminated Zeb2 expression in myeloid, but not lymphoid progenitors, producing mice that completely lacked pre-cDC2 specification and mature cDC2 in vivo. These mice failed to make an appropriate TH2 response against H. polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths. Notably, Zeb2 expression is not required to maintain cDC2 identity, but acts only to block cDC1 specification. Thus, the competition between C/EBPs and NFIL3 binding at the −165 kb Zeb2 enhancer determines CDP divergence.

Abstract As a cell-based cancer vaccine, dendritic cells (DC), derived from peripheral blood monocytes or bone marrow (BM) treated with GM-CSF (GMDC), were initially thought to induce antitumor immunity by presenting tumor antigens directly to host T cells. Subsequent work revealed that GMDCs do not directly prime tumor-specific T cells, but must transfer their antigens to host DCs. This reduces their advantage over strictly antigen-based strategies proposed as cancer vaccines. Type 1 conventional DCs (cDC1) have been reported to be superior to GMDCs as a cancer vaccine, but whether they act by transferring antigens to host DCs is unknown. To test this, we compared antitumor responses induced by GMDCs and cDC1 in Irf8 +32–/– mice, which lack endogenous cDC1 and cannot reject immunogenic fibrosarcomas. Both GMDCs and cDC1 could cross-present cell-associated antigens to CD8+ T cells in vitro. However, injection of GMDCs into tumors in Irf8 +32–/– mice did not induce antitumor immunity, consistent with their reported dependence on host cDC1. In contrast, injection of cDC1s into tumors in Irf8 +32–/– mice resulted in their migration to tumor-draining lymph nodes, activation of tumor-specific CD8+ T cells, and rejection of the tumors. Tumor rejection did not require the in vitro loading of cDC1 with antigens, indicating that acquisition of antigens in vivo is sufficient to induce antitumor responses. Finally, cDC1 vaccination showed abscopal effects, with rejection of untreated tumors growing concurrently on the opposite flank. These results suggest that cDC1 may be a useful future avenue to explore for antitumor therapy. See related Spotlight by Hubert et al., p. 918

The divergence of the common dendritic cell progenitor1–3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors—such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer4,6—but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the –165 kb Zeb2 enhancer8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR–Cas9 targeting showed that these NFIL3–C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3–C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (TH2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting TH2 responses to helminths9–11. Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the –165 kb Zeb2 enhancer. The transcription factor NFIL3 acts antagonistically to C/EBP proteins by binding the Zeb2 enhancer to prevent Zeb2 expression and the development of the conventional type 2 dendritic cell lineage.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2(Delta-165)mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2(Delta-165) mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2(Delta-165) mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.